G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
neural cell adhesion molecule 1
G00000642 (Mus musculus)

Databases (8)

ENSG00000149294 (Ensembl human gene)
4684 (Entrez Gene)
285 (G2Cdb plasticity & disease)
NCAM1 (GeneCards)
116930 (OMIM)
Marker Symbol
HGNC:7656 (HGNC)
Protein Expression
142 (human protein atlas)
Protein Sequence
P13591 (UniProt)

Synonyms (2)

  • CD56
  • NCAM

Literature (130)

Pubmed - other

  • CD94 surface density identifies a functional intermediary between the CD56bright and CD56dim human NK-cell subsets.

    Yu J, Mao HC, Wei M, Hughes T, Zhang J, Park IK, Liu S, McClory S, Marcucci G, Trotta R and Caligiuri MA

    Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, OH 43210, USA.

    Human CD56(bright) natural killer (NK) cells possess little or no killer immunoglobulin-like receptors (KIRs), high interferon-gamma (IFN-gamma) production, but little cytotoxicity. CD56(dim) NK cells have high KIR expression, produce little IFN-gamma, yet display high cytotoxicity. We hypothesized that, if human NK maturation progresses from a CD56(bright) to a CD56(dim) phenotype, an intermediary NK cell must exist, which demonstrates more functional overlap than these 2 subsets, and we used CD94 expression to test our hypothesis. CD94(high)CD56(dim) NK cells express CD62L, CD2, and KIR at levels between CD56(bright) and CD94(low)CD56(dim) NK cells. CD94(high)CD56(dim) NK cells produce less monokine-induced IFN-gamma than CD56(bright) NK cells but much more than CD94(low)CD56(dim) NK cells because of differential interleukin-12-mediated STAT4 phosphorylation. CD94(high)CD56(dim) NK cells possess a higher level of granzyme B and perforin expression and CD94-mediated redirected killing than CD56(bright) NK cells but lower than CD94(low)CD56(dim) NK cells. Collectively, our data suggest that the density of CD94 surface expression on CD56(dim) NK cells identifies a functional and likely developmental intermediary between CD56(bright) and CD94(low)CD56(dim) NK cells. This supports the notion that, in vivo, human CD56(bright) NK cells progress through a continuum of differentiation that ends with a CD94(low)CD56(dim) phenotype.

    Funded by: NCI NIH HHS: CA68458, CA95426, P01 CA095426, R01 CA068458, R37 CA068458

    Blood 2010;115;2;274-81

  • Salmonella induced IL-23 and IL-1beta allow for IL-12 production by monocytes and Mphi1 through induction of IFN-gamma in CD56 NK/NK-like T cells.

    van de Wetering D, de Paus RA, van Dissel JT and van de Vosse E

    Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.

    Background: The type-1 cytokine pathway plays a pivotal role in immunity against intracellular bacterial pathogens such as Salmonellae and Mycobacteria. Bacterial stimulation of pattern recognition receptors on monocytes, macrophages and dendritic cells initiates this pathway, and results in the production of cytokines that activate lymphocytes to produce interferon (IFN)-gamma. Interleukin (IL)-12 and IL-23 are thought to be the key cytokines required for initiating a type-1 cytokine immune response to Mycobacteria and Salmonellae. The relative contribution of IL-23 and IL-12 to this process is uncertain.

    We show that various TLR agonists induce the production of IL-23 but not IL-12 in freshly isolated human monocytes and cultured human macrophages. In addition, type 1 pro-inflammatory macrophages (Mphi1) differentiated in the presence of GM-CSF and infected with live Salmonella produce IL-23, IL-1beta and IL-18, but not IL-12. Supernatants of Salmonella-infected Mphi1 contained more IL-18 and IL-1beta as compared with supernatants of Mphi1 stimulated with isolated TLR agonists, and induced IFN-gamma production in human CD56(+) cells in an IL-23 and IL-1beta-dependent but IL-12-independent manner. In addition, IL-23 together with IL-18 or IL-1beta led to the production of GM-CSF in CD56(+) cells. Both IFN-gamma and GM-CSF enhanced IL-23 production by monocytes in response to TLR agonists, as well as induced IL-12 production.

    The findings implicate a positive feedback loop in which IL-23 can enhance its release via induction of IFN-gamma and GM-CSF. The IL-23 induced cytokines allow for the subsequent production of IL-12 and amplify the IFN-gamma production in the type-1 cytokine pathway.

    PloS one 2009;4;12;e8396

  • Aberrant expression of class III beta-tubulin in basal cell carcinoma of the skin.

    Ishida M, Kushima R and Okabe H

    Department of Clinical Laboratory Medicine, Shiga University of Medical Science, Shiga, Japan.

    Tubulin is a major component of microtubules. Class III beta-tubulin (beta III) is a neuron-associated beta-tubulin isotype and expressed in the normal central and peripheral nervous systems. According to a previous study, beta III is not expressed in normal skin and squamous cell carcinoma. However, its expression has not been examined in basal cell carcinoma (BCC) of the skin. Expression of beta III was analyzed together with neural cell adhesion molecule (NCAM), chromogranin A, synaptophysin, epithelial membrane antigen (EMA) and cytokeratin (CK) 20 by immunohistochemical methods in 10 non-neoplastic skin tissues and 50 BCCs. In the normal skin, immunoreactivity to beta III was restricted to the nerve bundles in the dermis and subcutis, no positivity was shown in epithelial cells of the epidermis and skin appendages. beta III and NCAM were expressed in 50 and 68% of BCCs, respectively, predominantly periphery of tumor nests, although the distribution of both markers was not always identical. Chromogranin A, synaptophysin and CK 20 were not expressed in any of BCCs. EMA was focally expressed in only 8% of BCCs. beta III is a potential candidate for inclusion to the panel of immunohistochemical markers to distinguish small BCCs from non-neoplastic hair buds, because non-neoplastic hair follicles are not positive for beta III.

    Oncology reports 2009;22;4;733-7

  • Mucosal remodeling in long-standing ulcerative colitis with colorectal neoplasia: significant alterations of NCAM+ or alpha-SMA+ subepithelial myofibroblasts and interstitial cells.

    Okayasu I, Yoshida T, Mikami T, Hana K, Yokozawa M, Araki K, Mitsuhashi J, Kikuchi M, Adachi E and Sada M

    Department of Pathology, Kitasato University, Kanagawa, Japan. isaokaya@med.kitasato-u.ac.jp

    Evidence has been provided in ulcerative colitis (UC) that early genomic instability of both epithelial and stromal cells is important for colorectal tumorigenesis, as well as remodeling and morphological alterations of mucosal crypts. To clarify roles of stromal cells in tumor development in UC, the present study focused on heterogeneous phenotypes of subepithelial myofibroblasts and interstitial cells, in association with mucosal remodeling. To clarify the relationship of alterations to tumorigenesis, mucosa of resected rectae from patients with UC (n= 49) and sporadic cancer (n= 10) were analyzed on immunohistochemistry and also on immunoelectron microscopy. Heterogeneous phenotypes of neural cell adhesion molecule (NCAM)+ and/or alpha-smooth muscle actin (alpha-SMA)+ subepithelial myofibroblasts and interstitial cells were demonstrated, corresponding to colonic stellate cells. Decrease of NCAM+ subepithelial myofibroblasts and interstitial cells, and increase of alpha-SMA+ interstitial cells were significant in UC with neoplasia as compared to without neoplasia. alpha-SMA+ muscularis mucosae was significantly more thickened in tumor cases. Deposits of Masson's trichrome+ and type III and I collagen in the muscularis mucosae and lamina propria appeared to increase in relation to the numbers of alpha-SMA+ interstitial cells. Mucosal remodeling with alterations of NCAM+ or alpha-SMA+ subepithelial and interstitial cells may play a critical role in UC-associated tumorigenesis.

    Pathology international 2009;59;10;701-11

  • NCAM expression induces neurogenesis in vivo.

    Boutin C, Schmitz B, Cremer H and Diestel S

    Institut de Biologie du Développement de Marseille-Luminy, UMR 6216, CNRS/Université de la Méditeranée, Campus de Luminy-case 907, Marseille cedex 9, France.

    Neural cell adhesion molecule (NCAM) plays an important role during neural development and in the adult brain, whereby most functions of NCAM have been ascribed to its unique polysialic acid (PSA) modification. Recently we presented evidence suggesting that expression of NCAM in vivo interferes with the maintenance of forebrain neuronal stem cells. We here aimed at investigating the fate of cells generated from NCAM-overexpressing stem cells in postnatal mouse brain and at elucidating the functional domains of NCAM mediating this effect. We show that ectopic expression of the NCAM140 isoform in radial glia and type C cells induces an increase in cell proliferation and consequently the presence of additional neuronal type A cells in the rostral migratory stream. A mutant NCAM protein comprising only fibronectin type III repeats and immunoglobulin-like domain 5 was sufficient to induce this effect. Furthermore, we show that the neurogenic effect is independent of PSA, as transgenic NCAM is not polysialylated in radial glia and type C cells. These results suggest that heterophilic interactions of NCAM with other components of the cell membrane must be involved.

    The European journal of neuroscience 2009;30;7;1209-18

  • Identification of new putative susceptibility genes for several psychiatric disorders by association analysis of regulatory and non-synonymous SNPs of 306 genes involved in neurotransmission and neurodevelopment.

    Gratacòs M, Costas J, de Cid R, Bayés M, González JR, Baca-García E, de Diego Y, Fernández-Aranda F, Fernández-Piqueras J, Guitart M, Martín-Santos R, Martorell L, Menchón JM, Roca M, Sáiz-Ruiz J, Sanjuán J, Torrens M, Urretavizcaya M, Valero J, Vilella E, Estivill X, Carracedo A and Psychiatric Genetics Network Group

    CIBER en Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III, Madrid, Spain.

    A fundamental difficulty in human genetics research is the identification of the spectrum of genetic variants that contribute to the susceptibility to common/complex disorders. We tested here the hypothesis that functional genetic variants may confer susceptibility to several related common disorders. We analyzed five main psychiatric diagnostic categories (substance-abuse, anxiety, eating, psychotic, and mood disorders) and two different control groups, representing a total of 3,214 samples, for 748 promoter and non-synonymous single nucleotide polymorphisms (SNPs) at 306 genes involved in neurotransmission and/or neurodevelopment. We identified strong associations to individual disorders, such as growth hormone releasing hormone (GHRH) with anxiety disorders, prolactin regulatory element (PREB) with eating disorders, ionotropic kainate glutamate receptor 5 (GRIK5) with bipolar disorder and several SNPs associated to several disorders, that may represent individual and related disease susceptibility factors. Remarkably, a functional SNP, rs945032, located in the promoter region of the bradykinin receptor B2 gene (BDKRB2) was associated to three disorders (panic disorder, substance abuse, and bipolar disorder), and two additional BDKRB2 SNPs to obsessive-compulsive disorder and major depression, providing evidence for common variants of susceptibility to several related psychiatric disorders. The association of BDKRB2 (odd ratios between 1.65 and 3.06) to several psychiatric disorders supports the view that a common genetic variant could confer susceptibility to clinically related phenotypes, and defines a new functional hint in the pathophysiology of psychiatric diseases.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2009;150B;6;808-16

  • Case-control association study of 65 candidate genes revealed a possible association of a SNP of HTR5A to be a factor susceptible to bipolar disease in Bulgarian population.

    Yosifova A, Mushiroda T, Stoianov D, Vazharova R, Dimova I, Karachanak S, Zaharieva I, Milanova V, Madjirova N, Gerdjikov I, Tolev T, Velkova S, Kirov G, Owen MJ, O'Donovan MC, Toncheva D and Nakamura Y

    Laboratory for International Alliance, RIKEN Center for Genomic Medicine, Tsurumi-ku, Yokohama, Japan.

    Background: Bipolar affective disorder (BAD) is a psychiatric illness characterized by episodes of mania and depression. Although the etiology is not clear, epidemiological studies suggest it is a result of an interaction of genetic and environmental factors. Despite of enormous efforts and abundant studies conducted, none has yet been identified definitively a gene susceptible to bipolar disorder.

    Methods: Ninety-four Bulgarian patients diagnosed with bipolar disorder and 184 Bulgarian healthy individuals, were used for genotyping of 191 single nucleotide polymorphisms (SNPs) by TaqMan and/or Invader assays. Seventeen SNPs that revealed P value less than 0.05 in the first screening were genotyped using an additional independent set of samples, consisting of 78 BAD cases and 372 controls.

    Results: After applying the Bonferonni correction on genotyping results of 172 cases and 556 controls, only one SNP, rs1800883, in the HTR5A gene revealed a significant level of P value (P=0.000097; odds ratio=1.80 (95%CI, 1.27-2.54); corrected P=0.017).

    Conclusions: Our findings suggest that HTR5A gene could play an important role in the pathogenesis of bipolar disorder in our population. However these findings should be viewed with caution and replication studies in other populations are necessary in support of these findings.

    Funded by: Medical Research Council: G0800509

    Journal of affective disorders 2009;117;1-2;87-97

  • Developmental regulation of neural cell adhesion molecule in human prefrontal cortex.

    Cox ET, Brennaman LH, Gable KL, Hamer RM, Glantz LA, Lamantia AS, Lieberman JA, Gilmore JH, Maness PF and Jarskog LF

    Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

    Neural cell adhesion molecule (NCAM) is a membrane-bound cell recognition molecule that exerts important functions in normal neurodevelopment including cell migration, neurite outgrowth, axon fasciculation, and synaptic plasticity. Alternative splicing of NCAM mRNA generates three main protein isoforms: NCAM-180, -140, and -120. Ectodomain shedding of NCAM isoforms can produce an extracellular 105-115 kilodalton soluble neural cell adhesion molecule fragment (NCAM-EC) and a smaller intracellular cytoplasmic fragment (NCAM-IC). NCAM also undergoes a unique post-translational modification in brain by the addition of polysialic acid (PSA)-NCAM. Interestingly, both PSA-NCAM and NCAM-EC have been implicated in the pathophysiology of schizophrenia. The developmental expression patterns of the main NCAM isoforms and PSA-NCAM have been described in rodent brain, but no studies have examined NCAM expression across human cortical development. Western blotting was used to quantify NCAM in human postmortem prefrontal cortex in 42 individuals ranging in age from mid-gestation to early adulthood. Each NCAM isoform (NCAM-180, -140, and -120), post-translational modification (PSA-NCAM) and cleavage fragment (NCAM-EC and NCAM-IC) demonstrated developmental regulation in frontal cortex. NCAM-180, -140, and -120, as well as PSA-NCAM, and NCAM-IC all showed strong developmental regulation during fetal and early postnatal ages, consistent with their identified roles in axon growth and plasticity. NCAM-EC demonstrated a more gradual increase from the early postnatal period to reach a plateau by early adolescence, potentially implicating involvement in later developmental processes. In summary, this study implicates the major NCAM isoforms, PSA-NCAM and proteolytically cleaved NCAM in pre- and postnatal development of the human prefrontal cortex. These data provide new insights on human cortical development and also provide a basis for how altered NCAM signaling during specific developmental intervals could affect synaptic connectivity and circuit formation, and thereby contribute to neurodevelopmental disorders.

    Funded by: NIMH NIH HHS: K08 MH001752, K08 MH001752-01A2, MH-01752, MH-64065, MH001752-01A2, P50 MH064065, P50 MH064065-06

    Neuroscience 2009;162;1;96-105

  • Clinical relevance of neuroendocrine differentiation in non-small cell lung cancer assessed by immunohistochemistry: a retrospective study on 405 surgically resected cases.

    Sterlacci W, Fiegl M, Hilbe W, Auberger J, Mikuz G and Tzankov A

    Institute for Pathology, Medical University Innsbruck, Muellerstrasse 44, 6020 Innsbruck, Austria. william.sterlacci@i-med.ac.at

    Neuroendocrine differentiation in non-small cell lung cancer is a common feature, which has caused contradictory conclusions concerning survival estimates and responsiveness to therapy. Aiming to clarify this conflict, we analyzed neuroendocrine differentiation by immunohistochemistry in 405 surgically resected non-small cell lung carcinomas using standardized tissue microarray platform and the currently recommended antibody panel consisting of chromogranin-A, synaptophysin, and neural-cell adhesion molecule. Diagnostic criteria provided by the World Health Organization were applied. Histological subtypes were primarily reclassified according to current guidelines, assisted by auxiliary immunohistochemistry. Extensive clinical data was acquired, enabling detailed clinicopathological correlation. Importantly, neuroendocrine differentiation assessed by immunohistochemistry showed no significant relation to overall survival estimates, which remained unaffected by histological subtype, neuroendocrine marker type, adjuvant therapy, and recurring disease. The only exception was a small group consisting of three large cell carcinomas, each expressing all three neuroendocrine markers and demonstrating decreased survival. In conclusion, additional immunohistochemical detection of neuroendocrine differentiation in non-small cell lung cancer is presently not of prognostic importance and does not justify a distinct consideration.

    Virchows Archiv : an international journal of pathology 2009;455;2;125-32

  • Regional and duration of illness differences in the alteration of NCAM-180 mRNA expression within the cortex of subjects with schizophrenia.

    Gibbons AS, Thomas EA and Dean B

    Rebecca L. Cooper Research Laboratories, Mental Health Research Institute of Victoria, Parkville, Victoria, Australia. a.gibbons@mhri.edu.au

    Schizophrenia has been proposed to have a neurodevelopmental aetiology. Neural Cell Adhesion Molecule 1 (NCAM1) is involved in several neurodevelopmental processes and abnormal expression of this gene has been associated in the pathology of schizophrenia and, thus, altered NCAM1 expression may be characteristic of the early stages of the illness. Alternative splicing of the NCAM1 transcript produces 3 major isoforms. Using qPCR we analysed mRNA expression of one of these isoforms; the 180 kDa isoform of NCAM1 (NCAM-180), in Brodmann Area (BA) 46, BA10 and BA17, post-mortem, from 15 subjects with a short duration of illness of schizophrenia (<7 years) and 15 control subjects. NCAM-180 mRNA expression was increased in BA46 from subjects with schizophrenia compared to controls (p=0.013). By contrast, there were no significant differences in the expression of NCAM-180 mRNA in BA10 (p=0.575) or BA17 (p=0.772). We then analysed NCAM-180 mRNA expression in BA46 from 15 subjects with a longer duration of illness of schizophrenia (>22 years) and 15 controls. There was no significant difference in NCAM-180 mRNA expression in this second cohort. This data suggests NCAM-180 mRNA expression is altered in a regionally-specific manner in schizophrenia and these changes are associated with the early period following diagnosis.

    Funded by: NIMH NIH HHS: R01 MH069696, R01 MH069696-0, R01 MH069696-01A1

    Schizophrenia research 2009;112;1-3;65-71

  • Utility of CD56 immunohistochemical studies in follow-up of plasma cell myeloma.

    Harrington AM, Hari P and Kroft SH

    Department of Pathology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

    Although 70% to 80% of plasma cell myelomas (PCMs) express CD56, few data are available on the usefulness of CD56 immunohistochemical analysis in assessing residual disease. We retrospectively reviewed 127 PCM posttreatment bone marrow (BM) specimens, classifying them as positive or negative for residual disease (independent of CD56 immunohistochemical studies) based on abnormal plasma cell (PC) morphologic features or flow cytometry (FC) and/or light chain restriction by immunohistochemical studies (conventional criteria). CD56 immunohistochemical analysis was performed on these and 20 negative lymphoma staging BM specimens. Of 127 BM specimens, 74 were positive and 53 were negative for residual PCM by conventional criteria. Of 74 BM specimens positive by conventional criteria, 59 (80%) demonstrated CD56 (strong+) PCs in clusters and/or with cytologic atypia. Of the 53 BM specimens negative by conventional criteria, 3 showed CD56 (strong+) morphologically atypical PCs in clusters or scattered. CD56 immunohistochemical analysis is useful for detecting residual PCM, particularly in morphologically equivocal cases in which light chain restriction cannot be demonstrated, and may serve as a potential response criterion.

    American journal of clinical pathology 2009;132;1;60-6

  • [Two cases of subcutaneous panniculitis-like T-cell lymphoma (CD4- CD8+ CD56-)].

    Springinsfeld G, Guillaume JC, Boeckler P, Tortel MC and Cribier B

    Service de dermatologie, hôpital civil, hôpitaux universitaires de Strasbourg, faculté de médecine, ULP, 1, place de l'Hôpital, 67091 Strasbourg cedex, France. geraldine.springinsfeld@chru-strasbourg.fr

    Background: Subcutaneous T-cell lymphoma is a rare disease and diagnosis is often difficult. We report two cases of subcutaneous T-cell lymphoma simulating panniculitis and initially treated with systemic steroids.

    Case No. 1. A 75-year-old woman, otherwise asymptomatic, presented with plaques and nodules of the legs present for 4 months. Histological and immunohistochemical analysis revealed subcutaneous T-cell lymphoma of CD4- CD8+ CD56- phenotype. Improvement was observed with systemic steroids followed by radiotherapy. Case No. 2. A 53-year-old woman presented with a 10-year history of recurrent infiltrated plaques. Lupus panniculitis was evoked but not confirmed. In the context of new panniculitis lesions, histological examination showed a dense lymphocytic infiltrate involving the fat lobules. Lymphocyte immunophenotyping and genotyping led to a diagnosis of subcutaneous T-cell lymphoma of CD4- CD8+ CD56- phenotype. Molecular analysis showed T-cell monoclonality (alpha beta). The lesions improved after few months of systemic corticosteroids. Although new nodular lesions appeared from time to time, further therapy was not deemed necessary.

    Discussion: A distinction is currently made between two types of subcutaneous T-cell lymphomas and the prognosis and therapeutic consequences differ widely. In our two patients, subcutaneous CD8+ T-cell lymphoma was diagnosed. The clinical course was indolent in both cases. Differential diagnosis with regard to lupus panniculitis can be difficult because of its slow progression. In such cases of low-grade lymphomas, first-line therapy may be limited to systemic corticosteroids.

    Annales de dermatologie et de venereologie 2009;136;3;264-8

  • Absence of polysialylated NCAM is an unfavorable prognostic phenotype for advanced stage neuroblastoma.

    Korja M, Jokilammi A, Salmi TT, Kalimo H, Pelliniemi TT, Isola J, Rantala I, Haapasalo H and Finne J

    Department of Medical Biochemistry and Genetics, University of Turku, Turku, Finland. mkorja@utu.fi

    Background: The expression of a neural crest stem cell marker, polysialic acid (polySia), and its main carrier, neural cell adhesion molecule (NCAM), have been detected in some malignant tumors with high metastatic activity and unfavorable prognosis, but the diagnostic and prognostic value of polySia-NCAM in neuroblastoma is unclear.

    Methods: A tumor tissue microarray (TMA) of 36 paraffin-embedded neuroblastoma samples was utilized to detect polySia-NCAM expression with a polySia-binding fluorescent fusion protein, and polySia-NCAM expression was compared with clinical stage, age, MYCN amplification status, histology (INPC), and proliferation index (PI).

    Results: PolySia-NCAM-positive neuroblastoma patients had more often metastases at diagnosis, and polySia-NCAM expression associated with advanced disease (P = 0.047). Most interestingly, absence of polySia-NCAM-expressing tumor cells in TMA samples, however, was a strong unfavorable prognostic factor for overall survival in advanced disease (P = 0.0004), especially when MYCN was not amplified. PolySia-NCAM-expressing bone marrow metastases were easily detected in smears, aspirates and biopsies.

    Conclusion: PolySia-NCAM appears to be a new clinically significant molecular marker in neuroblastoma, hopefully with additional value in neuroblastoma risk stratification.

    BMC cancer 2009;9;57

  • Regulation of the CD56 promoter and its association with proliferation, anti-apoptosis and clinical factors in multiple myeloma.

    Damgaard T, Knudsen LM, Dahl IM, Gimsing P, Lodahl M and Rasmussen T

    Department of Haematology 54P4, Herlev University Hospital of Copenhagen, Herlev, Denmark. tdamgaard@sund.ku.dk

    Multiple myeloma (MM) is an incurable B-cell malignancy characterised by uncontrolled growth and accumulation of malignant plasma cells in the bone marrow. Aberrant expression of CD56 in patients with MM is thought to contribute to a worsened disease course and metastasis. We therefore investigated the regulation of the CD56 promoter in relation to typical clinical factors. We used qPCR and FACS to measure the expression levels of CD56, and potential regulatory factors in patients with MM and related these with MM progression/prognosis. The transcription factors BTBD3, Pax5, RUNX1 and MMSET were positively associated with CD56 expression, as was CYCLIN D1, which is involved in disease progression, anti-apoptosis and proliferation. RUNX1 was negatively associated with the survival of stem-cell transplanted patients. Our findings propose four potential activators of the CD56 promoter and for CD56 to be involved in proliferation and anti-apoptosis, leading to disease progression in MM.

    Leukemia & lymphoma 2009;50;2;236-46

  • Diagnostic utility of CD56 immunohistochemistry in papillary carcinoma of the thyroid.

    El Demellawy D, Nasr AL, Babay S and Alowami S

    University of Northern Ontario School of Medicine, Thunder Bay Regional Health Sciences Centre, Department of Pathology and Laboratory Medicine, Suite 1720, 980 Oliver Road, Thunder Bay, Ontario P7J1A7, Canada. eldemeld@tbh.ne

    Diagnosis of papillary thyroid carcinoma (PTC), in many but not all cases, is an easily achievable diagnosis with almost minimal interobservable variability between pathologists. However, some cases of PTC, particularly the follicular variant, are quite challenging and show wide interobservable variability even among expert thyroid pathologists. Since proper diagnosis of PTC is crucial as it affects patients' clinical management and prognosis, indications of PTC must be clearly apparent to be an objective rather than a subjective diagnosis. Unfortunately, to date, immunohistochemistry and molecular studies have failed to fully solve this problem. In this study, we assessed the protein expression and loss using antibodies against CD56 in normal follicular thyroid epithelium, follicular thyroid lesions, and follicular thyroid neoplasms in an attempt to evaluate its diagnostic value. A total of 185 cases were studied with tissues from 75 carcinomas (72 papillary, 2 follicular, 1 Hürthle cell) and 35 adenomas (32 follicular and 3 Hürthle cell) evaluated by immunohistochemistry for the expression of this marker. Non-neoplastic thyroids included 65 cases: nodular hyperplasia (n=25), thyrotoxic hyperplasia (Grave's disease) (n=5), lymphocytic thyroiditis (n=19), and Hashimoto's thyroiditis (n=6). Ten cases of normal thyroids from radical laryngectomies for laryngeal squamous cell carcinomas were also studied. The marker pattern and intensity of staining were scored. Positive expression of the markers in 10% or more of follicular epithelium within the tumor or lesional cells was considered positive. An expression of <10% was considered to be negative. Diffuse CD56 expression was consistently present in normal, lesional, and neoplastic follicular epithelium, except for PTC, including the follicular variant. We concluded that CD56 is of value to distinguish PTC from other thyroid follicular pathology/histology with a sensitivity of 100% and a specificity of 100%. We suggest that CD56 is extremely useful in the diagnosis of PTC, including the follicular variant, and to distinguish it from other follicular cell-derived thyroid tumors/lesions. Application of CD56 by a group of expert pathologists on a larger series of follicular thyroid neoplasms of uncertain malignant potentials may potentially provide an objective diagnostic tool.

