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Splice Variants (splice + variants)
Kinds of Splice Variants Selected AbstractsLocalization and functional characterization of the human NKCC2 isoformsACTA PHYSIOLOGICA, Issue 3 2010I. Carota Abstract Aim:, Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na+/K+/2Cl, cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2 isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. Methods:, RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were performed to characterize human NKCC2 isoforms. Results:, All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant isoform in humans; similarly, isoform-specific in situ hybridization showed high expression levels of human NKCC2A along the TAL. Compared to NKCC2B and NKCC2F, human NKCC2A had the lowest Cl, affinity as determined by 86Rb+ uptake studies in oocytes. All NKCC2 isoforms were more efficiently inhibited by bumetanide than by furosemide. A sequence analysis of the amino acids encoded by exon 4 variants revealed high similarities between human and rodent NKCC2 isoforms, suggesting that differences in ion transport characteristics between species may be related to sequence variations outside the highly conserved sequence encoded by exon 4. Conclusion:, The human NKCC2 is an example of how differential splicing forms the basis for a diversification of transporter protein function. [source] Differential expression of CaMK-II genes during early zebrafish embryogenesisDEVELOPMENTAL DYNAMICS, Issue 1 2007Sarah C. Rothschild Abstract CaMK-II is a highly conserved Ca2+/calmodulin-dependent protein kinase expressed throughout the lifespan of all vertebrates. During early development, CaMK-II regulates cell cycle progression and "non-canonical" Wnt-dependent convergent extension. In the zebrafish, Danio rerio, CaMK-II activity rises within 2 hr after fertilization. At the time of somite formation, zygotic expression from six genes (camk2a1, camk2b1, camk2g1, camk2g2, camk2d1, camk2d2) results in a second phase of increased activity. Zebrafish CaMK-II genes are 92,95% identical to their human counterparts in the non-variable regions. During the first three days of development, alternative splicing yields at least 20 splice variants, many of which are unique. Whole-mount in situ hybridization reveals that camk2g1 comprises the majority of maternal expression. All six genes are expressed strongly in ventral regions at the 18-somite stage. Later, camk2a1 is expressed in anterior somites, heart, and then forebrain. Camk2b1 is expressed in somites, mid- and forebrain, gut, retina, and pectoral fins. Camk2g1 appears strongly along the midline and then in brain, gut, and pectoral fins. Camk2g2 is expressed early in the midbrain and trunk and exhibits the earliest retinal expression. Camk2d1 is elevated early at somite boundaries, then epidermal tissue, while camk2d2 is expressed in discrete anterior locations, steadily increasing along either side of the dorsal midline and then throughout the brain, including the retina. These findings reveal a complex pattern of CaMK-II gene expression consistent with pleiotropic roles during development. Developmental Dynamics 236:295,305, 2007. © 2006 Wiley-Liss, Inc. [source] Transient expression of serotonin 5-HT4 receptors in the mouse developing thalamocortical projectionsDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2010Erin R. Slaten Abstract The serotonin 5-HT4 receptor (5-HT4 -R) is an unusually complex G-protein coupled receptor that is likely to play important roles in brain development and that may underlie the comorbidity of central and peripheral abnormalities in some developmental disorders. We studied the expression of 5-HT4 -Rs in the developing mouse forebrain at embryonic days 13, 15, 17, and at postnatal days 3 and 14 by using immunohistochemistry, tract tracing, and quantitative RT-PCR. The developing thalamocortical projections transiently expressed 5-HT4 -Rs in the embryonic brain and the 5-HT4 -R expression in the forebrain changed from axonal to somatic around birth. From embryonic days 13,17, the forebrain mRNA levels of the 5-HT4(a) -R and 5-HT4(b) -R splice variants increased nine- and fivefold, respectively, whereas the levels of the 5-HT4(e) -R and 5-HT4(f) -R variants remained relatively low throughout the studied period of embryonic development. These results suggest that during development 5-HT4 -R expression undergoes a dynamic regulation and that this regulation may be important for the normal development of sensory and limbic processing. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010. [source] Splice-isoform specific immunolocalization of neuronal nitric oxide synthase in mouse and rat brain reveals that the PDZ-complex-building nNOS, ,-finger is largely exposed to antibodiesDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2007Kristina Langnaese Abstract Knock out mice deficient for the splice-isoform ,, of neuronal nitric oxide synthase (nNOS,,) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, ,, and ,,, we generated isoform-specific anti-peptide antibodies against the nNOS,, specific ,,-finger motif involved in PDZ domain scaffolding and the nNOS,, specific N-terminus. The nNOS,, ,,-finger-specific antibody clearly recognized the 160-kDa band of recombinant nNOS,, on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild-type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan-nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ-domain proteins via the ,,-finger motif. This conclusion was confirmed by the inhibition of PSD-95/nNOS interaction by the nNOS,, ,,-finger antibody in pull-down assays. By contrast, nNOS,, ,,-finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOS,, knock out mice, nNOS,, was not detectable, whereas the pan-nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting ,,/,,-isoforms in these cells. The nNOS,, antibody clearly detected bacterial expressed nNOS,, fusion protein and nNOS,, in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild-type mice, indicating an upregulation of nNOS,, in nNOS,, deficient animals. