G2Cdb::Gene report

Gene id
G00001122
Gene symbol
Calm1 (MGI)
Species
Mus musculus
Description
calmodulin 1
Orthologue
G00002371 (Homo sapiens)

Databases (11)

Curated Gene
OTTMUSG00000016979 (Vega mouse gene)
Gene
ENSMUSG00000001175 (Ensembl mouse gene)
12313 (Entrez Gene)
5 (G2Cdb plasticity & disease)
Gene Expression
NM_009790 (Allen Brain Atlas)
g03111 (BGEM)
12313 (Genepaint)
calm1 (gensat)
Literature
114180 (OMIM)
Marker Symbol
MGI:88251 (MGI)
Protein Sequence
P62204 (UniProt)

Literature (59)

Pubmed - other

  • Stimulation-mediated translocation of calmodulin and neurogranin from soma to dendrites of mouse hippocampal CA1 pyramidal neurons.

    Huang KP, Huang FL and Shetty PK

    Program of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. huangk@mail.nih.gov

    Calmodulin (CaM) and neurogranin (Ng) are two abundant neuronal proteins in the forebrain whose interactions are implicated in the enhancement of synaptic plasticity. To gain further insight into the actions of these two proteins we investigated whether they co-localize in principle neurons and whether they respond to high frequency stimulation in a coordinated fashion. Immunohistochemical staining of CaM and Ng in mouse hippocampal slices revealed that CaM was highly concentrated in the nucleus of CA1 pyramidal neurons, whereas Ng was more broadly localized throughout the soma and dendrites. The asymmetrical localization of CaM in the nucleus of pyramidal neurons was in sharp contrast to the distribution observed in pyramidal cells of the neighboring subiculum, where CaM was uniformly localized throughout the soma and dendrites. The somatic concentrations of CaM and Ng in CA1 pyramidal neurons were approximately 10- and two-fold greater than observed in the dendrites, respectively. High frequency stimulation (HFS) of hippocampal slices promoted mobilization of CaM and Ng from soma to dendrites. These responses were spatially restricted to the area close to the site of stimulation and were inhibited by the N-methyl-D-asparate receptor antagonist 2-amino-5-phosphonopentanoic acid. Furthermore, HFS failed to promote translocation of CaM from soma to dendrites of slices from Ng knockout mice, which also exhibited deficits in HFS-induced long-term potentiation. Translocated CaM and Ng exhibited distinct puncta decorating the apical dendrites of pyramidal neurons and appeared to be concentrated in dendritic spines. These findings suggest that mobilization of CaM and Ng to stimulated dendritic spines may enhance synaptic efficacy by increasing and prolonging the Ca2+ transients and activation of Ca2+/CaM-dependent enzymes.

    Funded by: Intramural NIH HHS: ZIA HD000187-31

    Neuroscience 2011;178;1-12

  • The planar cell polarity gene Vangl2 is required for mammalian kidney-branching morphogenesis and glomerular maturation.

    Yates LL, Papakrivopoulou J, Long DA, Goggolidou P, Connolly JO, Woolf AS and Dean CH

    Mammalian Genetics Unit, Medical Research Council, Harwell, Oxfordshire, UK.

    The planar cell polarity (PCP) pathway, incorporating non-canonical Wnt signalling, controls embryonic convergent (CE) extension, polarized cell division and ciliary orientation. It also limits diameters of differentiating renal tubules, with mutation of certain components of the pathway causing cystic kidneys. Mutations in mouse Vangl genes encoding core PCP proteins cause neural tube defects (NTDs) and Vangl2 mutations also impair branching of embryonic mouse lung airways. Embryonic metanephric kidneys also undergo branching morphogenesis and Vangl2 is known to be expressed in ureteric bud/collecting duct and metanephric mesenchymal/nephron lineages. These observations led us to investigate metanephroi in Vangl2 mutant mice, Loop-tail (Lp). Although ureteric bud formation is normal in Vangl2(Lp/Lp) embryos, subsequent in vivo and in vitro branching morphogenesis is impaired. Null mutant kidneys are short, consistent with a CE defect. Differentiating glomerular epithelia express several PCP genes (Vangl1/2, Celsr1, Scrib, Mpk1/2 and Fat4) and glomeruli in Vangl2(Lp/Lp) fetuses are smaller and contain less prominent capillary loops than wild-type littermates. Furthermore, Vangl2(Lp/+) kidneys had modest reduction in glomerular numbers postnatally. Vangl2(Lp/Lp) metanephroi contained occasional dilated tubules but no overt cystic phenotype. These data show for the first time that a PCP gene is required for normal morphogenesis of both the ureteric bud and metanephric mesenchyme-derived structures. It has long been recognized that certain individuals with NTDs are born with malformed kidneys, and recent studies have discovered VANGL mutations in some NTD patients. On the basis of our mutant mouse study, we suggest that PCP pathway mutations should be sought when NTD and renal malformation co-exist.

    Funded by: Medical Research Council; Wellcome Trust

    Human molecular genetics 2010;19;23;4663-76

  • YB-1 binds to GluR2 mRNA and CaM1 mRNA in the brain and regulates their translational levels in an activity-dependent manner.

    Tanaka T, Ohashi S, Funakoshi T and Kobayashi S

    Research Unit of Biochemistry, School of Pharmacy, Nihon University, Funabashi, Chiba, Japan.

    The translational regulator YB-1 binds to mRNAs. In the brain, YB-1 is prominently expressed from the prenatal stage until the first week after birth, being associated with polysomes and distributed in neuronal dendrites, but its expression declines to a much lower level thereafter. It is therefore of interest to identify the mRNAs whose translation is controlled by YB-1 in the postnatal growing brain. In this study we found that YB-1 interacted with the mRNAs for glutamate receptor subunit 2 (GluR2) and calmodulin1 (CaM1) in both brain and NG108-15 cells. Overexpression or knockdown of YB-1 altered the levels of these proteins significantly in cultured cells without any change in their mRNA levels. When the cells were treated with neurotransmitters, translation of these proteins was induced within a short time, and a change in the amount of YB-1 on its target mRNAs was observed in the heavy-sedimenting polysome fractions on a sucrose gradient. Depletion of YB-1 expression by siRNA abrogated the translational activation. Furthermore, in the brain of kainic acid-treated mice, the distribution of YB-1 was shifted to much heavier fractions associated with polysomes within 30 min to 1 h after the treatment, and the distribution returned to lighter fractions within the following 2 h. The protein levels of GluR2 and CaM1 were also increased transiently when the distribution of YB-1 on the gradient changed. These results suggest that in the brain of growing mice, YB-1 binds to GluR2 and CaM1 mRNAs and regulates their translation in an activity-dependent manner.

    Cellular and molecular neurobiology 2010;30;7;1089-100

  • Defective calmodulin binding to the cardiac ryanodine receptor plays a key role in CPVT-associated channel dysfunction.

    Xu X, Yano M, Uchinoumi H, Hino A, Suetomi T, Ono M, Tateishi H, Oda T, Okuda S, Doi M, Kobayashi S, Yamamoto T, Ikeda Y, Ikemoto N and Matsuzaki M

    Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan.

    Calmodulin (CaM), one of the accessory proteins of the cardiac ryanodine receptor (RyR2), is known to play a significant role in the channel regulation of the RyR2. However, the possible involvement of calmodulin in the pathogenic process of catecholaminergic polymorphic ventricular tachycardia (CPVT) has not been investigated. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions using a knock-in (KI) mouse model with CPVT-linked RyR2 mutation (R2474S). Without added effectors, the affinity of CaM binding to the RyR2 was indistinguishable between KI and WT hearts. In response to cAMP (1 micromol/L), the RyR2 phosphorylation at Ser2808 increased in both WT and KI hearts to the same extent. However, cAMP caused a significant decrease of the CaM-binding affinity in KI hearts, but the affinity was unchanged in WT. Dantrolene restored a normal level of CaM-binding affinity in the cAMP-treated KI hearts, suggesting that defective inter-domain interaction between the N-terminal domain and the central domain of the RyR2 (the target of therapeutic effect of dantrolene) is involved in the cAMP-induced reduction of the CaM-binding affinity. In saponin-permeabilized cardiomyocytes, the addition of cAMP increased the frequency of spontaneous Ca(2+) sparks to a significantly larger extent in KI cardiomyocytes than in WT cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca(2+) sparks. In conclusion, CPVT mutation causes defective inter-domain interaction, significant reduction in the ability of CaM binding to the RyR2, spontaneous Ca(2+) leak, and then lethal arrhythmia.

    Funded by: NHLBI NIH HHS: HL072841, R01 HL072841, R01 HL072841-06

    Biochemical and biophysical research communications 2010;394;3;660-6

  • Ca2+-dependent release of Munc18-1 from presynaptic mGluRs in short-term facilitation.

    Nakajima Y, Mochida S, Okawa K and Nakanishi S

    Department of Biological Sciences, Faculty of Medicine, Kyoto University, Kyoto, Japan. yoshiaki@phy.med.kyoto-u.ac.jp

    Short-term synaptic facilitation plays an important role in information processing in the central nervous system. Although the crucial requirement of presynaptic Ca(2+) in the expression of this plasticity has been known for decades, the molecular mechanisms underlying the plasticity remain controversial. Here, we show that presynaptic metabotropic glutamate receptors (mGluRs) bind and release Munc18-1 (also known as rbSec1/nSec1), an essential protein for synaptic transmission, in a Ca(2+)-dependent manner, whose actions decrease and increase synaptic vesicle release, respectively. We found that mGluR4 bound Munc18-1 with an EC(50) for Ca(2+) of 168 nM, close to the resting Ca(2+) concentration, and that the interaction was disrupted by Ca(2+)-activated calmodulin (CaM) at higher concentrations of Ca(2+). Consistently, the Munc18-1-interacting domain of mGluR4 suppressed both dense-core vesicle secretion from permeabilized PC12 cells and synaptic transmission in neuronal cells. Furthermore, this domain was sufficient to induce paired-pulse facilitation. Obviously, the role of mGluR4 in these processes was independent of its classical function of activation by glutamate. On the basis of these experimental data, we propose the following model: When neurons are not active, Munc18-1 is sequestered by mGluR4, and therefore the basal synaptic transmission is kept low. After the action potential, the increase in the Ca(2+) level activates CaM, which in turn liberates Munc18-1 from mGluR4, causing short-term synaptic facilitation. Our findings unite and provide a new insight into receptor signaling and vesicular transport, which are pivotal activities involved in a variety of cellular processes.

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;43;18385-9

  • Calmodulin is a functional regulator of Cav1.4 L-type Ca2+ channels.

    Griessmeier K, Cuny H, Rötzer K, Griesbeck O, Harz H, Biel M and Wahl-Schott C

    Center for Integrated Protein Science (CIPS-M) and Zentrum für Pharmaforschung, Department Pharmazie, Ludwig-Maximilians-Universität München, D-81377 München.