    Pathology, research and practice 2009;205;5;303-9

  • A peptide motif from the second fibronectin module of the neural cell adhesion molecule, NCAM, NLIKQDDGGSPIRHY, is a binding site for the FGF receptor.

    Jacobsen J, Kiselyov V, Bock E and Berezin V

    Protein Laboratory, Department of Neuroscience and Pharmacology, Panum Institute, University of Copenhagen, Building 24.2, Blegdamsvej 3, 2200, Copenhagen N, Denmark.

    The mechanism of fibroblast growth factor receptor (FGFR) activation by the neural cell adhesion molecule (NCAM) is not well understood. A motif in the second NCAM fibronectin type III (FN3) module, termed FGL, has by means of nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) analyses been demonstrated to be involved in NCAM-FGFR interactions. An FGFR activation motif (FRM) in the first NCAM FN3 module also has been suggested to take part in NCAM interactions with FGFR. Here, we show for the first time that a peptide motif in the second NCAM FN3 module, different from the previously described FGL motif (NLIKQDDGGSPIRHY; termed BCL) binds and activates FGFR and induces FGFR-dependent neurite outgrowth in cultures of cerebellar granule neurons. Our results provide evidence that the BCL motif is one of the multiple FGFR binding sites in NCAM.

    Neurochemical research 2008;33;12;2532-9

  • Haplotypic variants in DRD2, ANKK1, TTC12, and NCAM1 are associated with comorbid alcohol and drug dependence.

    Yang BZ, Kranzler HR, Zhao H, Gruen JR, Luo X and Gelernter J

    Department of Psychiatry, Division of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06516, USA.

    Background: Each gene in the chromosome 11q23 cluster of NCAM1, TTC12, ANKK1, and DRD2 is functionally linked to dopamine in brain. Many association studies of DRD2 and substance dependence (SD), including alcohol dependence (AD) and drug dependence (DD), have been reported; the results have been inconsistent. Recent association studies have considered this cluster more comprehensively, examining the association of SD with several risk variants mapped to the other genes in the cluster. Because comorbid AD with DD (AD+DD) is common, we hypothesized that heterogeneity of the SD diagnoses studied might have contributed to the inconsistency of prior results.

    Methods: We conducted 2 separate association studies of AD+DD and AD without DD (AD-only) in 1,090 European-Americans using family-based and case-control designs and 43 single nucleotide polymorphisms mapped to this cluster. We used a generalized linear model and haplotype score tests for the case-control sample, and the family-based association test for the family sample.

    Results: For AD+DD, the risk regions centered on TTC12 exon 3 [optimal individual haplotype simulated p (p(oihs)) = 0.000015], and another extended from ANKK1 exon 8 to DRD2;C957T (p(oihs) = 0.0028), in both samples. NCAM1 exon 12 markers showed global significance in both designs, but were significant for specific haplotypes (p(oihs) = 0.0029) only for the family sample. For AD-only, NCAM1 intron 14 to 18 and the junction of ANKK1 and DRD2 were associated globally. Population stratification was excluded as the basis for these results. Linkage disequilibrium contrast tests supported selection at TTC12 exon 3 and ANKK1 exon 2.

    Conclusions: We conclude that variants in TTC12 exon 3, NCAM1 exon 12, and the two 3'-ends of ANKK1 and DRD2 co-regulate risk for comorbid AD and DD.

    Funded by: NCRR NIH HHS: M01 RR006192, M01 RR006192-155299, M01 RR06192, RR006192-155299; NIAAA NIH HHS: AA012870-06, AA013732-04, AA016015-05, K08 AA013732, K08 AA013732-04, K08 AA13732, K24 AA013736, K24 AA013736-09, K24 AA13736, P50 AA012870, P50 AA012870-06, P50 AA12870, P60 AA003510, P60 AA003510-34, R01 AA011330, R01 AA011330-11, R01 AA016015, R01 AA016015-05, R01 AA11330; NIDA NIH HHS: K24 DA015105, K24 DA015105-05, K24 DA15105, R01 DA012690, R01 DA012690-09, R01 DA012849, R01 DA012849-10, R01 DA12690, R01 DA12849; None: R01 DA012849-10

    Alcoholism, clinical and experimental research 2008;32;12;2117-27

  • Universal expression of cell adhesion molecule NCAM in neuroblastoma in contrast to L1: implications for different roles in tumor biology of neuroblastoma?

    Wachowiak R, Rawnaq T, Metzger R, Quaas A, Fiegel H, Kähler N, Rolle U, Izbicki JR, Kaifi J and Till H

    Department of Pediatric Surgery, University of Leipzig, Liebigstrasse 20A, 04103, Leipzig, Germany. robin.wachowiak@medizin.uni-leipzig.de

    Purpose: Neuroblastoma is a biological, genetic and morphological heterogeneous tumor with a variable clinical course. NCAM is a cell adhesion molecule belonging to the immunoglobulin superfamily with structural similarities to cell adhesion molecule L1. The aim of this study was to determine the expression of NCAM in neuroblastoma and to compare the results to the findings of a previous study which examined L1 expression in the same group of patients.

    NCAM expression was investigated on a tissue array with 66 surgically resected neuroblastoma samples by immunohistochemistry with a monoclonal antibody clone 1B6 and peroxidase method.

    Results: Strong expression of NCAM was detected in all of the 66 (100%) neuroblastoma tumors in contrast to L1 which was not expressed in all tumors.

    Conclusion: In contrast to L1, which was found to predict favorable outcome, NCAM is universally expressed in neuroblastoma. Therefore NCAM represents a marker for neuroblastomas irrespectively of their stages whereas L1 as an indicator for developing neuronal cells seems to identify more mature stages of this tumor. The high grade of NCAM expression might present a prerequisite for establishment of antibody-based therapies.

    Pediatric surgery international 2008;24;12;1361-4

  • Polysialylated-neural cell adhesion molecule (PSA-NCAM) in the human trigeminal ganglion and brainstem at prenatal and adult ages.

    Quartu M, Serra MP, Boi M, Ibba V, Melis T and Del Fiacco M

    Department of Cytomorphology, University of Cagliari, Cittadella Universitaria di Monserrato, Monserrato (Cagliari), Italy. quartu@unica.it

    Background: The polysialylated neuronal cell adhesion molecule (PSA-NCAM) is considered a marker of developing and migrating neurons and of synaptogenesis in the immature vertebrate nervous system. However, it persists in the mature normal brain in some regions which retain a capability for morphofunctional reorganization throughout life. With the aim of providing information relevant to the potential for dynamic changes of specific neuronal populations in man, this study analyses the immunohistochemical occurrence of PSA-NCAM in the human trigeminal ganglion (TG) and brainstem neuronal populations at prenatal and adult age.

    Results: Western blot analysis in human and rat hippocampus supports the specificity of the anti-PSA-NCAM antibody and the immunodetectability of the molecule in postmortem tissue. Immunohistochemical staining for PSA-NCAM occurs in TG and several brainstem regions during prenatal life and in adulthood. As a general rule, it appears as a surface staining suggestive of membrane labelling on neuronal perikarya and proximal processes, and as filamentous and dot-like elements in the neuropil. In the TG, PSA-NCAM is localized to neuronal perikarya, nerve fibres, pericellular networks, and satellite and Schwann cells; further, cytoplasmic perikaryal staining and positive pericellular fibre networks are detectable with higher frequency in adult than in newborn tissue. In the adult tissue, positive neurons are mostly small- and medium-sized, and amount to about 6% of the total ganglionic population. In the brainstem, PSA-NCAM is mainly distributed at the level of the medulla oblongata and pons and appears scarce in the mesencephalon. Immunoreactivity also occurs in discretely localized glial structures. At all ages examined, PSA-NCAM occurs in the spinal trigeminal nucleus, solitary nuclear complex, vestibular and cochlear nuclei, reticular formation nuclei, and most of the precerebellar nuclei. In specimens of different age, the distribution pattern remains fairly steady, whereas the density of immunoreactive structures and the staining intensity may change and are usually higher in newborn than in adult specimens.

    Conclusion: The results obtained show that, in man, the expression of PSA-NCAM in selective populations of central and peripheral neurons occurs not only during prenatal life, but also in adulthood. They support the concept of an involvement of this molecule in the structural and functional neural plasticity throughout life. In particular, the localization of PSA-NCAM in TG primary sensory neurons likely to be involved in the transmission of protopathic stimuli suggests the possible participation of this molecule in the processing of the relevant sensory neurotransmission.

    BMC neuroscience 2008;9;108

  • Placental expression of insulin-like growth factor-I, fibroblast growth factor-basic, and neural cell adhesion molecule in preeclampsia.

    Ozkan S, Vural B, Filiz S, Coştur P and Dalçik H

    Department of Obstetrics and Gynecology, School of Medicine, Kocaeli University, Kocaeli, Turkey. sozkan1972@yahoo.com

    Objective: To investigate placental expression of insulin-like growth factor-I (IGF-I), fibroblast growth factor-basic (FGF-b), and neural cell adhesion molecule (N-CAM) in preeclampsia.

    An immunohistochemical analysis using IGF-I, FGF-b, and N-CAM antibodies was conducted on 4% paraformaldehyde-fixed placental tissues of preeclamptic patients (N = 14) and normotensive pregnant subjects (N = 10). Immunostaining patterns of chorionic villi and amniochorionic membranes were assessed.

    Results: Significantly increased FGF-b and N-CAM immunoreactivities in cytotrophoblasts and increased FGF-b immunoreactivity in capillary endothelium of chorionic villi of preeclamptic subjects were noted. Significantly increased FGF-b and decreased N-CAM immunoreactivities in extravillous trophoblasts and decidual cells of amniochorionic membranes obtained from preeclamptic subjects were demonstrated. Additionally, a significantly increased IGF-I immunoreactivity was shown in decidual cells of preeclamptic cases.

    Conclusion: Investigation of the regional distribution of IGF-I, FGF-b, and N-CAM at the maternal-fetal interface establishes a better understanding of cell-specific altered growth processes, which may be associated with the pathogenesis of preeclampsia.

    The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians 2008;21;11;831-8

  • No genetic association between NCAM1 gene polymorphisms and schizophrenia in the Chinese population.

    Xu Z, He Z, Huang K, Tang W, Li Z, Tang R, Xu Y, Feng G, He L and Shi Y

    Bio-X Center, Institute of Neuropsychiatric Science and Systems Biological Medicine, Shanghai Jiao Tong University, Shanghai 200042, PR China.

    Background: The neural cell adhesion molecule 1(NCAM1, aliases NCAM and CD56) is a cell-surface molecule which makes homophilic adhesion between neural cells involved in cell migration, axon outgrowth and synaptic plasticity. Recent studies reported that NCAM1 might act as a candidate schizophrenia susceptibility gene.

    Method: We genotyped five SNPs (rs1943620, rs1836796, rs1821693, rs686050, rs584427) within the NCAM1 gene and conducted a case-control study in 288 schizophrenic patients and 288 healthy subjects in the Chinese Han population. We compared allele and genotype frequencies and haplotype distributions between cases and controls.

    Result: No significant differences in allele and genotype frequencies were found for each single SNP between schizophrenic patients and healthy subjects. Moreover, there were no significant differences in haplotype distributions between cases and controls (global chi2=1.318, P=0.725, df=3).

    Conclusion: Our study suggests that the five SNPs within NCAM1 gene we studied may not play a major role in the schizophrenia susceptibility in the Chinese Han population.

    Progress in neuro-psychopharmacology & biological psychiatry 2008;32;7;1633-6

  • CD56bright cells increase expression of {alpha}4 integrin at ovulation in fertile cycles.

    Peralta CG, Han VK, Horrocks J, Croy BA and van den Heuvel MJ

    Department of Pediatrics, University of Western Ontario, Stratford, ON, Canada N5A 6S6.

    Leukocyte content of human endometrium changes rapidly after ovulation, particularly as a result of gains in CD56(bright) uterine NK (uNK) cells. We have proposed that uNK precursor cells are found within the blood CD56(bright) pool and are recruited to decidualizing endometrium through functional changes in their adhesion molecules and chemokine receptors. This study sought to quantify alterations in adhesion molecules, cytokines, chemokines, and receptors induced in circulating CD56(+) cells of fertile and infertile women by ovulation. Blood was drawn from 12 fertile volunteers and six female-infertility patients at Menstrual Cycle Day (d) 5 and on the day following the preovulatory surge of luteinizing hormone (LH). CD56(bright), CD56(dim), and CD56(+)CD3(+) cell subsets were isolated and evaluated by flow cytometry, quantitative PCR, or Western blotting. In CD56(bright) cells from fertile but not infertile women, alpha(4) integrin increased between d5 and the preovulatory LH surge. CD56(dim) and NKT cells did not show a change in alpha(4) integrin but differed significantly between fertile and infertile donors, and infertile donors had reduced homing molecule expression in CD56(dim) and NKT cells, and at ovulation, their NKT cells showed elevated cytokine production. None of the circulating CD56(+) cell subsets had transcripts for receptors for estrogen, progesterone, LH, or prolactin. Thus, immunological events associated with the LH surge induce alterations in all subsets of CD56(+) cells, and the unique induction of alpha(4) integrin in CD56(bright) cells of fertile women constitutes a potential method to promote uterine homing.

    Funded by: Canadian Institutes of Health Research: 67956; None: 67956

    Journal of leukocyte biology 2008;84;4;1065-74

  • Neural cell adhesion molecule expression in the human carotid body.

    Porzionato A, Vigato E, Macchi V, Parenti A and De Caro R

    Section of Anatomy, Department of Human Anatomy and Physiology, University of Padova, Italy.

    We studied by immunocytochemistry the expression of NCAM in human carotid bodies, sampled at autopsy from 16 adult subjects (mean age +/- SD: 44.3 +/- 3.4 years). No NCAM immunoreactivity was visible in type II cells. An high percentage (78.3 +/- 7.2%) of type I cells showed positive anti-NCAM immunoreaction. Statistically significant differences were not found in anti-NCAM immunostaining of light and dark cells (80.2 +/- 6.2% vs 74.7 +/- 13.4%, P>0.05). The high expression level of NCAM in the carotid body indicates a role in regulating adhesion between type I cells. The ascertained role of NCAM in neural mechanisms of differentiation, survival and cell plasticity suggests a possible involvement in the development/differentiation process of the carotid body and in determining cellular/molecular changes due to chronic hypoxia.

    Italian journal of anatomy and embryology = Archivio italiano di anatomia ed embriologia 2008;113;4;249-56

  • Association between high expression of natural killer related-genes (NCAM/CD94) and early death during induction in children with acute myeloid leukemia.

    Lonigro L, Mirabile E, Munda S, Barchitta M, Bottino D, Fazzio A, Di Cataldo A, Agodi A and Schilirò G

    Leukemia 2008;22;9;1778-81

  • NCAM as a cystogenesis marker gene of PKD2 overexpression.

    Yoo KH, Lee TY, Yang MH, Park EY, Yook YJ, Lee HS and Park JH

    Department of Biological Science, Sookmyung Women's University, Seoul 140-742, Korea.

    ADPKD (Autosomal Dominant Polycystic Kidney Disease) is characterized by the progressive expansion of multiple cystic lesions in the kidneys. ADPKD is caused by mutations in Ed-pl. consider PKD1 and PKD2. Recently a relation between c-myc and the pathogenesis of ADPKD was reported. In addition, c-Myc is a downstream effector of PKD1. To identify the gene regulated by PKD2 and c-Myc, we performed gene expression profiling in PKD2 and c-Myc overexpressing cells using a human 8K cDNA microarray. NCAM (neuronal cell adhesion molecule) levels were significantly reduced in PKD2 overexpressing systems in vitro and in vivo. These results suggest that NCAM is an important molecule in the cystogenesis induced by PKD2 overexpression.

    BMB reports 2008;41;8;593-6

  • An unusual CD56(bright) CD16(low) NK cell subset dominates the early posttransplant period following HLA-matched hematopoietic stem cell transplantation.

    Dulphy N, Haas P, Busson M, Belhadj S, Peffault de Latour R, Robin M, Carmagnat M, Loiseau P, Tamouza R, Scieux C, Rabian C, Di Santo JP, Charron D, Janin A, Socié G and Toubert A

    Institut National de la Santé et de la Recherche Médicale, U662, Paris, France. nicolas.dulphy@univ-paris-diderot.fr

    The expansion of the cytokine-producing CD56(bright) NK cell subset is a main feature of lymphocyte reconstitution after allogeneic hematopoietic stem cell transplantation (HSCT). We investigated phenotypes and functions of CD56(bright) and CD56(dim) NK subsets from 43 HLA-matched non-T cell-depleted HSCT donor-recipient pairs. The early expansion of CD56(bright) NK cells gradually declined in the posttransplant period but still persisted for at least 1 year and was characterized by the emergence of an unusual CD56(bright)CD16(low) subset with an intermediate maturation profile. The activating receptors NKG2D and NKp46, but also the inhibitory receptor NKG2A, were overexpressed compared with donor CD56(bright) populations. Recipient CD56(bright) NK cells produced higher amounts of IFN-gamma than did their respective donors and were competent for degranulation. Intracellular perforin content was increased in CD56(bright) NK cells as well as in T cells compared with donors. IL-15, the levels of which were increased in the posttransplant period, is a major candidate to mediate these changes. IL-15 serum levels and intracellular T cell perforin were significantly higher in recipients with acute graft-vs-host disease. Altogether, CD56(bright) NK cells postallogeneic HSCT exhibit peculiar phenotypic and functional properties. Functional interactions between this subset and T cells may be important in shaping the immune response after HSCT.

    Journal of immunology (Baltimore, Md. : 1950) 2008;181;3;2227-37

  • Isopentenyl pyrophosphate-activated CD56+ {gamma}{delta} T lymphocytes display potent antitumor activity toward human squamous cell carcinoma.

    Alexander AA, Maniar A, Cummings JS, Hebbeler AM, Schulze DH, Gastman BR, Pauza CD, Strome SE and Chapoval AI

    Department of Otorhinolaryngology-Head and Neck Surgery, University of Maryland School of Medicine, MD 21201, USA.

    Purpose: The expression of CD56, a natural killer cell-associated molecule, on alphabeta T lymphocytes correlates with their increased antitumor effector function. CD56 is also expressed on a subset of gammadelta T cells. However, antitumor effector functions of CD56(+) gammadelta T cells are poorly characterized.

    To investigate the potential effector role of CD56(+) gammadelta T cells in tumor killing, we used isopentenyl pyrophosphate and interleukin-2-expanded gammadelta T cells from peripheral blood mononuclear cells of healthy donors.

    Results: Thirty to 70% of expanded gammadelta T cells express CD56 on their surface. Interestingly, although both CD56(+) and CD56(-) gammadelta T cells express comparable levels of receptors involved in the regulation of gammadelta T-cell cytotoxicity (e.g., NKG2D and CD94), only CD56(+) gammadelta T lymphocytes are capable of killing squamous cell carcinoma and other solid tumor cell lines. This effect is likely mediated by the enhanced release of cytolytic granules because CD56(+) gammadelta T lymphocytes expressed higher levels of CD107a compared with CD56(-) controls following exposure to tumor cell lines. Lysis of tumor cell lines is blocked by concanamycin A and a combination of anti-gammadelta T-cell receptor + anti-NKG2D monoclonal antibody, suggesting that the lytic activity of CD56(+) gammadelta T cells involves the perforin-granzyme pathway and is mainly gammadelta T-cell receptor/NKG2D dependent. Importantly, CD56-expressing gammadelta T lymphocytes are resistant to Fas ligand and chemically induced apoptosis.

    Conclusions: Our data indicate that CD56(+) gammadelta T cells are potent antitumor effectors capable of killing squamous cell carcinoma and may play an important therapeutic role in patients with head and neck cancer and other malignancies.

    Funded by: NCI NIH HHS: CA113261, R01 CA113261, R01 CA113261-01A2, R01 CA113261-02, R01 CA113261-02S1, R01 CA113261-03, R01 CA113261-04; NIAID NIH HHS: T32 AI007540

    Clinical cancer research : an official journal of the American Association for Cancer Research 2008;14;13;4232-40

  • Clinicopathological features of extramedullary recurrence/relapse of multiple myeloma.

    Cerny J, Fadare O, Hutchinson L and Wang SA

    Division of Hematology Oncology, Department of Internal Medicine, UMass Memorial Medical Center, University of Massachusetts School of Medicine, Worcester, MA 01605, USA.

    Extramedullary relapses of multiple myeloma (MM) during the course of disease are rare. We report a series of six patients with primary intramedullary MM that were treated with immunomodulatory therapy and/or stem cell transplant, and that later developed extramedullary relapses at various body sites. These six cases represent 3.9% of the 156 patients treated for MM at our institution over a 9-yr period (1999-2007). Five (83.3%) of the six cases showed immature/high-grade histology in the extramedullary relapses as compared with their antecedent MM. The neural cell adhesion molecule, CD56, was immunohistochemically demonstrable in 75% (three of four) of the original myelomas tested, but was absent in 83.3% (five of six) of their extramedullary relapses. The disease typically behaved aggressively and was rapidly fatal in all six patients even when therapy was administered. The median time of progression to extramedullary relapse was 29 months (range 9-64 months), and the median survival after diagnosis of the relapses was only 38 d (range 1-106 d). Our case series shows that extramedullary relapse of MM is characterized by high-grade histology, loss of CD56 expression, frequent resistance to current therapeutic regimens, aggressive biological behavior, and very short survival.

    Funded by: NCI NIH HHS: P30 CA016672

    European journal of haematology 2008;81;1;65-9

  • Neural cell adhesion molecule (NCAM) isoform expression is associated with neuroblastoma differentiation status.

    Winter C, Pawel B, Seiser E, Zhao H, Raabe E, Wang Q, Judkins AR, Attiyeh E and Maris JM

    Division of Oncology, Children's Hospital of Philadelphia and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

    Background: NCAM is a member of the immunoglobulin superfamily of cell adhesion molecules. While highly expressed on neuroblastoma cells, the relative contribution of the three major NCAM isoforms (120, 140, and 180 kDa) to neuroblastoma biology has not been investigated.

    Methods: NCAM protein expression was measured in a neuroblastic tumor tissue microarray (N = 185) by immunohistochemistry. Relative expression of NCAM mRNA isoforms was measured in a panel of 24 human neuroblastomas and compared to fetal and adult human brain using real-time quantitative PCR and Western blot analysis. Associations with clinical and tumor biological co-variates were performed.

    Results: NCAM protein was detected on all neuroblastic tumors and was highly expressed in all but 7/167 cases. The mRNA species predicted to encode the 120 kDa protein species was the most abundant isoform in adult brain, ganglioneuromas and ganglioneuroblastomas (P = 0.0007), but the mRNA predicted to encode the 180 kDa species was predominant in neuroblastomas (P = 0.043). Microdissected ganglion and neuroblast cells from human primary tumors confirmed these findings.

    Conclusion: Ganglioneuromas and ganglioneuroblastomas express the adhesive 120 kDa NCAM isoform, while neuroblastomas preferentially express the 180 kDa isoform classically involved in cell motility. These data suggest a mechanism for the enhanced metastatic potential of undifferentiated neuroblastomas.

    Pediatric blood & cancer 2008;51;1;10-6

  • Placental expression of insulin-like growth factor-I, fibroblast growth factor-basic and neural cell adhesion molecule in pregnancies with small for gestational age fetuses.

    Ozkan S, Vural B, Dalçik C, Taş A and Dalçik H

    Department of Obstetrics and Gynecology, School of Medicine, Kocaeli University, Kocaeli, Turkey. sozkan1972@yahoo.com

    Objective: To investigate placental expression of insulin-like growth factor-I (IGF-I), fibroblast growth factor-basic (FGF-b) and neural cell adhesion molecule (N-CAM) regarding the pathogenesis of pregnancies with small for gestational age (SGA) fetuses.

    An immunohistochemical analysis using anti-IGF-I, anti-FGF-b and anti-N-CAM antibodies was carried out on 4% paraformaldehyde-fixed placental tissues of third trimester pregnancies complicated with SGA fetuses (n=12) and subjects exhibiting appropriately grown fetuses (n=10). Immunostaining patterns of chorionic villi and amniochorionic membranes were assessed.

    Result: IGF-I, FGF-b and N-CAM immunostainings in chorionic villi demonstrated significantly increased immunoreactivities in cytotrophoblasts of SGA cases, whereas increased IGF-I immunostaining in syncitiotrophoblasts and increased N-CAM immunostaining in capillary endothelium were noted in the same group. IGF-I, FGF-b and N-CAM immunostainings in amniochorionic membranes revealed significantly decreased IGF-I immunoreactivities in extravillous trophoblasts and increased IGF-I immunoreactivities in decidual cells of SGA cases, while significantly decreased N-CAM immunoreactivities in both decidual cells and extravillous trophoblasts were noted. FGF-b immunostaining revealed no significant differences in both extravillous trophoblasts and decidual cells of SGA cases.

    Conclusion: Increased placental expression of IGF-I, FGF-b and N-CAM may act in an autocrine and/or paracrine manner to restore the impaired trophoblastic proliferation, migration and metabolism at all gestational stages by means of a positive feedback mechanism.

    Journal of perinatology : official journal of the California Perinatal Association 2008;28;7;468-74

  • Expression of CD56 and WT1 in ovarian stroma and ovarian stromal tumors.

    He H, Luthringer DJ, Hui P, Lau SK, Weiss LM and Chu PG

    Division of Pathology, City of Hope National Medical Center, Los Angeles, CA, USA.