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007 [source] Localization of KCNC1 (Kv3.1) potassium channel subunits in the avian auditory nucleus magnocellularis and nucleus laminaris during developmentDEVELOPMENTAL NEUROBIOLOGY, Issue 2 2003Suchitra Parameshwaran-Iyer Abstract The KCNC1 (previously Kv3.1) potassium channel, a delayed rectifier with a high threshold of activation, is highly expressed in the time coding nuclei of the adult chicken and barn owl auditory brainstem. The proposed role of KCNC1 currents in auditory neurons is to reduce the width of the action potential and enable neurons to transmit high frequency temporal information with little jitter. Because developmental changes in potassium currents are critical for the maturation of the shape of the action potential, we used immunohistochemical methods to examine the developmental expression of KCNC1 subunits in the avian auditory brainstem. The KCNC1 gene gives rise to two splice variants, a longer KCNC1b and a shorter KCNC1a that differ at the carboxy termini. Two antibodies were used: an antibody to the N-terminus that does not distinguish between KCNC1a and b isoforms, denoted as panKCNC1, and another antibody that specifically recognizes the C terminus of KCNC1b. A comparison of the staining patterns observed with the panKCNC1 and the KCNC1b specific antibodies suggests that KCNC1a and KCNC1b splice variants are differentially regulated during development. Although panKCNC1 immunoreactivity is observed from the earliest time examined in the chicken (E10), a subcellular redistribution of the immunoproduct was apparent over the course of development. KCNC1b specific staining has a late onset with immunostaining first appearing in the regions that map high frequencies in nucleus magnocellularis (NM) and nucleus laminaris (NL). The expression of KCNC1b protein begins around E14 in the chicken and after E21 in the barn owl, relatively late during ontogeny and at the time that synaptic connections mature morphologically and functionally. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 165,178, 2003 [source] Mass spectrometrical analysis of the mitochondrial carrier Aralar1 from mouse hippocampusELECTROPHORESIS, Issue 11 2010Seok Heo Abstract Aralar1 is a mitochondrial aspartate/glutamate carrier and a key component of the malate,aspartate NADH shuttle system. An analytical approach to obtain high sequence coverage is important to predict conformation, identify splice variants and binding partners or generate specific antibodies. Moreover, a method allowing determination of Aralar1 from brain samples is a prerequisite for evaluating a biological role. Sucrose gradient ultracentrifugation was applied to enrich native membrane protein fractions and these were run on blue-native PAGE, followed by multidimensional gel electrophoresis. Spots from the third-dimensional gel electrophoresis were in-gel digested with trypsin, chymotrypsin and subtilisin. Subsequently, peptides were analyzed by nano-ESI-LC-MS/MS using collision-induced dissociation and electron transfer dissociation modes. ModiroÔ v1.1 along with Mascot v2.2 software was used for data handling. Aralar1 could be clearly separated, unambiguously identified and characterized from protein extracts of mouse hippocampus by the use of the multidimensional gel electrophoretic steps. The combined sequence coverage of Aralar1 from trypsin, chymotrypsin and subtilisin digestions was 99.85%. The results provide the basis for future studies of Aralar1 at the protein chemical rather than at the immunochemical level in the brain and thus challenge and enable determination of Aralar1 levels required for understanding biological functions in health and disease. [source] Alternative splicing of MDM2 mRNA in lung carcinomas and lung cell linesENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2005Mao-Wen Weng Abstract The MDM2 gene is overexpressed in several human tumors and its product may be processed into various isoforms. Recently, alternative splicing forms of MDM2 mRNA have been detected in various types of tumors. In this study, lung tissue from human non small cell lung cancers was examined for MDM2 mRNA splicing variants by nested RT-PCR. Of the 117 lung cancer tissue samples analyzed, a total of 31 (26.5%) had splice variants for the MDM2 gene, while 59 (50.4%) had undetectable levels of MDM2 transcript. Further analysis indicated that the predominant variant for 26 of the 31 samples with alternative MDM2 splicing products was MDM2-657, a splice variant lacking exons 3,11. Significant associations were found between the frequency of alternative splicing and the gender and smoking habits of the patients. Approximately 36% of male patients had alternative splicing of MDM2 compared with only 9.5% of female patients (P = 0.008); 44.2% of the smoker patients had alternative MDM2 splice forms versus 16.2% of nonsmokers (P = 0.003). Furthermore, most normal lung cell lines examined possessed only full-length MDM2 mRNA, while among several lung cancer cell lines, only H1355 and CaLu-1 cells lacked alternatively spliced MDM2 transcripts. When H1355 cells were treated in vitro with the cigarette smoke carcinogen benzo[a]pyrene (B[a]P) or the B[a]P metabolite benzo[a]pyrene diolepoxide (BPDE), three MDM2 splicing products were