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Sample records for 2b subunit gene

  1. Localization of two potassium channel {beta} subunit genes, KCNA1B and KCNA2B

    SciTech Connect

    Schultz, D.; Smith, L.; Thayer, M.

    1996-02-01

    The gating properties and current amplitudes of mammalian voltage-activated Shaker potassium channels are modulated by at least two associated {beta} subunits (Kv{beta}1.1 and Kv{beta}1.2). The human Kv{beta}1.1 gene (KCNA1B) resides on chromosome 3, as indicated by somatic cell hybrid mapping. More precise localization of KCNA1B to 3q26.1 was obtained with fluorescence in situ hybridization (FISH) and was corroborated by PCR screening of the CEPH YAC library. The human Kv{beta}1.2 gene (KCNA2B) resides on chromosome 1, as indicated by somatic cell hybrid mapping, and has been localized by FISH to 1p36.3. 20 refs., 2 figs.

  2. ANKS1B Gene Product AIDA-1 Controls Hippocampal Synaptic Transmission by Regulating GluN2B Subunit Localization

    PubMed Central

    Tindi, Jaafar O.; Chávez, Andrés E.; Cvejic, Svetlana; Calvo-Ochoa, Erika; Castillo, Pablo E.

    2015-01-01

    NMDA receptors (NMDARs) are key mediators of glutamatergic transmission and synaptic plasticity, and their dysregulation has been linked to diverse neuropsychiatric and neurodegenerative disorders. While normal NMDAR function requires regulated expression and trafficking of its different subunits, the molecular mechanisms underlying these processes are not fully understood. Here we report that the amyloid precursor protein intracellular domain associated-1 protein (AIDA-1), which associates with NMDARs and is encoded by ANKS1B, a gene recently linked to schizophrenia, regulates synaptic NMDAR subunit composition. Forebrain-specific AIDA-1 conditional knock-out (cKO) mice exhibit reduced GluN2B-mediated and increased GluN2A-mediated synaptic transmission, and biochemical analyses show AIDA-1 cKO mice have low GluN2B and high GluN2A protein levels at isolated hippocampal synaptic junctions compared with controls. These results are corroborated by immunocytochemical and electrophysiological analyses in primary neuronal cultures following acute lentiviral shRNA-mediated knockdown of AIDA-1. Moreover, hippocampal NMDAR-dependent but not metabotropic glutamate receptor-dependent plasticity is impaired in AIDA-1 cKO mice, further supporting a role for AIDA-1 in synaptic NMDAR function. We also demonstrate that AIDA-1 preferentially associates with GluN2B and with the adaptor protein Ca2+/calmodulin-dependent serine protein kinase and kinesin KIF17, which regulate the transport of GluN2B-containing NMDARs from the endoplasmic reticulum (ER) to synapses. Consistent with this function, GluN2B accumulates in ER-enriched fractions in AIDA-1 cKO mice. These findings suggest that AIDA-1 regulates NMDAR subunit composition at synapses by facilitating transport of GluN2B from the ER to synapses, which is critical for NMDAR plasticity. Our work provides an explanation for how AIDA-1 dysfunction might contribute to neuropsychiatric conditions, such as schizophrenia. PMID:26085624

  3. Localization of eight additional genes in the human major histocompatibility complex, including the gene encoding the casein kinase II {beta} subunit (CSNK2B)

    SciTech Connect

    Albertella, M.R.; Jones, H.; Thomson, W.

    1996-09-01

    A wide range of autoimmune and other diseases are known to be associated with the major histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility antigens in the class I and class II regions, but some appear to be more strongly associated with genes in the central 1100-kb class III region, making it important to characterize this region fully for the presence of novel genes. An {approximately}220-kb segment of DNA in the class III region separating the Hsp70 (HSPA1L) and BAT1 (D6S8IE) genes, which was previously known to contain 14 genes. Genomic DNA fragments spanning the gaps between the known genes were used as probes to isolate cDNAs corresponding to five new genes within this region. Evidence from Northern blot analysis and exon trapping experiments that suggested the presence of at least two more new genes was also obtained. Partial cDNA and complete exonic genomic sequencing of one of the new genes has identified it as the casein kinase II{beta} subunit (CSNK2B). Two of the other novel genes lie within a region syntenic to that implicated in susceptibility to experimental allergic orchitis in the mouse, an autoimmune disease of the testis, and represent additional candidates for the Orch-1 locus associated with this disease. In addition, characterization of the 13-kb intergenic gap separating the RD (D6545) and G11 (D6S60E) genes has revealed the presence of a gene encoding a 1246-amino-acid polypeptide that shows significant sequence similarity to the yeast anti-viral Ski2p gene product. 49 refs., 8 figs.

  4. Identification of a Novel Rat NR2B Subunit Gene Promoter Region Variant and Its Association with Microwave-Induced Neuron Impairment.

    PubMed

    Wang, Li-Feng; Tian, Da-Wei; Li, Hai-Juan; Gao, Ya-Bing; Wang, Chang-Zhen; Zhao, Li; Zuo, Hong-Yan; Dong, Ji; Qiao, Si-Mo; Zou, Yong; Xiong, Lu; Zhou, Hong-Mei; Yang, Yue-Feng; Peng, Rui-Yun; Hu, Xiang-Jun

    2016-05-01

    Microwave radiation has been implicated in cognitive dysfunction and neuronal injury in animal models and in human investigations; however, the mechanism of these effects is unclear. In this study, single nucleotide polymorphism (SNP) sites in the rat GRIN2B promoter region were screened. The associations of these SNPs with microwave-induced rat brain dysfunction and with rat pheochromocytoma-12 (PC12) cell function were investigated. Wistar rats (n = 160) were exposed to microwave radiation (30 mW/cm(2) for 5 min/day, 5 days/week, over a period of 2 months). Screening of the GRIN2B promoter region revealed a stable C-to-T variant at nucleotide position -217 that was not induced by microwave exposure. The learning and memory ability, amino acid contents in the hippocampus and cerebrospinal fluid, and NR2B expression were then investigated in the different genotypes. Following microwave exposure, NR2B protein expression decreased, while the Glu contents in the hippocampus and CSF increased, and memory impairment was observed in the TT genotype but not the CC and CT genotypes. In PC12 cells, the effects of the T allele were more pronounced than those of the C allele on transcription factor binding ability, transcriptional activity, NR2B mRNA, and protein expression. These effects may be related to the detrimental role of the T allele and the protective role of the C allele in rat brain function and PC12 cells exposed to microwave radiation. PMID:25917873

  5. Setdb1 histone methyltransferase regulates mood-related behaviors and expression of the NMDA receptor subunit NR2B.

    PubMed

    Jiang, Yan; Jakovcevski, Mira; Bharadwaj, Rahul; Connor, Caroline; Schroeder, Frederick A; Lin, Cong L; Straubhaar, Juerg; Martin, Gilles; Akbarian, Schahram

    2010-05-26

    Histone methyltransferases specific for the histone H3-lysine 9 residue, including Setdb1 (Set domain, bifurcated 1)/Eset/Kmt1e are associated with repressive chromatin remodeling and expressed in adult brain, but potential effects on neuronal function and behavior remain unexplored. Here, we report that transgenic mice with increased Setdb1 expression in adult forebrain neurons show antidepressant-like phenotypes in behavioral paradigms for anhedonia, despair, and learned helplessness. Chromatin immunoprecipitation in conjunction with DNA tiling arrays (ChIP-chip) revealed that genomic occupancies of neuronal Setdb1 are limited to <1% of annotated genes, which include the NMDA receptor subunit NR2B/Grin2B and other ionotropic glutamate receptor genes. Chromatin conformation capture and Setdb1-ChIP revealed a loop formation tethering the NR2B/Grin2b promoter to the Setdb1 target site positioned 30 kb downstream of the transcription start site. In hippocampus and ventral striatum, two key structures in the neuronal circuitry regulating mood-related behaviors, Setdb1-mediated repressive histone methylation at NR2B/Grin2b was associated with decreased NR2B expression and EPSP insensitivity to pharmacological blockade of NR2B, and accelerated NMDA receptor desensitization consistent with a shift in NR2A/B subunit ratios. In wild-type mice, systemic treatment with the NR2B antagonist, Ro25-6981 [R-(R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propranol], and hippocampal small interfering RNA-mediated NR2B/Grin2b knockdown resulted in behavioral changes similar to those elicited by the Setdb1 transgene. Together, these findings point to a role for neuronal Setdb1 in the regulation of affective and motivational behaviors through repressive chromatin remodeling at a select set of target genes, resulting in altered NMDA receptor subunit composition and other molecular adaptations. PMID:20505083

  6. DAPK1 Interaction with NMDA Receptor NR2B Subunits Mediates Brain Damage in Stroke

    PubMed Central

    Tu, Weihong; Xu, Xin; Peng, Lisheng; Zhong, Xiaofen; Zhang, Wenfeng; Soundarapandian, Mangala M.; Balel, Cherine; Wang, Manqi; Jia, Nali; Zhang, Wen; Lew, Frank; Chan, Sic Lung; Chen, Yanfang; Lu, Youming

    2010-01-01

    SUMMARY N-methyl-D-aspartate (NMDA) receptors constitute a major subtype of glutamate receptors at extra-synaptic sites that link multiple intracellular catabolic processes responsible for irreversible neuronal death. Here, we report that cerebral ischemia recruits death-associated protein kinase 1 (DAPK1) into the NMDA receptor NR2B protein complex in the cortex of adult mice. DAPK1 directly binds with the NMDA receptor NR2B C-terminal tail consisting of amino acid 1292–1304 (NR2BCT). A constitutively active DAPK1 phosphorylates NR2B subunit at Ser-1303 and in turn enhances the NR1/NR2B receptor channel conductance. Genetic deletion of DAPK1 or administration of NR2BCT that uncouples an activated DAPK1 from an NMDA receptor NR2B subunit in vivo in mice blocks injurious Ca2+ influx through NMDA receptor channels at extrasynaptic sites and protects neurons against cerebral ischemic insults. Thus, DAPK1 physically and functionally interacts with the NMDA receptor NR2B subunit at extra-synaptic sites and this interaction acts as a central mediator for stroke damage. PMID:20141836

  7. Evidence that glutathione S-transferases B1B1 and B2B2 are the products of separate genes and that their expression in human liver is subject to inter-individual variation. Molecular relationships between the B1 and B2 subunits and other Alpha class glutathione S-transferases.

    PubMed Central

    Hayes, J D; Kerr, L A; Cronshaw, A D

    1989-01-01

    The Alpha class glutathione S-transferases (GSTs) in human liver are composed of polypeptides of Mr 25,900. These enzymes are dimeric, and two immunochemically distinct subunits, B1 and B2, have been described that combine to form GSTs B1B1, B1B2 and B2B2 [Stockman, Beckett & Hayes (1985) Biochem. J. 227, 457-465]. Gradient affinity elution from GSH-Sepharose has been used to resolve the three Alpha class GSTs, and this method has been applied to demonstrate marked inter-individual differences in the hepatic content of GSTs B1B1, B1B2 and B2B2. The B1 and B2 subunits can be resolved by reverse-phase h.p.l.c., and their elution positions suggest that they are equivalent to the alpha chi and alpha y h.p.l.c. peaks described by Ketterer and his colleagues [Ostlund Farrants, Meyer, Coles, Southan, Aitken, Johnson & Ketterer (1987) Biochem. J. 245, 423-428]. The B1 and B2 subunits have now been cleaved with CNBr and the fragments subjected to automated amino acid sequence analysis. The sequence data show that B1 and B2 subunits do not arise from post-translational modification, as had been previously believed for the hepatic Alpha class GSTs, but are instead the products of separate genes; B1 and B2 subunits were found to contain different amino acid residues at positions 88, 110, 111, 112, 116, 124 and 127. The relationship between the B1 and B2 subunits and the cloned GTH1 and GTH2 cDNA sequences [Rhoads, Zarlengo & Tu (1987) Biochem. Biophys. Res. Commun. 145, 474-481] is discussed. PMID:2604726

  8. Localization of sulfonylurea receptor subunits, SUR2A and SUR2B, in rat heart.

    PubMed

    Zhou, Ming; He, Hui-Jing; Suzuki, Ryoji; Liu, Ke-Xiang; Tanaka, Osamu; Sekiguchi, Masaki; Itoh, Hideaki; Kawahara, Katsumasa; Abe, Hiroshi

    2007-08-01

    To understand the possible functions and subcellular localizations of sulfonylurea receptors (SURs) in cardiac muscle, polyclonal anti-SUR2A and anti-SUR2B antisera were raised. Immunoblots revealed both SUR2A and SUR2B expression in mitochondrial fractions of rat heart and other cellular fractions such as microsomes and cell membranes. Immunostaining detected ubiquitous expression of both SUR2A and SUR2B in rat heart in the atria, ventricles, interatrial and interventricular septa, and smooth muscles and endothelia of the coronary arteries. Electron microscopy revealed SUR2A immunoreactivity in the cell membrane, endoplasmic reticulum (ER), and mitochondria. SUR2B immunoreactivity was mainly localized in the mitochondria as well as in the ER and cell membrane. Thus, SUR2A and SUR2B are not only the regulatory subunits of sarcolemmal K(ATP) channels but may also function as regulatory subunits in mitochondrial K(ATP) channels and play important roles in cardioprotection. PMID:17438353

  9. Functional contributions of synaptically localized NR2B subunits of the NMDA receptor to synaptic transmission and long-term potentiation in the adult mouse CNS

    PubMed Central

    Miwa, Hideki; Fukaya, Masahiro; Watabe, Ayako M; Watanabe, Masahiko; Manabe, Toshiya

    2008-01-01

    The NMDA-type glutamate receptor is a heteromeric complex composed of the NR1 and at least one of the NR2 subunits. Switching from the NR2B to the NR2A subunit is thought to underlie functional alteration of the NMDA receptor during synaptic maturation, and it is generally believed that it results in preferential localization of NR2A subunits on the synaptic site and that of NR2B subunits on the extracellular site in the mature brain. It has also been proposed that activation of the NR2A and NR2B subunits results in long-term potentiation (LTP) and long-term depression (LTD), respectively. Furthermore, recent reports suggest that synaptic and extrasynaptic receptors may have distinct roles in synaptic plasticity as well as in gene expression associated with neuronal death. Here, we have investigated whether NR2B subunit-containing receptors are present and functional at mature synapses in the lateral nucleus of the amygdala (LA) and the CA1 region of the hippocampus, comparing their properties between the two brain regions. We have found, in contrast to the above hypotheses, that the NR2B subunit significantly contributes to synaptic transmission as well as LTP induction. Furthermore, its contribution is greater in the LA than in the CA1 region, and biophysical properties of NMDA receptors and the NR2B/NR2A ratio are different between the two brain regions. These results indicate that NR2B subunit-containing NMDA receptors accumulate on the synaptic site and are responsible for the unique properties of synaptic function and plasticity in the amygdala. PMID:18372311

  10. Ribonucleotide reductase small subunit M2B prognoses better survival in colorectal cancer

    PubMed Central

    Liu, Xiyong; Lai, Lily; Wang, Xiaochen; Xue, Lijun; Leora, Sofia; Wu, Jun; Hu, Shuya; Zhang, Keqiang; Kuo, Mei-Ling; Zhou, Lun; Zhang, Hang; Wang, Yafan; Wang, Yan; Zhou, Bingsen; Nelson, Rebecca A; Zheng, Shu; Zhang, Suzhan; Chu, Peiguo; Yen, Yun

    2011-01-01

    Ribonucleotide reductase subunit RRM2B (p53R2) has been reported to suppress invasion and metastasis in colorectal cancer (CRC). Here we report that high levels of RRM2B expression is correlated with markedly better survival in CRC patients. In a fluorescence-labeled orthotopic mouse xenograft model, we confirmed that overexpression of RRM2B in non-metastatic CRC cells prevented lung and/or liver metastasis, relative to control cells that did metastasize. Clinical outcome studies were conducted on a training set with 103 CRCs and a validation set with 220 CRCs. All participants underwent surgery with periodic follow-up to determine survivability. A newly developed specific RRM2B antibody was employed to perform immunohistochemistry (IHC) for determining RRM2B expression levels on tissue arrays. In the training set, the Kaplan-Meier and multivariate COX analysis revealed that RRM2B is associated with better survival of CRCs, especially in stage IV patients (Hazard ratio, HR=0.40; 95% CI 0.18–0.86, p=0.016). In the validation set, RRM2B was negatively related to tumor invasion (odds ratio, OR=0.45, 95% CI 0.19–0.99, p=0.040) and lymph node involvement (OR=0.48, 95% CI 0.25–0.92, p=0.026). Further, elevated expression of RRM2B was associated with better prognosis in this set as determined by multivariate analyses (HR=0.48, 95% CI 0.26–0.91, p=0.030). Further investigations revealed that RRM2B was correlated with better survival of CRCs with advanced stage III–IV tumors rather than earlier stage I–II tumors. Taken together, our findings establish that RRM2B suppresses invasiveness of cancer cells and that its expression is associated with a better survival prognosis for CRC patients. PMID:21415168

  11. Archaeal translation initiation revisited: the initiation factor 2 and eukaryotic initiation factor 2B alpha-beta-delta subunit families

    NASA Technical Reports Server (NTRS)

    Kyrpides, N. C.; Woese, C. R.

    1998-01-01

    As the amount of available sequence data increases, it becomes apparent that our understanding of translation initiation is far from comprehensive and that prior conclusions concerning the origin of the process are wrong. Contrary to earlier conclusions, key elements of translation initiation originated at the Universal Ancestor stage, for homologous counterparts exist in all three primary taxa. Herein, we explore the evolutionary relationships among the components of bacterial initiation factor 2 (IF-2) and eukaryotic IF-2 (eIF-2)/eIF-2B, i.e., the initiation factors involved in introducing the initiator tRNA into the translation mechanism and performing the first step in the peptide chain elongation cycle. All Archaea appear to posses a fully functional eIF-2 molecule, but they lack the associated GTP recycling function, eIF-2B (a five-subunit molecule). Yet, the Archaea do posses members of the gene family defined by the (related) eIF-2B subunits alpha, beta, and delta, although these are not specifically related to any of the three eukaryotic subunits. Additional members of this family also occur in some (but by no means all) Bacteria and even in some eukaryotes. The functional significance of the other members of this family is unclear and requires experimental resolution. Similarly, the occurrence of bacterial IF-2-like molecules in all Archaea and in some eukaryotes further complicates the picture of translation initiation. Overall, these data lend further support to the suggestion that the rudiments of translation initiation were present at the Universal Ancestor stage.

  12. Association Study of N-Methyl-D-Aspartate Receptor Subunit 2B (GRIN2B) Polymorphisms and Schizophrenia Symptoms in the Han Chinese Population.

    PubMed

    Yang, Yongfeng; Li, Wenqiang; Zhang, Hongxing; Yang, Ge; Wang, Xiujuan; Ding, Minli; Jiang, Tianzi; Lv, Luxian

    2015-01-01

    Schizophrenia (SZ) is a common and complex psychiatric disorder that has a significant genetic component. The glutamatergic system is the major excitatory neurotransmitter system in the central nervous system, and is mediated by N-methyl-D-aspartate (NMDA) receptors. Disturbances in this system have been hypothesized to play a major role in SZ pathogenesis. Several studies have revealed that the NMDA receptor subunit 2B (GRIN2B) potentially associates with SZ and its psychiatric symptoms. In this study, we performed a case-control study to identify polymorphisms of the GRIN2B gene that may confer susceptibility to SZ in the Han Chinese population. Thirty-four single nucleotide polymorphisms (SNPs) were genotyped in 528 paranoid SZ patients and 528 control subjects. A significant association was observed in allele and genotype between SZ and controls at rs2098469 (χ2 = 8.425 and 4.994; p = 0.025 and 0.014, respectively). Significant associations were found in the allele at rs12319804 (χ2 = 4.436; p = 0.035), as well as in the genotype at rs12820037 and rs7298664 between SZ and controls (χ2 = 11.162 and 38.204; p = 0.003 and 4.27×10(-8), respectively). After applying the Bonferroni correction, rs7298664 still had significant genotype associations with SZ (p = 1.71×10(-7)). In addition, rs2098469 genotype and allele frequencies, and 12820037 allele frequencies were nominally associated with SZ. Three haplotypes, CGA (rs10845849-rs12319804-rs10845851), CC (rs12582848-rs7952915), and AAGAC (rs2041986-rs11055665-rs7314376-rs7297101-rs2098469), had significant differences between SZ and controls (χ2 = 4.324, 4.582, and 4.492; p = 0.037, 0.032, and 0.034, respectively). In addition, three SNPs, rs2098469, rs12820037, and rs7298664, were significantly associated with cognition factors PANSS subscores in SZ (F = 16.799, 7.112, and 13.357; p = 0.000, 0.017, and 0.000, respectively). In conclusion, our study provides novel evidence for an association between GRIN2B

  13. Association Study of N-Methyl-D-Aspartate Receptor Subunit 2B (GRIN2B) Polymorphisms and Schizophrenia Symptoms in the Han Chinese Population

    PubMed Central

    Zhang, Hongxing; Yang, Ge; Wang, Xiujuan; Ding, Minli; Jiang, Tianzi; Lv, Luxian

    2015-01-01

    Schizophrenia (SZ) is a common and complex psychiatric disorder that has a significant genetic component. The glutamatergic system is the major excitatory neurotransmitter system in the central nervous system, and is mediated by N-methyl-D-aspartate (NMDA) receptors. Disturbances in this system have been hypothesized to play a major role in SZ pathogenesis. Several studies have revealed that the NMDA receptor subunit 2B (GRIN2B) potentially associates with SZ and its psychiatric symptoms. In this study, we performed a case–control study to identify polymorphisms of the GRIN2B gene that may confer susceptibility to SZ in the Han Chinese population. Thirty-four single nucleotide polymorphisms (SNPs) were genotyped in 528 paranoid SZ patients and 528 control subjects. A significant association was observed in allele and genotype between SZ and controls at rs2098469 (χ2 = 8.425 and 4.994; p = 0.025 and 0.014, respectively). Significant associations were found in the allele at rs12319804 (χ2 = 4.436; p = 0.035), as well as in the genotype at rs12820037 and rs7298664 between SZ and controls (χ2 = 11.162 and 38.204; p = 0.003 and 4.27×10-8, respectively). After applying the Bonferroni correction, rs7298664 still had significant genotype associations with SZ (p = 1.71×10-7). In addition, rs2098469 genotype and allele frequencies, and 12820037 allele frequencies were nominally associated with SZ. Three haplotypes, CGA (rs10845849—rs12319804—rs10845851), CC (rs12582848—rs7952915), and AAGAC (rs2041986—rs11055665—rs7314376—rs7297101—rs2098469), had significant differences between SZ and controls (χ2 = 4.324, 4.582, and 4.492; p = 0.037, 0.032, and 0.034, respectively). In addition, three SNPs, rs2098469, rs12820037, and rs7298664, were significantly associated with cognition factors PANSS subscores in SZ (F = 16.799, 7.112, and 13.357; p = 0.000, 0.017, and 0.000, respectively). In conclusion, our study provides novel evidence for an association between

  14. Age- and Hormone-Regulation of N-Methyl-d-Aspartate Receptor Subunit NR2b in the Anteroventral Periventricular Nucleus of the Female Rat

    PubMed Central

    Maffucci, J. A.; Noel, M. L.; Gillette, R.; Wu, D.; Gore, A. C.

    2009-01-01

    Glutamate, acting through its N-methyl-d-aspartate (NMDA) and non-NMDA receptors in the hypothalamus, regulates reproductive neuroendocrine functions via direct and indirect actions upon gonadotrophin-releasing hormone (GnRH) neurones. Previous studies indicate that the NMDA receptor subunit NR2b undergoes changes in protein and gene expression in the hypothalamus in general, and on GnRH neurones in particular, during reproductive ageing. In the present study, we examined whether the NR2b-expressing cell population, both alone and in association with the NR1 subunit (i.e. the latter subunit is necessary for a functional NMDA receptor), is altered as a function of age and/or steroid hormone treatment. Studies focused on the anteroventral periventricular (AVPV) nucleus of the hypothalamus, a region critically involved in the control of reproduction. Young (3-5 months), middle-aged (9-12 months), and aged (approximately 22 months) female rats were ovariectomised and, 1 month later, they were treated sequentially with oestradiol plus progesterone, oestradiol plus vehicle, or vehicle plus vehicle, then perfused. Quantitative stereologic analysis of NR2b-immunoreactive cell numbers in the AVPV showed an age-associated decrease in the density of NR2b-immunoreactive cells, but no effect of hormone treatment. In a second study, immunofluorescent double labelling of NR2b and NR1 was analysed by confocal microscopy of fraction volume, a semi-quantitative measure of fluorescence intensity. No effect of ageing was detected for immunofluorescent NR1 or NR2b alone, whereas the NR2b fraction volume increased in the oestradiol plus vehicle group. With ageing, the fraction volume of the NR2b/NR1-colocalised subunits increased. Together with the stereology results, this suggests that, although fewer cells express the NR2b subunit in the ageing AVPV, a greater percentage of these subunits are co-expressed with NR1. Our results suggest that the subunit composition of NMDA receptors in

  15. Role for the NR2B Subunit of the NMDA Receptor in Mediating Light Input to the Circadian System

    PubMed Central

    Wang, LM; Schroeder, A; Loh, D; Smith, D; Lin, K; Han, JH; Michel, S; Hummer, DL; Ehlen, JC; Albers, HE; Colwell, CS

    2008-01-01

    Light information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinal ganglion cells that utilize glutamate as a neurotransmitter. A variety of evidence suggests that the release of glutamate then activates N-methyl-Daspartate (NMDA) receptors within the SCN and triggers a signaling cascade that ultimately leads to phase shifts in the circadian system. In this study, we first sought to explore the role of the NR2B subunit in mediating the effects of light on the circadian system. We found that localized microinjection of the NR2B subunit antagonist ifenprodil into the SCN region inhibits the magnitude of light-induced phase shifts of the circadian rhythm in wheel-running activity. Next, we found that the NR2B message and levels of phospho-NR2B levels vary with time of day in SCN tissue using semi-quantitative real-time PCR and Western blot analysis, respectively. Functionally, we found that blocking the NR2B subunit with ifenprodil significantly reduced the magnitude of NMDA currents recorded in SCN neurons. Ifenprodil also significantly reduced the magnitude of NMDA-induced calcium changes in SCN cells. Together, these results demonstrate that the NR2B subunit is an important component of NMDA receptor mediated responses within SCN neurons and that this subunit contributes to light-induced phase shifts of the mammalian circadian system. PMID:18380671

  16. Turning off of GluN2B subunits and turning on of CICR in hippocampal LTD induction after developmental GluN2 subunit switch.

    PubMed

    Yasuda, Hiroki; Mukai, Hideyuki

    2015-11-01

    NMDA receptors (NMDARs) are essential for the induction of synaptic plasticity that mediates activity-dependent refinement of neural circuits during development. GluN2B subunits of NMDARs are abundant at synapses in the immature hippocampus and begin to be replaced by GluN2A subunits with the help of casein kinase 2 activity in the second postnatal week, the critical period for the GluN2 subunit switch (Sanz-Clemente et al. (2000) Neuron 67:984-996). However, the physiological role of GluN2B subunits in the hippocampus during this critical period has not been elucidated. Here, we report that GluN2B subunits mediate the induction of long-term depression (LTD) in the CA1 region of the hippocampus only until this period. Ifenprodil and Ro25-6981, selective inhibitors of NMDARs containing GluN2B subunits, blocked LTD in postnatal Day 11-14 (P11-14) rat hippocampal slices but not in P18-22 hippocampus. Just a few days after P14, synaptic NMDAR currents became narrower than those at P11-14, and calcium influx through NMDARs must be reduced. We found that calcium-induced calcium release (CICR) through ryanodine receptors starts to support the induction of NMDAR-dependent LTD at P18-22. Intracellular application of thapsigargin and ryanodine, inhibitors of Ca2+ -ATP pumps on internal stores and ryanodine receptors, respectively, did not at all affect LTD in the hippocampus at P11-14 but completely blocked LTD in the P18-22 hippocampus. Therefore, calcium influx through NMDAR with GluN2B subunits is sufficient to induce LTD at P11-14, after which CICR compensates for the decrease in calcium influx during LTD induction. PMID:25727316

  17. Co-activation of NR2A and NR2B subunits induces resistance to fear extinction.

    PubMed

    Leaderbrand, Katherine; Corcoran, Kevin A; Radulovic, Jelena

    2014-09-01

    Unpredictable stress is known to profoundly enhance susceptibility to fear and anxiety while reducing the ability to extinguish fear when threat is no longer present. Accordingly, partial aversive reinforcement, via random exposure to footshocks, induces fear that is resistant to extinction. Here we sought to determine the hippocampal mechanisms underlying susceptibility versus resistance to context fear extinction as a result of continuous (CR) and partial (PR) reinforcement, respectively. We focused on N-methyl-D-aspartate receptor (NMDAR) subunits 2A and B (NR2A and NR2B) as well as their downstream signaling effector, extracellular signal-regulated kinase (ERK), based on their critical role in the acquisition and extinction of fear. Pharmacological inactivation of NR2A, but not NR2B, blocked extinction after CR, whereas inactivation of NR2A, NR2B, or both subunits facilitated extinction after PR. The latter finding suggests that co-activation of NR2A and NR2B contributes to persistent fear following PR. In contrast to CR, PR increased membrane levels of ERK and NR2 subunits after the conditioning and extinction sessions, respectively. In parallel, nuclear activation of ERK was significantly reduced after the extinction session. Thus, co-activation and increased surface expression of NR2A and NR2B, possibly mediated by ERK, may cause persistent fear. These findings suggest that patients with post-traumatic stress disorder (PTSD) may benefit from antagonism of specific NR2 subunits. PMID:24055686

  18. GluN2A and GluN2B subunit-containing NMDA receptors in hippocampal plasticity

    PubMed Central

    Shipton, Olivia A.; Paulsen, Ole

    2014-01-01

    N-Methyl-d-aspartate receptor (NMDAR)-dependent synaptic plasticity is a strong candidate to mediate learning and memory processes that require the hippocampus. This plasticity is bidirectional, and how the same receptor can mediate opposite changes in synaptic weights remains a conundrum. It has been suggested that the NMDAR subunit composition could be involved. Specifically, one subunit composition of NMDARs would be responsible for the induction of long-term potentiation (LTP), whereas NMDARs with a different subunit composition would be engaged in the induction of long-term depression (LTD). Unfortunately, the results from studies that have investigated this hypothesis are contradictory, particularly in relation to LTD. Nevertheless, current evidence does suggest that the GluN2B subunit might be particularly important for plasticity and may make a synapse bidirectionally malleable. In particular, we conclude that the presence of GluN2B subunit-containing NMDARs at the postsynaptic density might be a necessary, though not a sufficient, condition for the strengthening of individual synapses. This is owing to the interaction of GluN2B with calcium/calmodulin-dependent protein kinase II (CaMKII) and is distinct from its contribution as an ion channel. PMID:24298164

  19. MEF2B mutations in non-Hodgkin lymphoma dysregulate cell migration by decreasing MEF2B target gene activation

    PubMed Central

    Pon, Julia R.; Wong, Jackson; Saberi, Saeed; Alder, Olivia; Moksa, Michelle; Grace Cheng, S. -W.; Morin, Gregg B.; Hoodless, Pamela A.; Hirst, Martin; Marra, Marco A.

    2015-01-01

    Myocyte enhancer factor 2B (MEF2B) is a transcription factor with mutation hotspots at K4, Y69 and D83 in diffuse large B-cell lymphoma (DLBCL). To provide insight into the regulatory network of MEF2B, in this study, we analyse global gene expression and DNA-binding patterns. We find that candidate MEF2B direct target genes include RHOB, RHOD, CDH13, ITGA5 and CAV1, and that indirect target genes of MEF2B include MYC, TGFB1, CARD11, MEF2C, NDRG1 and FN1. MEF2B overexpression increases HEK293A cell migration and epithelial–mesenchymal transition, and decreases DLBCL cell chemotaxis. K4E, Y69H and D83V MEF2B mutations decrease the capacity of MEF2B to activate transcription and decrease its' effects on cell migration. The K4E and D83V mutations decrease MEF2B DNA binding. In conclusion, our map of the MEF2B regulome connects MEF2B to drivers of oncogenesis. PMID:26245647

  20. MEF2B mutations in non-Hodgkin lymphoma dysregulate cell migration by decreasing MEF2B target gene activation.

    PubMed

    Pon, Julia R; Wong, Jackson; Saberi, Saeed; Alder, Olivia; Moksa, Michelle; Grace Cheng, S-W; Morin, Gregg B; Hoodless, Pamela A; Hirst, Martin; Marra, Marco A

    2015-01-01

    Myocyte enhancer factor 2B (MEF2B) is a transcription factor with mutation hotspots at K4, Y69 and D83 in diffuse large B-cell lymphoma (DLBCL). To provide insight into the regulatory network of MEF2B, in this study, we analyse global gene expression and DNA-binding patterns. We find that candidate MEF2B direct target genes include RHOB, RHOD, CDH13, ITGA5 and CAV1, and that indirect target genes of MEF2B include MYC, TGFB1, CARD11, MEF2C, NDRG1 and FN1. MEF2B overexpression increases HEK293A cell migration and epithelial-mesenchymal transition, and decreases DLBCL cell chemotaxis. K4E, Y69H and D83V MEF2B mutations decrease the capacity of MEF2B to activate transcription and decrease its' effects on cell migration. The K4E and D83V mutations decrease MEF2B DNA binding. In conclusion, our map of the MEF2B regulome connects MEF2B to drivers of oncogenesis. PMID:26245647

  1. Effects of diazoxide on Aβ1-42-induced expression of the NR2B subunit in cultured cholinergic neurons.

    PubMed

    Zhu, Jin; Fu, Qingxi; Xia, Chunfeng; Ma, Guozhao

    2015-12-01

    The accumulation of amyloid-β protein (Aβ) is significant in the pathogenesis of Alzheimer's disease. Several previous studies indicate that the NR2B‑containing N‑methyl‑D‑aspartate receptors are critically involved in the Aβ mediated disruption of neuronal function. Diazoxide (DZ), a highly selective drug capable of opening mitochondrial ATP‑sensitive potassium channels, has neuroprotective effects against neuronal cell death. However, the mechanism by which DZ protects cholinergic neurons against Aβ‑induced cytotoxicity remains to be elucidated. The present study was designed to investigate the effects of DZ pretreatment against Aβ1‑42‑induced expression of NR2B in order to gain novel insights into the neuroprotective mechanisms. Following exposure to Aβ1‑42 for 24 h, the expression of the NR2B subunit remained unchanged compared with the control group. However, a significant increase in the expression of the NR2B subunit was observed following treatment with Aβ1‑42 for 72 h (P<0.05); and the upregulation of the expression of the NR2B subunit was reversed by pretreatment with DZ (P<0.05). These results suggested that DZ may counteract Aβ1‑42‑mediated cytotoxicity by alleviating the expression of NR2B. PMID:26496862

  2. NR2B subunit in the prefrontal cortex: A double-edged sword for working memory function and psychiatric disorders.

    PubMed

    Monaco, Sarah A; Gulchina, Yelena; Gao, Wen-Jun

    2015-09-01

    The prefrontal cortex (PFC) is a brain region featured with working memory function. The exact mechanism of how working memory operates within the PFC circuitry is unknown, but persistent neuronal firing recorded from prefrontal neurons during a working memory task is proposed to be the neural correlate of this mnemonic encoding. The PFC appears to be specialized for sustaining persistent firing, with N-methyl-D-aspartate (NMDA) receptors, especially slow-decay NR2B subunits, playing an essential role in the maintenance of sustained activity and normal working memory function. However, the NR2B subunit serves as a double-edged sword for PFC function. Because of its slow kinetics, NR2B endows the PFC with not only "neural psychic" properties, but also susceptibilities for neuroexcitotoxicity and psychiatric disorders. This review aims to clarify the interplay among working memory, the PFC, and NMDA receptors; demonstrate the importance of NR2B in the maintenance of persistent activity; understand the risks and vulnerabilities of how NR2B is related to the development of neuropsychiatric disorders; identify gaps that currently exist in our understanding of these processes; and provide insights regarding future directions that may clarify these issues. We conclude that the PFC is a specialized brain region with distinct delayed maturation, unique neuronal circuitry, and characteristic NMDA receptor function. The unique properties and development of NMDA receptors, especially enrichment of NR2B subunits, endow the PFC with not only the capability to generate sustained activity for working memory, but also serves as a major vulnerability to environmental insults and risk factors for psychiatric disorders. PMID:26143512

  3. The mediator complex subunit Med10 regulates heart valve formation in zebrafish by controlling Tbx2b-mediated Has2 expression and cardiac jelly formation.

    PubMed

    Just, Steffen; Hirth, Sofia; Berger, Ina M; Fishman, Mark C; Rottbauer, Wolfgang

    2016-09-01

    In search for novel key regulators of cardiac valve formation, we isolated the zebrafish cardiac valve mutant ping pong (png). We find that an insertional promoter mutation within the zebrafish mediator complex subunit 10 (med10) gene is leading to impaired heart valve formation. Expression of the T-box transcription factor 2b (Tbx2b), known to be essential in cardiac valve development, is severely reduced in png mutant hearts. We demonstrate here that transient reconstitution of Tbx2b expression rescues AV canal development in png mutant zebrafish. By contrast, overexpression of Forkhead box N4 (Foxn4), a known upstream regulator of Tbx2b, is not capable to reconstitute tbx2b expression and heart valve formation in Med10-deficient png mutant hearts. Interestingly, hyaluronan synthase 2 (has2), a known downstream target of Tbx2 and producer of hyaluronan (HA) - a major ECM component of the cardiac jelly and critical for proper heart valve development - is completely absent in ping pong mutant hearts. We propose here a rather unique role of Med10 in orchestrating cardiac valve formation by mediating Foxn4 dependent tbx2b transcription, expression of Has2 and subsequently proper development of the cardiac jelly. PMID:27343557

  4. Dual contribution of NR2B subunit of NMDA receptor and SK3 Ca(2+)-activated K+ channel to genetic predisposition to anorexia nervosa.

    PubMed

    Koronyo-Hamaoui, Maya; Frisch, Amos; Stein, Daniel; Denziger, Yardena; Leor, Shani; Michaelovsky, Elena; Laufer, Neil; Carel, Cynthia; Fennig, Silvana; Mimouni, Mark; Ram, Anca; Zubery, Eynat; Jeczmien, Pablo; Apter, Alan; Weizman, Abraham; Gak, Eva

    2007-01-01

    Since identification of the genetic component in anorexia nervosa (AN), genes that partake in serotonergic and dopaminergic systems and in hormonal and weight regulation have been suggested as potential candidates for AN susceptibility. We propose another set of candidate genes. Those are genes that are involved in the signaling pathway using NMDA-R and SK channels and have been suggested as possible effectors of NMDA-R driven signaling. The role of NMDA-R in the etiology of schizophrenia has already been substantiated on various levels. Several studies based on population and family groups have implicated SK3 in schizophrenia and more recently in AN as well. Our study group consisted of 90 AN family trios. We examined the transmission of two potentially functional polymorphisms, 5073T>G polymorphism in the gene encoding the NR2B subunit of NMDA-R and CAG repeats in the coding region of SK3 channel gene. Using HHRR and TDT approaches, we found that both polymorphisms were preferentially transmitted to AN offspring (TDT yielded chi(2)=5.01, p=0.025 for NR2B 5073G alleles and chi(2)=11.75, p<0.001 for SK3 L alleles including >19 repeats). Distribution analysis of the combined NR2B/SK3 genotypes suggests that the contribution of both polymorphisms to AN risk is independent and cumulative (OR=2.44 for NR2B GG genotype and OR=3.01 for SK3 SL and LL genotypes, and OR=6.8 for the combined NR2B/SK3 genotypes including high-risk alleles). These findings point to the contribution of genes associated with the NMDA-R signaling pathway to predisposition and development of AN. PMID:16157352

  5. NMDA receptor subunits and associated signaling molecules mediating antidepressant-related effects of NMDA-GluN2B antagonism

    PubMed Central

    Kiselycznyk, Carly; Jury, Nicholas; Halladay, Lindsay; Nakazawa, Kazu; Mishina, Masayoshi; Sprengel, Rolf; Grant, Seth G.N.; Svenningsson, Per; Holmes, Andrew

    2015-01-01

    Drugs targeting the glutamate N-methyl-D-aspartate receptor (NMDAR) may be efficacious for treating mood disorders, as exemplified by the rapid antidepressant effects produced by single administration of the NMDAR antagonist ketamine. Though the precise mechanisms underlying the antidepressant-related effects of NMDAR antagonism remain unclear, recent studies implicate specific NMDAR subunits, including GluN2A and GluN2B, as well as the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunit glutamate receptor interacting molecule, PSD-95. Here, integrating mutant and pharmacological in mice, we investigated the contribution of these subunits and molecules to antidepressant-related behaviors and the antidepressant-related effects of the GluN2B blocker, Ro 25-6981. We found that global deletion of GluA1 or PSD-95 reduced forced swim test (FST) immobility, mimicking the antidepressant-related effect produced by systemically administered Ro 25-6981 in C57BL/6J mice. Moreover, the FST antidepressant-like effects of systemic Ro 25-6981 were intact in mutants with global GluA1 deletion or GluN1 deletion in forebrain interneurons, but were absent in mutants constitutively lacking GluN2A or PSD-95. Next, we found that microinfusing Ro 25-6981 into the medial prefrontal cortex (mPFC), but not basolateral amygdala, of C57BL/6J mice was sufficient to produce an antidepressant-like effect. Together, these findings extend and refine current understanding of the mechanisms mediating antidepressant-like effects produced by NMDAR-GluN2B antagonists, and may inform the development of a novel class of medications for treating depression that target the GluN2B subtype of NMDAR. PMID:25800971

  6. Regulation of Postsynaptic Function by the Dementia-Related ESCRT-III Subunit CHMP2B

    PubMed Central

    Chassefeyre, Romain; Martínez-Hernández, José; Bertaso, Federica; Bouquier, Nathalie; Blot, Béatrice; Laporte, Marine; Fraboulet, Sandrine; Couté, Yohann; Devoy, Anny; Isaacs, Adrian M.; Pernet-Gallay, Karin; Fagni, Laurent

    2015-01-01

    The charged multivesicular body proteins (Chmp1–7) are an evolutionarily conserved family of cytosolic proteins that transiently assembles into helical polymers that change the curvature of cellular membrane domains. Mutations in human CHMP2B cause frontotemporal dementia, suggesting that this protein may normally control some neuron-specific process. Here, we examined the function, localization, and interactions of neuronal Chmp2b. The protein was highly expressed in mouse brain and could be readily detected in neuronal dendrites and spines. Depletion of endogenous Chmp2b reduced dendritic branching of cultured hippocampal neurons, decreased excitatory synapse density in vitro and in vivo, and abolished activity-induced spine enlargement and synaptic potentiation. To understand the synaptic effects of Chmp2b, we determined its ultrastructural distribution by quantitative immuno-electron microscopy and its biochemical interactions by coimmunoprecipitation and mass spectrometry. In the hippocampus in situ, a subset of neuronal Chmp2b was shown to concentrate beneath the perisynaptic membrane of dendritic spines. In synaptoneurosome lysates, Chmp2b was stably bound to a large complex containing other members of the Chmp family, as well as postsynaptic scaffolds. The supramolecular Chmp assembly detected here corresponds to a stable form of the endosomal sorting complex required for transport-III (ESCRT-III), a ubiquitous cytoplasmic protein complex known to play a central role in remodeling of lipid membranes. We conclude that Chmp2b-containing ESCRT-III complexes are also present at dendritic spines, where they regulate synaptic plasticity. We propose that synaptic ESCRT-III filaments may function as a novel element of the submembrane cytoskeleton of spines. PMID:25698751

  7. GluN2B subunit-containing NMDA receptor antagonists prevent Abeta-mediated synaptic plasticity disruption in vivo.

    PubMed

    Hu, Neng-Wei; Klyubin, Igor; Anwyl, Roger; Anwy, Roger; Rowan, Michael J

    2009-12-01

    Currently, treatment with the relatively low-affinity NMDA receptor antagonist memantine provides limited benefit in Alzheimer's disease (AD). One probable dose-limiting factor in the use of memantine is the inhibition of NMDA receptor-dependent synaptic plasticity mechanisms believed to underlie certain forms of memory. Moreover, amyloid-beta protein (Abeta) oligomers that are implicated in causing the cognitive deficits of AD potently inhibit this form of plasticity. Here we examined if subtype-preferring NMDA receptor antagonists could preferentially protect against the inhibition of NMDA receptor-dependent plasticity of excitatory synaptic transmission by Abeta in the hippocampus in vivo. Using doses that did not affect control plasticity, antagonists selective for NMDA receptors containing GluN2B but not other GluN2 subunits prevented Abeta(1-42) -mediated inhibition of plasticity. Evidence that the proinflammatory cytokine TNFalpha mediates this deleterious action of Ass was provided by the ability of TNFalpha antagonists to prevent Abeta(1-42) inhibition of plasticity and the abrogation of a similar disruptive effect of TNFalpha using a GluN2B-selective antagonist. Moreover, at nearby synapses that were resistant to the inhibitory effect of TNFalpha, Abeta(1-42) did not significantly affect plasticity. These findings suggest that preferentially targeting GluN2B subunit-containing NMDARs may provide an effective means of preventing cognitive deficits in early Alzheimer's disease. PMID:19918059

  8. Mutations of the human interferon alpha-2b (hIFNα-2b) gene in cancer patients receiving radiotherapy

    PubMed Central

    Shahid, Saman; Chaudhry, Muhammad Nawaz; Mahmood, Nasir

    2015-01-01

    This research aimed to find out the impact of ionizing radiations on the hIFNα-2b gene of radiotherapy treated cancer patients. The gene hIFNα-2b synthesizes a protein which is an important anticancerous and antiviral protein. The cancer patients (breast, lung, thyroid, oral and prostate) who were undergoing a radiotherapy treatment were selected. A molecular analysis was performed for DNA isolation and gene amplification through PCR, to identify gene mutations. Further, by bioinformatics tools we concluded that how mutations identified in gene sequences have led to the alterations in the hINFα-2b protein in radiotherapy receiving cancer patients. The 32% mutations in the hINFα-2b gene were identified and all were frameshift mutations. Radiotherapy can impact the immune system and cancer patients may modulate their immunity. Understaning the mechanisms of radiotherapy-elicited immune response may be helpful in the development of those therapeutic interventions that can enhance the efficacy of radiotherapy. PMID:26396921

  9. Acute hypoxia differentially affects the NMDA receptor NR1, NR2A and NR2B subunit mRNA levels in the developing chick optic tectum: stage-dependent plasticity in the 2B-2A ratio.