    The immunophenotype of ovarian stroma and spindle cell tumors derived from ovarian stroma has not been well studied. We studied the expression of CD56, WT1, estrogen receptor-beta (ER-beta), progesterone receptor (PR), smooth muscle actin, S-100, CD34, and muscle specific actin in 16 normal ovaries, 17 ovarian fibromas, 11 ovarian cellular fibromas, 10 ovarian fibrothecomas, and 11 ovarian leiomyomas. In addition, we studied CD56 and WT1 expression in 7 cases of normal endometrium, 8 uterine smooth muscle tumors, 5 endometrial stromal tumors and 64 nongynecologic (GYN) spindle cell sarcomas. All normal ovaries, ovarian fibromas, fibrothecomas, and ovarian leiomyomas were positive for CD56 and WT1. Most of the normal ovaries, ovarian fibromas, ovarian fibrothecomas, and ovarian leiomyomata also expressed ER-beta and PR. Eight of 17 ovarian fibromas, 5 of 11 ovarian cellular fibromas, and 4 of 10 ovarian fibrothecoma with focal fibroblastic differentiation were positive for smooth muscle actin. A few cases of these tumors also expressed S-100 and CD34. Only rare cases of non-GYN spindle cell sarcomas expressed WT1. Our study results show that ovarian fibromas, fibrothecomas, and leiomyomas have a similar immunophenotype (positive for CD56, WT1, ER-beta, and PR) to that of ovarian stromal cells, supporting an ovarian stromal origin for these neoplasms. However, unlike normal ovarian stromal cells, ovarian fibromas, fibrothecomas, and leiomyomas can also show fibroblastic, smooth muscle, Schwannian, and solitary fibrous tumorlike differentiation. WT1 is a fairly specific marker for spindle cell tumors of gynecologic organs, including ovarian spindle cell tumors, endometrial stromal tumors, and uterine smooth muscle tumors. Non-GYN spindle cell sarcomas rarely express WT1. CD56 is strongly expressed in ovarian stromal cells but not in endometrial stromal cells. CD56 is often expressed by a wide variety of spindle cell sarcomas, thus, it has no value in differentiating GYN from non-GYN spindle cell tumors.

    The American journal of surgical pathology 2008;32;6;884-90

  • Insights into GFRalpha1 regulation of neural cell adhesion molecule (NCAM) function from structure-function analysis of the NCAM/GFRalpha1 receptor complex.

    Sjöstrand D and Ibáñez CF

    Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, S-171-77 Stockholm, Sweden.

    The neural cell adhesion molecule NCAM binds glial cell line-derived neurotrophic factor (GDNF) through specific determinants located in its third immunoglobulin (Ig) domain. However, high affinity GDNF binding and downstream signaling depend upon NCAM co-expression with the GDNF co-receptor GFRalpha1. GFRalpha1 promotes high affinity GDNF binding to NCAM and down-regulates NCAM-mediated homophilic cell adhesion, but the mechanisms underlying these effects are unknown. NCAM and GFRalpha1 interact at the plasma membrane, but the molecular determinants involved have not been characterized nor is it clear whether their interaction is required for GFRalpha1 regulation of NCAM function. We have investigated the structure-function relationships underlying GFRalpha1 binding to NCAM in intact cells. The fourth Ig domain of NCAM was both necessary and sufficient for the interaction of NCAM with GFRalpha1. Moreover, although the N-terminal domain of GFRalpha1 had previously been shown to be dispensable for GDNF binding, we found that it was both necessary and sufficient for the efficient interaction of this receptor with NCAM. GFRalpha1 lacking its N-terminal domain was still able to potentiate GDNF binding to NCAM and assemble into a tripartite receptor complex but showed a reduced capacity to attenuate NCAM-mediated cell adhesion. On its own, the GFRalpha1 N-terminal domain was sufficient to decrease NCAM-mediated cell adhesion. These results indicate that direct receptor-receptor interactions are not required for high affinity GDNF binding to NCAM but play an important role in the regulation of NCAM-mediated cell adhesion by GFRalpha1.

    The Journal of biological chemistry 2008;283;20;13792-8

  • Polysialylated NCAM represses E-cadherin-mediated cell-cell adhesion in pancreatic tumor cells.

    Schreiber SC, Giehl K, Kastilan C, Hasel C, Mühlenhoff M, Adler G, Wedlich D and Menke A

    Institute of Zoology 2, University Karlsruhe, Karlsruhe, Germany.

    Inhibition of cell-cell adhesion between epithelial cells represents an early step during tumor metastasis. Down-regulation or perturbation of E-cadherin-mediated adherens junctions is an essential requirement in this process.

    Methods: The interaction between polysialylated neural cell adhesion molecule (PSA-NCAM) and the E-cadherin adhesion complex was studied by coimmunoprecipitation assays. The presence of PSA-NCAM was correlated with tumor invasion by using cell-cell aggregation and cell migration assays. The importance of polysialic acid (PSA) in the interaction of NCAM with E-cadherin and inhibition of cell-cell adhesion was confirmed by enzymatic removal of PSA from NCAM and down-regulation of PSA-transferases by siRNA.

    Results: Expression of oncogenic K-Ras(V12) in pancreatic carcinoma cells resulted in induction of PSA-NCAM expression and reduced E-cadherin-mediated cellular adhesion. The association of PSA-NCAM with the E-cadherin adhesion complex correlated with decreased cell-cell aggregation and elevated cell migration of pancreatic carcinoma cells. Enzymatic removal of PSA from NCAM or reduction of polysialyltransferase expression led to reduced association between NCAM and E-cadherin and subsequently increased E-cadherin-mediated cell-cell aggregation and reduced cell migration.

    Conclusions: Our data suggest the induction of PSA-NCAM by oncogenic K-Ras as a novel molecular mechanism by which E-cadherin-mediated cellular adhesion is reduced and dissemination of tumor cells is facilitated.

    Gastroenterology 2008;134;5;1555-66

  • Aberrant co-expression of CD19 and CD56 as surrogate markers of acute myeloid leukemias with t(8;21) in Taiwan.

    Chen SW, Li CF, Chuang SS, Tzeng CC, Hsieh YC, Lee PS, Chen CH, Huang WT, Hwang WS and Tsao CJ

    Division of Hemato-Oncology, Department of Internal Medicine, Lioying Chi-Mei Hospital, Tainan, Taiwan.

    Aberrant antigen expression in acute myeloid leukemia (AML) has been extensively studied in the West with limited reports from Taiwan. We carried out this retrospective study to characterize the frequency and significance of aberrant antigen expression of AML in Taiwan. Among 111 cases, 58 (52%) showed aberrant antigen expression, most frequently CD7 (27%) and CD56 (23%). Aberrant CD7 expression was observed in all non-AML-M3 subtypes, most frequently in AML-M7 (4/6, 67%); while CD19 expression was only observed in AML-M2 (5/36, 14%). CD56 expression was most common in AML-M5 (4/8, 50%). The relative frequency of CD19 and CD56 expression in AML with t(8;21) was higher than those with other chromosomal abnormalities or normal karyotype (P = 0.011 and 0.005, respectively). In non-M3 AML, aberrant antigen expression was identified in 56/96 (58%) cases, in contrast to 2/15 (13%) AML-M3 cases (P = 0.001). CD7, CD19 and CD56 expression was not correlated with remission rate. We concluded that aberrant immunophenotype was more frequent in non-M3 leukemias in Taiwan. The relative frequency of CD19 and/or CD56 expression in AML with t(8;21) was significantly higher than those without this translocation and co-expression of these two antigens may serve as the surrogate markers for AML with t(8;21).

    International journal of laboratory hematology 2008;30;2;133-8

  • Developmental regulation of GABAergic interneuron branching and synaptic development in the prefrontal cortex by soluble neural cell adhesion molecule.

    Brennaman LH and Maness PF

    Department of Biochemistry and Biophysics, and Silvio Conte Center for Schizophrenia Research, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.

    Neural cell adhesion molecule, NCAM, is an important regulator of neuronal process outgrowth and synaptic plasticity. Transgenic mice that overexpress the soluble NCAM extracellular domain (NCAM-EC) have reduced GABAergic inhibitory and excitatory synapses, and altered behavioral phenotypes. Here, we examined the role of dysregulated NCAM shedding, modeled by overexpression of NCAM-EC, on development of GABAergic basket interneurons in the prefrontal cortex. NCAM-EC overexpression disrupted arborization of basket cells during the major period of axon/dendrite growth, resulting in decreased numbers of GAD65- and synaptophysin-positive perisomatic synapses. NCAM-EC transgenic protein interfered with interneuron branching during early postnatal stages when endogenous polysialylated (PSA) NCAM was converted to non-PSA isoforms. In cortical neuron cultures, soluble NCAM-EC acted as a dominant inhibitor of NCAM-dependent neurite branching and outgrowth. These findings suggested that excess soluble NCAM-EC reduces perisomatic innervation of cortical neurons by perturbing axonal/dendritic branching during cortical development.

    Funded by: NIMH NIH HHS: MH064065, MH064065-01A10003, P50 MH064065, P50 MH064065-01A10003

    Molecular and cellular neurosciences 2008;37;4;781-93

  • Structure of the tandem fibronectin type 3 domains of neural cell adhesion molecule.

    Carafoli F, Saffell JL and Hohenester E

    Department of Life Sciences, Biophysics Section, Blackett Laboratory, Imperial College London, London SW7 2AZ, UK.

    Activation of the fibroblast growth factor receptor (FGFR) by neural cell adhesion molecule (NCAM) is essential for NCAM-mediated neurite outgrowth. Previous peptide studies have identified two regions in the fibronectin type 3 (FN3)-like domains of NCAM as being important for these activities. Here we report the crystal structure of the NCAM FN3 domain tandem, which reveals an acutely bent domain arrangement. Mutation of a non-conserved surface residue (M610R) led to a second crystal form showing a substantially different conformation. Thus, the FN3 domain linker is highly flexible, suggesting that it corresponds to the hinge seen in electron micrographs of NCAM. The two putative FGFR1-binding segments, one in each NCAM FN3 domain, are situated close to the domain interface. They form a contiguous patch in the more severely bent conformation but become separated upon straightening of the FN3 tandem, suggesting that conformational changes within NCAM may modulate FGFR1 activation. Surface plasmon resonance experiments demonstrated only a very weak interaction between the NCAM FN3 tandem and soluble FGFR1 proteins expressed in mammalian cells (dissociation constant >100 muM). Thus, the NCAM-FGFR1 interaction at the cell surface is likely to depend upon avidity effects due to receptor clustering.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/D524840/1, BBD5248401; Wellcome Trust: 054334

    Journal of molecular biology 2008;377;2;524-34

  • Induction of CD56 and TCR-independent activation of T cells with aging.

    Lemster BH, Michel JJ, Montag DT, Paat JJ, Studenski SA, Newman AB and Vallejo AN

    Department of Pediatrics, University of Pittsburgh, PA 15213, USA.

    Degeneration of the thymus and severe contraction of the T cell repertoire with aging suggest that immune homeostasis in old age could be mediated by distinct effectors. Therefore, receptors expressed on T cells as they undergo senescence in vitro, as well as those displayed by circulating T cells during normal chronologic aging, were examined. Monitoring of T cells driven to senescence showed de novo induction of CD56, the prototypic receptor of NK cells. Analysis of fresh T cells in peripheral blood showed an age-dependent induction of CD56. These unusual T cells expressed high levels of Bcl2, p16, and p53, and had limited, or completely lost, ability to undergo cell division, properties consistent with senescence. CD56 cross-linking without TCR ligation on CD56(+) T cells resulted in extensive protein phosphorylation, NF-kappaB activation, and Bax down-regulation. CD56 cross-linking was also sufficient to drive production of various humoral factors. These data suggest that the immunologic environment in old age is functionally distinct, rather than being a dysfunctional version of that seen at a young age. CD56(+) T cells are unique effectors capable of mediating TCR-independent immune cascades that could be harnessed to enhance protective immunity in the elderly.

    Funded by: NCI NIH HHS: P20CA103730; NCRR NIH HHS: C06-RR14489; NIA NIH HHS: AG022379-06, P30 AG024827, P30AG024827, R01 AG022379, R01 AG022379-06, R01AG022379, R01AG023629, T35-AG026778

    Journal of immunology (Baltimore, Md. : 1950) 2008;180;3;1979-90

  • Clinicopathological correlates of plasma cell CD56 (NCAM) expression in multiple myeloma.

    Kraj M, Sokołowska U, Kopeć-Szlezak J, Pogłód R, Kruk B, Woźniak J and Szpila T

    Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland. mkraj@ihit.waw.pl

    The aim of this prospective, long-term study was to define the flow cytometric characteristics of plasma cell CD56 expression as well as determine the clinical characteristics of 204 multiple myeloma (MM) patients and 26 plasma cell leukemia (PCL) patients with regard to CD56 expression. CD56 expression intensity was determined by measurement of antigen molecules on the cell defined as Antibodies Bound per Cell (ABC) and calculation of Relative Fluorescence Intensity (RFI). CD56 expression was found in 66% of MM and 54% of PCL cases. The RFI values for individual MM patients ranged from 7.6 to 27.4 while ABC values on MM plasma cells from 2255 to 58469. There was a correlation between the proportion of all bone marrow CD38(++)/CD138(+) cells with CD56 expression and ABC and RFI indices. With regard to CD56 expression positive patients, the CD56(-) MM patients presented lower frequency of osteolysis (p = 0.01). The median survival was 48 months in CD56(+) patients and 43 months (p = 0.84) in CD56(-) cases. In conclusion, CD56 expression carries no distinct adverse prognosis and the lack of CD56 expression does not define a unique clinicopathological or prognostic entity in MM. A remarkable heterogeneity of CD56 expression intensity in CD56(+) patients imposes the necessity of determining CD56 expression intensity in candidates to antibody-based therapy.

    Leukemia & lymphoma 2008;49;2;298-305

  • Neural cell adhesion molecule-180-mediated homophilic binding induces epidermal growth factor receptor (EGFR) down-regulation and uncouples the inhibitory function of EGFR in neurite outgrowth.

    Povlsen GK, Berezin V and Bock E

    ENKAM Pharmaceuticals, Copenhagen, Denmark.

    The neural cell adhesion molecule (NCAM) plays important roles in neuronal development, regeneration, and synaptic plasticity. NCAM homophilic binding mediates cell adhesion and induces intracellular signals, in which the fibroblast growth factor receptor plays a prominent role. Recent studies on axon guidance in Drosophila suggest that NCAM also regulates the epidermal growth factor receptor (EGFR) (Molecular and Cellular Neuroscience, 28, 2005, 141). A possible interaction between NCAM and EGFR in mammalian cells has not been investigated. The present study demonstrates for the first time a functional interaction between NCAM and EGFR in mammalian cells and investigates the molecular mechanisms underlying this interaction. First, NCAM and EGFR are shown to play opposite roles in neurite outgrowth regulation in cerebellar granular neurons. The data presented indicate that negative regulation of EGFR is one of the mechanisms underlying the neuritogenic effect of NCAM. Second, it is demonstrated that expression of the NCAM-180 isoform induces EGFR down-regulation in transfected cells and promotes EGFR down-regulation induced by EGF stimulation. It is demonstrated that the mechanism underlying this NCAM-180-induced EGFR down-regulation involves increased EGFR ubiquitination and lysosomal EGFR degradation. Furthermore, NCAM-180-mediated EGFR down-regulation requires NCAM homophilic binding and interactions of the cytoplasmic domain of NCAM-180 with intracellular interaction partners, but does not require NCAM-mediated fibroblast growth factor receptor activation.

    Journal of neurochemistry 2008;104;3;624-39

  • Association of haplotypic variants in DRD2, ANKK1, TTC12 and NCAM1 to alcohol dependence in independent case control and family samples.

    Yang BZ, Kranzler HR, Zhao H, Gruen JR, Luo X and Gelernter J

    Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA.

    There have been many conflicting reports concerning the association of the DRD2 locus with alcohol dependence (AD). To investigate whether these findings could be reconciled by considering the genomic region of DRD2 in greater detail, we conducted two separate association studies of AD in 1220 European-American subjects using family-based (488 subjects) and case-control (318 cases and 414 controls) designs, and 43 single nucleotide polymorphisms mapped to the gene cluster of NCAM1, TTC12, ANKK1 and DRD2. We used a generalized linear model and haplotype score tests for the case-control sample, and the family-based association test for the family sample. Haplotype associations centered on TTC12 exon 3 [rs1893699-rs723077; optimal individual haplotype simulated P-value (P(oihs)) = 0.00021] in both independent samples (family and case-control). Additional AD-associated haplotypes centered around NCAM1 exon 12 in the family sample (P(oihs) = 0.0032), and at exons 2 and 5 of ANKK1 in the case-control sample (P(oihs) = 0.00058). LD contrasts between cases and controls support selection at TTC12 exon 3 and ANKK1 exon 2. The armadillo repeat domains encoded by TTC12 and dopamine interact in the Wnt pathway and may have effects on dopamine cell development in the ventral midbrain. We conclude that risk for AD is attributable in part to variants in four regions within this cluster: exon 3 of TTC12, exon 12/intron13 of NCAM1 and exons 2 and 5 of ANKK1. The complexity of these relationships, many of which replicate between our independent samples, may explain prior inconsistent results.

    Funded by: NCRR NIH HHS: M01 RR06192; NIAAA NIH HHS: K08 AA13732, K24 AA13736, P50 AA012870, P50 AA12870, R01 AA011330, R01 AA016015, R01 AA11330; NIDA NIH HHS: K24 DA015105, K24 DA15105, R01 DA012849, R01 DA12690, R01 DA12849

    Human molecular genetics 2007;16;23;2844-53

  • Expression of neural cell adhesion molecule in salivary adenoid cystic carcinoma and its correlation with perineural invasion.

    Shang J, Sheng L, Wang K, Shui Y and Wei Q

    Department of Radiation Oncology, The Second Affiliated Hospital, Cancer Institute, Zhejiang University School of Medicine, Hangzhou 310009, PR China.

    The expression of neural cell adhesion molecule (NCAM) was analyzed in immunohistochemical preparations from adenoid cystic carcinoma. The goal was to evaluate whether NCAM expression could be used as a biological marker for the perineural invasion of adenoid cystic carcinoma in the head and neck. The presence of perineural invasion and NCAM expression was evaluated in samples from 49 patients. Perineural invasion was identified in 33 of them (67%). A high incidence of perineural invasion was found in adenoid cystic carcinoma in the parotid, hard palate, maxillary sinus and oral cavity. Positive NCAM staining was observed in 28 of 49 patients (57%). Of the 28 patients with NCAM staining, perineural invasion was identified in 24 (86%). In contrast, only 9 (43%) of the 21 tumors without NCAM staining had perineural invasion. The difference in NCAM expression between cases with and without perineural invasion was statistically significant (p<0.01). When positive NCAM staining was used to estimate the presence of perineural invasion, the sensitivity was 73 and the specificity 75%. Histopathologic nodal involvement was found in 6 of 18 cases in which neck dissection had been performed. All 6 cases displayed positive NCAM staining, and 5 displayed perineural invasion in the primary adenoid cystic carcinoma. In conclusion, NCAM expression can, to a certain extent, be used as a predictor of perineural invasion in adenoid cystic carcinoma. Moreover, lymph node metastases could serve as a clinical indicator for perineural invasion and for NCAM expression.

    Oncology reports 2007;18;6;1413-6

  • Lack of CD56 expression on myeloma cells is not a marker for poor prognosis in patients treated by high-dose chemotherapy and is associated with translocation t(11;14).

    Hundemer M, Klein U, Hose D, Raab MS, Cremer FW, Jauch A, Benner A, Heiss C, Moos M, Ho AD and Goldschmidt H

    Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany.

    Lack of CD56 expression was reported to be associated with a poor prognosis in multiple myeloma (MM) patients treated with conventional chemotherapy. Aim of our retrospective study was to analyse whether CD56 expression on MM cells reveals as a prognostic factor in patients treated with high-dose chemotherapy. MM cells of 99 patients prior to treatment with high-dose chemotherapy were analysed for CD56 expression by flow cytometry. Multivariable analysis of event-free survival in these patients showed no statistically significant difference between the CD56(-) (n=28) and the CD56(+) (n=71) group. The lack of CD56 expression on MM cells of these patients correlated significantly with the presence of translocation (11;14) (t(11;14)) (estimated correlation coefficient=0.655 95%, confidence interval (0.481; 0.779)). In summary, our results indicate that lack of CD56 expression on MM cells is not a prognostic marker in patients treated with high-dose chemotherapy, but is associated with t(11;14).

    Bone marrow transplantation 2007;40;11;1033-7

  • NCAM is ubiquitylated, endocytosed and recycled in neurons.

    Diestel S, Schaefer D, Cremer H and Schmitz B

    Institute of Animal Sciences, Department of Biochemistry, University of Bonn, Katzenburgweg 9a, 53115 Bonn, Germany.

    The neural cell adhesion molecule NCAM plays an important role during neural development and in the adult brain. To study the intracellular trafficking of NCAM in neurons, two major isoforms, NCAM140 or NCAM180, were expressed in primary cortical neurons and in the rat B35 neuroblastoma cell line. NCAM was endocytosed and subsequently recycled to the plasma membrane, whereas only a minor fraction was degraded in lysosomes. In cortical neurons, endocytosis of NCAM was detected in the soma, neurites and growth cones in a developmentally regulated fashion. Furthermore, we found that NCAM is mono-ubiquitylated at the plasma membrane and endocytosis was significantly increased in cells overexpressing ubiquitin. Therefore, we propose that ubiquitylation represents an endocytosis signal for NCAM.

    Journal of cell science 2007;120;Pt 22;4035-49

  • Down-regulation of the axonal polysialic acid-neural cell adhesion molecule expression coincides with the onset of myelination in the human fetal forebrain.

    Jakovcevski I, Mo Z and Zecevic N

    Neuroscience Department, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3401, USA.

    The polysialic acid (PSA) modification of neural cell adhesion molecule, which reduces neural cell adhesion molecule (NCAM) - mediated cell adhesion, is involved in several developmental processes, such as cell migration, axonal growth, path finding, and synaptic plasticity. It has been suggested that PSA-NCAM expression may inhibit myelination. To clarify the relationship between myelination and the expression of PSA-NCAM we systematically investigated its expression in the human forebrain from embryonic stage to midgestation (19-24 gestation weeks, gw). Immunofluorescence on cryosections showed that PSA-NCAM is expressed at the earliest stage studied (5.5 gw) in the primordial plexiform layer of the telencephalon, which mainly consists of neuronal processes. At midgestation, cortical axonal tracts in the emerging white matter were PSA-NCAM+, but they were not yet myelinated, based on the lack of myelin basic protein (MBP) immunoreaction. To follow the progression of myelination we developed organotypic slice cultures that included the subventricular and intermediate zones of the fetal forebrain. In freshly prepared slices, similar to cryosections, axonal tracts were PSA-NCAM+ but did not express MBP. After 5 days in culture there was a dramatic increase in MBP expression around the axons of the intermediate zone, which suggested the onset of myelination. Simultaneously with MBP up-regulation PSA-NCAM expression in axons was completely lost, as demonstrated both with immunofluorescence and Western blot analysis. These results support the idea that in the human fetal forebrain axonal PSA-NCAM expression is inversely related to primary myelination.

    Funded by: NINDS NIH HHS: R01 NS041489, R01 NS041489-07

    Neuroscience 2007;149;2;328-37

  • Phenotypic studies of natural killer cell subsets in human transporter associated with antigen processing deficiency.

    Zimmer J, Bausinger H, Andrès E, Donato L, Hanau D, Hentges F, Moretta A and de la Salle H

    Laboratoire d'Immunogénétique-Allergologie, Centre de Recherche Public de la Santé, Luxembourg City, Luxembourg. jacques.zimmer@crp-sante.lu

    Peripheral blood natural killer (NK) cells from patients with transporter associated with antigen processing (TAP) deficiency are hyporesponsive. The mechanism of this defect is unknown, but the phenotype of TAP-deficient NK cells is almost normal. However, we noticed a high percentage of CD56(bright) cells among total NK cells from two patients. We further investigated TAP-deficient NK cells in these patients and compared them to NK cells from two other TAP-deficient patients with no clinical symptoms and to individuals with chronic inflammatory diseases other than TAP deficiency (chronic lung diseases or vasculitis). Peripheral blood mononuclear cells isolated from venous blood were stained with fluorochrome-conjugated antibodies and the phenotype of NK cells was analyzed by flow cytometry. In addition, (51)Chromium release assays were performed to assess the cytotoxic activity of NK cells. In the symptomatic patients, CD56(bright) NK cells represented 28% and 45%, respectively, of all NK cells (higher than in healthy donors). The patients also displayed a higher percentage of CD56(dim)CD16(-) NK cells than controls. Interestingly, this unusual NK cell subtype distribution was not found in the two asymptomatic TAP-deficient cases, but was instead present in several of the other patients. Over-expression of the inhibitory receptor CD94/NKG2A by TAP-deficient NK cells was confirmed and extended to the inhibitory receptor ILT2 (CD85j). These inhibitory receptors were not involved in regulating the cytotoxicity of TAP-deficient NK cells. We conclude that expansion of the CD56(bright) NK cell subtype in peripheral blood is not a hallmark of TAP deficiency, but can be found in other diseases as well. This might reflect a reaction of the immune system to pathologic conditions. It could be interesting to investigate the relative distribution of NK cell subsets in various respiratory and autoimmune diseases.

    PloS one 2007;2;10;e1033

  • The CD16- CD56(bright) NK cell subset is resistant to reactive oxygen species produced by activated granulocytes and has higher antioxidative capacity than the CD16+ CD56(dim) subset.

    Harlin H, Hanson M, Johansson CC, Sakurai D, Poschke I, Norell H, Malmberg KJ and Kiessling R

    Department of Oncology and Pathology, Cancer Center Karolinska, Karolinska Institutet, Stockholm, Sweden.