detected by nested RT-PCR. Finally, with the use of several specific inhibitors, we found that BPDE-induced MDM2 mRNA alternative splicing in H1355 cells may occur through the PI3K or MAPK pathway. Overall, our results suggest that carcinogens present in cigarette smoke increase the risk of alternative MDM2 splicing, which is highly associated with lung cancer. Environ. Mol. Mutagen., 2005. © 2005 Wiley-Liss, Inc. [source] Interleukin-5 does not influence differential transcription of transmembrane and soluble isoforms of IL-5R, in vivoEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 3 2006Jonas Byström Abstract:, Interleukin-5 (IL-5) promotes signal transduction and expansion of eosinophil colonies in bone marrow via interactions with its heterodimeric receptor (IL-5R). Two variants encoding soluble forms of the alpha subunit (sIL-5R,) have been described, although the signals promoting and/or limiting differential transcription remain to be clarified. Objectives:,Our intent was to explore the role of IL-5 in regulating differential transcription of these splice variants in vivo. Methods:,We have designed a quantitative reverse transcriptase-polymerase chain reaction assay to detect transcripts encoding the transmembrane, soluble 1 and 2 forms of IL-5R, in two strains of wild-type (BALB/c and C57BL/6) and corresponding IL-5 gene-deleted mice. Wild-type mice respond to S. mansoni infection with a gradual increase in serum IL-5 and eosinophilia, which is not observed in IL-5 gene-deleted mice. Results and conclusions:,We find that IL-5 is not necessary for differential splicing to occur in vivo, as all three forms of the IL-5R, are detected in both strains of IL-5 gene-deleted mice, with ratios of transcript expression (transmembrane : soluble 1 : soluble 2) that were indistinguishable from their wild-type counterparts. Differential splicing does vary markedly between strains, potentially because of local effects of strain-specific polymorphisms. [source] Manipulation of NK cytotoxicity by the IAP family member LivinEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2007Boaz Nachmias Abstract Natural killer (NK) cells are part of the innate immune system, capable of killing tumor and virally infected cells. NK cells induce apoptosis in the target cell by either granule- or receptor-mediated pathways. A set of inhibitory and activation ligands governs NK cell activation. As transformed cells often attempt to evade NK cell killing, up-regulation of a potential anti-apoptotic factor should provide a survival advantage. The inhibitor of apoptosis protein (IAP) family can inhibit apoptosis induced by a variety of stimuli. We have previously described a new IAP family member, termed Livin, which has two splice variants (, and ,) with differential anti-apoptotic activities. In this study, we explore the ability of Livin to inhibit NK cell-induced killing. We demonstrate that Livin,, moderately protects against NK cell killing whereas Livin,, augments killing. We show that Livin,, inhibition in Jurkat cells is apparent upon concomitant activation of an inhibitory signal, suggesting that Livin augments an extrinsic inhibitory signal rather than functioning as an independent inhibitory mechanism. Finally, we demonstrate that detection of both Livin isoforms in melanoma cells correlates with a low killing rate. To date, this is the first evidence that directly demonstrates the ability of IAP to protect against NK cell-induced apoptosis. [source] Distribution and functional characterization of human Nav1.3 splice variantsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2005R. Thimmapaya Abstract The focus of the present study is the molecular and functional characterization of four splice variants of the human Nav1.3 , subunit. These subtypes arise due to the use of alternative splice donor sites of exon 12, which encodes a region of the , subunit that resides in the intracellular loop between domains I and II. This region contains several important phosphorylation sites that modulate Na+ channel kinetics in related sodium channels, i.e. Nav1.2. While three of the four Nav1.3 isoforms, 12v1, 12v3 and 12v4 have been previously identified in human, 12v2 has only been reported in rat. Herein, we evaluate the distribution of these splice variants in human tissues and the functional characterization of each of these subtypes. We demonstrate by reverse transcriptase-polymerase chain reaction (RT-PCR) that each subtype is expressed in the spinal cord, thalamus, amygdala, cerebellum, adult and fetal whole brain and heart. To investigate the functional properties of these different splice variants, each , subunit isoform was cloned by RT-PCR from human fetal brain and expressed in Xenopus oocytes. Each isoform exhibited functional voltage-dependent Na+ channels with similar sensitivities to tetrodotoxin (TTX) and comparable current amplitudes. Subtle shifts in the V1/2 of activation and inactivation (2,3 mV) were observed among the four isoforms, although the functional significance of these differences remains unclear. This study has demonstrated that all four human splice variants of the Nav1.3 channel , subunit are widely expressed and generate functional TTX-sensitive Na+ channels that likely modulate cellular excitability. [source] Contribution of Kir3.1, Kir3.2A and Kir3.2C subunits to native G protein-gated inwardly rectifying potassium currents in cultured hippocampal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2003Joanne L. Leaney Abstract G protein-gated inwardly rectifying potassium (GIRK) channels are found in neurons, atrial myocytes and neuroendocrine cells. A characteristic feature is their activation by stimulation of Gi/o -coupled