    PubMed

    Vacotto, Marina; Rapacioli, Melina; Flores, Vladimir; de Plazas, Sara Fiszer

    2010-10-01

    It is known that the NMDA-R NR1 subunit is needed for the receptor activity and that under hypoxia the evolution toward apoptosis or neuronal survival depends on the balance NR2A/NR2B subunits. This paper analyzes the effect of acute hypoxia on the above mentioned subunits mRNAs during development. The mean percentage of NR1+ neurons displayed the higher plasticity during development while the NR2A+ neurons the higher stability. Acute hypoxia increased the mean percentage of NR1+ and NR2B+ neurons at ED12 but only that of NR1+ neurons at ED18. Acute hypoxia increased the levels of expression of NR1 and NR2B mRNAs at ED12 without changes in the NR2A mRNA. During early stages there is a higher sensitivity to change the subunits mRNA levels under a hypoxic treatment. At ED12 acute hypoxia increased the probability of co-expression of the NR1-NR2A and NR1-NR2B subunits combinations, the level of NR1 and NR2B and the ratio NR2B/NR2A. These conditions facilitate the evolution towards apoptosis. PMID:20596770

  10. SNARE Protein Syntaxin-1 Colocalizes Closely with NMDA Receptor Subunit NR2B in Postsynaptic Spines in the Hippocampus

    PubMed Central

    Hussain, Suleman; Ringsevjen, Håvard; Egbenya, Daniel L.; Skjervold, Torstein L.; Davanger, Svend

    2016-01-01

    Syntaxins are a family of membrane-integrated proteins that are instrumental in exocytosis of vesicles. Syntaxin-1 is an essential component of the presynaptic exocytotic fusion machinery in the brain and interacts with several other proteins. Syntaxin-1 forms a four-helical bundle complex with proteins SNAP-25 and VAMP2 that drives fusion of vesicles with the plasma membrane in the active zone (AZ). Little is known, however, about the ultrastructural localization of syntaxin-1 at the synapse. We have analyzed the intrasynaptic expression of syntaxin-1 in glutamatergic hippocampal synapses in detail by using quantitative postembedding immunogold labeling. Syntaxin-1 was present in highest concentrations at the presynaptic AZ, supporting its role in transmitter release. Presynaptic plasma membrane lateral to the AZ, as well as presynaptic cytoplasmic (PreCy) vesicles were also labeled. However, syntaxin-1 was also significantly expressed in postsynaptic spines, where it was localized at the postsynaptic density (PSD), at postsynaptic lateral membranes and in postsynaptic cytoplasm. Postsynaptically, syntaxin-1 colocalized in the nanometer range with the N-methyl-D-aspartate (NMDA) receptor subunit NR2B, but only weakly with the AMPA receptor subunits GluA2/3. This observation points to the possibility that syntaxin-1 may be involved with NR2B vesicular trafficking from cytoplasmic stores to the postsynaptic plasma membrane, thus facilitating synaptic plasticity. Confocal immunofluorescence double labeling with PSD-95 and ultrastructural fractionation of synaptosomes also confirm localization of syntaxin-1 at the PSD. PMID:26903802

  11. Phosphorylation of NR2B NMDA subunits by protein kinase C in arcuate nucleus contributes to inflammatory pain in rats

    PubMed Central

    Bu, Fan; Tian, Huiyu; Gong, Shan; Zhu, Qi; Xu, Guang-Yin; Tao, Jin; Jiang, Xinghong

    2015-01-01

    The arcuate nucleus (ARC) of the hypothalamus plays a key role in pain processing. Although it is well known that inhibition of NMDA receptor (NMDAR) in ARC attenuates hyperalgesia induced by peripheral inflammation, the underlying mechanism of NMDAR activation in ARC remains unclear. Protein kinase C (PKC) is involved in several signalling cascades activated in physiological and pathological conditions. Therefore, we hypothesised that upregulation of PKC activates NMDARs in the ARC, thus contributing to inflammatory hyperalgesia. Intra-ARC injection of chelerythrine (CC), a specific PKC inhibitor, attenuated complete Freund’s adjuvant (CFA) induced thermal and mechanical hyperalgesia in a dose-dependent manner. In vivo extracellular recordings showed that microelectrophoresis of CC or MK-801 (a NMDAR antagonist) significantly reduced the enhancement of spontaneous discharges and pain-evoked discharges of ARC neurons. In addition, CFA injection greatly enhanced the expression of total and phosphorylated PKCγ in the ARC. Interestingly, CFA injection also remarkably elevated the level of phosphorylated NR2B (Tyr1472) without affecting the expression of total NR2B. Importantly, intra-ARC injection of CC reversed the upregulation of phosphorylated NR2B subunits in the ARC. Taken together, peripheral inflammation leads to an activation of NMDARs mediated by PKC activation in the ARC, thus producing thermal and mechanical hyperalgesia. PMID:26515544

  12. Copy Number Variation of UGT 2B Genes in Indian Families Using Whole Genome Scans

    PubMed Central

    Veerappa, Avinash M.; Padakannaya, Prakash; Ramachandra, Nallur B.

    2016-01-01

    Background and Objectives. Uridine diphospho-glucuronosyltransferase 2B (UGT2B) is a family of genes involved in metabolizing steroid hormones and several other xenobiotics. These UGT2B genes are highly polymorphic in nature and have distinct polymorphisms associated with specific regions around the globe. Copy number variations (CNVs) status of UGT2B17 in Indian population is not known and their disease associations have been inconclusive. It was therefore of interest to investigate the CNV profile of UGT2B genes. Methods. We investigated the presence of CNVs in UGT2B genes in 31 members from eight Indian families using Affymetrix Genome-Wide Human SNP Array 6.0 chip. Results. Our data revealed >50% of the study members carried CNVs in UGT2B genes, of which 76% showed deletion polymorphism. CNVs were observed more in UGT2B17 (76.4%) than in UGT2B15 (17.6%). Molecular network and pathway analysis found enrichment related to steroid metabolic process, carboxylesterase activity, and sequence specific DNA binding. Interpretation and Conclusion. We report the presence of UGT2B gene deletion and duplication polymorphisms in Indian families. Network analysis indicates the substitutive role of other possible genes in the UGT activity. The CNVs of UGT2B genes are very common in individuals indicating that the effect is neutral in causing any suspected diseases. PMID:27092269

  13. Structure of the Zinc-Bound Amino-Terminal Domain of the NMDA Receptor NR2B Subunit

    SciTech Connect

    Karakas, E.; Simorowski, N; Furukawa, H

    2009-01-01

    N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino-terminal domain (ATD) distinct from the L-glutamate-binding domain. The molecular basis for the ATD-mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc-free and zinc-bound states. The structures reveal the overall clamshell-like architecture distinct from the non-NMDA receptor ATDs and molecular determinants for the zinc-binding site, ion-binding sites, and the architecture of the putative phenylethanolamine-binding site.

  14. Tight Binding of the Phosphorylated α Subunit of Initiation Factor 2 (eIF2α) to the Regulatory Subunits of Guanine Nucleotide Exchange Factor eIF2B Is Required for Inhibition of Translation Initiation

    PubMed Central

    Krishnamoorthy, Thanuja; Pavitt, Graham D.; Zhang, Fan; Dever, Thomas E.; Hinnebusch, Alan G.

    2001-01-01

    Translation initiation factor 2 (eIF2) is a heterotrimeric protein that transfers methionyl-initiator tRNAMet to the small ribosomal subunit in a ternary complex with GTP. The eIF2 phosphorylated on serine 51 of its α subunit [eIF2(αP)] acts as competitive inhibitor of its guanine nucleotide exchange factor, eIF2B, impairing formation of the ternary complex and thereby inhibiting translation initiation. eIF2B is comprised of catalytic and regulatory subcomplexes harboring independent eIF2 binding sites; however, it was unknown whether the α subunit of eIF2 directly contacts any eIF2B subunits or whether this interaction is modulated by phosphorylation. We found that recombinant eIF2α (glutathione S-transferase [GST]–SUI2) bound to the eIF2B regulatory subcomplex in vitro, in a manner stimulated by Ser-51 phosphorylation. Genetic data suggest that this direct interaction also occurred in vivo, allowing overexpressed SUI2 to compete with eIF2(αP) holoprotein for binding to the eIF2B regulatory subcomplex. Mutations in SUI2 and in the eIF2B regulatory subunit GCD7 that eliminated inhibition of eIF2B by eIF2(αP) also impaired binding of phosphorylated GST-SUI2 to the eIF2B regulatory subunits. These findings provide strong evidence that tight binding of phosphorylated SUI2 to the eIF2B regulatory subcomplex is crucial for the inhibition of eIF2B and attendant downregulation of protein synthesis exerted by eIF2(αP). We propose that this regulatory interaction prevents association of the eIF2B catalytic subcomplex with the β and γ subunits of eIF2 in the manner required for GDP-GTP exchange. PMID:11438658

  15. Novel approach to probe subunit-specific contributions to N-methyl-D-aspartate (NMDA) receptor trafficking reveals a dominant role for NR2B in receptor recycling.

    PubMed

    Tang, Tina Tze-Tsang; Badger, John D; Roche, Paul A; Roche, Katherine W

    2010-07-01

    N-Methyl-d-aspartate (NMDA) receptors are expressed at excitatory synapses throughout the brain and are essential for neuronal development and synaptic plasticity. Functional NMDA receptors are tetramers, typically composed of NR1 and NR2 subunits (NR2A-D). NR2A and NR2B are expressed in the forebrain and are thought to assemble as diheteromers (NR1/NR2A, NR1/NR2B) and triheteromers (NR1/NR2A/NR2B). NR2A and NR2B contain cytosolic domains that regulate distinct postendocytic sorting events, with NR2A sorting predominantly into the degradation pathway, and NR2B preferentially trafficking through the recycling pathway. However, the interplay between these two subunits remains an open question. We have now developed a novel approach based on the dimeric feature of the alpha- and beta-chains of the human major histocompatibility complex class II molecule. We created chimeras of alpha- and beta-chains with the NR2A and NR2B C termini and evaluated endocytosis of dimers. Like chimeric proteins containing only a single NR2A or NR2B C-terminal domain, major histocompatibility complex class II-NR2A homodimers sort predominantly to late endosomes, whereas NR2B homodimers traffic to recycling endosomes. Interestingly, NR2A/NR2B heterodimers traffic preferentially through the recycling pathway, and NR2B is dominant in regulating dimer trafficking in both heterologous cells and neurons. In addition, the recycling of NR2B-containing NMDARs in wild-type neurons is not significantly different from NR2A(-/-) neurons. These data support a dominant role for NR2B in regulating the trafficking of triheteromeric NMDARs in vivo. Furthermore, our molecular approach allows for the direct and selective evaluation of dimeric assemblies and can be used to define dominant trafficking domains in other multisubunit protein complexes. PMID:20427279

  16. Multiple domains in the C-terminus of NMDA receptor GluN2B subunit contribute to neuronal death following in vitro ischemia.

    PubMed

    Vieira, Marta M; Schmidt, Jeannette; Ferreira, Joana S; She, Kevin; Oku, Shinichiro; Mele, Miranda; Santos, Armanda E; Duarte, Carlos B; Craig, Ann Marie; Carvalho, Ana Luísa

    2016-05-01

    Global cerebral ischemia induces selective degeneration of specific subsets of neurons throughout the brain, particularly in the hippocampus and cortex. One of the major hallmarks of cerebral ischemia is excitotoxicity, characterized by overactivation of glutamate receptors leading to intracellular Ca(2+) overload and ultimately neuronal demise. N-methyl-d-aspartate receptors (NMDARs) are considered to be largely responsible for excitotoxic injury due to their high Ca(2+) permeability. In the hippocampus and cortex, these receptors are most prominently composed of combinations of two GluN1 subunits and two GluN2A and/or GluN2B subunits. Due to the controversy regarding the differential role of GluN2A and GluN2B subunits in excitotoxic cell death, we investigated the role of GluN2B in the activation of pro-death signaling following an in vitro model of global ischemia, oxygen and glucose deprivation (OGD). For this purpose, we used GluN2B(-/-) mouse cortical cultures and observed that OGD-induced damage was reduced in these neurons, and partially prevented in wild-type rat neurons by a selective GluN2B antagonist. Notably, we found a crucial role of the C-terminal domain of the GluN2B subunit in triggering excitotoxic signaling. Indeed, expression of YFP-GluN2B C-terminus mutants for the binding sites to post-synaptic density protein 95 (PSD95), Ca(2+)-calmodulin kinase IIα (CaMKIIα) or clathrin adaptor protein 2 (AP2) failed to mediate neuronal death in OGD conditions. We focused on the GluN2B-CaMKIIα interaction and found a determinant role of this interaction in OGD-induced death. Inhibition or knock-down of CaMKIIα exerted a neuroprotective effect against OGD-induced death, whereas overexpression of this kinase had a detrimental effect. Importantly, in comparison with neurons overexpressing wild-type CaMKIIα, neurons overexpressing a mutant form of the kinase (CaMKII-I205K), unable to interact with GluN2B, were partially protected against OGD-induced damage

  17. Association between the NMDA glutamate receptor GRIN2B gene and obsessive–compulsive disorder

    PubMed Central

    Alonso, Pino; Gratacós, Mónica; Segalàs, Cinto; Escaramís, Georgia; Real, Eva; Bayés, Mónica; Labad, Javier; López-Solà, Clara; Estivill, Xavier; Menchón, José M.

    2012-01-01

    Background Recent data from neuroimaging, genetic and clinical trials and animal models suggest a role for altered glutamatergic neurotransmission in the pathogenesis of obsessive–compulsive disorder (OCD). The aim of this study was to investigate whether variants in the GRIN2B gene, the gene encoding the NR2 subunit of the N-methyl-d-aspartate (NMDA) glutamate receptor, may contribute to genetic susceptibility to OCD or to different OCD subphenotypes. Methods Between 2003 and 2008, we performed a case–control association study in which we genotyped 10 tag single-nucleotide polymorphisms (SNPs) in the 3′ untranslated region (3′ UTR) of GRIN2B. We performed SNP association and haplotype analysis considering the OCD diagnosis and different OCD subphenotypes: early-onset OCD, comorbid tic disorders and OCD clinical symptom dimensions. Results We enrolled 225 patients with OCD and 279 controls recruited from the OCD Clinic at Bellvitge Hospital (Barcelona, Spain). No significant difference in the distribution of alleles or genotypes was detected between patients with OCD and controls. Nonetheless, on analyzing OCD subphenotypes, the rs1805476 SNP in male patients (95% confidence interval [CI] 1.37–4.22, p = 0.002) and a 4-SNP haplotype in the whole sample (rs1805476, rs1805501, rs1805502 and rs1805477; odds ratio 1.92, 95% CI 1.22–3.01; permutation p = 0.023) were significantly associated with the presence of contamination obsessions and cleaning compulsions. Limitations Study limitations included the risk of population stratification associated with the case–control design, use of psychiatrically unscreened blood donors as the control group, reduced sample size of participants with certain OCD subphenotypes and tested polymorphisms limited to 3′ UTR and exon 13 of GRIN2B. Conclusion Our results converge with recent data suggesting a possible contribution of glutamatergic variants to the genetic vulnerability to OCD or at least to certain OCD

  18. Dual response of the KATP channels to staurosporine: a novel role of SUR2B, SUR1 and Kir6.2 subunits in the regulation of the atrophy in different skeletal muscle phenotypes.

    PubMed

    Mele, Antonietta; Camerino, Giulia M; Calzolaro, Sara; Cannone, Maria; Conte, Diana; Tricarico, Domenico

    2014-09-15

    We investigated on the role of the genes encoding for the ATP-sensitive K(+)-channel (KATP) subunits (SUR1-2A/B, Kir6.2) in the atrophy induced "in vitro" by staurosporine (STS) in different skeletal muscle phenotypes of mouse. Patch-clamp and gene expression experiments showed that the expression/activity of the sarcolemma KATP channel subunits was higher in the fast-twitch than in the slow-twitch fibers. After 1 to 3h of incubation time, the STS (2.14×10(-6)M) treatment enhanced the expression/activity of the SUR2B, SUR1 and Kir6.2 subunit genes, but not SUR2A, in the slow-twitch muscle fibers, induced the caspase-3-9, Atrogin-1 and Murf-1 gene expression without affecting protein content. After 3 to 6h, the STS-related atrophy markedly down-regulated the SUR2B, SUR1 and Kir6.2 genes reducing the KATP currents and reduced the protein content/muscle weight ratio of the slow-twitch muscle by -36.4±6% (p<0.05). After 6 to 24h, no additional changes of the SUR1-2B and Kir6.2 gene expression and muscle protein were observed. In the fast-twitch muscles, STS mildly affected the atrophic genes and protein content, but potentiated the KATP currents down-regulating the Bnip-3 gene. Diazoxide (250-500×10(-6)M), a SUR1-2B/Kir6.2 channel opener, prevented the protein loss induced by STS in the slow-twitch muscle after 6h showing an EC50 of 1.35×10(-7)M and Emax of 75%, down-regulated the caspase-9 gene and enhanced the KATP currents. The enhanced expression/activity of the SUR2B, SUR1 and Kir6.2 genes are cytoprotective against STS-induced atrophy in the slow-twitch muscle; their reduced expression/activity is associated with proteolysis and atrophy in skeletal muscle. PMID:24998494

  19. The selectivity of conantokin-G for ion channel inhibition of NR2B subunit-containing NMDA receptors is regulated by amino acid residues in the S2 region of NR2B.

    PubMed

    Sheng, Zhenyu; Liang, Zhong; Geiger, James H; Prorok, Mary; Castellino, Francis J

    2009-08-01

    The conantokins are short, naturally occurring peptides that inhibit ion flow through N-methyl-d-aspartate receptor (NMDAR) channels. One member of this peptide family, conantokin-G (con-G), shows high selectivity for antagonism of NR2B-containing NMDAR channels, whereas other known conantokins are less selective inhibitors with regard to the nature of the NR2 subunit of the NMDAR complex. In order to define the molecular determinants of NR2B that govern con-G selectivity, we evaluated the ability of con-G to inhibit NMDAR ion channels expressed in human embryonic kidney (HEK)293 cells transfected with NR1, in combination with various NR2A/2B chimeras and point mutants, by electrophysiology using cells voltage-clamped in the whole-cell configuration. We found that a variant of the con-G-insensitive subunit, NR2A, in which the 158 residues comprising the S2 peptide segment (E(657)-I(814)) were replaced by the corresponding S2 region of NR2B (E(658)-I(815)), results in receptors that are highly sensitive to inhibition by con-G. Of the 22 amino acids that are different between the NR2A-S2 and the NR2B-S2 regions, exchange of one of these, M(739) of NR2B for the equivalent K(738) of NR2A, was sufficient to completely import the inhibitory activity of con-G into NR1b/NR2A-containing NMDARs. Some reinforcement of this effect was found by substitution of a second amino acid, K(755) of NR2B for Y(754) of NR2A. The discovery of the molecular determinants of NR2B selectivity with con-G has implications for the design of subunit-selective neurobiological probes and drug therapies, in addition to advancing our understanding of NR2B- versus NR2A-mediated neurological processes. PMID:19427876

  20. Gene Expression Switching of Receptor Subunits in Human Brain Development

    PubMed Central

    Bar-Shira, Ossnat; Maor, Ronnie; Chechik, Gal

    2015-01-01

    Synaptic receptors in the human brain consist of multiple protein subunits, many of which have multiple variants, coded by different genes, and are differentially expressed across brain regions and developmental stages. The brain can tune the electrophysiological properties of synapses to regulate plasticity and information processing by switching from one protein variant to another. Such condition-dependent variant switch during development has been demonstrated in several neurotransmitter systems including NMDA and GABA. Here we systematically detect pairs of receptor-subunit variants that switch during the lifetime of the human brain by analyzing postmortem expression data collected in a population of donors at various ages and brain regions measured using microarray and RNA-seq. To further detect variant pairs that co-vary across subjects, we present a method to quantify age-corrected expression correlation in face of strong temporal trends. This is achieved by computing the correlations in the residual expression beyond a cubic-spline model of the population temporal trend, and can be seen as a nonlinear version of partial correlations. Using these methods, we detect multiple new pairs of context dependent variants. For instance, we find a switch from GLRA2 to GLRA3 that differs from the known switch in the rat. We also detect an early switch from HTR1A to HTR5A whose trends are negatively correlated and find that their age-corrected expression is strongly positively correlated. Finally, we observe that GRIN2B switch to GRIN2A occurs mostly during embryonic development, presumably earlier than observed in rodents. These results provide a systematic map of developmental switching in the neurotransmitter systems of the human brain. PMID:26636753

  1. Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation through activating the NR2B subunits of NMDA receptors

    SciTech Connect

    Shi, Wen-Zhu; Miao, Yu-Liang; Guo, Wen-Zhi; Wu, Wei; Li, Bao-Wei; An, Li-Na; Fang, Wei-Wu; Mi, Wei-Dong

    2014-04-25

    Highlights: • Leptin promotes the proliferation of neural stem cells isolated from embryonic mouse hippocampus. • Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation. • The effects of leptin are partially mediated by upregulating NR2B subunits. - Abstract: Corticosterone inhibits the proliferation of hippocampal neural stem cells (NSCs). The removal of corticosterone-induced inhibition of NSCs proliferation has been reported to contribute to neural regeneration. Leptin has been shown to regulate brain development, improve angiogenesis, and promote neural regeneration; however, its effects on corticosterone-induced inhibition of NSCs proliferation remain unclear. Here we reported that leptin significantly promoted the proliferation of hippocampal NSCs in a concentration-dependent pattern. Also, leptin efficiently reversed the inhibition of NSCs proliferation induced by corticosterone. Interestingly, pre-treatment with non-specific NMDA antagonist MK-801, specific NR2B antagonist Ro 25-6981, or small interfering RNA (siRNA) targeting NR2B, significantly blocked the effect of leptin on corticosterone-induced inhibition of NSCs proliferation. Furthermore, corticosterone significantly reduced the protein expression of NR2B, whereas pre-treatment with leptin greatly reversed the attenuation of NR2B expression caused by corticosterone in cultured hippocampal NSCs. Our findings demonstrate that leptin reverses the corticosterone-induced inhibition of NSCs proliferation. This process is, at least partially mediated by increased expression of NR2B subunits of NMDA receptors.

  2. Crystal Structure of the C-terminal Domain of the Subunit of Human Translation Initiation Factor eIF2B

    SciTech Connect

    Wei, J.; Jia, M; Zheng, C; Wang, M; Gao, F; Xu, H; Gong, W

    2010-01-01

    Eukaryotic translation initiation factor eIF2B, the guanine nucleotide exchange factor (GEF) for eIF2, catalyzes conversion of eIF2 {center_dot} GDP to eIF2 {center_dot} GTP. The eIF2B is composed of five subunits, {alpha}, {beta}, {gamma}, {delta} and {var_epsilon}, within which the {var_epsilon} subunit is responsible for catalyzing the guanine exchange reaction. Here we present the crystal structure of the C-terminal domain of human eIF2B{var_epsilon} (eIF2B{var_epsilon}-CTD) at 2.0-{angstrom} resolution. The structure resembles a HEAT motif and three charge-rich areas on its surface can be identified. When compared to yeast eIF2B{var_epsilon}-CTD, one area involves highly conserved AA boxes while the other two are only partially conserved. In addition, the previously reported mutations in human eIF2B{var_epsilon}-CTD, which are related to the loss of the GEF activity and human VWM disease, have been discussed. Based on the structure, most of such mutations tend to destabilize the HEAT motif.

  3. Reduced levels of NR2A and NR2B subunits of NMDA receptor and PSD-95 in the prefrontal cortex in major depression

    PubMed Central

    Feyissa, Anteneh M.; Zyga, Agata; Stockmeier, Craig A.; Karolewicz, Beata

    2009-01-01

    Recent neuroimaging and postmortem studies have demonstrated abnormalities in glutamatergic transmission in major depression. Glutamate NMDA (N-methyl-D-aspartate) receptors are one of the major mediators of excitatory neurotransmission in the central nervous system. At synaptic sites, NMDA receptors are linked with postsynaptic density protein-95 (PSD-95) that plays a key role in mediating trafficking, clustering, and downstream signaling events, following receptor activation. In this study, we examined the expression of NMDA receptor subunits NR1, NR2A, and NR2B as well as PSD-95 in the anterior prefrontal cortex (PFC) using Western blot method. Cortical samples were obtained from age, gender and postmortem interval matched depressed and psychiatrically healthy controls. The results revealed that there was a reduced expression of the NMDA receptor subunits NR2A (−54%) and NR2B (−48%), and PSD-95 protein level (−40%) in the PFC of depressed subjects relative to controls, with no change in the NR1 subunit. The alterations in NMDA receptor subunits, especially the NR2A and NR2B, as well as PSD-95 suggest an abnormality in the NMDA receptor signaling in the PFC in major depression. Our findings in conjunction with recent clinical, cellular, and neuroimaging studies further implicate the involvement of glutamate neurotransmission in the pathophysiology of depression. This study provides additional evidence that NMDA receptor complex is a target for discovery of novel antidepressants. PMID:18992785

  4. Propofol effectively inhibits lithium-pilocarpine- induced status epilepticus in rats via downregulation of N-methyl-D-aspartate receptor 2B subunit expression

    PubMed Central

    Wang, Henglin; Wang, Zhuoqiang; Mi, Weidong; Zhao, Cong; Liu, Yanqin; Wang, Yongan; Sun, Haipeng

    2012-01-01

    Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine. The inhibitory effects of propofol on status epilepticus in rats were judged based on observation of behavior, electroencephalography and 24-hour survival rate. Propofol (12.5–100 mg/kg) improved status epilepticus in a dose-dependent manner, and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection. Western blot results showed that, 24 hours after induction of status epilepticus, the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly increased in rat cerebral cortex and hippocampus. Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels, but not the increase in N-methyl-D-aspartate receptor 2A subunit levels. The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine. This effect may be associated with downregulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures. PMID:25737709

  5. Over-expression of the GluN2B subunit in the forebrain facilitates the acquisition of morphine-related positive and aversive memory in rats.

    PubMed

    Li, Yijing; Ping, Xingjie; Yu, Peng; Liang, Jing; Shen, Fang; Han, Jisheng; Cui, Cailian

    2016-09-15

    GluN2B-containing N-methyl-d-aspartate (NMDA) receptors in the brain are known to have an important role in drug-associated learning and memory. Selective blockage of GluN2B-containing NMDA receptors (GluN2B-NMDARs) has been shown to impair morphine-induced conditioned place preference (CPP) without affecting natural reward-induced CPP. In the present study, GluN2B transgenic rats with over-expressed GluN2B-subunits in the forebrain were used to assess the susceptibility to CPP induced by morphine and natural rewards as well as to naloxone-induced conditioned place aversion (CPA). The results showed that GluN2B transgenic rats exhibited a relatively higher susceptibility to morphine-induced CPP and naloxone-induced CPA than their wild-type littermates did, while they retained the similar sensitivity as wild-type rats to CPP induced by natural reinforcers (food and sucrose). These findings suggest that increased level of GluN2B-NMDARs in forebrain facilitates formation of drug-related memory, but not that associated with natural rewards. GluN2B-NMDARs might be a potential target for the treatment of drug abuse. PMID:27217103

  6. Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157:H7.

    PubMed

    Gomes, P A D P; Bentancor, L V; Paccez, J D; Sbrogio-Almeida, M E; Palermo, M S; Ferreira, R C C; Ferreira, L C S

    2009-04-01

    No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) produced by enterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains. PMID:24031368

  7. Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157:H7

    PubMed Central

    Gomes, P.A.D.P.; Bentancor, L.V.; Paccez, J.D.; Sbrogio-Almeida, M.E.; Palermo, M.S.; Ferreira, R.C.C.; Ferreira, L.C.S.

    2009-01-01

    No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) produced by enterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains. PMID:24031368

  8. Structure and expression of the gene (HNRPA2B1) encoding the human hnRNP protein A2/B1

    SciTech Connect

    Kozu, Tomoko; Henrich, B.; Schaefer, K.P.

    1995-01-20

    Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a major nuclear protein and one of the major components of the hnRNP core complex in mammalian cells. We first determined the complete sequence of the human gene for hnRNP protein A2 (HNRPA2B1). The human HNRPA2B1 gene exists in a single copy over 9 kb in length. The gene was split into 12 exons, including a 36-nucleotide mini-exon, which was specific to the hnRNP protein B1, providing genetic evidence that the B1 mRNA was generated from the primary HNRPA2B1 transcript by alternative splicing. The 5{prime} region of HNRPA2B1 was GC-rich and contained several DNA motifs for the binding of several transcription factors, which included 2 CCAAT boxes and no TATA sequences. The 5{prime} ends of the mRNA were mapped to multiple positions. These structural features are characteristic of promoter regions of housekeeping genes. Northern blot and RT-PCR analyses of the HNRPA2B1 transcripts revealed levels of B1 mRNA from 2 to 5% of total A2/B1 transcripts and showed that both A2 and B1 mRNAs were transcribed in all human cell lines and mouse tissues studied. The structural and evolutionary characteristics of the A2 and A1 proteins as they relate to each other are discussed. 38 refs., 5 figs.

  9. Bisphenol-A rapidly promotes dynamic changes in hippocampal dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDA receptor subunit NR2B

    SciTech Connect

    Xu Xiaohong Ye Yinping; Li Tao; Chen Lei; Tian Dong; Luo Qingqing; Lu Mei

    2010-12-01

    Bisphenol-A (BPA) is known to be a potent endocrine disrupter. Evidence is emerging that estrogen exerts a rapid influence on hippocampal synaptic plasticity and the dendritic spine density, which requires activation of NMDA receptors. In the present study, we investigated the effects of BPA (ranging from 1 to 1000 nM), focusing on the rapid dynamic changes in dendritic filopodia and the expressions of estrogen receptor (ER) {beta} and NMDA receptor, as well as the phosphorylation of NMDA receptor subunit NR2B in the cultured hippocampal neurons. A specific ER antagonist ICI 182,780 was used to examine the potential involvement of ERs. The results demonstrated that exposure to BPA (ranging from 10 to 1000 nM) for 30 min rapidly enhanced the motility and the density of dendritic filopodia in the cultured hippocampal neurons, as well as the phosphorylation of NR2B (pNR2B), though the expressions of NMDA receptor subunits NR1, NR2B, and ER{beta} were not changed. The antagonist of ERs completely inhibited the BPA-induced increases in the filopodial motility and the number of filopodia extending from dendrites. The increased pNR2B induced by BPA (100 nM) was also completely eliminated. Furthermore, BPA attenuated the effects of 17{beta}-estradiol (17{beta}-E{sub 2}) on the dendritic filopodia outgrowth and the expression of pNR2B when BPA was co-treated with 17{beta}-E{sub 2}. The present results suggest that BPA, like 17{beta}-E{sub 2}, rapidly results in the enhanced motility and density of dendritic filopodia in the cultured hippocampal neurons with the concomitant activation of NMDA receptor subunit NR2B via an ER-mediated signaling pathway. Meanwhile, BPA suppressed the enhancement effects of 17{beta}-E{sub 2} when it coexists with 17{beta}-E{sub 2}. These results provided important evidence suggesting the neurotoxicity of the low levels of BPA during the early postnatal development of the brain.

  10. Cloning and chromosomal localization of the human A{sub 2b} adenosine receptor gene (ADORA2B) and its pseudogene

    SciTech Connect

    Jacobson, M.A.; Johnson, R.G.; Luneau, C.J.

    1995-05-20

    To determine the chromosomal localization of the human A{sub 2b} adenosine receptor, the corresponding genomic clone was isolated and used as a probe for fluorescence in situ hybridization to metaphase chromosomes. Partial sequence analysis of the A{sub 2b} gene (ADORA2B) revealed an intron that interrupted the coding region corresponding to the second intracellular loop similar to that reported for A{sub 1} and A{sub 2a} adenosine receptor genes. A pseudogene for the A{sub 2b} receptor was also identified; it exhibited 79% identity to the A{sub 2b} adenosine receptor cDNA coding sequence and contained multiple deletions, point mutations, and frame shifts and two in-frame stops. These changes would result in the inability to encode a functional receptor. The genomic clones were utilized to localize the A{sub 2b} receptor to chromosome 17p12 and the A{sub 2b} pseudogene to chromosome 1q32. 15 refs., 1 fig.

  11. Protection of Mice against Shiga Toxin 2 (Stx2)-Associated Damage by Maternal Immunization with a Brucella Lumazine Synthase-Stx2 B Subunit Chimera

    PubMed Central

    Mejias, María Pilar; Cabrera, Gabriel; Fernández-Brando, Romina Jimena; Baschkier, Ariela; Ghersi, Giselle; Abrey-Recalde, Maria Jimena; Miliwebsky, Elizabeth; Meiss, Roberto; Goldbaum, Fernando; Zylberman, Vanesa; Rivas, Marta

    2014-01-01

    Hemolytic-uremic syndrome (HUS) is defined as the triad of anemia, thrombocytopenia, and acute kidney injury. Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC), which causes a prodromal hemorrhagic enteritis, remains the most common etiology of the typical or epidemic form of HUS. Because no licensed vaccine or effective therapy is presently available for human use, we recently developed a novel immunogen based on the B subunit of Shiga toxin 2 (Stx2B) and the enzyme lumazine synthase from Brucella spp. (BLS) (BLS-Stx2B). The aim of this study was to analyze maternal immunization with BLS-Stx2B as a possible approach for transferring anti-Stx2 protection to the offspring. BALB/c female mice were immunized with BLS-Stx2B before mating. Both dams and pups presented comparable titers of anti-Stx2B antibodies in sera and fecal extracts. Moreover, pups were totally protected against a lethal dose of systemic Stx2 injection up to 2 to 3 months postpartum. In addition, pups were resistant to an oral challenge with an Stx2-producing EHEC strain at weaning and did not develop any symptomatology associated with Stx2 toxicity. Fostering experiments demonstrated that anti-Stx2B neutralizing IgG antibodies were transmitted through breast-feeding. Pups that survived the EHEC infection due to maternally transferred immunity prolonged an active and specific immune response that protected them against a subsequent challenge with intravenous Stx2. Our study shows that maternal immunization with BLS-Stx2B was very effective at promoting the transfer of specific antibodies, and suggests that preexposure of adult females to this immunogen could protect their offspring during the early phase of life. PMID:24421050

  12. Complex Alternative Splicing of the Smarca2 Gene Suggests the Importance of Smarca2-B Variants

    PubMed Central

    Yang, Min; Sun, Yuan; Ma, Ling; Wang, Chenguang; Wu, Jian-min; Bi, Anding; Liao, D. Joshua

    2011-01-01

    BRM is an ATPase component of the SWI/SNF complex that regulates chromatin remodeling and cell proliferation and is considered a tumor suppressor. In this study we characterized transcripts from the Smarca2 gene that encodes the BRM protein. We found that the human Smarca2 gene (hSmarca2), like its mouse counterpart (mSmarca2), also initiated a short transcript from intron 27 of the long transcript. We name the long and short transcripts as Smarca2-a and Smarca2-b, respectively. Like its human counterpart, mSmarca2-a also underwent alternative splicing at the 54-bp exon 29. The hSmarca2-b had two alternative initiation sites and underwent alternative splicing at three different 3' sites of exon 1 and at exons 2, 3 and/or 5. We identified nine hSmarca2-b mRNA variants that might produce five different proteins. mSmarca2-b also underwent alternative splicing at exon 3 and/or exon 5, besides alternatively retaining part of intron 1 in exon 1. Smarca2-b was expressed more abundantly than Smarca2-a in many cell lines and was more sensitive to serum starvation. Moreover, cyclin D1 also regulated the expression of both Smarca2-a and Smarca2-b in a complex manner. These data suggest that the functions of the Smarca2 gene may be very complex, not just simply inhibiting cell proliferation, and in certain situations may be elicited mainly by expressing the much less known Smarca2-b, not the better studied Smarca2-a and its products BRM proteins. PMID:21811517

  13. Metformin inhibits histone H2B monoubiquitination and downstream gene transcription in human breast cancer cells.

    PubMed

    DU, Yu; Zheng, Haiyan; Wang, Jiang; Ren, Ye; Li, Mi; Gong, Chen; Xu, Fei; Yang, Caihong

    2014-08-01

    Metformin, one of the most widely prescribed antihyperglycemic drugs, has recently received increasing attention for its potential effects with regard to cancer prevention and treatment. However, the mechanisms behind the suppression of cancer cell growth by metformin remain far from completely understood. The aim of the present study was to investigate whether metformin could regulate histone modification and its downstream gene transcription, and its potential function in inhibiting breast cancer cell proliferation. A T47D cell proliferation curve was determined by cell counting following metformin treatment with differing doses or time courses. The cell cycle was analyzed by flow cytometry with propidium iodide staining. Histone H2B monoubiquitination was evaluated by western blotting subsequent to histone extraction. The histone H2B monoubiquitination downstream gene expression level was determined by quantitative PCR. The results showed that metformin changed the cell-cycle check-point and inhibited breast cancer cell proliferation in a dose-dependent manner. AMPK was activated and histone H2B monoubiquitination and downstream gene transcription were inhibited following metformin treatment in the T47D cells. The effect of metformin on T47D cell proliferation was dependent on AMPK activity. It was concluded that metformin can suppress breast cancer cell growth by the activation of AMPK and the inhibition of histone H2B monoubiquitination and downstream gene transcription. This study reveals a novel potential mechanism of cancer cell growth suppression by metformin. PMID:25009658

  14. Cloning and characterization of a third type of human alpha-amylase gene, AMY2B.

    PubMed

    Yokouchi, H; Horii, A; Emi, M; Tomita, N; Doi, S; Ogawa, M; Mori, T; Matsubara, K

    1990-06-15

    We have previously reported concerning the existence of a third type of human alpha-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229-235; Tomita et al., Gene 76 (1989) 11-18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human alpha-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively. Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197-1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5' region; hence the promoter lies far upstream relative to the other two AMY genes. PMID:2401405

  15. Conantokins Derived from the Asprella Clade Impart ConRl-B, an NMDA Receptor Antagonist with a Unique Selectivity Profile for NR2B Subunits

    PubMed Central

    Gowd, Konkallu Hanumae; Han, Tiffany S.; Twede, Vernon; Gajewiak, Joanna; Smith, Misty D.; Watkins, Maren; Platt, Randall J.; Toledo, Gabriela; White, H. Steve; Olivera, Baldomero M.; Bulaj, Grzegorz

    2014-01-01

    Using molecular phylogeny has accelerated the discovery of peptidic ligands targeted to ion channels and receptors. One clade of venomous cone snails, Asprella, appears to be significantly enriched in conantokins, antagonists of N-Methyl D-Asparate receptors (NMDARs). Here, we describe the characterization of two novel conantokins from Conus rolani, including conantokin conRl-B that has shown an unprecedented selectivity for blocking NMDARs that contain NR2B subunits. ConRl-B shares only some sequence similarity to the most studied NR2B-selective conantokin, conG. The divergence between conRl-B and conG in the second inter-Gla loop was used to design analogs for structure-activity studies; the presence of Pro10 was found to be key to the high potency of conRl-B for NR2B, whereas the ε-amino group of Lys8 contributed to discrimination in blocking NR2B- and NR2A-containing NMDARs. In contrast to previous findings from Tyr5 substitutions in other conantokins, conRl-B [L5Y] showed potencies on the four NR2 NMDA receptor subtypes that were similar to those of the native conRl-B. When delivered into the brain, conRl-B was active in suppressing seizures in the model of epilepsy in mice, consistent with NR2B-containing NMDA receptors being potential targets for antiepileptic drugs. Circular dichroism experiments confirmed that the helical conformation of conRl-B is stabilized by divalent metal ions. Given the clinical applications of NMDA antagonists, conRl-B provides a potentially important pharmacological tool for understanding the differential roles of NMDA receptor subtypes in the nervous system. This work shows the effectiveness of coupling molecular phylogeny, chemical synthesis and pharmacology for discovering new bioactive natural products. PMID:22594498

  16. Mutations in the GCD7 subunit of yeast guanine nucleotide exchange factor eIF-2B overcome the inhibitory effects of phosphorylated eIF-2 on translation initiation.

    PubMed Central

    Vazquez de Aldana, C R; Hinnebusch, A G

    1994-01-01

    Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) impairs translation initiation by inhibiting the guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In Saccharomyces cerevisiae, phosphorylation of eIF-2 alpha by the protein kinase GCN2 specifically stimulates translation of GCN4 mRNA in addition to reducing general protein synthesis. We isolated mutations in several unlinked genes that suppress the growth-inhibitory effect of eIF-2 alpha phosphorylation catalyzed by mutationally activated forms of GCN2. These suppressor mutations, affecting eIF-2 alpha and the essential subunits of eIF-2B encoded by GCD7 and GCD2, do not reduce the level of eIF-2 alpha phosphorylation in cells expressing the activated GCN2c kinase. Four GCD7 suppressors were shown to reduce the derepression of GCN4 translation in cells containing wild-type GCN2 under starvation conditions or in GCN2c strains. A fifth GCD7 allele, constructed in vitro by combining two of the GCD7 suppressors mutations, completely impaired the derepression of GCN4 translation, a phenotype characteristic of deletions in GCN1, GCN2, or GCN3. This double GCD7 mutation also completely suppressed the lethal effect of expressing the mammalian eIF-2 alpha kinase dsRNA-PK in yeast cells, showing that the translational machinery had been rendered completely insensitive to phosphorylated eIF-2. None of the GCD7 mutations had any detrimental effect on cell growth under nonstarvation conditions, suggesting that recycling of eIF-2 occurs efficiently in the suppressor strains. We propose that GCD7 and GCD2 play important roles in the regulatory interaction between eIF-2 and eIF-2B and that the suppressor mutations we isolated in these genes decrease the susceptibility of eIF-2B to the inhibitory effects of phosphorylated eIF-2 without impairing the essential catalytic function of eIF-2B in translation initiation. Images PMID:8164676

  17. Copy number variations in the amylase gene (AMY2B) in Japanese native dog breeds.

    PubMed

    Tonoike, A; Hori, Y; Inoue-Murayama, M; Konno, A; Fujita, K; Miyado, M; Fukami, M; Nagasawa, M; Mogi, K; Kikusui, T

    2015-10-01

    A recent study suggested that increased copy numbers of the AMY2B gene might be a crucial genetic change that occurred during the domestication of dogs. To investigate AMY2B expansion in ancient breeds, which are highly divergent from modern breeds of presumed European origins, we analysed copy numbers in native Japanese dog breeds. Copy numbers in the Akita and Shiba, two ancient breeds in Japan, were higher than those in wolves. However, compared to a group of various modern breeds, Akitas had fewer copy numbers, whereas Shibas exhibited the same level of expansion as modern breeds. Interestingly, average AMY2B copy numbers in the Jomon-Shiba, a unique line of the Shiba that has been bred to maintain their appearance resembling ancestors of native Japanese dogs and that originated in the same region as the Akita, were lower than those in the Shiba. These differences may have arisen from the earlier introduction of rice farming to the region in which the Shiba originated compared to the region in which the Akita and the Jomon-Shiba originated. Thus, our data provide insights into the relationship between the introduction of agriculture and AMY2B expansion in dogs. PMID:26358734

  18. A Vibrio cholerae ghost-based subunit vaccine induces cross-protective chlamydial immunity that is enhanced by CTA2B, the nontoxic derivative of cholera toxin

    PubMed Central

    Ekong, Eno E.; Okenu, Daniel N.; Mania-Pramanik, Jayanti; He, Qing; Igietseme, Joseph U.; Ananaba, Godwin A.; Lyn, Deborah; Black, Carolyn; Eko, Francis O.

    2011-01-01

    The Vibrio cholerae ghost (rVCG) platform is an effective carrier and delivery system for designing efficacious Chlamydia vaccines. We investigated whether CTA2B, the nontoxic derivative of cholera toxin, can augment protective immunity conferred by an rVCG-based chlamydial vaccine and enhance cross-protection against heterologous chlamydial strains. An rVCG vaccine coexpressing chlamydial major outer membrane protein and CTA2B was genetically constructed and antigens were targeted to the inner membrane of V. cholerae before ghost production by gene E-mediated lysis. Effective immunomodulation by CTA2B was demonstrated by the ability of the vaccine construct to enhance the activation and maturation of dendritic cells in vitro. Also, C57BL/6 mice immunized via mucosal and systemic routes showed increased specific mucosal and systemic antibody and T-helper type-1 (Th1) responses, irrespective of the route. The enhanced production of IFN-γ, but not IL-4 by genital mucosal and splenic T cells, indicated a predominantly Th1 response. Clearance of the Chlamydia muridarum vaginal infection was significantly enhanced by codelivery of the vaccine with CTA2B, with the intravaginal route showing a moderate advantage. These results indicate that the rVCG-based vaccine is capable of inducing cross-protection against heterologous chlamydial serovars and that incorporation of mucosal adjuvants, such as CTA2B in the rVCG delivery platform, may enhance protective immunity. PMID:19040663

  19. Induction of the CYP2B genes by triphenyldioxane treatment in the rat liver.

    PubMed

    Grigorieva, E; Soshilov, A; Surovtseva, Y; Schwartz, E L; Duzhak, T G; Gulyaeva, L F; Lyakhovich, V V

    2002-08-01

    Triphenyldioxane (TPD) is a potent phenobarbital-type (PB) inducer of the CYP2B cytochrome isoforms, the inducing effect of which is one order of magnitude higher than PB. The fact that TPD is unable to induce CYP2B genes having the proximal promoter disrupted (mouse Cyp2b10) suggests an existence of the proximal promoter-dependent mechanism of the CYP2B induction. So a TPD-dependent activation of the nuclear proteins to the binding with Barbie-box sequence (the most conservative part of the proximal promoter) was studied. In the nuclear extracts from the intact rat liver there were detected five proteins that could be activated to the Barbie-box binding by the TPD treatment in vitro (II, III, NI, NII and NIII). The first three were effected also by another PB-like inducers tested (PB and TCPOBOP), when NII and NIII complexes were formed under the influence of TPD only. It is possible that a direct activation of the NII and NIII proteins by TPD exists as (3)H-labeled TPD was detected in the composition of NII and NIII complexes. However, both of them disappeared from the nuclear extracts after the long exposure time with TPD (6 h or more). A short induction by the direct intra-liver delivery of TPD (15-30 min) led to the stabile activation of one TPD-specific protein. Apart from the activation of the Barbie-box-binding protein, the short TPD treatment caused the activation of three nuclear proteins being able to interact with the NR1 sequence of the distal promoter PBREM element. These findings suggest that TPD is really the first member of the PB-like inducers family for which a special mechanism of CYP2B induction may exist. PMID:12110287

  20. Impact of UGT2B17 gene deletion on the steroid profile of an athlete.

    PubMed

    Martín-Escudero, Pilar; Muñoz-Guerra, Jesús; Del Prado, Nayade; Galindo Canales, Mercedes; Fuentes Ferrer, Manuel; Vargas, Soledad; Soldevilla, Ana B; Serrano-Garde, Ester; Miguel-Tobal, Francisco; Maestro de Las Casas, Marisa; Fernandez-Pérez, Cristina

    2015-12-01

    The measurement of the testosterone to epitestosterone ratio (T/E ratio) in urine is often used as a marker for testosterone administration in the doping control field. This study examines the frequencies of the different expression forms of the UGT2B17 gene, and assesses their effects on this marker in volunteer subjects. The sample for this descriptive study was composed of male and female athletes aged between 16 and 55 years old who practiced different sports disciplines. All participants underwent a sports-medical physical examination, and subsequently provided 10 urine samples consecutively over a period of 48 h. The dependent variable examined was T/E and the main independent variable was the UGT2B17 gene polymorphism. During 1 year, 1410 urine samples were obtained from 141 athletes. The frequencies of the three genotypes were as follows: wt homozygotes (ins/ins) 48.2% (n = 68), mutant homozygotes (del/del) 12.1% (n = 17), and heterozygotes (ins/del) 39.7% (n = 56). Genotype distributions varied significantly (P < 0.001) according to ethnicity, 80% of Asian subjects being homozygous for the gene deletion (del/del) compared to 6.9% of Caucasian subjects. A multivariate analysis adjusted for genotype, age, sex, and sports discipline revealed that athletes with the del/del polymorphism showed a significantly lower mean T/E than heterozygotes (ins/del). In contrast, homozygous athletes for the gene insertion (ins/ins) showed higher mean T/E ratios than heterozygotes (ins/del). UGT2B17 gene deletion has a strong influence on the T/E ratio in urine, which is the most efficient indicator of testosterone prohormone misuse. Others factors studied seem not to have such an impact. The genotyping of UGT2B17 is an important source of information for understanding steroid profiling in the doping control field; therefore it is suggested that it be included in the Athletes Biological Passport. PMID:26668303

  1. The Trypanosoma brucei protein phosphatase gene: polycistronic transcription with the RNA polymerase II largest subunit gene.