    Human NK cells can be divided into CD56(dim) and CD56(bright) subsets. These two types of NK cells respond to different types of stimuli, with CD56(dim) NK cells having direct cytotoxic ability and CD56(bright) NK cells having mainly an immunoregulatory function. We show that the CD16+ CD56(dim) NK subset is characterized by sensitivity to cell death induced by activated granulocytes. We identified hydrogen peroxide (H2O2) as the major effector molecule responsible for the cytotoxic effect of granulocytes on CD56(dim) NK cells, because the ability of granulocytes to kill CD56(dim) NK cells was completely abrogated in the presence of the hydrogen peroxide scavenger catalase. When exposing NK cells to H2O2, CD56(dim) cells showed rapid mitochondrial depolarization and down-regulation of activating NKRs, eventually resulting in cell death, whereas CD56(bright) cells remained unaffected. The difference in sensitivity to H2O2 was mirrored by a difference in intracellular oxidation levels between CD56(dim) and CD56(bright) NK cells, and cell lysates from the latter subset possessed a greater ability to block H2O2-mediated oxidation. Our data may explain the preferential accumulation of CD56(bright) NK cells often seen in environments rich in reactive oxygen species, such as at sites of chronic inflammation and in tumors.

    Funded by: NCI NIH HHS: CA102280

    Journal of immunology (Baltimore, Md. : 1950) 2007;179;7;4513-9

  • Impact of polysialylated CD56 on natural killer cell cytotoxicity.

    Moebius JM, Widera D, Schmitz J, Kaltschmidt C and Piechaczek C

    Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.

    Background: Siglec-7, a sialic acid binding inhibitory receptor expressed by NK cells is masked in vivo by a so far unknown ligand. It shows a strong binding prevalence for alpha-2,8-linked disialic acids in vitro.

    Results: Here we describe the expression of PSA-NCAM (alpha-2,8-linked polysialic acid modified NCAM) on functional adult peripheral blood natural killer cells and examine its possible role in masking Siglec-7. Unmasking of Siglec-7 using Clostridium perfringens neuraminidase massively reduces NK cell cytotoxicity. By contrast a specific removal of PSA using Endo-NF does not lead to a reduction of NK cell cytotoxicity.

    Conclusion: The results presented here therefore indicate that PSA-NCAM is not involved in masking Siglec-7.

    BMC immunology 2007;8;13

  • HIV-1 Nef impairs the dynamic of DC/NK crosstalk: different outcome of CD56(dim) and CD56(bright) NK cell subsets.

    Quaranta MG, Napolitano A, Sanchez M, Giordani L, Mattioli B and Viora M

    Department of Drug Research and Evaluation, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy.

    Dendritic cells (DCs) and natural killer (NK) cells are essential components of the innate immunity and play a critical role in the first phase of host defense against infection. Interactions between DCs and NK cells have been demonstrated in a variety of settings, with evidence emerging of complex bidirectional crosstalk between the two cell types. The accessory HIV-1 Nef protein is a crucial determinant for viral replication and pathogenesis. We previously demonstrated that Nef, hijacking DC functional activity, subverts the DC arm of immune response to escape the adaptive immune attack. Here, we monitor the effect of Nef on the outcome of the innate immune response, focusing on the impact of Nef on DC/NK crosstalk. We demonstrate that Nef up-regulates the ability of DCs to stimulate the immunoregulatory NK cells (CD56(bright)) as assessed by the activated phenotype, up-regulation of their proliferative response and INF-gamma release. On the other hand, Nef-pulsed DCs inhibit cytotoxic NK cells (CD56(dim)), as assessed by the reduced HLA-DR surface expression, reduced proliferation and cytotoxic activity. Moreover, in the presence of Nef-pulsed DCs, we found a significant up-regulation of TNF-alpha secretion and a significant reduction of IL-10, GM-CSF, MIP-1alpha and RANTES secretion. Our findings suggest that the Nef-induced dysregulation in the DC/NK cell crosstalk may represent a potential mechanism through which HIV escapes innate immune surveillance.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2007;21;10;2323-34

  • Neural cell adhesion molecule (CD56)-positive B-cell lymphoma.

    Isobe Y, Sugimoto K, Takeuchi K, Ando J, Masuda A, Mori T and Oshimi K

    Department of Hematology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan. yisobe@med.juntendo.ac.jp

    Expression of neural cell-adhesion molecule CD56 is a rare event in B-cell lymphoma. We described four cases of CD56-positive B-cell lymphoma, including follicular lymphoma and diffuse large B-cell lymphoma. These lymphoma cells expressed CD10 and bcl-6, which suggests germinal center-stage phenotype. Immunohistochemistry showed that CD56-positive cells aggregated and displayed a cohesive growth pattern, indicating that homotypic adhesion through the molecules might affect the manner of tumor growth and expansion. Although CD56 expression level varies among the cases, this molecule might play some roles in the manner of growth and expansion of CD56-positive B-cell lymphomas.

    European journal of haematology 2007;79;2;166-9

  • PSA-NCAM expression in the human prefrontal cortex.

    Varea E, Castillo-Gómez E, Gómez-Climent MA, Blasco-Ibáñez JM, Crespo C, Martínez-Guijarro FJ and Nàcher J

    Neurobiology Unit and Program in Basic and Applied Neurosciences, Cell Biology Department, Universitat de València, Dr. Moliner 50, Burjassot 46100, Spain.

    The prefrontal cortex (PFC) of adult rodents is capable of undergoing neuronal remodeling and neuroimaging studies in humans have revealed that the structure of this region also appears affected in different psychiatric disorders. However, the cellular mechanisms underlying this plasticity are still unclear. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) may mediate these structural changes through its anti-adhesive properties. PSA-NCAM participates in neurite outgrowth and synaptogenesis and changes in its expression occur parallel to neuronal remodeling in certain regions of the adult brain. PSA-NCAM is expressed in the hippocampus and temporal cortex of adult humans, but it has not been studied in the PFC. Employing immunohistochemistry on sections from the rostromedial superior frontal gyrus we have found that PSA-NCAM is expressed in the human PFC neuropil following a laminated pattern and in a subpopulation of mature neurons, which lack doublecortin expression. Most of these cells have been identified as interneurons expressing calbindin. The expression of PSA-NCAM in the human PFC is similar to that of rodents. Since this molecule has been linked to the neuronal remodeling found in experimental models of depression, it may also participate in the structural plasticity described in the PFC of depressed patients.

    Funded by: NIDDK NIH HHS: DK4130

    Journal of chemical neuroanatomy 2007;33;4;202-9

  • Alteration of serum and tumoral neural cell adhesion molecule (NCAM) isoforms in patients with brain tumors.

    Todaro L, Christiansen S, Varela M, Campodónico P, Pallotta MG, Lastiri J, Sacerdote de Lustig E, Bal de Kier Joffé E and Puricelli L

    Research Area of the Institute of Oncology Angel H. Roffo, University of Buenos Aires, Av. San Martin 5481, Buenos Aires, C1417DTB, Argentina.

    The neural cell adhesion molecule (NCAM) is involved in the intercellular junctions of neurons and glial cells. We investigated its relevance as a biomarker in gliomas which main characteristic is their high invasiveness. We studied by Western blot the pattern of serum NCAM bands in patients with gliomas (n = 34), with brain metastasis of different primary cancers (n = 27) and with benign brain tumors (n = 22)] compared with healthy controls (n = 69). For densitometric analysis NCAM bands > or = 130 kDa (HMW) and <130 kDa (LMW) were clustered. We observed that glioma patients presented higher NCAM HMW and lower NCAM LMW levels than control subjects (P < 0.01). A similar pattern was found in patients with brain metastasis or brain benign tumors, suggesting that the pattern of serum NCAM bands would be useful to detect brain tumor pathology. On the other hand, serum NCAM expression was not associated with the main clinicopathological features of gliomas, including overall survival. Interestingly, we found that 9/12 patients with glioma showed a significant decrease in NCAM HMW/LMW ratio between 1-3 months after successful tumor removal. Thus, serum NCAM could be a useful marker for monitoring treatment.NCAM expression was also analyzed at tissular level in 59 glioma sections from paraffined tumors. We observed that NCAM immunostaining was inversely correlated with the histological grade of malignancy, remaining this association in a multivariate analysis. Besides, loss of NCAM staining was significantly associated with bad prognosis in an univariate analysis.

    Journal of neuro-oncology 2007;83;2;135-44

  • Neural cell adhesion molecule expression on renal interstitial cells.

    Marković-Lipkovski J, Müller CA, Klein G, Flad T, Klatt T, Blaschke S, Wessels JT and Müller GA

    Institute of Pathology, University of Belgrade, 11000 Belgrade, Dr Subotića 1, Serbia. acal@matf.bg.ac.yu

    Background: At early stages of kidney development, the neural cell adhesion molecule (NCAM) is highly expressed on cells of the metanephrogenic mesenchyme. During maturation of the fetal kidney, NCAM gradually disappears. So far, it has been widely accepted that NCAM in the adult kidney is only expressed by nerves, and not by other cell types.

    Methods: NCAM expression was analysed in human adult healthy and diseased kidneys by immunohistochemistry and western blot analysis. NCAM+ renal interstitial cells were further characterized by double immunofluorescent staining using antibodies against neurofilaments, alpha smooth muscle actin, vimentin, alpha5beta1 integrin, CD68, CD11c, HLA-DR and the potential progenitor cell markers CD34, CD117, CD133, CD24, nestin and cadherin-11.

    Results: In adult human kidneys, NCAM expression is restricted to rare interstitial cells with dendritic morphology, which are neurofilament-negative and predominantly localized on the corticomedullary junction. They are also negative for fibroblast cell markers, but co-express the haematopoietic stem cell markers CD34 and CD133. The number of NCAM+ interstitial cells increased in the initial phases of interstitial fibrosis. Western blot analysis of renal tissues with incipient interstitial fibrosis tissues showed the expression of the 140 kDa NCAM isoform.

    Conclusions: These data indicate that a rare subpopulation of NCAM+ interstitial cells could represent renal progenitors, and that NCAM+ interstitial cells can participate in the initial phase of interstitial fibrosis.

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2007;22;6;1558-66

  • NCAM1 and neurocognition in schizophrenia.

    Sullivan PF, Keefe RS, Lange LA, Lange EM, Stroup TS, Lieberman J and Maness PF

    Department of Genetics, University of North Carolina, Chapel Hill, North Carolina 27599-7264, USA. pfsulliv@med.unc.edu

    Background: Alterations in neurocognition may be fundamental to schizophrenia and may be endophenotypes. Neural cell adhesion molecule 1 (NCAM1, aliases NCAM and CD56) may be a candidate gene for schizophrenia or for neurocognition in schizophrenia as supported by linkage and functional findings.

    Methods: Subjects were 641 patients with schizophrenia who participated in the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) clinical trial. Neurocognition was assessed at study baseline. Nine NCAM1 single nucleotide polymorphisms (SNPs) were blindly genotyped. Analysis of covariance was used to test for single SNP associations and haplotype regression for multilocus associations.

    Results: As there were suggestions of population stratification, all analyses were conducted stratified by inferred ancestry. In the "Europe only" stratum, there were nominally significant associations with five contiguous SNPs (rs1943620, rs1836796, rs1821693, rs686050, rs584427) with the strongest association at rs1836796 (p = .007). Via permutation testing, the probability of obtaining five consecutive statistically significant SNPs with p-values <or= .05 was p = .0044. These results were robust to examination of model assumptions. Haplotype analyses did not identify significant haplotype associations.

    Conclusions: Although it is essential to see if these findings replicate in additional samples, we suggest that NCAM1 deserves further scrutiny for its relevance to clinical and etiological aspects of schizophrenia.

    Funded by: NIMH NIH HHS: N01 MH90001, R01 MH074027

    Biological psychiatry 2007;61;7;902-10

  • NCAM1 association study of bipolar disorder and schizophrenia: polymorphisms and alternatively spliced isoforms lead to similarities and differences.

    Atz ME, Rollins B and Vawter MP

    Department of Psychiatry and Human Behavior, University of California, Irvine, California, USA.

    Objective: The neural cell adhesion molecule (NCAM1) is a multifunction transmembrane protein involved in synaptic plasticity, neurodevelopment, and neurogenesis. Multiple NCAM1 proteins were differentially altered in bipolar disorder and schizophrenia. Single nucleotide polymorphisms (SNPs) in the NCAM1 gene were significantly associated with bipolar disorder in the Japanese population. Bipolar disorder and schizophrenia may share common vulnerability or susceptibility risk factors for shared features in each disorder.

    Methods: Both SNPs and splice variants in the NCAM1 gene were analysed in bipolar disorder and schizophrenia. A case-control study design for association of SNPs and differential exon expression in the NCAM1 gene was used.

    Results: A genotypic association between bipolar disorder and SNP b (rs2303377 near mini-exon b) and a suggestive association between schizophrenia and SNP 9 (rs646558) were found. Three of the two marker haplotypes for SNP 9 and SNP b showed varying frequencies between bipolar and controls (P<0.0001) as well as between schizophrenia and controls (P<0.0001). There were nine NCAM1 transcripts present in postmortem brain samples that involve alternative splicing of NCAM1 mini-exons (a, b, c) and the secreted (SEC) exon. Significant differences in the amounts of four alternatively spliced isoforms were found between NCAM1 SNP genotypes. In exploratory analysis, the c-SEC alternative spliced isoform was significantly decreased in bipolar disorder compared to controls for NCAM1 SNP b heterozygotes (P=0.013).

    Conclusions: Diverse NCAM1 transcripts were found with possibly different functions. The results suggest that SNPs within NCAM1 contribute differential risk for both bipolar disorder and schizophrenia possibly by alternative splicing of the gene.

    Funded by: NCI NIH HHS: F32 CA090073; NIMH NIH HHS: P50 MH060398, P50 MH60398, R01 MH085801, R21 MH074307, R21 MH074307-01

    Psychiatric genetics 2007;17;2;55-67

  • Role of GPI-anchored NCAM-120 in rabies virus infection.

    Hotta K, Motoi Y, Okutani A, Kaku Y, Noguchi A, Inoue S and Yamada A

    The United Graduate school of Veterinary Science, Gifu University, Ynagido 1-1, Gifu 501-1193, Japan.

    Although the neural cell adhesion molecule (NCAM) -140 and -180 have been shown to serve as a receptor for rabies virus (RV), it was not known whether the other major isoform of NCAM, GPI-anchored NCAM-120 functions as RV receptor. In this study, we have established HEp-2 cells stably expressing NCAM-120 or NCAM-140, and their susceptibilities to RV infection were compared. The results demonstrated that NCAM-120 served as virus attachment protein; however, the cells expressing NCAM-120 did not support efficient RV replication. Furthermore, the level of IFN-ss mRNA was apparently elevated in NCAM-120 expressing cells but not in NCAM-140 expressing cells, suggesting that GPI-anchored NCAM-120 suppressed RV replication via induction of IFN-ss even though NCAM-120 was able to promote virus penetration into the cells.

    Microbes and infection 2007;9;2;167-74

  • Chemoresistance induces enhanced adhesion and transendothelial penetration of neuroblastoma cells by down-regulating NCAM surface expression.

    Blaheta RA, Daher FH, Michaelis M, Hasenberg C, Weich EM, Jonas D, Kotchetkov R, Doerr HW and Cinatl J

    Zentrum der Chirurgie, Klinik für Urologie und Kinderurologie, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany. blaheta@em.uni-frankfurt.de

    Background: Drug resistance to chemotherapy is often associated with increased malignancy in neuroblastoma (NB). One explanation for the link between resistance and malignancy might be that resistance facilitates cancer progression and invasion. To investigate this hypothesis, adhesion, transendothelial penetration and NCAM (CD56) adhesion receptor expression of drug-resistant versus drug-sensitive NB tumor cells were evaluated.

    Methods: Acquired drug resistance was mimicked by exposing parental UKF-NB-2, UKF-NB-3 or IMR-32 tumor cells to increasing concentrations of vincristine- (VCR) or doxorubicin (DOX) to establish the resistant tumor cell sublines UKF-NB-2VCR, UKF-NB-2DOX, UKF-NB-3VCR, UKF-NB-3DOX, IMR-32VCR and IMR-32DOX. Additionally, the malignant behaviour of UKF-NB-4, which already possessed the intrinsic multidrug resistance (MDR) phenotype, was analyzed. UKF-NB-4 exposed to VCR or DOX were designated UKF-NB-4VCR or UKF-NB-4DOX. Combined phase contrast - reflection interference contrast microscopy was used to separately evaluate NB cell adhesion and penetration. NCAM was analyzed by flow cytometry, western blot and RT-PCR.

    Results: VCR and DOX resistant tumor sublines showed enhanced adhesion and penetration capacity, compared to their drug naïve controls. Strongest effects were seen with UKF-NB-2VCR, UKF-NB-3VCR and IMR-32DOX. DOX or VCR treatment also evoked increased invasive behaviour of UKF-NB-4. The process of accelerated tumor invasion was accompanied by decreased NCAM surface and protein expression, and down-regulation of NCAM coding mRNA. Transfection of UKF-NB-4VCR cells with NCAM cDNA led to a significant receptor up-regulation, paralleled by diminished adhesion to an endothelial cell monolayer.

    Conclusion: It is concluded that NB cells resistant to anticancer drugs acquire increased invasive capacity relative to non-resistant parental cells, and that enhanced invasion is caused by strong down-regulation of NCAM adhesion receptors.

    BMC cancer 2006;6;294

  • Haplotype spanning TTC12 and ANKK1, flanked by the DRD2 and NCAM1 loci, is strongly associated to nicotine dependence in two distinct American populations.

    Gelernter J, Yu Y, Weiss R, Brady K, Panhuysen C, Yang BZ, Kranzler HR and Farrer L

    Division of Human Genetics, Department of Psychiatry, Yale University School of Medicine, West Haven, CT 06516, USA. joel.gelernter@yale.edu

    Nicotine dependence (ND) is a moderately heritable trait. We ascertained a set of 1615 subjects in 632 families [319 African-American (AA) and 313 European-American (EA)] based on affected sibling pairs with cocaine or opioid dependence. Subjects were interviewed with the Semi-Structured Assessment for Drug Dependence and Alcoholism (SSADDA). Previously, we identified a modest linkage peak (LOD score =1.97) for ND in the EA part of the sample on chromosome 11q23, a region that includes the NCAM1-TTC12-ANKK1-DRD2 gene cluster. DRD2 and NCAM1 are functional candidate genes for substance dependence; the TTC12 and ANKK1 loci are not well characterized. We genotyped a set of 43 single nucleotide polymorphisms (SNPs) spanning this region, and performed family-based association and haplotype analysis. There was relatively weak evidence for association of the flanking DRD2 and NCAM1 markers to ND, but very strong evidence of association of multiple SNPs at TTC12 and ANKK1 in both populations (minimal P=0.0007 in AAs and minimal P=0.00009 in EAs), and in the pooled sample, as well as strong evidence for highly significant association of a single haplotype spanning TTC12 and ANKK1 to ND in the pooled sample (P=0.0000001). We conclude that a risk locus for ND, important both in AAs and EAs, maps to a region that spans TTC12 and ANKK1. Functional studies of these loci are warranted. These results provide additional information useful in evaluating the many earlier discrepant findings regarding association of DRD2 with substance dependence.

    Funded by: NCRR NIH HHS: M01 RR06192; NIDA NIH HHS: K02 DA00326, K24 DA15105, R01 DA12690, R01 DA12849

    Human molecular genetics 2006;15;24;3498-507

  • A novel alpha-helix in the first fibronectin type III repeat of the neural cell adhesion molecule is critical for N-glycan polysialylation.

    Mendiratta SS, Sekulic N, Hernandez-Guzman FG, Close BE, Lavie A and Colley KJ

    Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60607, USA.

    Polysialic acid is a developmentally regulated, anti-adhesive glycan that is added to the neural cell adhesion molecule, NCAM. Polysialylated NCAM is critical for brain development and plays roles in synaptic plasticity, axon guidance, and cell migration. The first fibronectin type III repeat of NCAM, FN1, is necessary for the polysialylation of N-glycans on the adjacent immunoglobulin domain. This repeat cannot be replaced by other fibronectin type III repeats. We solved the crystal structure of human NCAM FN1 and found that, in addition to a unique acidic surface patch, it possesses a novel alpha-helix that links strands 4 and 5 of its beta-sandwich structure. Replacement of the alpha-helix did not eliminate polysialyltransferase recognition, but shifted the addition of polysialic acid from the N-glycans modifying the adjacent immunoglobulin domain to O-glycans modifying FN1. Other experiments demonstrated that replacement of residues in the acidic surface patch alter the polysialylation of both N- and O-glycans in the same way, while the alpha-helix is only required for the polysialylation of N-glycans. Our data are consistent with a model in which the FN1 alpha-helix is involved in an Ig5-FN1 interaction that is critical for the correct positioning of Ig5 N-glycans for polysialylation.

    Funded by: NIGMS NIH HHS: R01 GM063843, R01 GM63843

    The Journal of biological chemistry 2006;281;47;36052-9

  • The fibroblast growth factor receptor acid box is essential for interactions with N-cadherin and all of the major isoforms of neural cell adhesion molecule.

    Sanchez-Heras E, Howell FV, Williams G and Doherty P

    Wolfson Centre for Age-Related Diseases, King's College London, London SE1 1UL, United Kingdom.

    Interactions between the neural cell adhesion molecules NCAM and N-cadherin with the fibroblast growth factor receptor (FGFR) are important for a number of developmental events and have also been implicated in tumor progression. The factors regulating these interactions are not known. We have used co-immunoprecipitation and co-clustering paradigms to show that both adhesion molecules can interact with the 3Ig IIIC isoform of the FGFR1 in a number of cell types. Interestingly, whereas the interaction can be seen over most of the cell surface, it is not seen at points of cell-cell contact where the adhesion molecules accumulate at stable junctions. We also demonstrate for the first time that all of the major isoforms of NCAM can interact with the FGFR. Using deletion mutagenesis we have found that the adhesion molecule/FGFR interaction can withstand the removal of most of any one of the FGFR immunoglobulin-like domains (D1-D3). In contrast, the FGFR interaction with N-cadherin and NCAM (but not FGF) is absolutely dependant on the presence of the acid box motif that can be found in the linker region between D1 and D2. As this motif can be spliced out of all four FGFRs, it suggests that this is one mechanism that can regulate the interaction of the receptor with different ligand classes.

    Funded by: Wellcome Trust

    The Journal of biological chemistry 2006;281;46;35208-16

  • Concomitant aberrant overexpression of RUNX1 and NCAM in regenerating bone marrow of myeloid leukemia of Down's syndrome.

    Langebrake C, Klusmann JH, Wortmann K, Kolar M, Puhlmann U and Reinhardt D

    Medizinische Hochschule Hannover, Department of Pediatric Hematology and Oncology, Carl-Neuberg-Strasse 1, 30625 Hannover. aml.bfm@mh-hannover.de

    Myeloid leukemia of Down's syndrome (ML-DS) has characteristic biological features (e.g. expression of the truncated GATA1s), which are different from those of non-DS childhood acute myeloid leukemias (AML). The objective of this study was to investigate factors predisposing to the development of ML-DS.

    We analyzed 134 bone marrow specimens from 64 children with ML-DS and non-DS AML during chemotherapy and 7 specimens from DS children with- out leukemia,who did not receive any chemotherapy,The specimens were analyzed by multiparameter flow cytometry and quantitative reverse transcriptase polymerase chain reaction for transcription factors involved in hematopoiesis.

    Results: Samples taken from children with ML-DS in complete remission during chemotherapy aberrantly expressed CD56 (NCAM) at the surface of monocytic and granulocytic cells. Compared to non-DS AML cases,children with ML-DS had a statistically significant higher proportion of CD56+ cells in the CD33+ fraction: 71%+/-6% vs. 4%+/-1% (p<0.00001). A significant decrease of the amount of CD33+/CD56+ cells was observed during and after maintenance therapy. An increased number of CD33+/CD56+ cells was also present (>85%) in children with DS who did not receive chemotherapy, but showed a left-shift (due to infection), compared with DS children without left-shift (<10% CD33+/CD56+ cells). Within the CD33+/CD56+ fraction, RUNX1 was overexpressed more than 5-fold (p<0.02) compared to CD33+/CD56- cells, whereas there were no differences regarding GATA1, SPI1, ERG or ETS-2 levels.

    The combined overexpression of RUNX1 and NCAM during stress hematopoiesis in children with DS might be a key factor in the development of overt leukemia and/or in the growth advantage of the malignant GATA1s clone in ML- DS.

    Haematologica 2006;91;11;1473-80

  • Identical twins discordant for type 1 diabetes show a different pattern of in vitro CD56+ cell activation.

    Goodier MR, Nawroly N, Beyan H, Hawa M, Leslie RD and Londei M

    Department of Immunology Imperial College London, Faculty of Medicine, Chelsea and Westminster Hospital, UK. m.goodier@imperial.ac.uk

    Background: Recent studies in animal models indicate a role for natural killer (NK) cells in the protection against type 1 diabetes. In humans, a reduction of NK cell numbers has been reported in identical twins discordant for type 1 diabetes, irrespective of whether they have the disease. Here we have tested whether the activation and expansion of human NK cells with lipopolysaccharide (LPS) reveals differences between these twins.

    Methods: Proportions of CD56(+) NK cells and T-cells and Va24Vb11(+) NK-T cells from diabetic and non-diabetic twins was assessed before and after activation using flow cytometry. NK receptor usage was monitored by PCR and flow cytometry.

    Results: The profile of the expressed Killer Cell immunoglobulin-like receptor (KIR) repertoire (using mRNA) in freshly isolated NK cells was identical in pairs of identical twins, despite marked variation among individual twins as well as controls. Basal numbers of CD56(+) and CD94(+) (CD3(-) and CD3(+)) cells and Valpha24(+)Vbeta11(+) NK-T cells were similarly strongly correlated between identical twins (p < 0.006 for all correlations). Following LPS stimulation, the pattern of KIR mRNA expression remained unaltered in twins and the proportion of NK cells and Valpha24(+)Vbeta11(+) NK-T cells remained correlated between pairs of twins. However, there was a significant reduction in the proportion of CD56(+) cells and CD94(+) cells (whether defined as CD3(-) or CD3(+)) responding to LPS in the diabetic compared to the non-diabetic twin (p = 0.031 and 0.025, respectively).

    Conclusion: This reduction in NK cell expansion in response to LPS in patients with type 1 diabetes is consistent with a non-genetically determined alteration in the innate immune response either predisposing to or resulting from the disease.

    Funded by: Wellcome Trust

    Diabetes/metabolism research and reviews 2006;22;5;367-75

  • The phenotypes of pluripotent human hepatic progenitors.