receptors. In central neurons, for example, they are activated by adenosine and GABA and, as such, they play an important role in neurotransmitter-mediated regulation of membrane excitability. The channels are tetrameric assemblies of Kir3.x subunits (Kir3.1,3.4 plus splice variants). In this study I have attempted to identify the channel subunits which contribute to the native GIRK current recorded from primary cultured rat hippocampal pyramidal neurons. Reverse transcriptase,polymerase chain reaction revealed the expression of mRNA for Kir3.1, 3.2A, 3.2C and 3.3 subunits and confocal immunofluorescence microscopy was used to investigate their expression patterns. Diffuse staining was observed on both cell somata and dendrites for Kir3.1 and Kir3.2A yet that for Kir3.2C was weaker and punctate. Whole-cell patch clamp recordings were used to record GIRK currents from hippocampal pyramidal neurons which were identified on the basis of inward rectification, dependence of reversal potential on external potassium concentration and sensitivity to tertiapin. The GIRK currents were enhanced by the stimulation of a number of Gi/o -coupled receptors and were inhibited by pertussis toxin. In order to ascertain which Kir3.x subunits were responsible for the native GIRK current I compared the properties with those of the cloned Kir3.1 + 3.2A and Kir3.1 + 3.2C channels heterologously expressed in HEK293 cells. [source] Expression of PRiMA in the mouse brain: membrane anchoring and accumulation of ,tailed' acetylcholinesteraseEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2003Noël A. Perrier Abstract We analysed the expression of PRiMA (proline-rich membrane anchor), the membrane anchor of acetylcholinesterase (AChE), by in situ hybridization in the mouse brain. We compared the pattern of PRiMA transcripts with that of AChE transcripts, as well as those of choline acetyltransferase and M1 muscarinic receptors which are considered pre- and postsynaptic cholinergic markers. We also analysed cholinesterase activity and its molecular forms in several brain structures. The results suggest that PRiMA expression is predominantly or exclusively related to the cholinergic system and that anchoring of cholinesterases to cell membranes by PRiMA represents a limiting factor for production of the AChE tailed splice variant (AChET),PRiMA complex, which represents the major AChE component in the brain. This enzyme species is mostly associated with cholinergic neurons because the pattern of PRiMA mRNA expression largely coincides with that of ChAT. We also show that, in both mouse and human, PRiMA proteins exist as two alternative splice variants which differ in their cytoplasmic regions. [source] Differential modulation of AMPA receptors by cyclothiazide in two types of striatal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2000Vladimir S. Vorobjev Abstract The modulation of ,-amino-3-hydroxy-5-methyl-4-isoxazol-propionate (AMPA) receptor-mediated currents by cyclothiazide was investigated in acutely isolated cells from rat striatum with whole-cell patch-clamp recording. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) was used to identify medium spiny and giant aspiny neurons and to determine their AMPA receptor subunit composition mostly in separate experiments. After pretreatment with cyclothiazide, kainate-induced AMPA responses were more strongly potentiated in medium spiny than in giant aspiny neurons; cyclothiazide induced a ninefold leftward shift in the kainate concentration,response curve for medium spiny neurons (not giant aspiny neurons). The EC50s for the cyclothiazide potentiation did not differ substantially between medium spiny neurons and giant aspiny neurons. The recovery of kainate-activated currents from modulation by cyclothiazide was slower for medium spiny neurons than for giant aspiny neurons. Medium spiny neurons expressed GluR-A, GluR-B and GluR-C, but not GluR-D subunits in both flip and flop splice variants. All giant aspiny neurons expressed GluR-A and GluR-D, exclusively in the flop form, half of them also expressed GluR-B and GluR-C. This is in keeping with slow and fast desensitization kinetics in medium spiny neurons and giant aspiny neurons, respectively, and differences in cyclothiazide modulation. The rate of cyclothiazide dissociation from the AMPA receptor, activated by glutamate, was ,,90 times slower in medium spiny neurons than in giant aspiny neurons. In giant aspiny neurons (not medium spiny neurons) this rate was strongly dependent on the presence of an agonist; 1 m m glutamate increased it 30-fold. Thus, two major cell groups in the striatum display distinct AMPA receptor compositions carrying specific properties of glutamate responses. Excitatory transmission will thus be differentially affected by cyclothiazide-type compounds. [source] Rescue of ,2 subunit-deficient mice by transgenic overexpression of the GABAA receptor ,2S or ,2L subunit isoformsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2000Kristin Baer Abstract The ,2 subunit is an important functional determinant of GABAA receptors and is essential for formation of high-affinity benzodiazepine binding sites and for synaptic clustering of major GABAA receptor subtypes along with gephyrin. There are two splice variants of the ,2 subunit, ,2 short (,2S) and ,2 long (,2L), the latter carrying in the cytoplasmic domain an additional eight amino acids with a putative phosphorylation site. Here, we show that transgenic mice expressing either the ,2S or ,2L subunit on a ,2 subunit-deficient background are phenotypically indistinguishable from wild-type. They express nearly normal levels of ,2 subunit protein and [3H]flumazenil binding sites. Likewise, the