    Cav1.4 channels are unique among the high voltage-activated Ca2+ channel family because they completely lack Ca2+-dependent inactivation and display very slow voltage-dependent inactivation. Both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained Ca2+ influx through Cav1.4 channels is required to couple slow graded changes of the membrane potential with tonic glutamate release. Loss of Cav1.4 function causes severe impairment of retinal circuitry function and has been linked to night blindness in humans and mice. Recently, an inhibitory domain (ICDI: inhibitor of Ca2+-dependent inactivation) in the C-terminal tail of Cav1.4 has been discovered that eliminates Ca2+-dependent inactivation by binding to upstream regulatory motifs within the proximal C terminus. The mechanism underlying the action of ICDI is unclear. It was proposed that ICDI competitively displaces the Ca2+ sensor calmodulin. Alternatively, the ICDI domain and calmodulin may bind to different portions of the C terminus and act independently of each other. In the present study, we used fluorescence resonance energy transfer experiments with genetically engineered cyan fluorescent protein variants to address this issue. Our data indicate that calmodulin is preassociated with the C terminus of Cav1.4 but may be tethered in a different steric orientation as compared with other Ca2+ channels. We also find that calmodulin is important for Cav1.4 function because it increases current density and slows down voltage-dependent inactivation. Our data show that the ICDI domain selectively abolishes Ca2+-dependent inactivation, whereas it does not interfere with other calmodulin effects.

    The Journal of biological chemistry 2009;284;43;29809-16

  • Ca(2+) and calmodulin initiate all forms of endocytosis during depolarization at a nerve terminal.

    Wu XS, McNeil BD, Xu J, Fan J, Xue L, Melicoff E, Adachi R, Bai L and Wu LG

    National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, USA.

    Although endocytosis maintains synaptic transmission, how endocytosis is initiated is unclear. We found that calcium influx initiated all forms of endocytosis at a single nerve terminal in rodents, including clathrin-dependent slow endocytosis, bulk endocytosis, rapid endocytosis and endocytosis overshoot (excess endocytosis), with each being evoked with a correspondingly higher calcium threshold. As calcium influx increased, endocytosis gradually switched from very slow endocytosis to slow endocytosis to bulk endocytosis to rapid endocytosis and to endocytosis overshoot. The calcium-induced endocytosis rate increase was a result of the speeding up of membrane invagination and fission. Pharmacological experiments suggested that the calcium sensor mediating these forms of endocytosis is calmodulin. In addition to its role in recycling vesicles, calcium/calmodulin-initiated endocytosis facilitated vesicle mobilization to the readily releasable pool, probably by clearing fused vesicle membrane at release sites. Our findings provide a unifying mechanism for the initiation of various forms of endocytosis that are critical in maintaining exocytosis.

    Funded by: Intramural NIH HHS: Z01 NS003009-04, Z01 NS003009-05

    Nature neuroscience 2009;12;8;1003-1010

  • MicroRNA-1 negatively regulates expression of the hypertrophy-associated calmodulin and Mef2a genes.

    Ikeda S, He A, Kong SW, Lu J, Bejar R, Bodyak N, Lee KH, Ma Q, Kang PM, Golub TR and Pu WT

    Department of Cardiology, Children's Hospital Boston, Department of Genetics, Harvard Medical School, Boston, Massachusetts 021151, USA.

    Calcium signaling is a central regulator of cardiomyocyte growth and function. Calmodulin is a critical mediator of calcium signals. Because the amount of calmodulin within cardiomyocytes is limiting, the precise control of calmodulin expression is important for the regulation of calcium signaling. In this study, we show for the first time that calmodulin levels are regulated posttranscriptionally in heart failure. The cardiomyocyte-restricted microRNA miR-1 inhibited the translation of calmodulin-encoding mRNAs via highly conserved target sites within their 3' untranslated regions. In keeping with its effect on calmodulin expression, miR-1 downregulated calcium-calmodulin signaling through calcineurin to NFAT. miR-1 also negatively regulated the expression of Mef2a and Gata4, key transcription factors that mediate calcium-dependent changes in gene expression. Consistent with the downregulation of these hypertrophy-associated genes, miR-1 attenuated cardiomyocyte hypertrophy in cultured neonatal rat cardiomyocytes and in the intact adult heart. Our data indicate that miR-1 regulates cardiomyocyte growth responses by negatively regulating the calcium signaling components calmodulin, Mef2a, and Gata4.

    Funded by: Howard Hughes Medical Institute; NHLBI NIH HHS: P01 HL074734, P50 HL074734

    Molecular and cellular biology 2009;29;8;2193-204

  • Ca extrusion by NCX is compromised in olfactory sensory neurons of OMP mice.

    Kwon HJ, Koo JH, Zufall F, Leinders-Zufall T and Margolis FL

    Department of Anatomy and Neurobiology, School of Medicine, University of Maryland, Baltimore, Maryland, United States of America.

    Background: The role of olfactory marker protein (OMP), a hallmark of mature olfactory sensory neurons (OSNs), has been poorly understood since its discovery. The electrophysiological and behavioral phenotypes of OMP knockout mice indicated that OMP influences olfactory signal transduction. However, the mechanism by which this occurs remained unknown.

    We used intact olfactory epithelium obtained from WT and OMP(-/-) mice to monitor the Ca(2+) dynamics induced by the activation of cyclic nucleotide-gated channels, voltage-operated Ca(2+) channels, or Ca(2+) stores in single dendritic knobs of OSNs. Our data suggested that OMP could act to modulate the Ca(2+)-homeostasis in these neurons by influencing the activity of the plasma membrane Na(+)/Ca(2+)-exchanger (NCX). Immunohistochemistry verifies colocalization of NCX1 and OMP in the cilia and knobs of OSNs. To test the role of NCX activity, we compared the kinetics of Ca(2+) elevation by stimulating the reverse mode of NCX in both WT and OMP(-/-) mice. The resulting Ca(2+) responses indicate that OMP facilitates NCX activity and allows rapid Ca(2+) extrusion from OSN knobs. To address the mechanism by which OMP influences NCX activity in OSNs we studied protein-peptide interactions in real-time using surface plasmon resonance technology. We demonstrate the direct interaction of the XIP regulatory-peptide of NCX with calmodulin (CaM).

    Conclusions: Since CaM also binds to the Bex protein, an interacting protein partner of OMP, these observations strongly suggest that OMP can influence CaM efficacy and thus alters NCX activity by a series of protein-protein interactions.

    Funded by: NIDCD NIH HHS: R01 DC003112, R01 DC005249, R01-DC-003112, R01-DC-005249, R01-DC-02227, T32 DC-00054, T32 DC000054

    PloS one 2009;4;1;e4260

  • Molecular networks involved in mouse cerebral corticogenesis and spatio-temporal regulation of Sox4 and Sox11 novel antisense transcripts revealed by transcriptome profiling.

    Ling KH, Hewitt CA, Beissbarth T, Hyde L, Banerjee K, Cheah PS, Cannon PZ, Hahn CN, Thomas PQ, Smyth GK, Tan SS, Thomas T and Scott HS

    Molecular Medicine Division, The Walter and Eliza Hall Institute of Medical Research, Royal Parade, Parkville, Victoria 3052, Australia. michael.ling@imvs.sa.gov.au

    Background: Development of the cerebral cortex requires highly specific spatio-temporal regulation of gene expression. It is proposed that transcriptome profiling of the cerebral cortex at various developmental time points or regions will reveal candidate genes and associated molecular pathways involved in cerebral corticogenesis.

    Results: Serial analysis of gene expression (SAGE) libraries were constructed from C57BL/6 mouse cerebral cortices of age embryonic day (E) 15.5, E17.5, postnatal day (P) 1.5 and 4 to 6 months. Hierarchical clustering analysis of 561 differentially expressed transcripts showed regionalized, stage-specific and co-regulated expression profiles. SAGE expression profiles of 70 differentially expressed transcripts were validated using quantitative RT-PCR assays. Ingenuity pathway analyses of validated differentially expressed transcripts demonstrated that these transcripts possess distinctive functional properties related to various stages of cerebral corticogenesis and human neurological disorders. Genomic clustering analysis of the differentially expressed transcripts identified two highly transcribed genomic loci, Sox4 and Sox11, during embryonic cerebral corticogenesis. These loci feature unusual overlapping sense and antisense transcripts with alternative polyadenylation sites and differential expression. The Sox4 and Sox11 antisense transcripts were highly expressed in the brain compared to other mouse organs and are differentially expressed in both the proliferating and differentiating neural stem/progenitor cells and P19 (embryonal carcinoma) cells.

    Conclusions: We report validated gene expression profiles that have implications for understanding the associations between differentially expressed transcripts, novel targets and related disorders pertaining to cerebral corticogenesis. The study reports, for the first time, spatio-temporally regulated Sox4 and Sox11 antisense transcripts in the brain, neural stem/progenitor cells and P19 cells, suggesting they have an important role in cerebral corticogenesis and neuronal/glial cell differentiation.

    Genome biology 2009;10;10;R104

  • Proteomics analysis identifies phosphorylation-dependent alpha-synuclein protein interactions.

    McFarland MA, Ellis CE, Markey SP and Nussbaum RL

    National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20891, USA.

    Mutations and copy number variation in the SNCA gene encoding the neuronal protein alpha-synuclein have been linked to familial Parkinson disease (Thomas, B., and Beal, M. F. (2007) Parkinson's disease. Hum. Mol. Genet. 16, R183-R194). The carboxyl terminus of alpha-synuclein can be phosphorylated at tyrosine 125 and serine 129, although only a small fraction of the protein is phosphorylated under normal conditions (Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P. J., and Haass, C. (2000) Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein. J. Biol. Chem. 275, 390-397). Under pathological conditions, such as in Parkinson disease, alpha-synuclein is a major component of Lewy bodies, a pathological hallmark of Parkinson disease, and is mostly phosphorylated at Ser-129 (Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J. P., Kling, K., Lee, M., Diep, L., Keim, P. S., Shen, X. F., Chataway, T., Schlossmacher, M. G., Seubert, P., Schenk, D., Sinha, S., Gai, W. P., and Chilcote, T. J. (2006) Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739-29752). Controversy exists over the extent to which phosphorylation of alpha-synuclein and/or the visible protein aggregation in Lewy bodies are steps in disease pathogenesis, are protective, or are neutral markers for the disease process. Here we used the combination of peptide pulldown assays and mass spectrometry to identify and compare protein-protein interactions of phosphorylated and non-phosphorylated alpha-synuclein. We showed that non-phosphorylated alpha-synuclein carboxyl terminus pulled down protein complexes that were highly enriched for mitochondrial electron transport proteins, whereas alpha-synuclein carboxyl terminus phosphorylated on either Ser-129 or Tyr-125 did not. Instead the set of proteins pulled down by phosphorylated alpha-synuclein was highly enriched in certain cytoskeletal proteins, in vesicular trafficking proteins, and in a small number of enzymes involved in protein serine phosphorylation. This targeted comparative proteomics approach for unbiased identification of protein-protein interactions suggests that there are functional consequences when alpha-synuclein is phosphorylated.

    Funded by: Intramural NIH HHS; NIMH NIH HHS: Z01 MH000279

    Molecular & cellular proteomics : MCP 2008;7;11;2123-37

  • Identification of essential interacting elements in K-Ras/calmodulin binding and its role in K-Ras localization.

    Lopez-Alcalá C, Alvarez-Moya B, Villalonga P, Calvo M, Bachs O and Agell N

    Departament de Biologia Cellular i Anatomia Patològica, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spain.