    Schmelzer E, Wauthier E and Reid LM

    Department of Cell and Molecular Biology, University of North Carolina School of Medicine, Campus Box 7038, Glaxo Building Rooms 32-35, Chapel Hill, 27599, USA. stemcell@med.unc.edu

    Human livers contain two pluripotent hepatic progenitors, hepatic stem cells and hepatoblasts, with size, morphology, and gene expression profiles distinct from that of mature hepatocytes. Hepatic stem cells, the precursors to hepatoblasts, persist in stable numbers throughout life, and those isolated from the livers of all age donors from fetal to adult are essentially identical in their gene and protein expression profiles. The gene expression profile of hepatic stem cells throughout life consists of high levels of expression of cytokeratin 19 (CK19), neuronal cell adhesion molecule (NCAM), epithelial cell adhesion molecule (EpCAM), and claudin-3 (CLDN-3); low levels of albumin; and a complete absence of expression of alpha-fetoprotein (AFP) and adult liver-specific proteins. By contrast, hepatoblasts, the dominant cell population in fetal and neonatal livers, decline in numbers with age and are found as <0.1% of normal adult livers. They express high levels of AFP, elevated levels of albumin, low levels of expression of adult liver-specific proteins, low levels of CK19, and a loss of NCAM and CLDN-3. Mature hepatocytes lack expression altogether of EpCAM, NCAM, AFP, CLDN-3, cytokeratin 19, and have acquired the well-known adult-specific profile that includes expression of high levels of albumin, cytochrome P4503A4, connexins, phosphoenolpyruvate carboxykinase, and transferrin. Thus, hepatic stem cells have a unique stem cell phenotype, whereas hepatoblasts have low levels of expression of both stem cell genes and genes expressed in high levels in mature hepatocytes.

    Funded by: NIAAA NIH HHS: AA014243; NIDDK NIH HHS: DK52851, IP30-DK065933

    Stem cells (Dayton, Ohio) 2006;24;8;1852-8

  • Neural-cell adhesion molecule (NCAM) expression by immature and tumor-derived endothelial cells favors cell organization into capillary-like structures.

    Bussolati B, Grange C, Bruno S, Buttiglieri S, Deregibus MC, Tei L, Aime S and Camussi G

    Cattedra di Nefrologia, Dipartimento di Medicina Interna and Centro Ricerca Medicina Sperimentale (CeRMS), University of Torino, Ospedale Maggiore S. Giovanni Battista, Corso Dogliotti 14, 10126 Torino, Italy.

    The neural cell adhesion molecule (NCAM) is widely expressed during embryogenesis, down-regulated in the course of differentiation to be re-expressed during progression of some tumors. We here found that renal tumor-derived endothelial cells (TEC) but not normal endothelial cells (HMEC) expressed NCAM. In TEC, NCAM expression was regulated by the renal embryonic transcription factor PAX2, as transfection with PAX2 antisense abrogated NCAM expression. NCAM stimulation with an agonistic synthetic NCAM peptide enhanced apoptosis resistance and increased ability of TEC to organize in vessel-like structures. The angiogenic effect of NCAM peptide was, at least in part, mediated by the association of NCAM and FGFR1. HMEC transiently acquired NCAM when organized in vessel-like structures after VEGF stimulation or when transfected with PAX2 gene. During the process of VEGF-induced endothelial differentiation of renal stem cells and of circulating endothelial progenitors, NCAM was transiently expressed to disappear at complete endothelial maturation. Targeting NCAM with a saporin-conjugated peptide induced a cytotoxic effect on TEC but not on HMEC. In conclusion, we identified a new role of NCAM in tumor neo-angiogenesis relevant for endothelial cell organization into capillary-like structures. In addition, we found that NCAM expression was associated with an immature phenotype of endothelial cells.

    Experimental cell research 2006;312;6;913-24

  • NCAM is hyposialylated in hereditary inclusion body myopathy due to GNE mutations.

    Ricci E, Broccolini A, Gidaro T, Morosetti R, Gliubizzi C, Frusciante R, Di Lella GM, Tonali PA and Mirabella M

    Department of Neuroscience, Catholic University, Rome, Italy.

    The authors found that the neural cell adhesion molecule (NCAM) is hyposialylated in hereditary inclusion body myopathy (HIBM) muscle, as suggested by its decreased molecular weight by Western blot. This abnormality represented the only pathologic feature differentiating HIBM due to GNE mutations from other myopathies with similar clinical and pathologic characteristics. If further confirmed in larger series of patients, this may be a useful diagnostic marker of GNE-related HIBM.

    Neurology 2006;66;5;755-8

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • Human CD56bright and CD56dim natural killer cell subsets respond differentially to direct stimulation with Mycobacterium bovis bacillus Calmette-Guérin.

    Batoni G, Esin S, Favilli F, Pardini M, Bottai D, Maisetta G, Florio W and Campa M

    Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, University of Pisa, Italy. batoni@biomed.unipi.it

    Mycobacterium bovis bacillus Calmette-Guérin (BCG) is capable of directly stimulating several effector functions of human natural killer (NK) cells in the absence of interleukin-12 and professional antigen presenting cells. To assess the contribution of two main human NK-cell subsets (CD56(dim) and CD56(bright)) to the overall in vitro NK-cell response to BCG, peripheral blood mononuclear cells depleted of nylon wool-adherent cells or purified NK cells were stimulated with live BCG. By combining intranuclear bromodeoxyuridine (BrdU) staining and analysis of CD56 marker intensity, statistically higher percentages of BrdU(+) cells were found among the CD56(bright) subset than the CD56(dim) subset after 6 days of stimulation with BCG. Similarly, evaluation of intracellular interferon-gamma (IFN-gamma) revealed that CD56(bright) cells were those mainly involved in IFN-gamma production in response to BCG. In contrast, the CD56(dim) subset contained higher levels of perforin and granzyme A, two key molecules for exocytosis-mediated cytotoxicity, than the CD56(bright) subset. Although 16-20-h stimulation with BCG did not substantially alter the expression of cytotoxic molecules by the two subsets, a decrease in perforin content was observed in the CD56(dim), but not in the CD56(bright) subset, following 4-h incubation with the NK-sensitive target K562 cell line. This decrease in perforin content correlated with the induction by BCG-stimulated NK cells, of early markers of apoptosis on target cells to a greater extent than unstimulated cells suggesting a major role for the CD56(dim) subset in cytotoxic activity in response to BCG. Taken together, these results demonstrate that CD56(bright) and CD56(dim) human NK-cell subsets exert different functional activities in response to a live bacterial pathogen.

    Scandinavian journal of immunology 2005;62;6;498-506

  • NKG2A and CD56 are coexpressed on activated TH2 but not TH1 lymphocytes.

    Freishtat RJ, Mitchell LW, Ghimbovschi SD, Meyers SB and Hoffman EP

    Division of Emergency Medicine, Children's National Medical Center, and Department of Pediatrics, George Washington University School of Medicine and Health Sciences, Washington, DC 20010-2970, USA. rfreishtat@cnmcresearch.org

    NKG2A is commonly expressed on cytotoxic cells but has been found on activated T helper (TH) cells. In identifying novel markers differentiating between TH1 and TH2 lymphocytes, we focused on NKG2A expression. TH1 and TH2 cells were negatively isolated from healthy volunteers for microarray analysis and reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry of quiescent and activated TH1 and TH2 cells was performed. Isolates were >95% pure CD3+CD4+ cells (TH1=90.3% and TH2=84.1%). Microarrays revealed differential expression of NKG2A and NKG2C isoforms between TH1 and TH2 cells. RT-PCR indicated greater expression of NKG2A in TH2 cells (4-fold) and NKG2C in TH1 cells (3-fold). Flow studies revealed tripling of TH2 NKG2A with activation to 10.76+/-4.01% (p=0.05), a 23-fold increase in CD56 to 35+/-14.54% (p=0.03), and an increase in NKG2A+CD56+ double-positive cells to 3.04+/-1.38% (p=0.04). TH1 lymphocytes did not differ with activation. We identified co-induction of NKG2A and CD56 on activation of TH2 cells. These cells would likely bind more HLA-E and exhibit increased effector inhibition. Given that certain viruses are known to decrease MHC class I and thus HLA-E production by antigen-presenting cells, activated TH2 cells would bind less HLA-E in this scenario. This would likely result in less effector inhibition and a relatively robust TH2 response.

    Funded by: NCRR NIH HHS: K23 RR020069, L40 RR020507, L40 RR020507-01, M01 RR020359, M01 RR020359-026201, M01-RR-020359; NHLBI NIH HHS: L40 HL074778, L40 HL074778-02; NICHD NIH HHS: K12 HD001399, K12-HD-01399

    Human immunology 2005;66;12;1223-34

  • Degree of polymerization (DP) of polysialic acid (polySia) on neural cell adhesion molecules (N-CAMS): development and application of a new strategy to accurately determine the DP of polySia chains on N-CAMS.

    Nakata D and Troy FA

    Department of Biochemistry and Molecular Medicine, University of California School of Medicine, Davis, California 95616.

    Alpha2,8-linked polysialic acid (polySia) is a structurally unique antiadhesive glycotope that covalently modifies N-linked glycans on neural cell adhesion molecules (N-CAMs). These sugar chains play a key role in modulating cell-cell interactions, principally during embryonic development, neural plasticity, and tumor metastasis. The degree of polymerization (DP) of polySia chains on N-CAM is postulated to be of critical importance in regulating N-CAM function. There are limitations, however, in the conventional methods to accurately determine the DP of polySia on N-CAM, the most serious being partial acid hydrolysis of internal alpha2,8-ketosidic linkages that occur during fluorescent derivatization, a step necessary to enhance chromatographic detection. To circumvent this problem, we have developed a facile method that combines the use of Endo-beta-galactosidase to first release linear polySia chains from N-CAM, with high resolution high pressure liquid chromatography profiling. This strategy avoids acid hydrolysis prior to chromatographic profiling and thus provides an accurate determination of the DP and distribution of polySia on N-CAM. The potential of this new method was evaluated using a nonpolysialylated construct of N-CAM that was polysialylated in vitro using a soluble construct of ST8Sia II or ST8Sia IV. Whereas most of the oligosialic acid/polySia chains consisted of DPs approximately 50-60 or less, a subpopulation of chains with DPs approximately 150 to approximately 180 and extending to DP approximately 400 were detected. The DP of this subpopulation is considerably greater than reported previously for N-CAM. Endo-beta-galactosidase can also release polySia chains from polysialylated membranes expressed in the neuroblastoma cell line, Neuro2A, and native N-CAM from embryonic chick brains.

    Funded by: NIGMS NIH HHS: GM55703

    The Journal of biological chemistry 2005;280;46;38305-16

  • Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry.

    Liu T, Qian WJ, Gritsenko MA, Camp DG, Monroe ME, Moore RJ and Smith RD

    Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, USA.

    The enormous complexity, wide dynamic range of relative protein abundances of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies. Here, we describe an approach for broad analysis of human plasma N-glycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F (PNGase F). Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 nonredundant N-glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein (approximately 200 pg/mL), cathepsin L (approximately 1 ng/mL), and transforming growth factor beta 1 (approximately 2 ng/mL). A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high-resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated.

    Funded by: NCRR NIH HHS: P41 RR018522, RR18522; NIGMS NIH HHS: U54 GM-62119-02, U54 GM062119

    Journal of proteome research 2005;4;6;2070-80

  • Induction of apoptosis in human salivary gland tumor cells by anti-NCAM antibody.

    Fukuda M, Horiuchi Y, Oku Y, Ishikawa M, Suka N, Suzuki S, Kusama K and Sakashita H

    Second Division of Oral and Maxillofacial Surgery, Department of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado, Saitama 350-0283, Japan. fukudam@dent.meikai.ac.jp

    Neural cell adhesion molecule (NCAM) is a type of cell surface glycoprotein and a member of the immunoglobulin superfamily. It has been reported that NCAM may be associated with perineural invasion by malignant salivary gland tumors such as adenoid cystic carcinoma. We have previously demonstrated that NCAM is constitutively expressed in the human salivary gland tumor cell line HSG, in vitro. In the present study, we have aimed to clarify the hypothesis that NCAM-mediated inhibition of salivary gland tumor proliferation is caused by homophilic binding and involves the prevention of signal transduction for perineural invasion using HSG cells. NCAM mRNA and protein expression was found to decrease in a dose-dependent manner upon treatment with the anti-NCAM antibody (MAb NCAM) for 24 h. The MTT assay showed a significant reduction in the number of viable HSG cells. Confocal laser microscopy showed that HSG cells underwent apoptosis after treatment with MAb NCAM. The activation of caspases 3, 7 and 9 was observed in HSG cells after treatment with MAb NCAM, thus confirming that apoptosis was induced by the activated caspases. Apaf-1 activity was also detected in HSG cells in a dose-dependent manner after treatment with MAb NCAM. The up-regulation of TGF-beta1-mediated NCAM expression appeared to lead to the activation of homophilic NCAM binding, further accelerating HSG cell proliferation. In addition, the localization of NCAM in adenoid cystic carcinomas (ACCs) was examined using an immunohistochemical method. NCAM was slightly to moderately positive in 9 of 13 cases (69.2%) of ACC. These findings suggest that NCAM is associated not only with a cell-to-cell adhesion mechanism, but also with tumorigenesis, including growth, development and perineural invasion in human salivary gland tumors.

    Oncology reports 2005;14;5;1143-9

  • Specific amino acids in the first fibronectin type III repeat of the neural cell adhesion molecule play a role in its recognition and polysialylation by the polysialyltransferase ST8Sia IV/PST.

    Mendiratta SS, Sekulic N, Lavie A and Colley KJ

    Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago College of Medicine, 60607, USA.

    Polysialic acid is an anti-adhesive protein modification that promotes cell migration and the plasticity of cell interactions. Because so few proteins carry polysialic acid, we hypothesized that polysialylation is a protein-specific event and that a specific polysialyltransferase-substrate interaction is the basis of this specificity. The major substrate for the polysialyltransferases is the neural cell adhesion molecule, NCAM. Previous work demonstrates that the first fibronectin type III repeat of NCAM (FN1) was necessary for the polysialylation of the N-glycans on the adjacent immunoglobulin domain (Ig5) (Close, B. E., Mendiratta, S. S., Geiger, K. M., Broom, L. J., Ho, L. L., and Colley, K. J. (2003) J. Biol. Chem. 278, 30796-30805). This suggested that FN1 may be a recognition site for the polysialyltransferases. In this study, we showed that the second fibronectin type III repeat (FN2) of NCAM cannot replace FN1. Arg substitution of three unique acidic amino acids on the surface of FN1 eliminated polysialylation not only of a minimal Ig5-FN1 substrate but also of full-length NCAM. Ala substitution of these residues eliminated Ig5-FN1 polysialylation but not that of full-length NCAM, suggesting that the two proteins are interacting differently with the enzymes and that multiple residues are involved in the enzyme-NCAM interaction. By using another truncated protein, Ig5-FN1-FN2, we confirmed the importance of enzyme-substrate positioning for optimal recognition and polysialylation. In sum, we have found that acidic residues on the surface of FN1 are part of a larger protein interaction region that is critical for NCAM recognition and polysialylation by the polysialyltransferases.

    Funded by: NIGMS NIH HHS: R01 GM063843, R01 GM63483

    The Journal of biological chemistry 2005;280;37;32340-8

  • Neuroprotection mediated by subtoxic dose of NMDA in SH-SY5Y neuroblastoma cultures: activity-dependent regulation of PSA-NCAM expression.

    Singh J and Kaur G

    Department of Biotechnology, Neurochemistry and Neuroendocrinology Lab, Guru Nanak Dev University, Amritsar, Punjab, India.

    The NMDA class of glutamate receptors plays a critical role in CNS, such as synaptic plasticity, axonal sprouting, growth, and migration. NMDA receptor stimulation has been shown to regulate polysialylated neural cell adhesion molecule (PSA-NCAM) expression in glial cell cultures and in hippocampal slice cultures. There is also growing evidence that molecular chaperons and ROS are related to the synaptic plasticity phenomena. We have examined the neuroprotective effect of subtoxic dose of NMDA in retinoic acid differentiated SH-SY5Y neuroblastoma cells. SH-SY5Y cell line differentiated with retinoic acid (10 muM) was exposed to NMDA (100 microM) or to antagonist MK-801 (200 nM) + NMDA and cells harvested after 24 h of treatment for PSA-NCAM, NCAM, and HSP70 expression study and for biochemical analysis. A significant increase was observed in PSA-NCAM, NCAM-180, NCAM-140, and HSP70 expression as seen by Western blotting and immunocytofluorescent studies in NMDA-treated cultures. Biochemical analysis revealed a significant increase in the activities of glutathione peroxidase (GPx) and copper zinc-superoxide dismutase (CuZnSOD) upon exposure to NMDA. No significant change was observed in the level of lipid peroxidation. All the changes observed reverted back to the control values upon pretreatment of cultures with MK-801, a non-competitive NMDA receptor antagonist, prior to NMDA exposure indicating the involvement of NMDA receptor in these changes. These results illustrate the neuroprotective role of subtoxic dose of NMDA in SH-SY5Y neuroblastoma cells.

    Brain research. Molecular brain research 2005;137;1-2;223-34

  • CD56 reactivity in small cell carcinoma of the uterine cervix.

    Albores-Saavedra J, Latif S, Carrick KS, Alvarado-Cabrero I and Fowler MR

    Department of Pathology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130, USA. JAlbor@lsuhsc.edu

    Small cell carcinoma (SCC) of the uterine cervix, like its pulmonary counterpart, is a rare but distinctive neoplasm that should be separated from nonendocrine carcinomas because of its highly aggressive clinical course and response to chemotherapy and irradiation. CD56 (neural cell adhesion molecule) has recently been shown to be the best marker for the diagnosis of pulmonary SCC. In this study, we assessed the sensitivity and specificity of CD56 in the diagnosis of SCC of the uterine cervix compared with those of chromogranin and synaptophysin. Twenty-two (88%) of 25 SCCs of the uterine cervix labeled with CD56 in a predominantly membranous and diffuse pattern, whereas 16 of 25 (64%) stained with synaptophysin in a predominantly diffuse pattern and 8 of 25 (32%) showed predominantly focal immunoreactivity for chromogranin. In contrast, 3 of 21 (14%) moderately to poorly differentiated squamous cell carcinomas and 1 of 16 (6%) moderately differentiated adenocarcinomas showed focal immunoreactivity for CD56. Although not specific, CD56 seems to be the most sensitive marker for the diagnosis of SCC of the uterine cervix. Moreover, its diffuse reactivity reduces the possibility of obtaining negative results in small biopsy samples.

    International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists 2005;24;2;113-7

  • Phosphoproteomic analysis of synaptosomes from human cerebral cortex.

    DeGiorgis JA, Jaffe H, Moreira JE, Carlotti CG, Leite JP, Pant HC and Dosemeci A

    Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Protein phosphorylation is a crucial post-translational modification mechanism in the regulation of synaptic organization and function. Here, we analyzed synaptosome fractions from human cerebral cortex obtained during therapeutic surgery. To minimize changes in the phosphorylation state of proteins, the tissue was homogenized within two minutes of excision. Synaptosomal proteins were digested with trypsin and phosphopeptides were isolated by immobilized metal affinity chromatography and analyzed by liquid chromatography and tandem mass spectrometry. The method allowed the detection of residues on synaptic proteins that were presumably phosphorylated in the intact cell, including synapsin 1, syntaxin 1, and SNIP, PSD-93, NCAM, GABA-B receptor, chaperone molecules, and protein kinases. Some of the residues identified are the same or homologous to sites that had been previously described to be phosphorylated in mammals whereas others appear to be novel sites which, to our knowledge, have not been reported previously. The study shows that new phosphoproteomic strategies can be used to analyze subcellular fractions from small amounts of tissue for the identification of phosphorylated residues for research and potentially for diagnostic purposes.

    Journal of proteome research 2005;4;2;306-15

  • A novel epitope of N-CAM defines precursors of human adherent NK cells.

    Li S, Xu J, Makarenkova VP, Tjandrawan T, Vakkila J, Reichert T, Gooding W, Lagenaur CF, Achim CL, Chambers WH, Herberman RB, Whiteside TL and Vujanovic NL

    Department of Pathology, University of Pittsburgh School of Medicine, PA 15213-1863, USA.

    Activated, adherent natural killer (A-NK) cells represent a distinct subpopulation of interleukin (IL)-2-stimulated NK cells, which are selectively endowed with the increased expression of integrins and ability to adhere to solid surfaces, migrate into, infiltrate, and destroy cancerous tissues. The present study defines the phenotype and functions of precursors of A-NK (pre-A-NK) cells in humans. Peripheral blood pre-A-NK cells, in contrast to the rest of NK cells, express a novel epitope of CD56 neuronal cell adhesion molecule, termed ANK-1, and increased cell-surface levels of integrins. Pre-A-NK cells also express low levels of CD56 and CD161, and some express CD162 receptor, do not express CD25 or activation markers, and are effective mediators of NK cytotoxicity. Thus, pre-A-NK cells are generally similar to CD56(dim) NK cells. However, pre-A-NK cells differ from the main NK cell subpopulation by having a lower expression level of CD16 and a lower ability to mediate redirected antibody-dependent, cell-mediated cytotoxicity. More importantly, pre-A-NK cells are preferentially endowed with the ability to rapidly respond to IL-2 by integrin-mediated adherence to endothelial cells, extracellular matrix, and plastic. This early, specific response of pre-A-NK cells to IL-2 is followed by their activation, vigorous proliferation, and differentiation into phenotypically and functionally similar A-NK cells. Pre-A-NK cells represent only approximately 26% of peripheral blood NK cells but encompass the majority of NK cells in normal and cancerous, solid tissues. We conclude that pre-A-NK cells represent a distinct subset of resting, mature NK cells with the characteristics indicative of their ability to migrate and reside in solid tissues.

    Funded by: NIDCR NIH HHS: 1-P60 DE13059, P0-1 DE12321, R01 DE14775

    Journal of leukocyte biology 2004;76;6;1187-99

  • CD56bright NK cells are enriched at inflammatory sites and can engage with monocytes in a reciprocal program of activation.

    Dalbeth N, Gundle R, Davies RJ, Lee YC, McMichael AJ and Callan MF

    Division of Medicine, Imperial College London, Commonwealth Building, Hammersmith Campus, Du Cane Road, London W12 0NN, UK.

    Human NK cells may be divided into a CD56(dim) subset and a CD56(bright) subset. In peripheral blood, CD56(dim) NK cells dominate, whereas in lymph nodes, CD56(bright) NK cells are more common. In this study we show that CD56(bright) NK cells accumulate within inflammatory lesions in a wide variety of clinical diseases affecting several different anatomical sites. We demonstrate that when activated by the monokines IL-12, IL-15, and IL-18, these NK cells promote TNF-alpha production by CD14(+) monocytes in a manner that is dependent on cell:cell contact. Conversely, CD14(+) monocytes synergize with monokines to promote IFN-gamma production by these NK cells. Again, this interaction is dependent on cell:cell contact. The experiments show that CD56(bright) NK cells accumulate in inflammatory lesions and, in the appropriate cytokine environment, can engage with CD14(+) monocytes in a reciprocal activatory fashion, thereby amplifying the inflammatory response. Such a positive feedback loop is likely to be important in the pathogenesis of chronic inflammatory conditions such as rheumatoid arthritis.

    Funded by: Medical Research Council: G116/121

    Journal of immunology (Baltimore, Md. : 1950) 2004;173;10;6418-26

  • Acute lymphoblastic leukemia with coexpression of CD56 and CD57: case report.

    Matsubara K, Yura K, Hirata T, Nigami H, Harigaya H, Nozaki H, Fukaya T and Baba K

    Department of Pediatrics, Nishi-Kobe Medical Center, 5-7-1 Kojidai, Nishi-ku, Kobe 651-2273, Japan. kskmatsu@s4.dion.ne.jp

    The authors present the clinical profile of a 6-year-old girl with an unusual immunophenotype of acute lymphoblastic leukemia (ALL). At the initial presentation, massive hepatosplenomegaly developed. The leukemic cells were myeloperoxidase-negative and morphologically lymphoblastic. These cells were positive for B-precursor-cell (CD10, CD19) antigens and natural killer cells (CD56, CD57). Rearrangements of both immunoglobulin heavy chain alleles and monoallelic rearrangement of T-cell receptors (TCRs)-beta and -delta genes, but not that of TCR-gamma gene, were detected, suggesting that these cells being of B-precursor origin. The patient received chemotherapy for extremely high-risk ALL with a good response. To the authors' knowledge, this is the first pediatric case describing coexpression of CD56 and CD57 on B-lineage ALL.

    Pediatric hematology and oncology 2004;21;7;677-82

  • Pituitary tumor-derived fibroblast growth factor receptor 4 isoform disrupts neural cell-adhesion molecule/N-cadherin signaling to diminish cell adhesiveness: a mechanism underlying pituitary neoplasia.

    Ezzat S, Zheng L and Asa SL

    Department of Medicine, University of Toronto, Canada.

    We previously identified pituitary tumor-derived fibroblast growth factor receptor 4 (ptd-FGFR4), an alternatively transcribed N-terminally truncated cytoplasmic receptor isoform. Unlike wild-type FGFR4, ptd-FGFR4 facilitates cell transformation and results in pituitary tumor formation in transgenic mice. To investigate differences in the tumorigenic properties of FGFR4 and ptd-FGFR4, we examined their abilities to modulate cell adhesiveness. Introduction of ptd-FGFR4 into GH4 pituitary cells or NIH 3T3 fibroblasts resulted in significant reduction in cell adhesion to a collagen IV matrix compared with FGFR4- or empty vector-transfected cells. This adhesive difference was evident in the absence or presence of FGF stimulation. Furthermore, treatment with beta1-integrin neutralizing antibody markedly reduced adhesiveness in FGFR4-transfected cells but had little effect on the depressed adhesiveness of ptd-FGFR4-transfected cells. Unlike wild-type FGFR4, ptd-FGFR4 does not associate with neural cell-adhesion molecule (NCAM). Cells expressing FGFR4 demonstrate membranous N-cadherin with a noninvasive growth pattern identical to control GH4 cells when injected into immunodeficient mice. In contrast, ptd-FGFR4-expressing cells develop invasive tumors in vivo with marked loss of N-cadherin that localizes to the cytoplasm. Consistent with these changes, beta-catenin expression was diminished and its interaction with N-cadherin was disrupted in the presence of ptd-FGFR4, but both were intact in the presence of wild-type FGFR4. These data highlight the importance of membrane-anchored FGFR4 in assembling a multiprotein FGFR4 complex with NCAM and N-cadherin playing pivotal functions in maintaining normal cell adhesion. Disruption of distinct NCAM/N-cadherin proadhesive complexes by a tumor-derived FGFR4 isoform provides a novel mechanism beyond ligand independence that explains the pathobiology of proliferative and infiltrative but nonmetastatic neoplasms.