distribution, number and size of GABAA receptor clusters colocalized with gephyrin are similar to wild-type in both juvenile and adult mice. Our results indicate that the two ,2 subunit splice variants can substitute for each other and fulfil the basic functions of GABAA receptors, allowing in vivo studies that address isoform-specific roles in phosphorylation-dependent regulatory mechanisms. [source] The interferon alpha induced protein ISG12 is localized to the nuclear membraneFEBS JOURNAL, Issue 22 2001Pia M. Martensen Interferons exert their biological function mainly through the activation of interferon-stimulated genes (ISGs). ISG12 (originally designated p27) belongs to a family of small, interferon , inducible genes of unknown function. We have determined the 5, end sequence of ISG12 cDNA from the human cell lines HeLa and AMA by RACE. Comparing this sequence to ISG12 sequences in the expressed sequence tag (EST) database revealed the presence of two alternative splice variants of ISG12 in human cells exhibiting the same open reading frame. We have sequenced the promoter region of the ISG12 gene and found ISRE, IRF1/IRF2, and STAT elements correlating to the interferon , inducibility of the gene. Subsequently, we have expressed human ISG12, a 12-kDa hydrophobic protein in the baculovirus expression system and with a C-terminal FLAG-tag in the human cell line 293. Recombinant ISG12 sediments in the nuclear envelope in both cell types. Finally, we have been able to demonstrate the prevalence of the ISG12 gene product in the nuclear envelope of HeLa cells treated with interferon , by immunocytochemical analyses. ISG12 is the first interferon induced protein found localizing to the nuclear envelope. [source] Identification of novel splice variants of the human catalytic subunit c, of cAMP-dependent protein kinaseFEBS JOURNAL, Issue 19 2001Sigurd Ørstavik Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed C,, C,, C, and PrKX have been identified. Here we demonstrate that the human C, gene encodes six splice variants, designated C,1, C,2, C,3, C,4, C,4ab and C,4abc. The C, splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1, 1-2, 1-3, 1-4 and three exons designated a, b and c. All these exons are located upstream of exon 2 in the C, gene. The previously identified human C, variant has been termed C,1, and is similar to the C, isoform identified in the mouse, ox, pig and several other mammals. Human C,2, which is the homologue of bovine C,2, has no homologue in the mouse. Human C,3 and C,4 are homologous to the murine C,3 and C,2 splice variants, whereas human C,4ab and C,4abc represent novel isofoms previously not identified in any other species. At the mRNA level, the C, splice variants reveal tissue specific expression. C,1 was most abundantly expressed in the brain, with low-level expression in several other tissues. The C,3 and C,4 splice variants were uniquely expressed in human brain in contrast to C,2, which was most abundantly expressed in tissues of the immune system, with no detectable expression in brain. We suggest that the various C, splice variants when complexed with regulatory subunits may give rise to novel holoenzymes of protein kinase A that may be important for mediating specific effects of cAMP. [source] Identification of rat cyclic nucleotide phosphodiesterase 11A (PDE11A)FEBS JOURNAL, Issue 16 2001Comparison of rat, human PDE11A splicing variants ,We have isolated and characterized rat cyclic nucleotide phosphodiesterase (PDE)11A, which exhibits properties of a dual-substrate PDE, and its splice variants (RNPDE11A2, RNPDE11A3, and RNPDE11A4). The deduced amino-acid sequence of the longest form of rat PDE11A splice variant, RNPDE11A4, was 94% identical with that of the human variant (HSPDE11A4). Rat PDE11A splice variants were expressed in a tissue-specific manner. RNPDE11A4 showed unique tissue distribution distinct from HSPDE11A4, which is specifically expressed in the prostate. Rat PDE11A splice variants were expressed in COS-7 cells, and their enzymatic characteristics were compared. Although the Km values for cAMP and cGMP were similar for all of them (1.3,1.6 and 2.1,3.9 µm, respectively), the Vmax values differed significantly (RNPDE11A4 >> RNPDE11A2 > RNPDE11A3). Human PDE11A variants also displayed very similar Km values and significantly different Vmax values (HSPDE11A4 >> HSPDE11A2 > HSPDE11A3 >> HSPDE11A1). The Vmax values of HSPDE11A4 for cAMP and cGMP were at least 100 times higher than those of HSPDE11A1. These observations indicate unique characteristics of PDE11A splicing variants. [source] Ability of human CDC25B phosphatase splice variants to replace the function of the fission yeast Cdc25 cell cycle regulatorFEMS YEAST RESEARCH, Issue 3 2004Matthieu Lemaire Abstract CDC25 phosphatases are essential and evolutionary-conserved actors of the eukaryotic cell cycle control. To examine and compare the properties of three splicing variants of human CDC25B, recombinant fission yeast strains expressing the human proteins in place of the endogenous Cdc25 were generated and characterized. We report, that the three CDC25B variants: (i) efficiently replace the yeast counterpart in vegetative growth, (ii) partly restore the , and UV radiation DNA damage-activated checkpoint, (iii) fail to restore the DNA replication checkpoint activated by hydroxyurea. Although these yeast strains do not reveal the specific functions of the human CDC25B variants, they should provide useful screening tools for the identification of new cell cycle regulators and pharmacological inhibitors of CDC25 phosphatase. [source] Alterations of pre-mRNA splicing in cancerGENES, CHROMOSOMES AND CANCER, Issue 4 2005Zane Kalnin