    We previously showed that K-Ras is a calmodulin-binding protein. Involvement of this interaction in anterograde and retrograde transport of K-Ras was then suggested. To test this we have analyzed here the domains of K-Ras essential for the interaction with calmodulin. At least three different regions in the K-Ras molecule were important; they are the hypervariable region, the alpha-helix between amino acids 151 and 166, and the Switch II. Within the hypervariable region, both the hydrophobic farnesyl group and the positive-charged amino acids were essential for the interaction between K-Ras and calmodulin in cellular extracts. Consistently, K-Ras S181D, which mimics phosphorylation of Ser-181 of K-Ras, also completely abolished binding to calmodulin. K-Ras mutants correctly farnesylated that did not bind calmodulin were all located at plasma membrane, showing that calmodulin interaction was not required for the transport of K-Ras to plasma membrane. In NIH3T3 cells, K-Ras and calmodulin colocalized mainly in the plasma membrane even after the addition of Ca(2+) ionophore, indicating that interaction did not directly lead to K-Ras internalization. Furthermore, using a K-Ras with impaired binding to calmodulin but with membrane localization, we could demonstrate in striatal neurones that interaction between K-Ras and calmodulin was not required for Golgi K-Ras translocation induced by Ca(2+) influx.

    The Journal of biological chemistry 2008;283;16;10621-31

  • Identification of transcripts with enriched expression in the developing and adult pancreas.

    Hoffman BG, Zavaglia B, Witzsche J, Ruiz de Algara T, Beach M, Hoodless PA, Jones SJ, Marra MA and Helgason CD

    Department of Cancer Endocrinology, BC Cancer Research Center, West 10th Ave, Vancouver, BC V5Z 1L3, Canada. bhoffman@bccrc.ca

    Background: Despite recent advances, the transcriptional hierarchy driving pancreas organogenesis remains largely unknown, in part due to the paucity of comprehensive analyses. To address this deficit we generated ten SAGE libraries from the developing murine pancreas spanning Theiler stages 17-26, making use of available Pdx1 enhanced green fluorescent protein (EGFP) and Neurog3 EGFP reporter strains, as well as tissue from adult islets and ducts.

    Results: We used a specificity metric to identify 2,536 tags with pancreas-enriched expression compared to 195 other mouse SAGE libraries. We subsequently grouped co-expressed transcripts with differential expression during pancreas development using K-means clustering. We validated the clusters first using quantitative real time PCR and then by analyzing the Theiler stage 22 pancreas in situ hybridization staining patterns of over 600 of the identified genes using the GenePaint database. These were then categorized into one of the five expression domains within the developing pancreas. Based on these results we identified a cascade of transcriptional regulators expressed in the endocrine pancreas lineage and, from this, we developed a predictive regulatory network describing beta-cell development.

    Conclusion: Taken together, this work provides evidence that the SAGE libraries generated here are a valuable resource for continuing to elucidate the molecular mechanisms regulating pancreas development. Furthermore, our studies provide a comprehensive analysis of pancreas development, and insights into the regulatory networks driving this process are revealed.

    Genome biology 2008;9;6;R99

  • Role of calmodulin and myosin light chain kinase in the activation of carbachol-activated cationic current in murine ileal myocytes.

    Kim BJ, Jeon JH, Kim SJ and So I

    Center for Bio-Artificial Muscle and Department of Physiology, Seoul National University College of Medicine, Seoul 110-799, Korea.

    We investigated the effect of calmodulin (CaM) and myosin light chain kinase (MLCK) on murine ileal myocytes using the whole-cell patch-clamp technique. Under the voltage clamp, at the holding potential of -60 mV, 50 micromol/L carbachol (CCh) induced inward currents (I CCh), and spontaneous decay of I CCh occurred. The peak inward currents induced by the repetitive application of CCh (50 micromol/L) tended to decrease in amplitude. Intracellular application of 0.2 mmol/L guanosine 5'-O-(gamma-thio)triphosphate (GTP gammaS) from the patch electrode induced an inward current at a holding potential of -60m V, and the peak inward currents induced by the repetitive application of Cs tended to decrease slightly in amplitude. The amplitude of I CCh was reduced by pretreatment either with W-7, trifluoroperazine, W-5, and melittin (CaM inhibitors) or with ML-7 and ML-9 (selective MLCK inhibitors), and the inhibitory effects were reversible. However, when we pretreated with 50 micromol/L W-7 or 5 micromol/L ML-7 on GTP gammaS-induced inward currents, almost no inhibition was observed in the inward currents. Application of both Rho kinase inhibitor and MLCK inhibitor inhibited GTP gammaS-induced currents. We conclude that CaM and MLCK modulate the activation process of I CCh in murine ileal myocytes and suggest that the classical type transient receptor potential (TRPC) channel 5 might be a candidate for nonselective cationic currents (NSCC) activated by muscarinic stimulation in gastrointestinal smooth muscle cells.

    Canadian journal of physiology and pharmacology 2007;85;12;1254-62

  • Calmodulin binds and stabilizes the regulatory enzyme, CTP: phosphocholine cytidylyltransferase.

    Chen BB and Mallampalli RK

    Department of Internal Medicine, Roy J. and Lucille A. Carver college of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.

    CTP:phosphocholine cytidylyltransferase (CCTalpha) is a proteolytically sensitive enzyme essential for production of phosphatidylcholine, the major phospholipid of animal cell membranes. The molecular signals that govern CCTalpha protein stability are unknown. An NH(2)-terminal PEST sequence within CCTalpha did not serve as a degradation signal for the proteinase, calpain. Calmodulin (CaM) stabilized CCTalpha from calpain proteolysis. Adenoviral gene transfer of CaM in cells protected CCTalpha, whereas CaM small interfering RNA accentuated CCTalpha degradation by calpains. CaM bound CCTalpha as revealed by fluorescence resonance energy transfer and two-hybrid analysis. Mapping and site-directed mutagenesis of CCTalpha uncovered a motif (LQERVDKVK) harboring a vital recognition site, Gln(243), whereby CaM directly binds to the enzyme. Mutagenesis of CCTalpha Gln(243) not only resulted in loss of CaM binding but also led to complete calpain resistance in vitro and in vivo. Thus, calpains and CaM both access CCTalpha using a structurally similar molecular signature that profoundly affects CCTalpha levels. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTalpha that stabilizes the enzyme under proinflammatory stress.

    Funded by: NHLBI NIH HHS: HL068135, HL071040, HL080229, HL081784

    The Journal of biological chemistry 2007;282;46;33494-506

  • cAMP-responsive element-binding protein expression and regulation in the mouse preimplantation embryo.

    Jin XL and O'Neill C

    Human Reproduction Unit, Discipline of Physiology and Discipline of Medicine, Royal North Shore Hospital, University of Sydney, Sydney, New South Wales 2065, Australia.

    Gene expression from the new embryonic genome is required for normal preimplantation embryo development. Two members of the cAMP-responsive element-binding protein (Creb) family of transcription factors, Creb1 and activating transcription factor 1 (Atf1), are essential for normal preimplantation development. These transcription factors are activated by phosphorylation. Creb1 mRNA was expressed throughout the preimplantation phase. Cytoplasmic immunolocalization of Creb1 was detected in all preimplantation embryo stages. The antigen was largely excluded from the pronuclei/nuclei at embryonic stages except in the mid-cycle two-cell and compacted eight-cell embryo. Activation-state-specific antibodies showed serine 133 phosphorylated Creb1 localization was similar to Creb1 staining, except that there was no increase in staining at the eight-cell stage. Increased staining of phosphorylated Creb1 was observed in the nucleus of mid-cycle two-cell embryos. Increased expression of phosphorylated Creb1 in the two-cell embryo was induced by brief exposure of embryos to ionomycin, but not by a dibutyryl cAMP. This was blocked by buffering intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester), but not by a cAMP antagonist, Rp-cyclic 3',5'-hydrogen phosphorothioate adenosine. Calmodulin is an intracellular receptor for calcium. Calmodulin mRNA was expressed throughout the preimplantation phase of development. The calmodulin antagonist, W-7, inhibited the ionomycin-induced localization of phosphorylated Creb1 in the nucleus. Treatment of embryos with W-7 caused a dose-dependent inhibition of normal development of zygotes to the blastocysts stage. The study shows Creb1 expression and nuclear localization was dynamically regulated in the early embryo. The marked nuclear accumulation and phosphorylation of Creb1 at the two-cell stage occurred at the time of transcription from the embryonic genome and was regulated in a calcium- and calmodulin-dependent manner.

    Reproduction (Cambridge, England) 2007;134;5;667-75

  • Effect of calcium on calmodulin bound to the IQ motifs of myosin V.

    Trybus KM, Gushchin MI, Lui H, Hazelwood L, Krementsova EB, Volkmann N and Hanein D

    Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405, USA. kathleen.trybus@uvm.edu

    The long neck of unconventional myosin V is composed of six tandem "IQ motifs," which are fully occupied by calmodulin (CaM) in the absence of calcium. Calcium regulates the activity, the folded-to-extended conformational transition, and the processive run length of myosin V, and thus, it is important to understand how calcium affects CaM binding to the IQ motifs. Here we used electron cryomicroscopy together with computer-based docking of crystal structures into three-dimensional reconstructions of actin decorated with a motor domain-two IQ complex to provide an atomic model of myosin V in the presence of calcium. Calcium causes a major rearrangement of the bound CaMs, dissociation of CaM bound to IQ motif 2, and propagated changes in the motor domain. Tryptophan fluorescence spectroscopy showed that calcium-CaM binds to IQ motifs 1, 3, and 5 in a different conformation than apoCaM. Proteolytic cleavage was consistent with CaM preferentially dissociating from the second IQ motif. The enzymatic and mechanical functions of myosin V can, therefore, be modulated both by calcium-dependent conformational changes of bound CaM as well as by CaM dissociation.

    Funded by: NHLBI NIH HHS: HL38113; NIAMS NIH HHS: AR47199; NIGMS NIH HHS: GM076503

    The Journal of biological chemistry 2007;282;32;23316-25

  • Mapping of QTLs for oral alcohol self-administration in B6.C and B6.I quasi-congenic RQI strains.

    Vadasz C, Saito M, Gyetvai BM, Oros M, Szakall I, Kovacs KM, Prasad VV, Morahan G and Toth R

    Laboratory of Neurobehavioral Genetics, Nathan S. Kline Institute for Psychiatric Research, 140 Old Orangeburg Road, Orangeburg, NY 10962, USA. vadasz@nki.rfmh.org

    One strategy to identify neurochemical pathways of addiction is to map the relevant genes. In the present study we used 43 B6.C and 35 B6.I inbred RQI mouse strains, carrying <3% donor genome on C57BL/6ByJ background, for gene mapping. The strains were phenotyped for consumption of alcohol (12% v/v) in a two-bottle-choice paradigm, and genotyped for 396 microsatellite markers. The current mapping study extends our earlier experiment scanning five mouse chromosomes (Vadasz et al. (2000) Scanning of five chromosomes for alcohol consumption loci. Alcohol 22:25-34) to a whole-genome study, and discusses the differences and limitations. Data were analyzed with composite interval (CIM) and multiple interval (MIM) QTL mapping methods. CIM of B6.C strains detected significant QTLs on chrs. 6 and 12. A suggestive, but not significant, locus was detected in the B6.I strains on chr. 12. The best MIM model for B6.C strains confirmed one QTL on chr. 6 and one QTL on chr. 12, while the MIM model for the B6.I strains confirmed the suggestive locus on chr. 12. Some of the QTLs for alcohol consumption are new, while others confirm previously reported QTLs for alcohol preference, and alcohol acceptance.

    Funded by: NIAAA NIH HHS: AA11031, R01 AA011031, R01 AA011031-06

    Neurochemical research 2007;32;7;1099-112

  • Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria.

    Hoogaars WM, Engel A, Brons JF, Verkerk AO, de Lange FJ, Wong LY, Bakker ML, Clout DE, Wakker V, Barnett P, Ravesloot JH, Moorman AF, Verheijck EE and Christoffels VM

    Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.