    PubMed Central

    Evers, R; Cornelissen, A W

    1990-01-01

    We have previously described the trypanosomal gene encoding the largest subunit of RNA polymerase II (RNAP II) and found that two almost identical genes are encoded within the Trypanosoma brucei genome. Here we show by Southern analyses that the 5' breakpoint between both loci is located approximately 7.5 kb upstream of the RNAP II genes. Northern analyses revealed that the 5' duplicated segment contains at least four other genes, which are transcribed in both bloodstream and procyclic trypanosomes. The gene located immediately upstream of the RNAP II gene in both loci was characterized by sequence analyses. The deduced amino acid sequences show a high degree of similarity to the catalytic subunit of protein phosphatase class 1 (PP1) genes. S1 mapping provided strong evidence in support of the fact that the PP1 and RNAP II genes belong to a single transcription unit. Images PMID:2169604

  2. Behavioral phenotype in five individuals with de novo mutations within the GRIN2B gene

    PubMed Central

    2013-01-01

    Background Intellectual disability (ID) is often associated with behavioral problems or disorders. Mutations in the GRIN2B gene (MRD6, MIM613970) have been identified as a common cause of ID (prevalence of 0.5 – 1% in individuals with ID) associated with EEG and behavioral problems. Methods We assessed five GRIN2B mutation carriers aged between 3 and 14 years clinically and via standardized questionnaires to delineate a detailed behavioral phenotype. Parents and teachers rated problem behavior of their affected children by completing the Developmental Behavior Checklist (DBC) and the Conners’ Rating Scales Revised (CRS-R:L). Results All individuals had mild to severe ID and needed guidance in daily routine. They showed characteristic behavior problems with prominent hyperactivity, impulsivity, distractibility and a short attention span. Stereotypies, sleeping problems and a friendly but boundless social behavior were commonly reported. Conclusion Our observations provide an initial delineation of the behavioral phenotype of GRIN2B mutation carriers. PMID:23718928

  3. Characterization of the human rod transducin alpha-subunit gene.

    PubMed Central

    Fong, S L

    1992-01-01

    The human rod transducin alpha subunit (Tr alpha) gene has been cloned. A cDNA clone, HG14, contained a 1.1 kb insertion when compared with the human Tr alpha cDNA published by Van Dop et al. (1). Based on two overlapping clones isolated from a human genomic library, the human Tr alpha gene is 4.9 kb in length and consists of nine exons interrupted by eight introns. Northern blots of human retina total RNA showed that the gene is transcribed by rod photoreceptors into two species of mRNA, 1.3 kb and 2.4 kb in size. Apparently, this is the result of alternative splicing. Two putative transcription initiation sites were determined by primer extension and S1 nuclease protection assays. The putative promoter regions of the human and mouse Tr alpha genes have an identity of 78.1%. As found in the mouse gene (2), no TATA consensus sequence is present in the human gene. Images PMID:1614872

  4. PON2 and ATP2B2 gene polymorphisms with noise-induced hearing loss

    PubMed Central

    Li, Xiuting; Cao, Jinglian; Wang, Jun; Song, Haiyan; Ji, Guixiang; Dong, Qiu; Wei, Chunlong; Cao, Ying; Wang, Boshen

    2016-01-01

    Background Noise-induced hearing loss (NIHL) is a complex disease induced by a combination of genetic and environmental factors. Paraoxonase2 (PON2) gene involved in the regulation of reactive oxygen species, and affecting the vulnerability of cochlea to NIHL, and ATPase, calcium-transporting, plasma membrane 2 (ATP2B2) gene which encodes plasma membrane calcium-transporting ATPase isoform 2 (PMCA2) are the candidate genes relating to the attack of NIHL. In this study, we investigated whether ATP2B2 and PON2 polymorphisms were associated with NIHL in Chinese of Han nationality population. Methods We performed a case-control study between six single nucleotide polymorphisms (SNPs) (rs1719571, rs3209637 and rs4327369 within ATP2B2, rs12026, rs7785846 and rs12704796 within PON2) and NIHL in 454 subjects. All the SNPs were genotypes, using the TaqMan MGB probe assay. Odds ratios (ORs) were calculated with 95% confidence intervals (95% CIs) with logistic regression analysis to test the level of association for SNPs. Results In our study, 221 subjects with hearing loss and 233 subjects without hearing loss were recruited. The frequencies of the CG and CG + GG genotype of rs12026 (PON2) conferred risk factors for NIHL with adjusted OR values of 2.62 (95% CI, 1.69–4.06) and 2.48 (95% CI, 1.63–3.78), respectively. This kind of significance was also found at locus rs7785846, where genotypes CT and CT + TT were the risk types, with adjusted ORs of 2.52 (95% CI, 1.62–3.93) and 2.35 (95% CI, 1.54–3.58), respectively. We performed stratified analysis per noise exposure level, when it came to rs7785846 and rs12026 in the >92 dB(A) noise exposure group, the subjects who carried heterozygote were of significantly (P<0.01) higher susceptibility to NIHL than homozygote carriers. By contrast, no significantly higher risk was found for any rs12704796 genotypes or any genotypes in ATP2B2 (P>0.05), which may suggest that these SNPs did not have significant effects on noise

  5. Carbohydrate-to-carbohydrate interactions between α2,3-linked sialic acids on α2 integrin subunits and asialo-GM1 underlie the bone metastatic behaviour of LNCAP-derivative C4-2B prostate cancer cells

    PubMed Central

    Van Slambrouck, Séverine; Groux-Degroote, Sophie; Krzewinski-Recchi, Marie-Ange; Cazet, Aurélie; Delannoy, Philippe; Steelant, Wim F. A.

    2014-01-01

    Complex interplays among proteins, lipids and carbohydrates can alter the phenotype and are suggested to have a crucial role in tumour metastasis. Our previous studies indicated that a complex of the GSLs (glycosphingolipids), AsGM1 (asialo-GM1), which lacks α2,3-linked sialic acid, and α2β1 integrin receptors is responsible for the metastatic behaviour of C4-2B prostate cancer cells. Herein, we identified and addressed the functional significance of changes in sialylation during prostate cancer progression. We observed an increase in α2,3-linked sialic acid residues on α2 subunits of α2β1 integrin receptors, correlating with increased gene expression of α2,3-STs (sialyltransferases), particularly ST3GAL3. Cell surface α2,3-sialylation of α2 subunits was required for the integrin α2β1-dependent cell adhesion to collagen type I and the same α2,3-linked sialic acid residues on the integrin receptor were responsible for the interaction with the carbohydrate moiety of AsGM1, explaining the complex formation between AsGM1 and α2β1 integrin receptors. These results provide novel insights into the role of sialic acids in the organization and function of important membrane components in invasion and metastatic processes. PMID:25137483

  6. Impact of UGT2B17 Gene Deletion on the Pharmacokinetics of 17-Hydroexemestane in Healthy Volunteers.

    PubMed

    Chen, Shanly M; Atchley, Daniel H; Murphy, Michael A; Gurley, Bill J; Kamdem, Landry K

    2016-07-01

    Exemestane is an aromatase inhibitor drug used for the treatment of hormone-dependent breast cancer. 17-Hydroexemestane, the major and biologically active metabolite of exemestane in humans, is eliminated via glucuronidation by the polymorphic UGT2B17 phase II drug-metabolizing enzyme. Previous microsomal studies have shown that UGT2B17 gene deletion affects the intrinsic hepatic clearances of 17-hydroexemestane in vitro. In this open-label study we set out to assess the effect of UGT2B17 gene deletion on the pharmacokinetics of 17-hydroexemestane in healthy female volunteers with and without UGT2B17. To achieve this goal, 14 healthy postmenopausal women (8 carriers of the homozygous UGT2B17 wild-type allele and 6 carriers of the homozygous UGT2B17 gene-deletion allele) were enrolled and invited to receive a single 25-mg oral dose of exemestane. Pharmacokinetics was assessed over 72 hours postdosing. Our results showed that there were statistically significant differences in plasma 17-hydroexemestane AUC0-∞ (P = .0007) and urine 17-hydroexemestane C24h (P = .001) between UGT2B17 genotype groups. Our data suggest that UGT2B17 gene deletion influences 17-hydroexemestane pharmacokinetics in humans. PMID:26608382

  7. Expansion of transducin subunit gene families in early vertebrate tetraploidizations.

    PubMed

    Lagman, David; Sundström, Görel; Ocampo Daza, Daniel; Abalo, Xesús M; Larhammar, Dan

    2012-10-01

    Hundreds of gene families expanded in the early vertebrate tetraploidizations including many gene families in the phototransduction cascade. We have investigated the evolution of the heterotrimeric G-proteins of photoreceptors, the transducins, in relation to these events using both phylogenetic analyses and synteny comparisons. Three alpha subunit genes were identified in amniotes and the coelacanth, GNAT1-3; two of these were identified in amphibians and teleost fish, GNAT1 and GNAT2. Most tetrapods have four beta genes, GNB1-4, and teleosts have additional duplicates. Finally, three gamma genes were identified in mammals, GNGT1, GNG11 and GNGT2. Of these, GNGT1 and GNGT2 were found in the other vertebrates. In frog and zebrafish additional duplicates of GNGT2 were identified. Our analyses show all three transducin families expanded during the early vertebrate tetraploidizations and the beta and gamma families gained additional copies in the teleost-specific genome duplication. This suggests that the tetraploidizations contributed to visual specialisations. PMID:22814267

  8. Regulation of expression of a soybean storage protein subunit gene. Progress report

    SciTech Connect

    Thompson, J.F.; Madison, J.T.

    1984-04-23

    We have found that the methionine repression of the ..beta..-subunit gene expression is not due to degradation of the ..beta..-subunit but is due to an effect on synthesis of the ..beta..-subunit. The effect of methionine on the synthesis of the ..beta..-is due to an inhibition of ..beta..-subunit mRNA synthesis. 3 references, 1 figure.

  9. Okadaic acid induces epileptic seizures and hyperphosphorylation of the NR2B subunit of the NMDA receptor in rat hippocampus in vivo.

    PubMed

    Arias, Clorinda; Montiel, Teresa; Peña, Fernando; Ferrera, Patricia; Tapia, Ricardo

    2002-09-01

    Overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors is closely related to epilepsy and excitotoxicity, and the phosphorylation of these receptors may facilitate glutamate-mediated synaptic transmission. Here we show that in awake rats the microinjection into the hippocampus of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, induces in about 20 min intense electroencephalographic and behavioral limbic-type seizures, which are suppressed by the systemic administration of the NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine hydrogen maleate and by the intrahippocampal administration of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinases. Two hours after okadaic acid, when the EEG seizures were intense, an increased serine phosphorylation of some hippocampal proteins, including an enhancement of the serine phosphorylation of the NMDA receptor subunit NR2B, was detected by immunoblotting. Twenty-four hours after okadaic acid a marked destruction of hippocampal CA1 region was observed, which was not prevented by the receptor antagonists. These findings suggest that hyperphosphorylation of glutamate receptors in vivo may result in an increased sensitivity to the endogenous transmitter and therefore induce neuronal hyperexcitability and epilepsy. PMID:12429230

  10. Fyn Kinase regulates GluN2B subunit-dominant NMDA receptors in human induced pluripotent stem cell-derived neurons

    PubMed Central

    Zhang, Wen-Bo; Ross, P. Joel; Tu, YuShan; Wang, Yongqian; Beggs, Simon; Sengar, Ameet S.; Ellis, James; Salter, Michael W.

    2016-01-01

    NMDA receptor (NMDAR)-mediated fast excitatory neurotransmission is implicated in a broad range of physiological and pathological processes in the mammalian central nervous system. The function and regulation of NMDARs have been extensively studied in neurons from rodents and other non-human species, and in recombinant expression systems. Here, we investigated human NMDARs in situ by using neurons produced by directed differentiation of human induced pluripotent stem cells (iPSCs). The resultant cells showed electrophysiological characteristics demonstrating that they are bona fide neurons. In particular, human iPSC-derived neurons expressed functional ligand-gated ion channels, including NMDARs, AMPA receptors, GABAA receptors, as well as glycine receptors. Pharmacological and electrophysiological properties of NMDAR-mediated currents indicated that these were dominated by receptors containing GluN2B subunits. The NMDAR currents were suppressed by genistein, a broad-spectrum tyrosine kinase inhibitor. The NMDAR currents were also inhibited by a Fyn-interfering peptide, Fyn(39–57), but not a Src-interfering peptide, Src(40–58). Together, these findings are the first evidence that tyrosine phosphorylation regulates the function of NMDARs in human iPSC-derived neurons. Our findings provide a basis for utilizing human iPSC-derived neurons in screening for drugs targeting NMDARs in neurological disorders. PMID:27040756

  11. Common Variants in the ATP2B1 Gene Are Associated With Susceptibility to Hypertension

    PubMed Central

    Tabara, Yasuharu; Kohara, Katsuhiko; Kita, Yoshikuni; Hirawa, Nobuhito; Katsuya, Tomohiro; Ohkubo, Takayoshi; Hiura, Yumiko; Tajima, Atsushi; Morisaki, Takayuki; Miyata, Toshiyuki; Nakayama, Tomohiro; Takashima, Naoyuki; Nakura, Jun; Kawamoto, Ryuichi; Takahashi, Norio; Hata, Akira; Soma, Masayoshi; Imai, Yutaka; Kokubo, Yoshihiro; Okamura, Tomonori; Tomoike, Hitonobu; Iwai, Naoharu; Ogihara, Toshio; Inoue, Itsuro; Tokunaga, Katsushi; Johnson, Toby; Caulfield, Mark; Munroe, Patricia; Umemura, Satoshi; Ueshima, Hirotsugu; Miki, Tetsuro

    2016-01-01

    Hypertension is one of the most common complex genetic disorders. We have described previously 38 single nucleotide polymorphisms (SNPs) with suggestive association with hypertension in Japanese individuals. In this study we extend our previous findings by analyzing a large sample of Japanese individuals (n=14 105) for the most associated SNPs. We also conducted replication analyses in Japanese of susceptibility loci for hypertension identified recently from genome-wide association studies of European ancestries. Association analysis revealed significant association of the ATP2B1 rs2070759 polymorphism with hypertension (P=5.3×10−5; allelic odds ratio: 1.17 [95% CI: 1.09 to 1.26]). Additional SNPs in ATP2B1 were subsequently genotyped, and the most significant association was with rs11105378 (odds ratio: 1.31 [95% CI: 1.21 to 1.42]; P=4.1×10−11). Association of rs11105378 with hypertension was cross-validated by replication analysis with the Global Blood Pressure Genetics consortium data set (odds ratio: 1.13 [95% CI: 1.05 to 1.21]; P=5.9×10−4). Mean adjusted systolic blood pressure was highly significantly associated with the same SNP in a meta-analysis with individuals of European descent (P=1.4×10−18). ATP2B1 mRNA expression levels in umbilical artery smooth muscle cells were found to be significantly different among rs11105378 genotypes. Seven SNPs discovered in published genome-wide association studies were also genotyped in the Japanese population. In the combined analysis with replicated 3 genes, FGF5 rs1458038, CYP17A1, rs1004467, and CSK rs1378942, odds ratio of the highest risk group was 2.27 (95% CI: 1.65 to 3.12; P=4.6×10−7) compared with the lower risk group. In summary, this study confirmed common genetic variation in ATP2B1, as well as FGF5, CYP17A1, and CSK, to be associated with blood pressure levels and risk of hypertension. PMID:20921432

  12. Phosphorylation of Tyrosine 1070 at the GluN2B Subunit Is Regulated by Synaptic Activity and Critical for Surface Expression of N-Methyl-D-aspartate (NMDA) Receptors.

    PubMed

    Lu, Wen; Fang, Weiqing; Li, Jian; Zhang, Bin; Yang, Qian; Yan, Xunyi; Peng, Lin; Ai, Heng; Wang, Jie-jie; Liu, Xiao; Luo, Jianhong; Yang, Wei

    2015-09-18

    The number and subunit composition of synaptic N-methyl-d-aspartate receptors (NMDARs) play critical roles in synaptic plasticity, learning, and memory and are implicated in neurological disorders. Tyrosine phosphorylation provides a powerful means of regulating NMDAR function, but the underling mechanism remains elusive. In this study we identified a tyrosine site on the GluN2B subunit, Tyr-1070, which was phosphorylated by a proto-oncogene tyrosine-protein (Fyn) kinase and critical for the surface expression of GluN2B-containing NMDARs. The phosphorylation of GluN2B at Tyr-1070 was required for binding of Fyn kinase to GluN2B, which up-regulated the phosphorylation of GluN2B at Tyr-1472. Moreover, our results revealed that the phosphorylation change of GluN2B at Tyr-1070 accompanied the Tyr-1472 phosphorylation and Fyn associated with GluN2B in synaptic plasticity induced by both chemical and contextual fear learning. Taken together, our findings provide a new mechanism for regulating the surface expression of NMDARs with implications for synaptic plasticity. PMID:26229100

  13. A GmAOX2b antisense gene compromises vegetative growth and seed production in soybean.

    PubMed

    Chai, Tsun-Thai; Simmonds, Daina; Day, David A; Colmer, Timothy D; Finnegan, Patrick M

    2012-07-01

    The alternative oxidase mediates the cyanide-resistant respiratory pathway in plant mitochondria. In non-thermogenic plants, the role of alternative oxidase in plant growth and development is not well understood. Soybean (Glycine max) lines carrying a GmAOX2b antisense gene had compromised vegetative growth and reproductive performance under typical glasshouse growth conditions. The reduction in vegetative growth was demonstrated by reduction in shoot height, the number of leaves per plant and the green leaf area. Antisense plants also had decreased pod formation and seed to pod ratios, which together led to a reduction in the number and total mass of seed produced. The negative effects of the antisense gene on pod set, seed set, ovule availability and total seed mass were primarily confined to the branches, rather than the main stem. The preferential effect of alternative oxidase suppression in the branches is discussed in relation to the reproductive potential of soybean under stress. Taken together, these results demonstrate that alternative oxidase provides the benefit of sustaining plant vegetative growth and reproductive capacity in soybean. PMID:22307678

  14. Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice.

    PubMed

    Holland, Ruby A; Leonard, John J; Kensey, Nicholas A; Hannikainen, Paavali A; De Jonghe, Bart C

    2014-09-01

    Although rats and mice do not vomit, these species are widely studied as models of energy balance and sickness behavior. Previous work has shown that rats exhibit similar neuroanatomical activation of brain and visceral afferent pathways following cisplatin chemotherapy compared to vomiting species. However, the neural response to cisplatin in mice is understudied. Here, food intake, body weight, and central c-Fos immunofluorescence were analyzed in the hindbrains of male C57BL/6 mice following IP saline or cisplatin (5mg/kg, and 20mg/kg doses). As glutamate receptor signaling is classically linked to inhibitory feeding pathways in the rodent, gene expression of selected α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartic acid (NMDA) receptor subunits were assessed in the dorsal vagal complex (DVC), parabrachial nucleus (PBN), amygdala, and bed nucleus of the stria terminalis (BNST). Our results show dose-dependent reductions in food intake and body weight following cisplatin treatment, as well as increases in cisplatin-induced c-Fos in the PBN and throughout the DVC. Quantitative PCR analysis shows cisplatin-induced increases in NMDA receptor subunit expression, particularly NR2B, in the DVC, PBN, BNST, and amygdala. In addition, upregulation of AMPA receptor subunits (GluA1 and/or GluA2) were observed in all regions examined except the amygdala. Taken together, these results suggest similar neural pathways mediating cisplatin effects in mice compared to other well-studied species, which are likely mediated by central upregulation of AMPA and NMDA receptors. PMID:24582677

  15. Characterization of rainbow trout myostatin-2 genes (rtMSTN-2a & -2b): genomic organization, differential expression and pseudogenization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myostatin is an extremely potent negative regulator of vertebrate skeletal muscle development. A phylogenetic analysis suggests that salmonids should possess four distinct genes, although only MSTN-1 orthologs have been characterized. Described herein are the rainbow trout (rt) MSTN-2a and -2b genes...

  16. Identification of Significant Association and Gene-Gene Interaction of GABA Receptor Subunit Genes in Autism

    PubMed Central

    Ma, D. Q.; Whitehead, P. L.; Menold, M. M.; Martin, E. R.; Ashley-Koch, A. E.; Mei, H.; Ritchie, M. D.; DeLong, G. R.; Abramson, R. K.; Wright, H. H.; Cuccaro, M. L.; Hussman, J. P.; Gilbert, J. R.; Pericak-Vance, M. A.

    2005-01-01

    Autism is a common neurodevelopmental disorder with a significant genetic component. Existing research suggests that multiple genes contribute to autism and that epigenetic effects or gene-gene interactions are likely contributors to autism risk. However, these effects have not yet been identified. Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, has been implicated in autism etiology. Fourteen known autosomal GABA receptor subunit genes were studied to look for the genes associated with autism and their possible interactions. Single-nucleotide polymorphisms (SNPs) were screened in the following genes: GABRG1, GABRA2, GABRA4, and GABRB1 on chromosome 4p12; GABRB2, GABRA6, GABRA1, GABRG2, and GABRP on 5q34-q35.1; GABRR1 and GABRR2 on 6q15; and GABRA5, GABRB3, and GABRG3 on 15q12. Intronic and/or silent mutation SNPs within each gene were analyzed in 470 white families with autism. Initially, SNPs were used in a family-based study for allelic association analysis—with the pedigree disequilibrium test and the family-based association test—and for genotypic and haplotypic association analysis—with the genotype-pedigree disequilibrium test (geno-PDT), the association in the presence of linkage (APL) test, and the haplotype family-based association test. Next, with the use of five refined independent marker sets, extended multifactor-dimensionality reduction (EMDR) analysis was employed to identify the models with locus joint effects, and interaction was further verified by conditional logistic regression. Significant allelic association was found for markers RS1912960 (in GABRA4; P = .01) and HCV9866022 (in GABRR2; P = .04). The geno-PDT found significant genotypic association for HCV8262334 (in GABRA2), RS1912960 and RS2280073 (in GABRA4), and RS2617503 and RS12187676 (in GABRB2). Consistent with the allelic and genotypic association results, EMDR confirmed the main effect at RS1912960 (in GABRA4). EMDR also identified a

  17. Dopamine receptor D5 deficiency results in a selective reduction of hippocampal NMDA receptor subunit NR2B expression and impaired memory.

    PubMed

    Moraga-Amaro, Rodrigo; González, Hugo; Ugalde, Valentina; Donoso-Ramos, Juan Pablo; Quintana-Donoso, Daisy; Lara, Marcelo; Morales, Bernardo; Rojas, Patricio; Pacheco, Rodrigo; Stehberg, Jimmy

    2016-04-01

    Pharmacological evidence associates type I dopamine receptors, including subtypes D1 and D5, with learning and memory. Analyses using genetic approaches have determined the relative contribution of dopamine receptor D1 (D1R) in cognitive tasks. However, the lack of drugs that can discriminate between D1R and D5R has made the pharmacological distinction between the two receptors difficult. Here, we aimed to determine the role of D5R in learning and memory. In this study we tested D5R knockout mice and wild-type littermates in a battery of behavioral tests, including memory, attention, locomotion, anxiety and motivational evaluations. Our results show that genetic deficiency of D5R significantly impairs performance in the Morris water maze paradigm, object location and object recognition memory, indicating a relevant role for D5R in spatial memory and recognition memory. Moreover, the lack of D5R resulted in decreased exploration and locomotion. In contrast, D5R deficiency had no impact on working memory, anxiety and depressive-like behavior, measured using the spontaneous alternation, open-field, tail suspension test, and forced swimming test. Electrophysiological analyses performed on hippocampal slices showed impairment in long-term-potentiation in mice lacking D5R. Further analyses at the molecular level showed that genetic deficiency of D5R results in a strong and selective reduction in the expression of the NMDA receptor subunit NR2B in the hippocampus. These findings demonstrate the relevant contribution of D5R in memory and suggest a functional interaction of D5R with hippocampal glutamatergic pathways. PMID:26714288

  18. Enhancement of GluN2B Subunit-Containing NMDA Receptor Underlies Serotonergic Regulation of Long-Term Potentiation after Critical Period in the Rat Visual Cortex

    PubMed Central

    Joo, Kayoung; Rhie, Duck-Joo

    2015-01-01

    Serotonin [5-hydroxytryptamine (5-HT)] regulates synaptic plasticity in the visual cortex. Although the effects of 5-HT on plasticity showed huge diversity depending on the ages of animals and species, it has been unclear how 5-HT can show such diverse effects. In the rat visual cortex, 5-HT suppressed long-term potentiation (LTP) at 5 weeks but enhanced LTP at 8 weeks. We speculated that this difference may originate from differential regulation of neurotransmission by 5-HT between the age groups. Thus, we investigated the effects of 5-HT on apha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-, γ-aminobutyric acid receptor type A (GABAAR)-, and N-methyl-D-aspartic acid receptor (NMDAR)-mediated neurotransmissions and their involvement in the differential regulation of plasticity between 5 and 8 weeks. AMPAR-mediated currents were not affected by 5-HT at both 5 and 8 weeks. GABAAR-mediated currents were enhanced by 5-HT at both age groups. However, 5-HT enhanced NMDAR-mediated currents only at 8 weeks. The enhancement of NMDAR-mediated currents appeared to be mediated by the enhanced function of GluN2B subunit-containing NMDAR. The enhanced GABAAR- and NMDAR-mediated neurotransmissions were responsible for the suppression of LTP at 5 weeks and the facilitation of LTP at 8 weeks, respectively. These results indicate that the effects of 5-HT on neurotransmission change with development, and the changes may underlie the differential regulation of synaptic plasticity between different age groups. Thus, the developmental changes in 5-HT function should be carefully considered while investigating the 5-HT-mediated metaplastic control of the cortical network. PMID:26557019

  19. Subunits of the Schizosaccharomyces pombe RNA polymerase II: enzyme purification and structure of the subunit 3 gene.

    PubMed Central

    Azuma, Y; Yamagishi, M; Ishihama, A

    1993-01-01

    To improve our understanding of the structure and function of eukaryotic RNA polymerase II, we purified the enzyme from the fission yeast Schizosaccharomyces pombe. The highly purified RNA polymerase II contained more than eleven polypeptides. The sizes of the largest the second-, and the third-largest polypeptides as measured by SDS-polyacrylamide gel electrophoresis were about 210, 150, and 40 kilodaltons (kDa), respectively, and are similar to those of RPB1, 2, and 3 subunits of Saccharomyces cerevisiae RNA polymerase II. Using the degenerated primers designed after amino acid micro-sequencing of the 40 kDa third-largest polypeptide (subunit 3), we cloned the subunit 3 gene (rpb3) and determined its DNA sequence. Taken together with the sequence of parts of PCR-amplified cDNA, the predicted coding sequence of rpb3, interrupted by two introns, was found to encode a polypeptide of 297 amino acid residues in length with a molecular weight of 34 kDa. The S. pombe subunit 3 contains four structural domains conserved for the alpha-subunit family of RNA polymerase from both eukaryotes and prokaryotes. A putative leucine zipper motif was found to exist in the C-terminal proximal conserved region (domain D). Possible functions of the conserved domains are discussed. Images PMID:8367291

  20. Transcription factor assembly on the nicotinic receptor beta4 subunit gene promoter.

    PubMed

    Scofield, Michael D; Brüschweiler-Li, Lei; Mou, Zhongming; Gardner, Paul D

    2008-04-16

    Nicotinic acetylcholine receptors are involved in a plethora of fundamental biological processes ranging from muscle contraction to formation of memories. The receptors are pentameric proteins whose subunits are encoded by distinct genes. Subunit composition of a mature nicotinic receptor is governed in part by the transcriptional regulation of each subunit gene. Here, using chromatin immunoprecipitation assays, we report the interaction of the transcription factors Sp1, Sp3, c-Jun and Sox10 with the beta4 subunit gene promoter in neuronal-like cell lines and rodent brain tissue. Our results corroborate previous in-vitro data demonstrating that these transcription factors interact with the beta4 promoter. Taken together, these data suggest that Sp1, Sp3, c-Jun and Sox10 regulate expression of the beta4 subunit gene in the mammalian brain. PMID:18382288

  1. Bigenomic transcriptional regulation of all thirteen cytochrome c oxidase subunit genes by specificity protein 1

    PubMed Central

    Dhar, Shilpa S.; Johar, Kaid; Wong-Riley, Margaret T. T.

    2013-01-01

    Cytochrome c oxidase (COX) is one of only four known bigenomic proteins, with three mitochondria-encoded subunits and 10 nucleus-encoded ones derived from nine different chromosomes. The mechanism of regulating this multi-subunit, bigenomic enzyme is not fully understood. We hypothesize that specificity protein 1 (Sp1) functionally regulates the 10 nucleus-encoded COX subunit genes directly and the three mitochondrial COX subunit genes indirectly by regulating mitochondrial transcription factors A and B (TFAM, TFB1M and TFB2M) in neurons. By means of in silico analysis, electrophoretic mobility shift and supershift assays, chromatin immunoprecipitation, RNA interference and over-expression experiments, the present study documents that Sp1 is a critical regulator of all 13 COX subunit genes in neurons. This regulation is intimately associated with neuronal activity. Silencing of Sp1 prevented the upregulation of all COX subunits by KCl, and over-expressing Sp1 rescued all COX subunits from being downregulated by tetrodotoxin. Thus, Sp1 and our previously described nuclear respiratory factors 1 and 2 are the three key regulators of all 13 COX subunit genes in neurons. The binding sites for Sp1 on all 10 nucleus-encoded COX subunits, TFAM, TFB1M and TFB2M are highly conserved among mice, rats and humans. PMID:23516108

  2. A Case of Congenital Central Hypoventilation Syndrome with PHOX2B Gene Mutation in a Korean Neonate

    PubMed Central

    Kwon, Kyoung-Ah; Byun, Shin-Yun; Kim, Shine-Young; Hwang, Sang-Hyoun

    2010-01-01

    Congenital central hypoventilation syndrome (CCHS) is a life-threatening disorder with apnea and cyanosis during sleep requiring immediate endotracheal intubation during the first day of life. The PHOX2B gene has been identified as the major gene involved in CCHS. This is the first report of a Korean neonate with CCHS confirmed to have a PHOX2B mutation with expanded alleles containing 20 polyalanine repeats that is a relatively small number compared to previous cases. The patient required intermittent ventilator support during sleep only and did not suffer from any other disorders of the autonomic nerve system. He consistently needs ventilator support during sleep and remains alive. Analysis of PHOX2B gene is useful for diagnosis and appropriate therapeutic intervention of CCHS patients. PMID:20676341

  3. Mutations in Human Interferon α2b Gene and Potential as Risk Factor Associated with Female Breast Cancer.

    PubMed

    Ahmed, Fayyaz; Mahmood, Nasir; Shahid, Saman; Hussain, Zahid; Ahmed, Ishtiaq; Jalal, Amir; Ijaz, Bushra; Shahid, Abubaker; Mujtaba, Ghulam; Mustafa, Tanveer

    2016-08-01

    The current study explored the potential links between breast cancer and human interferon α-2b (hIFNα-2b) gene mutations. The hIFNα-2b gene was amplified from breast cancer tumor tissue samples (N = 60) by polymerase chain reaction (PCR) and the products were subjected to gene sequencing. A total of 38 (63.3%) samples showed positive PCR amplification results. Several of these also exhibited frequent alterations (mutations) after 400 bp and, in particular, adenine was replaced by other bases. A total of 19 selected mutated amino acids were analyzed for local/general fold pattern changes. Human IFNα-2b receptor (IFNAR): ligand (hIFNα-2b protein) interactions through a Z-DOCK (3.0.2) server were also evaluated to assess the binding patterns of each ligand to receptor to induce Janus-Kinase-signal transducer and activator of transcription antiproliferative signal transduction pathway inside the cancer cells. Certain local structural and conformational changes were predicted to be induced by mutations in the ligand. The variant models of the hIFNα-2b displayed structural and conformational changes that signified that changes to hIFNα-2b may be a risk factor in addition to other known factors associated with onset/progression of female breast carcinoma. It was hoped that others might build upon the research in this study evaluating protein structural models with mutations and their consequent interactions with receptors in the development of potent immune therapeutic drugs for breast cancer that are based on recombinant hIFNα-2b. PMID:27403569

  4. Evolution of the primate cytochrome c oxidase subunit II gene.

    PubMed

    Adkins, R M; Honeycutt, R L

    1994-03-01

    We examined the nucleotide and amino acid sequence variation of the cytochrome c oxidase subunit II (COII) gene from 25 primates (4 hominoids, 8 Old World monkeys, 2 New World monkeys, 2 tarsiers, 7 lemuriforms, 2 lorisiforms). Marginal support was found for three phylogenetic conclusions: (1) sister-group relationship between tarsiers and a monkey/ape clade, (2) placement of the aye-aye (Daubentonia) sister to all other strepsirhine primates, and (3) rejection of a sister-group relationship of dwarf lemurs (i.e., Cheirogaleus) with lorisiform primates. Stronger support was found for a sister-group relationship between the ring-tail lemur (Lemur catta) and the gentle lemurs (Hapalemur). In congruence with previous studies on COII, we found that the monkeys and apes have undergone a nearly two-fold increase in the rate of amino acid replacement relative to other primates. Although functionally important amino acids are generally conserved among all primates, the acceleration in amino acid replacements in higher primates is associated with increased variation in the amino terminal end of the protein. Additionally, the replacement of two carboxyl-bearing residues (glutamate and aspartate) at positions 114 and 115 may provide a partial explanation for the poor enzyme kinetics in cross-reactions between the cytochromes c and cytochrome c oxidases of higher primates and other mammals. PMID:8006990

  5. Relationship between Human Evolution and Neurally Mediated Syncope Disclosed by the Polymorphic Sites of the Adrenergic Receptor Gene α2B-AR

    PubMed Central

    Komiyama, Tomoyoshi; Oka, Akira; Kamiguchi, Hiroshi; Nagata, Eiichiro; Sakura, Hiroshi; Otsuka, Kuniaki; Kobayashi, Hiroyuki

    2015-01-01

    The objective of this study was to clarify the effects of disease on neurally mediated syncope (NMS) during an acute stress reaction. We analyzed the mechanism of the molecular interaction and the polymorphisms of the alpha-2 adrenoreceptor (α2B-AR) gene as the potential psychiatric cause of incentive stress. We focused on the following three genotypes of the repeat polymorphism site at Glu 301–303 in the α2B-AR gene: Glu12/12, Glu12/9, and Glu9/9. On the basis of our clinical research, NMS is likely to occur in people with the Glu12/9 heterotype. To verify this, we assessed this relationship with the interaction of Gi protein and adenylate cyclase by in silico analysis of the Glu12/9 heterotype. By measuring the difference in the dissociation time of the Gi-α subunit twice, we found that the Glu12/9 heterotype suppressed the action of adenylate cyclase longer than the Glu homotypes. As this difference in the Glu repeat number effect is thought to be one of the causes of NMS, we investigated the evolutionary significance of the Glu repeat number. Glu8 was originally repeated in simians, while the Glu12 repeats occurred over time during the evolution of bipedalism in humans. Taken with the Glu12 numbers, NMS would likely become a defensive measure to prevent significant blood flow to the human brain. PMID:25860977

  6. Relationship between human evolution and neurally mediated syncope disclosed by the polymorphic sites of the adrenergic receptor gene α2B-AR.

    PubMed

    Komiyama, Tomoyoshi; Hirokawa, Takatsugu; Sato, Kyoko; Oka, Akira; Kamiguchi, Hiroshi; Nagata, Eiichiro; Sakura, Hiroshi; Otsuka, Kuniaki; Kobayashi, Hiroyuki

    2015-01-01

    The objective of this study was to clarify the effects of disease on neurally mediated syncope (NMS) during an acute stress reaction. We analyzed the mechanism of the molecular interaction and the polymorphisms of the alpha-2 adrenoreceptor (α2B-AR) gene as the potential psychiatric cause of incentive stress. We focused on the following three genotypes of the repeat polymorphism site at Glu 301-303 in the α2B-AR gene: Glu12/12, Glu12/9, and Glu9/9. On the basis of our clinical research, NMS is likely to occur in people with the Glu12/9 heterotype. To verify this, we assessed this relationship with the interaction of Gi protein and adenylate cyclase by in silico analysis of the Glu12/9 heterotype. By measuring the difference in the dissociation time of the Gi-α subunit twice, we found that the Glu12/9 heterotype suppressed the action of adenylate cyclase longer than the Glu homotypes. As this difference in the Glu repeat number effect is thought to be one of the causes of NMS, we investigated the evolutionary significance of the Glu repeat number. Glu8 was originally repeated in simians, while the Glu12 repeats occurred over time during the evolution of bipedalism in humans. Taken with the Glu12 numbers, NMS would likely become a defensive measure to prevent significant blood flow to the human brain. PMID:25860977

  7. Network analysis of neurotransmitter related human kinase genes: possible role of SRC, RAF1, PTK2B?

    PubMed

    Brys, Zoltan; Pluhar, Andras; Kis, Janos Tibor; Buda, Bela; Szabo, Attila

    2013-09-01

    Previous co-expression analysis of human kinase genes highlighted 119 genes in neurotransmitter-related activity (based on Go:Terms). Using a merged interactome dataset, we analyzed the network of these Neurotransmitter Related Human Kinase Genes. Using the full interactome dataset we extended the network and calculating degrees and closeness centralities we identified SRC, MAPK1, RAF1, PTK2B and AKT1 kinase genes as potentially relevant nodes which did not show relevant activity in the original experimental study. As AKT1 and MAPK1 have already been indicated in various neuronal functions, we hypothesize a potential direct or indirect role for SRC, RAF1, PTK2B genes in neurotransmission and in central nervous system signaling processes. PMID:24108181

  8. Higher levels of phosphorylated Y1472 on GluN2B subunits in the frontal cortex of aged mice are associated with good spatial reference memory, but not cognitive flexibility.

    PubMed

    Zamzow, Daniel R; Elias, Val; Acosta, Varinia A; Escobedo, Emily; Magnusson, Kathy R

    2016-06-01

    The N-methyl-D-aspartate receptor (NMDAr) is particularly vulnerable to aging. The GluN2B subunit of the NMDAr, compared to other NMDAr subunits, suffers the greatest losses of expression in the aging brain, especially in the frontal cortex. While expression levels of GluN2B mRNA and protein in the aged brain are well documented, there has been little investigation into age-related posttranslational modifications of the subunit. In this study, we explored some of the mechanisms that may promote differences in the NMDAr complex in the frontal cortex of aged animals. Two ages of mice, 3 and 24 months, were behaviorally tested in the Morris water maze. The frontal cortex and hippocampus from each mouse were subjected to differential centrifugation followed by solubilization in Triton X-100. Proteins from Triton-insoluble membranes, Triton-soluble membranes, and intracellular membranes/cytosol were examined by Western blot. Higher levels of GluN2B tyrosine 1472 phosphorylation in frontal cortex synaptic fractions of old mice were associated with better reference learning but poorer cognitive flexibility. Levels of GluN2B phosphotyrosine 1336 remained steady, but there were greater levels of the calpain-induced 115 kDa GluN2B cleavage product on extrasynaptic membranes in these old good learners. There was an age-related increase in calpain activity, but it was not associated with better learning. These data highlight a unique aging change for aged mice with good spatial learning that might be detrimental to cognitive flexibility. This study also suggests that higher levels of truncated GluN2B on extrasynaptic membranes are not deleterious to spatial memory in aged mice. PMID:27094400

  9. Cytochrome oxidase subunit V gene of Neurospora crassa: DNA sequences, chromosomal mapping, and evidence that the cya-4 locus specifies the structural gene for subunit V.

    PubMed Central

    Sachs, M S; Bertrand, H; Metzenberg, R L; RajBhandary, U L

    1989-01-01

    The sequences of cDNA and genomic DNA clones for Neurospora cytochrome oxidase subunit V show that the protein is synthesized as a 171-amino-acid precursor containing a 27-amino-acid N-terminal extension. The subunit V protein sequence is 34% identical to that of Saccharomyces cerevisiae subunit V; these proteins, as well as the corresponding bovine subunit, subunit IV, contain a single hydrophobic domain which most likely spans the inner mitochondrial membrane. The Neurospora crassa subunit V gene (cox5) contains two introns, 398 and 68 nucleotides long, which share the conserved intron boundaries 5'GTRNGT...CAG3' and the internal consensus sequence ACTRACA. Two short sequences, YGCCAG and YCCGTTY, are repeated four times each in the cox5 gene upstream of the mRNA 5' termini. The cox5 mRNA 5' ends are heterogeneous, with the major mRNA 5' end located 144 to 147 nucleotides upstream from the translational start site. The mRNA contains a 3'-untranslated region of 186 to 187 nucleotides. Using restriction-fragment-length polymorphism, we mapped the cox5 gene to linkage group IIR, close to the arg-5 locus. Since one of the mutations causing cytochrome oxidase deficiency in N. crassa, cya-4-23, also maps there, we transformed the cya-4-23 strain with the wild-type cox5 gene. In contrast to cya-4-23 cells, which grow slowly, cox5 transformants grew quickly, contained cytochrome oxidase, and had 8- to 11-fold-higher levels of subunit V in their mitochondria. These data suggest (i) that the cya-4 locus in N. crassa specifies structural information for cytochrome oxidase subunit V and (ii) that, in N. crassa, as in S. cerevisiae, deficiencies in the production of nuclearly encoded cytochrome oxidase subunits result in deficiency in cytochrome oxidase activity. Finally, we show that the lower levels of subunit V in cya-4-23 cells are most likely due to substantially reduced levels of translatable subunit V mRNA. Images PMID:2540423

  10. Global Gene Expression Profiling of Myeloid Immune Cell Subsets in Response to In Vitro Challenge with Porcine Circovirus 2b

    PubMed Central

    Mavrommatis, Bettina; Offord, Victoria; Patterson, Robert; Watson, Mick; Kanellos, Theo; Steinbach, Falko; Grierson, Sylvia; Werling, Dirk

    2014-01-01

    Compelling evidence suggests that the early interaction between porcine circovirus 2 (PCV-2) and the innate immune system is the key event in the pathogenesis of Post-Weaning Multisystemic Wasting Syndrome (PMWS). Furthermore, PCV2 has been detected in bone-marrow samples, potentially enabling an easy spread and reservoir for the virus. To assess the gene-expression differences induced by an in-vitro PCV2b infection in different three different myeloid innate immune cell subsets generated from the same animal, we used the Agilent Porcine Gene Expression Microarray (V2). Alveolar macrophages (AMØs), monocyte-derived dendritic cells (MoDCs) and bone-marrow cells (BMCs) were generated from each animal, and challenged with a UK-isolate of a PCV2 genotype b-strain at a MOI of 0.5. Remarkably, analysis showed a highly distinct and cell-type dependent response to PCV2b challenge. Overall, MoDCs showed the most marked response to PCV2b challenge in vitro and revealed a key role for TNF in the interaction with PCV2b, whereas only few genes were affected in BMCs and AMØs. These observations were further supported by an enrichment of genes in the downstream NF-κB Signalling pathway as well as an up regulation of genes with pro-apoptotic functions post-challenge. PCV2b challenge increases the expression of a large number of immune-related and pro-apoptotic genes mainly in MoDC, which possibly explain the increased inflammation, granulomatous inflammation and lymphocyte depletion seen in PMWS-affected pigs. PMID:24618842

  11. Reconstitutable Charged Polymeric (PLGA)2-b-PEI Micelles for Gene Therapeutics Delivery

    PubMed Central

    Mishra, Deepa; Kang, Han Chang; Bae, You Han

    2011-01-01

    This study investigated the potential of creating a charged polymeric micelle-based nucleic acid delivery system that could easily be reconstituted by the addition of water. (PLGA36kDa)2-b-bPEI25kDa (PLGA MW 36kDa, bPEI Mw 25kDa, PLGA:bPEI block ratio = 2) was synthesized and used to prepare cationic micelles. The copolymer retained proton-buffering capability from the bPEI block within the endosomal pH range. Micelle/pDNA complexes retained their particle size (100–150 nm) and surface charge (30–40 mV) following reconstitution. It was found that adding a small amount of low molecular weight bPEI (1.8 kDa) completely shielded pDNA in the micelle/pDNA complexes and enhanced transfection efficiency 50–100 fold for both fresh and reconstituted complexes without affecting complex size. Transfection efficiency for “reconstituted” micelle/pDNA/bPEI1.8kDa (WR 1) complexes was 16-fold higher than its “fresh” counterpart. Although transfection levels achieved using “reconstituted” micelle/pDNA/bPEI1.8kDa complexes were 3.6-fold lower than control “fresh” bPEI25kDa/pDNA (N/P 5) complexes, transfection levels were 39-fold higher than “reconstituted” bPEI25kDa/pDNA (N/P 5) complexes. The micelle/pDNA/bPEI1.8kDa system showed very low cytotoxicity in MCF7 cells even with pDNA doses up to 20 μg, and transfection levels increased linearly with increasing pDNA dose. These results indicate that this PLGA-b-bPEI polymeric micelle-based system is well suited as a reconstitutable gene delivery system, and has high potential for use as a delivery system for gene therapy applications. PMID:21354616

  12. The 5-HT2B receptor gene maps to 2q36.3-2q37.1

    SciTech Connect

    Le Coniat, M.; Berger, R.; Choi, Doo-Sup; Maroteaux, L.

    1996-02-15

    This article reports on the localization of the serotonin 5-HT2B receptor to human chromosome 2q36.3-2q37.1 using fluorescence in situ hybridization. The structure and function of this gene, as well as its expression, remain to be investigated in the human. 9 refs.

  13. Slice orientation and muscarinic acetylcholine receptor activation determine the involvement of N-methyl D-aspartate receptor subunit GluN2B in hippocampal area CA1 long-term depression

    PubMed Central

    2011-01-01

    Background The contribution of different GluN2 subunits of the N-methyl D-aspartate (NMDA) receptor to the induction of bidirectional hippocampal synaptic plasticity is a controversial topic. As both supporting and refuting evidence for the hypothesis of subunit specialization in opposing directions of plasticity has accumulated since it was first proposed a few years ago, we hypothesize that differences in experimental conditions may have in part contributed to some of the inconsistent results from these studies. Here we investigate the controversial hypothesis that long-term depression (LTD) is preferentially induced by GluN2B-containing NMDA receptors in area CA1 of hippocampal slices. Results We find that brain slices from 2-3 week old rats prepared in the sagittal orientation have GluN2B-independent LTD whereas slices prepared in the coronal orientation have GluN2B-dependent LTD. There was no difference between the orientations in the fraction of the NMDAR EPSC sensitive to a GluN2B-selective antagonist, leading us to believe that the intracellular signaling properties of the NMDARs were different in the two preparations. Coronal slices had greater association of LTD-related intracellular signaling protein RasGRF1 with GluN2B relative to sagittal slices. Antagonism of muscarinic acetylcholine receptors (mAChRs) in the sagittal slices returned LTD to a GluN2B-dependent form and increased the association of GluN2B with RasGRF1. Conclusions These results suggest a novel form of NMDAR modulation by mAChRs and clarify some disagreement in the literature. PMID:22082088

  14. A Special Extract of Bacopa monnieri (CDRI-08) Restores Learning and Memory by Upregulating Expression of the NMDA Receptor Subunit GluN2B in the Brain of Scopolamine-Induced Amnesic Mice

    PubMed Central

    Rai, Rakesh; Singh, Hemant K.; Prasad, S.