    Molecular endocrinology (Baltimore, Md.) 2004;18;10;2543-52

  • Clinical and biological significance of CD56 antigen expression in acute promyelocytic leukemia.

    Ito S, Ishida Y, Oyake T, Satoh M, Aoki Y, Kowata S, Uchiyama T, Enomoto S, Sugawara T, Numaoka H, Suzuki K and Murai K

    Department of Hematology/Oncology, Iwate Medical University School of Medicine, Morioka, Japan. shigei@iwate-med.ac.jp

    The biological significance of CD56 antigen expression in patients with acute promyelocytic leukemia (APL) has been under investigation. We investigated the clinical and biologic features of CD56+APL. In our series, CD56 antigen was positive in 4 of 28 (14%) APL patients. No differences were found regarding age, gender, performance status (PS), initial leukocyte and platelet counts, lactate dehydrogenase (LDH) and fibrinogen (Fbg) levels according to CD56 expression. CD34 antigen was co-expressed in 3 of the 4 patients with CD56+ APL, in contrast to 2 of the 24 patients with CD56- APL (P = .01). Extramedullary relapse occurred in 3 of the 4 patients with CD56+ APL, in contrast to none of the 24 patients with CD56- APL (P = .001). Median remission duration was 4 months in CD56+ APL and was not reached in CD56- APL. The CD56+ population had a shorter remission duration (P < .0001) and disease-free survival (P < .0001). In contrast, no difference was found in overall survival. These results suggested that CD56 expression was associated with the leukemogenetic mutation at the primitive hematopoietic progenitor cell level and extramedullary relapse in APL patients treated with ATRA and chemotherapy.

    Leukemia & lymphoma 2004;45;9;1783-9

  • A Wilms tumor cell line, HFWT, can greatly stimulate proliferation of CD56+ human natural killer cells and their novel precursors in blood mononuclear cells.

    Harada H, Watanabe S, Saijo K, Ishiwata I and Ohno T

    RIKEN Cell Bank, RIKEN (The Institute of Physical and Chemical Research), Tsukuba Science City, Japan.

    Objective: A Wilms tumor cell line, HFWT, selectively stimulates expansion of natural killer (NK) cells from human peripheral blood mononuclear cells (PBMC). In this study, we attempted to identify NK precursors in PBMC or in cord blood mononuclear cells (CBMC) that preferentially respond to feeder HFWT cells.

    Human NK cells or candidate precursor cells were fractionated from PBMC or CBMC by magnetic antibody cell sorting or by flow cytometry and applied to limiting dilution analysis to determine the proportion of NK/NK precursor cells, which are able to proliferate on irradiated HFWT cells. NK and NK precursor cells were cultured in medium containing interleukin-2 (IL-2). Expansion of NK cells from both resting NK cells and NK precursor cells was examined using proliferation from single cells, expression of NK cell markers, and cytotoxic activity.

    Results: In the limiting dilution analysis, NK cells expanded on irradiated HFWT cells not only from CD3-CD56bright and CD3-CD56dim NK cells, but also from CD16+/-CD122+ cells in the lineage-negative (Lin-, CD3-CD14-CD19-CD56-) cell fraction. The feeder HFWT cells stimulated Lin-CD122+ cell proliferation more strongly than feeder cells from the well-known human NK target cell line K562. CBMC contained significantly higher percentages of Lin-CD122+ cells than PBMC.

    Conclusion: CD3-CD14-CD19-CD56- cells expressing CD122+ (a subunit of the IL-2 receptor) preferentially respond to HFWT feeder cells and are novel precursors of CD3-CD56+ NK cells in human PBMC and CBMC.

    Experimental hematology 2004;32;7;614-21

  • Time-controlled transcardiac perfusion cross-linking for the study of protein interactions in complex tissues.

    Schmitt-Ulms G, Hansen K, Liu J, Cowdrey C, Yang J, DeArmond SJ, Cohen FE, Prusiner SB and Baldwin MA

    Institute for Neurodegenerative Disease, San Francisco, California 94143, USA. g.schmittulms@utoronto.ca

    Because of their sensitivity to solubilizing detergents, membrane protein assemblies are difficult to study. We describe a protocol that covalently conserves protein interactions through time-controlled transcardiac perfusion cross-linking (tcTPC) before disruption of tissue integrity. To validate tcTPC for identifying protein-protein interactions, we established that tcTPC allowed stringent immunoaffinity purification of the gamma-secretase complex in high salt concentrations and detergents and was compatible with mass spectrometric identification of cross-linked aph-1, presenilin-1 and nicastrin. We then applied tcTPC to identify more than 20 proteins residing in the vicinity of the cellular prion protein (PrPC), suggesting that PrP is embedded in specialized membrane regions with a subset of molecules that, like PrP, use a glycosylphosphatidylinositol anchor for membrane attachment. Many of these proteins have been implicated in cell adhesion/neuritic outgrowth, and harbor immunoglobulin C2 and fibronectin type III-like motifs.

    Funded by: NCRR NIH HHS: NCRR RR01614; NIA NIH HHS: AG010770, AG02132

    Nature biotechnology 2004;22;6;724-31

  • Association of neural cell adhesion molecule 1 gene polymorphisms with bipolar affective disorder in Japanese individuals.

    Arai M, Itokawa M, Yamada K, Toyota T, Arai M, Haga S, Ujike H, Sora I, Ikeda K and Yoshikawa T

    Department of Schizophrenia Research, Tokyo Metropolitan Organization for Medical Research, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156-8585, Japan.

    Background: Although the pathogenesis of mood disorders remains unclear, heritable factors have been shown to be involved. Neural cell adhesion molecule 1 (NCAM1) is known to play important roles in cell migration, neurite growth, axonal guidance, and synaptic plasticity. Disturbance of these neurodevelopmental processes is proposed as one etiology for mood disorder. We therefore undertook genetic analysis of NCAM1 in mood disorders.

    Methods: We determined the complete genomic organization of human NCAM1 gene by comparing complementary deoxyribonucleic acid and genomic sequences; mutation screening detected 11 polymorphisms. The genotypic, allelic, and haplotype distributions of these variants were analyzed in unrelated control individuals (n = 357) and patients with bipolar disorder (n = 151) and unipolar disorder (n = 78), all from central Japan.

    Results: Three single nucleotide polymorphisms, IVS6+32T>C, IVS7+11G>C and IVS12+21C>A, displayed significant associations with bipolar disorder (for allelic associations, nominal p =.04, p =.02, and p =.004, respectively, all p >.05 after Bonferroni corrections). Furthermore, the haplotype located in a linkage disequilibrium block was strongly associated with bipolar disorder (the p value of the most significant three-marker haplotype is .005).

    Conclusions: Our results suggest that genetic variations in NCAM1 or nearby genes could confer risks associated with bipolar affective disorder in Japanese individuals.

    Biological psychiatry 2004;55;8;804-10

  • Neural cell adhesion molecule in human serum. Increased levels in dementia of the Alzheimer type.

    Todaro L, Puricelli L, Gioseffi H, Guadalupe Pallotta M, Lastiri J, Bal de Kier Joffé E, Varela M and Sacerdote de Lustig E

    Research Area of the Institute of Oncology Angel H. Roffo, University of Buenos Aires, Buenos Aires, Argentina. ltodaro@fmed.uba.ar

    Memory impairment is a process associated with alterations in neuronal plasticity, synapses formation, and stabilization. As the neural cell adhesion molecule (NCAM) plays a key role in synaptic bond stabilization, we analyzed the usefulness of soluble NCAM isoforms in the diagnosis of patients with dementia of the Alzheimer type (DAT). NCAM was measured in the sera of 70 control subjects and 43 DAT patients (with different severity of cognitive impairment, GDS), employing Western blot and densitometric quantification. LMW-NCAM bands (100-130 kDa) decreased significantly with age independently of sex. DAT patients presented values of LMW-NCAM and HMW-NCAM significantly higher than healthy controls of similar age (higher than 130 kDa). Only LMW-NCAM was associated with GDS. Our results suggest that NCAM could be involved in the pathogenesis of DAT disorder and that serum NCAM levels could be useful as differential diagnostic markers of the disease.

    Neurobiology of disease 2004;15;2;387-93

  • The IL-12 signature: NK cell terminal CD56+high stage and effector functions.

    Loza MJ and Perussia B

    Department of Microbiology and Immunology, Kimmel Cancer Center, Jefferson Medical College, 233 South 10th Street, Philadelphia, PA 19107, USA.

    We report that human peripheral NK cells expressing high CD56 levels (CD56(+high)) are terminally differentiated cells indistinguishable from mature NK cells recently activated in the presence of IL-12, and not a functionally distinct NK-cell subset or progenitors to mature CD56(+low) NK cells. CD56(+high) NK cells coexpress all differentiation Ags constitutive or inducible in mature (CD56(+)) NK cells, except CD16, present at lower level than on most mature NK cells. Also, activation markers, activating receptors and adhesion molecules, and most inducible receptors are expressed exclusively and constitutively and are inducible at higher levels on CD56(+high) than on CD56(+low) NK cells. Consistent with their activated phenotype, many CD56(+high) NK cells are cycling and mediate heightened effector functions (proliferation, IFN-gamma and IL-10 but not IL-13 production) in response to IL-12 and other NK cell-specific stimuli. Conversely, IL-12 induces on CD56(+low) NK cells all markers constitutively expressed on the CD56(+high) NK cells, concomitantly preventing the IL-2 (and IL-15)-inducible expression of NKp44 and CD16 re-expression after immune complex-induced down-modulation, and CD56(-/+low) NK cells acquire a CD56(+high) NK cell phenotype in short term in vitro culture with IL-12. The significance of these findings to the NK cell-mediated regulation of immune responses and NK cell development is discussed.

    Funded by: NCI NIH HHS: CA 56036; NIAID NIH HHS: AI 055842

    Journal of immunology (Baltimore, Md. : 1950) 2004;172;1;88-96

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • Prognostic significance of surface markers expressed in multiple myeloma: CD56 and other antigens.

    Sahara N and Takeshita A

    Internal Medicine III, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu-shi, 431-3192, Japan. sahara@hama-med.ac.jp

    Multiple myeloma (MM) is characterized by increased numbers of malignant plasma cells. Plasma cells, that represent the terminal differentiation of B lymphocytes, have considerable heterogeneity of surface markers expressed on them. Some studies showed the prognostic significance of several immunophenotypic molecules on MM cells. Here, we review several surface markers related to their prognostic significance in MM patients. We also report that CD56-negative MM is the unique entity characterized by poor prognosis with high incidence of extramedullary disease, Bence Jones protein, renal insufficiency, thrombocytopenia and plasmablastic morphology.

    Leukemia & lymphoma 2004;45;1;61-5

  • [The overexpression of NCAM (CD56) in human hearts is specific for ischemic damage].

    Gattenlöner S, Waller C, Ertl G, Bültmann BD, Müller-Hermelink HK and Marx A

    Institut für Pathologie der Universitat Würzburg, Würzburg, Germany.

    The main reason for myocardial dysfunction is chronical myocardial ischemia. Recently we could show, that NCAM (CD56), a neural cell adhesion molecule and member of the immunoglobuline superfamily, and the transcription factor AML1 (RUNX1) are overexpressed in chronic ischemic human heart failure compaired to normal hearts. Here we demonstrate, that the overexpression of NCAM (CD56) is specific for ischemic damage as compaired to other heart diseases including congestive cardiomyopathy, hypertrophic obstrutive cardiomyopathy, myocarditis and sarcoidosis. Concerning the transcriptional regulation of NCAM (CD56) by AML1 (RUNX1) we isolated 3 novel isoforms of AML 1 (RUNX1) with different transactivating function, that might be a regulatory element of the NCAM (CD56) overexpression in chronical myocardial ischemia.

    Verhandlungen der Deutschen Gesellschaft fur Pathologie 2004;88;246-51

  • Expression patterns of PSA-NCAM in the human ganglionic eminence and its vicinity: role of PSA-NCAM in neuronal migration and axonal growth?

    Ulfig N and Chan WY

    Neuroembryonic Research Laboratory, Department of Anatomy, University of Rostock, Rostock, Germany. norbert.ulfig@med.uni-rostock.de

    The ganglionic eminence (GE) is a transient but conspicuous structure of the developing forebrain which not only gives rise to a large number of precursor neurons and glial cells for various structures of the forebrain, but in addition serves as an intermediate target for growing axons to the cerebral cortex. To investigate the roles of the highly polysialylated isoform of the neural cell adhesion molecule (PSA-NCAM) in cell migration and axonal growth within the GE and its neighbouring structures, the spatio-temporal expression pattern of PSA-NCAM was examined in human fetal forebrains between 14 and 36 weeks of gestation with a specific immunohistochemical method. Scattered PSA-NCAM-positive cells were found in the centre but more frequently in the marginal zone of the GE. Intensely labelled cells were also identified in the gangliothalamic body, basolateral nuclei of the amygdala and the subventricular and intermediate zone adjacent to the GE. This cellular immunoreactivity started to appear in various structures during the period from 14 to 19 weeks and gradually diminished after 25-28 weeks. Strong immunoreactivity was also detected in fibres running from the intermediate zone of the neocortex to the internal capsule from 16 weeks onwards, and after 24 weeks, the immunoreactivity was gradually decreased. In the vicinity of the GE, between 16 and 22 weeks, short fibre bundles were observed to leave the longitudinally oriented axons of the internal capsule to reach the marginal zone of the GE. Our results suggest a close relationship between PSA-NCAM expression and neuronal migration (over short distances) and transitory axonal projections (target recognition and axonal fasciculation) in the region of the GE.

    Cells, tissues, organs 2004;177;4;229-36

  • The expression pattern of CD56 (N-CAM) in human bone marrow biopsies infiltrated by acute leukemia.

    Khanlari B, Buser A, Lugli A, Tichelli A and Dirnhofer S

    Institute of Pathology, University Hospital Basel, Schoenbeinstrasse 40, 4031 Basel, Switzerland.

    In hematological neoplasms CD56 (N-CAM) is expressed by T/natural killer (NK) cell lymphoma, by most neoplastic plasma cells in multiple myeloma and also in a subset of acute myelogenous leukemias (AML). In the latter, it is an indicator of poor clinical outcome. Most of the data on CD56 expression in acute leukemia have been obtained by flow cytometric analysis. Up to now, no systematic analysis of the expression pattern of CD56 in formalin fixed paraffin embedded bone marrow biopsies of acute leukemias has been performed. We immunohistochemically studied the expression of CD56 in a series of 141 bone marrow biopsies fixed in Sublimat Mercury II Chloride (SUSA) including 100 cases of AML FAB M0-M7, 11 cases of AML not further specified, 3 cases of biphenotypical leukemia, 20 cases of acute lymphoblastic leukemia (ALL) and 7 cases of reactive bone marrow biopsies. Overall, 14 of 134 (10%) leukemia cases were positive for CD56. Detail analysis revealed positivity in 5/13 cases of AML M5 (38%), 3/9 AML M1 (33%), 1/8 AML M0 (13%), 1/11 AML not specified (9%), 2/31 AML M2 (7%) and 2/26 AML M4 (8%). All cases of ALL and biphenotypic leukemias were CD56 negative. The CD56 expression in AML M5 was statistically significant (p = 0.003). On paraffin embedded bone marrow biopsies CD56 expression occurs in de novo AML with an overall frequency of 13%. It is significantly correlated with AML M5, which is positive in 38% of the cases. Cases of ALL are consistently CD56 negative.

    Leukemia & lymphoma 2003;44;12;2055-9

  • CD56-positive cells with or without synaptophysin expression are recognized in the pancreatic duct epithelium: a study with adult and fetal tissues and specimens from chronic pancreatitis.

    Fujisawa M, Notohara K, Tsukayama C, Mizuno R and Okada S

    Department of Pathological Research, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan.

    We observed the distribution of CD56+ epithelial cells in the pancreatic duct system using 25 fetal, one infantile, 3 normal adult, 4 diabetic, and 8 chronically inflamed pancreatic tissue samples. In the early stage of gestation (12 to 17 weeks), CD56+ cells were commonly seen in the immature tubular structures. They were often continuous to pancreatic islets, and their distribution was similar to that of synaptophysin (Syn)+ cells, suggesting that they are precursors of islet neogenesis. Their number decreased in proportion to gestational age. Instead, from 24 weeks of gestation, luminal cell clusters that were common in interlobular ducts revealed CD56+. These cell clusters were unrelated to islet neogenesis and Syn expression. Similar CD56+ luminal cell clusters were also observed in cases of chronic pancreatitis, whereas they were scarce in normal adult and diabetic tissues. CD56+ cells were also occasionally seen in intralobular ducts, intercalated ducts, and centroacinar cells in cases of chronic pancreatitis. We conclude that there are two types of CD56+ epithelial cells in the pancreatic duct system: CD56+ endocrine cells are numerous during the early stage of gestation, when islet neogenesis appears, while CD56+ luminal cells may represent developmental and regenerative changes of pancreatic ducts.

    Acta medica Okayama 2003;57;6;279-84

  • NCAM(CD56) and RUNX1(AML1) are up-regulated in human ischemic cardiomyopathy and a rat model of chronic cardiac ischemia.

    Gattenlöhner S, Waller C, Ertl G, Bültmann BD, Müller-Hermelink HK and Marx A

    Institute of Pathology, University of Würzburg, Josef-Schneiderstrasse 2, 97080 Würzburg, Germany. stefan.gattenloehner@mail.uni-wuerzburg.de

    Chronic myocardial ischemia is the leading cause of impaired myocardial contractility and heart failure. To identify differentially expressed genes in human ischemic cardiomyopathy (ICM), we constructed a subtracted cDNA library using specimens of ICM compared to normal human heart. Among 100 randomly sequenced clones, seven sequences represented recently identified candidate genes for differential expression in cardiac hypertrophy. A further clone without a known hypertrophy-association coded for the adhesion molecule NCAM(CD56). RNase protection assay, immunohistochemistry, and Western blotting revealed strong overexpression of NCAM(CD56) in all hearts with ICM (n = 14) compared to normal hearts (n = 8), whereas in congestive cardiomyopathy (CCM) (n = 8), hypertrophic obstructive cardiomyopathy (n = 2), myocarditis (n = 4), and sarcoidosis (n = 2), at most slight overexpression of NCAM(CD56) was observed. NCAM(CD56) overexpression abnormally involved the whole cell membrane and the cytoplasma of cardiomyocytes only inside and adjacent to ischemia-induced cardiac scars. Normal or hypertrophic fibers at a distance from ischemic scars were devoid of NCAM overexpression. Identical alterations were observed in an experimental rat ICM model, but not in normal nor in spontaneously hypertensive rat hearts. In search of NCAM(CD56)-related transcription factors we found RUNX1(AML1) up-regulation in ICM and detected RUNX1(AML1) binding within the NCAM(CD56) promoter by electromobility shift assay. We concluded that strong overexpression of NCAM(CD56) and RUNX1(AML1) is a constant and characteristic feature of cardiomyocytes within or adjacent to scars in ICM.

    The American journal of pathology 2003;163;3;1081-90

  • The minimal structural domains required for neural cell adhesion molecule polysialylation by PST/ST8Sia IV and STX/ST8Sia II.

    Close BE, Mendiratta SS, Geiger KM, Broom LJ, Ho LL and Colley KJ

    Department of Biochemistry and Molecular Genetics, University of Illinois, College of Medicine, Chicago, Illinois 60612, USA.

    A limited number of mammalian proteins are modified by polysialic acid, with the neural cell adhesion molecule (NCAM) being the most abundant of these. We hypothesize that polysialylation is a protein-specific glycosylation event and that an initial protein-protein interaction between polysialyltransferases and glycoprotein substrates mediates this specificity. To evaluate the regions of NCAM required for recognition and polysialylation by PST/ST8Sia IV and STX/ST8Sia II, a series of domain deletion proteins were generated, co-expressed with each enzyme, and their polysialylation analyzed. A protein consisting of the fifth immunoglobulin-like domain (Ig5), which contains the reported sites of polysialylation, and the first fibronectin type III repeat (FN1) was polysialylated by both enzymes, whereas a protein consisting of Ig5 alone was not polysialylated by either enzyme. This demonstrates that the Ig5 domain of NCAM and FN1 are sufficient for polysialylation, and suggests that the FN1 may constitute an enzyme recognition and docking site. Two other NCAM mutants, NCAM-6 (Ig1-5) and NCAM-7 (FN1-FN2), were weakly polysialylated by PST/ST8Sia IV, suggesting that a weaker enzyme recognition site may exist within the Ig domains, and that glycans in the FN region are polysialylated. Further analysis indicated that O-linked oligosaccharides in NCAM-7, and O-linked and N-linked glycans in full-length NCAM, are polysialylated when these proteins are co-expressed with the polysialyltransferases in COS-1 cells. Our data support a model in which the polysialyltransferases bind to the FN1 of NCAM to polymerize polysialic acid chains on appropriately presented glycans in adjacent regions.

    Funded by: NIGMS NIH HHS: R01 GM 48134, R01 GM 63483, R01 GM063843

    The Journal of biological chemistry 2003;278;33;30796-805

  • The neural cell adhesion molecule NCAM is an alternative signaling receptor for GDNF family ligands.

    Paratcha G, Ledda F and Ibáñez CF

    Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, Retzius väg 8, A2:2, 17177, Stockholm, Sweden.

    Intercellular communication involves either direct cell-cell contact or release and uptake of diffusible signals, two strategies mediated by distinct and largely nonoverlapping sets of molecules. Here, we show that the neural cell adhesion molecule NCAM can function as a signaling receptor for members of the GDNF ligand family. Association of NCAM with GFRalpha1, a GPI-anchored receptor for GDNF, downregulates NCAM-mediated cell adhesion and promotes high-affinity binding of GDNF to p140(NCAM), resulting in rapid activation of cytoplasmic protein tyrosine kinases Fyn and FAK in cells lacking RET, a known GDNF signaling receptor. GDNF stimulates Schwann cell migration and axonal growth in hippocampal and cortical neurons via binding to NCAM and activation of Fyn, but independently of RET. These results uncover an unexpected intersection between short- and long-range mechanisms of intercellular communication and reveal a pathway for GDNF signaling that does not require the RET receptor.

    Cell 2003;113;7;867-79

  • Neural cell adhesion molecule (NCAM) association with PKCbeta2 via betaI spectrin is implicated in NCAM-mediated neurite outgrowth.

    Leshchyns'ka I, Sytnyk V, Morrow JS and Schachner M

    Zentrum für Molekulare Neurobiologie, Universität Hamburg, Germany.

    In hippocampal neurons and transfected CHO cells, neural cell adhesion molecule (NCAM) 120, NCAM140, and NCAM180 form Triton X-100-insoluble complexes with betaI spectrin. Heteromeric spectrin (alphaIbetaI) binds to the intracellular domain of NCAM180, and isolated spectrin subunits bind to both NCAM180 and NCAM140, as does the betaI spectrin fragment encompassing second and third spectrin repeats (betaI2-3). In NCAM120-transfected cells, betaI spectrin is detectable predominantly in lipid rafts. Treatment of cells with methyl-beta-cyclodextrin disrupts the NCAM120-spectrin complex, implicating lipid rafts as a platform linking NCAM120 and spectrin. NCAM140/NCAM180-betaI spectrin complexes do not depend on raft integrity and are located both in rafts and raft-free membrane domains. PKCbeta2 forms detergent-insoluble complexes with NCAM140/NCAM180 and spectrin. Activation of NCAM enhances the formation of NCAM140/NCAM180-spectrin-PKCbeta2 complexes and results in their redistribution to lipid rafts. The complex is disrupted by the expression of dominant-negative betaI2-3, which impairs binding of spectrin to NCAM, implicating spectrin as the bridge between PKCbeta2 and NCAM140 or NCAM180. Redistribution of PKCbeta2 to NCAM-spectrin complexes is also blocked by a specific fibroblast growth factor receptor inhibitor. Furthermore, transfection with betaI2-3 inhibits NCAM-induced neurite outgrowth, showing that formation of the NCAM-spectrin-PKCbeta2 complex is necessary for NCAM-mediated neurite outgrowth.

    The Journal of cell biology 2003;161;3;625-39

  • CD56: a useful marker for diagnosing Merkel cell carcinoma.

    Kurokawa M, Nabeshima K, Akiyama Y, Maeda S, Nishida T, Nakayama F, Amano M, Ogata K and Setoyama M

    Department of Dermatology, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. mkurokawa@post1.miyazaki-med.ac.jp

    Background: Merkel cell carcinoma (MCC) of the skin is an aggressive but rare malignant neuroendocrine tumor. For its pathological diagnosis, we use a panel of immunohistochemical markers, such as cytokeratin 20 (CK 20), epithelial membrane antigen (EMA), chromogranin A, neuron specific enolase (NSE), synaptophysin, and Leu7 (CD57) to demonstrate its epithelial and neuroendocrine features. CD56, or neural cell adhesion molecule (NCAM), has been demonstrated recently as the tumor marker of the pulmonary neuroendocrine cell system. Its expression in MCC, however, has still rarely been investigated. Furthermore, in such very few previous studies on NCAM expression in MCC, all the tumor cells were not necessarily demonstrated to express NCAM.

    Objectives: To study the immunoreactivity of CD56 in MCC, especially using a monoclonal antibody of a clone 1B6, different from those adopted in the previous reports.

    Methods: We reexamined CD56 expression immunohistochemically in five MCC cases, along with the conventional panel of markers described above, using paraffin-embedded tissue sections.

    Results: CD56 revealed the most diffuse and intense positive staining, which was noted along the cell borders, in all specimens compared with other neuroendocrine tumor markers.

    Conclusions: The results of our study indicate that CD56, especially a new monoclonal antibody (clone 1B6), is a useful immunohistochemical marker for MCC.