    The sinoatrial node initiates the heartbeat and controls the rate and rhythm of contraction, thus serving as the pacemaker of the heart. Despite the crucial role of the sinoatrial node in heart function, the mechanisms that underlie its specification and formation are not known. Tbx3, a transcriptional repressor required for development of vertebrates, is expressed in the developing conduction system. Here we show that Tbx3 expression delineates the sinoatrial node region, which runs a gene expression program that is distinct from that of the bordering atrial cells. We found lineage segregation of Tbx3-negative atrial and Tbx3-positive sinoatrial node precursor cells as soon as cardiac cells turn on the atrial gene expression program. Tbx3 deficiency resulted in expansion of expression of the atrial gene program into the sinoatrial node domain, and partial loss of sinoatrial node-specific gene expression. Ectopic expression of Tbx3 in mice revealed that Tbx3 represses the atrial phenotype and imposes the pacemaker phenotype on the atria. The mice displayed arrhythmias and developed functional ectopic pacemakers. These data identify a Tbx3-dependent pathway for the specification and formation of the sinoatrial node, and show that Tbx3 regulates the pacemaker gene expression program and phenotype.

    Genes & development 2007;21;9;1098-112

  • Myocardium defects and ventricular hypoplasia in mice homozygous null for the Forkhead Box M1 transcription factor.

    Ramakrishna S, Kim IM, Petrovic V, Malin D, Wang IC, Kalin TV, Meliton L, Zhao YY, Ackerson T, Qin Y, Malik AB, Costa RH and Kalinichenko VV

    Department of Medicine, University of Chicago, Chicago, Illinois.

    The Forkhead Box m1 (Foxm1) transcription factor is expressed in cardiomyocytes and cardiac endothelial cells during heart development. In this study, we used a novel Foxm1 -/- mouse line to demonstrate that Foxm1-deletion causes ventricular hypoplasia and diminished DNA replication and mitosis in developing cardiomyocytes. Proliferation defects in Foxm1 -/- hearts were associated with a reduced expression of Cdk1-activator Cdc25B phosphatase and NFATc3 transcription factor, and with abnormal nuclear accumulation of the Cdk-inhibitor p21(Cip1) protein. Depletion of Foxm1 levels by siRNA caused altered expression of these genes in cultured HL-1 cardiomyocytes. Endothelial-specific deletion of the Foxm1 fl/fl allele in Tie2-Cre Foxm1 fl/fl embryos did not influence heart development and cardiomyocyte proliferation. Foxm1 protein binds to the -9,259/-9,288-bp region of the endogenous mouse NFATc3 promoter, indicating that Foxm1 is a transcriptional activator of the NFATc3 gene. Foxm1 regulates expression of genes essential for the proliferation of cardiomyocytes during heart development.

    Funded by: NHLBI NIH HHS: HL 84151-01

    Developmental dynamics : an official publication of the American Association of Anatomists 2007;236;4;1000-13

  • BGEM: an in situ hybridization database of gene expression in the embryonic and adult mouse nervous system.

    Magdaleno S, Jensen P, Brumwell CL, Seal A, Lehman K, Asbury A, Cheung T, Cornelius T, Batten DM, Eden C, Norland SM, Rice DS, Dosooye N, Shakya S, Mehta P and Curran T

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States.

    Funded by: NINDS NIH HHS: 5R37NS036558, N01-NS-0-2331, R37 NS036558

    PLoS biology 2006;4;4;e86

  • Identification of naturally processed peptides bound to the class I MHC molecule H-2Kd of normal and TAP-deficient cells.

    Suri A, Walters JJ, Levisetti MG, Gross ML and Unanue ER

    Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, USA.

    This report details the biochemical features of natural peptides selected by the H-2Kd class I MHC molecule. In normal cell lines, the length of the naturally processed peptides ranged from 8 to 18 amino acids, although the majority were 9-mers (16% were longer than nine residues). The binding motif for the 9-mer peptides was dominated by the presence of a tyrosine at P2 and an isoleucine/leucine at the P9 position. The P2 residue contributed most towards binding; and the short peptides bound better and formed longer-lived cell surface complexes than the long peptides, which bound poorly and dissociated rapidly. The longer peptides did not exhibit this strictly defined motif. Trimming the long peptides to their shorter forms did not enhance binding and conversely, extending the 9-mer peptides did not decrease binding. The long peptides were present on the cell-surface bound to H-2Kd (Kd) and were not intermediate products of the class I MHC processing pathway. Finally, in two different TAP-deficient cells the long peptides were the dominant species, which suggested that TAP-independent pathways selected for long peptides by class I MHC molecules.

    European journal of immunology 2006;36;3;544-57

  • Non-equivalence of embryonic and somatic cell nuclei affecting spindle composition in clones.

    Miyara F, Han Z, Gao S, Vassena R and Latham KE

    The Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA.

    Cloning by nuclear transfer remains inefficient but is more efficient when nuclei from embryonic cells or embryonic stem cells (ECNT) are employed as compared with somatic cells (SCNT). The factors determining efficiency have not been elucidated. We find that somatic and embryonic nuclei differ in their ability to organize meiotic and mitotic spindles of normal molecular composition. Calmodulin, a component of meiotic and mitotic spindle chromosome complexes (SCCs), displays sharply reduced association with the SCC forming after SCNT but not ECNT. This defect persists in mitotic spindles at least through the second mitosis, despite abundant calmodulin expression in the cell, and correlates with slow chromosome congression. We propose that somatic cell nuclei lack factors needed to direct normal SCC formation in oocytes and early embryos. These results reveal a striking control of SCC formation by the transplanted nucleus and provide the first identified molecular correlate of donor stage-dependent restriction in nuclear potency.

    Funded by: NCI NIH HHS: CA088261; NICHD NIH HHS: HD43092

    Developmental biology 2006;289;1;206-17

  • Overexpression of calmodulin induces cardiac hypertrophy by a calcineurin-dependent pathway.

    Obata K, Nagata K, Iwase M, Odashima M, Nagasaka T, Izawa H, Murohara T, Yamada Y and Yokota M

    Department of Cardiovascular Genome Science, Nagoya University School of Medicine, Nagoya, Japan.

    The possible role of calcineurin in cardiac hypertrophy induced by calmodulin (CaM) overexpression in the heart was investigated. CaM transgenic (CaM-TG) mice developed marked cardiac hypertrophy and exhibited up-regulation of atrial natriuretic factor (ANF) and beta-myosin heavy chain gene expression in the heart during the first 2 weeks after birth. The activity of calcineurin in the heart was also significantly increased in CaM-TG mice compared with wild-type littermates. Treatment of CaM-TG mice with the calcineurin inhibitor FK506 (1mg/kg per day) prevented the increase in the heart-to-body weight ratio as well as that in cardiomyocyte width. FK506 also inhibited the induction of fetal-type cardiac gene expression in CaM-TG mice. Overexpression of CaM in cultured rat cardiomyocytes activated the ANF gene promoter in a manner sensitive to FK506. Activation of a calcineurin-dependent pathway thus contributes to the development of cardiac hypertrophy induced by CaM overexpression in the heart.

    Biochemical and biophysical research communications 2005;338;2;1299-305

  • Calmodulin physically interacts with the erythropoietin receptor and enhances Jak2-mediated signaling.

    Kakihana K, Yamamoto M, Iiyama M and Miura O

    Department of Hematology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Tokyo 113-8519, Japan.

    Stimulation of the erythropoietin receptor (EpoR) induces a transient increase in intracellular Ca2+ level as well as activation of the Jak2 tyrosine kinase to stimulate various downstream signaling pathways. Here, we demonstrate that the universal Ca2+ receptor calmodulin (CaM) binds EpoR in a Ca2+-dependent manner in vitro. Binding studies using various EpoR mutants in hematopoietic cells showed that CaM binds the membrane-proximal 65-amino-acid cytoplasmic region (amino acids 258-312) of EpoR that is critical for activation of Jak2-mediated EpoR signaling. Structurally unrelated CaM antagonists, W-13 and CMZ, inhibited activation of Jak2-mediated EpoR signaling pathways, whereas W-12, a W-13 analog, did not show any significant inhibitory effect. Moreover, overexpression of CaM augmented Epo-induced tyrosine phosphorylation of the EpoR. W-13, but not W-12, also inhibited Epo-induced proliferation and survival. Together, these results indicate that CaM binds to the membrane-proximal EpoR cytoplasmic region and plays an essential role in activation of Jak2-mediated EpoR signaling.

    Biochemical and biophysical research communications 2005;335;2;424-31

  • Gonadotropin-releasing hormone induction of extracellular-signal regulated kinase is blocked by inhibition of calmodulin.

    Roberson MS, Bliss SP, Xie J, Navratil AM, Farmerie TA, Wolfe MW and Clay CM

    Department of Biomedical Sciences, Cornell University, T3-004d Veterinary Research Tower, Ithaca, New York 14853, usa. msr14@cornell.edu

    Our previous studies demonstrate that GnRH-induced ERK activation required influx of extracellular Ca2+ in alphaT3-1 and rat pituitary cells. In the present studies, we examined the hypothesis that calmodulin (Cam) plays a fundamental role in mediating the effects of Ca2+ on ERK activation. Cam inhibition using W7 was sufficient to block GnRH-induced reporter gene activity for the c-Fos, murine glycoprotein hormone alpha-subunit, and MAPK phosphatase (MKP)-2 promoters, all shown to require ERK activation. Inhibition of Cam (using a dominant negative) was sufficient to block GnRH-induced ERK but not c-Jun N-terminal kinase activity activation. The Cam-dependent protein kinase (CamK) II inhibitor KN62 did not recapitulate these findings. GnRH-induced phosphorylation of MAPK/ERK kinase 1 and c-Raf kinase was blocked by Cam inhibition, whereas activity of phospholipase C was unaffected, suggesting that Ca2+/Cam modulation of the ERK cascade potentially at the level of c-Raf kinase. Enrichment of Cam-interacting proteins using a Cam agarose column revealed that c-Raf kinase forms a complex with Cam. Reconstitution studies reveal that recombinant c-Raf kinase can associate directly with Cam in a Ca2+-dependent manner and this interaction is reduced in vitro by addition of W7. Cam was localized in lipid rafts consistent with the formation of a Ca2+-sensitive signaling platform including the GnRH receptor and c-Raf kinase. These data support the conclusion that Cam may have a critical role as a Ca2+ sensor in specifically linking Ca2+ flux with ERK activation within the GnRH signaling pathway.

    Funded by: NICHD NIH HHS: R01 HD34722; PHS HHS: F3244379

    Molecular endocrinology (Baltimore, Md.) 2005;19;9;2412-23

  • Binding of calmodulin to the carboxy-terminal region of p21 induces nuclear accumulation via inhibition of protein kinase C-mediated phosphorylation of Ser153.

    Rodríguez-Vilarrupla A, Jaumot M, Abella N, Canela N, Brun S, Díaz C, Estanyol JM, Bachs O and Agell N

    Department de Biologia Cel . lular, Fac. Medicina, Barcelona, Spain.