    2015-01-01

    In the present communication, we have investigated effects of the CDRI-08, a well characterized extract of Bacopa monnieri, on expression of the GluN2B subunit of NMDAR in various brain regions of the scopolamine-induced amnesic mice. Our behavioral data reveal that scopolamine-treated amnesic mice exhibit significant decline in the spatial memory compared to the normal control mice. Our RT-PCR and immunoblotting data revealed that the scopolamine treatment resulted in a significant downregulation of the NMDAR GluN2B subunit expression in prefrontal cortex and hippocampus. Our enzyme assay data revealed that scopolamine caused a significant increase in the acetylcholinesterase activity in both the brain regions. Further, oral administration of the CDRI-08 to scopolamine-treated amnesic mice restored the spatial memory which was found to be associated with significant upregulation of the GluN2B subunit expression and decline in the acetylcholinesterase activity in prefrontal cortex as well as hippocampus towards their levels in the normal control mice. Our study provides the evidence for the mechanism underlying role of the Bacopa monnieri extract (CDRI-08) in restoring spatial memory in amnesic mice, which may have therapeutic implications. PMID:26413117

  15. The role of GluN2A and GluN2B NMDA receptor subunits in AgRP and POMC neurons on body weight and glucose homeostasis

    PubMed Central

    Üner, Aykut; Gonçalves, Gabriel H.M.; Li, Wenjing; Porceban, Matheus; Caron, Nicole; Schönke, Milena; Delpire, Eric; Sakimura, Kenji; Bjørbæk, Christian

    2015-01-01

    Objective Hypothalamic agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) expressing neurons play critical roles in control of energy balance. Glutamatergic input via n-methyl-d-aspartate receptors (NMDARs) is pivotal for regulation of neuronal activity and is required in AgRP neurons for normal body weight homeostasis. NMDARs typically consist of the obligatory GluN1 subunit and different GluN2 subunits, the latter exerting crucial differential effects on channel activity and neuronal function. Currently, the role of specific GluN2 subunits in AgRP and POMC neurons on whole body energy and glucose balance is unknown. Methods We used the cre-lox system to genetically delete GluN2A or GluN2B only from AgRP or POMC neurons in mice. Mice were then subjected to metabolic analyses and assessment of AgRP and POMC neuronal function through morphological studies. Results We show that loss of GluN2B from AgRP neurons reduces body weight, fat mass, and food intake, whereas GluN2B in POMC neurons is not required for normal energy balance control. GluN2A subunits in either AgRP or POMC neurons are not required for regulation of body weight. Deletion of GluN2B reduces the number of AgRP neurons and decreases their dendritic length. In addition, loss of GluN2B in AgRP neurons of the morbidly obese and severely diabetic leptin-deficient Lepob/ob mice does not affect body weight and food intake but, remarkably, leads to full correction of hyperglycemia. Lepob/ob mice lacking GluN2B in AgRP neurons are also more sensitive to leptin's anti-obesity actions. Conclusions GluN2B-containing NMDA receptors in AgRP neurons play a critical role in central control of body weight homeostasis and blood glucose balance via mechanisms that likely involve regulation of AgRP neuronal survival and structure, and modulation of hypothalamic leptin action. PMID:26500840

  16. Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats.

    PubMed

    Kobayashi, Misato; Hoshinaga, Yukiko; Miura, Natsuko; Tokuda, Yuki; Shigeoka, Shigeru; Murai, Atsushi; Horio, Fumihiko

    2014-01-01

    The mechanisms underlying the decrease in hepatic cytochrome P-450 (CYP) content in ascorbic acid deficiency was investigated in scurvy-prone ODS rats. First, male ODS rats were fed a diet containing sufficient ascorbic acid (control) or a diet without ascorbic acid (deficient) for 18 days, with or without the intraperitoneal injection of phenobarbital. Ascorbic acid deficiency decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial cytochrome oxidase (COX) complex IV subunit I protein, and simultaneously increased heme oxygenase-1 protein in microsomes and mitochondria. Next, heme oxygenase-1 inducers, that is lipopolysaccharide and hemin, were administered to phenobaribital-treated ODS rats fed sufficient ascorbic acid. The administration of these inducers decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial COX complex IV subunit I protein. These results suggested that the stimulation of hepatic heme oxygenase-1 expression by ascorbic acid deficiency caused the decrease in CYP content in liver. PMID:25036135

  17. Dexmedetomidine protects against learning and memory impairments caused by electroconvulsive shock in depressed rats: Involvement of the NMDA receptor subunit 2B (NR2B)-ERK signaling pathway.

    PubMed

    Gao, Xin; Zhuang, Fu-Zhi; Qin, Shou-Jun; Zhou, Li; Wang, Yun; Shen, Qing-Feng; Li, Mei; Villarreal, Michelle; Benefield, Lauren; Gu, Shu-Ling; Ma, Teng-Fei

    2016-09-30

    Cognitive impairment is a common adverse effect of electroconvulsive therapy (ECT) during treatment for severe depression. Dexmedetomidine (DEX), a sedative-anesthetic drug, is used to treat post-ECT agitation. However, it is not known if DEX can protect against ECT-induced cognitive impairments. To address this, we used chronic unpredictable mild stress (CUMS) to establish a model of depression for ECT treatment. Our Morris water maze and sucrose preference test results suggest that DEX alleviates ECT-induced learning and memory impairments without altering the antidepressant efficacy of ECT. To further investigate the underlying mechanisms of DEX, hippocampal expression of NR2B, p-ERK/ERK, p-CREB/CREB, and BDNF were quantified by western blotting. These results show that DEX suppresses over-activation of NR2B and enhances phosphorylation of ERK1/2 in the hippocampus of ECT-treated depressed rats. Furthermore, DEX had no significant effect on ECT-induced increases in p-CREB and BDNF. Overall, our findings suggest that DEX ameliorates ECT-induced learning and memory impairments in depressed rats via the NR2B-ERK signaling cascade. Moreover, CREB/BDNF seems not appear to participate in the cognitive protective mechanisms of DEX during ECT treatment. PMID:27455425

  18. Blocking GluN2B subunits reverses the enhanced seizure susceptibility after prolonged febrile seizures with a wide therapeutic time-window.

    PubMed

    Chen, Bin; Feng, Bo; Tang, Yangshun; You, Yi; Wang, Yi; Hou, Weiwei; Hu, Weiwei; Chen, Zhong

    2016-09-01

    Febrile seizures (FSs), the most common type of convulsive events in infants, are closely associated with temporal lobe epilepsy (TLE) in adulthood. It is urgent to investigate how FSs promote epileptogenesis and find the potential therapeutic targets. In the present study, we showed that the phosphorylation of GluN2B Tyr1472 gradually reached peak level at 24h after prolonged FSs and remained elevated during 7days thereafter. IL-1β treatment alone, which in previous study mimicked the effect of prolonged FSs on adult seizure susceptibility, increased GluN2B Tyr1472 phosphorylation. Both IL-1 receptor antagonist (IL-1Ra) and IL-1R1 deletion were sufficient to reverse the prolonged FSs induced hyper-phosphorylation of GluN2B Tyr1472. GluN2B antagonist ifenprodil showed a wide therapeutic time-window (3days) to reverse the enhanced seizure susceptibility after prolonged FSs or IL-1β treatment. Our study demonstrated that GluN2B phosphorylation at Tyr1472 site mediated by the transient increase of IL-1β was involved in the enhanced adult seizure susceptibility after prolonged FSs, implicating GluN2B-containing NMDAR is a new potential drug target with a wide therapeutic time window to prevent epileptogenesis in patients with infantile FSs. PMID:27240522

  19. Effect of Histone Acetylation on N-Methyl-D-Aspartate 2B Receptor Subunits and Interleukin-1 Receptors in Association with Nociception-Related Somatosensory Cortex Dysfunction in a Mouse Model of Sepsis.

    PubMed

    Imamura, Yukio; Yoshikawa, Nao; Murkami, Yuki; Mitani, Satoko; Matsumoto, Naoya; Matsumoto, Hisatake; Yamada, Tomoki; Yamakawa, Kazuma; Nakagawa, Junichiro; Ogura, Hiroshi; Shimazu, Takeshi; Jin, Takashi

    2016-06-01

    Whole-body inflammation (i.e., sepsis) often results in brain-related sensory dysfunction. We previously reported that interleukin (IL)-1 resulted in synaptic dysfunction of septic encephalopathy, but the underlying molecular mechanisms remain unknown, as do effective treatments. Using mice, we examined immunohistochemistry, co-immunoprecipitation, enzyme-linked immunosorbent assay, and behavior analyses, and investigated the role of the N-methyl-D-aspartate 2B subunit (NR2B) of NMDA receptor, IL-1 receptor, and histone acetylation in the pathophysiology underlying sensory dysfunction induced by lipopolysaccharide (LPS). Mice groups of sham-operated, LPS, LPS with an NR2B antagonist, or LPS with resveratrol (a histone acetylation activator) were analyzed. We found that LPS increased NR2B and interleukin-1 receptor (IL-1R) immunoreactivity. The expression of Iba1, a marker for microglia and/or macrophages, increased more significantly in the brain than in the spinal cord, implicating NR2B and IL-1R in brain inflammation. Immunoprecipitation with NR2B and IL-1R revealed related antibodies. Blood levels of IL-1β (i.e., the IL-1R ligand) increased, though not significantly, suggesting that inflammation peaked at 20 h. Behavioral assessments of central (CNS) and peripheral sensory (PNS) function indicated that LPS delayed CNS but not PNS escape latency. Finally, NR2B antagonist or resveratrol in the lateral ventricle antagonized the effects of LPS in the brain and improved animal survival. In summary, histone acetylation may control expression of NR2B and IL-1R, alleviating inflammation-induced sensory neuronal dysfunction caused by LPS. PMID:26682951

  20. Chronic brain inflammation causes a reduction in GluN2A and GluN2B subunits of NMDA receptors and an increase in the phosphorylation of mitogen-activated protein kinases in the hippocampus

    PubMed Central

    2014-01-01

    Neuroinflammation plays a key role in the initiation and progression of neurodegeneration in Alzheimer’s disease (AD). Chronic neuroinflammation results in diminished synaptic plasticity and loss of GluN1 N-methyl-D-aspartate (NMDA) receptors in the hippocampus, leading to the cognitive deficits that are the most common symptoms of AD. Therefore, it is suggested that chronic inflammation may alter expression levels of GluN2A and GluN2B subunits of NMDA receptors and associated intracellular signalling. Chronic neuroinflammation was induced by chronic infusion of lipopolysaccharide (LPS) into the fourth ventricle in Fischer-344 rats. The status of hippocampus-dependent memory was evaluated in control rats and rats chronically infused with LPS. Microglial activation in the hippocampus was examined using immunohistochemical staining. Western blot analysis was used to measure membrane levels of GluN2A and GluN2B subunits of NMDA receptors and mitogen-activated protein kinase (MAPK) in the hippocampi of these rats, and immunofluorescent double labeling was used to assess the cellular location of MAPK. Microglial activation was observed in the hippocampi of rats that showed memory impairments with chronic LPS infusion. Chronic LPS infusion reduced the levels of GluN2A and GluN2B and increased the levels of phosphorylated MAPKs in the hippocampus. MAPK-positive immunoreactivity was observed mostly in the neurons and also in non-neuronal cells. Reductions in GluN2A and GluN2B subunits of NMDA receptors coupled with altered MAPK signaling, in response to inflammatory stimuli may be related to the cognitive deficits observed in AD. PMID:24761931

  1. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.

    PubMed

    Blatt, C; Eversole-Cire, P; Cohn, V H; Zollman, S; Fournier, R E; Mohandas, L T; Nesbitt, M; Lugo, T; Jones, D T; Reed, R R

    1988-10-01

    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding alpha-subunit proteins, two different beta subunits, and one gamma subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The beta subunits were also assigned--GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins. PMID:2902634

  2. Genomic cloning and characterization of the rat glutathione S-transferase-A3-subunit gene.

    PubMed Central

    Fotouhi-Ardakani, N; Batist, G

    1999-01-01

    The rat glutathione S-transferase-A3-subunit (GSTA3) gene is a member of the class Alpha GSTs, which we have previously reported to be overexpressed in anti-cancer-drug-resistant cells. In this study, we report the isolation and characterization of the entire rat GSTA3 (rGST Yc1) subunit gene. The rat GSTA3 subunit gene is approximately 15 kb in length and consists of seven exons interrupted by introns of different lengths. Exon 1, with a length of 219 bp, contains only the 5'-untranslated region of the gene. Each exon-intron splicing junction exhibited the consensus sequence for a mammalian splice site. The transcription start site and exon 1 of rat GSTA3 were characterized by a combination of primer extension and rapid amplification of the cDNA ends. Position +1 was identified 219 bp upstream of the first exon-intron splicing junction. The proximal promoter region of the rat GSTA3 subunit gene does not contain typical TATA or CAAT boxes. A computer-based search for potential transcription-factor binding sites revealed the existence of a number of motifs such as anti-oxidant-responsive element, ras-response element, activator protein-1, nuclear factor-kappaB, cAMP-response-element-binding protein, Barbie box and E box. The functional activity of the regulatory region of the rat GSTA3 subunit gene was shown by its ability to drive the expression of a chloramphenicol acetyltransferase reporter gene in rat mammary carcinoma cells, and its activity was greater in melphalan-resistant cells known to have transcriptional activation of this gene by previous studies. The structure of the gene, with a large intron upstream of the translation-initiation site, may explain why the isolation of this promoter has been so elusive. This information will provide the opportunity to examine the involvement of the rat GSTA3 subunit gene in drug resistance and carcinogenesis. PMID:10215608

  3. Genomic cloning and characterization of the rat glutathione S-transferase-A3-subunit gene.

    PubMed

    Fotouhi-Ardakani, N; Batist, G

    1999-05-01

    The rat glutathione S-transferase-A3-subunit (GSTA3) gene is a member of the class Alpha GSTs, which we have previously reported to be overexpressed in anti-cancer-drug-resistant cells. In this study, we report the isolation and characterization of the entire rat GSTA3 (rGST Yc1) subunit gene. The rat GSTA3 subunit gene is approximately 15 kb in length and consists of seven exons interrupted by introns of different lengths. Exon 1, with a length of 219 bp, contains only the 5'-untranslated region of the gene. Each exon-intron splicing junction exhibited the consensus sequence for a mammalian splice site. The transcription start site and exon 1 of rat GSTA3 were characterized by a combination of primer extension and rapid amplification of the cDNA ends. Position +1 was identified 219 bp upstream of the first exon-intron splicing junction. The proximal promoter region of the rat GSTA3 subunit gene does not contain typical TATA or CAAT boxes. A computer-based search for potential transcription-factor binding sites revealed the existence of a number of motifs such as anti-oxidant-responsive element, ras-response element, activator protein-1, nuclear factor-kappaB, cAMP-response-element-binding protein, Barbie box and E box. The functional activity of the regulatory region of the rat GSTA3 subunit gene was shown by its ability to drive the expression of a chloramphenicol acetyltransferase reporter gene in rat mammary carcinoma cells, and its activity was greater in melphalan-resistant cells known to have transcriptional activation of this gene by previous studies. The structure of the gene, with a large intron upstream of the translation-initiation site, may explain why the isolation of this promoter has been so elusive. This information will provide the opportunity to examine the involvement of the rat GSTA3 subunit gene in drug resistance and carcinogenesis. PMID:10215608

  4. The Ad5 [E1-, E2b-]-based vector: a new and versatile gene delivery platform

    NASA Astrophysics Data System (ADS)

    Jones, Frank R.; Gabitzsch, Elizabeth S.; Balint, Joseph P.

    2015-05-01

    Based upon advances in gene sequencing and construction, it is now possible to identify specific genes or sequences thereof for gene delivery applications. Recombinant adenovirus serotype-5 (Ad5) viral vectors have been utilized in the settings of gene therapy, vaccination, and immunotherapy but have encountered clinical challenges because they are recognized as foreign entities to the host. This recognition leads to an immunologic clearance of the vector that contains the inserted gene of interest and prevents effective immunization(s). We have reported on a new Ad5-based viral vector technology that can be utilized as an immunization modality to induce immune responses even in the presence of Ad5 vector immunity. We have reported successful immunization and immunotherapy results to infectious diseases and cancers. This improved recombinant viral platform (Ad5 [E1-, E2b-]) can now be utilized in the development of multiple vaccines and immunotherapies.

  5. Increased efficacy of an adenovirus-vectored foot-and-mouth disease capsid subunit vaccine expressing nonstructural protein 2B is associated with a specific T cell response

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We previously demonstrated that an adenovirus-based FMDV serotype A24 subunit vaccine, Ad5-A24, expressed under the control of a cytomegalovirus promoter (CMV) can protect swine and bovines against homologous challenge, but swine vaccinated with an Ad5-vectored FMDV O1 Campos vaccine, Ad5-O1Campos (...

  6. Structure and sequence of the gene for the largest subunit of trypanosomal RNA polymerase III.

    PubMed Central

    Köck, J; Evers, R; Cornelissen, A W

    1988-01-01

    As the first step in the analysis of the transcription process in the African trypanosome, Trypanosoma brucei, we have started to characterise the trypanosomal RNA polymerases. We have previously described the gene encoding the largest subunit of RNA polymerase II and found that two almost identical RNA polymerase II genes are encoded within the genome of T. brucei. Here we present the identification, cloning and sequence analysis of the gene encoding the largest subunit of RNA polymerase III. This gene contains a single open reading frame encoding a polypeptide with a Mr of 170 kD. In total, eight encoding a polypeptide with a Mr of 170 kD. In total, eight highly conserved regions with significant homology to those previously reported in other eukaryotic RNA polymerase largest subunits were identified. Some of these domains contain functional sites, which are conserved among all eukaryotic largest subunit genes analysed thus far. Since these domains make up a large part of each polypeptide, independent of the RNA polymerase class, these data strongly support the hypothesis that these domains provide a major part of the transcription machinery of the RNA polymerase complex. The additional domains which are uniquely present in the largest subunit of RNA polymerase I and II, respectively, two large hydrophylic insertions and a C-terminal extension, might be a determining factor in specific transcription of the gene classes. Images PMID:3174432

  7. Direct retroviral delivery of human cytochrome P450 2B6 for gene-directed enzyme prodrug therapy of cancer.

    PubMed

    Kan, O; Griffiths, L; Baban, D; Iqball, S; Uden, M; Spearman, H; Slingsby, J; Price, T; Esapa, M; Kingsman, S; Kingsman, A; Slade, A; Naylor, S

    2001-07-01

    Human cytochrome P450 2B6 (CYP2B6) metabolizes the prodrug cyclophosphamide (CPA) to produce phosphoramide mustard that cross-links DNA leading to cell death. We have constructed a novel retroviral vector encoding CYP2B6 (designated "MetXia-P450") and used it to transduce the human tumor cell lines HT29 and T47D. MetXia-P450 transduction sensitised these cells to the cytotoxic effects of the prodrug CPA. Results from in vitro experiments demonstrated adverse effects on the clonogenic survival of cyclophosphamide-treated cells transduced with MetXia-P450. Cytotoxic activity accompanied by bystander effect was particularly evident in 3-D multicellular spheroid models suggesting that this in vitro system may be a more appropriate model for assessing the efficacy of gene directed-enzyme prodrug therapy (GDEPT). We have applied this approach in a clinically relevant gene therapy protocol on established subcutaneous tumor xenografts. These studies show for the first time the efficacy of a P450-based GDEPT strategy mediated by a direct retroviral gene transfer in vivo. PMID:11498768

  8. A novel subunit vaccine co-expressing GM-CSF and PCV2b Cap protein enhances protective immunity against porcine circovirus type 2 in piglets.

    PubMed

    Zhang, Huawei; Qian, Ping; Peng, Bo; Shi, Lin; Chen, Huanchun; Li, Xiangmin

    2015-05-15

    Porcine circovirus type 2 (PCV2) causes porcine circovirus-associated disease. Capsid (Cap) protein of PCV2 is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. GM-CSF is an immune adjuvant that enhances responses to vaccines. In this study, recombinant baculoviruses Ac-Cap and Ac-Cap-GM-CSF expressing the Cap protein alone and co-expressing the Cap protein and porcine GM-CSF, respectively, were constructed successfully. The target proteins were analyzed by western blotting and IFA. Further, these proteins were confirmed by electron microscopy, which showed that Cap proteins could self-assemble into virus-like particles having diameters of 17-25nm. Animal experiments showed that pigs immunized with Cap-GM-CSF subunit vaccine showed significantly higher levels of PCV2-specific antibodies and neutralizing antibodies than pigs immunized with the Cap subunit vaccine and a commercial vaccine (Ingelvac CircoFLEX; P<0.05). After PCV2 wild strain challenged, Pigs receiving the Cap-GM-CSF subunit vaccine showed significantly higher average daily weight gain after wild-type PCV2 challenge than pigs receiving the other three vaccines (P<0.05). None of PCV2 DNA was detected in all immunized animals, except control animals immunized with phosphate-buffered saline. These results indicated that GM-CSF was a powerful immunoadjuvant for PCV2 subunit vaccines because it enhanced humoral immune response and improved immune protection against PCV2 infection in pigs. Thus, the novel Cap-GM-CSF subunit vaccine has the potential to be used as an effective and safe vaccine candidate against PCV2 infection. PMID:25863115

  9. Nuclear respiratory factor 2 regulates the expression of the same NMDA receptor subunit genes as NRF-1: both factors act by a concurrent and parallel mechanism to couple energy metabolism and synaptic transmission.

    PubMed

    Priya, Anusha; Johar, Kaid; Wong-Riley, Margaret T T

    2013-01-01

    Neuronal activity and energy metabolism are tightly coupled processes. Previously, we found that nuclear respiratory factor 1 (NRF-1) transcriptionally co-regulates energy metabolism and neuronal activity by regulating all 13 subunits of the critical energy generating enzyme, cytochrome c oxidase (COX), as well as N-methyl-d-aspartate (NMDA) receptor subunits 1 and 2B, GluN1 (Grin1) and GluN2B (Grin2b). We also found that another transcription factor, nuclear respiratory factor 2 (NRF-2 or GA-binding protein) regulates all subunits of COX as well. The goal of the present study was to test our hypothesis that NRF-2 also regulates specific subunits of NMDA receptors, and that it functions with NRF-1 via one of three mechanisms: complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation of mouse neuroblastoma cells and rat visual cortical tissue, promoter mutations, real-time quantitative PCR, and western blot analysis, NRF-2 was found to functionally regulate Grin1 and Grin2b genes, but not any other NMDA subunit genes. Grin1 and Grin2b transcripts were up-regulated by depolarizing KCl, but silencing of NRF-2 prevented this up-regulation. On the other hand, over-expression of NRF-2 rescued the down-regulation of these subunits by the impulse blocker TTX. NRF-2 binding sites on Grin1 and Grin2b are conserved among species. Our data indicate that NRF-2 and NRF-1 operate in a concurrent and parallel manner in mediating the tight coupling between energy metabolism and neuronal activity at the molecular level. PMID:23085505

  10. The cyclope gene of Drosophila encodes a cytochrome c oxidase subunit VIc homolog.

    PubMed

    Szuplewski, S; Terracol, R

    2001-08-01

    Cytochrome c oxidase is the terminal enzyme of the mitochondrial electron transfer chain. In eukaryotes, the enzyme is composed of 3 mitochondrial DNA-encoded subunits and 7-10 (in mammals) nuclear DNA-encoded subunits. This enzyme has been extensively studied in mammals and yeast but, in Drosophila, very little is known and no mutant has been described so far. Here we report the genetic and molecular characterization of mutations in cyclope (cype) and the cloning of the gene encoding a cytochrome c oxidase subunit VIc homolog. cype is an essential gene whose mutations are lethal and show pleiotropic phenotypes. The 77-amino acid peptide encoded by cype is 46% identical and 59% similar to the human subunit (75 amino acids). The transcripts are expressed maternally and throughout development in localized regions. They are found predominantly in the central nervous system of the embryo; in the central region of imaginal discs; in the germarium, follicular, and nurse cells of the ovary; and in testis. A search in the Genome Annotation Database of Drosophila revealed the absence of subunit VIIb and the presence of 9 putative nuclear cytochrome c oxidase subunits with high identity scores when compared to the 10 human subunits. PMID:11514451

  11. The cyclope gene of Drosophila encodes a cytochrome c oxidase subunit VIc homolog.

    PubMed Central

    Szuplewski, S; Terracol, R

    2001-01-01

    Cytochrome c oxidase is the terminal enzyme of the mitochondrial electron transfer chain. In eukaryotes, the enzyme is composed of 3 mitochondrial DNA-encoded subunits and 7-10 (in mammals) nuclear DNA-encoded subunits. This enzyme has been extensively studied in mammals and yeast but, in Drosophila, very little is known and no mutant has been described so far. Here we report the genetic and molecular characterization of mutations in cyclope (cype) and the cloning of the gene encoding a cytochrome c oxidase subunit VIc homolog. cype is an essential gene whose mutations are lethal and show pleiotropic phenotypes. The 77-amino acid peptide encoded by cype is 46% identical and 59% similar to the human subunit (75 amino acids). The transcripts are expressed maternally and throughout development in localized regions. They are found predominantly in the central nervous system of the embryo; in the central region of imaginal discs; in the germarium, follicular, and nurse cells of the ovary; and in testis. A search in the Genome Annotation Database of Drosophila revealed the absence of subunit VIIb and the presence of 9 putative nuclear cytochrome c oxidase subunits with high identity scores when compared to the 10 human subunits. PMID:11514451

  12. Coordinate inhibition of expression of several genes for protein subunits of human nuclear RNase P

    PubMed Central

    Kovrigina, Elizaveta; Wesolowski, Donna; Altman, Sidney

    2003-01-01

    The deliberate inhibition of expression of one of the protein subunits (Rpp38) of human nuclear RNase P is achievable by using external guide sequence (EGS) technology. Both the protein product and the mRNA are greatly reduced 24 h after transient transfection with a gene coding for an appropriate EGS. Control experiments indicated that four other protein subunits of RNase P and their RNAs are also inhibited with no external manipulation. The remaining RNase P proteins, their mRNAs, and the RNA subunit of RNase P all are unchanged. Several short nucleotide sequences adjacent to the ORFs for the inhibited genes are similar and could be targets for transcriptional repression. The explanation of coordinate inhibition of the expression of the product of one particular gene by the transfection of an EGS (or RNA interference) requires some care in terms of interpreting phenotypic effects because, in our case, several gene products that are not targeted are also inhibited. PMID:12552092

  13. Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms

    NASA Technical Reports Server (NTRS)

    Kopczynski, E. D.; Bateson, M. M.; Ward, D. M.

    1994-01-01

    When PCR was used to recover small-subunit (SSU) rRNA genes from a hot spring cyanobacterial mat community, chimeric SSU rRNA sequences which exhibited little or no secondary structural abnormality were recovered. They were revealed as chimeras of SSU rRNA genes of uncultivated species through separate phylogenetic analysis of short sequence domains.

  14. The SHORT-ROOT-like gene PtSHR2B is involved in Populus phellogen activity.

    PubMed

    Miguel, Andreia; Milhinhos, Ana; Novák, Ondřej; Jones, Brian; Miguel, Célia M

    2016-03-01

    SHORT-ROOT (SHR) is a GRAS transcription factor first characterized for its role in the specification of the stem cell niche and radial patterning in Arabidopsis thaliana (At) roots. Three SHR-like genes have been identified in Populus trichocarpa (Pt). PtSHR1 shares high similarity with AtSHR over the entire length of the coding sequence. The two other Populus SHR-like genes, PtSHR2A and PtSHR2B, are shorter in their 5' ends when compared with AtSHR. Unlike PtSHR1, that is expressed throughout the cambial zone of greenhouse-grown Populus trees, PtSHR2Bprom:uidA expression was detected in the phellogen. Additionally, PtSHR1 and PtSHR2B expression patterns markedly differ in the shoot apex and roots of in vitro plants. Transgenic hybrid aspen expressing PtSHR2B under the 35S constitutive promoter showed overall reduced tree growth while the proportion of bark increased relative to the wood. Reverse transcription-quantitative PCR (RT-qPCR) revealed increased transcript levels of cytokinin metabolism and response-related genes in the transgenic plants consistent with an increase of total cytokinin levels. This was confirmed by cytokinin quantification by LC-MS/MS. Our results indicate that PtSHR2B appears to function in the phellogen and therefore in the regulation of phellem and periderm formation, possibly acting through modulation of cytokinin homeostasis. Furthermore, this work points to a functional diversification of SHR after the divergence of the Populus and Arabidopsis lineages. This finding may contribute to selection and breeding strategies of cork oak in which, unlike Populus, the phellogen is active throughout the entire tree lifespan, being at the basis of a highly profitable cork industry. PMID:26709311

  15. Delivery of a Foot-and-Mouth Disease Virus Empty Capsid Subunit Antigen with Nonstructural Protein 2B Improves Protection of Swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously demonstrated that a replication-defective human adenovirus serotype 5 (Ad5) vector carrying the capsid (P1-2A) and 3C protease coding regions as well as a portion of the 2B coding region of foot-and-mouth disease virus (FMDV) (Ad5-A24) protects cattle and swine from direct inocula...

  16. Identification of MEF2B and TRHDE Gene Polymorphisms Related to Growth Traits in a New Ujumqin Sheep Population

    PubMed Central

    Xuan, Junli; Wang, Huihua; Yuan, Zehu; Wu, Mingming; Liu, Ruizao; Zhu, Caiye; Wei, Caihong; Zhao, Fuping; Du, Lixin

    2016-01-01

    2 SNPs were discovered in our previous genome-wide association study (GWAS): s58995.1 (rs420767326 A>G) in MEF2B gene and OAR3_115712045.1 (rs401775061 A>C) in TRHDE gene, which were significantly associated with post-weaning gain in sheep. Herein, we performed a replication experiment to investigate single nucleotide polymorphisms (SNPs) within the MEF2B and TRHDE gene exons, the 5′untranslated regions (within 1000bp), the 3′ untranslated regions (within 1000bp) and their associations with Ujumqin sheep growth traits in 4-month age and 6-month age, respectively. Finally,3 SNPs were selected to be investigated including 1 SNP in 3′untranslated regions in MEF2B gene (rs417014745 A>G) and 2 SNPs in TRHDE gene (rs426980328 T>C and rs430810656 G>A).The χ2 test showed all the 3 variations were in Hardy–Weinberg equilibrium (P>0.05) status. Association analysis suggested that rs426980328 T>C was significantly associated with body weight and chest girth in 4-month age (P<0.05). rs430810656 G>A exhibited extremely significant association with body weight and chest girth in 4-month age (P<0.01). rs417014745 A>G was extremely significantly associated with body weight and chest girth in 4-month age and chest girth in 6-month age (P<0.01), and it was also significantly associated with body weight in 6-month age (P<0.05). Combined effect analysis indicated significant associations between the combinations of rs426980328-rs417014745, rs430810656-rs417014745 and several growth traits (P<0.05). These results suggested MEF2B and TRHDE genes affected growth traits in Ujumqin sheep and the combination effect of the two genes also played a significant effective role. These SNPs might have potential value as genetic markers for growth traits and it could be used in Ujumqin sheep breeding in future. Further studies are necessary to confirm our findings. PMID:27472808

  17. Mammalian mitochondrial ribosomal small subunit (MRPS) genes: A putative role in human disease.

    PubMed

    Gopisetty, Gopal; Thangarajan, Rajkumar

    2016-09-01

    Mitochondria are prominently understood as power houses producing ATP the primary energy currency of the cell. However, mitochondria are also known to play an important role in apoptosis and autophagy, and mitochondrial dysregulation can lead to pathological outcomes. Mitochondria are known to contain 1500 proteins of which only 13 are coded by mitochondrial DNA and the rest are coded by nuclear genes. Protein synthesis in mitochondria involves mitochondrial ribosomes which are 55-60S particles and are composed of small 28S and large 39S subunits. A feature of mammalian mitoribosome which differentiate it from bacterial ribosomes is the increased protein content. The human mitochondrial ribosomal protein (MRP) gene family comprises of 30 genes which code for mitochondrial ribosomal small subunit and 50 genes for the large subunit. The present review focuses on the mitochondrial ribosomal small subunit genes (MRPS), presents an overview of the literature and data gleaned from publicly available gene and protein expression databases. The survey revealed aberrations in MRPS gene expression patterns in varied human diseases indicating a putative role in their etiology. PMID:27170550

  18. Targeted mutagenesis of aryl hydrocarbon receptor 2a and 2b genes in Atlantic killifish (Fundulus heteroclitus).

    PubMed

    Aluru, Neelakanteswar; Karchner, Sibel I; Franks, Diana G; Nacci, Diane; Champlin, Denise; Hahn, Mark E

    2015-01-01

    Understanding molecular mechanisms of toxicity is facilitated by experimental manipulations, such as disruption of function by gene targeting, that are especially challenging in non-standard model species with limited genomic resources. While loss-of-function approaches have included gene knock-down using morpholino-modified oligonucleotides and random mutagenesis using mutagens or retroviruses, more recent approaches include targeted mutagenesis using zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. These latter methods provide more accessible opportunities to explore gene function in non-traditional model species. To facilitate evaluation of toxic mechanisms for important categories of aryl hydrocarbon pollutants, whose actions are known to be receptor mediated, we used ZFN and CRISPR-Cas9 approaches to generate aryl hydrocarbon receptor 2a (AHR2a) and AHR2b gene mutations in Atlantic killifish (Fundulus heteroclitus) embryos. This killifish is a particularly valuable non-traditional model, with multiple paralogs of AHR whose functions are not well characterized. In addition, some populations of this species have evolved resistance to toxicants such as halogenated aromatic hydrocarbons. AHR-null killifish will be valuable for characterizing the role of the individual AHR paralogs in evolved resistance, as well as in normal development. We first used five-finger ZFNs targeting exons 1 and 3 of AHR2a. Subsequently, CRISPR-Cas9 guide RNAs were designed to target regions in exon 2 and 3 of AHR2a and AHR2b. We successfully induced frameshift mutations in AHR2a exon 3 with ZFN and CRISPR-Cas9 guide RNAs, with mutation frequencies of 10% and 16%, respectively. In AHR2b, mutations were induced using CRISPR-Cas9 guide RNAs targeting sites in both exon 2 (17%) and exon 3 (63%). We screened AHR2b exon 2 CRISPR-Cas9-injected embryos for off

  19. Targeted mutagenesis of aryl hydrocarbon receptor 2a and 2b genes in Atlantic killifish (Fundulus heteroclitus)

    PubMed Central

    Aluru, Neelakanteswar; Karchner, Sibel I.; Franks, Diana G.; Nacci, Diane; Champlin, Denise; Hahn, Mark E.

    2014-01-01

    Understanding molecular mechanisms of toxicity is facilitated by experimental manipulations, such as disruption of function by gene targeting, that are especially challenging in non-standard model species with limited genomic resources. While loss-of-function approaches have included gene knock-down using morpholino-modified oligonucleotides and random mutagenesis using mutagens or retroviruses, more recent approaches include targeted mutagenesis using zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. These latter methods provide more accessible opportunities to explore gene function in non-traditional model species. To facilitate evaluations of toxic mechanisms for important categories of aryl hydrocarbon pollutants, whose actions are known to be receptor mediated, we used ZFN and CRISPR-Cas9 approaches to generate aryl hydrocarbon receptor 2a (AHR2a) and AHR2b gene mutations in Atlantic killifish (Fundulus heteroclitus) embryos. This killifish is a particularly valuble non-traditional model for this study, with multiple paralogs of AHR whose functions are not well characterized. In addition, some populations of this species have evolved resistance to toxicants such as halogenated aromatic hydrocarbons. AHR-null killifish will be valuable for characterizing the role of the individual AHR paralogs in evolved resistance, as well as in normal development. We first used five-finger ZFNs targeting exons 1 and 3 of AHR2a. Subsequently, CRISPR-Cas9 guide RNAs were designed to target regions in exon 2 and 3 of AHR2a and AHR2b. We successfully induced frameshift mutations in AHR2a exon 3 with ZFN and CRISPR-Cas9 guide RNAs, with mutation frequencies of 10% and 16%, respectively. In AHR2b, mutations were induced using CRISPR-Cas9 guide RNAs targeting sites in both exon 2 (17%) and exon 3 (63%). We screened AHR2b exon 2 CRISPR-Cas9-injected embryos for

  20. Mas-Related Gene (Mrg) C Activation Attenuates Bone Cancer Pain via Modulating Gi and NR2B

    PubMed Central

    Lu, Cui’e; Lei, Yishan; Ma, Zhengliang; Gu, Xiaoping

    2016-01-01

    Objective This study is to investigate the role of Mas-related gene (Mrg) C in the pathogenesis and treatment of bone cancer pain (BCP). Methods BCP mouse model was established by osteosarcoma cell inoculation. Pain-related behaviors were assessed with the spontaneous lifting behavior test and mechanical allodynia test. Expression levels of MrgC, Gi, and NR2B in the spinal cord were detected with Western blot analysis and immunohistochemistry. Results Pain-related behavior tests showed significantly increased spontaneous flinches (NSF) and decreased paw withdrawal mechanical threshold (PWMT) in mouse models of BCP. Western blot analysis showed that, compared with the control group and before modeling, all the expression levels of MrgC, Gi, and NR2B in the spinal cord of BCP mice were dramatically elevated, which were especially increased at day 7 after operation and thereafter, in a time-dependent manner. Moreover, the treatment of MrgC agonist BAM8-22 significantly up-regulated Gi and down-regulated NR2B expression levels, in the spinal cord of BCP mice, in a time-dependent manner. On the other hand, anti-MrgC significantly down-regulated Gi expression, while dramatically up-regulated NR2B expression, in the BCP mice. Similar results were obtained from the immunohistochemical detection. Importantly, BAM8-22 significantly attenuated the nociceptive behaviors in the BCP mice. Conclusion Our results indicated the MrgC-mediated Gi and NR2B expression alterations in the BCP mice, which might contribute to the pain hypersensitivity. These findings may provide a novel strategy for the treatment of BCP in clinic. PMID:27152740

  1. Overexpression of a CYP94 family gene CYP94C2b increases internode length and plant height in rice

    PubMed Central

    Kurotani, Ken-Ich; Hattori, Tsukaho; Takeda, Shin

    2015-01-01

    Plant growth is controlled by intrinsic developmental programmes and environmental cues. Jasmonate (JA) has important roles in both processes, by regulating cell division and differentiation, as well as in defense responses and senescence. We report an increase in rice plant height caused by overexpression of a gene encoding a cytochrome P450 enzyme, CYP94C2b, which promoted deactivation of JA-Ile. The height increase occurred through enhanced elongation of internodes in the absence of concomitant cell elongation, unlike previous findings with coi1 knock-down plants. Thus, modulating JA metabolism can increase the number of elongated cells in an internode. Based on these and previous findings, we discuss the difference in the effects of CYP94C2b overexpression vs. coi1 knock-down. PMID:26251886

  2. Creation of transgenic Brassica napus L. plants expressing human alpha 2b interferon gene.

    PubMed

    Sakhno, L O; Kvasko, O Y; Olevinska, Z M; Spivak, M Y; Kuchuk, M V

    2012-01-01

    Spring rapeseed transgenic lines expressing human interferon alpha 2b were created by Agrobacterium-mediated transformation of aseptic plant leaf explants. The maximum antiviral activity of the leaf extracts reached 4500 IU/g fresh weight. It was determined that the antioxidant activity and the activity of an enzyme of plant antioxidant system--superoxide dismutase (SOD)--in the leaf tissues of transgenic plants increased compared to controls. There were no correlations between the interferon and antioxidant activities, as well as between SOD and interferon activities. Using the obtained transgenic rapeseed plants with high interferon and antioxidant activities as a feed additive for animals might have preventive effect on their body, increasing resistance to infections of various origins. PMID:23285745

  3. Disruption of genes encoding subunits of yeast vacuolar H(+)-ATPase causes conditional lethality.

    PubMed Central

    Nelson, H; Nelson, N

    1990-01-01

    The main function of vacuolar H(+)-ATPases in eukaryotic cells is to generate proton and electrochemical gradients across the membranes of the vacuolar system. The enzyme is composed of a catalytic sector with five subunits (A-E) and a membrane sector containing at least two subunits (a and c). We disrupted two genes of this enzyme, in yeast cells, one encoding a subunit of the membrane sector (subunit c) and another encoding a subunit of the catalytic sector (subunit B). The resulting mutants did not grow in medium with a pH value higher than 6.5 and grew well only within a narrow pH range around 5.5. Transformation of the mutants with plasmids containing the corresponding genes repaired the mutations. Thus failure to lower the pH in the vacuolar system of yeast, and probably other eukaryotic cells, is lethal and the mutants may survive only if a low external pH allows for this acidification by fluid-phase endocytosis. Images PMID:2139726

  4. MULTIPLEX PCR ASSAY FOR DETECTION OF HUMAN SOMATOTROPIN AND INTERFERON ALPHA2b GENES IN PLANT MATERIAL.

    PubMed

    Gerasymenko, I M; Mazur, M G; Sheludko, Y V; Kuchuk, N V

    2015-01-01

    Using transgenic plants as factories for production of physiologically active human proteins arouses special concern because occasional escape of such transgenes into environment may cause health problems. Creation of plant varieties producing pharmaceutically valuable proteins should be accompanied by development of detection methods suitable for controlling the transgene behavior. Here we describe a multiplex PCR protocol for revealing of two human genes (encoding growth hormone and interferon alpha2b) that have been successfully introduced into plant genomes. The primer pair designed for detection of human growth hormone coding sequence amplifies fragments of different size from the full-length gene in the human genome and the intronless coding sequence usually used for plant transformation. Application of this primer pair may be recommended for ruling out false positive results due to sample contamination with human DNA. Such a control may be useful also in PCR analysis during establishing of transgenic plants carrying genes of human origin. PMID:26214899

  5. Nuclear respiratory factor 2 regulates the expression of the same NMDA receptor subunit genes as NRF-1: Both factors act by a concurrent and parallel mechanism to couple energy metabolism and synaptic transmission

    PubMed Central

    Priya, Anusha; Johar, Kaid; Wong-Riley, Margaret

    2012-01-01

    Neuronal activity and energy metabolism are tightly coupled processes. Previously, we found that nuclear respiratory factor 1 (NRF-1) transcriptionally co-regulates energy metabolism and neuronal activity by regulating all 13 subunits of the critical energy generating enzyme, cytochrome c oxidase (COX), as well as GluN1 (Grin1) and GluN2B (Grin2b) subunits of NMDA receptors. We also found that another transcription factor, nuclear respiratory factor 2 (NRF-2 or GA-binding protein) regulates all subunits of COX as well. The goal of the present study was to test our hypothesis that NRF-2 also regulates specific subunits of NMDA receptors, and that it functions with NRF-1 via one of three mechanisms: complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation of mouse neuroblastoma cells and rat visual cortical tissue, promoter mutations, real-time quantitative PCR, and western blot analysis, NRF-2 was found to functionally regulate Grin1 and Grin2b genes, but not any other NMDA subunit genes. Grin1 and Grin2b transcripts were up-regulated by depolarizing KCl, but silencing of NRF-2 prevented this up-regulation. On the other hand, over-expression of NRF-2 rescued the down-regulation of these subunits by the impulse blocker TTX. NRF-2 binding sites on Grin1 and Grin2b are conserved among species. Our data indicate that NRF-2 and NRF-1 operate in a concurrent and parallel manner in mediating the tight coupling between energy metabolism and neuronal activity at the molecular level. PMID:23085505

  6. Experimental evidence that overexpression of NR2B glutamate receptor subunit is associated with brain vacuolation in adult glutaryl-CoA dehydrogenase deficient mice: A potential role for glutamatergic-induced excitotoxicity in GA I neuropathology.

    PubMed

    Rodrigues, Marília Danyelle Nunes; Seminotti, Bianca; Amaral, Alexandre Umpierrez; Leipnitz, Guilhian; Goodman, Stephen Irwin; Woontner, Michael; de Souza, Diogo Onofre Gomes; Wajner, Moacir

    2015-12-15

    Glutaric aciduria type I (GA I) is biochemically characterized by accumulation of glutaric and 3-hydroxyglutaric acids in body fluids and tissues, particularly in the brain. Affected patients show progressive cortical leukoencephalopathy and chronic degeneration of the basal ganglia whose pathogenesis is still unclear. In the present work we investigated parameters of bioenergetics and redox homeostasis in various cerebral structures (cerebral cortex, striatum and hippocampus) and heart of adult wild type (Gcdh(+/+)) and glutaryl-CoA dehydrogenase deficient knockout (Gcdh(-/-)) mice fed a baseline chow. Oxidative stress parameters were also measured after acute lysine overload. Finally, mRNA expression of NMDA subunits and GLT1 transporter was determined in cerebral cortex and striatum of these animals fed a baseline or high lysine (4.7%) chow. No significant alterations of bioenergetics or redox status were observed in these mice. In contrast, mRNA expression of the NR2B glutamate receptor subunit and of the GLT1 glutamate transporter was higher in cerebral cortex of Gcdh(-/-) mice. Furthermore, NR2B expression was markedly elevated in striatum of Gcdh(-/-) animals receiving chronic Lys overload. These data indicate higher susceptibility of Gcdh(-/-) mice to excitotoxic damage, implying that this pathomechanism may contribute to the cortical and striatum alterations observed in GA I patients. PMID:26671102

  7. [Antiviral activity of extracts of transgenic cichory and lettuce plants with the human interferon alpha-2b gene].