Recent genomewide analyses of alternative splicing (AS) indicate that up to 70% of human genes may have alternative splice forms, suggesting that AS together with various posttranslational modifications plays a major role in the production of proteome complexity. Splice-site selection under normal physiological conditions is regulated in the developmental stage in a tissue type-specific manner by changing the concentrations and the activity of splicing regulatory proteins. Whereas spliceosomal errors resulting in the production of aberrant transcripts rarely occur in normal cells, they seem to be an intrinsic property of cancer cells. Changes in splice-site selection have been observed in various types of cancer and may affect genes implicated in tumor progression (for example, CD44, MDM2, and FHIT) and in susceptibility to cancer (for example, BRCA1 and APC). Splicing defects can arise from inherited or somatic mutations in cis -acting regulatory elements (splice donor, acceptor and branch sites, and exonic and intronic splicing enhancers and silencers) or variations in the composition, concentration, localization, and activity of regulatory proteins. This may lead to altered efficiency of splice-site recognition, resulting in overexpression or down-regulation of certain splice variants, a switch in splice-site usage, or failure to recognize splice sites correctly, resulting in cancer-specific splice forms. At least in some cases, changes in splicing have been shown to play a functionally significant role in tumorigenesis, either by inactivating tumor suppressors or by gain of function of proteins promoting tumor development. Moreover, cancer-specific splicing events may generate novel epitopes that can be recognized by the host's immune system as cancer specific and may serve as targets for immunotherapy. Thus, the identification of cancer-specific splice forms provides a novel source for the discovery of diagnostic or prognostic biomarkers and tumor antigens suitable as targets for therapeutic intervention. © 2005 Wiley-Liss, Inc. [source] Expression of T-type calcium channel splice variants in human gliomaGLIA, Issue 2 2004Isabelle Latour Abstract In humans, three isoforms of the T-type (Cav3.1) calcium-channel ,1 subunit have been reported as a result of alternate splicing of exons 25 and 26 in the III,IV linker region (Cav3.1a, Cav3.1b or Cav3.1bc). In the present study, we report that human glioma express Cav3.1 channels in situ, that splicing of these exons is uniquely regulated and that there is expression of a glioma-specific novel T-type variant (Cav3.1ac). Seven human glioma samples were collected at surgery, RNA was extracted, and cDNA was produced for RT-PCR analysis. In addition, three glioma cell lines (U87, U563, and U251N), primary cultures of human fetal astrocytes, as well as adult and fetal human brain cDNA were used. Previously described Cav3.1 splice variants were present in glioma samples, cultured cells and whole brain. Consistent with the literature, our results reveal that in the normal adult brain, Cav3.1a transcripts predominate, while Cav3.1b is mostly fetal-specific. RT-PCR results on glioma and glioma cell lines showed that Cav3.1 expression in tumor cells resemble fetal brain expression pattern as Cav3.1bc is predominantly expressed. In addition, we identified a novel splice variant, Cav3.1ac, expressed in three glioma biopsies and one glioma cell line, but not in normal brain or fetal astrocytes. Transient expression of this variant demonstrates that Cav3.1ac displays similar current-voltage and steady-state inactivation properties compared with Cav3.1b, but a slower recovery from inactivation. Taken together, our data suggest glioma-specific Cav3.1 gene regulation, which could possibly contribute to tumor pathogenesis. © 2004 Wiley-Liss, Inc. [source] Human inhibitor of growth 1 inhibits hepatoma cell growth and influences p53 stability in a variant-dependent manner,HEPATOLOGY, Issue 2 2009Zhi Zhu Inhibitor of growth 1 (ING1) is a type II tumor suppressor that affects cell function by altering chromatin structure and regulating transcription. Recently, three ING1 splice variants have been cloned, but their roles in apoptosis and p53 regulation in human hepatocellular carcinoma (HCC) have not been fully elucidated. The present study found that ING1, in a variant-dependent manner, inhibited hepatoma cell proliferation and colony formation, induced apoptosis and cell cycle arrest at G0/G1 phase, and postponed tumor formation in nude mice. Expression of p33ING1b and p24ING1c variants, but not p47ING1a, was markedly reduced in HCC samples. Reverse transcription polymerase chain reaction and western blotting analysis revealed that ectopic overexpression of p33ING1b or p24ING1c variant increased the expression of p53 downstream genes such as p21waf1 and bax, and repressed bcl-2 expression (P < 0.01), whereas p47ING1a inactivated p21waf1 promoter (P < 0.01). Furthermore, we found that p33ING1b and p24ING1c repressed Mdm2 expression (P < 0.01) and competed with Mdm2 for binding to p53. Interestingly, p33ING1band p24ING1c did not directly bind to Mdm2 protein but strongly increased p14arf expression (P < 0.01) and interacted with p14arf protein to stimulate p53. Moreover, we found that ectopic overexpression of p33ING1b or p24ING1c significantly induced p53 protein acetylation at Lys-373/Lys-382 residue, but did not alter the phosphorylation status of p53. Conclusion: ING1 variants p33ING1b and p24ING1c may modulate p53 activity and subsequently inhibit hepatoma cell growth by at least two possible mechanisms: interacting with Mdm2 and p14arf to stabilize and activate p53, or increasing p53 acetylation. (HEPATOLOGY 