    Intracellular localization plays an important role in the functional regulation of the cell cycle inhibitor p21. We have previously shown that calmodulin binds to p21 and that calmodulin is essential for the nuclear accumulation of p21. Here, we analyze the mechanism of this regulation. We show that calmodulin inhibits in vitro phosphorylation of p21 by protein kinase C (PKC) and that this inhibition is dependent upon calmodulin binding to p21. Two-dimensional electrophoresis analysis of cells expressing the p21 wild type or p21S153A, a nonphosphorylatable mutant of p21 at position 153, indicates that Ser153 of p21 is a phosphorylable residue in vivo. Furthermore, Western blot analysis using phospho-Ser153-specific antibodies indicates that Ser153 phosphorylation in vivo is induced when PKC is activated and calmodulin is inhibited. The mutation of Ser153 to aspartate, a pseudophosphorylated residue, inhibits the nuclear accumulation of p21. Finally, whereas wild-type p21 translocates to the cytoplasm after PKC activation in the presence of calmodulin inhibitors, p21 carrying a nonphosphorylatable residue at position 153 remains in the nucleus. We propose that calmodulin binding to p21 prevents its phosphorylation by PKC at Ser153 and consequently allows its nuclear localization. When phosphorylated at Ser153, p21 is located at the cytoplasm and disrupts stress fibers.

    Molecular and cellular biology 2005;25;16;7364-74

  • Calmodulin and calmodulin-dependent kinase IIalpha regulate osteoblast differentiation by controlling c-fos expression.

    Zayzafoon M, Fulzele K and McDonald JM

    Department of Pathology, University of Alabama at Birmingham, USA.

    Ca(2+)/calmodulin-dependent protein kinase IIalpha (alpha-CaMKII) was once thought to be exclusively expressed in neuronal tissue, but it is becoming increasingly evident that CaMKII is also expressed in various extraneural cells. CaMKII plays a critical role in regulating various signaling pathways leading to modulation of several aspects of cellular functions, including proliferation, differentiation, cytoskeletal structure, and gene expression. The purpose of this study was to examine the expression of CaMKII in osteoblast-like cells (MC4) and to elucidate its role in osteoblast differentiation. We demonstrated that CaMKII, specifically the alpha isoform, is expressed in osteoblasts both in vitro and in vivo. Inhibition of CaMKII by the calmodulin antagonist trifluoperazine or the CaMKII antagonist KN93 reduces alkaline phosphatase activity and mineralization, as well as causes 85 and 56% decreases in alkaline phosphatase and osteocalcin gene expression, respectively. CaM and CaMKII antagonists, using the newborn mouse calvaria in vivo model, cause a 50% decrease in osteoblast number (N.Ob-BS) and a 32% decrease in mineralization (BV/TV). Pharmacologic and genetic inhibition of alpha-CaMKII by using trifluoperazine, KN93, and alpha-CaMKII small interfering RNA decreases the phosphorylation of ERK and of cAMP-response element-binding protein, leading to a significant decrease in the transactivation of serum response element and cAMP-response element. Inhibition of alpha-CaMKII decreases the expression of c-fos, AP-1 transactivation, and AP-1 DNA binding activity. Our findings demonstrated that alpha-CaMKII is expressed in osteoblasts and is involved in c-fos expression via regulation of serum response element and cAMP-response element. Inhibition of alpha-CaMKII results in a decrease in c-fos expression and AP-1 activation, leading to inhibition of osteoblast differentiation.

    Funded by: NIAMS NIH HHS: 1R01-AR050235, P30-AR46031

    The Journal of biological chemistry 2005;280;8;7049-59

  • Electron paramagnetic resonance spectroscopy and molecular modelling of the interaction of myelin basic protein (MBP) with calmodulin (CaM)-diversity and conformational adaptability of MBP CaM-targets.

    Polverini E, Boggs JM, Bates IR, Harauz G and Cavatorta P

    Istituto Nazionale per la Fisica della Materia, Dipartimento di Fisica, Università di Parma, Parco Area delle Scienze 7/A, 43100 Parma, Italy. eugenia.polverini@fis.unipr.it

    The classic 18.5 kDa isoform of murine myelin basic protein (mMBP) has been shown to bind calmodulin (CaM) strongly and specifically in vitro. Here, we have used site-directed spin labelling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy to map more precisely the sites of interaction of recombinant mMBP (rmMBP) with CaM. On the basis of these and previous experimental data, and the predictions of CaM-binding motifs using the Calmodulin Target Database (), three main segments of MBP were suggested for the interaction. The first site is located at the C-terminus; the second one lies in the central portion of the protein and forms an amphipathic alpha-helix in reconstituted myelin-mimetic systems; the third is quite close to the N-terminus. The murine Golli-MBP isoform J37 has also been shown to bind CaM in vitro, and an interaction site was predicted in the N-terminal Golli-specific portion of the protein. From these four segments, we selected peptide fragments of 12-14 residues in length, chosen on the bases of their amphipathicity and CaM-target characteristics. We modelled each of these peptides as alpha-helices, and performed docking simulations to investigate their interactions with the CaM peptide-binding tunnel. Different yet almost equally favourable CaM-binding modes were found for each of them. The experimental SDSL/EPR and theoretical modelling results were in good agreement, and supported the conjecture that there are several plausible CaM-binding sites in MBP, that could be induced into an alpha-helical conformation by their interaction with CaM and account for strong immobilisation of spin-labeled residues in all three segments. Phosphorylation and deimination were also emulated and simulated for known sites of MBP post-translational modification. The results obtained confirmed the appropriate utilisation of simple residue substitutions to mimic the natural modifications, and demonstrated molecular mechanisms by which MBP-CaM interactions could be modulated in vivo.

    Journal of structural biology 2004;148;3;353-69

  • Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas.

    Watahiki A, Waki K, Hayatsu N, Shiraki T, Kondo S, Nakamura M, Sasaki D, Arakawa T, Kawai J, Harbers M, Hayashizaki Y and Carninci P

    Genome Science Laboratory, RIKEN, Wako main campus, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan.

    It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line-specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development.

    Nature methods 2004;1;3;233-9

  • Mice lacking calsarcin-1 are sensitized to calcineurin signaling and show accelerated cardiomyopathy in response to pathological biomechanical stress.

    Frey N, Barrientos T, Shelton JM, Frank D, Rütten H, Gehring D, Kuhn C, Lutz M, Rothermel B, Bassel-Duby R, Richardson JA, Katus HA, Hill JA and Olson EN

    Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9148, USA. norbert.frey@med.uni-heidelberg.de

    Signaling by the calcium-dependent phosphatase calcineurin profoundly influences the growth and gene expression of cardiac and skeletal muscle. Calcineurin binds to calsarcins, a family of muscle-specific proteins of the sarcomeric Z-disc, a focal point in the pathogenesis of human cardiomyopathies. We show that calsarcin-1 negatively modulates the functions of calcineurin, such that calcineurin signaling was enhanced in striated muscles of mice that do not express calsarcin-1. As a consequence of inappropriate calcineurin activation, mice with a null mutation in calsarcin-1 showed an excess of slow skeletal muscle fibers. The absence of calsarcin-1 also activated a hypertrophic gene program, despite the absence of hypertrophy, and enhanced the cardiac growth response to pressure overload. In contrast, cardiac adaptation to other hypertrophic stimuli, such as chronic catecholamine stimulation or exercise, was not affected. These findings show important roles for calsarcins as modulators of calcineurin signaling and the transmission of a specific subset of stress signals leading to cardiac remodeling in vivo.

    Funded by: NHLBI NIH HHS: R01 HL072016

    Nature medicine 2004;10;12;1336-43

  • A network of control mediated by regulator of calcium/calmodulin-dependent signaling.

    Rakhilin SV, Olson PA, Nishi A, Starkova NN, Fienberg AA, Nairn AC, Surmeier DJ and Greengard P

    Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, NY 10021, USA.

    Calmodulin (CaM) is a major effector for the intracellular actions of Ca2+ in nearly all cell types. We identified a CaM-binding protein, designated regulator of calmodulin signaling (RCS). G protein-coupled receptor (GPCR)-dependent activation of protein kinase A (PKA) led to phosphorylation of RCS at Ser55 and increased its binding to CaM. Phospho-RCS acted as a competitive inhibitor of CaM-dependent enzymes, including protein phosphatase 2B (PP2B, also called calcineurin). Increasing RCS phosphorylation blocked GPCR- and PP2B-mediated suppression of L-type Ca2+ currents in striatal neurons. Conversely, genetic deletion of RCS significantly increased this modulation. Through a molecular mechanism that amplifies GPCR- and PKA-mediated signaling and attenuates GPCR- and PP2B-mediated signaling, RCS synergistically increases the phosphorylation of key proteins whose phosphorylation is regulated by PKA and PP2B.

    Funded by: NIDA NIH HHS: DA10044, DA12958, P01 DA010044; NIMH NIH HHS: MH40899; NINDS NIH HHS: NS34696

    Science (New York, N.Y.) 2004;306;5696;698-701

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Calmodulin-mediated activation of Akt regulates survival of c-Myc-overexpressing mouse mammary carcinoma cells.

    Deb TB, Coticchia CM and Dickson RB

    Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.

    c-Myc-overexpressing mammary epithelial cells are proapoptotic; their survival is strongly promoted by epidermal growth factor (EGF). We now demonstrate that EGF-induced Akt activation and survival in transgenic mouse mammary tumor virus-c-Myc mouse mammary carcinoma cells are both calcium/calmodulin-dependent. Akt activation is abolished by the phospholipase C-gamma inhibitor U-73122, by the intracellular calcium chelator BAPTA-AM, and by the specific calmodulin antagonist W-7. These results implicate calcium/calmodulin in the activation of Akt in these cells. In addition, Akt activation by serum and insulin is also inhibited by W-7. EGF-induced and calcium/calmodulin-mediated Akt activation occurs in both tumorigenic and non-tumorigenic mouse and human mammary epithelial cells, independent of their overexpression of c-Myc. These results imply that calcium/calmodulin may be a common regulator of Akt activation, irrespective of upstream receptor activator, mammalian species, and transformation status in mammary epithelial cells. However, only c-Myc-overexpressing mouse mammary carcinoma cells (but not normal mouse mammary epithelial cells) undergo apoptosis in the presence of the calmodulin antagonist W-7, indicating the vital selective role of calmodulin for survival of these cells. Calcium/calmodulin-regulated Akt activation is mediated directly by neither calmodulin kinases nor phosphatidylinositol 3-kinase (PI-3 kinase). Pharmacological inhibitors of calmodulin kinase kinase and calmodulin kinases II and III do not inhibit EGF-induced Akt activation, and calmodulin antagonist W-7 does not inhibit phosphotyrosine-associated PI-3 kinase activation. Akt is, however, co-immunoprecipitated with calmodulin in an EGF-dependent manner, which is inhibited by calmodulin antagonist W-7. We conclude that calmodulin may serve a vital regulatory function to direct the localization of Akt to the plasma membrane for its activation by PI-3 kinase.

    Funded by: NCI NIH HHS: R01-CA72460

    The Journal of biological chemistry 2004;279;37;38903-11

  • GenePaint.org: an atlas of gene expression patterns in the mouse embryo.

    Visel A, Thaller C and Eichele G

    Max Planck Institute of Experimental Endocrinology, Feodor-Lynen-Strasse 7, D-30625 Hannover, Germany.

    High-throughput instruments were recently developed to determine gene expression patterns on tissue sections by RNA in situ hybridization. The resulting images of gene expression patterns, chiefly of E14.5 mouse embryos, are accessible to the public at http://www.genepaint.org. This relational database is searchable for gene identifiers and RNA probe sequences. Moreover, patterns and intensity of expression in approximately 100 different embryonic tissues are annotated and can be searched using a standardized catalog of anatomical structures. A virtual microscope tool, the Zoom Image Server, was implemented in GenePaint.org and permits interactive zooming and panning across approximately 15,000 high-resolution images.