    PubMed

    Matveeva, N A; Kudriavets, Iu I; Likhova, A A; Shakhovskiĭ, A M; Bezdenezhnykh, N A; Kvasko, E Iu

    2012-01-01

    Biological activity of protein extracts from transgenic plants of chicory Cichorium intybus L. and lettuce Lactuca sativa L. with human interferon alpha2b gene was investigated against vesicular stomatitis virus. It was shown that the extracts from the hairy roots of chicory and lettuce transformed by A. rhizogenes possess the antiviral activity 1620...5400 IU/g weight, and the extracts from leaves of the plants transformed by A. tumefaciens--till 9375 IU/g weight. Dependence of plant extract biological activity on the transformation vector was shown. PMID:23342646

  8. Structural analysis of the α subunit of Na(+)/K(+) ATPase genes in invertebrates.

    PubMed

    Thabet, Rahma; Rouault, J-D; Ayadi, Habib; Leignel, Vincent

    2016-01-01

    The Na(+)/K(+) ATPase is a ubiquitous pump coordinating the transport of Na(+) and K(+) across the membrane of cells and its role is fundamental to cellular functions. It is heteromer in eukaryotes including two or three subunits (α, β and γ which is specific to the vertebrates). The catalytic functions of the enzyme have been attributed to the α subunit. Several complete α protein sequences are available, but only few gene structures were characterized. We identified the genomic sequences coding the α-subunit of the Na(+)/K(+) ATPase, from the whole-genome shotgun contigs (WGS), NCBI Genomes (chromosome), Genomic Survey Sequences (GSS) and High Throughput Genomic Sequences (HTGS) databases across distinct phyla. One copy of the α subunit gene was found in Annelida, Arthropoda, Cnidaria, Echinodermata, Hemichordata, Mollusca, Placozoa, Porifera, Platyhelminthes, Urochordata, but the nematodes seem to possess 2 to 4 copies. The number of introns varied from 0 (Platyhelminthes) to 26 (Porifera); and their localization and length are also highly variable. Molecular phylogenies (Maximum Likelihood and Maximum Parsimony methods) showed some clusters constituted by (Chordata/(Echinodermata/Hemichordata)) or (Plathelminthes/(Annelida/Mollusca)) and a basal position for Porifera. These structural analyses increase our knowledge about the evolutionary events of the α subunit genes in the invertebrates. PMID:26812300

  9. Human and rodent MaxiK channel beta-subunit genes: cloning and characterization.

    PubMed

    Jiang, Z; Wallner, M; Meera, P; Toro, L

    1999-01-01

    Voltage- and Ca2+-sensitive K+ (MaxiK) channels play key roles in controlling neuronal excitability and vascular tone. We cloned and analyzed human and rodent genes for the modulatory beta subunit, KCNMB1. The human and mouse beta-subunit genes are approximately 11 and approximately 9 kb in length, respectively, and have a four exon-three intron structure. Primer extension assay localized the transcription initiation site at 442 (human) or 440 (mouse) bp upstream of the translation initiation codon, agreeing with the transcript size in Northern blots. Both genes have a TATA-less putative promoter region, with a transcription initiator-like region, and motifs characteristic of regulated promoters, including muscle-specific enhancing factors-1 and -2. Consistent with a tissue-specific expression of KCNMB1, regulated at the transcriptional level, beta-subunit transcripts are abundant in smooth muscle and heart, but scarce in lymphatic tissues, brain, and liver. Expressed rat and mouse beta subunits increase the apparent Ca2+ sensitivity of the human MaxiK channel alpha subunit. PMID:9888999

  10. Cytochrome oxidase subunit II gene in mitochondria of Oenothera has no intron

    PubMed Central

    Hiesel, Rudolf; Brennicke, Axel

    1983-01-01

    The cytochrome oxidase subunit II gene has been localized in the mitochondrial genome of Oenothera berteriana and the nucleotide sequence has been determined. The coding sequence contains 777 bp and, unlike the corresponding gene in Zea mays, is not interrupted by an intron. No TGA codon is found within the open reading frame. The codon CGG, as in the maize gene, is used in place of tryptophan codons of corresponding genes in other organisms. At position 742 in the Oenothera sequence the TGG of maize is changed into a CGG codon, where Trp is conserved as the amino acid in other organisms. Homologous sequences occur more than once in the mitochondrial genome as several mitochondrial DNA species hybridize with DNA probes of the cytochrome oxidase subunit II gene. ImagesFig. 5. PMID:16453484

  11. Chronic Intermittent Ethanol Regulates Hippocampal GABA(A) Receptor Delta Subunit Gene Expression

    PubMed Central

    Follesa, Paolo; Floris, Gabriele; Asuni, Gino P.; Ibba, Antonio; Tocco, Maria G.; Zicca, Luca; Mercante, Beniamina; Deriu, Franca; Gorini, Giorgio

    2015-01-01

    Chronic ethanol consumption causes structural and functional reorganization in the hippocampus and induces alterations in the gene expression of gamma-aminobutyric acid type A receptors (GABAARs). Distinct forced intermittent exposure models have been used previously to investigate changes in GABAAR expression, with contrasting results. Here, we used repeated cycles of a Chronic Intermittent Ethanol paradigm to examine the relationship between voluntary, dependence-associated ethanol consumption, and GABAAR gene expression in mouse hippocampus. Adult male C57BL/6J mice were exposed to four 16-h ethanol vapor (or air) cycles in inhalation chambers alternated with limited-access two-bottle choice between ethanol (15%) and water consumption. The mice exposed to ethanol vapor showed significant increases in ethanol consumption compared to their air-matched controls. GABAAR alpha4 and delta subunit gene expression were measured by qRT-PCR at different stages. There were significant changes in GABAAR delta subunit transcript levels at different time points in ethanol-vapor exposed mice, while the alpha4 subunit levels remained unchanged. Correlated concurrent blood ethanol concentrations suggested that GABAAR delta subunit mRNA levels fluctuate depending on ethanol intoxication, dependence, and withdrawal state. Using a vapor-based Chronic Intermittent Ethanol procedure with combined two-bottle choice consumption, we corroborated previous evidences showing that discontinuous ethanol exposure affects GABAAR delta subunit expression but we did not observe changes in alpha4 subunit. These findings indicate that hippocampal GABAAR delta subunit expression changes transiently over the course of a Chronic Intermittent Ethanol paradigm associated with voluntary intake, in response to ethanol-mediated disturbance of GABAergic neurotransmission. PMID:26617492

  12. The neuronal nicotinic acetylcholine receptor {alpha}7 subunit gene: Cloning, mapping, structure, and targeting in mouse

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1994-09-01

    The neuronal nicotinic acetylcholine receptor {alpha}7 subunit is a member of a family of ligand-gated ion channels, and is the only subunit know to bind {alpha}-bungarotoxin in mammalian brain. {alpha}-Bungarotoxin binding sites are known to be more abundant in the hippocampus of mouse strains that are particularly sensitive to nicotine-induced seizures. The {alpha}7 receptor is highly permeable to calcium, which could suggest a role in synaptic plasticity in the nervous system. Auditory gating deficiency, an abnormal response to a second auditory stimulus, is characteristic of schizophrenia. Mouse strains that exhibit a similar gating deficit have reduced hippocampal expression of the {alpha}7 subunit. We have cloned and sequenced the full length cDNA for the mouse {alpha}7 gene (Acra-7) and characterized its gene structure. The murine {alpha}7 shares amino acid identity of 99% and 93% with the rat and human {alpha}7 subunits, respectively. Using an interspecies backcross panel, the murine gene was mapped to chromosome 7 near the p locus, a region syntenic with human chromosome 15; the human gene (CHRNA7) was confirmed to map to 15q13-q14 by FISH. To generate a mouse {alpha}7 mutant by homologous recombination, we have constructed a replacement vector which will delete transmembrane domains II-IV and the cytoplasmic domain from the gene product. Recombinant embryonic stem (ES) cell clones were selected and used to develop mouse chimeras that are currently being bred to obtain germline transmission.

  13. Two EPR-detectable [4Fe-4S] clusters, N2a and N2b, are bound to the NuoI (TYKY) subunit of NADH:ubiquinone oxidoreductase (Complex I) from Rhodobacter capsulatus.

    PubMed

    Chevallet, Mireille; Dupuis, Alain; Issartel, Jean-Paul; Lunardi, Joël; van Belzen, Ronald; Albracht, Simon P J

    2003-03-01

    NADH:ubiquinone oxidoreductases (Complex I) contain a subunit, TYKY in the bovine enzyme and NuoI in the enzyme from Rhodobacter capsulatus, which is assumed to bind two [4Fe-4S] clusters because it contains two sets of conserved cysteine motifs similar to those found in the 2[4Fe-4S] ferredoxins. It was recently shown that the TYKY subunit is not an ordinary 2[4Fe-4S] ferredoxin, but has a unique amino acid sequence, which is only found in NAD(P)H:quinone oxidoreductases and certain membrane-bound [NiFe]-hydrogenases expected to be involved in redox-linked proton translocation [FEBS Lett. 485 (2000) 1]. We have generated a set of R. capsulatus mutants in which five out of the eight conserved cysteine residues in NuoI were replaced by other amino acids. The resulting mutants fell into three categories with virtually no, intermediate or quite normal Complex I activities. EPR-spectroscopic analysis of the membranes of the C67S and C106S mutants, two mutants belonging to the second and third group, respectively, showed a specific 50% decrease of the EPR signal attributed to cluster N2. It is concluded that the NuoI (TYKY) subunit binds two clusters N2, called N2a and N2b, which exhibit very similar spectral features when analyzed by X-band EPR spectroscopy. PMID:12615348

  14. ATXN2 and Its Neighbouring Gene SH2B3 Are Associated with Increased ALS Risk in the Turkish Population

    PubMed Central

    Uyan, Özgün; Ağım, Zeynep Sena; Özoğuz, Aslihan; Parman, Yeşim; Deymeer, Feza; Oflazer, Piraye; Koç, Filiz; Özçelik, Hilmi; Auburger, Georg; Başak, A. Nazlı

    2012-01-01

    Expansions of the polyglutamine (polyQ) domain (≥34) in Ataxin-2 (ATXN2) are the primary cause of spinocerebellar ataxia type 2 (SCA2). Recent studies reported that intermediate-length (27–33) expansions increase the risk of Amyotrophic Lateral Sclerosis (ALS) in 1–4% of cases in diverse populations. This study investigates the Turkish population with respect to ALS risk, genotyping 158 sporadic, 78 familial patients and 420 neurologically healthy controls. We re-assessed the effect of ATXN2 expansions and extended the analysis for the first time to cover the ATXN2 locus with 18 Single Nucleotide Polymorphisms (SNPs) and their haplotypes. In accordance with other studies, our results confirmed that 31–32 polyQ repeats in the ATXN2 gene are associated with risk of developing ALS in 1.7% of the Turkish ALS cohort (p = 0.0172). Additionally, a significant association of a 136 kb haplotype block across the ATXN2 and SH2B3 genes was found in 19.4% of a subset of our ALS cohort and in 10.1% of the controls (p = 0.0057, OR: 2.23). ATXN2 and SH2B3 encode proteins that both interact with growth receptor tyrosine kinases. Our novel observations suggest that genotyping of SNPs at this locus may be useful for the study of ALS risk in a high percentage of individuals and that ATXN2 and SH2B3 variants may interact in modulating the disease pathway. PMID:22916186

  15. ATXN2 and its neighbouring gene SH2B3 are associated with increased ALS risk in the Turkish population.

    PubMed

    Lahut, Suna; Ömür, Özgür; Uyan, Özgün; Ağım, Zeynep Sena; Özoğuz, Aslihan; Parman, Yeşim; Deymeer, Feza; Oflazer, Piraye; Koç, Filiz; Özçelik, Hilmi; Auburger, Georg; Başak, A Nazlı

    2012-01-01

    Expansions of the polyglutamine (polyQ) domain (≥ 34) in Ataxin-2 (ATXN2) are the primary cause of spinocerebellar ataxia type 2 (SCA2). Recent studies reported that intermediate-length (27-33) expansions increase the risk of Amyotrophic Lateral Sclerosis (ALS) in 1-4% of cases in diverse populations. This study investigates the Turkish population with respect to ALS risk, genotyping 158 sporadic, 78 familial patients and 420 neurologically healthy controls. We re-assessed the effect of ATXN2 expansions and extended the analysis for the first time to cover the ATXN2 locus with 18 Single Nucleotide Polymorphisms (SNPs) and their haplotypes. In accordance with other studies, our results confirmed that 31-32 polyQ repeats in the ATXN2 gene are associated with risk of developing ALS in 1.7% of the Turkish ALS cohort (p=0.0172). Additionally, a significant association of a 136 kb haplotype block across the ATXN2 and SH2B3 genes was found in 19.4% of a subset of our ALS cohort and in 10.1% of the controls (p=0.0057, OR: 2.23). ATXN2 and SH2B3 encode proteins that both interact with growth receptor tyrosine kinases. Our novel observations suggest that genotyping of SNPs at this locus may be useful for the study of ALS risk in a high percentage of individuals and that ATXN2 and SH2B3 variants may interact in modulating the disease pathway. PMID:22916186

  16. Inducer effect on the complex formation between rat liver nuclear proteins and cytochrome P450 2B gene regulatory elements.

    PubMed

    Duzhak, T G; Schwartz, E I; Gulyaeva, L F; Lyakhovich, V V

    2002-09-01

    DNA gel retardation assay has been applied to the investigation of complexes between rat liver nuclear proteins and Barbie box positive regulatory element of cytochrome P450 2B (CYP2B) genes. The intensities of B1 and B2 bands detected in the absence of an inducer increased after 30 min protein incubation with phenobarbital (PB) or triphenyldioxane (TPD), but not with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOPOB). In addition, a new complex (B3 band) was for the first time detected under induction by PB, TPD, and TCPOPOB. Increase in the incubation time up to 2 h facilitated the formation of other new complexes (B4 and B5 bands), which were detected only in the presence of TPD. The use of [3H]TPD in hybridization experiments revealed that this inducer, capable of binding to Barbie box DNA, is also present in B4 and B5 complexes. It is probable that the investigated compounds activate the same proteins at the initial induction steps, which correlates with the formation of B1, B2, and B3 complexes. The further induction step might be inducer-specific, as indicated by the formation of B4 and B5 complexes in the presence of TPD only. Thus, the present data suggest the possibility of specific gene activation signaling pathways that are dependent on a particular inducer. PMID:12387719

  17. Missense mutation in the Chlamydomonas chloroplast gene that encodes the Rubisco large subunit

    SciTech Connect

    Spreitzer, R.J.; Brown, T.; Chen, Zhixiang; Zhang, Donghong; Al-Abed, S.R. )

    1988-04-01

    The 69-12Q mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase activity, but retains holoenzyme protein. It results from a mutation in the chloroplast large-subunit gene that causes an isoleucine-for-threonine substitution at amino-acid residue 173. Considering that lysine-175 is involved in catalysis, it appears that mutations cluster at the active site.

  18. Identification of co-expressed gene signatures in mouse B1, marginal zone and B2 B-cell populations

    PubMed Central

    Mabbott, Neil A; Gray, David

    2014-01-01

    In mice, three major B-cell subsets have been identified with distinct functionalities: B1 B cells, marginal zone B cells and follicular B2 B cells. Here, we used the growing body of publicly available transcriptomics data to create an expression atlas of 84 gene expression microarray data sets of distinct mouse B-cell subsets. These data were subjected to network-based cluster analysis using BioLayout Express3D. Using this analysis tool, genes with related functions clustered together in discrete regions of the network graph and enabled the identification of transcriptional networks that underpinned the functional activity of distinct cell populations. Some gene clusters were expressed highly by most of the cell populations included in this analysis (such as those with activity related to house-keeping functions). Others contained genes with expression patterns specific to distinct B-cell subsets. While these clusters contained many genes typically associated with the activity of the cells they were specifically expressed in, many novel B-cell-subset-specific candidate genes were identified. A large number of uncharacterized genes were also represented in these B-cell lineage-specific clusters. Further analysis of the activities of these uncharacterized candidate genes will lead to the identification of novel B-cell lineage-specific transcription factors and regulators of B-cell function. We also analysed 36 microarray data sets from distinct human B-cell populations. These data showed that mouse and human germinal centre B cells shared similar transcriptional features, whereas mouse B1 B cells were distinct from proposed human B1 B cells. PMID:24032749

  19. Characterization of the gene encoding the largest subunit of Plasmodium falciparum RNA polymerase III.

    PubMed

    Li, W B; Bzik, D J; Tanaka, M; Gu, H M; Fox, B A; Inselburg, J

    1991-06-01

    We report here the isolation, sequence analysis, structure, and expression of the gene encoding the largest subunit of RNA polymerase III (RPIII) from Plasmodium falciparum. The P. falciparum RPIII gene consists of 5 exons and 4 introns, is expressed in all of the asexual erythrocytic stages of the parasite as a 8.5-kb mRNA, and is present in a single copy on chromosome 13. The predicted 2339 amino acid residue RPIII subunit contained 5 regions that were conserved between different eukaryotic RPIII subunits, and 4 variable regions that separated the conserved regions. Three of the variable regions were greatly enlarged in comparison to the corresponding variable regions in other RPIII subunits. Variable region C' represented nearly one-third of the P. falciparum RPIII subunit (750 amino acid residues), included a unique repeated decapeptide sequence, and had some homology with yeast DNA topoisomerase II. Noteworthy amino acid sequences and structures were identified in both the conserved regions and in the enlarged variable regions, and their possible role(s) as domains that regulate RPIII enzyme activity is discussed. PMID:1656254

  20. The expression profile of acid-sensing ion channel (ASIC) subunits ASIC1a, ASIC1b, ASIC2a, ASIC2b, and ASIC3 in the esophageal vagal afferent nerve subtypes

    PubMed Central

    Dusenkova, Svetlana; Ru, Fei; Surdenikova, Lenka; Nassenstein, Christina; Hatok, Jozef; Dusenka, Robert; Banovcin, Peter; Kliment, Jan; Tatar, Milos

    2014-01-01

    Acid-sensing ion channels (ASICs) have been implicated in esophageal acid sensing and mechanotransduction. However, insufficient knowledge of ASIC subunit expression profile in esophageal afferent nerves hampers the understanding of their role. This knowledge is essential because ASIC subunits form heteromultimeric channels with distinct functional properties. We hypothesized that the esophageal putative nociceptive C-fiber nerves (transient receptor potential vanilloid 1, TRPV1-positive) express multiple ASIC subunits and that the ASIC expression profile differs between the nodose TRPV1-positive subtype developmentally derived from placodes and the jugular TRPV1-positive subtype derived from neural crest. We performed single cell RT-PCR on the vagal afferent neurons retrogradely labeled from the esophagus. In the guinea pig, nearly all (90%–95%) nodose and jugular esophageal TRPV1-positive neurons expressed ASICs, most often in a combination (65–75%). ASIC1, ASIC2, and ASIC3 were expressed in 65–75%, 55–70%, and 70%, respectively, of both nodose and jugular TRPV1-positive neurons. The ASIC1 splice variants ASIC1a and ASIC1b and the ASIC2 splice variant ASIC2b were similarly expressed in both nodose and jugular TRPV1-positive neurons. However, ASIC2a was found exclusively in the nodose neurons. In contrast to guinea pig, ASIC3 was almost absent from the mouse vagal esophageal TRPV1-positive neurons. However, ASIC3 was similarly expressed in the nonnociceptive TRPV1-negative (tension mechanoreceptors) neurons in both species. We conclude that the majority of esophageal vagal nociceptive neurons express multiple ASIC subunits. The placode-derived nodose neurons selectively express ASIC2a, known to substantially reduce acid sensitivity of ASIC heteromultimers. ASIC3 is expressed in the guinea pig but not in the mouse vagal esophageal TRPV1-positive neurons, indicating species differences in ASIC expression. PMID:25190475

  1. Intracellular gene transfer: Reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase

    PubMed Central

    Daley, Daniel O.; Clifton, Rachel; Whelan, James

    2002-01-01

    Subunit 2 of cytochrome c oxidase (Cox2) in legumes offers a rare opportunity to investigate factors necessary for successful gene transfer of a hydrophobic protein that is usually mitochondrial-encoded. We found that changes in local hydrophobicity were necessary to allow import of this nuclear-encoded protein into mitochondria. All legume species containing both a mitochondrial and nuclear encoded Cox2 displayed a similar pattern, with a large decrease in hydrophobicity evident in the first transmembrane region of the nuclear encoded protein compared with the organelle-encoded protein. Mitochondrial-encoded Cox2 could not be imported into mitochondria under the direction of the mitochondrial targeting sequence that readily supports the import of nuclear encoded Cox2. Removal of the first transmembrane region promotes import ability of the mitochondrial-encoded Cox2. Changing just two amino acids in the first transmembrane region of mitochondrial-encoded Cox2 to the corresponding amino acids in the nuclear encoded Cox2 also promotes import ability, whereas changing the same two amino acids in the nuclear encoded Cox2 to what they are in the mitochondrial-encoded copy prevents import. Therefore, changes in amino acids in the mature protein were necessary and sufficient for gene transfer to allow import under the direction of an appropriate signal to achieve the functional topology of Cox2. PMID:12142462

  2. A model for the transcriptional regulation of the CYP2B1/B2 gene in rat liver.

    PubMed

    Prabhu, L; Upadhya, P; Ram, N; Nirodi, C S; Sultana, S; Vatsala, P G; Mani, S A; Rangarajan, P N; Surolia, A; Padmanaban, G

    1995-10-10

    The phenobarbitone-responsive minimal promoter has been shown to lie between nt -179 and nt + 1 in the 5' (upstream) region of the CYP2B1/B2 gene in rat liver, on the basis of the drug responsiveness of the sequence linked to human growth hormone gene as reporter and targeted to liver as an asialoglycoprotein-DNA complex in vivo. Competition analyses of the nuclear protein-DNA complexes formed in gel shift assays with the positive (nt -69 to -98) and negative (nt -126 to -160) cis elements (PE and NE, respectively) identified within this region earlier indicate that the same protein may be binding to both the elements. The protein species purified on PE and NE affinity columns appear to be identical based on SDS/PAGE analysis, where it migrates as a protein of 26-28 kDa. Traces of a high molecular weight protein (94-100 kDa) are also seen in the preparation obtained after one round of affinity chromatography. The purified protein stimulates transcription of a minigene construct containing the 179 nt on the 5' side of the CYP2B1/B2 gene linked to the I exon in a cell-free system from liver nuclei. The purified protein can give rise to all the three complexes (I, II, and III) with the PE, just as the crude nuclear extract, under appropriate conditions. Manipulations in vitro indicate that the NE has a significantly higher affinity for the dephosphorylated form than for the phosphorylated form of the protein. The PE binds both forms. Phenobarbitone treatment of the animal leads to a significant increase in the phosphorylation of the 26- to 28-kDa and 94-kDa proteins in nuclear labeling experiments followed by isolation on a PE affinity column. We propose that the protein binding predominantly to the NE in the dephosphorylated state characterizes the basal level of transcription of the CYP2B1/B2 gene. Phenobarbitone treatment leads to phosphorylation of the protein, shifting the equilibrium toward binding to the PE. This can promote interaction with an upstream

  3. A model for the transcriptional regulation of the CYP2B1/B2 gene in rat liver.

    PubMed Central

    Prabhu, L; Upadhya, P; Ram, N; Nirodi, C S; Sultana, S; Vatsala, P G; Mani, S A; Rangarajan, P N; Surolia, A; Padmanaban, G

    1995-01-01

    The phenobarbitone-responsive minimal promoter has been shown to lie between nt -179 and nt + 1 in the 5' (upstream) region of the CYP2B1/B2 gene in rat liver, on the basis of the drug responsiveness of the sequence linked to human growth hormone gene as reporter and targeted to liver as an asialoglycoprotein-DNA complex in vivo. Competition analyses of the nuclear protein-DNA complexes formed in gel shift assays with the positive (nt -69 to -98) and negative (nt -126 to -160) cis elements (PE and NE, respectively) identified within this region earlier indicate that the same protein may be binding to both the elements. The protein species purified on PE and NE affinity columns appear to be identical based on SDS/PAGE analysis, where it migrates as a protein of 26-28 kDa. Traces of a high molecular weight protein (94-100 kDa) are also seen in the preparation obtained after one round of affinity chromatography. The purified protein stimulates transcription of a minigene construct containing the 179 nt on the 5' side of the CYP2B1/B2 gene linked to the I exon in a cell-free system from liver nuclei. The purified protein can give rise to all the three complexes (I, II, and III) with the PE, just as the crude nuclear extract, under appropriate conditions. Manipulations in vitro indicate that the NE has a significantly higher affinity for the dephosphorylated form than for the phosphorylated form of the protein. The PE binds both forms. Phenobarbitone treatment of the animal leads to a significant increase in the phosphorylation of the 26- to 28-kDa and 94-kDa proteins in nuclear labeling experiments followed by isolation on a PE affinity column. We propose that the protein binding predominantly to the NE in the dephosphorylated state characterizes the basal level of transcription of the CYP2B1/B2 gene. Phenobarbitone treatment leads to phosphorylation of the protein, shifting the equilibrium toward binding to the PE. This can promote interaction with an upstream

  4. Coupling of energy metabolism and synaptic transmission at the transcriptional level: Role of nuclear respiratory factor 1 in regulating both cytochrome c oxidase and NMDA glutamate receptor subunit genes

    PubMed Central

    Dhar, Shilpa S.; Wong-Riley, Margaret T. T.

    2009-01-01

    Neuronal activity and energy metabolism are tightly coupled processes. Regions high in neuronal activity, especially of the glutamatergic type, have high levels of cytochrome c oxidase (COX). Perturbations in neuronal activity affect the expressions of COX and glutamatergic N-methyl-D-aspartate receptor subunit 1 (NR1). The present study sought to test our hypothesis that the coupling extends to the transcriptional level, whereby NR1 and possibly other NR subunits and COX are co-regulated by the same transcription factor, nuclear respiratory factor 1 (NRF-1), which regulates all COX subunit genes. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation, promoter mutations, and real-time quantitative PCR, NRF-1 was found to functionally bind to the promoters of Grin 1 (NR1), Grin 2b (NR2b) and COX subunit genes, but not of Grin2a and Grin3a genes. These transcripts were up-regulated by KCl and down-regulated by TTX in cultured primary neurons. However, silencing of NRF-1 with small interference RNA blocked the up-regulation of Grin1, Grin2b, and COX induced by KCl, and over-expression of NRF-1 rescued these transcripts that were suppressed by TTX. NRF-1 binding sites on Grin1 and Grin2b genes are also highly conserved among mice, rats, and humans. Thus, NRF-1 is an essential transcription factor critical in the co-regulation of NR1, NR2b, and COX, and coupling exists at the transcriptional level to ensure coordinated expressions of proteins important for synaptic transmission and energy metabolism. PMID:19144849

  5. A remarkably stable TipE gene cluster: evolution of insect Para sodium channel auxiliary subunits

    PubMed Central

    2011-01-01

    Background First identified in fruit flies with temperature-sensitive paralysis phenotypes, the Drosophila melanogaster TipE locus encodes four voltage-gated sodium (NaV) channel auxiliary subunits. This cluster of TipE-like genes on chromosome 3L, and a fifth family member on chromosome 3R, are important for the optional expression and functionality of the Para NaV channel but appear quite distinct from auxiliary subunits in vertebrates. Here, we exploited available arthropod genomic resources to trace the origin of TipE-like genes by mapping their evolutionary histories and examining their genomic architectures. Results We identified a remarkably conserved synteny block of TipE-like orthologues with well-maintained local gene arrangements from 21 insect species. Homologues in the water flea, Daphnia pulex, suggest an ancestral pancrustacean repertoire of four TipE-like genes; a subsequent gene duplication may have generated functional redundancy allowing gene losses in the silk moth and mosquitoes. Intronic nesting of the insect TipE gene cluster probably occurred following the divergence from crustaceans, but in the flour beetle and silk moth genomes the clusters apparently escaped from nesting. Across Pancrustacea, TipE gene family members have experienced intronic nesting, escape from nesting, retrotransposition, translocation, and gene loss events while generally maintaining their local gene neighbourhoods. D. melanogaster TipE-like genes exhibit coordinated spatial and temporal regulation of expression distinct from their host gene but well-correlated with their regulatory target, the Para NaV channel, suggesting that functional constraints may preserve the TipE gene cluster. We identified homology between TipE-like NaV channel regulators and vertebrate Slo-beta auxiliary subunits of big-conductance calcium-activated potassium (BKCa) channels, which suggests that ion channel regulatory partners have evolved distinct lineage-specific characteristics

  6. Nucleotide sequence of the gene for the b subunit of human factor XIII

    SciTech Connect

    Bottenus, R.E.; Ichinose, A.; Davie, E.W. )

    1990-12-01

    Factor XIII (M{sub r} 320 000) is a blood coagulation factor that stabilizes and strengthens the fibrin clot. It circulates in blood as a tetramer composed of two a subunits (M{sub r} 75 000 each) and two b subunits (M{sub r} 80 000 each). The b subunit consists of 641 amino acids and includes 10 tandem repeats of 60 amino acids known as GP-I structures, short consensus repeats (SCR), or sushi domains. In the present study, the human gene for the b subunit has been isolated from three different genomic libraries prepared in {lambda} phage. Fifteen independent phage with inserts coding for the entire gene were isolated and characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene was found to be 28 kilobases in length and consisted of 12 exons (I-XII) separated by 11 intervening sequences. The leader sequence was encoded by exon I, while the carbonyl-terminal region of the protein was encoded by exon XII. Exons II-XI each coded for a single sushi domain, suggesting that the gene evolved through exon shuffling and duplication. The 12 exons in the gene ranged in size from 64 to 222 base pairs, while the introns ranged in size from 87 to 9970 nucleotides and made up 92{percent} of the gene. One nucleotide change was found in the coding region of the gene when its sequence was compared to that of the cDNA. This difference, however, did not result in a change in the amino acid sequence of the protein.

  7. Structural Changes of Regulatory Domain Heterodimer of N-Methyl-d-aspartate Receptor Subunits GluN1 and GluN2B through the Binding of Spermine and Ifenprodil

    PubMed Central

    Tomitori, Hideyuki; Suganami, Akiko; Saiki, Ryotaro; Mizuno, Satomi; Yoshizawa, Yuki; Masuko, Takashi; Tamura, Yutaka; Nishimura, Kazuhiro; Toida, Toshihiko; Williams, Keith; Kashiwagi, Keiko

    2012-01-01

    Modeling the binding sites for spermine and ifenprodil on the regulatory (R) domains of the N-methyl-d-aspartate receptor GluN1 and GluN2B subunits was carried out after measuring spermine stimulation and ifenprodil inhibition at receptors containing GluN1 and GluN2B R domain mutants. Models were constructed based on the published crystal structure of the GluN1 and GluN2B R domains, which form a heterodimer (Nature 475:249–253, 2011). The experimental results and modeling suggest that a binding site for spermine was formed by the residues near the cleft between the R1 and R2 lobes of the GluN1 R domain (GluN1R) together with residues on the surface of the R2 (C-terminal side) lobe of the GluN2B R domain (GluN2BR). The ifenprodil binding site included residues on the surface of the R1 lobe (N-terminal side) of GluN1R together with residues near the cleft between the R1 and R2 lobes of GluN2BR. It was confirmed using a Western blot analysis that GluN1R and GluN2BR formed a heterodimer. Models of spermine and ifenprodil binding to the heterodimer were constructed. The modeling suggests that an open space between the two R1 lobes of GluN1R and GluN2BR is promoted through spermine binding and that the R1 lobes of GluN1R and GluN2BR approach each other through ifenprodil binding—an effect opposite to that seen with the binding of spermine. PMID:22743575

  8. Antisense expression of the peptide transport gene AtPTR2-B delays flowering and arrests seed development in transgenic Arabidopsis plants.

    PubMed Central

    Song, W; Koh, S; Czako, M; Marton, L; Drenkard, E; Becker, J M; Stacey, G

    1997-01-01

    Previously, we identified a peptide transport gene, AtPTR2-B, from Arabidopsis thaliana that was constitutively expressed in all plant organs, suggesting an important physiological role in plant growth and development. To evaluate the function of this transporter, transgenic Arabidopsis plants were constructed expressing antisense or sense AtPTR2-B. Genomic Southern analysis indicated that four independent antisense and three independent sense AtPTR2-B transgenic lines were obtained, which was confirmed by analysis of the segregation of the kanamycin resistance gene carried on the T-DNA. RNA blot data showed that the endogenous AtPTR2-B mRNA levels were significantly reduced in transgenic leaves and flowers, but not in transgenic roots. Consistent with this reduction in endogenous AtPTR2-B mRNA levels, all four antisense lines and one sense line exhibited significant phenotypic changes, including late flowering and arrested seed development. These phenotypic changes could be explained by a defect in nitrogen nutrition due to the reduced peptide transport activity conferred by AtPTR2-B. These results suggest that AtPTR2-B may play a general role in plant nutrition. The AtPTR2-B gene was mapped to chromosome 2, which is closely linked to the restriction fragment length polymorphism marker m246. PMID:9232875

  9. Mouse muscle nicotinic acetylcholine receptor gamma subunit: cDNA sequence and gene expression.

    PubMed Central

    Yu, L; LaPolla, R J; Davidson, N

    1986-01-01

    Clones coding for the mouse nicotinic acetylcholine receptor (AChR) gamma subunit precursor have been selected from a cDNA library derived from a mouse myogenic cell line and sequenced. The deduced protein sequence consists of a signal peptide of 22 amino acid residues and a mature gamma subunit of 497 amino acid residues. There is a high degree of sequence conservation between this mouse sequence and published human and calf AChR gamma subunits and, after allowing for functional amino acid substitutions, also to the more distantly related chicken and Torpedo AChR gamma subunits. The degree of sequence conservation is especially high in the four putative hydrophobic membrane spanning regions, supporting the assignment of these domains. RNA blot hybridization showed that the mRNA level of the gamma subunit increases by 30 fold or more upon differentiation of the two mouse myogenic cell lines, BC3H-1 and C2C12, suggesting that the primary controls for changes in gene expression during differentiation are at the level of transcription. One cDNA clone was found to correspond to a partially processed nuclear transcript containing two as yet unspliced intervening sequences. Images PMID:3010242

  10. Association analysis of GABA receptor subunit genes on 5q33 with heroin dependence in a Chinese male population.

    PubMed

    Loh, E W; Tang, N L S; Lee, D T S; Liu, S I; Stadlin, Alfreda

    2007-06-01

    GABAA receptor subunit genes clustered on 5q33 play a role in the development of alcoholism and methamphetamine use disorder without psychosis. The present study explored the possible contribution of the same subunit genes to the development of heroin dependence. Single nucleotide polymorphisms (SNPs) of the GABAA receptor subunits GABRB2, GABRA6, GABRA1, and GABRG2 were examined in 178 male Han Chinese heroin-dependent and 170 male control subjects. A significant difference in allele frequency for the SNP rs211014 in the GABAAgamma2 receptor subunit gene between cases and controls was identified (P = 0.015). A possible mechanism for the involvement of the GABA receptor subunit genes on 5q33 in the development of heroin dependence is discussed. PMID:17440936

  11. Cloning and characterization of the 2B4 gene encoding a molecule associated with non-MHC-restricted killing mediated by activated natural killer cells and T cells

    SciTech Connect

    Mathew, P.A.; Garni-Wagner, B.A.; Land, K.; Takashima, A.; Stoneman, E.; Bennett, M.; Kumar, V. )

    1993-11-15

    The authors have recently described a signal transducing molecule, 2B4, expressed on all NK and T cells that mediate non-MHC-restricted killing. The gene encoding this molecule was cloned and its nucleotide sequence determined. The encoded protein of 398 amino acids has a leader peptide of 18 amino acids and a transmembrane region of 24 amino acids. The predicted protein has eight N-linked glycosylation sites, suggesting that it is highly glycosylated. Comparison of 2B4 with sequences in the databanks indicates that 2B4 is a member of the Ig supergene family, and it shows homology to murine and rat CD48 and human LFA-3. Northern blot analysis has shown at least three transcripts for 2B4 in adherent lymphokine-activated killer cells of several mouse strains and TCR-[gamma]/[delta] dendritic epidermal T cell lines but not in allospecific T cell clones. These three mRNA are the products of differential splicing of heterogeneous nuclear RNA. Southern blot analysis of genomic DNA from several mouse strains revealed that 2B4 belongs to a family of closely related genes. The 2B4 gene has been mapped to mouse chromosome 1 by analysis of 2B4 expression in recombinant inbred mouse strains. 48 refs., 6 figs., 2 tabs.

  12. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

    NASA Technical Reports Server (NTRS)

    Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

  13. Activation of spinal MrgC-Gi-NR2B-nNOS signaling pathway by Mas oncogene-related gene C receptor agonist bovine adrenal medulla 8-22 attenuates bone cancer pain in mice

    PubMed Central

    Sun, Yu’e; Zhang, Juan; Lei, Yishan; Lu, Cui’e; Hou, Bailing; Ma, Zhengliang; Gu, Xiaoping

    2016-01-01

    Objectives: In the present study, we investigate the effects of Mas oncogene-related gene (Mrg) C receptors (MrgC) on the expression and activation of spinal Gi protein, N-methyl-D-aspartate receptor subunit 2B (NR2B), and neuronal nitric oxide synthase (nNOS) in mouse model of bone cancer pain. Methods: The number of spontaneous foot lift (NSF) and paw withdrawal mechanical threshold (PWMT) were measured after inoculation of tumor cells and intrathecal injection of MrgC agonist bovine adrenal medulla 8-22 (BAM8-22) or MrgC antagonist anti-MrgC for 14 days after operation. Expression of spinal MrgC, Gi protein, NR2B and nNOS and their phosphorylated forms after inoculation was examined by immunohistochemistry and Western blotting. Double labeling was used to identify the co-localization of NR2B or nNOS with MrgC in spinal cord dorsal horn (SCDH) neurons. The effects of intrathecal injection of BAM8-22 or anti-MrgC on nociceptive behaviors and the corresponding expression of spinal MrgC, Gi protein, NR2B and nNOS were also investigated. Results: The expression of spinal MrgC, Gi protein, NR2B, and nNOS was higher in tumor-bearing mice in comparison to sham mice or normal mice. Intrathecal injection of MrgC agonist BAM8-22 significantly alleviated bone cancer pain, up-regulated MrgC and Gi protein expression, and down-regulated the expression of spinal p-NR2B, t-nNOS and p-nNOS in SCDH on day 14 after operation, whereas administration of anti-MrgC produced the opposite effect. Meanwhile, MrgC-like immunoreactivity (IR) co-localizes with NR2B-IR or nNOS-IR in SCDH neurons. Conclusions: The present study demonstrates that MrgC-activated spinal Gi-NR2B-nNOS signaling pathway plays important roles in the development of bone cancer pain. These findings may provide a novel strategy for the treatment of bone cancer pain. PMID:27158400

  14. Tight linkage of genes that encode the two glutamate synthase subunits of Escherichia coli K-12.

    PubMed Central

    Lozoya, E; Sanchez-Pescador, R; Covarrubias, A; Vichido, I; Bolivar, F

    1980-01-01

    A hybrid deoxyribonucleic acid molecule, plasmid pRSP20, which was isolated from the Clarke and Carbon Escherichia coli gene bank, was shown to complement the gltB31 mutation, which affects the synthesis of glutamate synthase in E. coli strain PA340. We present evidence which demonstrates that plasmid pRSP20 carries an 8-megadalton E. coli chromosomal fragment, including the genes encoding the two unequal glutamate synthase subunits. Polypeptides with molecular weights of about 135,000 and 53,000, which comigrated with purified E. coli glutamate synthase subunit polypeptides and immunoprecipitated with antibodies to E. coli glutamate synthase, were synthesized by minicells carrying the pRSP20 plasmid. Images PMID:6107287

  15. Regulation of expression of a soybean storage protein subunit gene. Progress report

    SciTech Connect

    Thompson, J.F.; Madison, J.T.

    1984-07-16

    We have found that soybean cotyledons could be cultured in vitro and that the storage proteins were formed essentially as on a plant. When methionine was added to the medium, the cotyledons grew faster, and the methionine content of the protein fraction was increased by over 20 percent. The high methionine content of the protein fraction was found to be due to a shift in the relative amounts of the two major storage proteins. The later effect was the result of methionine treatment suppressing the expression of one storage protein subunit gene. The goal was to determine the mechanism by which methionine is able to regulate the expression of the ..beta..-subunit gene.

  16. DNA Promoter Methylation-dependent Transcription of the Double C2-like Domain β (DOC2B) Gene Regulates Tumor Growth in Human Cervical Cancer*

    PubMed Central

    Kabekkodu, Shama Prasada; Bhat, Samatha; Radhakrishnan, Raghu; Aithal, Abhijit; Mascarenhas, Roshan; Pandey, Deeksha; Rai, Lavanya; Kushtagi, Pralhad; Mundyat, Gopinath Puthiya; Satyamoorthy, Kapaettu

    2014-01-01

    Double C2-like domain β (DOC2B) gene encodes for a calcium-binding protein, which is involved in neurotransmitter release, sorting, and exocytosis. We have identified the promoter region of the DOC2B gene as hypermethylated in pre-malignant, malignant cervical tissues, and cervical cancer cell lines by methylation-sensitive dimethyl sulfoxide-polymerase chain reaction and bisulfite genome sequencing; whereas, it was unmethylated in normal cervical tissues (p < 0.05). The promoter hypermethylation was inversely associated with mRNA expression in SiHa, CaSki, and HeLa cells and treatment with demethylating agent 5-aza-2-deoxycytidine restored DOC2B expression. The region −630 to +25 bp of the DOC2B gene showed robust promoter activity by a luciferase reporter assay and was inhibited by in vitro artificial methylation with Sss1 methylase prior to transient transfections. Overexpression of the DOC2B gene in SiHa cells when compared with controls showed significantly reduced colony formation, cell proliferation, induced cell cycle arrest, and repressed cell migration and invasion (p < 0.05). Ectopic expression of DOC2B resulted in anoikis-mediated cell death and repressed tumor growth in a nude mice xenograft model (p < 0.05). DOC2B expressing cells showed a significant increase in intracellular calcium level (p < 0.05), impaired AKT1 and ERK1/2 signaling, and induced actin cytoskeleton remodeling. Our results show that promoter hypermethylation and silencing of the DOC2B gene is an early and frequent event during cervical carcinogenesis and whose reduced expression due to DNA promoter methylation may lead to selective cervical tumor growth. PMID:24570007

  17. Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms

    PubMed Central

    Li, Ben-Wen; Rush, Amy C.; Weil, Gary J.

    2015-01-01

    Acetylcholine receptors (AChRs) are required for body movement in parasitic nematodes and are targets of “classical” anthelmintic drugs such as levamisole and pyrantel and of newer drugs such as tribendimidine and derquantel. While neurotransmission explains the effects of these drugs on nematode movement, their effects on parasite reproduction are unexplained. The levamisole AChR type (L-AChRs) in Caenorhabditis elegans is comprised of five subunits: Cel-UNC-29, Cel-UNC-38, Cel-UNC-63, Cel-LEV-1 and Cel-LEV-8. The genome of the filarial parasite Brugia malayi contains nine AChRs subunits including orthologues of Cel-unc-29, Cel-unc-38, and Cel-unc-63. We performed in situ hybridization with RNA probes to localize the expression of five AChR genes (Bm1_35890-Bma-unc-29, Bm1_20330-Bma-unc-38, Bm1_38195-Bma-unc-63, Bm1_48815-Bma-acr-26 and Bm1_40515-Bma-acr-12) in B. malayi adult worms. Four of these genes had similar expression patterns with signals in body muscle, developing embryos, spermatogonia, uterine wall adjacent to stretched microfilariae, wall of Vas deferens, and lateral cord. Three L-AChR subunit genes (Bma-unc-29, Bma-unc-38 and Bma-unc-63) were expressed in body muscle, which is a known target of levamisole. Bma-acr-12 was co-expressed with these levamisole subunit genes in muscle, and this suggests that its protein product may form receptors with other alpha subunits. Bma-acr-26 was expressed in male muscle but not in female muscle. Strong expression signals of these genes in early embryos and gametes in uterus and testis suggest that AChRs may have a role in nervous system development of embryogenesis and spermatogenesis. This would be consistent with embryotoxic effects of drugs that target these receptors in filarial worms. Our data show that the expression of these receptor genes is tightly regulated with regard to localization in adult worms and developmental stage in embryos and gametes. These results may help to explain the broad effects

  18. Expression, purification, and crystallization of Schizosaccharomyces pombe eIF2B.

    PubMed

    Kashiwagi, Kazuhiro; Shigeta, Tomoaki; Imataka, Hiroaki; Ito, Takuhiro; Yokoyama, Shigeyuki

    2016-03-01

    Tight control of protein synthesis is necessary for cells to respond and adapt to environmental changes rapidly. Eukaryotic translation initiation factor (eIF) 2B, the guanine nucleotide exchange factor for eIF2, is a key target of translation control at the initiation step. The nucleotide exchange activity of eIF2B is inhibited by the stress-induced phosphorylation of eIF2. As a result, the level of active GTP-bound eIF2 is lowered, and protein synthesis is attenuated. eIF2B is a large multi-subunit complex composed of five different subunits, and all five of the subunits are the gene products responsible for the neurodegenerative disease, leukoencephalopathy with vanishing white matter. However, the overall structure of eIF2B has remained unresolved, due to the difficulty in preparing a sufficient amount of the eIF2B complex. To overcome this problem, we established the recombinant expression and purification method for eIF2B from the fission yeast Schizosaccharomyces pombe. All five of the eIF2B subunits were co-expressed and reconstructed into the complex in Escherichia coli cells. The complex was successfully purified with a high yield. This recombinant eIF2B complex contains each subunit in an equimolar ratio, and the size exclusion chromatography analysis suggests it forms a heterodecamer, consistent with recent reports. This eIF2B increased protein synthesis in the reconstituted in vitro human translation system. In addition, disease-linked mutations led to subunit dissociation. Furthermore, we crystallized this functional recombinant eIF2B, and the crystals diffracted to 3.0 Å resolution. PMID:27023709

  19. Excess adenosine A2B receptor signaling contributes to priapism through HIF-1α mediated reduction of PDE5 gene expression

    PubMed Central

    Ning, Chen; Wen, Jiaming; Zhang, Yujin; Dai, Yingbo; Wang, Wei; Zhang, Weiru; Qi, Lin; Grenz, Almut; Eltzschig, Holger K.; Blackburn, Michael R.; Kellems, Rodney E.; Xia, Yang

    2014-01-01

    Priapism is featured with prolonged and painful penile erection and is prevalent among males with sickle cell disease (SCD). The disorder is a dangerous urological and hematological emergency since it is associated with ischemic tissue damage and erectile disability. Here we report that phosphodiesterase-5 (PDE5) gene expression and PDE activity is significantly reduced in penile tissues of two independent priapic models: SCD mice and adenosine deaminase (ADA)-deficient mice. Moreover, using ADA enzyme therapy to reduce adenosine or a specific antagonist to block A2B adenosine receptor (ADORA2B) signaling, we successfully attenuated priapism in both ADA−/− and SCD mice by restoring penile PDE5 gene expression to normal levels. This finding led us to further discover that excess adenosine signaling via ADORA2B activation directly reduces PDE5 gene expression in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. Overall, we reveal that excess adenosine-mediated ADORA2B signaling underlies reduced penile PDE activity by decreasing PDE5 gene expression in a HIF-1α-dependent manner and provide new insight for the pathogenesis of priapism and novel therapies for the disease.—Ning, C., Wen, J., Zhang, Y., Dai, Y., Wang, W., Zhang, W., Qi, L., Grenz, A., Eltzschig, H. K., Blackburn, M. R., Kellems, R. E., Xia, Y. Excess adenosine A2B receptor signaling contributes to priapism through HIF-1α mediated reduction of PDE5 gene expression. PMID:24614760

  20. Multiple pathways regulate the expression of genes encoding sodium channel subunits in developing neurons.

    PubMed

    Giraud, P; Alcaraz, G; Jullien, F; Sampo, B; Jover, E; Couraud, F; Dargent, B

    1998-05-01

    In primary cultures of fetal neurons, activation of sodium channels with either alpha-scorpion toxin or veratridine caused a rapid and persistent decrease of mRNAs encoding beta2 and different sodium channel alpha mRNAs. In contrast, beta1 subunit mRNA was up-regulated by sodium channel activation. This phenomenon was calcium-independent. The effects of activating toxins on mRNAs of different sodium channel subunits were mimicked by membrane depolarization. An important aspect of this study was the demonstration that cAMP also caused rapid reduction of alphaI, alphaII and alphaIII mRNA levels whereas beta1 subunit mRNA was up regulated and beta2 subunit mRNA was not affected. Sodium channel activation by veratridine was shown to increase cAMP immunoreactivity in cultured neurons, but alphaII mRNA down-regulation induced by activating toxins was not reversed by protein kinase A antagonists, indicating that this phenomenon is not protein kinase A dependent. The effects of cAMP and membrane depolarisation were antagonized by the PKA inhibitor H89. These results are indicative of the existence of multiple and independent regulatory pathways modulating the expression of sodium channel genes in the developing central nervous system. PMID:9602139

  1. Rice Ribosomal Protein Large Subunit Genes and Their Spatio-temporal and Stress Regulation

    PubMed Central

    Moin, Mazahar; Bakshi, Achala; Saha, Anusree; Dutta, Mouboni; Madhav, Sheshu M.; Kirti, P. B.

    2016-01-01

    Ribosomal proteins (RPs) are well-known for their role in mediating protein synthesis and maintaining the stability of the ribosomal complex, which includes small and large subunits. In the present investigation, in a genome-wide survey, we predicted that the large subunit of rice ribosomes is encoded by at least 123 genes including individual gene copies, distributed throughout the 12 chromosomes. We selected 34 candidate genes, each having 2–3 identical copies, for a detailed characterization of their gene structures, protein properties, cis-regulatory elements and comprehensive expression analysis. RPL proteins appear to be involved in interactions with other RP and non-RP proteins and their encoded RNAs have a higher content of alpha-helices in their predicted secondary structures. The majority of RPs have binding sites for metal and non-metal ligands. Native expression profiling of 34 ribosomal protein large (RPL) subunit genes in tissues covering the major stages of rice growth shows that they are predominantly expressed in vegetative tissues and seedlings followed by meiotically active tissues like flowers. The putative promoter regions of these genes also carry cis-elements that respond specifically to stress and signaling molecules. All the 34 genes responded differentially to the abiotic stress treatments. Phytohormone and cold treatments induced significant up-regulation of several RPL genes, while heat and H2O2 treatments down-regulated a majority of them. Furthermore, infection with a bacterial pathogen, Xanthomonas oryzae, which causes leaf blight also induced the expression of 80% of the RPL genes in leaves. Although the expression of RPL genes was detected in all the tissues studied, they are highly responsive to stress and signaling molecules indicating that their encoded proteins appear to have roles in stress amelioration besides house-keeping. This shows that the RPL gene family is a valuable resource for manipulation of stress tolerance in

  2. Rice Ribosomal Protein Large Subunit Genes and Their Spatio-temporal and Stress Regulation.

    PubMed

    Moin, Mazahar; Bakshi, Achala; Saha, Anusree; Dutta, Mouboni; Madhav, Sheshu M; Kirti, P B

    2016-01-01

    Ribosomal proteins (RPs) are well-known for their role in mediating protein synthesis and maintaining the stability of the ribosomal complex, which includes small and large subunits. In the present investigation, in a genome-wide survey, we predicted that the large subunit of rice ribosomes is encoded by at least 123 genes including individual gene copies, distributed throughout the 12 chromosomes. We selected 34 candidate genes, each having 2-3 identical copies, for a detailed characterization of their gene structures, protein properties, cis-regulatory elements and comprehensive expression analysis. RPL proteins appear to be involved in interactions with other RP and non-RP proteins and their encoded RNAs have a higher content of alpha-helices in their predicted secondary structures. The majority of RPs have binding sites for metal and non-metal ligands. Native expression profiling of 34 ribosomal protein large (RPL) subunit genes in tissues covering the major stages of rice growth shows that they are predominantly expressed in vegetative tissues and seedlings followed by meiotically active tissues like flowers. The putative promoter regions of these genes also carry cis-elements that respond specifically to stress and signaling molecules. All the 34 genes responded differentially to the abiotic stress treatments. Phytohormone and cold treatments induced significant up-regulation of several RPL genes, while heat and H2O2 treatments down-regulated a majority of them. Furthermore, infection with a bacterial pathogen, Xanthomonas oryzae, which causes leaf blight also induced the expression of 80% of the RPL genes in leaves. Although the expression of RPL genes was detected in all the tissues studied, they are highly responsive to stress and signaling molecules indicating that their encoded proteins appear to have roles in stress amelioration besides house-keeping. This shows that the RPL gene family is a valuable resource for manipulation of stress tolerance in rice

  3. Molecular cloning and characterization of two nicotinic acetylcholine receptor β subunit genes from Apis cerana cerana.