    Journal of dermatological science 2003;31;3;219-24

  • Preferential apoptosis of CD56dim natural killer cell subset in patients with cancer.

    Bauernhofer T, Kuss I, Henderson B, Baum AS and Whiteside TL

    University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA.

    Natural killer (NK) cells (CD56(+)/CD3(-)) in the circulation of cancer patients were reported to have low NK activity and undergo spontaneous apoptosis. A possible relationship between apoptosis and impaired NK activity was studied by Annexin V-binding and NK-cell assays performed with peripheral blood mononuclear cells of patients with head and neck cancer (HNC), breast cancer (BC) and normal controls (NC). Cells stained with Annexin V (Anx) and antibodies to CD56, CD3, CD95, CD25, CD122 or CD132 were examined by flow cytometry. NK activity was tested against K562 targets in 4-h (51)Cr-release assays. The ratio of CD56(dim)/CD56(bright) NK cells was significantly different in patients vs. controls (10 vs. 16; p<0.01). A significantly greater percentage of CD56(dim) NK cells bound Anx in HNC patients (27+/-17%, median +/- SD) or BC (46+/-18%) than in NC (15+/-18%, p<0.04 and p<0.0002, respectively). CD56(dim) NK cells were preferentially targeted for apoptosis. NK activity was significantly lower in patients with HNC and BC than in NC (p<0.009). An inverse correlation between NK activity and the percent of Anx(+)CD56(dim) NK cells was observed in cancer patients (p =0.002) but not in NC. In patients, circulating CD56(dim) NK cells were targeted for apoptosis, leading to low levels of NK activity.

    Funded by: NCI NIH HHS: 1R01-CA82016; NIDCR NIH HHS: P01-1-DE12321

    European journal of immunology 2003;33;1;119-24

  • Costimulation of T cell receptor-triggered IL-2 production by Jurkat T cells via fibroblast growth factor receptor 1 upon its engagement by CD56.

    Kos FJ and Chin CS

    Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia, USA. fjkos@beckman.com

    Recent studies have demonstrated that neural cell adhesion molecule (NCAM) is involved in multiple adhesive interactions with several different classes of ligands on the cell surface and in the extracellular matrix. One of these ligands is fibroblast growth factor receptor (FGFR) that is expressed on neural cells. While it is known that CD56 is a molecular isoform of NCAM expressed on human NK cells and a subset of T cells, it remains poorly characterized, with its ligand unidentified. Therefore, we were prompted to examine if CD56 molecules on NK cells interact with FGFR expressed on T cells. We demonstrate that ligation of FGFR1 beta on J.C2-14 Jurkat T cells by CD56 on fixed NK-92 cells costimulates TCR/CD3-triggered IL-2 production. CD56-binding mAbs inhibited the costimulatory effect of NK-92 cells in 50-75%. Flow cytometric analysis and cell adhesion assays showed that FGFR1 beta/Fc and FGFR2 beta/Fc chimeric proteins bind to NK-92 cells. The binding of FGFR1 beta/Fc protein to CD56 molecules was verified by immunoprecipitation of CD56 with anti-CD56 mAb followed by Western blotting with FGFR1 beta/Fc. These findings suggest that ligation of FGFR1 by CD56 may contribute to the interaction between NK cells and T cells that we have postulated in our previous studies.

    Immunology and cell biology 2002;80;4;364-9

  • CD56 expression predicts occurrence of CNS disease in acute lymphoblastic leukemia.

    Ravandi F, Cortes J, Estrov Z, Thomas D, Giles FJ, Huh YO, Pierce S, O'Brien S, Faderl S and Kantarjian HM

    Department of Hematology/Oncology, University of Illinois, Chicago, IL, USA.

    We examined the pre-treatment bone marrow samples from 200 consecutive adult patients with acute lymphoblastic leukemia (ALL) treated on various protocols at the University of Texas, M.D. Anderson Cancer Center between 1986 and 1998. Standard MFC techniques were used to determine CD56 expression on the leukemia blasts cells. The expression of CD56 was correlated with clinical characteristics at diagnosis, response to therapy, survival and disease-free survival. Blast expression of CD56 (> or = 20% of leukemic blasts) was seen in 16 (8%) of patients, with a median expression of 67% (range 20-99%). CD56 expression was associated with a higher incidence of central nervous system (CNS) disease at diagnosis (19% versus 4%; P=0.016). Incidence of CNS disease at any time was higher in patients with CD56+ disease (31% versus 14%; P=0.057). Among the 109 patients uniformly treated with the hyperCVAD regimen, CD56 expression was associated with a statistically significant higher incidence of CNS disease (33% versus 9%; P=0.026). CD56 expression in ALL is uncommon but may predict a higher risk for CNS disease. If these results are confirmed, CD56 expression could be used in combination with other high-risk features (e.g. lactate dehydrogenase (LDH), S-phase fraction, mature B-cell phenotype) to design a risk-oriented approach to CNS prophylaxis.

    Leukemia research 2002;26;7;643-9

  • PSA-NCAM immunoreactivity in chandelier cell axon terminals of the human temporal cortex.

    Arellano JI, DeFelipe J and Muñoz A

    Instituto Cajal, CSIC, E-28002 Madrid, Spain.

    In the adult central nervous system, the expression of polysialylated forms of the cell-surface glycoprotein NCAM (PSA-NCAM) is thought to be confined to areas particularly susceptible to plastic changes. In the present study, PSA-NCAM was found to be expressed in the somata, dendrites and axonal processes of some neurons, including cartridge-like elements, which according to our criteria, were identified as chandelier cell axon terminals (chandelier terminals), in the adult human entorhinal cortex and neocortex. These chandelier terminals were very numerous in layers II and III, whereas in deeper layers they were found only occasionally. Double immunocytochemical staining for PSA-NCAM with parvalbumin (PV), with GABA transporter (GAT-1) or with the 5-HT(1A) serotonin receptor allowed us to verify them as true chandelier terminals. Nearly all (92-95%) PV-immunoreactive (-ir) and GAT-1-ir chandelier terminals in layers II and III coexpressed PSA-NCAM. Most of the PSA-NCAM-ir chandelier terminals (89-98%) were also labeled for PV and GAT-1. The results suggest that chandelier terminals in layers II and III of the human entorhinal cortex and temporal neocortex might be particularly susceptible to plastic changes.

    Cerebral cortex (New York, N.Y. : 1991) 2002;12;6;617-24

  • [Neural cell adhesion molecules in lipid microdomains].

    Itoh K and Kamiguchi H


    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 2002;47;4 Suppl;338-43

  • NCAM regulates cell motility.

    Prag S, Lepekhin EA, Kolkova K, Hartmann-Petersen R, Kawa A, Walmod PS, Belman V, Gallagher HC, Berezin V, Bock E and Pedersen N

    Protein Laboratory, Institute of Molecular Pathology, University of Copenhagen, Denmark.

    Cell migration is required during development of the nervous system. The regulatory mechanisms for this process, however, are poorly elucidated. We show here that expression of or exposure to the neural cell adhesion molecule (NCAM) strongly affected the motile behaviour of glioma cells independently of homophilic NCAM interactions. Expression of the transmembrane 140 kDa isoform of NCAM (NCAM-140) caused a significant reduction in cellular motility, probably through interference with factors regulating cellular attachment, as NCAM-140-expressing cells exhibited a decreased attachment to a fibronectin substratum compared with NCAM-negative cells. Ectopic expression of the cytoplasmic part of NCAM-140 also inhibited cell motility, presumably via the non-receptor tyrosine kinase p59(fyn) with which NCAM-140 interacts. Furthermore, we showed that the extracellular part of NCAM acted as a paracrine inhibitor of NCAM-negative cell locomotion through a heterophilic interaction with a cell-surface receptor. As we showed that the two N-terminal immunoglobulin modules of NCAM, which are known to bind to heparin, were responsible for this inhibition, we presume that this receptor is a heparan sulfate proteoglycan. A model for the inhibitory effect of NCAM is proposed, which involves competition between NCAM and extracellular components for the binding to membrane-associated heparan sulfate proteoglycan.

    Journal of cell science 2002;115;Pt 2;283-92

  • Alternative mRNA splicing in colon cancer causes loss of expression of neural cell adhesion molecule.

    Huerta S, Srivatsan ES, Venkatesan N, Peters J, Moatamed F, Renner S and Livingston EH

    Department of Surgery, VA Greater Los Angeles Health Care System/UCLA School of Medicine, Los Angeles, CA 90073, USA.

    Background: The neural cell adhesion molecule (NCAM) has numerous isoforms resulting from alternative splicing of mRNA. The 3 major isoforms found in adult tissue are (1) a 120-kDa protein that is linked to the plasma membrane by glycosylphosphatidylinositol; (2) a 140-kDa form that has a transmembrane component and a cytoplasmic tail with unknown function; and (3) a 180-kDa isoform that has an intracellular protein that binds the cytoskeleton. NCAM is capable of homotypic binding and therefore plays a role in cell-cell adhesion for cells expressing the 180-kDa isoform by anchoring groups of cells into epithelial sheets. NCAM-180 is the isoform found in colonocytes, and loss of expression is associated with clinically aggressive colon cancers.

    Methods: Western blotting and reverse transcriptase-polymerase chain reaction were used to screen commercially available cell lines for NCAM-180 expression. For cell-line pairs with differential NCAM-180 expression, exon analysis was performed with reverse transcriptase-polymerase chain reaction to determine where the molecule was spliced, culminating in failed expression. These results were confirmed with exon analysis in colon cancers harvested at the time of laparotomy.

    Results: Analysis of a SW480 cell line (derived from a patient's primary colon cancer lesion) revealed NCAM-180 expression, whereas no expression was found in the SW620 cell line (derived from a metastatic lesion from the same patient). Exon analysis of NCAM mRNA transcripts from SW620 revealed that the transcripts were truncated after exon 12. This region correlates to an area between 2 fibronectin-III domains on the NCAM protein.

    Conclusions: The most common site for NCAM alternative splicing is between the 2 fibronectin-III domains corresponding to the border between exons 12 and 13 of the NCAM gene. Loss of NCAM-180 expression in aggressive colon carcinoma results from a splice defect in the same area, which may result in defective intracellular adhesion between colonocytes.

    Surgery 2001;130;5;834-43

  • Characterization of human cleaved N-CAM and association with schizophrenia.

    Vawter MP, Usen N, Thatcher L, Ladenheim B, Zhang P, VanderPutten DM, Conant K, Herman MM, van Kammen DP, Sedvall G, Garver DL and Freed WJ

    Development and Plasticity Section, Cellular Neurobiology Research Branch, National Institute on Drug Abuse, 5500 Nathan Shock Drive, Baltimore, Maryland 21224, USA.

    The neural cell adhesion molecule (N-CAM) is a cell recognition molecule involved in cellular migration, synaptic plasticity, and CNS development. A 105- to 115-kDa isoform of N-CAM (cleaved N-CAM or cN-CAM) is increased in schizophrenia in hippocampus, prefrontal cortex, and CSF. We purified and partially characterized cN-CAM, a putative novel isoform, and confirmed that the first 9 amino acids were identical to exon 1 of N-CAM, without the signal sequence. Analysis of trypsin-digested cN-CAM fragments by matrix-assisted laser desorption ionization on a time-of-flight mass spectrometer (MALDI-TOF) yielded peptides that could be identified as being derived from the first 548 amino acid residues of the expected N-CAM amino acid sequence. Immunological identification with four specific N-CAM antisera directed toward cytoplasmic, secreted, variable alternative spliced exon, or GPI epitopes failed to indicate other known splice variants. Neuraminidase treatment of cN-CAM produced a minor alteration resulting in a faster migrating immunoreactive band, indicating partial glycosylation of cN-CAM. Membranous particles from cytosolic brain extract containing cN-CAM were obtained by ultracentrifugation; however, CSF contained few such particles. cN-CAM and synaptophysin were colocalized on these particles. Both cN-CAM and N-CAM 180 were present in synaptosomal preparations of human brain. Following incubation of synaptosomes or brain tissue without protease inhibitors, N-CAM 180 was degraded and cN-CAM was increased. A cN-CAM-like band was present in human fetal neuronal cultures, but not in fetal astrocyte cultures. Thus, cN-CAM represents a protease- and neuraminidase-susceptible fragment possibly derived by proteolytic cleavage of N-CAM 180. An enlargement in ventricular volume in a group of adult patients with schizophrenia over a 2-year interval was found to be correlated with CSF cN-CAM levels as measured at the time of the initial MRI scan (r = 0.53, P = 0.01). cN-CAM is associated with ventricular enlargement; thus, the release of N-CAM fragments may be part of the pathogenic mechanism of schizophrenia in vulnerable brain regions such as the hippocampus and prefrontal cortex. Alternatively, the increases in cN-CAM in schizophrenia may be a reflection of a more general abnormality in the regulation of proteolysis or of extracellular matrix stability.

    Experimental neurology 2001;172;1;29-46

  • Significance of cell adhesion molecules, CD56/NCAM in particular, in human tumor growth and spreading.

    Zeromski J, Nyczak E and Dyszkiewicz W

    Department of Clinical Immunology, Karol Marcinkowski University of Medical Sciences, Poznań, Poland. jzeromski@eucalyptus.usoms.poznan.pl

    Cell adhesion molecules (CAM) represent a large group of cell surface protein moieties with distinctive biological functions. In physiological terms they ascertain cell to cell contact such as cell cohesion of epithelia, condition cell migration and transmigration via biological membranes such as blood vessel walls, provide means for homing cells in a new microenvironment etc. These features of CAM are exploited by tumor cells to grow and spread in a tumor bearing host. CD56/N-CAM antigen is 140 kD isoform of neural cell adhesion molecule. N-CAM belongs to the large Ig superfamily of CAMs. CD56 can be traced at various sites, including nervous tissue, neuro-muscular junctions, neuroendocrine and endocrine organs. It is well known as a differentiation antigen of natural killer (NK) cells. Its role and function are far from clear, but its adhesion properties are evident in cell-cell (homophilic) interactions. CD56 has been, however, demonstrated the cells various human malignancies. Tumors of the nervous system such as neuroblastoma, are well known to express this marker. Malignant lymphomas of T-NK cell origin bear CD56, as well as multiple myeloma, melanoma and some cancers of epithelial origin. These data suggest that CD56/N-CAM antigen is, in some unknown manner involved in tumor biology.

    Folia histochemica et cytobiologica 2001;39 Suppl 2;36-7

  • Overexpression of murine Pax3 increases NCAM polysialylation in a human medulloblastoma cell line.

    Mayanil CS, George D, Mania-Farnell B, Bremer CL, McLone DG and Bremer EG

    Pediatric Brain Tumor Research Program, Department of Neurosurgery, Children's Memorial Institute of Education and Research, Chicago, Illinois 60614, USA.

    Polysialic acid (PSA) is a developmentally regulated carbohydrate found primarily on neural cell adhesion molecules (NCAM) in embryonic tissues. The majority of NCAM in adult tissues lacks this unique carbohydrate, but polysialylated NCAM (PSA-NCAM) is present in adult brain regions where neural regeneration persists and in some pediatric brain tumors such as medulloblastoma, which show greater propensity for leptomeningeal spread. Pax3, a developmentally regulated paired homeodomain transcription factor, is thought to be involved in the regulation of neural cell adhesion molecules. Overexpression of murine Pax3 into a human medulloblastoma cell line (DAOY) resulted in an increase in NCAM polysialylation and a 2-4-fold increase in alpha2, 8-polysialyltransferase type II mRNA levels. No difference was observed in alpha2,8-polysialyltransferase type IV message. The addition of PSA to NCAM changed the adhesive behavior of these Pax3 transfectants. Transfectants expressing high PSA-NCAM show much less NCAM-dependent aggregation than those with less PSA-NCAM. In addition, Pax3 transfectants having high PSA-NCAM show heterophilic adhesion involving polysialic acid to heparan sulfate proteoglycan and agrin. These observations suggest that a developmentally regulated transcription factor, Pax3, could affect NCAM polysialylation and subsequently cell-cell and cell-substratum interaction.

    Funded by: NINDS NIH HHS: NS33383

    The Journal of biological chemistry 2000;275;30;23259-66

  • Differential biosynthesis of polysialic acid on neural cell adhesion molecule (NCAM) and oligosaccharide acceptors by three distinct alpha 2,8-sialyltransferases, ST8Sia IV (PST), ST8Sia II (STX), and ST8Sia III.

    Angata K, Suzuki M, McAuliffe J, Ding Y, Hindsgaul O and Fukuda M

    Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, California 92037, USA.

    Polysialylated neural cell adhesion molecule (NCAM) is thought to play a critical role in neural development. Polysialylation of NCAM was shown to be achieved by two alpha2,8-polysialyltransferases, ST8Sia IV (PST) and ST8Sia II (STX), which are moderately related to another alpha2,8-sialyltransferase, ST8Sia III. Here we describe that all three alpha2,8-sialyltransferases can utilize oligosaccharides as acceptors but differ in the efficiency of adding polysialic acid on NCAM. First, we found that ST8Sia III can form polysialic acid on the enzyme itself (autopolysialylation) but not on NCAM. These discoveries prompted us to determine if ST8Sia IV and ST8Sia II share the property of ST8Sia III in utilizing low molecular weight oligosaccharides as acceptors. By using a newly established method, we found that ST8Sia IV, ST8Sia II, and ST8Sia III all add oligosialic and polysialic acid on various sialylated N-acetyllactosaminyl oligosaccharides, including NCAM N-glycans, fetuin N-glycans, synthetic sialylated N-acetyllactosamines, and on alpha(2)-HS-glycoprotein. Our results also showed that monosialyl and disialyl N-acetyllactosamines can serve equally as an acceptor, suggesting that no initial addition of alpha2,8-sialic acid is necessary for the action of polysialyltransferases. Polysialylation of NCAM by ST8Sia IV and ST8Sia II is much more efficient than polysialylation of N-glycans isolated from NCAM. Moreover, ST8Sia IV and ST8Sia II catalyze polysialylation of NCAM much more efficiently than ST8Sia III. These results suggest that no specific acceptor recognition is involved in polysialylation of low molecular weight sialylated oligosaccharides, whereas the enzymes exhibit pronounced acceptor specificities if glycoproteins are used as acceptors.

    Funded by: NCI NIH HHS: P01 CA71932, R01 CA33595

    The Journal of biological chemistry 2000;275;24;18594-601

  • LDL receptor-related protein as a component of the midkine receptor.

    Muramatsu H, Zou K, Sakaguchi N, Ikematsu S, Sakuma S and Muramatsu T

    Department of Biochemistry, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.

    Midkine (MK) is a heparin-binding growth factor with migration-promoting and survival-promoting activities. To identify signaling receptor(s) of MK, membrane glycoproteins with MK-binding activity were isolated from day 13 mouse embryos by lectin- and MK-affinity chromatography. SDS-PAGE followed by protein sequence analysis revealed the presence of LDL receptor-related protein (LRP) and NCAM in the fraction. The dissociation constant of binding between LRP and MK was 3.5 nM. Receptor-associated protein (RAP), which interfered with the binding, inhibited MK-dependent survival of embryonic neurons. Brushin/megalin, which is also a high molecular weight protein belonging to the LDL receptor family, bound to MK less strongly than LRP. These findings suggest that LRP is a component of the receptor complex for MK.

    Biochemical and biophysical research communications 2000;270;3;936-41

  • Brain-derived neurotrophic factor restores long-term potentiation in polysialic acid-neural cell adhesion molecule-deficient hippocampus.

    Muller D, Djebbara-Hannas Z, Jourdain P, Vutskits L, Durbec P, Rougon G and Kiss JZ

    Neuropharmacology and Department of Morphology, Centre Médical Universitaire, 1211 Geneva 4, Switzerland. Dominique.Muller@medecine.unige.ch

    The neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) contribute to long-term potentiation (LTP) in the CA1 hippocampus. Here we report that the deficient LTP found in slices prepared from NCAM knockout mice and in organotypic slice cultures treated with Endo-N, an enzyme that cleaves the PSA moiety of NCAM, can be rescued by brain-derived neurotrophic factor (BDNF). This effect is not reproduced by nerve growth factor, but can be obtained with high concentrations of NT4/5. The effect of BDNF cannot be accounted for by modifications of N-methyl-D-aspartate receptor-dependent responses or of high-frequency bursts. PSA-NCAM, however, could directly interact with BDNF. Exogenous application of PSA residues or recombinant PSA-NCAM also prevents LTP. Furthermore trkB phosphorylation, and thus BDNF signaling, is reduced in both NCAM knockout mice and Endo-N-treated slice cultures. These results suggest that one action of PSA-NCAM could be to sensitize pyramidal neurons to BDNF, thereby modulating activity-dependent synaptic plasticity.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;8;4315-20

  • Homophilic NCAM interactions interfere with L1 stimulated neurite outgrowth.

    Kristiansen LV, Marques FA, Soroka V, Ronn LC, Kiselyov V, Pedersen N, Berezin V and Bock E

    Protein Laboratory, Institute of Molecular Pathology, University of Copenhagen, Panum Institute, Blegdamsvej 3C, Bld. 6.2, DK-2200, Copenhagen N, Denmark.

    The cell adhesion molecules NCAM and L1 are considered to play key roles in neuronal development and plasticity. L1 has been shown to interact with NCAM, possibly through NCAM binding to oligomannosidic glycans present in L1. We investigated the effect of recombinant immunoglobulin (Ig) modules of NCAM involved in homophilic NCAM binding, on L1 induced neurite outgrowth from PC12-E2 cells and found a complete inhibition of L1 induced neurite outgrowth after addition of Ig-modules 1, 2 and 3 of NCAM, suggesting that the ligation state of NCAM is crucial for normal L1 signaling.

    FEBS letters 1999;464;1-2;30-4

  • The neural cell adhesion molecule (NCAM) in development and plasticity of the nervous system.

    Rønn LC, Hartz BP and Bock E

    Protein Laboratory, Institute for Molecular Pathology, University of Copenhagen, Denmark. lcr@plab.ku.dk

    The neural cell adhesion molecule (NCAM) is a member of the immunoglobulin superfamily and is strongly expressed in the nervous system. NCAM is found in three major forms, of which two--NCAM-140 and NCAM-180--are transmembrane proteins, while the third--NCAM-120--is attached to the membrane via a glycosylphosphatidyl inositol anchor. In addition, soluble NCAM forms exist in brain, cerebrospinal fluid, and plasma. NCAM mediates cell adhesion through homophilic as well as through heterophilic interactions. Following NCAM binding, transmembrane signalling is believed to be activated, resulting in increased intracellular calcium. By mediating cell adhesion to other cells and to the extracellular matrix and by activating intracellular signaling pathways, NCAM influences cell migration, neurite extension, and fasciculation, and possibly formation of synapses in the brain. From studies on NCAM knock-out mice, NCAM have been shown to be crucial for the formation of the olfactory bulb and the mossy fiber system in the hippocampus. In addition, NCAM is important for neuronal plasticity in the adult brain associated with learning and regeneration.

    Experimental gerontology 1998;33;7-8;853-64

  • Differential and cooperative polysialylation of the neural cell adhesion molecule by two polysialyltransferases, PST and STX.

    Angata K, Suzuki M and Fukuda M

    Glycobiology Program, La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037, USA.

    PST and STX are polysialyltransferases that form polysialic acid in the neural cell adhesion molecule (NCAM), and these two polysialyltransferases often exist together in the same tissues. To determine the individual and combined roles of PST and STX in polysialic acid synthesis, in the present study we asked if PST and STX differ in the acceptor requirement and if PST and STX act together in polysialylation of NCAM. We first examined whether PST and STX differ in the requirement of sialic acid and core structures of N-glycans attached to NCAM. Polysialic acid was formed well on Lec4 and Lec13 cells, which are defective in N-acetylglucosaminyltransferase V and GDP-fucose synthesis, respectively, demonstrating that a side chain elongating from GlcNAcbeta1-->6Manalpha1-->6R and alpha-1,6-linked fucose are not required. PST and STX were found to add polysialic acid on NCAM.Fc molecules sialylated by alpha-2,3- or alpha-2,6-linkage in vitro, but not on NCAM.Fc lacking either sialic acid. These results indicate that both PST and STX have relatively broad specificity on N-glycan core structures in NCAM and no remarkable difference exists between PST and STX for the requirement of core structures and sialic acid attached to the N-glycans of NCAM. We then, using various N-glycosylation site mutants of NCAM, discovered that PST strongly prefer the sixth N-glycosylation site, which is the closest to the transmembrane domain, over the fifth site. STX slightly prefer the sixth N-glycosylation site over the fifth N-glycosylation site. The results also demonstrated that polysialic acid synthesized by PST is larger than that synthesized by STX in vitro. Moreover, a mixture of PST and STX more efficiently synthesized polysialic acid on NCAM than PST or STX alone. These results suggest that polysialylation of NCAM is influenced by the difference between PST and STX in their preference for N-glycosylation sites on NCAM. The results also suggest that PST and STX form polysialylated NCAM in a synergistic manner.

    Funded by: NCI NIH HHS: R01 CA 33895

    The Journal of biological chemistry 1998;273;43;28524-32

  • NCAM140 interacts with the focal adhesion kinase p125(fak) and the SRC-related tyrosine kinase p59(fyn).

    Beggs HE, Baragona SC, Hemperly JJ and Maness PF

    Department of Biochemistry, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.

    Axonal growth cones respond to adhesion molecules and extracellular matrix components by rapid morphological changes and growth rate modification. Neurite outgrowth mediated by the neural cell adhesion molecule (NCAM) requires the src family tyrosine kinase p59(fyn) in nerve growth cones, but the molecular basis for this interaction has not been defined. The NCAM140 isoform, which is found in migrating growth cones, selectively co-immunoprecipitated with p59(fyn) from nonionic detergent (Brij 96) extracts of early postnatal mouse cerebellum and transfected rat B35 neuroblastoma and COS-7 cells. p59(fyn) did not associate significantly with the NCAM180 isoform, which is found at sites of stable neural cell contacts, or with the glycophosphatidylinositol-linked NCAM120 isoform. pp60(c-)src, a tyrosine kinase that promotes neurite growth on the neuronal cell adhesion molecule L1, did not interact with any NCAM isoform. Whereas p59(fyn) was constitutively associated with NCAM140, the focal adhesion kinase p125(fak), a nonreceptor tyrosine kinase known to mediate integrin-dependent signaling, became recruited to the NCAM140-p59(fyn) complex when cells were reacted with antibodies against the extracellular region of NCAM. Treatment of cells with a soluble NCAM fusion protein or with NCAM antibodies caused a rapid and transient increase in tyrosine phosphorylation of p125(fak) and p59(fyn). These results suggest that NCAM140 binding interactions at the cell surface induce the assembly of a molecular complex of NCAM140, p125(fak), and p59(fyn) and activate the catalytic function of these tyrosine kinases, initiating a signaling cascade that may modulate growth cone migration.