    Nucleic acids research 2004;32;Database issue;D552-6

  • Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention.

    Zambrowicz BP, Abuin A, Ramirez-Solis R, Richter LJ, Piggott J, BeltrandelRio H, Buxton EC, Edwards J, Finch RA, Friddle CJ, Gupta A, Hansen G, Hu Y, Huang W, Jaing C, Key BW, Kipp P, Kohlhauff B, Ma ZQ, Markesich D, Payne R, Potter DG, Qian N, Shaw J, Schrick J, Shi ZZ, Sparks MJ, Van Sligtenhorst I, Vogel P, Walke W, Xu N, Zhu Q, Person C and Sands AT

    Lexicon Genetics, 8800 Technology Forest Place, The Woodlands, TX 77381, USA. brian@lexgen.com

    The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in approximately 60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;24;14109-14

  • The role of calmodulin in the regulation of osteoclastogenesis.

    Zhang L, Feng X and McDonald JM

    Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

    Calmodulin plays an important role in regulating the function of mature osteoclasts. However, its role in osteoclastogenesis has not been investigated. In the present study, we examined the role of calmodulin in osteoclastogenesis using in vivo and in vitro systems. Calmodulin antagonists, trifluoperazine (TFP), W7, and tamoxifen, dose-dependently inhibited osteoclast formation, which occurred only in the last 24 h of a 4-d osteoclastogenesis culture using mouse bone marrow macrophages. Inhibitory effects were quantitated by measuring tartrate-resistant acid phosphatase activity and counting osteoclast numbers. In contrast, bis indolylmaleimide, a protein kinase C inhibitor, showed no such inhibitory effect even when applied at a concentration that was 10-fold greater than its IC50. Overexpressing calmodulin by recombinant retrovirus reversed the inhibitory effect of TFP on osteoclast-like differentiation in RAW264.7 cells. Furthermore, administration of TFP to mice was as effective as estrogen in abolishing the ovariectomy-induced increment of osteoclastogenesis as determined by quantitative assessment of tartrate-resistant acid phosphatase activity in tibias, which led to the recovery of the ovariectomy-induced decrement in trabecular bone volume. To investigate potential cellular and molecular mechanisms by which calmodulin antagonists inhibit osteoclastogenesis, Z-VAD-FMK, a broad caspase inhibitor, failed to block the inhibitory effect of TFP on mouse osteoclast formation, indicating that apoptosis is not the underlying mechanism. Pretreatment of RAW264.7 cells with different concentrations of TFP dose-dependently inhibited receptor activator of nuclear factor kappaB ligand-stimulated phosphorylation of c-Jun N-terminal kinase and inhibitory kappaBalpha but not that of p38. Taken together, our data indicate that calmodulin mediates osteoclast differentiation, possibly via modulating specific receptor activator of NF-kappaB-signaling pathways.

    Funded by: NIAMS NIH HHS: 1T32AR47512, AR43225, AR46031, AR47512

    Endocrinology 2003;144;10;4536-43

  • BayGenomics: a resource of insertional mutations in mouse embryonic stem cells.

    Stryke D, Kawamoto M, Huang CC, Johns SJ, King LA, Harper CA, Meng EC, Lee RE, Yee A, L'Italien L, Chuang PT, Young SG, Skarnes WC, Babbitt PC and Ferrin TE

    Department of Pharmaceutical Chemistry, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA.

    The BayGenomics gene-trap resource (http://baygenomics.ucsf.edu) provides researchers with access to thousands of mouse embryonic stem (ES) cell lines harboring characterized insertional mutations in both known and novel genes. Each cell line contains an insertional mutation in a specific gene. The identity of the gene that has been interrupted can be determined from a DNA sequence tag. Approximately 75% of our cell lines contain insertional mutations in known mouse genes or genes that share strong sequence similarities with genes that have been identified in other organisms. These cell lines readily transmit the mutation to the germline of mice and many mutant lines of mice have already been generated from this resource. BayGenomics provides facile access to our entire database, including sequence tags for each mutant ES cell line, through the World Wide Web. Investigators can browse our resource, search for specific entries, download any portion of our database and BLAST sequences of interest against our entire set of cell line sequence tags. They can then obtain the mutant ES cell line for the purpose of generating knockout mice.

    Funded by: NCRR NIH HHS: P41 RR001081, P41 RR01081; NHLBI NIH HHS: U01 HL066621, U01 HL66621

    Nucleic acids research 2003;31;1;278-81

  • A family of MHC class I-like genes located in the vicinity of the mouse leukocyte receptor complex.

    Kasahara M, Watanabe Y, Sumasu M and Nagata T

    Department of Biosystems Science, School of Advanced Sciences, Graduate University for Advanced Studies (Sokendai), Shonan Village, Hayama 240-0193, Japan. kasahara@soken.ac.jp

    Some members of the major histocompatibility complex (MHC) class I gene family are encoded outside the MHC. Here we describe a family of mouse class I-like genes mapping to the vicinity of the leukocyte receptor complex (LRC) on chromosome 7. This family, which we call Mill (MHC class I-like located near the LRC), has two members designated Mill1 and Mill2. Both genes are predicted to encode membrane glycoproteins with domain organization essentially similar to that of MHC class I heavy chains. The following features of Mill are noteworthy. (i) The deduced MILL proteins lack most of the residues known to be involved in the docking of peptides in classical MHC class I molecules. (ii) Among the known members of the class I gene family, MILL1 and MILL2 are related most closely to MICA/MICB encoded in the human MHC. (iii) Unlike all other known members of the class I gene family, Mill1 and Mill2 have an exon between those coding for the signal peptide and the alpha1 domain. (iv) Mill1 has a more restricted expression profile than Mill2. (v) The gene orthologous to Mill1 or Mill2 apparently is absent in the human. (vi) Mill1 and Mill2 show a limited degree of polymorphism in laboratory mice. The observation that the Mill family is related most closely to the MIC family, together with its apparent absence in the human, suggests its involvement in innate immunity.

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;21;13687-92

  • Calmodulin regulates intracellular trafficking of epidermal growth factor receptor and the MAPK signaling pathway.

    Tebar F, Villalonga P, Sorkina T, Agell N, Sorkin A and Enrich C

    Departament de Biologia Cel.lular, Facultat de Medicina, Institut d'Investigacions August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain 08036.

    The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family involved in signal transduction and the regulation of cellular proliferation and differentiation. It is also a calmodulin-binding protein. To examine the role of calmodulin in the regulation of EGFR, the effect of calmodulin antagonist, W-13, on the intracellular trafficking of EGFR and the MAPK signaling pathway was analyzed. W-13 did not alter the internalization of EGFR but inhibited its recycling and degradation, thus causing the accumulation of EGF and EGFR in enlarged early endosomal structures. In addition, we demonstrated that W-13 stimulated the tyrosine phosphorylation of EGFR and consequent recruitment of Shc adaptor protein with EGFR, presumably through inhibition of the calmodulin-dependent protein kinase II (CaM kinase II). W-13-mediated EGFR phosphorylation was blocked by metalloprotease inhibitor, BB94, indicating a possible involvement of shedding in this process. However, MAPK activity was decreased by W-13; dissection of this signaling pathway showed that W-13 specifically interferes with Raf-1 activity. These data are consistent with the regulation of EGFR by calmodulin at several steps of the receptor signaling and trafficking pathways.

    Molecular biology of the cell 2002;13;6;2057-68

  • Chromosomal mapping of calmodulin 1 (CALM1) and alpha-globin 1 genes (HBA1) in the bovine.

    Muramatsu Y, Taniguchi Y, Yamada T, Konfortov BA, Yasue H and Sasaki Y

    Department of Food Science, Shizuoka Eiwa College, Ikeda, Japan.

    Chromosomal mapping of the bovine calmodulin 1 and alpha-globin 1 genes was performed by analyzing bovine/murine somatic cell hybrid DNAs with PCR using primers specific for 3'-untranslated regions of those bovine genes. The calmodulin 1 and alpha-globin 1 genes were assigned to bovine chromosomes 25 and 29, respectively. Results from the present study should contribute to improvement in map resolution of bovine chromosomes and increase comparative information available on bovine chromosomes.

    Animal biotechnology 2001;12;2;137-40

  • New Dyscalc loci for myocardial cell necrosis and calcification (dystrophic cardiac calcinosis) in mice.

    Ivandic BT, Utz HF, Kaczmarek PM, Aherrahrou Z, Axtner SB, Klepsch C, Lusis AJ and Katus HA

    Department of Medicine II, University of Luebeck, 23538 Luebeck, Germany. ivandic@medinf.mu-luebeck.de

    Dystrophic cardiac calcinosis (DCC) occurs among certain inbred strains of mice and involves necrosis and subsequent calcification as response of myocardial tissue to injury. Using a complete linkage map approach, we investigated the genetics of DCC in an F(2) intercross of resistant C57BL/6J and susceptible C3H/HeJ inbred strains and identified previously a major predisposing quantitative trait locus (QTL), Dyscalc1, on proximal chromosome 7. Analysis of inheritance suggested, however, that DCC is influenced by additional modifier QTL, which have as yet not been mapped. Here, we report the identification by composite interval mapping of the DCC loci Dyscalc2, Dyscalc3, and Dyscalc4 on chromosomes 4, 12 and 14, respectively. Together, the four Dyscalc loci explained 47% of the phenotypic variance of DCC, which was induced by a high-fat diet. Additive epistasis between Dyscalc1 and Dyscalc2 enhanced DCC. Examining recombinant inbred strains, we propose a 10-cM interval containing Dyscalc1 and discuss potential candidate genes.

    Funded by: NHLBI NIH HHS: HL-30568

    Physiological genomics 2001;6;3;137-44

  • A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens.

    Thut CJ, Rountree RB, Hwa M and Kingsley DM

    Department of Developmental Biology, Stanford University School of Medicine, 279 Campus Drive, Beckman Center B300, Stanford, California 94305-5329, USA.

    The anterior segment of the vertebrate eye includes the cornea, iris, ciliary body, trabecular meshwork, and lens. Although malformations of these structures have been implicated in many human eye diseases, little is known about the molecular mechanisms that control their development. To identify genes involved in anterior segment formation, we developed a large-scale in situ hybridization screen and examined the spatial and temporal expression of over 1000 genes during eye development. This screen identified 62 genes with distinct expression patterns in specific eye structures, including several expressed in novel patterns in the anterior segment. Using these genes as developmental markers, we tested for the presence of inductive signals that control the differentiation of anterior segment tissues. Organ culture recombination experiments showed that a chick lens is capable of inducing the expression of markers of the presumptive iris and ciliary body in the developing mouse neural retina. The inducing activity from the lens acts only over short ranges and is present at multiple stages of eye development. These studies provide molecular evidence that an evolutionarily conserved signal from the lens controls tissue specification in the developing optic cup.

    Developmental biology 2001;231;1;63-76

  • A proteomics approach to the identification of mammalian mitochondrial small subunit ribosomal proteins.

    Koc EC, Burkhart W, Blackburn K, Moseley A, Koc H and Spremulli LL

    Department of Chemistry and School of Public Health, Environmental Science and Engineering, University of North Carolina, Chapel Hill, North Carolina 27599-3290, USA.

    Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins in six individual spots were subjected to in-gel tryptic digestion. Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by electrospray tandem mass spectrometry. The peptide sequences obtained were used to screen human expressed sequence tag data bases, and complete consensus cDNAs were assembled. Mammalian mitochondrial small subunit ribosomal proteins from six different classes of ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins correspond to Escherichia coli S10 and S14. Homologs of two human mitochondrial proteins not found in prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans. A homolog of one of these proteins was observed in D. melanogaster but not in C. elegans, while a homolog of the other was present in C. elegans but not in D. melanogaster. A homolog of one of the ribosomal proteins not found in prokaryotes was tentatively identified in the yeast genome. This latter protein is the first reported example of a ribosomal protein that is shared by mitochondrial ribosomes from lower and higher eukaryotes that does not have a homolog in prokaryotes.

    Funded by: NIGMS NIH HHS: GM32734

    The Journal of biological chemistry 2000;275;42;32585-91

  • Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray.

    Tanaka TS, Jaradat SA, Lim MK, Kargul GJ, Wang X, Grahovac MJ, Pantano S, Sano Y, Piao Y, Nagaraja R, Doi H, Wood WH, Becker KG and Ko MS

    Laboratory of Genetics and DNA Array Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224-6820, USA.

    cDNA microarray technology has been increasingly used to monitor global gene expression patterns in various tissues and cell types. However, applications to mammalian development have been hampered by the lack of appropriate cDNA collections, particularly for early developmental stages. To overcome this problem, a PCR-based cDNA library construction method was used to derive 52,374 expressed sequence tags from pre- and peri-implantation embryos, embryonic day (E) 12.5 female gonad/mesonephros, and newborn ovary. From these cDNA collections, a microarray representing 15,264 unique genes (78% novel and 22% known) was assembled. In initial applications, the divergence of placental and embryonic gene expression profiles was assessed. At stage E12.5 of development, based on triplicate experiments, 720 genes (6.5%) displayed statistically significant differences in expression between placenta and embryo. Among 289 more highly expressed in placenta, 61 placenta-specific genes encoded, for example, a novel prolactin-like protein. The number of genes highly expressed (and frequently specific) for placenta has thereby been increased 5-fold over the total previously reported, illustrating the potential of the microarrays for tissue-specific gene discovery and analysis of mammalian developmental programs.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;16;9127-32

  • Calmodulin functions as an activator of Pur alpha binding to single-stranded purine-rich DNA elements (PUR elements).

    Kuo CH, Nishikawa E, Ichikawa H, Sadakata T, Niu SY and Miki N

    Department of Pharmacology, Medical School, Osaka University, Japan. kuo@pharmal.med.osaka-u.ac.jp

    Pur alpha is a single stranded DNA-binding protein and binds to a consensus sequence (GGN)n. We have reported that the DNA-binding activity of a single stranded cyclic AMP response element-binding protein (ssCRE-BP) is suppressed in cerebellum treated chronically with morphine, ssCRE-BP is identical to Pur alpha and the DNA binding activity of Pur alpha is markedly enhanced by a heat stable activator in the nuclear extract. In this report, we purified this activator. The amino acid composition and partial amino acid sequence were determined to be identical to those of calmodulin (CaM), which enhanced the binding of GST-Pur alpha to various PUR elements in the 5' non-coding regions of the neuropeptide Y, myelin basic protein and nicotinic Ach receptor beta 4 subunit genes. The data suggest a novel gene expression pathway mediated by Ca/CaM-Pur alpha which may regulate a variety of genes in addition to those regulated through the CREB pathway.

    Biochemical and biophysical research communications 1999;255;2;406-11

  • Genome-wide mapping of unselected transcripts from extraembryonic tissue of 7.5-day mouse embryos reveals enrichment in the t-complex and under-representation on the X chromosome.

    Ko MS, Threat TA, Wang X, Horton JH, Cui Y, Wang X, Pryor E, Paris J, Wells-Smith J, Kitchen JR, Rowe LB, Eppig J, Satoh T, Brant L, Fujiwara H, Yotsumoto S and Nakashima H

    Center for Molecular Medicine and Genetics and Department of Internal Medicine, Wayne State University School of Medicine, 5047 Gullen Mall, Detroit, MI 48202, USA. msko@cmb.biosci.wayne.edu

    Mammalian embryos can only survive if they attach to the uterus (implantation) and establish proper maternal-fetal interactions. To understand this complex implantation pathway, we have initiated genomic analysis with a systematic study of the cohort of genes expressed in extraembryonic cells that are derived from the conceptus and play a major role in this process. A total of 2103 cDNAs from the extraembryonic portion of 7.5-day post-conception mouse embryos yielded 3186 expressed sequence tags, approximately 40% of which were novel to the sequence databases. Furthermore, when 155 of the cDNA clones with no homology to previously detected genes were genetically mapped, apparent clustering of these expressed genes was detected in subregions of chromosomes 2, 7, 9 and 17, with 6.5% of the observed genes localized in the t-complex region of chromosome 17, which represents only approximately 1.5% of the mouse genome. In contrast, X-linked genes were under-represented. Semi-quantitative RT-PCR analyses of the mapped genes demonstrated that one third of the genes were expressed solely in extraembryonic tissue and an additional one third of the genes were expressed predominantly in the extraembryonic tissues. The over-representation of extraembryonic-expressed genes in dosage-sensitive autosomal imprinted regions and under-representation on the dosage-compensated X chromosome may reflect a need for tight quantitative control of expression during development.

    Funded by: NHGRI NIH HHS: HG00941; NICHD NIH HHS: HD32243

    Human molecular genetics 1998;7;12;1967-78

  • Isolation of genes identified in mouse renal proximal tubule by comparing different gene expression profiles.

    Takenaka M, Imai E, Kaneko T, Ito T, Moriyama T, Yamauchi A, Hori M, Kawamoto S and Okubo K

    First Department of Medicine, Osaka University School of Medicine, Japan.

    An expression profile is a list based on a large scale sequencing of 1000 cDNA clones, showing the expressed genes and the abundance of their transcripts in a given cell or tissue (Okubo K et al: Nature Genet 2:173, 1992). We constructed an expression profile of mouse renal proximal tubules (PT) carefully isolated by microdissection in order to characterize its gene expression. Altogether 1000 clones were analyzed; there were 646 types of transcripts in PT, among which 196 were identical or homologous to the previously reported genes. The most abundant transcript was kidney-androgen regulated protein. By comparing the expression profile of PT with those obtained from other sources, several genes were identified only in PT. They included known transcripts and transcripts that were not homologous to the known genes. Three (GS4001, 3991, and 4059) of the non-homologous genes were analyzed by Northern blotting and in situ hybridization, and GS4001 and 4059 were predominantly expressed in the kidney, whereas GS3991 was detected in the liver as well as in the kidney. The sequence analysis of the full-size cDNAs demonstrated that GS4001 was a new member of aspartic proteinases and GS4059 was a novel gene. It also revealed that GS3991 was a mouse homologue of SA gene known to be expressed in PT. The expression profile of mouse PT and its comparison with those of other tissues and cells provide an alternate way of isolating genes predominantly expressed in PT, and also provides probes to study the molecular mechanisms of gene expression in the kidney.

    Kidney international 1998;53;3;562-72

  • Genetic mapping of 262 loci derived from expressed sequences in a murine interspecific cross using single-strand conformational polymorphism analysis.

    Brady KP, Rowe LB, Her H, Stevens TJ, Eppig J, Sussman DJ, Sikela J and Beier DR

    Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

    We have demonstrated previously that noncoding sequences of genes are a robust source of polymorphisms between mouse species when tested using single-strand conformation polymorphism (SSCP) analysis, and that these polymorphisms are useful for genetic mapping. In this report we demonstrate that presumptive 3'-untranslated region sequence obtained from expressed sequence tags (ESTs) can be analyzed in a similar fashion, and we have used this approach to map 262 loci using an interspecific backcross. These results demonstrate SSCP analysis of genes or ESTs is a simple and efficient means for the genetic localization of transcribed sequences, and is furthermore an approach that is applicable to any system for which there is sufficient sequence polymorphism.

    Funded by: NHGRI NIH HHS: HG00941, HG00951

    Genome research 1997;7;11;1085-93

  • Phosphorylation-dependent regulation of N-methyl-D-aspartate receptors by calmodulin.

    Hisatsune C, Umemori H, Inoue T, Michikawa T, Kohda K, Mikoshiba K and Yamamoto T

    Department of Oncology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan.

    The N-methyl-D-aspartate (NMDA) receptor plays important roles in synaptic plasticity and brain development. The NMDA receptor subunits have large intracellular domains in the COOH-terminal region that may interact with signal-transducing proteins. By using the yeast two-hybrid system, we found that calmodulin interacts with the COOH terminus of the NR1 subunit and inactivates the channels in a Ca2+-dependent manner. Here we show that protein kinase C (PKC)-mediated phosphorylation on serine residues of NR1 decreases its affinity for calmodulin. This suggests that PKC-mediated phosphorylation of NR1 prevents calmodulin from binding to the NR1 subunit and thereby inhibits the inactivation of NMDA receptors by calmodulin. In addition, we show that stimulation of metabotropic glutamate receptor 1alpha, which potentiates NMDA channels through PKC, decreases the ability of NR1 to bind to calmodulin. Thus, our data provide clues to understanding the basis of cross-talk between two types of receptors, metabotropic glutamate receptors and the NR1 subunit, in NMDA channel potentiation.

    The Journal of biological chemistry 1997;272;33;20805-10

  • A novel pair of immunoglobulin-like receptors expressed by B cells and myeloid cells.

    Kubagawa H, Burrows PD and Cooper MD

    Department of Pathology, Division of Developmental and Clinical Immunology, University of Alabama, Birmingham, AL 35294-3300, USA.

    An Fcalpha receptor probe of human origin was used to identify novel members of the Ig gene superfamily in mice. Paired Ig-like receptors, named PIR-A and PIR-B, are predicted from sequence analysis of the cDNAs isolated from a mouse splenic library. Both type I transmembrane proteins possess similar ectodomains with six Ig-like loops, but have different transmembrane and cytoplasmic regions. The predicted PIR-A protein has a short cytoplasmic tail and a charged Arg residue in the transmembrane region that, by analogy with the FcalphaR relative, suggests the potential for association with an additional transmembrane protein to form a signal transducing unit. In contrast, the PIR-B protein has an uncharged transmembrane region and a long cytoplasmic tail containing four potential immunoreceptor tyrosine-based inhibitory motifs. These features are shared by the related killer inhibitory receptors. PIR-A proteins appear to be highly variable, in that predicted peptide sequences differ for seven randomly selected PIR-A clones, whereas PIR-B cDNA clones are invariant. Southern blot analysis with PIR-B and PIR-A-specific probes suggests only one PIR-B gene and multiple PIR-A genes. The PIR-A and PIR-B genes are expressed in B lymphocytes and myeloid lineage cells, wherein both are expressed simultaneously. The characteristics of the highly-conserved PIR-A and PIR-B genes and their coordinate cellular expression suggest a potential regulatory role in humoral, inflammatory, and allergic responses.

    Funded by: NIAID NIH HHS: AI34568, AI39816, R01 AI039816, R37 AI039816

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;10;5261-6

  • Isolation of novel tissue-specific genes from cDNA libraries representing the individual tissue constituents of the gastrulating mouse embryo.

    Harrison SM, Dunwoodie SL, Arkell RM, Lehrach H and Beddington RS

    National Institute for Medical Research, London, UK.