    PubMed

    Yu, Xiaoli; Wang, Mian; Kang, Mingjiang; Liu, Li; Guo, Xingqi; Xu, Baohua

    2011-08-01

    Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission in the insect nervous system and are important targets for insecticides. In this study, we identified and characterized two novel β subunit genes (Accβ1 and Accβ2) from Apis cerana cerana. Homology analysis indicated that Accβ1 and Accβ2 possess characteristics that are typical of nAChR subunits although Accβ2 was distinct from Accβ1 and the other nAChR subunits, due to its unusual transmembrane structure and uncommon exon-intron boundary within the genomic region encoding the TM1 transmembrane domain. Analysis of the 5' flanking regions indicated that Accβ1 and Accβ2 possess different regulatory elements, suggesting that the genes might exhibit various expression and regulatory patterns. RT-PCR analysis demonstrated that Accβ2 was expressed at a much higher level than Accβ1 in the tissues of adult bees. During development, Accβ1 was highly expressed at the pupal stages, whereas Accβ2 was abundantly expressed at the larval stages. Furthermore, Accβ1 and Accβ2 were both induced by exposure to various insecticides and environmental stresses although Accβ2 was more responsive than Accβ1. These results indicate that Accβ1 and Accβ2 may have distinct roles in insect growth and development and that they may belong to separate regulatory pathways involved in the response to insecticides and environmental stresses. This report is the first description of the differences between the nAChR β subunit genes in the Chinese honey bee and establishes an initial foundation for further study. PMID:21618599

  4. Cloning and expression of the gene encoding catalytic subunit of thermostable glucose dehydrogenase from Burkholderia cepacia in Escherichia coli.

    PubMed

    Inose, Ken; Fujikawa, Masako; Yamazaki, Tomohiko; Kojima, Katsuhiro; Sode, Koji

    2003-02-21

    We have cloned a 1620-nucleotide gene encoding the catalytic subunit (alpha subunit) of a thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia. The FAD binding motif was found in the N-terminal region of the alpha subunit. The deduced primary structure of the alpha subunit showed about 48% identity to the catalytic subunits of sorbitol dehydrogenase (SDH) from Gluconobacter oxydans and 2-keto-D-gluconate dehydrogenases (2KGDH) from Erwinia herbicola and Pantoea citrea. The alpha subunit of B. cepacia was expressed in Escherichia coli in its active water-soluble form, showing maximum dye-mediated GDH activity at 70 degrees C, retaining high thermal stability. A putative open reading frame (ORF) of 507 nucleotides was also found upstream of the alpha subunit encoding an 18-kDa peptide, designated as gamma subunit. The deduced primary structure of gamma subunit showed about 30% identity to the small subunits of the SDH from G. oxydans and 2KGDHs from E. herbicola and P. citrea. PMID:12573242

  5. Genetic Differentiation of the Mitochondrial Cytochrome Oxidase c Subunit I Gene in Genus Paramecium (Protista, Ciliophora)

    PubMed Central

    Zhao, Yan; Gentekaki, Eleni; Yi, Zhenzhen; Lin, Xiaofeng

    2013-01-01

    Background The mitochondrial cytochrome c oxidase subunit I (COI) gene is being used increasingly for evaluating inter- and intra-specific genetic diversity of ciliated protists. However, very few studies focus on assessing genetic divergence of the COI gene within individuals and how its presence might affect species identification and population structure analyses. Methodology/Principal findings We evaluated the genetic variation of the COI gene in five Paramecium species for a total of 147 clones derived from 21 individuals and 7 populations. We identified a total of 90 haplotypes with several individuals carrying more than one haplotype. Parsimony network and phylogenetic tree analyses revealed that intra-individual diversity had no effect in species identification and only a minor effect on population structure. Conclusions Our results suggest that the COI gene is a suitable marker for resolving inter- and intra-specific relationships of Paramecium spp. PMID:24204730

  6. Immunochemical Proof that a Novel Rearranging Gene Encodes the T Cell Receptor δ Subunit

    NASA Astrophysics Data System (ADS)

    Band, Hamid; Hochstenbach, Frans; McLean, Joanne; Hata, Shingo; Krangel, Michael S.; Brenner, Michael B.

    1987-10-01

    The T cell receptor (TCR) δ protein is expressed as part of a heterodimer with TCR γ , in association with the CD3 polypeptides on a subset of functional peripheral blood T lymphocytes, thymocytes, and certain leukemic T cell lines. A monoclonal antibody directed against TCR δ was produced that binds specifically to the surface of several TCR γ δ cell lines and immunoprecipitates the TCR γ δ as a heterodimer from Triton X-100 detergent lysates and also immunoprecipitates the TCR δ subunit alone after chain separation. A candidate human TCR δ complementary DNA clone (IDP2 O-240/38), reported in a companion paper, was isolated by the subtractive library approach from a TCR γ δ cell line. This complementary DNA clone was used to direct the synthesis of a polypeptide that is specifically recognized by the monoclonal antibody to TCR δ . This complementary DNA clone thus corresponds to the gene that encodes the TCR δ subunit.

  7. Characterization of an ADP-glucose Pyrophosphorylase Small Subunit Gene Expressed in Developing Cotton (Gossypium hirsutum) Fibers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ADP-glucose pyrophosphorylase (ADPGp) plays a rate limiting role in the biosynthesis of starch and has been shown to be involved in cell expansion of tobacco sepals. A cotton gene encoding ADPGp small subunit was isolated and sequenced. The gene contains 8 introns similar to other ADPGp genes. The o...

  8. Glucocorticoid regulation of branched-chain alpha-ketoacid dehydrogenase E2 subunit gene expression.

    PubMed Central

    Costeas, P A; Chinsky, J M

    2000-01-01

    Regulation of the mammalian branched-chain alpha-ketoacid dehydrogenase complex (BCKAD) occurs under a variety of stressful conditions associated with changes in circulating glucocorticoids. Multiple levels of regulation in hepatocytes, including alteration of the levels of the structural subunits available for assembly (E1, alpha-ketoacid decarboxylase; E2, dihydrolipoamide acyltransferase; and E3, dihydrolipoamide dehydrogenase), as well as BCKAD kinase, which serves to phosphorylate the E1alpha subunit and inactivate complex activity, have been proposed. The direct role of glucocorticoids in regulating the expression of the murine gene encoding the major BCKAD subunit E2, upon which the other BCKAD subunits assemble, was therefore examined. Deletion analysis of the 5' proximal 7.0 kb of the murine E2 promoter sequence, using E2 promoter/luciferase expression minigene plasmids introduced into the hepatic H4IIEC3 cell line, suggested a promoter proximal region responsive to glucocorticoid regulation. Linker-scanning mutagenesis combined with deletion analysis established this functional glucocorticoid-responsive unit (GRU) to be located near the murine E2 proximal promoter site at -140 to -70 bp upstream from the transcription initiation site. The presence of this region in plasmid minigenes, containing varying amounts of the murine genomic sequence 5' upstream from proximal E2 promoter sequences, conferred 2-10 fold increases in luciferase reporter gene expression in H4IIEC3 cells, whether introduced by transient transfection or following co-selection for stable transfectants. The GRU region itself appeared to contain multiple interacting elements that combine to regulate overall E2 promoter activity in response to changing physiological conditions associated with varying concentrations of glucocorticoids and likely other hormonal effectors. PMID:10749674

  9. Fibrinogen {alpha} genes: Conservation of bipartite transcripts and carboxy-terminal-extended {alpha} subunits in vertebrates

    SciTech Connect

    Fu, Y.; Cao, Y.; Hertzberg, K.M.; Grieninger, G.

    1995-11-01

    All three well-studied subunits of the clotting protein fibrinogen ({alpha}, {beta}, {gamma}) share N-terminal structural homologies, but until recently only the {beta} and {gamma} chains were recognized as having similar globular C-termini. With the discovery of an extra exon in the human fibrinogen {alpha} gene (exon VI), a minor form of the {alpha} subunit ({alpha}{sub E}) with an extended {beta}- and {gamma}-like C-terminus has been identified. In the present study, the polymerase chain reaction has been used to identify sequences that encode counterparts to {alpha}{sub E} in chicken, rabbit, rat, and baboon. The basic six-exon structure of the fibrinogen {alpha} genes is shown to be conserved among mammals and birds, as are the intron positions. Bipartite transcripts - still bearing an intron prior to the last exon - are found among the products of the various vertebrate fibrinogen {alpha} genes. The last exon represents the largest conserved segment of the gene and, in each species examined, encodes exactly 236 amino acids. The C-termini of these {alpha}{sub E} chains align without a single gap and are between 76 and 99% identical. Since the exon VI-encoded domain of {alpha}{sub E} is as well conserved as the corresponding regions of the {beta} and {gamma} chains, it follows that it is equally important and that {alpha}{sub E}-fibrinogen plays a vital, if as-yet unrecognized physiological role. 21 refs., 7 figs., 1 tab.

  10. An approach for isolating high-molecular-weight glutenin subunit genes using monoclonal antibodies.

    PubMed

    Wang, H Q; Zhang, X Y

    2006-02-01

    High-molecular-weight glutenin subunits (HMW-GSs) play an important role in the breadmaking quality of wheat flour. In China, cultivars such as Triticum aestivum 'Xiaoyan No. 6' carrying the 1Bx14 and 1By15 glutenin subunits usually have attributes that result in high-quality bread and noodles. HMW-GS 1Bx14 and 1By15 were isolated by preparative sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and used as an antigen to immunize BALB/c mice. A resulting monoclonal antibody belonging to the IgG1 subclass was shown to bind to all HMW-GSs of Triticum aestivum cultivars, but did not bind to other storage proteins of wheat seeds in a Western blot analysis. After screening a complementary DNA expression library from immature seeds of 'Xiaoyan No. 6' using the monoclonal antibody, the HMW-GS 1By15 gene was isolated and fully sequenced. The deduced amino acid sequence showed an extra stretch of 15 amino acid repeats consisting of a hexapeptide and a nonapeptide in the repetitive domain of this y-type HMW subunit. Bacterial expression of a modified 1By15 gene, in which the coding sequence for the signal peptide was removed and a BamHI site eliminated, gave rise to a protein with mobility identical to that of HMW-GSs extracted from seeds of 'Xiaoyan No. 6' via SDS-PAGE. This approach for isolating genes using specific monoclonal antibody against HMW-GS genes is a good alternative to the extensively used polymerase chain reaction (PCR) technology based on sequence homology of HMW-GSs in wheat and its relatives. PMID:16498468

  11. Diverse gene-silencing mechanisms with distinct requirements for RNA polymerase subunits in Zea mays.

    PubMed

    Sloan, Amy E; Sidorenko, Lyudmila; McGinnis, Karen M

    2014-11-01

    In Zea mays, transcriptional regulation of the b1 (booster1) gene requires a distal enhancer and MEDIATOR OF PARAMUTATION1 (MOP1), MOP2, and MOP3 proteins orthologous to Arabidopsis components of the RNA-dependent DNA methylation pathway. We compared the genetic requirements for MOP1, MOP2, and MOP3 for endogenous gene silencing by two hairpin transgenes with inverted repeats of the a1 (anthocyaninless1) gene promoter (a1pIR) and the b1 gene enhancer (b1IR), respectively. The a1pIR transgene induced silencing of endogenous A1 in mop1-1 and mop3-1, but not in Mop2-1 homozygous plants. This finding suggests that transgene-derived small interfering RNAs (siRNAs) circumvented the requirement for MOP1, a predicted RNA-dependent RNA polymerase, and MOP3, the predicted largest subunit of RNA polymerase IV (Pol IV). Because the Arabidopsis protein orthologous to MOP2 is the second largest subunit of Pol IV and V, our results may indicate that hairpin-induced siRNAs cannot bypass the requirement for the predicted scaffolding activity of Pol V. In contrast to a1pIR, the b1IR transgene silenced endogenous B1 in all three homozygous mutant genotypes--mop1-1, Mop2-1, and mop3-1--suggesting that transgene mediated b1 silencing did not involve MOP2-containing Pol V complexes. Based on the combined results for a1, b1, and three previously described loci, we propose a speculative hypothesis of locus-specific deployment of Pol II, MOP2-containing Pol V, or alternative versions of Pol V with second largest subunits other than MOP2 to explain the mechanistic differences in silencing at specific loci, including one example associated with paramutation. PMID:25164883

  12. Cloning of eight Rhopalosiphum padi (Hemiptera: Aphididae) nAChR subunit genes and mutation detection of the β1 subunit in field samples from China.

    PubMed

    Zhang, Meng; Qiao, Xianfeng; Li, Yuting; Fang, Bing; Zuo, Yayun; Chen, Maohua

    2016-09-01

    The bird cherry-oat aphid, Rhopalosiphum padi (L.), is one of the most important wheat pests. This aphid damages through direct feeding and by transmitting the Barley yellow dwarf virus (BYDV). Both types of damage significantly reduce the quality and yield of wheat crops globally. Insecticides are the primary method of controlling the bird cherry-oat aphid in China, yet this aphid species has developed resistance to different types of insecticides, especially organophosphates and carbamates. In the last decade, control of R. padi depends primarily on the spray of neonicotinoid insecticides, however, research on the resistance of R. padi to neonicotinoids has been limited. In this study, the full lengths of seven α-subunit (Rpα1, Rpα2, Rpα3, Rpα4, Rpα5, Rpα7-1, and Rpα7-2) and one β-subunit (Rpβ1) genes from R. padi were obtained with RT-PCR and RACE techniques. Sequence analysis showed that these genes had all the characteristics of the nAChR gene family and were highly homologous with the reported nAChR genes from other insects, and alternative splicing was detected in Rpα3 and Rpα5 subunits. Analysis of the cDNA sequence of the extracellular region of the nicotinic acetylcholine receptor β1 subunit gene from 120 R. padi field samples collected in 11 Provinces revealed 17 single nucleotides polymorphism (SNP) sites, of which seven were amino acid polymorphism sites (V53I, V53G, N54T, A60T, F61L, W79C, and V83I) and two were in the loop D region (W79C and V83I). The current study will facilitate further studies on the molecular mechanisms of targeted resistance of the aphid to neonicotinoid insecticides. PMID:27521918

  13. The structures of the human calcium channel {alpha}{sub 1} subunit (CACNL1A2) and {beta} subunit (CACNLB3) genes

    SciTech Connect

    Yamada, Yuichiro; Masuda, Kazuhiro; Li, Qing

    1995-05-20

    Calcium influx in pancreatic {beta}-cells is regulated mainly by L-type voltage-dependent calcium channels (VDCCs) and triggers insulin secretion. The {alpha}{sub 1} subunit (CACN4) and the {beta} subunit ({beta}{sub 3}) of VDCCs, both of which are expressed in pancreatic islets, are major components for the VDCC activity, and so they may play a critical role in the regulation of insulin secretion. The authors have determined the structures of the human CACN4 (CACNL1A2) and the human {beta}{sub 3} (CACNLB3) genes. The CACNL1A2 gene spans more than 155 kb and has 49 exons. Most of the positions interrupted by introns are well conserved between the CACNL1A2 gene and the previously reported L-type VDCC {alpha}{sub 1} subunit, CACNL1A1, gene. On the other hand, the CACNLB3 gene distributes in {approximately} 8 kb and comprises 13 exons, most of which are located together within {approximately} 5 kb. Comparisons of the genomic sequences of CACNL1A2 with the previously reported cDNA sequences indicate that there are a number of polymorphisms in the human CACNL1A2 gene. In addition, the PCR-SSCP procedure of exon 1 of CACNL1A2 revealed a change from 7 to 8 ATG trinucleotide repeats in a patient with noninsulin-dependent diabetes mellitus (NIDDM), resulting in an addition of methionine at the amino-terminus of CACN4. The determination of the structures of the human CACNL1A2 and CACNLB3 genes should facilitate study of the role of these genes in the development of NIDDM and also other genetic diseases such as long QT syndrome. 39 refs., 3 figs., 3 tabs.

  14. The D-isoAsp-25 variant of histone H2B is highly enriched in active chromatin: potential role in the regulation of gene expression?

    PubMed

    Qin, Zhenxia; Zhu, Jeff X; Aswad, Dana W

    2016-02-01

    Approximately 12 % of histone H2B in mammalian brain contains an unusual D-aspartate residue in its N-terminal tail. Most of this D-aspartate is linked to the C-flanking glycine via an isopeptide bond. To explore the possible significance of these modifications, we generated an antibody to the D-isoaspartyl form of H2B, and used it to assess its levels in H2B associated with "active" vs. "silent" chromatin. We found that the D-isoaspartyl form of H2B appears to be highly enriched in the former. This irreversible modification could serve a novel regulatory function in gene expression. PMID:26666674

  15. Multiple Thyrotropin β-Subunit and Thyrotropin Receptor-Related Genes Arose during Vertebrate Evolution

    PubMed Central

    Maugars, Gersende; Dufour, Sylvie; Cohen-Tannoudji, Joëlle; Quérat, Bruno

    2014-01-01

    Thyroid-stimulating hormone (TSH) is composed of a specific β subunit and an α subunit that is shared with the two pituitary gonadotropins. The three β subunits derive from a common ancestral gene through two genome duplications (1R and 2R) that took place before the radiation of vertebrates. Analysis of genomic data from phylogenetically relevant species allowed us to identify an additional Tshβ subunit-related gene that was generated through 2R. This gene, named Tshβ2, present in cartilaginous fish, little skate and elephant shark, and in early lobe-finned fish, coelacanth and lungfish, was lost in ray-finned fish and tetrapods. The absence of a second type of TSH receptor (Tshr) gene in these species suggests that both TSHs act through the same receptor. A novel Tshβ sister gene, named Tshβ3, was generated through the third genomic duplication (3R) that occurred early in the teleost lineage. Tshβ3 is present in most teleost groups but was lostin tedraodontiforms. The 3R also generated a second Tshr, named Tshrb. Interestingly, the new Tshrb was translocated from its original chromosomic position after the emergence of eels and was then maintained in its new position. Tshrb was lost in tetraodontiforms and in ostariophysians including zebrafish although the latter species have two TSHs, suggesting that TSHRb may be dispensable. The tissue distribution of duplicated Tshβs and Tshrs was studied in the European eel. The endocrine thyrotropic function in the eel would be essentially mediated by the classical Tshβ and Tshra, which are mainly expressed in the pituitary and thyroid, respectively. Tshβ3 and Tshrb showed a similar distribution pattern in the brain, pituitary, ovary and adipose tissue, suggesting a possible paracrine/autocrine mode of action in these non-thyroidal tissues. Further studies will be needed to determine the binding specificity of the two receptors and how these two TSH systems are interrelated. PMID:25386660

  16. Multiple thyrotropin β-subunit and thyrotropin receptor-related genes arose during vertebrate evolution.

    PubMed

    Maugars, Gersende; Dufour, Sylvie; Cohen-Tannoudji, Joëlle; Quérat, Bruno

    2014-01-01

    Thyroid-stimulating hormone (TSH) is composed of a specific β subunit and an α subunit that is shared with the two pituitary gonadotropins. The three β subunits derive from a common ancestral gene through two genome duplications (1R and 2R) that took place before the radiation of vertebrates. Analysis of genomic data from phylogenetically relevant species allowed us to identify an additional Tshβ subunit-related gene that was generated through 2R. This gene, named Tshβ2, present in cartilaginous fish, little skate and elephant shark, and in early lobe-finned fish, coelacanth and lungfish, was lost in ray-finned fish and tetrapods. The absence of a second type of TSH receptor (Tshr) gene in these species suggests that both TSHs act through the same receptor. A novel Tshβ sister gene, named Tshβ3, was generated through the third genomic duplication (3R) that occurred early in the teleost lineage. Tshβ3 is present in most teleost groups but was lostin tedraodontiforms. The 3R also generated a second Tshr, named Tshrb. Interestingly, the new Tshrb was translocated from its original chromosomic position after the emergence of eels and was then maintained in its new position. Tshrb was lost in tetraodontiforms and in ostariophysians including zebrafish although the latter species have two TSHs, suggesting that TSHRb may be dispensable. The tissue distribution of duplicated Tshβs and Tshrs was studied in the European eel. The endocrine thyrotropic function in the eel would be essentially mediated by the classical Tshβ and Tshra, which are mainly expressed in the pituitary and thyroid, respectively. Tshβ3 and Tshrb showed a similar distribution pattern in the brain, pituitary, ovary and adipose tissue, suggesting a possible paracrine/autocrine mode of action in these non-thyroidal tissues. Further studies will be needed to determine the binding specificity of the two receptors and how these two TSH systems are interrelated. PMID:25386660

  17. Decrease of gene expression of astrocytic 5-HT2B receptors parallels development of depressive phenotype in a mouse model of Parkinson's disease.

    PubMed

    Zhang, Xique; Song, Dan; Gu, Li; Ren, Yan; Verkhratsky, Alexei; Peng, Liang

    2015-01-01

    Astrocytes contribute to pathogenesis of neuropsychiatric disorders, including major depression. Stimulation of astroglial 5-HT2B receptors transactivates epidermal growth factor receptors (EGFRs) and regulates gene expression. Previously we reported that expression of 5-HT2B receptors in cortical astrocytes is down-regulated in animals, which developed anhedonia in response to chronic stress; moreover this down-regulation as well as anhedonia, are reversed by chronic treatment with fluoxetine. In this study we have investigated whether astrocytic 5-HT2B receptor is involved in anhedonia in C57BL/6 mice model of Parkinson' disease (PD) induced by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 7 days. The MPTP treatment induced anhendonia in 66.7% of animals. The appearance of depressive behavior was accompanied with motor deficiency and decrease of tyrosine hydroxylase (TH) expression. Expression of mRNA and protein of 5-HT2B receptor in animals that became anhedonic decreased to 77.3 and 79.3% of control groups, respectively; in animals that received MPTP but did not develop anhedonia the expression of 5-HT2B receptor did not change. Experiments with FACS-sorted isolated cells demonstrated that decrease in 5-HT2B receptor expression was confined to astrocytes, and did not occur in neurons. Fluoxetine corrected MPTP-induced decrease of 5-HT2B receptor expression and depressive behavior. Our findings indicate that regulation of gene expression of 5-HT2B receptors in astroglia may be associated with pathophysiological evolution of PD-induced depression. PMID:26500493

  18. Decrease of gene expression of astrocytic 5-HT2B receptors parallels development of depressive phenotype in a mouse model of Parkinson’s disease

    PubMed Central

    Zhang, Xique; Song, Dan; Gu, Li; Ren, Yan; Verkhratsky, Alexei; Peng, Liang

    2015-01-01

    Astrocytes contribute to pathogenesis of neuropsychiatric disorders, including major depression. Stimulation of astroglial 5-HT2B receptors transactivates epidermal growth factor receptors (EGFRs) and regulates gene expression. Previously we reported that expression of 5-HT2B receptors in cortical astrocytes is down-regulated in animals, which developed anhedonia in response to chronic stress; moreover this down-regulation as well as anhedonia, are reversed by chronic treatment with fluoxetine. In this study we have investigated whether astrocytic 5-HT2B receptor is involved in anhedonia in C57BL/6 mice model of Parkinson’ disease (PD) induced by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 7 days. The MPTP treatment induced anhendonia in 66.7% of animals. The appearance of depressive behavior was accompanied with motor deficiency and decrease of tyrosine hydroxylase (TH) expression. Expression of mRNA and protein of 5-HT2B receptor in animals that became anhedonic decreased to 77.3 and 79.3% of control groups, respectively; in animals that received MPTP but did not develop anhedonia the expression of 5-HT2B receptor did not change. Experiments with FACS-sorted isolated cells demonstrated that decrease in 5-HT2B receptor expression was confined to astrocytes, and did not occur in neurons. Fluoxetine corrected MPTP-induced decrease of 5-HT2B receptor expression and depressive behavior. Our findings indicate that regulation of gene expression of 5-HT2B receptors in astroglia may be associated with pathophysiological evolution of PD-induced depression. PMID:26500493

  19. Generation of BAC transgenic tadpoles enabling live imaging of motoneurons by using the urotensin II-related peptide (ust2b) gene as a driver.

    PubMed

    Bougerol, Marion; Auradé, Frédéric; Lambert, François M; Le Ray, Didier; Combes, Denis; Thoby-Brisson, Muriel; Relaix, Frédéric; Pollet, Nicolas; Tostivint, Hervé

    2015-01-01

    Xenopus is an excellent tetrapod model for studying normal and pathological motoneuron ontogeny due to its developmental morpho-physiological advantages. In mammals, the urotensin II-related peptide (UTS2B) gene is primarily expressed in motoneurons of the brainstem and the spinal cord. Here, we show that this expression pattern was conserved in Xenopus and established during the early embryonic development, starting at the early tailbud stage. In late tadpole stage, uts2b mRNA was detected both in the hindbrain and in the spinal cord. Spinal uts2b+ cells were identified as axial motoneurons. In adult, however, the uts2b expression was only detected in the hindbrain. We assessed the ability of the uts2b promoter to drive the expression of a fluorescent reporter in motoneurons by recombineering a green fluorescent protein (GFP) into a bacterial artificial chromosome (BAC) clone containing the entire X. tropicalis uts2b locus. After injection of this construction in one-cell stage embryos, a transient GFP expression was observed in the spinal cord of about a quarter of the resulting animals from the early tailbud stage and up to juveniles. The GFP expression pattern was globally consistent with that of the endogenous uts2b in the spinal cord but no fluorescence was observed in the brainstem. A combination of histological and electrophysiological approaches was employed to further characterize the GFP+ cells in the larvae. More than 98% of the GFP+ cells expressed choline acetyltransferase, while their projections were co-localized with α-bungarotoxin labeling. When tail myotomes were injected with rhodamine dextran amine crystals, numerous double-stained GFP+ cells were observed. In addition, intracellular electrophysiological recordings of GFP+ neurons revealed locomotion-related rhythmic discharge patterns during fictive swimming. Taken together our results provide evidence that uts2b is an appropriate driver to express reporter genes in larval motoneurons of the

  20. Evolutionary Analysis of the B56 Gene Family of PP2A Regulatory Subunits

    PubMed Central

    Sommer, Lauren M.; Cho, Hyuk; Choudhary, Madhusudan; Seeling, Joni M.

    2015-01-01

    Protein phosphatase 2A (PP2A) is an abundant serine/threonine phosphatase that functions as a tumor suppressor in numerous cell-cell signaling pathways, including Wnt, myc, and ras. The B56 subunit of PP2A regulates its activity, and is encoded by five genes in humans. B56 proteins share a central core domain, but have divergent amino- and carboxy-termini, which are thought to provide isoform specificity. We performed phylogenetic analyses to better understand the evolution of the B56 gene family. We found that B56 was present as a single gene in eukaryotes prior to the divergence of animals, fungi, protists, and plants, and that B56 gene duplication prior to the divergence of protostomes and deuterostomes led to the origin of two B56 subfamilies, B56αβε and B56γδ. Further duplications led to three B56αβε genes and two B56γδ in vertebrates. Several nonvertebrate B56 gene names are based on distinct vertebrate isoform names, and would best be renamed. B56 subfamily genes lack significant divergence within primitive chordates, but each became distinct in complex vertebrates. Two vertebrate lineages have undergone B56 gene loss, Xenopus and Aves. In Xenopus, B56δ function may be compensated for by an alternatively spliced transcript, B56δ/γ, encoding a B56δ-like amino-terminal region and a B56γ core. PMID:25950761

  1. Induction of UDP-glucuronosyltransferase 2B15 gene expression by the major active metabolites of tamoxifen, 4-hydroxytamoxifen and endoxifen, in breast cancer cells.

    PubMed

    Chanawong, Apichaya; Hu, Dong Gui; Meech, Robyn; Mackenzie, Peter I; McKinnon, Ross A

    2015-06-01

    We previously reported upregulation of UGT2B15 by 17β-estradiol in breast cancer MCF7 cells via binding of the estrogen receptor α (ERα) to an estrogen response unit (ERU) in the proximal UGT2B15 promoter. In the present study, we show that this ERα-mediated upregulation was significantly reduced by two ER antagonists (fulvestrant and raloxifene) but was not affected by a third ER antagonist, 4-hydroxytamoxifen (4-OHTAM), a major active tamoxifen (TAM) metabolite. Furthermore, we found that, similar to 17β-estradiol, 4-OHTAM and endoxifen (another major active TAM metabolite) elevated UGT2B15 mRNA levels, and that this stimulation was significantly abrogated by fulvestrant. Further experiments using 4-OHTAM revealed a critical role for ERα in this regulation. Specifically; knockdown of ERα expression by anti-ERα small interfering RNA reduced the 4-OHTAM-mediated induction of UGT2B15 expression; 4-OHTAM activated the wild-type but not the ERU-mutated UGT2B15 promoter; and chromatin immunoprecipitation assays showed increased ERα occupancy at the UGT2B15 ERU in MCF7 cells upon exposure to 4-OHTAM. Together, these data indicate that both 17β-estradiol and the antiestrogen 4-OHTAM upregulate UGT2B15 in MCF7 cells via the same ERα-signaling pathway. This is consistent with previous observations that both 17β-estradiol and TAM upregulate a common set of genes in MCF7 cells via the ER-signaling pathway. As 4-OHTAM is a UGT2B15 substrate, the upregulation of UGT2B15 by 4-OHTAM in target breast cancer cells is likely to enhance local metabolism and inactivation of 4-OHTAM within the tumor. This represents a potential mechanism that may reduce TAM therapeutic efficacy or even contribute to the development of acquired TAM resistance. PMID:25795461

  2. The homeodomain Pbx2-Prep1 complex modulates hepatocyte nuclear factor 1alpha-mediated activation of the UDP-glucuronosyltransferase 2B17 gene.

    PubMed

    Gregory, Philip A; Mackenzie, Peter I

    2002-07-01

    UDP glucuronosyltransferases (UGT) are expressed in a wide range of tissues in which their levels of expression and distribution are dependent on cell-type specific regulatory mechanisms. The presence of a hepatocyte nuclear factor (HNF) 1 binding site in the proximal promoters of several UGT2B genes has been shown to contribute to their expression in liver cells and possibly other HNF1-containing cell types. In some of these UGT2B genes, a putative pre-B cell homeobox (Pbx) transcription factor binding site is found directly adjacent to the functional HNF1 site. To determine whether this putative Pbx site contributes to the regulation of UGT2B expression, we chose the UGT2B17 gene and investigated the capacity of its Pbx site to bind specific transcription factors and alter promoter activity. The UGT2B17 Pbx site matches a consensus Pbx site known to bind members of the Pbx, Hox, Meis, and Prep1 families of homeodomain-containing proteins and has previously been shown to bind nuclear proteins in DNaseI footprint assays. In this study, we used gel shift and functional assays to show that a Pbx2-Prep1 heterodimer can bind to the UGT2B17 Pbx site and interfere with the binding of HNF1alpha to its site adjacent to the Pbx site. This interaction of Pbx2-Prep1 and HNF1alpha results in down-regulation of HNF1alpha-mediated activation of the UGT2B17 promoter. Modulation of transcription by restricting the binding of transcriptional effectors to their target site is a novel role for Pbx2-Prep1 complexes. PMID:12065766

  3. Cloning DPB3, the gene encoding the third subunit of DNA polymerase II of Saccharomyces cerevisiae.

    PubMed Central

    Araki, H; Hamatake, R K; Morrison, A; Johnson, A L; Johnston, L H; Sugino, A

    1991-01-01

    DNA polymerase II purified from Saccharomyces cerevisiae contains polypeptides with apparent molecular masses of greater than 200, 80, 34, 30 and 29 kDa, the two largest of which (subunits A and B) are encoded by the essential genes POL2 and DPB2. By probing a lambda gt11 expression library of yeast DNA with antiserum against DNA polymerase II, we isolated a single gene, DPB3, that encodes both the 34- and 30-kDa polypeptides (subunit C and C'). The nucleotide sequence of DPB3 contained an open reading frame encoding a 23-kDa protein, significantly smaller than the observed molecular masses, 34- or 30-kDa, which might represent post-translationally modified forms of the DPB3 product. The predicted amino acid sequence contained a possible NTP-binding motif and a glutamate-rich region. NTP-binding motif and a glutamate-rich region. A dpb3 deletion mutant (dpb3 delta) was viable and yielded a DNA polymerase II lacking the 34- and 30-kDa polypeptides. dpb3 delta strains exhibited an increased spontaneous mutation rate, suggesting that the DPB3 product is required to maintain fidelity of chromosomal replication. Since a fifth, 29-kDa polypeptide was present in DNA polymerase II preparations from wild-type cell extracts throughout purification, the subunit composition appears to be A, B, C (or C and C') and D. The 5' nontranscribed region of DPB3 contained the MulI-related sequence ACGCGA, while the 0.9-kb DPB3 transcript accumulated periodically during the cell cycle and peaked at the G1/S boundary. The level of DPB3 transcript thus appears to be under the same cell cycle control as those of POL2, DPB2 and other DNA replication genes. DPB3 was mapped to chromosome II, 30 cM distal to his7. Images PMID:1923754

  4. Sucrose regulation of ADP-glucose pyrophosphorylase subunit genes transcript levels in leaves and fruits

    NASA Technical Reports Server (NTRS)

    Li, Xiangyang; Xing, Jinpeng; Gianfagna, Thomas J.; Janes, Harry W.

    2002-01-01

    ADP-glucose pyrophosphorylase (AGPase, EC2.7.7.27) is a key regulatory enzyme in starch biosynthesis. The enzyme is a heterotetramer with two S and two B subunits. In tomato, there are three multiple forms of the S subunit gene. Agp S1, S2 and B are highly expressed in fruit from 10 to 25 days after anthesis. Agp S3 is only weakly expressed in fruit. Sucrose significantly elevates expression of Agp S1, S2 and B in both leaves and fruits. Agp S1 exhibits the highest degree of regulation by sucrose. In fact, sucrose may be required for Agp S1 expression. For excised leaves incubated in water, no transcripts for Agp S1 could be detected in the absence of sucrose, whereas it took up to 16 h in water before transcripts were no longer detectable for Agp S2 and B. Neither Agp S3 nor the tubulin gene is affected by sucrose, demonstrating that this response is specifically regulated by a carbohydrate metabolic signal, and is not due to a general increase in metabolism caused by sucrose treatment. Truncated versions of the promoter for Agp S1 indicate that a specific region 1.3-3.0 kb upstream from the transcription site is responsible for sucrose sensitivity. This region of the S1 promoter contains several cis-acting elements present in the promoters of other genes that are also regulated by sucrose. c2002 Elsevier Science Ireland Ltd. All rights reserved.

  5. Activity, Expression and Function of a Second Drosophila Protein Kinase a Catalytic Subunit Gene

    PubMed Central

    Melendez, A.; Li, W.; Kalderon, D.

    1995-01-01

    The DC2 gene was isolated previously on the basis of sequence similarity to DCO, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 did not affect the viability or phenotype of imaginal disc cells lacking DC0 activity or embryonic hatching of animals with reduced DC0 activity. Furthermore, transgenes expressing DC2 from a DC0 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. PMID:8601490

  6. [Association of the polymorphisms of the FTO, KCNJ11, SLC30A8 and CDKN2B genes with type 2 diabetes].

    PubMed

    Nikitin, A G; Potapov, V A; Brovkin, A N; Lavrikova, E Iu; Hodyrev, D S; Shamkhalova, M Sh; Smetanina, S A; Suplotova, L N; Shestakova, M V; Nosikov, V V; Aver'ianov, A V

    2015-01-01

    To study the association with diabetes mellitus type 2 we performed anal- ysis of the distribution of frequencies of alleles and genotypes of polymorphic markers of FTO, KCNJ11, SIC30A8 and CDKN2B genes. The study included groups of T2DM patients and unrelated controls of Russian origin. Analysis of the distribution of frequencies of alleles and genotypes of the polymorphic markers of KCNJ11, SLC30A8 and CDKN2B genes showed the presence of association with T2DM in Russian population, while for the FTO gene was not found statistically significant associations with type 2 diabetes. We can conclude that in Russian population main role in the development of type 2 diabetes play genes, affecting the level of syn- thesis and secretion of the insulin in beta-cells of the pancreas. PMID:25916116

  7. Expression profile of G-protein βγ subunit gene transcripts in the mouse olfactory sensory epithelia

    PubMed Central

    Sathyanesan, Aaron; Feijoo, Adrian A.; Mehta, Saloni T.; Nimarko, Akua F.; Lin, Weihong

    2013-01-01

    Heterotrimeric G-proteins mediate a variety of cellular functions, including signal transduction in sensory neurons of the olfactory system. Whereas the Gα subunits in these neurons are well characterized, the gene transcript expression profile of Gβγ subunits is largely missing. Here we report our comprehensive expression analysis to identify Gβ and Gγ subunit gene transcripts in the mouse main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Our reverse transcriptase PCR (RT-PCR) and realtime qPCR analyses of all known Gβ (β1,2,3,4,5) and Gγ (γ1,2,2t,3,4,5,7,8,10,11,12,13) subunits indicate presence of multiple Gβ and Gγ subunit gene transcripts in the MOE and the VNO at various expression levels. These results are supported by our RNA in situ hybridization (RISH) experiments, which reveal the expression patterns of two Gβ subunits and four Gγ subunits in the MOE as well as one Gβ and four Gγ subunits in the VNO. Using double-probe fluorescence RISH and line intensity scan analysis of the RISH signals of two dominant Gβγ subunits, we show that Gγ13 is expressed in mature olfactory sensory neurons (OSNs), while Gβ1 is present in both mature and immature OSNs. Interestingly, we also found Gβ1 to be the dominant Gβ subunit in the VNO and present throughout the sensory epithelium. In contrast, we found diverse expression of Gγ subunit gene transcripts with Gγ2, Gγ3, and Gγ13 in the Gαi2-expressing neuronal population, while Gγ8 is expressed in both layers. Further, we determined the expression of these Gβγ gene transcripts in three post-natal developmental stages (p0, 7, and 14) and found their cell-type specific expression remains largely unchanged, except the transient expression of Gγ2 in a single basal layer of cells in the MOE during P7 and P14. Taken together, our comprehensive expression analyses reveal cell-type specific gene expression of multiple Gβ and Gγ in sensory neurons of the olfactory system. PMID:23759900

  8. Expression profile of G-protein βγ subunit gene transcripts in the mouse olfactory sensory epithelia.

    PubMed

    Sathyanesan, Aaron; Feijoo, Adrian A; Mehta, Saloni T; Nimarko, Akua F; Lin, Weihong

    2013-01-01

    Heterotrimeric G-proteins mediate a variety of cellular functions, including signal transduction in sensory neurons of the olfactory system. Whereas the Gα subunits in these neurons are well characterized, the gene transcript expression profile of Gβγ subunits is largely missing. Here we report our comprehensive expression analysis to identify Gβ and Gγ subunit gene transcripts in the mouse main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Our reverse transcriptase PCR (RT-PCR) and realtime qPCR analyses of all known Gβ (β1,2,3,4,5) and Gγ (γ1,2,2t,3,4,5,7,8,10,11,12,13) subunits indicate presence of multiple Gβ and Gγ subunit gene transcripts in the MOE and the VNO at various expression levels. These results are supported by our RNA in situ hybridization (RISH) experiments, which reveal the expression patterns of two Gβ subunits and four Gγ subunits in the MOE as well as one Gβ and four Gγ subunits in the VNO. Using double-probe fluorescence RISH and line intensity scan analysis of the RISH signals of two dominant Gβγ subunits, we show that Gγ13 is expressed in mature olfactory sensory neurons (OSNs), while Gβ1 is present in both mature and immature OSNs. Interestingly, we also found Gβ1 to be the dominant Gβ subunit in the VNO and present throughout the sensory epithelium. In contrast, we found diverse expression of Gγ subunit gene transcripts with Gγ2, Gγ3, and Gγ13 in the Gαi2-expressing neuronal population, while Gγ8 is expressed in both layers. Further, we determined the expression of these Gβγ gene transcripts in three post-natal developmental stages (p0, 7, and 14) and found their cell-type specific expression remains largely unchanged, except the transient expression of Gγ2 in a single basal layer of cells in the MOE during P7 and P14. Taken together, our comprehensive expression analyses reveal cell-type specific gene expression of multiple Gβ and Gγ in sensory neurons of the olfactory system. PMID:23759900

  9. Gene structure and chromosome localization to 7q21.3 of the human rod photoreceptor transducin {gamma}-subunit gene (GNGT1)

    SciTech Connect

    Scherer, S.W.; Tsui, Lap-Chee |; Feinstein, D.S.

    1996-07-01

    The transducin {gamma}-subunit gene (GNGT1) encodes a member ({gamma}{sub 1}) of the family of heterotrimeric G-protein {gamma}-subunits that is specific to rod photoreceptors. In this report we have determined the complete structure of the GNGT1 gene and have localized it to human chromosome 7q21.3 using somatic cell hybrid and yeast artificial chromosome analysis. 16 refs., 2 figs.

  10. Regulation of CDKN2B expression by interaction of Arnt with Miz-1 - a basis for functional integration between the HIF and Myc gene regulatory pathways

    PubMed Central

    2014-01-01

    Background Hypoxia- and Myc-dependent transcriptional regulatory pathways are frequently deregulated in cancer cells. These pathways converge in many cellular responses, but the underlying molecular mechanisms are unclear. Methods The ability of Miz-1 and Arnt to interact was identified in a yeast two-hybrid screen. The mode of interaction and the functional consequences of complex formation were analyzed by diverse molecular biology methods, in vitro. Statistical analyses were performed by Student’s t-test and ANOVA. Results In the present study we demonstrate that the aryl hydrocarbon receptor nuclear translocator (Arnt), which is central in hypoxia-induced signaling, forms a complex with Miz-1, an important transcriptional regulator in Myc-mediated transcriptional repression. Overexpression of Arnt induced reporter gene activity driven by the proximal promoter of the cyclin-dependent kinase inhibitor 2B gene (CDKN2B), which is an established target for the Myc/Miz-1 complex. In contrast, mutated forms of Arnt, that were unable to interact with Miz-1, had reduced capability to activate transcription. Moreover, repression of Arnt reduced endogenous CDKN2B expression, and chromatin immunoprecipitation demonstrated that Arnt interacts with the CDKN2B promoter. The transcriptional activity of Arnt was counteracted by Myc, but not by a mutated variant of Myc that is unable to interact with Miz-1, suggesting mutually exclusive interaction of Arnt and Myc with Miz-1. Our results also establish CDKN2B as a hypoxia regulated gene, as endogenous CDKN2B mRNA and protein levels were reduced by hypoxic treatment of U2OS cells. Conclusions Our data reveal a novel mode of regulation by protein-protein interaction that directly ties together, at the transcriptional level, the Myc- and hypoxia-dependent signaling pathways and expands our understanding of the roles of hypoxia and cell cycle alterations during tumorigenesis. PMID:24618291

  11. Gene regulation, alternative splicing, and posttranslational modification of troponin subunits in cardiac development and adaptation: a focused review

    PubMed Central

    Sheng, Juan-Juan; Jin, Jian-Ping

    2014-01-01

    Troponin plays a central role in regulating the contraction and relaxation of vertebrate striated muscles. This review focuses on the isoform gene regulation, alternative RNA splicing, and posttranslational modifications of troponin subunits in cardiac development and adaptation. Transcriptional and posttranscriptional regulations such as phosphorylation and proteolysis modifications, and structure-function relationships of troponin subunit proteins are summarized. The physiological and pathophysiological significances are discussed for impacts on cardiac muscle contractility, heart function, and adaptations in health and diseases. PMID:24817852

  12. Negative dominance in gene lamB: random assembly of secreted subunits issued from different polysomes.

    PubMed Central

    Marchal, C; Hofnung, M

    1983-01-01

    lamB is the structural gene for the lambda receptor, an oligomeric outer membrane protein from Escherichia coli K12 involved in phage lambda adsorption. We show that, under certain conditions, in a strain diploid for gene lamB, all the missense lamB mutations conferring lambda resistance that we have tested are dominant with respect to wild-type. We propose a model which allows a quantitative interpretation of the data. It is based on negative complementation at the level of oligomerisation. Wild-type and mutant subunits would assemble at random forming homo- and hetero-oligomers. Only wild-type homo-oligomers would be efficient for phage inactivation. For some classes of missense mutations the hetero-oligomers would have the capacity to bind, but not to inactivate the phage. The model confirms that active lambda receptor is a trimer and implies that for this secreted protein there is no preferential assembly of subunits originating from the same polysome. Images Fig. 2. PMID:11894914

  13. Activity, expression and function of a second Drosophila protein kinase a catalytic subunit gene

    SciTech Connect

    Melendez, A.; Li, W.; Kalderon, D.

    1995-12-01

    The DC2 was isolated previously on the basis of sequence similarity to DC0, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. 62 refs., 10 figs., 2 tabs.

  14. Characterization of a Low-Molecular-Weight Glutenin Subunit Gene from Bread Wheat and the Corresponding Protein That Represents a Major Subunit of the Glutenin Polymer1

    PubMed Central

    Masci, Stefania; D'Ovidio, Renato; Lafiandra, Domenico; Kasarda, Donald D.

    1998-01-01

    Both high- and low-molecular-weight glutenin subunits (LMW-GS) play the major role in determining the viscoelastic properties of wheat (Triticum aestivum L.) flour. To date there has been no clear correspondence between the amino acid sequences of LMW-GS derived from DNA sequencing and those of actual LMW-GS present in the endosperm. We have characterized a particular LMW-GS from hexaploid bread wheat, a major component of the glutenin polymer, which we call the 42K LMW-GS, and have isolated and sequenced the putative corresponding gene. Extensive amino acid sequences obtained directly for this 42K LMW-GS indicate correspondence between this protein and the putative corresponding gene. This subunit did not show a cysteine (Cys) at position 5, in contrast to what has frequently been reported for nucleotide-based sequences of LMW-GS. This Cys has been replaced by one occurring in the repeated-sequence domain, leaving the total number of Cys residues in the molecule the same as in various other LMW-GS. On the basis of the deduced amino acid sequence and literature-based assignment of disulfide linkages, a computer-generated molecular model of the 42K subunit was constructed. PMID:9847089

  15. Virus-induced gene silencing of RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In eukaryotic cells, RNA polymerase III is highly conserved, contains 17 subunits and transcribes housekeeping genes such as ribosomal 50S rRNA, tRNA and other small RNAs. Functional roles of the RPC5 are poorly characterized in the literature. In this work, we report that virus-induced gene silenci...