    Funded by: NINDS NIH HHS: NS26620

    The Journal of biological chemistry 1997;272;13;8310-9

  • Analysis of neurocan structures interacting with the neural cell adhesion molecule N-CAM.

    Retzler C, Göhring W and Rauch U

    Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany. rauch@vms.biochem.mpg.de

    Neurocan is a brain-specific chondroitin sulfate proteoglycan, which has been shown to bind to the neural cell adhesion molecule N-CAM and to inhibit its homophilic interaction. To study in more detail the structures of neurocan responsible for this interaction, various recombinant neurocan fragments were generated. The ability of these fragments to interact with N-CAM was investigated in several different in vitro assay systems, enzyme-linked immunosorbent assay-type binding assays, Covasphere-aggregation assays, and assays based on an optical biosensor (BIAcoreTM) system. The analysis of the homophilic N-CAM interaction in the BIAcore system revealed a KD of 64 nM. This homophilic interaction could be reduced by preincubation of soluble N-CAM with neurocan. Direct binding of N-CAM to immobilized neurocan core protein and recombinant neurocan fragments could also be demonstrated, and KD values between 25 and 100 nM were obtained. In addition, direct binding of N-CAM to chondroitin sulfate could be demonstrated. Binding of N-CAM to the immobilized neurocan core protein could be inhibited with all recombinant fragments containing chondroitin sulfate or major parts of the mucin-like central region of neurocan. For the inhibition of homophilic N-CAM interactions, however, a combination of globular and extended structures was required.

    The Journal of biological chemistry 1996;271;44;27304-10

  • Characterization of mouse ST8Sia II (STX) as a neural cell adhesion molecule-specific polysialic acid synthase. Requirement of core alpha1,6-linked fucose and a polypeptide chain for polysialylation.

    Kojima N, Tachida Y, Yoshida Y and Tsuji S

    Department of Molecular Glycobiology, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan.

    We previously showed that mouse ST8Sia II (STX) exhibits polysialic acid (PSA) synthase activity in vivo as well as in vitro (Kojima, N., Yoshida, Y., and Tsuji, S. (1995) FEBS Lett. 373, 119-122, 1995). In this paper, we reported that the neural cell adhesion molecule (NCAM) was specifically polysialylated by a single enzyme, ST8Sia II. PSA-expressing Neuro2a cells (N2a-STX) were established by stable transfection of the mouse ST8Sia II gene. Only the 140- and 180-kDa isoforms of NCAM in N2a-STX cells were specifically polysialylated in vivo, although other membrane proteins of N2a-STX were polysialylated in vitro. A recombinant soluble mouse ST8Sia II synthesized PSA on a recombinant soluble NCAM fused with the Fc region of human IgG1 (NCAM-Fc) as well as fetuin. However, NCAM-Fc served as a 1500-fold better acceptor for ST8Sia II than fetuin. Treatment of NCAM-Fc with Charonia lampas alpha-fucosidase, which is able to cleave alpha1,6-linked fucose, clearly reduced the polysialylation of NCAM-Fc by ST8Sia II. PSA was not synthesized on the N-glycanase-treated NCAM-Fc polypeptide or the free N-glycans of NCAM-Fc. When fetuin and its glycopeptide and N-glycans of fetuin were used as substrates for ST8Sia II, PSA was found to be synthesized on native fetuin and its glycopeptide but not on free N-glycans. These results strongly suggested that core alpha1, 6-fucose on N-glycans as well as the antennary structures of N-glycans and the polypeptide regions are required for the polysialylation by ST8Sia II. Furthermore, oligo and single alpha2, 8-sialylated glycoproteins were no longer polysialylated by mouse ST8Sia II. Therefore, the single enzyme, ST8Sia II, directly transferred all alpha2,8-sialic acid residues on the alpha2,3-linked sialic acids of N-glycans of specific NCAM isoforms to yield PSA-NCAM. Polysialylation did not require any initiator alpha2, 8-sialyltransferase but did depend on the carbohydrate and protein structures of NCAM.

    The Journal of biological chemistry 1996;271;32;19457-63

  • Expression cloning of a human polysialyltransferase that forms the polysialylated neural cell adhesion molecule present in embryonic brain.

    Nakayama J, Fukuda MN, Fredette B, Ranscht B and Fukuda M

    Glycobiology and Neurobiology Program, La Jolla Cancer Research Foundation, Cancer Research Center, CA 92037, USA.

    Polysialic acid is a developmentally regulated posttranslational modification of the neural cell adhesion molecule (N-CAM). It has been suggested that this large anionic carbohydrate modulates the adhesive property of N-CAM, but the precise function of polysialic acid is not known. Here we describe the isolation and functional expression of a cDNA encoding a human polysialyltransferase. For this expression cloning, COS-1 cells were cotransfected with a human fetal brain cDNA library and a cDNA encoding human N-CAM. Transfected COS-1 cells were stained with a monoclonal antibody specific for polysialic acid and enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA clone that directs the expression of polysialic acid on the cell surface. The deduced amino acid sequence indicates that the polysialyltransferase shares a common sequence motif with other sialyltransferases cloned so far. The polysialyltransferase is, however, distinct by having two clusters of basic amino acids. The amount of the polysialyltransferase transcripts correlates well with the formation of polysialic acid in various human tissues, and is abundant in the fetal brain but not in the adult brain. Moreover, HeLa cells stably expressing polysialic acid and N-CAM promoted neurite outgrowth and sprouting. These results indicate that the cloned polysialyltransferase forms polysialylated, embryonic N-CAM, which is critical for plasticity of neural cells.

    Funded by: NCI NIH HHS: CA33895; NICHD NIH HHS: HD25938; NIDDK NIH HHS: DK37016

    Proceedings of the National Academy of Sciences of the United States of America 1995;92;15;7031-5

  • Localization of NCAM on NCAM-B-expressing cells with inhibited migration in collagen.

    Meyer MB, Bastholm L, Nielsen MH, Elling F, Rygaard J, Chen W, Obrink B, Bock E and Edvardsen K

    Department of Electron Microscopy, University Institute of Pathological Anatomy, Copenhagen, Denmark.

    The extracellular matrix is a key element in neuronal development and tumour invasion, providing a substratum which sustains the adhesion and migration of cells. In order to study interactions between the neural cell adhesion molecule (NCAM) and collagen, we transfected mouse L cells with cDNA encoding the human transmembrane NCAM isoform of 140 kDa (NCAM-B). An L-cell/collagen type I system was used to study the influence of NCAM expression on in vitro invasion. We here report that migration of NCAM-expressing cells in collagen was inhibited compared to that of NCAM-negative cells transfected with the empty vector. Immunofluorescence confocal laser scanning microscopy (CLSM) and immunogold electron microscopy using anti-human NCAM antibodies demonstrated a heterogeneous distribution of NCAM on the plasma membrane of transfected L cells grown on collagen. NCAM was preferentially located at the surface of broad cytoplasmic protrusions and slender extensions, some of which were facing the collagen. This was in contrast to the homogeneous surface distribution of NCAM on cells grown on plastic. These data suggest that NCAM and collagen type I interact, and that this might lead to the migration inhibition of NCAM-expressing cells.

    APMIS : acta pathologica, microbiologica, et immunologica Scandinavica 1995;103;3;197-208

  • Molecular characterization of eukaryotic polysialyltransferase-1.

    Eckhardt M, Mühlenhoff M, Bethe A, Koopman J, Frosch M and Gerardy-Schahn R

    Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Germany.

    Polysialic acid (PSA) is a dynamically regulated product of post-translational modification of the neural cell adhesion molecule, NCAM. Presence of the large anionic carbohydrate modulates NCAM binding properties and, by increasing the intercellular space, influences interactions between other cell surface molecules. PSA expression underlies cell type- and developmental-specific alterations and correlates with stages of cellular motility. In the adult, PSA becomes restricted to regions of permanent neural plasticity and regenerating neural and muscle tissues. Recent data implicate its important function in spatial learning and memory, and in tumour biology. Here we describe the molecular characterization of polysialyltransferase-1, the key enzyme of eukaryotic PSA synthesis. In reconstitution experiments, the newly cloned enzyme induces PSA synthesis in all NCAM-expressing cell lines. Our data therefore represent convincing evidence that the polycondensation of alpha-2,8-linked sialic acids in mammals is the result of a single enzymatic activity and provide a new basis for studying the functional role of PSA in neuro- and tumour biology.

    Nature 1995;373;6516;715-8

  • Complementary DNA sequence encoding the major neural cell adhesion molecule isoform in a human small cell lung cancer cell line.

    Saito S, Tanio Y, Tachibana I, Hayashi S, Kishimoto T and Kawase I

    Department of Internal Medicine III, Osaka University Medical School, Japan.

    The neural cell adhesion molecule (N-CAM), a member of the immunoglobulin gene super-family mediating homophilic cell-cell adhesion in a neuroendocrine system, is preferentially expressed in human small cell lung cancer (SCLC). Immunoprecipitation of a panel of SCLC cell lines by monoclonal antibodies (mAbs) specific for N-CAM detects mainly the 145-kDa isoform. This result was correlated with Northern blotting where a single 6.2-kb mRNA was detected in nine SCLC cell lines. To determine cDNA sequence encoding the N-CAM isoform, we selected several cDNA clones encoding N-CAM isolated from OS2-R, a SCLC cell line established in our laboratory. Based on the analysis of the full-length cDNA obtained from two clones, the sequence of this 145-kDa isoform was shown to be essentially identical to that of the 140-kDa N-CAM isoform of neuroblastoma except for a single base pair changed at position 1620 without changing amino acid encoded.

    Lung cancer (Amsterdam, Netherlands) 1994;10;5-6;307-18

  • The fourth immunoglobulin-like domain of NCAM contains a carbohydrate recognition domain for oligomannosidic glycans implicated in association with L1 and neurite outgrowth.

    Horstkorte R, Schachner M, Magyar JP, Vorherr T and Schmitz B

    Department of Neurobiology, Swiss Federal Institute of Technology, Zürich.

    We have previously shown that the neural adhesion molecules L1 and NCAM interact with each other to form a complex which binds more avidly to L1 than L1 to L1 alone (Kadmon, G., A. Kowitz, P. Altevogt, and M. Schachner. 1990a. J. Cell Biol. 110:193-208). This cis-association between L1 and NCAM is carbohydrate-dependent (Kadmon, G., A. Kowitz, P. Altevogt, and M. Schachner. 1990b. J. Cell Biol. 110:209-218). In the present study, we report that L1 and NCAM bind to each other via oligomannosidic carbohydrates expressed by L1, but not by NCAM, as shown in several experiments: (a) complex formation between L1 and NCAM is inhibited by a mAb to oligomannosidic carbohydrates and by the oligosaccharides themselves; (b) NCAM binds to oligomannosidic carbohydrates; (c) within the L1/NCAM complex, the oligomannosidic carbohydrates are hidden from accessibility to a mAb against oligomannosidic carbohydrates; (d) the recombinant protein fragment of NCAM containing the immunoglobulin-like domains and not the fragment containing the fibronectin type III homologous repeats binds to oligomannosidic glycans. Furthermore, the fourth immunoglobulin-like domain of NCAM shows sequence homology with carbohydrate recognition domains of animal C-type lectins and, surprisingly, also with plant lectins. A peptide comprising part of the C-type lectin consensus sequence in the fourth immunoglobulin-like domain of NCAM interferes with the association between L1 and NCAM. The functional importance of oligomannosidic glycans at the cell surface was shown for neurite outgrowth in vitro. When neurons from early postnatal mouse cerebellum were maintained on laminin or poly-L-lysine, neurite outgrowth was inhibited by oligomannosidic glycans, by glycopeptides, glycoproteins, or neoglycolipids containing oligomannosidic glycans, but not by nonrelated oligosaccharides or oligosaccharide derivates. Neurite outgrowth was also inhibited by the peptide comprising part of the C-type lectin consensus sequence in the fourth immunoglobulin-like domain of NCAM. The combined results suggest that carbohydrate-mediated cis-associations between adhesion molecules at the cell surface modulate their functional properties.

    The Journal of cell biology 1993;121;6;1409-21

  • Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2.

    Chardin P, Camonis JH, Gale NW, van Aelst L, Schlessinger J, Wigler MH and Bar-Sagi D

    Institut de Pharmacologie Moleculaire et Cellulaire, CNRS, Valbonne, France.

    A human complementary DNA was isolated that encodes a widely expressed protein, hSos1, that is closely related to Sos, the product of the Drosophila son of sevenless gene. The hSos1 protein contains a region of significant sequence similarity to CDC25, a guanine nucleotide exchange factor for Ras from yeast. A fragment of hSos1 encoding the CDC25-related domain complemented loss of CDC25 function in yeast. This hSos1 domain specifically stimulated guanine nucleotide exchange on mammalian Ras proteins in vitro. Mammalian cells overexpressing full-length hSos1 had increased guanine nucleotide exchange activity. Thus hSos1 is a guanine nucleotide exchange factor for Ras. The hSos1 interacted with growth factor receptor-bound protein 2 (GRB2) in vivo and in vitro. This interaction was mediated by the carboxyl-terminal domain of hSos1 and the Src homology 3 (SH3) domains of GRB2. These results suggest that the coupling of receptor tyrosine kinases to Ras signaling is mediated by a molecular complex consisting of GRB2 and hSos1.

    Funded by: NCI NIH HHS: CA46370, CA55360

    Science (New York, N.Y.) 1993;260;5112;1338-43

  • A discordance between the human muscle NCAM sequence and those seen in other NCAM cDNA clones.

    Rossell RJ, Malik A, Helfrich W, Kemshead JT and Patel K

    ICRF, Frenchay Hospital, Bristol, UK.

    A cDNA clone, designated NC7, has been isolated from human foetal kidney that partially codes for the 140 kDa isoform of human NCAM. This clone contains a 6 bp insert that is not present in the human muscle cDNA clone lambda 4.4. This same sequence has also been found in both a cDNA clone obtained from a human Small Cell Lung Carcinoma (SCLC) line and in human genomic DNA. Furthermore, an equivalent sequence to the 6 bp region identified in the above samples is present in mouse, rat and chicken NCAM. The 6 bp insertion does not lie at a predicted intron/exon boundary as extrapolated by homology studies with the chicken and, therefore, the mechanism by which the sequence is deleted from the human muscle clone lambda 4.4 remains unclear.

    Biochimica et biophysica acta 1992;1130;1;95-6

  • Splicing of the VASE exon of neural cell adhesion molecule (NCAM) in human small-cell lung carcinoma (SCLC).

    van Duijnhoven HL, Helfrich W, de Leij L, Roebroek AJ, van de Ven WJ, Healey K, Culverwell A, Rossell RJ, Kemshead JT and Patel K

    Department of Biochemistry, University of Nijmegen, The Netherlands.

    Expression of the neural cell adhesion molecule (NCAM) on small-cell lung carcinoma (SCLC) cell lines and tumour tissue has been investigated. Cell lines were found to express highly sialylated NCAM. Neuraminidase treatment revealed the presence of the 140- and 120-kDa isoforms with differential expression of a 95-kDa protein. Similar data were obtained with SCLC tumour tissues. These results were corroborated by Northern blotting where mRNA of 6.7 and 5.5 kb coding for the 140- and 120-kDa isoforms, respectively, were identified. In a few tumours, a weaker band of 7.4-kb mRNA coding for the 180-kDa NCAM was also identified. This result could not be confirmed biochemically due to shortage of material. Finally, a 5-kb transcript was identified in all SCLC samples examined. The NCAM isoform coded by this mRNA remains unknown. Using the polymerase chain reaction (PCR), we have demonstrated the presence of the VASE mini-exon in some isoforms of SCLC NCAM. The VASE mini-exon sequence in human SCLC differs from the published murine sequence by only one base change. This substitution does not result in altered amino-acid sequence.

    International journal of cancer 1992;50;1;118-23

  • Molecular and functional analysis of human natural killer cell-associated neural cell adhesion molecule (N-CAM/CD56).

    Lanier LL, Chang C, Azuma M, Ruitenberg JJ, Hemperly JJ and Phillips JH

    Becton Dickinson Immunocytometry Systems, San Jose, CA 95131.

    The neural cell adhesion molecule (N-CAM/CD56) is a member of the Ig supergene family that has been shown to mediate homophilic binding. Several isoforms of N-CAM have been identified that are expressed preferentially in different tissues and stages of embryonic development. To examine the primary structure of N-CAM expressed in leukocytes, N-CAM cDNA were generated by polymerase chain reaction from RNA isolated from normal human NK cells and the KG1a hematopoietic leukemia cell line. The sequence of leukocyte-derived N-CAM cDNA was essentially identical with N-CAM cDNA from human neuroblastoma cells that encode the 140-kDa isoform of N-CAM. Inasmuch as N-CAM is preferentially expressed on human NK cells and a subset of T lymphocytes that mediate MHC-unrestricted cell-mediated cytotoxicity, we examined the potential role of N-CAM in cell-mediated cytotoxicity and heterotypic lymphocyte-tumor cell adhesion. N-CAM loss mutants were established from the human N-CAM+ KG1a leukemia cell line, and N-CAM cDNA was transfected into a human colon carcinoma cell line and murine L cells. Using this panel of mutants and transfectants, it was determined that expression of N-CAM on these target cells does not affect susceptibility to resting or IL-2-activated NK cell-mediated cytotoxicity. Moreover, expression of N-CAM in these transfectants failed to induce homotypic or heterotypic cellular adhesion. Collectively, these studies indicate that homophilic N-CAM interactions probably do not mediate a major role in the cytolytic interaction between NK cells and N-CAM+ tumor cell targets.

    Journal of immunology (Baltimore, Md. : 1950) 1991;146;12;4421-6

  • Precise in situ localization of NCAM, ETS1, and D11S29 on human meiotic chromosomes.

    Bello MJ, Salagnon N, Rey JA, Guichaoua MR, Bergé-Lefranc JL, Jordan BR and Luciani JM

    Laboratoire d'Embryologie et de Cytogénétique, Faculté de Médecine, Secteur Nord, France.

    In order to sublocalize NCAM, ETS1, and the anonymous DNA fragment D11S29 within 11q23, in situ hybridization was performed on pachytene bivalents. Analysis of the grain distribution within the band 11q23 indicated that the chromosomal sublocalization of both NCAM and D11S29 was in 11q23.1, whereas ETS1 was found to be localized in 11q23.3. These results clearly demonstrate the usefulness of in situ hybridization applied to pachytene bivalents to obtain accurate gene sublocalization.

    Cytogenetics and cell genetics 1989;52;1-2;7-10

  • Alternative splicing generates a secreted form of N-CAM in muscle and brain.

    Gower HJ, Barton CH, Elsom VL, Thompson J, Moore SE, Dickson G and Walsh FS

    Institute of Neurology, London, England.

    A number of different membrane associated isoforms of the neural cell adhesion molecule (N-CAM) have previously been identified. Here the structure of a novel secreted isoform of N-CAM is established by analysis of a cDNA corresponding to an N-CAM mRNA from human skeletal muscle. The mRNA incorporates a novel sequence block into the extracellular domain, which introduces an in-frame stop codon and thus prematurely terminates the coding sequence, generating a truncated N-CAM polypeptide. Analysis of genomic clones indicates that the inserted sequence is present as a discrete exon within the human N-CAM gene, and Northern analysis shows it to be associated specifically with a 5.2 kb mRNA species from skeletal muscle and brain. Stable transfectants expressing the secreted isoform accumulate it in the cytoplasm and release it to the culture medium. In contrast, cells transfected with cDNA encoding lipid-tailed N-CAM express it predominantly at the cell surface. The existence of a secreted isoform may further expand the spectrum of N-CAM function beyond its known involvement in intercellular adhesion to extracellular matrix interactions.

    Funded by: Wellcome Trust

    Cell 1988;55;6;955-64

  • Complete sequence and in vitro expression of a tissue-specific phosphatidylinositol-linked N-CAM isoform from skeletal muscle.

    Barton CH, Dickson G, Gower HJ, Rowett LH, Putt W, Elsom V, Moore SE, Goridis C and Walsh FS

    Institute of Neurology, London, UK.

    Neural cell adhesion molecules (N-CAMs) are a family of cell surface sialoglycoproteins encoded by a single copy gene. A full-length cDNA clone that encodes a nontransmembrane phosphatidylinositol (PI) linked N-CAM of Mr 125 x 10(3) has been isolated from a human skeletal muscle cDNA library. The deduced protein sequence encodes a polypeptide of 761 amino acids and is highly homologous to the N-CAM isoform in brain of Mr 120 x 10(3). The size difference between the 125 x 10(3). The size difference between the 125 x 10(3) Mr skeletal muscle form and the 120 x 10(3) Mr N-CAM form from brain is accounted for by the insertion of a block of 37 amino acids called MSD1, in the extracellular domain of the muscle form. Transient expression of the human cDNA in COS cells results in cell surface N-CAM expression via a putative covalent attachment to PI-containing phospholipid. Linked in vitro transcription and translation experiments followed by immunoprecipitation with anti-N-CAM antibodies demonstrate that the full-length clone of 761 amino acid coding potential produces a core polypeptide of Mr 110 x 10(3) which is processed by microsomal membranes to yield a 122 x 10(3) Mr species. Taken together, these results demonstrate that the cloned cDNA sequence encodes a lipid-linked, PI-specific phospholipase C releasable surface isoform of N-CAM with core glycopeptide molecular weight corresponding to the authentic muscle 125 x 10(3) Mr N-CAM isoform. This is the first direct correlation of cDNA and deduced protein sequence with a known PI-linked N-CAM isoform from skeletal muscle.

    Funded by: Wellcome Trust

    Development (Cambridge, England) 1988;104;1;165-73

  • The neural cell adhesion molecule (NCAM) as a regulator of cell-cell interactions.

    Rutishauser U, Acheson A, Hall AK, Mann DM and Sunshine J

    Department of Developmental Genetics and Anatomy, Case Western Reserve University School of Medicine, Cleveland, OH 44106.

    The neural cell adhesion molecule (NCAM) can influence a number of diverse intercellular events, including junctional communication, the association of axons with pathways and targets, and signals that alter levels of neurotransmitter enzymes. These pleiotropic effects appear to reflect the ability of NCAM to regulate membrane-membrane contact required to initiate specific interactions between other molecules. Such regulation can occur through changes in either NCAM expression or the molecule's content of polysialic acid (PSA). When NCAM with a low PSA content is expressed, adhesion is increased and contact-dependent events are triggered. In contrast, the large excluded volume of NCAM PSA can inhibit cell-cell interactions through hindrance of overall membrane apposition.

    Funded by: NEI NIH HHS: EY06107; NICHD NIH HHS: HD18369

    Science (New York, N.Y.) 1988;240;4848;53-7

  • Human muscle neural cell adhesion molecule (N-CAM): identification of a muscle-specific sequence in the extracellular domain.

    Dickson G, Gower HJ, Barton CH, Prentice HM, Elsom VL, Moore SE, Cox RD, Quinn C, Putt W and Walsh FS

    cDNA clones encoding neural cell adhesion molecule (N-CAM) mRNAs of 6.7, 5.2, and 4.3 kb from human skeletal muscle cells were isolated. A 6.7 kb mRNA encodes a transmembrane N-CAM isoform, present predominantly in mononucleate myoblasts, that shows sequence homology with chick brain N-CAM-140 and is down-regulated during myotube formation. In contrast, the 5.2 and 4.3 kb mRNAs encode nontransmembrane N-CAM isoforms that greatly increase during myoblast fusion. Furthermore, a discrete muscle-specific sequence domain (MSD1) was detected in the extracellular coding regions of the 5.2 and 4.3 kb mRNAs. This encodes a unique run of 37 amino acids and is not expressed in 7.2 and 6.7 kb mRNAs from human or chick brain or in the corresponding 6.7 kb muscle transcript. The MSD1 is also absent from chick and mouse brain mRNAs of 4.0 and 2.9 kb. These results show that diversity in N-CAM primary structure can be found in the extracellular domain in a tissue-specific manner.

    Cell 1987;50;7;1119-30

  • Neural cell adhesion molecule: structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing.

    Cunningham BA, Hemperly JJ, Murray BA, Prediger EA, Brackenbury R and Edelman GM

    The neural cell adhesion molecule, N-CAM, appears on early embryonic cells and is important in the formation of cell collectives and their boundaries at sites of morphogenesis. Later in development it is found on various differentiated tissues and is a major CAM mediating adhesion among neurons and between neurons and muscle. To provide a molecular basis for understanding N-CAM function, the complete amino acid sequences of the three major polypeptides of N-CAM and most of the noncoding sequences of their messenger RNA's were determined from the analysis of complementary DNA clones and were verified by amino acid sequences of selected CNBr fragments and proteolytic fragments. The extracellular region of each N-CAM polypeptide includes five contiguous segments that are homologous in sequence to each other and to members of the immunoglobulin superfamily, suggesting that interactions among immunoglobulin-like domains form the basis for N-CAM homophilic binding. Although different in their membrane-associated and cytoplasmic domains, the amino acid sequences of the three polypeptides appear to be identical throughout this extracellular region (682 amino acids) where the binding site is located. Variations in N-CAM activity thus do not occur by changes in the amino acid sequence that alter the specificity of binding. Instead, regulation is achieved by cell surface modulation events that alter N-CAM affinity, prevalence, mobility, and distribution on the surface. A major mechanism for modulation is alternative RNA splicing resulting in N-CAM's with different cytoplasmic domains that differentially interact with the cell membrane. Such regulatory mechanisms may link N-CAM binding function with other primary cellular processes during the embryonic development of pattern.

    Funded by: NIADDK NIH HHS: AM-04256; NICHD NIH HHS: HD-09635, HD-16550

    Science (New York, N.Y.) 1987;236;4803;799-806

Gene lists (8)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000011 G2C Homo sapiens Human clathrin Human orthologues of mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000012 G2C Homo sapiens Human Synaptosome Human orthologues of mouse synaptosome adapted from Collins et al (2006) 152
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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