2009.) [source] Expanded mutational spectrum in Cohen syndrome, tissue expression, and transcript variants of COH1,HUMAN MUTATION, Issue 2 2009Wenke Seifert Abstract Cohen syndrome is characterised by mental retardation, postnatal microcephaly, facial dysmorphism, pigmentary retinopathy, myopia, and intermittent neutropenia. Mutations in COH1 (VPS13B) have been found in patients with Cohen syndrome from diverse ethnic origins. We have carried out mutation analysis in twelve novel patients with Cohen syndrome from nine families. In this series, we have identified 13 different mutations in COH1, twelve of these are novel including six frameshift mutations, four nonsense mutations, two splice site mutations, and a one-codon deletion. Since different transcripts of COH1 have been reported previously, we have analysed the expression patterns of COH1 splice variants. The transcript variant NM_152564 including exon 28b showed ubiquitous expression in all examined human tissues. In contrast, human brain and retina showed differential splicing of exon 28 (NM_017890). Moreover, analysis of mouse tissues revealed ubiquitous expression of Coh1 homologous to human NM_152564 in all examined tissues but no prevalent alternative splicing. © 2008 Wiley-Liss, Inc. [source] BRCA1 and BRCA2 in Indian breast cancer patients,,HUMAN MUTATION, Issue 6 2002Sunita Saxena Abstract Incidence of breast cancer in Indian women is not as high as in Western countries, nonetheless age-adjusted incidence rates (AAR) have risen from 17.9 to 24.9 per 100,000 from 1965 to 1985. Although these rates are still approximately one quarter to one third of incidence rates in North America and Europe, respectively, due to the large population of women at risk, nearly 80,000 new cases were diagnosed in India in 2000. Although identification of BRCA1 and BRCA2 has greatly increased our understanding of breast cancer genetics in populations of Western European descent, the role of these genes in Indian populations remains unexplored. Analysis of a series of 20 breast cancer patients from North India with either family history of breast and/or ovarian cancer (2 or more affected first degree relatives) or early age of onset (<35 years) led to identification of two novel splice variants (331+1G>T; 4476+2T>C) in BRCA1 (10%). In addition, two BRCA2 missense variants were each identified in more than one patient (two unrelated individuals each) and likely represent population-specific polymorphisms. © 2002 Wiley-Liss, Inc. [source] Human B cells express the orphan chemokine receptor CRAM-A/B in a maturation-stage-dependent and CCL5-modulated mannerIMMUNOLOGY, Issue 2 2008Tanja N. Hartmann Summary Chemokines orchestrate the organization of leucocyte recruitment during inflammation and homeostasis. Despite growing knowledge of chemokine receptors, some orphan chemokine receptors are still not characterized. The gene CCRL2 encodes such a receptor that exists in two splice variants, CRAM-A and CRAM-B. Here, we report that CRAM is expressed by human peripheral blood and bone marrow B cells, and by different B-cell lines dependent on the B-cell maturation stage. Intriguingly, CRAM surface expression on the pre-B-cell lines Nalm6 and G2 is specifically upregulated in response to the inflammatory chemokine CCL5 (RANTES), a chemokine that is well known to play an important role in modulating immune responses. Although Nalm6 cells do not express any of the known CCL5 binding receptors, extracellular signal-regulated kinases 1 and 2 (ERK1/2) are phosphorylated upon CCL5 stimulation, suggesting a direct effect of CCL5 through the CRAM receptor. However, no calcium mobilization or migratory responses upon CCL5 stimulation are induced in B-cell lines or in transfected cells. Also, ERK1/2 phosphorylation cannot be inhibited by pertussis toxin, suggesting that CRAM does not couple to Gi proteins. Our results describe the expression of a novel, non-classical chemokine receptor on B cells that is potentially involved in immunomodulatory functions together with CCL5. [source] Identification and function of Abdominal-A in the silkworm, Bombyx moriINSECT MOLECULAR BIOLOGY, Issue 2 2009M-H. Pan Abstract Abdominal-A (adb-A) is a key gene in the development of insects. To understand its function in the silkworm, we cloned 1193 bp of the abd-A gene of Bombyx mori (Bmabd-A), including the complete coding sequence and part of the 3, untranslated region sequence. Bmabd-A has at least three mRNA splice variants with coding sequences of lengths 1032, 1044 and 1059 bp, encoding 343, 347 and 352 amino acids, respectively. Each splice variant of Bmabd-A has three exons and differs only in second exon size. Bmabd-A was expressed at low levels in unfertilized eggs, but increased gradually in fertilized eggs after laying 22 h. Bmabd-A expression decreased in ant silkworms (newly hatched silkworms). After RNA interference for Bmabd-A, the embryos had two mutant phenotypes, either completely or partially absent abdominal feet from the third to sixth abdominal segments, suggesting that Bmabd-A is responsible for normal development of the third to sixth abdominal segments during embryonic development. [source] Characterization of Phosphatase and Tensin Homolog expression in the mosquito Aedes aegypti: Six splice variants with developmental and tissue specificityINSECT MOLECULAR BIOLOGY, Issue 3 2007Michael A. Riehle Abstract Phosphatase and tensin homologue (PTEN), an inhibitor of insulin signalling, was characterized in Aedes aegypti. Surprisingly, six splice variants were identified: three with alternative terminal exons (AaegPTEN2 : 3 : 6) and three formed by intron retention (AaegPTEN1 : 4 : 5). All variants encoded active phosphatase