  16. Agrobacterium-mediated transformation of the β-subunit gene in 7S globulin protein in soybean using RNAi technology.

    PubMed

    Qu, J; Liu, S Y; Wang, P W; Guan, S Y; Fan, Y G; Yao, D; Zhang, L; Dai, J L

    2016-01-01

    The objective of this study was to use RNA interference (RNAi) to improve protein quality and decrease anti-nutritional effects in soybean. Agrobacterium tumefaciens-mediated transformation was conducted using RNAi and an expression vector containing the 7S globulin β-subunit gene. The BAR gene was used as the selective marker and cotyledonary nodes of soybean genotype Jinong 27 were chosen as explant material. Regenerated plants were detected by molecular biology techniques. Transformation of the β-subunit gene in the 7S protein was detected by PCR, Southern blot, and q-PCR. Positive plants (10 T0, and 6 T1, and 13 T2) were tested by PCR. Hybridization bands were detected by Southern blot analysis in two of the T1 transgenic plants. RNAi expression vectors containing the soybean 7S protein β-subunit gene were successfully integrated into the genome of transgenic plants. qRT-PCR analysis in soybean seeds showed a clear decrease in expression of the soybean β-subunit gene. The level of 7S protein β-subunit expression in transgenic plants decreased by 77.5% as compared to that of the wild-type plants. This study has established a basis for the application of RNAi to improve the anti-nutritional effects of soybean. PMID:27173254

  17. Abnormal epigenetic regulation of the gene expression levels of Wnt2b and Wnt7b: Implications for neural tube defects

    PubMed Central

    BAI, BAOLING; CHEN, SHUYUAN; ZHANG, QIN; JIANG, QIAN; LI, HUILI

    2016-01-01

    The association between Wnt genes and neural tube defects (NTDs) is recognized, however, it remains to be fully elucidated. Our previous study demonstrated that epigenetic mechanisms are affected in human NTDs. Therefore, the present study aimed to evaluate whether Wnt2b and Wnt7b are susceptible to abnormal epigenetic modification in NTDs, using chromatin immunoprecipitation assays to evaluate histone enrichments and the MassARRAY platform to detect the methylation levels of target regions within Wnt genes. The results demonstrated that the transcriptional activities of Wnt2b and Wnt7b were abnormally upregulated in mouse fetuses with NTDs and, in the GC-rich promoters of these genes, histone 3 lysine 4 (H3K4) acetylation was enriched, whereas H3K27 trimethylation was reduced. Furthermore, several CpG sites in the altered histone modification of target regions were significantly hypomethylated. The present study also detected abnormal epigenetic modifications of these Wnt genes in human NTDs. In conclusion, the present study detected abnormal upregulation in the levels of Wnt2b and Wnt7b, and hypothesized that the alterations may be due to the ectopic opening of chromatin structure. These results improve understanding of the dysregulation of epigenetic modification of Wnt genes in NTDs. PMID:26548512

  18. Traxoprodil, a selective antagonist of the NR2B subunit of the NMDA receptor, potentiates the antidepressant-like effects of certain antidepressant drugs in the forced swim test in mice.

    PubMed

    Poleszak, Ewa; Stasiuk, Weronika; Szopa, Aleksandra; Wyska, Elżbieta; Serefko, Anna; Oniszczuk, Anna; Wośko, Sylwia; Świąder, Katarzyna; Wlaź, Piotr

    2016-08-01

    One of the newest substances, whose antidepressant activity was shown is traxoprodil, which is a selective antagonist of the NR2B subunit of the NMDA receptor. The main goal of the present study was to evaluate the effect of traxoprodil on animals' behavior using the forced swim test (FST), as well as the effect of traxoprodil (10 mg/kg) on the activity of antidepressants, such as imipramine (15 mg/kg), fluoxetine (5 mg/kg), escitalopram (2 mg/kg) and reboxetine (2.5 mg/kg). Serotonergic lesion and experiment using the selective agonists of serotonin receptors 5-HT1A and 5-HT2 was conducted to evaluate the role of the serotonergic system in the antidepressant action of traxoprodil. Brain concentrations of tested agents were determined using HPLC. The results showed that traxoprodil at a dose of 20 and 40 mg/kg exhibited antidepressant activity in the FST and it was not related to changes in animals' locomotor activity. Co-administration of traxoprodil with imipramine, fluoxetine or escitalopram, each in subtherapeutic doses, significantly affected the animals' behavior in the FST and, what is important, these changes were not due to the severity of locomotor activity. The observed effect of traxoprodil is only partially associated with serotonergic system and is independent of the effect on the 5-HT1A and 5-HT2 serotonin receptors. The results of an attempt to assess the nature of the interaction between traxoprodil and the tested drugs show that in the case of joint administration of traxoprodil and fluoxetine, imipramine or escitalopram, there were interactions in the pharmacokinetic phase. PMID:26924124

  19. Assignment of the gene for the. beta. subunit of thyroid-stimulating hormone to the short arm of human chromosome 1

    SciTech Connect

    Dracopoli, N.C.; Rettig, W.J.; Whitfield, G.K.; Darlington, G.J.; Spengler, B.A.; Biedler, J.L.; Old, L.J.; Kourides, I.A.

    1986-03-01

    The chromosomal locations of the genes for the ..beta.. subunit of human thyroid-stimulating hormone (TSH) and the glycoprotein hormone ..cap alpha.. subunit have been determined by restriction enzyme analysis of DNA extracted from rodent-human somatic cell hybrids. Human chorionic gonadotropin (CG) ..cap alpha..-subunit cDNA and a cloned 0.9-kilobase (kb) fragment of the human TSH ..beta..-subunit gene were used as hybridization probes in the analysis of Southern blots of DNA extracted from rodent-human hybrid clones. Analysis of the segregation of 5- and 10-kb EcoRI fragments hybridizing to CG ..cap alpha..-subunit cDNA confirmed the previous assignment of this gene to chromosome 6. Analysis of the patterns of segregation of a 2.3-kb EcoRI fragment containing human TSH ..beta..-subunit sequences permitted the assignment of the TSH ..beta..-subunit gene to human chromosome 1. The subregional assignment of TSH ..beta.. subunit to chromosome 1p22 was made possible by the additional analysis of a set of hybrids containing partially overlapping segments of this chromosome. Human TSH ..beta.. subunit is not syntenic with genes encoding the ..beta.. subunits of CG, luteinizing hormone, or follicle-stimulating hormone and is assigned to a conserved linkage group that also contains the structural genes for the ..beta.. subunit of nerve growth factor (NGFB) and the proto-oncogene N-ras (NRAS).

  20. Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

    PubMed Central

    Chopra, Sameer S; Watanabe, Hiroshi; Zhong, Tao P; Roden, Dan M

    2007-01-01

    Background Action potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further insight into the evolution of electrical signaling in vertebrates, we investigated beta subunit genes in the teleost Danio rerio (zebrafish). Results We identified and cloned single zebrafish gene homologs for beta1-beta3 (zbeta1-zbeta3) and duplicate genes for beta4 (zbeta4.1, zbeta4.2). Sodium channel beta subunit loci are similarly organized in fish and mammalian genomes. Unlike their mammalian counterparts, zbeta1 and zbeta2 subunit genes display extensive alternative splicing. Zebrafish beta subunit genes and their splice variants are differentially-expressed in excitable tissues, indicating tissue-specific regulation of zbeta1-4 expression and splicing. Co-expression of the genes encoding zbeta1 and the zebrafish sodium channel alpha subunit Nav1.5 in Chinese Hamster Ovary cells increased sodium current and altered channel gating, demonstrating functional interactions between zebrafish alpha and beta subunits. Analysis of the synteny and phylogeny of mammalian, teleost, amphibian, and avian beta subunit and related genes indicated that all extant vertebrate beta subunits are orthologous, that beta2/beta4 and beta1/beta3 share common ancestry, and that beta subunits are closely related to other proteins sharing the V-type immunoglobulin domain structure. Vertebrate sodium channel beta subunit genes were not identified in the genomes of invertebrate chordates and are unrelated to known subunits of the para sodium channel in Drosophila. Conclusion The identification of conserved

  1. Effects of sex and chronic neonatal nicotine treatment on Na²⁺/K⁺/Cl⁻ co-transporter 1, K⁺/Cl⁻ co-transporter 2, brain-derived neurotrophic factor, NMDA receptor subunit 2A and NMDA receptor subunit 2B mRNA expression in the postnatal rat hippocampus.

    PubMed

    Damborsky, J C; Winzer-Serhan, U H

    2012-12-01

    Chronic exposure to nicotine during the first postnatal week in rats, a developmental period that corresponds to the third trimester of human gestation, results in sexually dimorphic long-term functional defects in the adult hippocampus. One potential cause could be the sex-specific differences in the maturation of GABA(A) receptor-mediated responses from excitatory to inhibitory, which depends on the expression of the Na(2+)/K(+)/Cl(-) co-transporter 1 (NKCC1) and the K(+)/Cl(-) co-transporter 2 (KCC2). In the rat hippocampus, this switch occurs during the first and second postnatal week in females and males, respectively, and is regulated by nicotinic receptor activation. Excitatory GABAergic signaling can increase brain-derived neurotrophic factor (BDNF) expression, which might exacerbate sex differences by impacting synaptogenesis. We hypothesized that chronic neonatal nicotine (CNN) exposure differentially regulates the expression of these co-transporters and BDNF in males and females. We use quantitative isotopic in situ hybridization to examine the expression of mRNAs for NKCC1, KCC2, BDNF, and NMDA receptor subunit 2A (NR2A) and NMDA receptor subunit 2B (NR2B) in the postnatal day (P) 5 and 8 rat hippocampi in both sexes that were either control-treated or with 6mg/kg/day nicotine in milk formula (CNN) via gastric intubation starting at P1. In line with prolonged GABAergic excitation, we found that at P5 males had significantly higher mRNA expression of NKCC1 and BDNF than females. CNN treatment resulted in a significant increase in KCC2 and BDNF mRNA expression in male but not female hippocampus (p<0.05). Males also had higher expression of NR2A and lower expression of NR2B at P5 compared to females (p<0.05). At P8, there were neither sex nor treatment effects on mRNA expression, indicating the end of a critical period for sensitivity to nicotine. These results suggest that differential maturation of GABA(A)R-mediated responses result in sex

  2. Deletion variant in the ADRA2B gene increases coupling between emotional responses at encoding and later retrieval of emotional memories.

    PubMed

    Todd, R M; Müller, D J; Palombo, D J; Robertson, A; Eaton, T; Freeman, N; Levine, B; Anderson, A K

    2014-07-01

    A deletion variant of the ADRA2B gene that codes for the α2b adrenoceptor has been linked to greater susceptibility to traumatic memory as well as attentional biases in perceptual encoding of negatively valenced stimuli. The goal of the present study was to examine whether emotional enhancements of memory associated with the ADRA2B deletion variant were predicted by encoding, as indexed by the subjectively perceived emotional salience (i.e., arousal) of events at the time of encoding. Genotyping was performed on 186 healthy young adults who rated positive, negative, and neutral scenes for level of emotional arousal and subsequently performed a surprise recognition memory task 1 week later. Experience of childhood trauma was also measured, as well as additional genetic variations associated with emotional biases and episodic memory. Results showed that subjective arousal was linked to memory accuracy and confidence for ADRA2B deletion carriers but not for non-carriers. Our results suggest that carrying the ADRA2B deletion variant enhances the relationship between arousal at encoding and subsequent memory for moderately arousing events. PMID:24149058

  3. Polymorphism and structure of the gene coding for the alpha 1 subunit of the Artemia franciscana Na/K-ATPase.

    PubMed Central

    García-Sáez, A; Perona, R; Sastre, L

    1997-01-01

    Genomic clones coding for one of the two identified Artemia franciscana Na/K-ATPase alpha subunits, the alpha 1 subunit, have been isolated. Several overlapping clones were obtained, although their restriction maps showed a large heterogeneity. Sequencing of their exons showed that they differ in up to 3.46% of their nucleotides in translated regions and 8.18% in untranslated regions. Southern blot analysis of DNA purified from different lots of A. franciscana cysts and from isolated individuals suggests that the variation is due to the existence of multiple Na/K-ATPase alpha 1 subunit alleles in A. franciscana. The Na/K-ATPase alpha 1 subunit gene is divided into 15 exons. Ten of the 14 introns are located in identical positions in this gene as in the human Na/K-ATPase alpha 3 subunit gene. Analysis of the 5' flanking region of the gene has allowed identification of the transcription-initiation sites. The adjacent upstream region has been shown to have functional promoter activity in cultured mammalian cells, suggesting the evolutionary conservation of some of the promoter regulatory sequences. PMID:9020888

  4. Structure of the gene encoding the 14.5 kDa subunit of human RNA polymerase II.

    PubMed Central

    Acker, J; Wintzerith, M; Vigneron, M; Kedinger, C

    1993-01-01

    The structure of the gene encoding the 14.5 kDa subunit of the human RNA polymerase II (or B) has been elucidated. The gene consists of six exons, ranging from 52 to over 101 bp, interspaced with five introns ranging from 84 to 246 bp. It is transcribed into three major RNA species, present at low abundance in exponentially growing HeLa cells. The corresponding messenger RNAs contain the same open reading frame encoding a 125 amino acid residue protein, with a calculated molecular weight of 14,523 Da. This protein (named hRPB14.5) shares strong homologies with the homologous polymerase subunits encoded by the Drosophila (RpII15) and yeast (RPB9) genes. Cysteines characteristic of two zinc fingers are conserved in all three corresponding sequences and, like the yeast protein, the hRPB14.5 subunit exhibits zinc-binding activity. Images PMID:8265347

  5. Vorinostat, a histone deacetylase inhibitor, facilitates fear extinction and enhances expression of the hippocampal NR2B-containing NMDA receptor gene.

    PubMed

    Fujita, Yosuke; Morinobu, Shigeru; Takei, Shiro; Fuchikami, Manabu; Matsumoto, Tomoya; Yamamoto, Shigeto; Yamawaki, Shigeto

    2012-05-01

    Histone acetylation, which alters the compact chromatin structure and changes the accessibility of DNA to regulatory proteins, is emerging as a fundamental mechanism for regulating gene expression. Histone deacetylase (HDAC) inhibitors increase histone acetylation and enhance fear extinction. In this study, we examined whether vorinostat, an HDAC inhibitor, facilitates fear extinction, using a contextual fear conditioning (FC) paradigm, in Sprague-Dawley rats. We found that vorinostat facilitated fear extinction. Next, the levels of global acetylated histone H3 and H4 were measured by Western blotting. We also assessed the effect of vorinostat on the hippocampal levels of NMDA receptor mRNA by real-time quantitative PCR (RT-PCR) and protein by Western blotting. 2 h after vorinostat administration, the levels acetylated histones and NR2B mRNA, but not NR1 or NR2A mRNA, were elevated in the hippocampus. The NR2B protein level was elevated 4 h after vorinostat administration. Last, we investigated the levels of acetylated histones and phospho-CREB (p-CREB) binding at the promoter of the NR2B gene using the chromatin immunoprecipitation (ChIP) assay followed by RT-PCR. The ChIP assay revealed increases in the levels of acetylated histones and they were accompanied by enhanced binding of p-CREB to its binding site at the promoter of the NR2B gene 2 h after vorinostat administration. These findings suggest that vorinostat increases the expression of NR2B in the hippocampus by enhancing histone acetylation, and this process may be implicated in fear extinction. PMID:22364833

  6. Exon junction complex subunits are required to splice Drosophila MAP kinase, a large heterochromatic gene

    PubMed Central

    Roignant, Jean-Yves; Treisman, Jessica E.

    2010-01-01

    Summary The exon junction complex (EJC) is assembled on spliced mRNAs upstream of exon-exon junctions, and can regulate their subsequent translation, localization, or degradation. We isolated mutations in Drosophila mago nashi (mago), which encodes a core EJC subunit, based on their unexpectedly specific effects on photoreceptor differentiation. Loss of Mago prevents Epidermal growth factor receptor signaling, due to a large reduction in MAPK mRNA levels. MAPK expression also requires the EJC subunits Y14 and eIF4AIII, and EJC-associated splicing factors. Mago depletion does not affect the transcription or stability of MAPK mRNA, but alters its splicing pattern. MAPK expression from an exogenous promoter requires Mago only when the template includes introns. MAPK is the primary functional target of mago in eye development; in cultured cells, Mago knockdown disproportionately affects other large genes located in heterochromatin. These data support a nuclear role for EJC components in splicing a specific subset of introns. PMID:20946982

  7. Mutations in the Gene Encoding the Ancillary Pilin Subunit of the Streptococcus suis srtF Cluster Result in Pili Formed by the Major Subunit Only

    PubMed Central

    Fittipaldi, Nahuel; Takamatsu, Daisuke; la Cruz Domínguez-Punaro, María de; Lecours, Marie-Pier; Montpetit, Diane; Osaki, Makoto; Sekizaki, Tsutomu; Gottschalk, Marcelo

    2010-01-01

    Pili have been shown to contribute to the virulence of different Gram-positive pathogenic species. Among other critical steps of bacterial pathogenesis, these structures participate in adherence to host cells, colonization and systemic virulence. Recently, the presence of at least four discrete gene clusters encoding putative pili has been revealed in the major swine pathogen and emerging zoonotic agent Streptococcus suis. However, pili production by this species has not yet been demonstrated. In this study, we investigated the functionality of one of these pili clusters, known as the srtF pilus cluster, by the construction of mutant strains for each of the four genes of the cluster as well as by the generation of antibodies against the putative pilin subunits. Results revealed that the S. suis serotype 2 strain P1/7, as well as several other highly virulent invasive S. suis serotype 2 isolates express pili from this cluster. However, in most cases tested, and as a result of nonsense mutations at the 5′ end of the gene encoding the minor pilin subunit (a putative adhesin), pili were formed by the major pilin subunit only. We then evaluated the role these pili play in S. suis virulence. Abolishment of the expression of srtF cluster-encoded pili did not result in impaired interactions of S. suis with porcine brain microvascular endothelial cells. Furthermore, non-piliated mutants were as virulent as the wild type strain when evaluated in a murine model of S. suis sepsis. Our results show that srtF cluster-encoded, S. suis pili are atypical compared to other Gram-positive pili. In addition, since the highly virulent strains under investigation are unlikely to produce other pili, our results suggest that pili might be dispensable for critical steps of the S. suis pathogenesis of infection. PMID:20052283

  8. Synthesis of the small subunit of ribulose-bisphosphate carboxylase from genes cloned into plasmids containing the SP6 promoter.

    PubMed Central

    Anderson, S; Smith, S M

    1986-01-01

    DNA sequences encoding ribulose 1,5-bisphosphate carboxylase small subunit precursor from Pisum sativum L. have been transcribed from plasmids containing the SP6 promoter, and translated in a wheat germ cell-free system. The small subunit precursor polypeptide, its N-terminal leader sequence (transit peptide) and the mature small subunit have each been synthesized independently from three different plasmid constructs. The precursor polypeptide is imported into isolated pea chloroplasts and processed to the mature small subunit by a stromal proteinase. The mature polypeptide is neither imported, nor subject to proteolysis by stromal extracts. The transit peptide alone is very rapidly degraded by a stromal proteinase activity which can be inhibited by EDTA or 1,10-phenanthroline. The use of these gene constructs helps to establish the crucial role of the transit peptide in protein import into the chloroplast. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3827863

  9. Case-control association study of polymorphisms in the voltage-gated sodium channel genes SCN1A, SCN2A, SCN3A, SCN1B, and SCN2B and epilepsy.

    PubMed

    Baum, Larry; Haerian, Batoul Sadat; Ng, Ho-Keung; Wong, Virginia C N; Ng, Ping Wing; Lui, Colin H T; Sin, Ngai Chuen; Zhang, Chunbo; Tomlinson, Brian; Wong, Gary Wing-Kin; Tan, Hui Jan; Raymond, Azman Ali; Mohamed, Zahurin; Kwan, Patrick

    2014-05-01

    High-frequency action potentials are mediated by voltage-gated sodium channels, composed of one large α subunit and two small β subunits, encoded mainly by SCN1A, SCN2A, SCN3A, SCN1B, and SCN2B genes in the brain. These play a key role in epilepsy, with the most commonly mutated gene in epilepsy being SCN1A. We examined whether polymorphisms in the above genes affect epilepsy risk in 1,529 epilepsy patients and 1,935 controls from four ethnicities or locations: Malay, Indian, and Chinese, all from Malaysia, and Chinese from Hong Kong. Of patients, 19 % were idiopathic, 42 % symptomatic, and 40 % cryptogenic. We genotyped 43 polymorphisms: 27 in Hong Kong, 28 in Malaysia, and 12 in both locations. The strongest association with epilepsy was rs3812718, or SCN1A IVS5N+5G>A: odds ratio (OR) = 0.85 for allele G (p = 0.0009) and 0.73 for genotype GG versus AA (p = 0.003). The OR was between 0.76 and 0.87 for all ethnicities. Meta-analysis confirmed the association (OR = 0.81 and p = 0.002 for G, and OR = 0.67 and p = 0.007 for GG versus AA), which appeared particularly strong for Indians and for febrile seizures. Allele G affects splicing and speeds recovery from inactivation. Since SCN1A is preferentially expressed in inhibitory neurons, G may decrease epilepsy risk. SCN1A rs10188577 displayed OR = 1.20 for allele C (p = 0.003); SCN2A rs12467383 had OR = 1.16 for allele A (p = 0.01), and displayed linkage disequilibrium with rs2082366 (r (2) = 0.67), whose genotypes tended toward association with SCN2A brain expression (p = 0.10). SCN1A rs2298771 was associated in Indians (OR = 0.56, p = 0.005) and SCN2B rs602594 with idiopathic epilepsy (OR = 0.62, p = 0.002). Therefore, sodium channel polymorphisms are associated with epilepsy. PMID:24337656

  10. DHA down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes by attenuating CAR translocation

    SciTech Connect

    Li, C.-C.; Lii, C.-K.; Liu, K.-L.; Yang, J.-J.; Chen, H.-W.

    2007-12-15

    The constitutive androstane receptor (CAR) plays an important role in regulating the expression of detoxifying enzymes, including cytochrome P450 2B (CYP 2B). Phenobarbital (PB) induction of human CYP 2B6 and mouse CYP 2b10 has been shown to be mediated by CAR. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA); however, the mechanism for this down-regulation by DHA was previously unknown. The objective of the present study was to determine whether change in CAR translocation is involved in the down-regulation by n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression in rat primary hepatocytes. We used 100 {mu}M arachidonic acid, linoleic acid, eicosapentaenoic acid, and DHA to test this hypothesis. PB triggered the translocation of CAR from the cytosol into the nucleus in a dose-dependent and time-dependent manner in our hepatocyte system, and the CAR distribution in rat primary hepatocytes was significantly affected by DHA. DHA treatment decreased PB-inducible accumulation of CAR in the nuclear fraction and increased it in the cytosolic fraction in a dose-dependent manner. The down-regulation of CYP 2B1 expression by DHA occurred in a dose-dependent manner, and a similar pattern was found for the nuclear accumulation of CAR. The results of immunoprecipitation showed a CAR/RXR heterodimer bound to nuclear receptor binding site 1 (NR-1) of the PB-responsive enhancer module (PBREM) of the CYP 2B1gene. The EMSA results showed that PB-induced CAR binding to NR-1 was attenuated by DHA. Taken together, these results suggest that attenuation of CAR translocation and decreased subsequent binding to NR-1 are involved in DHA's down-regulation of PB-induced CYP 2B1 expression.

  11. Genomic organization of the murine G protein beta subunit genes and related processed pseudogenes.

    PubMed

    Kitanaka, J; Wang, X B; Kitanaka, N; Hembree, C M; Uhl, G R

    2001-12-01

    The functional significance of heterotrimeric guanine nucleotide binding protein (G protein) for the many physiological processes including the molecular mechanisms of drug addiction have been described. In investigating the changes of mRNA expression after acute psychostimulant administration, we previously identified a cDNA encoding a G protein beta1 subunit (Gbeta1) that was increased up to four-fold in certain brain regions after administration of psychostimulants. The mouse Gbeta1 gene (the mouse genetic symbol, GNB1) was mapped to chromosome 4, but little was known of its genetic features. To characterize the GNB1 gene further, we have cloned and analyzed the genomic structures of the mouse GNBI gene and its homologous sequences. The GNBI gene spans at least 50 kb, and consists of 12 exons and 11 introns. The exon/intron boundaries were determined and found to follow the GT/AG rule. Exons 3-11 encode the Gbeta1 protein, and the exon 2 is an alternative, resulting in putative two splicing variants. Although intron 11 is additional for GNBI compared with GNB2 and GNB3, the intron positions within the protein coding region of GNB1, GNB2 and GNB3 are identical, suggesting that GNB1 should have diverged from the ancestral gene family earlier than the genes for GNB2 and GNB3. We also found the 5'-truncated processed pseudogenes with 71-89% similarities to GNBI mRNA sequence, suggesting that the truncated cDNA copies, which have been reverse-transcribed from a processed mRNA for GNB1, might have been integrated into several new locations in the mouse genome. PMID:11913780

  12. Small-subunit ribosomal RNA gene sequences of Phaeodarea challenge the monophyly of Haeckel's Radiolaria.

    PubMed

    Polet, Stephane; Berney, Cédric; Fahrni, José; Pawlowski, Jan

    2004-03-01

    In his grand monograph of Radiolaria, Ernst Haeckel originally included Phaeodarea together with Acantharea and Polycystinea, all three taxa characterized by the presence of a central capsule and the possession of axopodia. Cytological and ultrastructural studies, however, questioned the monophyly of Radiolaria, suggesting an independent evolutionary origin of the three taxa, and the first molecular data on Acantharea and Polycystinea brought controversial results. To test further the monophyly of Radiolaria, we sequenced the complete small subunit ribosomal RNA gene of three phaeodarians and three polycystines. Our analyses reveal that phaeodarians clearly branch among the recently described phylum Cercozoa, separately from Acantharea and Polycystinea. This result enhances the morphological variability within the phylum Cercozoa, which already contains very heterogeneous groups of protists. Our study also confirms the common origin of Acantharea and Polycystinea, which form a sister-group to the Cercozoa, and allows a phylogenetic reinterpretation of the morphological features of the three radiolarian groups. PMID:15144058

  13. Promoter Structure of the RNA Polymerase II Large Subunit Gene in Caenorhabditis elegans and C. briggsae

    PubMed Central

    Bird, D. McK.; Kaloshian, I.; Molinari, S.

    1997-01-01

    The 5'-end of the Caenorhabditis elegans ama-1 gene transcript, which encodes the largest subunit of RNA polymerase II, was cloned. Sequencing revealed that the message is trans-spliced. To characterize the Ce-ama-1 promoter, DNA sequence spanning 3 kb upstream from the initiation codon was determined. Typical elements, such as TATA and Spl sites, were absent. The homologue of ama-1 in C. briggsae, Cb-ama-1, was isolated and its 5' flanking sequence compared with that of Ce-ama-1, revealing only limited similarity, although both sequences included a potential initiator-class transcriptional regulator and phased repeats of an AT₃C motif. The latter elements are postulated to facilitate DNA bending and may play a role in transcription regulation. PMID:19274143

  14. Association between GABAA Receptor Subunit Gene Cluster and Zolpidem-Induced Complex Sleep Behaviors in Han Chinese

    PubMed Central

    Tsai, Jui-Hsiu; Yang, Pinchen; Lin, Hung-Hsun; Cheng, Kuang-hung; Yang, Yi-Hsin; Wu, Ming-Tsang; Chen, Cheng-Chung

    2013-01-01

    Study Objectives: To investigate and elucidate the role of GABAA receptor subunits, specifically the 2 genetic markers at the GABAA α1 and GABAA α6 receptors, in zolpidem-induced complex sleep behaviors (CSBs). Design: Genetic association study. Setting: Kaohsiung Medical University-affiliated hospitals, Kaohsiung, Taiwan. Patients: 30 zolpidem-induced CSB subjects and 37 controls. Interventions: N/A. Measurements and Results: The χ2 test demonstrated an association between the A15G variant at the GABAA α1 receptor subunit gene and zolpidem-induced CSBs (P = 0.007). The adjusted odds ratio of the GABAA α1 receptor subunit genotype for the risk of zolpidem-induced CSBs was approximately 10 (OR = 9.99, 95% CI = 1.82, 74.87; P = 0.013). Conclusions: The finding reveals that the A15G variant at the GABAA α1 receptor subunit gene confers a high risk of zolpidem-induced CSBs and may be considered in clinical services. Citation: Tsai JH; Yang P; Lin HH; Cheng Kh; Yang YH; Wu MT; Chen CC. Association between GABAA receptor subunit gene cluster and zolpidem-induced complex sleep behaviors in Han Chinese. SLEEP 2013;36(2):197–202. PMID:23372267

  15. Profiles of embryonic nuclear protein binding to the proximal promoter region of the soybean β-conglycinin α subunit gene.

    PubMed

    Yoshino, M; Tsutsumi, K; Kanazawa, A

    2015-01-01

    β-Conglycinin, a major component of seed storage protein in soybean, comprises three subunits: α, α' and β. The expression of genes for these subunits is strictly controlled during embryogenesis. The proximal promoter region up to 245 bp upstream of the transcription start site of the α subunit gene sufficiently confers spatial and temporal control of transcription in embryos. Here, the binding profile of nuclear proteins in the proximal promoter region of the α subunit gene was analysed. DNase I footprinting analysis indicated binding of proteins to the RY element and DNA regions including box I, a region conserved in cognate gene promoters. An electrophoretic mobility shift assay (EMSA) using different portions of box I as a probe revealed that multiple portions of box I bind to nuclear proteins. In addition, an EMSA using nuclear proteins extracted from embryos at different developmental stages indicated that the levels of major DNA-protein complexes on box I increased during embryo maturation. These results are consistent with the notion that box I is important for the transcriptional control of seed storage protein genes. Furthermore, the present data suggest that nuclear proteins bind to novel motifs in box I including 5'-TCAATT-3' rather than to predicted cis-regulatory elements. PMID:24943483

  16. MED23: a new Mediator of H2B monoubiquitylation.

    PubMed

    Streubel, Gundula; Bracken, Adrian P

    2015-12-01

    The Mediator multiprotein complex physically links transcription factors to RNA polymerase II and the basal transcription machinery. While the Mediator complex has been shown to be required for transcriptional initiation and elongation, the understanding of its interplay with histone modifying enzymes and post‐translational modifications remains elusive. In this issue of The EMBO Journal, Yao et al (2015) report that the MED23 subunit of the Mediator complex physically associates with the heterodimeric RNF20/40 E3‐ligase complex to facilitate the monoubiquitylation of histone H2B on gene bodies of actively transcribed genes. PMID:26438725

  17. Molecular cloning and expression analysis of a novel BCCP subunit gene from Aleurites moluccana.

    PubMed

    Xuan, W Y; Zhang, Y; Liu, Z Q; Feng, D; Luo, M Y

    2015-01-01

    Aleurites moluccana L. is grown as a roadside tree in southern China and the oil content of its seed is higher than other oil plants, such as Jatropha curcas and Camellia oleifera. A. moluccana is considered a promising energy plant because its seed oil could be used to produce biodiesel and bio-jet fuel. In addition, the bark, leaves, and kernels of A. moluccana have various medical and commercial uses. Here, a novel gene coding the biotin carboxyl carrier protein subunit (BCCP) was cloned from A. moluccana L. using the homology cloning method combined with rapid amplification of cDNA end (RACE) technology. The isolated full-length cDNA sequence (designated AM-accB) was 1188 bp, containing a 795-bp open reading frame coding for 265 amino acids. The deduced amino acid sequence of AM-accB contained a biotinylated domain located between amino acids 190 and 263. A. moluccana BCCP shows high identity at the amino acid level to its homologues in other higher plants, such as Vernicia fordii, J. curcas, and Ricinus communis (86, 77, and 70%, respectively), which all contain conserved domains for ACCase activity. The expression of the AM-accB gene during the middle stage of development and maturation in A. moluccana seeds was higher than that in early and later stages. The expression pattern of the AM-accB gene is very similar to that of the oil accumulation rate. PMID:26345927

  18. Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB)

    PubMed Central

    Jung, Lenice Roteia Cardoso; Bomfim, Maria Rosa Quaresma; Kroon, Erna Geessien; Nunes, Álvaro Cantini

    2015-01-01

    Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars. PMID:26273261

  19. Length variations of i-type low-molecular-weight glutenin subunit genes in diploid wheats.

    PubMed

    Long, H; Huang, Z; Wei, Y-M; Yan, Z-H; Ma, Z-C; Zheng, Y-L

    2008-04-01

    Allelic variation of the low-molecular-weight glutenin subunit (LMW-GS) is associated with the significant differences of dough quality in bread and durum wheat, and has been widely evaluated at protein level in wheat and its relatives. In this study, a PCR primer set, targeting the high variable repetitive domains, was employed to assay the length variation of i-type LMW-GS genes in the A-genomes of diploid wheats, the diploid progenitors of tetraploid and hexaploid wheat. A total of 71 accessions of diploid wheats, belonging to two wild and one cultivated species, were investigated. The higher variations of repetitive length in i-type LMW-GS genes were found in diploid wheats with Nei's genetic variation index (H) of 0.834. The two wild species, T. boeoticum and T. urartu, were found to possess the similar degree of variability, with the Nei's genetic variation index of 0.806 and 0.783, respectively. Less variations were detected in T. monococcum (H = 0.680), a cultivated species domesticated from T. boeoticum. The sufficient variations found in this study could be used as valuable sources for the enrichment of the genetic variations and the alteration of flour-processing properties of the cultivated wheat. To our knowledge, it was the first time that an analysis of length variation targeting a particular group of genes of LMW-GS complex multigene families was conducted. PMID:18666554

  20. Alanine Scanning of Cucumber Mosaic Virus (CMV) 2B Protein Identifies Different Positions for Cell-To-Cell Movement and Gene Silencing Suppressor Activity

    PubMed Central

    Nemes, Katalin; Gellért, Ákos; Balázs, Ervin; Salánki, Katalin

    2014-01-01

    The multifunctional 2b protein of CMV has a role in the long distance and local movement of the virus, in symptom formation, in evasion of defense mediated by salicylic acid as well as in suppression of RNA silencing. The role of conserved amino acid sequence domains were analyzed previously in the protein function, but comprehensive analysis of this protein was not carried out until recently. We have analyzed all over the 2b protein by alanine scanning mutagenesis changing three consecutive amino acids (aa) to alanine. We have identified eight aa triplets as key determinants of the 2b protein function in virus infection. Four of them (KKQ/22-24/AAA, QNR/31-33/AAA, RER/34-36/AAA, SPS/40-42/AAA) overlap with previously determined regions indispensable in gene silencing suppressor function. We have identified two additional triplets necessary for the suppressor function of the 2b protein (LPF/55-57/AAA, NVE/10-12/AAA), and two other positions were required for cell-to-cell movement of the virus (MEL/1-3/AAA, RHV/70-72/AAA), which are not essential for suppressor activity. PMID:25380036

  1. Molecular characterization and gene expression of the channel catfish Ferritin H subunit after bacterial infection and iron treatment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ferritins are the major iron storage protein in the cytoplasm of cells, responsible for regulating levels of intracellular iron. Ferritin genes are widely distributed in both prokaryotes and eukaryotes. In mammals, ferritin molecules are composed of heavy- (H) and light- (L) chain subunits; amphibia...

  2. Characterization of the low-molecular-weight glutenin subunit gene family members using a PCR-based marker approach

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Low-molecular-weight glutenin subunits (LMW-GS) are a class of seed storage proteins that play a major role in the determination of the processing quality of wheat flour. The LMW-GS are encoded by multi-gene families located on the short arms of the homoeologous group 1 chromosomes, at the Glu-A3, G...

  3. A novel mutation in the succinate dehydrogenase subunit D gene in siblings with the hereditary paraganglioma–pheochromocytoma syndrome

    PubMed Central

    Oakley, Gerard J; Yip, Linwah; Coyne, Christopher; Rangaswamy, Balasubramanya; Dixit, Sanjay B

    2014-01-01

    Germline mutations in the succinate dehydrogenase complex subunit D gene are now known to be associated with hereditary paraganglioma–pheochromocytoma syndromes. Since the initial succinate dehydrogenase complex subunit D gene mutation was identified about a decade ago, more than 131 unique variants have been reported. We report the case of two siblings presenting with multiple paragangliomas and pheochromocytomas; they were both found to carry a mutation in the succinate dehydrogenase complex subunit D gene involving a substitution of thymine to guanine at nucleotide 236 in exon 3. This particular mutation of the succinate dehydrogenase complex subunit D gene has only been reported in one previous patient in Japan; this is, therefore, the first report of this pathogenic mutation in siblings and the first report of this mutation in North America. With continued screening of more individuals, we will be able to create a robust mutation database that can help us understand disease patterns associated with particular variants and may be a starting point in the development of new therapies for familial paraganglioma syndromes. PMID:27489656

  4. Cloning and Expression of Beta Subunit Gene of Phycocyanin From Spirulina platensis in Escherichia coli

    PubMed Central

    Shoja, Zahra; Rajabi Memari, Hamid; Roayaei Ardakani, Mohammd

    2015-01-01

    Background: C-Phycocyanin (C-PC) from blue-green algae such as Spirulina has been reported to have various pharmacological characteristics, including anti-inflammatory and anti-tumor activities. Recombinant β-subunit of C-PC (C-PC/β) is an inhibitor of cell proliferation and an inducer of cancer cell apoptosis. Objectives: Since C-PC/β has a big potential to be used as a promising cancer prevention or therapy agent, the purpose of this study was to clone and express Spirulina platensis cpcB gene in a bacterial expression system. This is a significant step for the production of this compound. Materials and Methods: The cpcB gene was amplified using specific primers and cloned in a bacterial expression vector, namely pET43.1a+. Gene expression of cpcB was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the dot blotting technique. Results: The SDS-PAGE analysis and dot blotting confirmed the production of recombinant C-PC/β in the bacterial expression system. Over-expression of cpcB gene was optimized in induction by 1 mM Isopropyl-β-D-Thiogalactoside (IPTG), after four hours of inoculation at 30°C. Conclusions: Over-expression of the synthetic CPC/β protein in the bacterial system (Escherichia coli BL-21) showed that E. coli can be used as a basis for further research to produce this desired protein in large quantities. PMID:26464761

  5. Accurate, Rapid Taxonomic Classification of Fungal Large-Subunit rRNA Genes

    PubMed Central

    Liu, Kuan-Liang; Porras-Alfaro, Andrea; Eichorst, Stephanie A.

    2012-01-01

    Taxonomic and phylogenetic fingerprinting based on sequence analysis of gene fragments from the large-subunit rRNA (LSU) gene or the internal transcribed spacer (ITS) region is becoming an integral part of fungal classification. The lack of an accurate and robust classification tool trained by a validated sequence database for taxonomic placement of fungal LSU genes is a severe limitation in taxonomic analysis of fungal isolates or large data sets obtained from environmental surveys. Using a hand-curated set of 8,506 fungal LSU gene fragments, we determined the performance characteristics of a naïve Bayesian classifier across multiple taxonomic levels and compared the classifier performance to that of a sequence similarity-based (BLASTN) approach. The naïve Bayesian classifier was computationally more rapid (>460-fold with our system) than the BLASTN approach, and it provided equal or superior classification accuracy. Classifier accuracies were compared using sequence fragments of 100 bp and 400 bp and two different PCR primer anchor points to mimic sequence read lengths commonly obtained using current high-throughput sequencing technologies. Accuracy was higher with 400-bp sequence reads than with 100-bp reads. It was also significantly affected by sequence location across the 1,400-bp test region. The highest accuracy was obtained across either the D1 or D2 variable region. The naïve Bayesian classifier provides an effective and rapid means to classify fungal LSU sequences from large environmental surveys. The training set and tool are publicly available through the Ribosomal Database Project (http://rdp.cme.msu.edu/classifier/classifier.jsp). PMID:22194300

  6. Molecular evolution of the cytochrome c oxidase subunit 5A gene in primates

    PubMed Central

    2008-01-01

    Background Many electron transport chain (ETC) genes show accelerated rates of nonsynonymous nucleotide substitutions in anthropoid primate lineages, yet in non-anthropoid lineages the ETC proteins are typically highly conserved. Here, we test the hypothesis that COX5A, the ETC gene that encodes cytochrome c oxidase subunit 5A, shows a pattern of anthropoid-specific adaptive evolution, and investigate the distribution of this protein in catarrhine brains. Results In a dataset comprising 29 vertebrate taxa, including representatives from all major groups of primates, there is nearly 100% conservation of the COX5A amino acid sequence among extant, non-anthropoid placental mammals. The most recent common ancestor of these species lived about 100 million years (MY) ago. In contrast, anthropoid primates show markedly elevated rates of nonsynonymous evolution. In particular, branch site tests identify five positively selected codons in anthropoids, and ancestral reconstructions infer that substitutions in these codons occurred predominantly on stem lineages (anthropoid, ape and New World monkey) and on the human terminal branch. Examination of catarrhine brain samples by immunohistochemistry characterizes for the first time COX5A protein distribution in the primate neocortex, and suggests that the protein is most abundant in the mitochondria of large-size projection neurons. Real time quantitative PCR supports previous microarray results showing COX5A is expressed in cerebral cortical tissue at a higher level in human than in chimpanzee or gorilla. Conclusion Taken together, these results suggest that both protein structural and gene regulatory changes contributed to COX5A evolution during humankind's ancestry. Furthermore, these findings are consistent with the hypothesis that adaptations in ETC genes contributed to the emergence of the energetically expensive anthropoid neocortex. PMID:18197981

  7. Localization of a gene for a glutamate binding subunit of a NMDA receptor (GRINA) to 8q24

    SciTech Connect

    Lewis, T.B.; DuPont, B.R.; Leach, R.

    1996-02-15

    This article reports on the localization of a gene for a glutamate binding subunit of an N-methyl-D-aspartate (NMDA) receptor, called GRINA, to human chromosome 8q24 using fluorescence in situ hybridization and radiation hybridization mapping. This gene mapped outside the critical region for benign familial neonatal convulsions (BFNC), a rare form of epilepsy; however, GRINA could be the causative genetic factor inducing idiopathic generalized epilepsy. Further studies need to be conducted. 15 refs., 2 figs.

  8. Nucleotide sequence of the gene encoding the two-subunit pilin of Bacteroides nodosus 265.

    PubMed Central

    Elleman, T C; Hoyne, P A; McKern, N M; Stewart, D J

    1986-01-01

    The nucleotide sequence of the gene encoding pilin from Bacteroides nodosus 265 has been determined. The pilin is encoded by a single-copy gene, from which can be predicted a prepilin comprising a single protein chain of Mr 16,637. The prepilin sequence differs in several respects from the mature protein sequence. Seven additional N-terminal amino acid residues are present in prepilin, whereas residue 8, phenylalanine, undergoes posttranslational modification to become the N-methylated amino-terminal residue of mature pilin. In addition, further processing occurs through internal cleavage to produce two noncovalently linked subunits characteristic of pilins from serogroup H of B. nodosus, of which strain 265 is a member. The position of cleavage has been identified between alanine residues at positions 72 and 73 of the mature 149-residue pilin protein. The predicted pilin sequence of B. nodosus 265 shows extensive N-terminal amino acid sequence homology with other pilins of the N-methylphenylalanine type. In addition this sequence also shows homology with these N-methylphenylalanine-type pilins in the C-terminal region of the molecule, especially with pilin from Pseudomonas aeruginosa PAK. Images PMID:2873127

  9. Resequencing of the auxiliary GABAB receptor subunit gene KCTD12 in chronic tinnitus

    PubMed Central

    Sand, P. G.; Langguth, B.; Itzhacki, J.; Bauer, A.; Geis, S.; Cárdenas-Conejo, Z. E.; Pimentel, V.; Kleinjung, T.

    2012-01-01

    Tinnitus is a common and often incapacitating hearing disorder marked by the perception of phantom sounds. Susceptibility factors remain largely unknown but GABAB receptor signaling has long been implicated in the response to treatment and, putatively, in the etiology of the disorder. We hypothesized that variation in KCTD12, the gene encoding an auxiliary subunit of GABAB receptors, could help to predict the risk of developing tinnitus. Ninety-five Caucasian outpatients with a diagnosis of chronic tinnitus were systematically screened for mutations in the KCTD12 open reading frame and the adjacent 3′ untranslated region by Sanger sequencing. Allele frequencies were determined for 14 known variants of which three (rs73237446, rs34544607, and rs41287030) were polymorphic. When allele frequencies were compared to data from a large reference population of European ancestry, rs34544607 was associated with tinnitus (p = 0.04). However, KCTD12 genotype did not predict tinnitus severity (p = 0.52) and the association with rs34544607 was weakened after screening 50 additional cases (p = 0.07). Pending replication in a larger cohort, KCTD12 may act as a risk modifier in chronic tinnitus. Issues that are yet to be addressed include the effects of neighboring variants, e.g., in the KCTD12 gene regulatory region, plus interactions with variants of GABAB1 and GABAB2. PMID:22654739

  10. OBTAINING OF THE TRANSGENIC HELIANTHUS TUBEROSUS L. PLANTS, CALLUS AND "HAIRY" ROOT CULTURES ABLE TO EXPRESS THE RECOMBINANT HUMAN INTERFERON ALPHA-2b GENE.

    PubMed

    Maistrenko, O M; Luchakivska, Yu S; Zholobak, N M; Spivak, M Ya; Kuchuk, M V

    2015-01-01

    This work is the first to our knowledge to describe the successful attempt of Agrobacterium rhizogenes-mediated transformation of topinambour in order to obtain the transgenic H. tuberosus plants, callus and "hairy" root cultures. The plasmid vectors contained the sequence of interferon gene fused with Nicotiana plumbagenifolia L. calreticulin apoplast targeting signal driven by 35S CaMV promoter or root-specific Mll promoter. Nearly 75% isolated Ri-root lines and callus cultures were proved (by PCR analysis) to contain HuINFa-2b transgene. We also managed to obtain H. tuberosus transgenic plants through somatic embryogenesis on the transgenic "hairy" root culture. The obtained transgenic H. tuberosus cultures exhibited high-level antiviral activity that ranged from 2000 to 54500 IU/g FW that makes this crop considered a promising source of recombinant interferon alpha 2b protein. PMID:26638495

  11. Genes involved in lactose catabolism and organic acid production during growth of Lactobacillus delbrueckii UFV H2b20 in skimmed milk.