    A total of 5 conventional, directionally cloned plasmid cDNA libraries have been constructed from the entire embryonic region of the mid-gastrulation mouse embryo and from its four principal tissue constituents (ectoderm, mesoderm, endoderm and primitive streak). These libraries have been validated with respect to the number of independent clones, insert-size and appropriate representation of diagnostic marker genes. Subtractive hybridisation has been used to remove clones common to the Endoderm and Mesoderm cDNA libraries resulting in an Endoderm minus Mesoderm subtracted library. Probe prepared from this subtracted library has been hybridised to a grid containing approximately 18,500 Embryonic Region library clones. Three novel clones have been recovered as well as expected genes already known to be highly expressed in the primitive endoderm lineage at this stage of development. In situ hybridisation to early postimplantation embryos has revealed the expression patterns of these novel genes. One is highly expressed exclusively in visceral endoderm, one is expressed in ectodermal and endodermal tissues, and the third proves to be an early marker of prospective and differentiated surface ectoderm as well as being expressed in endoderm and its derivatives.

    Development (Cambridge, England) 1995;121;8;2479-89

  • Three different calmodulin-encoding cDNAs isolated by a modified 5'-RACE using degenerate oligodeoxyribonucleotides.

    Skinner TL, Kerns RT and Bender PK

    Department of Biochemistry and Anaerobic Microbiology, Virginia Tech., Blacksburg 24061.

    In order to obtain the 5' ends of the three mouse calmodulin (CaM) cDNAs, we modified the standard 5' RACE (rapid amplification of cDNA ends) method to use degenerate synthetic oligodeoxyribonucleotides to prime cDNA synthesis of all three CaM mRNAs. In this modified method, the degenerate primers were annealed to mRNAs in an incubation step prior to the reverse transcription reaction. Separating the annealing step from the reverse transcription reaction allowed for greater stringency by using higher temperatures than could be tolerated if the reverse transcriptase were present. Annealing was also done with lower primer concentration and was driven by a longer incubation time. After the annealing step, cDNA synthesis was initiated by diluting the annealing mixture into a 42 degrees C buffer with reverse transcriptase. The synthesized cDNA was poly(dA)-tailed to allow PCR amplification of the first-strand cDNA with an anchor-dT17 primer and the degenerate primers. The CaM cDNAs were evident after this PCR. A second PCR, with nested gene-specific primers, was used to isolate the individual CaM cDNAs from the products of the first PCR. Three distinct CaM cDNAs were cloned and sequenced. By comparison of the 5' untranslated sequences between the mouse CaM DNAs and rat CaM cDNAs, the corresponding homologs were assigned. The results suggest that application of this modified RACE method could improve the success of isolating specific cDNAs in cases where use of a nested primer is not possible or when amino-acid sequence information is available and only degenerate primers can be designed for cloning cDNAs by the 5'-RACE method.

    Gene 1994;151;1-2;247-51

  • Genes newly identified as regulated by glucocorticoids in murine thymocytes.

    Baughman G, Harrigan MT, Campbell NF, Nurrish SJ and Bourgeois S

    Regulatory Biology Laboratory, Salk Institute for Biological Studies, San Diego, California 92186-5800.

    Glucocorticoids induce dramatic biochemical and morphological changes in lymphocytes through an unknown process that requires RNA and protein synthesis. In order to identify genes involved in this response, we previously isolated 11 cDNA clones from the murine WEHI-7TG thymoma cell line that correspond to mRNAs induced by glucocorticoids. We now report the isolation of two new cDNA clones whose gene expression is regulated by glucocorticoids in WEHI-7TG cells. We further characterize the two new cDNA clones, as well as those described previously, by examining the response of each of the corresponding mRNAs to glucocorticoids in murine thymocytes. With the exception of two, all cDNAs correspond to genes that are induced by glucocorticoids in murine thymocytes within 4 h of treatment. We previously identified two of the cDNAs as the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein. We have now identified four additional cDNA clones that correspond to the genes for calmodulin, mitochondrial phosphate carrier protein, immunoglobulin (Ig)-related glycoprotein (GP-70), and the 70 kilodalton autoantigen for Lupus and Graves diseases. Two other cDNA clones represent previously undescribed genes: one shares a high similarity to known sequences for the family of G-protein-coupled receptors and the other to a human placental-specific protein, PP11. Another cDNA appears to contain sequences for an unknown gene and the remnants of a mouse transposon. ETn. The remaining clones represent new, unidentified genes induced by glucocorticoids in murine thymocytes and in the WEHI-7TG cell line.

    Funded by: NCI NIH HHS: 5T32CA-09254, CA-14195, CA-36146

    Molecular endocrinology (Baltimore, Md.) 1991;5;5;637-44

  • A Collection of cDNA Clones with Specific Expression Patterns in Mouse Brain.

    Kato K

    MRC Molecular Genetics Unit, Hills Road, Cambridge CB2 2QH, UK.

    A total of 950 cDNA clones were randomly selected from mouse cerebellar cDNA libraries, and of these, about 130 clones were found to correspond to mRNAs which were expressed unequally between the cerebellum and other parts of mouse brain. Their distribution patterns in adult mouse brain were analysed by in situ hybridization, and eight clones were found restricted to specific regions of the brain, including four clones specific to cerebellar granule cells and one clone specific to Purkinje cells. Another 27 clones were preferentially expressed in a diverse, but distinctive subpopulation of brain cells. Among them seven clones were especially abundant in specific nuclei, and three in specific fibre bundles. These clones will be useful in defining new subpopulations of brain cells characterized by different gene expression.

    The European journal of neuroscience 1990;2;8;704-711

  • Cloning and expression of mouse-brain calmodulin as an activator of Bordetella pertussis adenylate cyclase in Escherichia coli.

    Danchin A, Sezer O, Glaser P, Chalon P and Caput D

    Régulation de l'Expression Génétique, Institut Pasteur, Paris, France.

    Cloning of higher eukaryotic genes has seldom been performed by complementation of a defective prokaryotic function. This is especially true in the case of functions that are normally absent from the prokaryotic host. We demonstrate here that it is possible to identify by complementation the cDNA from mouse brain, which encodes calmodulin (CaM) synthesis, in spite of the fact that the recipient strain, Escherichia coli, does not normally harbour a CaM function. A three-component cloning procedure in which a gene product requiring CaM for activity, adenylate cyclase from the pathogen Bordetella pertussis, was used to screen a cDNA library for cAMP synthesis in E. coli. The nucleotide sequence of the corresponding cDNA is also reported.

    Gene 1989;80;1;145-9

  • Calmodulin is required for cell-cycle progression during G1 and mitosis.

    Rasmussen CD and Means AR

    Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.

    In order to examine the consequences of a transient increase or decrease in intracellular calmodulin (CaM) levels, two bovine-papilloma-virus (BPV)-based expression vectors capable of inducibly synthesizing CaM sense (BPV-MCM) or anti-sense (BPV-CaMAS) RNA have been constructed and used to stably transform mouse C127 cells. Upon addition of Zn2+, cells containing the BPV-MCM vector have transiently increased CaM mRNA and protein levels. Cells carrying the BPV-CaMAS vector transiently produce CaM anti-sense RNA resulting in a significant decrease in intracellular CaM concentration. Increased CaM caused a transient acceleration of proliferation, while the anti-sense RNA induced decrease in CaM caused a transient cell cycle arrest. Flow cytometric analysis showed that progression through G1 and mitosis was affected by changes in CaM levels. These data indicate that CaM levels may limit the rate of cell-cycle progression under normal conditions of growth.

    The EMBO journal 1989;8;1;73-82

  • Opposite regulation of the mRNAs for parvalbumin and p19/6.8 in myotonic mouse muscle.

    Kluxen FW, Schöffl F, Berchtold MW and Jockusch H

    Developmental Biology Unit, University of Bielefeld, Federal Republic of Germany.

    The gene mutation in the mouse, 'arrested development of righting response', adr, causes a defect of chloride conductance of the muscle fibre membrane leading to the symptoms of myotonia [Mehrke, G., Brinkmeier, H. and Jockusch, H. (1988) Muscle & Nerve 11, 440-446]. In fast muscle, the myotonic phenotype is accompanied by a drastic reduction of the Ca2+-binding protein, parvalbumin. Messenger RNA levels in organs of myotonic (ADR) mice were analysed. In fast muscles of the mutant, in-vitro-translatable parvalbumin mRNA was strongly reduced, whereas the mRNA for the slow-muscle-specific protein, p19/6.8, was increased. In contrast, the parvalbumin mRNA in the cerebellum was not affected by the adr mutation. A reduction of the two parvalbumin mRNA species (700 and 1100 nucleotides) in ADR fast muscle and unaltered parvalbumin mRNA levels in mutant cerebella were demonstrated by cDNA/mRNA hybridisation, using a rat parvalbumin cDNA as a probe. The mRNA level for another Ca2+-binding protein, calmodulin, was low in muscle and high in the central nervous system but was unaffected by the mutation. When adr/adr mice were fed a diet containing the membrane-stabilising drug, tocainide, the levels in muscle of the mRNAs for parvalbumin and p19/6.8 were partially normalised.

    European journal of biochemistry 1988;176;1;153-8

  • The abundance of calmodulin mRNAs is regulated in phosphorylase kinase-deficient skeletal muscle.

    Bender PK, Dedman JR and Emerson CP

    Department of Biology, University of Virginia, Charlottesville 22901.

    In the I/Lyn mouse strain a mutation on the X chromosome results in a deficiency of the major calmodulin-regulated enzyme in skeletal muscle, phosphorylase kinase. Calmodulin has been identified as the delta-subunit of phosphorylase kinase, and it is estimated that approximately 40% of the total calmodulin in rabbit skeletal muscle is associated with the phosphorylase kinase hexadecamer (alpha, beta, gamma, delta)4. The absence of phosphorylase kinase in I/Lyn skeletal muscle results in a reduction in the total amount of calmodulin. The mechanisms affecting this reduction were investigated by comparing the abundance and heterogeneities in calmodulin mRNAs between normal and phosphorylase kinase-deficient skeletal muscles. The results demonstrate that in normal tissue there are four species of calmodulin mRNA distinguished by their molecular weight. All four of these species are present in the deficient tissue, and none of them are preferentially reduced. However, there is a 54% reduction in all four mRNAs as well as in calmodulin in the deficient skeletal muscle relative to normal skeletal muscle. These results indicate that the expression of calmodulin mRNAs is coordinated with the expression of its major enzyme target in skeletal muscle.

    Funded by: NIADDK NIH HHS: AM34213; NIDDK NIH HHS: DK34213

    The Journal of biological chemistry 1988;263;20;9733-7

Gene lists (9)

Gene List Source Species Name Description Gene count
L00000001 G2C Mus musculus Mouse PSD Mouse PSD adapted from Collins et al (2006) 1080
L00000003 G2C Mus musculus Mouse clathrin Mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000004 G2C Mus musculus Mouse Synaptosome Mouse Synaptosome adapted from Collins et al (2006) 152
L00000005 G2C Mus musculus Mouse mGluR5 Mouse mGluR5 complex adapted from Collins et al (2006) 52
L00000007 G2C Mus musculus Mouse NRC Mouse NRC adapted from Collins et al (2006) 186
L00000008 G2C Mus musculus Mouse PSP Mouse PSP adapted from Collins et al (2006) 1121
L00000019 G2C Mus musculus Pocklington M1 Cluster 1 (mouse) from Pocklington et al (2006) 21
L00000070 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list (ortho) 1461
L00000072 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list 1556
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EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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