domains. Variants with alternative terminal exons also encoded C2 and COOH-domains, and AaegPTEN6 encoded a PDZ binding motif. These three variants also had unique expression patterns. AaegPTEN2 was expressed primarily in the ovary. AaegPTEN3 was predominant in heads and midguts, and throughout development, except early embryogenesis. AaegPTEN6 was expressed in fat body, ovaries, and throughout development. Intron retention variants were weakly expressed in most samples. These expression patterns suggest that AaegPTEN variants play unique roles in regulating insulin's pleiotropic effects. [source] A splice variant of PGRP-LC required for expression of antimicrobial peptides in Anopheles gambiaeINSECT SCIENCE, Issue 3 2007HUI LIN Abstract Members of the peptidoglycan recognition protein (PGRP) family play essential roles in different manifestations of immune responses in insects. PGRP-LC, one of seven members of this family in the malaria vector Anopheles gambiae produced several spliced variants. Here we show that PGRP-LC, and not other members of the PGRP family nor the six members of the Gram-negative binding protein families, is required for the expression of antimicrobial peptide genes (such as CEC1 and GAM1) under the control of the Imd-Rel2 pathway in an A. gambiae cell line, 4a3A. PGRP-LC produces many splice variants that can be classified into three sub-groups (LC1, LC2 and LC3), based on the carboxyl terminal sequences. RNA interference against one LC1 sub-group resulted in dramatic reduction of CEC1 and GAM1. Over-expression of LC1a and to a lesser extent LC3a (a member of the LC1 and LC3 sub-group, respectively) in the 4a3A cell line enhances the expression of CEC1 and GAM1. These results demonstrate that the LC1-subgroup splice variants are essential for the expression of CEC1 and GAM1 in A. gambiae cell line. [source] Distinct expression patterns of the immunogenic differentiation antigen NY-BR-1 in normal breast, testis and their malignant counterpartsINTERNATIONAL JOURNAL OF CANCER, Issue 7 2008Jean-Philippe Theurillat Abstract NY-BR-1 is a differentiation antigen and a potential target for cancer immunotherapy. Its mRNA expression is restricted to breast, testis, prostate and breast cancer by RT-PCR. In this study, we correlated NY-BR-1 protein and mRNA expression on tissue microarrays of mammary, prostatic and testicular malignancies using immunohistochemisty and in situ hybridization with probes for exon 4,7 and 30,33. NY-BR-1 mRNA was confined to primary spermatocytes, suggesting a role in spermatogenesis. Exon 4,7 and 30,33 were equally expressed this cell type. However, NY-BR-1 was absent in all germ cell tumours analyzed (n = 475) and present in one of 56 (2%) prostate carcinomas. In breast, NY-BR-1 mRNA expression was detected in 307 of 442 (70%) primary carcinomas, with strong correlation to its protein expression (p < 0.0001). mRNA expression was significantly stronger and more frequently detected by the exon 30,33 probe than by the exon 4,7 probe (70% vs. 35%, p < 0.0001), indicating the presence of alternative splice variants that lack 5-prime sequences. A similar restricted mRNA pattern was also observed in the normal breast epithelium. NY-BR-1 protein and mRNA correlated significantly with estrogen receptor , (ER,) protein expression (p < 0.0001), with stronger association to NY-BR-1 mRNA than protein (odds ratio 7.7 compared to 4.6). We identified 4 estrogen response elements (ERE)-like sequences nearby the promoter region, suggesting that NY-BR-1 transcription might be controlled by ER,. Accordingly, analysis of matching pairs of primary tumors with their recurrences showed a marked decrease of NY-BR-1 expression in recurrences after tamoxifen treatment (p < 0.0001). © 2007 Wiley-Liss, Inc. [source] Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responsesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2008Manchao Zhang Abstract The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin. J. Cell. Biochem. 103: 162,181, 2008. © 2007 Wiley-Liss, Inc. [source] The four mammalian splice variants encoded by the p21-activated kinase 3 gene have different biological propertiesJOURNAL OF NEUROCHEMISTRY, Issue 3 2008Patricia Kreis Abstract The p21-activated kinases (PAK1), PAK2, and PAK3 are members of the PAK group I and share high sequence identity and common biochemical properties. PAK3 is specifically implicated in neuronal plasticity and also regulates cell cycle progression, neuronal migration, and apoptosis. Loss of function of PAK3 is responsible for X-linked non-syndromic mental retardation whereas gain of PAK3 function is associated with cancer. To understand the functional specificities of PAK3, we analyzed the structure of PAK3 gene products. We report here the characterization of a new alternatively spliced exon called c located upstream of the previously identified exon b. Exon b is detected in all tetrapods and not in fish, exon c is only present in mammals. Mammalian PAK3 genes encode four splice variants and the corresponding proteins were detected with specific antibodies in brain extracts. All PAK3 transcripts are specifically expressed in brain and in particular in neurons. The presence of the exons b and c renders the kinase constitutively active and decreases interaction with GTPases. The expression of the new splice variants in COS7 cells alters cell morphology and modifies the structure of focal adhesions. We propose that the appearance of new alternatively spliced exons during evolution and the resulting increase of complexity of PAK3 gene products may confer new functions to this kinase and contribute to its specific roles in neuronal signaling. [source] | ||||||||||||||||||||||||||||||||||