    PubMed

    Do Carmo, A P; De Oliveira, M N V; Da Silva, D F; Castro, S B; Borges, A C; De Carvalho, A F; De Moraes, C A

    2012-03-01

    There are three main reasons for using lactic acid bacteria (LAB) as starter cultures in industrial food fermentation processes: food preservation due to lactic acid production; flavour formation due to a range of organic molecules derived from sugar, lipid and protein catabolism; and probiotic properties attributed to some strains of LAB, mainly of lactobacilli. The aim of this study was to identify some genes involved in lactose metabolism of the probiotic Lactobacillus delbrueckii UFV H2b20, and analyse its organic acid production during growth in skimmed milk. The following genes were identified, encoding the respective enzymes: ldh - lactate dehydrogenase, adhE - Ldb1707 acetaldehyde dehydrogenase, and ccpA-pepR1 - catabolite control protein A. It was observed that L. delbrueckii UFV H2b20 cultivated in different media has the unexpected ability to catabolyse galactose, and to produce high amounts of succinic acid, which was absent in the beginning, raising doubts about the subspecies in question. The phylogenetic analyses showed that this strain can be compared physiologically to L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis, which are able to degrade lactose and can grow in milk. L. delbrueckii UFV H2b20 sequences have grouped with L. delbrueckii subsp. bulgaricus ATCC 11842 and L. delbrueckii subsp. bulgaricus ATCC BAA-365, strengthening the classification of this probiotic strain in the NCFM group proposed by a previous study. Additionally, L. delbrueckii UFV H2b20 presented an evolutionary pattern closer to that of probiotic Lactobacillus acidophilus NCFM, corroborating the suggestion that this strain might be considered as a new and unusual subspecies among L. delbrueckii subspecies, the first one identified as a probiotic. In addition, its unusual ability to metabolise galactose, which was significantly consumed in the fermentation medium, might be exploited to produce low-browning probiotic Mozzarella cheeses, a desirable property

  12. Nicotinic acetylcholine receptor β2 subunit gene implicated in a systems-based candidate gene study of smoking cessation

    PubMed Central

    Conti, David V.; Lee, Won; Li, Dalin; Liu, Jinghua; Van Den Berg, David; Thomas, Paul D.; Bergen, Andrew W.; Swan, Gary E.; Tyndale, Rachel F.; Benowitz, Neal L.; Lerman, Caryn

    2008-01-01

    Although the efficacy of pharmacotherapy for tobacco dependence has been previously demonstrated, there is substantial variability among individuals in treatment response. We performed a systems-based candidate gene study of 1295 single nucleotide polymorphisms (SNPs) in 58 genes within the neuronal nicotinic receptor and dopamine systems to investigate their role in smoking cessation in a bupropion placebo-controlled randomized clinical trial. Putative functional variants were supplemented with tagSNPs within each gene. We used global tests of main effects and treatment interactions, adjusting the P-values for multiple correlated tests. An SNP (rs2072661) in the 3′ UTR region of the β2 nicotinic acetylcholine receptor subunit (CHRNB2) has an impact on abstinence rates at the end of treatment (adjusted P = 0.01) and after a 6-month follow-up period (adjusted P = 0.0002). This latter P-value is also significant with adjustment for the number of genes tested. Independent of treatment at 6-month follow-up, individuals carrying the minor allele have substantially decreased the odds of quitting (OR = 0.31; 95% CI 0.18–0.55). Effect of estimates indicate that the treatment is more effective for individuals with the wild-type (OR = 2.14, 95% CI 1.20–3.81) compared with individuals carrying the minor allele (OR = 0.83, 95% CI 0.32–2.19), although this difference is only suggestive (P = 0.10). Furthermore, this SNP demonstrated a role in the time to relapse (P = 0.0002) and an impact on withdrawal symptoms at target quit date (TQD) (P = 0.0009). Overall, while our results indicate strong evidence for CHRNB2 in ability to quit smoking, these results require replication in an independent sample. PMID:18593715

  13. Polymorphisms of estrogen metabolism-related genes ESR1, UGT2B17, and UGT1A1 are not associated with osteoporosis in surgically menopausal Japanese women

    PubMed Central

    Yokota, Megumi; Makita, Kazuya; Akahane, Tomoko; Sakai, Kensuke; Makabe, Takeshi; Horiba, Yuko; Yamagami, Wataru; Ogawa, Mariko; Iwata, Takashi; Yanamoto, Shigehisa; Deshimaru, Ryota; Banno, Kouji; Susumu, Nobuyuki; Aoki, Daisuke

    2015-01-01

    Introduction Bilateral salpingo-oophorectomy (BSO) is a risk factor for osteoporosis. Previous studies have reported an association between genetic polymorphisms and the risk of developing osteoporosis. However, the relationship between osteoporosis and genetic polymorphisms in Japanese women treated with BSO is not well understood. To improve the quality of life for post-BSO patients, it is important to determine the genetic factors that influence their risk for osteoporosis. The aim of this study was to investigate the association between gene variations of estrogen metabolism-related genes and osteoporosis in surgically menopausal patients, which may improve their quality of life. Material and methods This study included 203 menopausal women treated with BSO because of gynecologic disorders. One hundred and twenty-six women with artificial (surgical) menopause, who had undergone BSO in the premenopausal period, were compared with 77 women with natural menopause, who had undergone BSO in the postmenopausal period. The women were tested for bone mineral density to diagnose osteoporosis. Polymorphisms of estrogen receptor 1 (ESR1) and UDP-glucuronosyl transferase (UGT) genes UGT2B17 and UGT1A1 were analyzed, and their association with bone mass and osteoporosis was statistically evaluated. Results No significant association was found between osteoporosis and polymorphisms in ESR1, UGT2B17, or UGT1A1 in both groups, suggesting that BSO might be a more significant physiological factor in influencing bone mass density compared to genetic variations. Conclusions These results suggest that the ESR1, UGT2B17, and UGT1A1 polymorphisms are not genetic factors affecting osteoporosis in postmenopausal Japanese women. PMID:26528103

  14. Promoter activity of the 5'-flanking regions of medaka fish soluble guanylate cyclase alpha1 and beta1 subunit genes.

    PubMed Central

    Yamamoto, Takehiro; Suzuki, Norio

    2002-01-01

    We examined the spatial expression pattern of medaka fish (Oryzias latipes) soluble guanylate cyclase alpha(1) and beta(1) subunit genes, OlGCS-alpha(1) and OlGCS-beta(1), and characterized the 5'-flanking region required for expression of both genes by introducing various promoter-luciferase fusion-gene constructs into COS-1 cells and medaka fish embryos. The OlGCS-alpha(1) and OlGCS-beta(1) gene transcripts were detected in whole brain and kidney in 7-day and 9-day embryos. Primer-extension analysis demonstrated that there were no differences among various adult organs (brain, eye, kidney, ovary and testis) in the transcription start site of the OlGCS-alpha(1) and OlGCS-beta(1) genes. Neither gene contained the functional TATA box within its 5'-flanking region, and the basal promoter activity was found between nucleotides +33 and +42 in the OlGCS-alpha(1) gene and between nucleotides +146 and +155 in the OlGCS-beta(1) gene. In the assay of medaka fish embryos, the 5'-flanking region of the OlGCS-beta(1) gene exhibited lower promoter activity than that of the OlGCS-alpha(1) gene. In the experiments on dual-luciferase fusion-gene constructs, the 5'-flanking region of the OlGCS-alpha(1) gene connected to the 5'-flanking region of the OlGCS-beta(1) gene was introduced into medaka fish embryos, and the 5'-flanking regions of both subunit genes were shown to mutually influence each other's promoter activity. PMID:11772405

  15. PHYLOGENY OF ANGIOSTRONGYLUS CANTONENSIS IN THAILAND BASED ON CYTOCHROME C OXIDASE SUBUNIT I GENE SEQUENCE.

    PubMed

    Apichat, Vitta; Narongrit, Srisongcram; Jittranuch, Thiproaj; Anucha, Wongma; Wilaiwan, Polsut; Chamaiporn, Fukruksa; Thatcha, Yimthin; Bandid, Mangkit; Aunchalee, Thanwisai; Paron, Dekumyoy

    2016-05-01

    Angiostrongylus cantonensis is an emerging infectious agent causing eosinophilic meningitis or meningoencephalitis in humans with clinical manifestation of severe headache. Molecular genetic studies on classification and phylogeny of A. cantonensis in Thailand are limited. This study surveyed A. cantonensis larvae prevalence in natural intermediate hosts across Thailand and analyzed their phylogenetic relationships. A total of 14,032 freshwater and land snails were collected from 19 provinces of Thailand. None of Filopaludina sp, Pomacea sp, and Cyclophorus sp were infected with Angiostrongylus larvae, whereas Achatina fulica, Cryptozona siamensis, and Megaustenia siamensis collected from Kalasin, Kamphaeng Phet, Phetchabun, Phitsanulok, and Tak Provinces were infected, with C. siamensis being the common intermediate host. Based on morphology, larvae isolated from 11 samples of these naturally infected snails preliminarily were identified as A. cantonensis. Comparison of partial nucleotide sequences of cytochrome c oxidase subunit I gene revealed that four sequences are identical to A. cantonensis haplotype ac4 from Bangkok and the other seven to that of A. cantonensis isolate AC Thai, indicating two independent lineages of A. cantonensis in Thailand. PMID:27405119

  16. Deep resequencing of the voltage-gated potassium channel subunit KCNE3 gene in chronic tinnitus

    PubMed Central

    2011-01-01

    Membrane-stabilizing drugs have long been used for the treatment of chronic tinnitus, suggesting an underlying disturbance of sensory excitability due to changes in ion conductance. The present study addresses the potassium channel subunit gene KCNE3 as a potential candidate for tinnitus susceptibility. 288 Caucasian outpatients with a diagnosis of chronic tinnitus were systematically screened for mutations in the KCNE3 open reading frame and in the adjacent region by direct sequencing. Allele frequencies were determined for 11 known variants of which two (F66F and R83H) were polymorphic but were not associated with the disorder. No novel variants were identified and only three carriers of R83H were noted. However, owing to a lack of power, our study can neither rule out effects of KCNE3 on the risk for developing chronic tinnitus, nor can it exclude a role in predicting the severity of tinnitus. More extensive investigations are invited, including tests for possible effects of variation in this ion channel protein on the response to treatment. PMID:21899751

  17. Complex I Subunit Gene Therapy With NDUFA6 Ameliorates Neurodegeneration in EAE

    PubMed Central

    Talla, Venu; Koilkonda, Rajeshwari; Porciatti, Vittorio; Chiodo, Vince; Boye, Sanford L.; Hauswirth, William W.; Guy, John

    2015-01-01

    Purpose. To address the permanent disability induced by mitochondrial dysfunction in experimental autoimmune encephalomyelitis (EAE). Methods. Mice sensitized for EAE were rescued by intravitreal injection of adeno-associated viral vector serotype 2 with the complex I subunit gene scAAV-NDUFA6Flag. Controls were injected with a mitochondrially targeted red fluorescent protein (scAAV-COX8-cherry). Another group received scAAV-COX8-cherry, but was not sensitized for EAE. Serial pattern electroretinograms (PERGs) and optical coherent tomography (OCT) evaluated visual function and structure of the retina at 1, 3, and 6 months post injection (MPI). Treated mice were killed 6 MPI for histopathology. Immunodetection of cleaved caspase 3 gauged apoptosis. Complex I activity was assessed spectrophotometrically. Expression of NDUFA6Flag in the retina and optic nerve were evaluated between 1 week to 1 month post injection by RT-PCR, immunofluorescence and immunoblotting. Results. Reverse transcription-PCR and immunoblotting confirmed NDUFA6Flag overexpression with immunoprecipitation and blue native PAGE showing integration into murine complex I. Overexpression of NDUFA6Flag in the visual system of EAE mice rescued retinal complex I activity completely, axonal loss by 73%, and retinal ganglion cell (RGC) loss by 88%, RGC apoptosis by 66%, and restored the 33% loss of complex I activity in EAE to normal levels; thereby, preventing loss of vision indicated by the 43% reduction in the PERG amplitudes of EAE mice. Conclusions. NDUFA6 gene therapy provided long-term suppression of neurodegeneration in the EAE animal model suggesting that it may also ameliorate the mitochondrial dysfunction associated with permanent disability in optic neuritis and MS patients. PMID:25613946

  18. Sequence of the nifD gene coding for the α subunit of dinitrogenase from the cyanobacterium Anabaena

    PubMed Central

    Lammers, Peter J.; Haselkorn, Robert

    1983-01-01

    The nucleotide sequence of nifD, the structural gene for the α subunit of dinitrogenase from Anabaena 7120, has been determined. The coding sequence contains 1,440 nucleotides, which predict an amino acid sequence of 480 residues and Mr of 54,283. The predicted sequence contains eight cysteines, of which five are conserved with respect to adjoining sequences and position relative to the α subunits of dinitrogenase from Azotobacter, Clostridium, and Klebsiella. Because there are also five conserved cysteines in the β subunit of Anabaena dinitrogenase [Mazur, B. J. & Chiu, C.-F. (1982) Proc. Natl. Acad. Sci. USA 79, 6782-6786], the number of cysteine residues participating as ligands to FeS clusters is likely to be 20 per α2β2 tetramer. This number is sufficient to accommodate the known four Fe4S4 clusters, leaving at least four cysteines to be shared among the two FeMo cofactors and the more poorly characterized two-iron center. Although the α- and β-subunit gene sequences are not recognizably homologous, their secondary structures, predicted from the sequences, indicate similar domains around three of the conserved cysteine residues. PMID:16593347

  19. The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes.

    PubMed

    Hemsley, Piers A; Hurst, Charlotte H; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R; De Cothi, Elizabeth A; Steele, John F; Knight, Heather

    2014-01-01

    The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation-induced freezing tolerance. In addition, these three subunits are required for low temperature-induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced. PMID:24415770

  20. Prevalence of the Prefoldin Subunit 5 Gene Deletion in Canine Mammary Tumors

    PubMed Central

    Bornemann-Kolatzki, Kirsten; Neumann, Stephan; Escobar, Hugo Murua; Nolte, Ingo; Hammer, Susanne Conradine; Hewicker-Trautwein, Marion; Junginger, Johannes; Kaup, Franz-Josef; Brenig, Bertram; Schütz, Ekkehard

    2015-01-01

    Background A somatic deletion at the proximal end of canine chromosome 27 (CFA27) was recently reported in 50% of malignant mammary tumors. This region harbours the tumor suppressor gene prefoldin subunit 5 (PFDN5) and the deletion correlated with a higher Ki-67 score. PFDN5 has been described to repress c-MYC and is, therefore, a candidate tumor-suppressor and cancer-driver gene in canine mammary cancer. Aim of this study was to confirm the recurrent deletion in a larger number of tumors. Methods Droplet digital PCR for PFDN5 was performed in DNA from 102 malignant, 40 benign mammary tumors/dysplasias, 11 non-neoplastic mammary tissues and each corresponding genomic DNA from leukocytes. The copy number of PFDN5 was normalized to a reference amplicon on canine chromosome 32 (CFA32). Z-scores were calculated, based on Gaussian distributed normalized PFDN5 copy numbers of the leukocyte DNA. Z-scores ≤ -3.0 in tissue were considered as being indicative of the PFDN5 deletion and called as such. The Ki-67 proliferation index was assessed in a subset of 79 tissue samples by immunohistochemistry. Results The deletion was confirmed in 24% of all malignant tumors, detected in only 7.5% of the benign tumors and was not present in any normal mammary tissue sample. The subgroup of solid carcinomas (n = 9) showed the highest frequency of the deletion (67%) and those malignomas without microscopical high fraction of benign tissue (n = 71) had a 32% frequency (p<0.01 vs. benign samples). The Ki-67 score was found to be significantly higher (p<0.05) in the PFDN5-deleted group compared to malignant tumors without the deletion. Conclusions A somatic deletion of the PFDN5 gene is recurrently present in canine mammary cancer, supporting a potential role in carcinogenesis. The association of this deletion with higher Ki-67 indicates an increased proliferation rate and thus a link to tumor aggressiveness can be hypothesized. The confirmation of earlier results warrants further studies

  1. Effects of Ghrelin on Sexual Behavior and Luteinizing Hormone Beta-subunit Gene Expression in Male Rats

    PubMed Central

    Babaei-Balderlou, Farrin; Khazali, Homayoun

    2016-01-01

    Background: The hormones of hypothalamo-pituitary-gonadal (HPG) axis have facilitative effects on reproductive behavior in mammals. Ghrelin as a starvation hormone has an inhibitory effect on HPG axis’ function. Hence, it is postulated that ghrelin may reduce the sexual behavior through inhibiting of HPG axis. The aim of this study was to examine the effects of ghrelin and its antagonist, [D-Lys3 ]-GHRP-6, on sexual behavior and LH beta-subunit gene expression in male rats. Methods: In this experimental study, 128 male Wistar rats were divided into two groups. Each group was further subdivided into eight subgroups (n=8 rats/subgroup) including the animals that received saline, ghrelin (2, 4 or 8 nmol), [D-Lys3 ]-GHRP-6 (5 or 10 nmol) or co-administration of ghrelin (4 nmol) and [D-Lys3 ]-GHRP-6 (5 or 10 nmol) through the stereotaxically implanted cannula into the third cerebral ventricle. The sexual behavior of male rats encountering with females and the hypo-physeal LH beta-subunit gene expression were evaluated at two different groups. Data were analyzed by ANOVA and p<0.05 was considered statistically significant. Results: Ghrelin injection (4 and 8 nmol) significantly (p<0.01) increased the latencies to the first mount, intromission and ejaculation as well as the post-ejaculatory interval. Also, 4 and 8 nmol ghrelin significantly (p<0.05) increased the number of mount and decreased the number of ejaculation. In co-administrated groups, [D-Lys3 ]-GHRP-6 antagonized the effects of ghrelin. Ghrelin injection (4 and 8 nmol) reduced the LH beta-subunit gene expression while pretreatment with [D-Lys3 ]-GHRP-6 improved the gene expression. Conclusion: Ghrelin decreased the sexual behavior and LH beta-subunit gene expression in male rats, whereas [D-Lys3 ]-GHRP-6 antagonizes these effects. PMID:27141463

  2. Evolution, Expression Differentiation and Interaction Specificity of Heterotrimeric G-Protein Subunit Gene Family in the Mesohexaploid Brassica rapa

    PubMed Central

    Arya, Gulab C.; Kumar, Roshan; Bisht, Naveen C.

    2014-01-01

    Heterotrimeric G-proteins, comprising of Gα, Gβ, and Gγ subunits, are important signal transducers which regulate many aspects of fundamental growth and developmental processes in all eukaryotes. Initial studies in model plants Arabidopsis and rice suggest that the repertoire of plant G-protein is much simpler than that observed in metazoans. In order to assess the consequence of whole genome triplication events within Brassicaceae family, we investigated the multiplicity of G-protein subunit genes in mesohexaploid Brassica rapa, a globally important vegetable and oilseed crop. We identified one Gα (BraA.Gα1), three Gβ (BraA.Gβ1, BraA.Gβ2, and BraA.Gβ3), and five Gγ (BraA.Gγ1, BraA.Gγ2, BraA.Gγ3, BraA.Gγ4, and BraA.Gγ5) genes from B. rapa, with a possibility of 15 Gαβγ heterotrimer combinations. Our analysis suggested that the process of genome triplication coupled with gene-loss (gene-fractionation) phenomenon have shaped the quantitative and sequence diversity of G-protein subunit genes in the extant B. rapa genome. Detailed expression analysis using qRT-PCR assays revealed that the G-protein genes have retained ubiquitous but distinct expression profiles across plant development. The expression of multiple G-protein genes was differentially regulated during seed-maturation and germination stages, and in response to various phytohormone treatments and stress conditions. Yeast-based interaction analysis showed that G-protein subunits interacted in most of the possible combinations, with some degree of subunit-specific interaction specificity, to control the functional selectivity of G-protein heterotrimer in different cell and tissue-types or in response to different environmental conditions. Taken together, this research identifies a highly diverse G-protein signaling network known to date from B. rapa, and provides a clue about the possible complexity of G-protein signaling networks present across globally important Brassica species. PMID

  3. Evolution, expression differentiation and interaction specificity of heterotrimeric G-protein subunit gene family in the mesohexaploid Brassica rapa.

    PubMed

    Arya, Gulab C; Kumar, Roshan; Bisht, Naveen C

    2014-01-01

    Heterotrimeric G-proteins, comprising of Gα, Gβ, and Gγ subunits, are important signal transducers which regulate many aspects of fundamental growth and developmental processes in all eukaryotes. Initial studies in model plants Arabidopsis and rice suggest that the repertoire of plant G-protein is much simpler than that observed in metazoans. In order to assess the consequence of whole genome triplication events within Brassicaceae family, we investigated the multiplicity of G-protein subunit genes in mesohexaploid Brassica rapa, a globally important vegetable and oilseed crop. We identified one Gα (BraA.Gα1), three Gβ (BraA.Gβ1, BraA.Gβ2, and BraA.Gβ3), and five Gγ (BraA.Gγ1, BraA.Gγ2, BraA.Gγ3, BraA.Gγ4, and BraA.Gγ5) genes from B. rapa, with a possibility of 15 Gαβγ heterotrimer combinations. Our analysis suggested that the process of genome triplication coupled with gene-loss (gene-fractionation) phenomenon have shaped the quantitative and sequence diversity of G-protein subunit genes in the extant B. rapa genome. Detailed expression analysis using qRT-PCR assays revealed that the G-protein genes have retained ubiquitous but distinct expression profiles across plant development. The expression of multiple G-protein genes was differentially regulated during seed-maturation and germination stages, and in response to various phytohormone treatments and stress conditions. Yeast-based interaction analysis showed that G-protein subunits interacted in most of the possible combinations, with some degree of subunit-specific interaction specificity, to control the functional selectivity of G-protein heterotrimer in different cell and tissue-types or in response to different environmental conditions. Taken together, this research identifies a highly diverse G-protein signaling network known to date from B. rapa, and provides a clue about the possible complexity of G-protein signaling networks present across globally important Brassica species. PMID

  4. Structure and expression analysis of genes encoding ADP-glucose pyrophosphorylase large subunit in wheat and its relatives.

    PubMed

    Zhang, Xiao-Wei; Li, Si-Yu; Zhang, Ling-Ling; Yang, Qiang; Jiang, Qian-Tao; Ma, Jian; Qi, Peng-Fei; Li, Wei; Chen, Guo-Yue; Lan, Xiu-Jin; Deng, Mei; Lu, Zhen-Xiang; Liu, Chunji; Wei, Yu-Ming; Zheng, You-Liang

    2016-07-01

    ADP-glucose pyrophosphorylase (AGP), which consists of two large subunits (AGP-L) and two small subunits (AGP-S), controls the rate-limiting step in the starch biosynthetic pathway. In this study, a full-length open reading frame (ORF) of AGP-L gene (named as Agp2) in wheat and a series of Agp2 gene sequences in wheat relatives were isolated. The coding region of Agp2 contained 15 exons and 14 introns including a full-length ORF of 1566 nucleotides, and the deduced protein contained 522 amino acids (57.8 kDa). Generally, the phylogenetic tree of Agp2 indicated that sequences from A- and D-genome donor species were most similar to each other and sequences from B-genome donor species contained more variation. Starch accumulation and Agp2 expression in wheat grains reached their peak at 21 and 15 days post anthesis (DPA), respectively. PMID:27299732

  5. Nucleotide sequence of the gene encoding the major subunit of CS3 fimbriae of enterotoxigenic Escherichia coli.

    PubMed Central

    Boylan, M; Smyth, C J; Scott, J R

    1988-01-01

    The complete nucleotide sequence of a 612-base-pair DNA fragment containing the gene for the major fimbrial subunit of CS3 of enterotoxigenic Escherichia coli is presented. A possible promoter region, a ribosome-binding site, and two potential signal peptidase cleavage sites are indicated. Unlike the best-studied fimbrial proteins, the predicted CS3 sequence has no Cys residues. PMID:2903130

  6. Deletion of the structural gene for the NADH-dehydrogenase subunit 4 of Synechocystis 6803 alters respiratory properties.

    PubMed Central

    Dzelzkalns, V A; Obinger, C; Regelsberger, G; Niederhauser, H; Kamensek, M; Peschek, G A; Bogorad, L

    1994-01-01

    Chloroplasts and cyanobacteria contain genes encoding polypeptides homologous to some subunits of the mitochondrial respiratory NADH-ubiquinol oxidoreductase complex (NADH dehydrogenase). Nothing is known of the role of the NADH dehydrogenase complex in photosynthesis, respiration, or other functions in chloroplasts, and little is known about the specific roles of the perhaps 42 subunits of this complex in the mitochondrion. Inactivation of a gene for subunit 4 (ndhD-2, ndh4) of this complex in the cyanobacterium Synechocystis 6803 has no effect on photosynthesis, judging from the rate of photoautotrophic growth of mutant cells, but the mutant's respiratory rate is about 6 times greater than that of wild-type cells. Respiratory electron transport activity in cyanobacteria is associated both with photosynthetic thylakoid membranes and with the outer cytoplasmic membrane of the cell. Cytoplasmic membranes of mutant cells have much greater NADH-dependent cytochrome reductase activity than preparations from wild-type cells; this activity remains at wild-type levels in isolated thylakoid membranes. It is suggested that the 56.6-kD product of ndhD-2 is not essential for the activity of a cytoplasmic membrane-bound NADH dehydrogenase but that it regulates the rate of electron flow through the complex, establishing a link between this ndh gene and respiration. The activity of the molecularly distinct thylakoid-bound NADH dehydrogenase is apparently unaffected by the loss of ndhD-2. PMID:7846157

  7. Biased expression, under the control of single promoter, of human interferon α-2b and Escherichia coli methionine amino peptidase genes in E. coli, irrespective of their distance from the promoter.

    PubMed

    Arif, Amina; Rashid, Naeem; Aslam, Farheen; Mahmood, Nasir; Akhtar, Muhammad

    2016-03-01

    Human interferon α-2b and Escherichia coli methionine amino peptidase genes were cloned independently as well as bicistronically in expression plasmid pET-21a (+). Production of human interferon α-2b was comparable to that of E. coli methionine amino peptidase when these genes were expressed independently in E. coli BL21-CodonPlus (DE3)-RIL. However, human interferon α-2b was produced in a much less amount whereas there was no difference in the production of methionine amino peptidase when the encoding genes were expressed bicistronically. It is important to note that human interferon α-2b was the first gene in order, after the promoter and E. coli methionine amino peptidase was the next with a linker sequence of 27 nucleotides between them. PMID:27087087

  8. Genetic association of bipolar disorder with the β3 nicotinic receptor subunit gene

    PubMed Central

    Hartz, Sarah M.; Lin, Peng; Edenberg, Howard J.; Xuei, Xiaoling; Rochberg, Nanette; Saccone, Scott; Berrettini, Wade; Nelson, Elliot; Nurnberger, John; Bierut, Laura J.; Rice, John P.

    2011-01-01

    Objective Owing to the clinical relationship between bipolar disorder and nicotine dependence, we investigated two research questions: (i) are genetic associations with nicotine dependence different in individuals with bipolar disorder as compared with individuals without bipolar disorder, and (ii) do loci earlier associated with nicotine dependence have pleiotropic effects on these two diseases. Method Our study consisted of 916 cases with bipolar disorder and 1028 controls. On the basis of known associations with nicotine dependence, we genotyped eight single-nucleotide polymorphisms (SNPs) on chromosome 8 (three bins) in the regions of CHRNB3 and CHRNA6, and six SNPs on chromosome 15 (three bins) in the regions of CHRNA5 and CHRNA3. Results To determine whether the genetic associations with nicotine dependence are different in bipolar disorder than in the general population, we compared allele frequencies of candidate SNPs between individuals with nicotine dependence only and individuals with both nicotine dependence and bipolar disorder. There were no statistical differences between these frequencies, indicating that genetic association with nicotine dependence is similar in individuals with bipolar disorder as in the general population. In the investigation of pleiotropic effects of these SNPs on bipolar disorder, two highly correlated synonymous SNPs in CHRNB3, rs4952 and rs4953, were significantly associated with bipolar disorder (odds ratio 1.7, 95% confidence interval: 1.2–2.4, P = 0.001). This association remained significant both after adjusting for a smoking covariate and analyzing the association in nonsmokers only. Conclusion Our results suggest that (i) bipolar disorder does not modify the association between nicotine dependence and nicotinic receptor subunit genes, and (ii) variants in CHRNB3/CHRNA6 are independently associated with bipolar disorder. Psychiatr Genet 00:000–000. PMID:21191315

  9. Biogenesis of mitochondria: the mitochondrial gene (aap1) coding for mitochondrial ATPase subunit 8 in Saccharomyces cerevisiae.

    PubMed Central

    Macreadie, I G; Novitski, C E; Maxwell, R J; John, U; Ooi, B G; McMullen, G L; Lukins, H B; Linnane, A W; Nagley, P

    1983-01-01

    A mitochondrial gene (denoted aap1) in Saccharomyces cerevisiae has been characterized by nucleotide sequence analysis of a region of mtDNA between the oxi3 and oli2 genes. The reading frame of the aap1 gene specifies a hydrophobic polypeptide containing 48 amino acids. The functional nature of this reading frame was established by sequence analysis of a series of mit- mutants and revertants. Evidence is presented that the aap1 gene codes for a mitochondrially synthesized polypeptide associated with the mitochondrial ATPase complex. This polypeptide (denoted subunit 8) is a proteolipid whose size has been previously assumed to be 10 kilodaltons based on its mobility on SDS-polyacrylamide gels, but the sequence of the aap1 gene predicts a molecular weight of 5,815 for this protein. PMID:6223276

  10. Physical linkage of a GABAA receptor subunit gene to the DXS374 locus in human Xq28.

    PubMed Central

    Bell, M V; Bloomfield, J; McKinley, M; Patterson, M N; Darlison, M G; Barnard, E A; Davies, K E

    1989-01-01

    We report the physical linkage of the gene encoding one of the subunits of the GABAA receptor (GABRA3) to the polymorphic locus DXS374 on the human X chromosome at Xq28. X-linked manic depression and other psychiatric disorders have been mapped to this region, and thus GABRA3 is a potential candidate gene for these disorders. DXS374--and therefore GABRA3--lies distal to the fragile X locus at a recombination fraction of approximately .15. Images Figure 1 Figure 2 PMID:2574000

  11. Both NR2A and NR2B Subunits of the NMDA Receptor Are Critical for Long-Term Potentiation and Long-Term Depression in the Lateral Amygdala of Horizontal Slices of Adult Mice

    ERIC Educational Resources Information Center

    Muller, Tobias; Albrecht, Doris; Gebhardt, Christine

    2009-01-01

    The lateral nucleus of the amygdala (LA) is implicated in emotional and social behaviors. We recently showed that in horizontal brain slices, activation of NMDA receptors (NMDARs) is a requirement for persistent synaptic alterations in the LA, such as long-term potentiation (LTP) and long-term depression (LTD). In the LA, NR2A- and NR2B-type NMDRs…

  12. The NADH-binding subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans: Gene cloning and deduced primary structure

    SciTech Connect

    Xu, Xuemin; Matsuno-Yagi, Akemi; Yagi, Takao )

    1991-07-02

    The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN, non-heme iron, and acid-labile sulfide. The NADH-binding subunit of this enzyme complex was identified by direct photoaffinity labeling with ({sup 32}P)NADH. primers were synthesized on the basis of the N-terminal amino acid sequency of this polypeptide, and these primers were used to synthesize an oligonucleotide probe by the polymerase chain reaction. This probe was utilized to isolate the gene encoding the NADH-binding subunit from a genomic library of P. denitrificans. The nucleotide sequence of the gene and the deduced amino acid sequence of the entire NADH-binding subunit were determined. The NADH-binding subunit has 431 amino acid residues and a calculated molecular weight of 47 191. The encoded protein contains a putative NAD(H)-binding and an iron-sulfur cluster-binding consensus sequence. The deduced amino acid sequence of the Paracoccus NADH-binding subunit shows remarkable similarity to the {alpha} subunit of the NAD-linked hydrogenase of Alcaligenes eutrophus H16. When partial DNA sequencing of the regions surrounding the gene encoding the NADH-binding subunit was carried out, sequences homologous to the 24-, 49-, and 75-kDa polypeptides of bovine complex 1 were detected, suggesting that the structural genes of the Paracoccus NADH dehydrogenase complex constitute a gene cluster.

  13. YY1 and Sp1 activate transcription of the human NDUFS8 gene encoding the mitochondrial complex I TYKY subunit.

    PubMed

    Lescuyer, Pierre; Martinez, Pascal; Lunardi, Joël

    2002-03-19

    Complex I is the most complicated of the multimeric enzymes that constitute the mitochondrial respiratory chain. It is encoded by both mitochondrial and nuclear genomes. We have previously characterized the human NDUFS8 gene that encodes the TYKY subunit. This essential subunit is thought to participate in the electron transfer and proton pumping activities of complex I. Here, we have analyzed the transcriptional regulation of the NDUFS8 gene. Using primer extension assays, we have identified two transcription start sites. The basal promoter was mapped to a 247 bp sequence upstream from the main transcription start site by reporter gene analysis in HeLa cells and in differentiated or non-differentiated C2C12 cells. Three Sp1 sites and one YY1 site were identified in this minimal promoter. Through gel shift analysis, all sites were shown to bind to their cognate transcription factors. Site-directed mutagenesis revealed that the YY1 site and two upstream adjacent Sp1 sites drive most of the promoter activity. This work represents the first promoter analysis for a complex I gene. Together with previous studies, our results indicate that YY1 and Sp1 control the expression of genes encoding proteins that are involved in almost all steps of the oxidative phosphorylation metabolism. PMID:11955626

  14. The sequence of the gene for cytochrome c oxidase subunit I, a frameshift containing gene for cytochrome c oxidase subunit II and seven unassigned reading frames in Trypanosoma brucei mitochrondrial maxi-circle DNA.

    PubMed Central

    Hensgens, L A; Brakenhoff, J; De Vries, B F; Sloof, P; Tromp, M C; Van Boom, J H; Benne, R

    1984-01-01

    A 9.2 kb segment of the maxi-circle of Trypanosoma brucei mitochondrial DNA contains the genes for cytochrome c oxidase subunits I and II (coxI and coxII) and seven Unassigned Reading Frames ("URFs"). The genes for coxI and coxII display considerable homology at the aminoacid level (38 and 25%, respectively) to the corresponding genes in fungal and mammalian mtDNA, the only striking point of divergence being an unusually high cysteine content (about 4.5%). The reading frame coding for cytochrome c oxidase subunit II is discontinuous: the C-terminal portion of about 40 aminoacids, is present in the DNA-sequence in a -1 reading frame with respect to the N-terminal moiety. URF5, 8 and 10, show a low but distinct homology (about 20%) to mammalian mitochondrial URF-1, 4 and 5, respectively. In URF5, the first AUG is found at codon 145, whereas extensive homology to mammalian URF-1 sequences occurs upstream of this position. The possibility exists that UUG can serve as an initiator codon. URF7 and URF9 have a highly unusual aminoacid composition and do not possess AUG or UUG initiator codons. These URFs probably do not have a protein-coding function. The segment does not contain conventional tRNA genes. Images PMID:6093040

  15. Differential gene expression profiles of two excitable rat cell lines after over-expression of WT- and C121W-β1 sodium channel subunits.

    PubMed

    Baroni, D; Moran, O

    2015-06-25

    Voltage-dependent sodium channels are membrane proteins essential for cell excitability. They are composed by a pore-forming α-subunit, encoded in mammals by up to nine different genes, and four different ancillary β-subunits. The expression pattern of the α subunit isoforms confers the distinctive functional and pharmacological properties to different excitable tissues. β-Subunits are important modulators of channel function and expression. Mutation C121W of the β1-subunit causes an autosomal dominant epileptic syndrome without cardiac symptoms. In neuroectoderm GH3 and cardiac H9C2 cells, the over-expression of β1 subunit augments α subunit mRNA and protein levels as well as sodium current density. Interestingly, the introduction of the epileptogenic C121W-β1 subunit produces additional changes in the α-subunit expression pattern of H9C2 cells, leaving unaltered the sodium channel isoform composition of GH3 cells. The challenge of the present work was to identify those genes that were differentially expressed in response to WT- or C121W-β1 subunit over-expression in the two rat cell lines under analysis. Hence, we analyzed the total mRNA extracted from control-untransfected and from WT- and C121W-β1-transfected GH3 and H9C2 cells by DNA-microarray. We found that, in agreement with their different embryonal origin, the over-expression of WT- and C121W-β1 subunits modifies the expression of different gene sets in GH3 and H9C2 cells. Focusing on the effects of the C121W mutation, we found that it causes the modification of 214 genes, most of them were down-regulated (202) in GH3 cells; on the contrary, it determined the up-regulation of only five genes in H9C2 cells. Interestingly, most genes modified by the C121W β1 subunit are involved in pivotal processes of the cell such as cellular communication and protein expression. Our results confirm the important role of the sodium channel β1 subunit in the control of NaCh gene expression, and highlight once

  16. Evidence for interaction between markers in GABA(A) receptor subunit genes in an Argentinean autism spectrum disorder population.

    PubMed

    Sesarini, Carla V; Costa, Lucas; Naymark, Muriel; Grañana, Nora; Cajal, Andrea R; García Coto, Miguel; Pallia, Roberto C; Argibay, Pablo F

    2014-02-01

    Autism spectrum disorders (ASD) can be conceptualized as a genetic dysfunction that disrupts development and function of brain circuits mediating social cognition and language. At least some forms of ASD may be associated with high level of excitation in neural circuits, and gamma-aminobutyric acid (GABA) has been implicated in its etiology. Single-nucleotide polymorphisms (SNP) located within the GABA receptor (GABAR) subunit genes GABRA1, GABRG2, GABRB3, and GABRD were screened. A hundred and thirty-six Argentinean ASD patients and 150 controls were studied, and the contribution of the SNPs in the etiology of ASD was evaluated independently and/or through gene-gene interaction using multifactor dimensionality reduction (MDR) method. From the 18 SNP studied, 11 were not present in our Argentinean population (patients and controls) and 1 SNP had minor allele frequency < 0.1%. For the remaining six SNPs, none provided statistical significant association with ASD when considering allelic or genotypic frequencies. Non-significant association with ASD was found for the haplotype analysis. MDR identified evidence for synergy between markers in GABRB3 (chromosome 15) and GABRD (chromosome 1), suggesting potential gene-gene interaction across chromosomes associated with increased risk for autism (testing balanced accuracy: 0.6081 and cross-validation consistency: 10/10, P < 0.001). Considering our Argentinean ASD sample, it can be inferred that GABRB3 would be involved in the etiology of autism through interaction with GABRD. These results support the hypothesis that GABAR subunit genes are involved in autism, most likely via complex gene-gene interactions. PMID:24249596

  17. Mutations in the PCCA gene encoding the {alpha} subunit of propionyl-CoA carboxylase in patients with propionic acidemia

    SciTech Connect

    Campeau, E.; Leon-Del-Rio, A.; Gravel, R.A.

    1994-09-01

    Propionic acidemia is a rare autosomal recessive disorder characterized by a deficiency of the mitochondrial biotin-dependent enzyme, propionyl-CoA carboxylase (PCC). PCC has the structure {alpha}{sub 4}{beta}{sub 4}, with the {alpha} subunit containing the biotin prosthetic group. This study is concerned with defining the spectrum of mutations occurring in the PCCA gene encoding the {alpha} subunit. Mutations were initially assigned to this gene through complementation experiments done after somatic fusion of patient fibroblasts. The analyses were performed on PCR-amplified reverse transcripts of fibroblast RNA. The mutations were identified by single strand conformational polymorphism analysis and direct sequencing of PCR products. Three candidate disease-causing mutations and one DNA polymorphism were identified in the {alpha} subunit sequence in different patients: (1) a 3 bp deletion {triangle}CTG{sub 2058-2060}, which eliminates Cys687 near the biotin binding site (Lys669); (2) T{sub 611}{r_arrow}A which converts Met204 to Lys in a highly conserved region matching that of an ATP binding site; (3) An {approximately}50 bp deletion near the 3{prime} end of the cDNA which likely corresponds to the loss of an exon due to a splicing defect; and (4) a 3 bp insertion, +CAG{sub 2203}, located downstream of the stop codon, which is likely a DNA polymorphism. In order to determine the effect of the Cys687 deletion on the biotinylation of PCC, we expressed the mutation in a 67 amino acid C-terminal fragment of the PCC {alpha} subunit in E. coli in which biotinylation is directed by the bacterial biotin ligase. While the mutant peptide was expressed at about half-normal levels, the biotinylation of the peptide that was present was reduced to only {approximately}20% normal. We suggest, therefore, that the absence of PCC activity due to {triangle}Cys687 results at least in part from defective biotinylation of an unstable protein.

  18. The absence of genes for cytochrome c oxidase and reductase subunits in maxicircle kinetoplast DNA of the respiration-deficient plant trypanosomatid Phytomonas serpens.

    PubMed

    Nawathean, P; Maslov, D A

    2000-08-01

    By completing the sequencing of the maxicircle conserved region in the kinetoplast DNA of Phytomonas serpens, we showed that the genes for subunits I and II (COI and COII) of cytochrome c oxidase in this organism were missing. We had previously shown that the genes for cytochrome c oxidase subunit III and apocytochrome b were also missing. These deletions did not affect the structure or expression of the remaining genes. Partial editing of the mRNA for NADH dehydrogenase subunit 8, previously found in strain IG from insects, was complete in two other strains isolated from plants. The appearance of a novel maxicircle gene for MURF2 block I gRNA, which substitutes for the gene missing due to the COII gene deletion, may illustrate a general mechanism for the origin of gRNAs. PMID:10975258

  19. Bilaterian phylogeny based on analyses of a region of the sodium-potassium ATPase beta-subunit gene.

    PubMed

    Anderson, Frank E; Córdoba, Alonso J; Thollesson, Mikael

    2004-03-01

    Molecular investigations of deep-level relationships within and among the animal phyla have been hampered by a lack of slowly evolving genes that are amenable to study by molecular systematists. To provide new data for use in deep-level metazoan phylogenetic studies, primers were developed to amplify a 1.3-kb region of the alpha subunit of the nuclear-encoded sodium-potassium ATPase gene from 31 bilaterians representing several phyla. Maximum parsimony, maximum likelihood, and Bayesian analyses of these sequences (combined with ATPase sequences for 23 taxa downloaded from GenBank) yield congruent trees that corroborate recent findings based on analyses of other data sets (e.g., the 18S ribosomal RNA gene). The ATPase-based trees support monophyly for several clades (including Lophotrochozoa, a form of Ecdysozoa, Vertebrata, Mollusca, Bivalvia, Gastropoda, Arachnida, Hexapoda, Coleoptera, and Diptera) but do not support monophyly for Deuterostomia, Arthropoda, or Nemertea. Parametric bootstrapping tests reject monophyly for Arthropoda and Nemertea but are unable to reject deuterostome monophyly. Overall, the sodium-potassium ATPase alpha-subunit gene appears to be useful for deep-level studies of metazoan phylogeny. PMID:15045481

  20. Influence of a cap site element on tissue-restricted expression of the glycoprotein hormone alpha-subunit gene.

    PubMed

    Cox, G S; Xiong, W

    1999-07-14

    Little is known of the transcriptional regulators important for expression of the glycoprotein hormone alpha-subunit (GPHalpha) gene in nonendocrine tumors, which secrete free alpha-subunit at an incidence of 25-80%. Consequently, attempts were made to define cis-regulatory elements and their cognate trans-acting factors that modulate promoter activity in epithelial cell types that do not normally express the glycoprotein hormones. DNA-mediated transient expression of promoter-reporter constructs was used to identify a novel negative regulatory element located at the GPHalpha gene transcription start site. Mutagenesis of this element produced a 2- to 10-fold increase in promoter activity, depending on the particular mutation and the transfected tumor cell line. Electrophoretic mobility shift analysis detected a protein that binds specifically to a DNA motif encompassing the cap site. It was present at different levels in a variety of cell types. Significantly, the degree to which activity of the wild-type promoter was suppressed relative to that of the mutant promoter was proportional to the level of cap site binding protein in the collection of cell lines examined. These results indicate that a negative regulatory element centered at the GPHalpha gene cap site and its cognate DNA-binding protein make a significant contribution to the production of alpha-subunit in a variety of tumor tissues. A detailed understanding of this cis/trans pair may further suggest a mechanism to explain, at least in part, how this gene becomes activated in nonendocrine tumors. PMID:10403838

  1. Step-wise and lineage-specific diversification of plant RNA polymerase genes and origin of the largest plant-specific subunits.

    PubMed

    Wang, Yaqiong; Ma, Hong

    2015-09-01

    Proteins often function as complexes, yet little is known about the evolution of dissimilar subunits of complexes. DNA-directed RNA polymerases (RNAPs) are multisubunit complexes, with distinct eukaryotic types for different classes of transcripts. In addition to Pol I-III, common in eukaryotes, plants have Pol IV and V for epigenetic regulation. Some RNAP subunits are specific to one type, whereas other subunits are shared by multiple types. We have conducted extensive phylogenetic and sequence analyses, and have placed RNAP gene duplication events in land plant history, thereby reconstructing the subunit compositions of the novel RNAPs during land plant evolution. We found that Pol IV/V have experienced step-wise duplication and diversification of various subunits, with increasingly distinctive subunit compositions. Also, lineage-specific duplications have further increased RNAP complexity with distinct copies in different plant families and varying divergence for subunits of different RNAPs. Further, the largest subunits of Pol IV/V probably originated from a gene fusion in the ancestral land plants. We propose a framework of plant RNAP evolution, providing an excellent model for protein complex evolution. PMID:25921392

  2. Suppressor analysis of temperature-sensitive mutations of the largest subunit of RNA polymerase I in Saccharomyces cerevisiae: a suppressor gene encodes the second-largest subunit of RNA polymerase I.

    PubMed Central

    Yano, R; Nomura, M

    1991-01-01

    The SRP3-1 mutation is an allele-specific suppressor of temperature-sensitive mutations in the largest subunit (A190) of RNA polymerase I from Saccharomyces cerevisiae. Two mutations known to be suppressed by SRP3-1 are in the putative zinc-binding domain of A190. We have cloned the SRP3 gene by using its suppressor activity and determined its complete nucleotide sequence. We conclude from the following evidence that the SRP3 gene encodes the second-largest subunit (A135) of RNA polymerase I. First, the deduced amino acid sequence of the gene product contains several regions with high homology to the corresponding regions of the second-largest subunits of RNA polymerases of various origins, including those of RNA polymerase II and III from S. cerevisiae. Second, the deduced amino acid sequence contains known amino acid sequences of two tryptic peptides from the A135 subunit of RNA polymerase I purified from S. cerevisiae. Finally, a strain was constructed in which transcription of the SRP3 gene was controlled by the inducible GAL7 promoter. When this strain, which can grow on galactose but not on glucose, was shifted from galactose medium to glucose medium, a large decrease in the cellular concentration of A135 was observed by Western blot analysis. We have also identified the specific amino acid alteration responsible for suppression by SRP3-1 and found that it is located within the putative zinc-binding domain conserved among the second-largest subunits of eucaryotic RNA polymerases. From these results, it is suggested that this putative zinc-binding domain is in physical proximity to and interacts with the putative zinc-binding domain of the A190 subunit. Images PMID:1990281

  3. Conserved cis-regulatory modules in promoters of genes encoding wheat high-molecular-weight glutenin subunits

    PubMed Central

    Ravel, Catherine; Fiquet, Samuel; Boudet, Julie; Dardevet, Mireille; Vincent, Jonathan; Merlino, Marielle; Michard, Robin; Martre, Pierre

    2014-01-01

    The concentration and composition of the gliadin and glutenin seed storage proteins (SSPs) in wheat flour are the most important determinants of its end-use value. In cereals, the synthesis of SSPs is predominantly regulated at the transcriptional level by a complex network involving at least five cis-elements in gene promoters. The high-molecular-weight glutenin subunits (HMW-GS) are encoded by two tightly linked genes located on the long arms of group 1 chromosomes. Here, we sequenced and annotated the HMW-GS gene promoters of 22 electrophoretic wheat alleles to identify putative cis-regulatory motifs. We focused on 24 motifs known to be involved in SSP gene regulation. Most of them were identified in at least one HMW-GS gene promoter sequence. A common regulatory framework was observed in all the HMW-GS gene promoters, as they shared conserved cis-regulatory modules (CCRMs) including all the five motifs known to regulate the transcription of SSP genes. This common regulatory framework comprises a composite box made of the GATA motifs and GCN4-like Motifs (GLMs) and was shown to be functional as the GLMs are able to bind a bZIP transcriptional factor SPA (Storage Protein Activator). In addition to this regulatory framework, each HMW-GS gene promoter had additional motifs organized differently. The promoters of most highly expressed x-type HMW-GS genes contain an additional box predicted to bind R2R3-MYB transcriptional factors. However, the differences in annotation between promoter alleles could not be related to their level of expression. In summary, we identified a common modular organization of HMW-GS gene promoters but the lack of correlation between the cis-motifs of each HMW-GS gene promoter and their level of expression suggests that other cis-elements or other mechanisms regulate HMW-GS gene expression. PMID:25429295

  4. Enhancement of Lipid Productivity in Oleaginous Colletotrichum Fungus through Genetic Transformation Using the Yeast CtDGAT2b Gene under Model-Optimized Growth Condition

    PubMed Central

    Dey, Prabuddha; Mall, Nikunj; Chattopadhyay, Atrayee; Chakraborty, Monami; Maiti, Mrinal K.

    2014-01-01

    Oleaginous fungi are of special interest among microorganisms for the production of lipid feedstocks as they can be cultured on a variety of substrates, particularly waste lingocellulosic materials, and few fungal strains are reported to accumulate inherently higher neutral lipid than bacteria or microalgae. Previously, we have characterized an endophytic filamentous fungus Colletotrichum sp. DM06 that can produce total lipid ranging from 34% to 49% of its dry cell weight (DCW) upon growing with various carbon sources and nutrient-stress conditions. In the present study, we report on the genetic transformation of this fungal strain with the CtDGAT2b gene, which encodes for a catalytically efficient isozyme of type-2 diacylglycerol acyltransferase (DGAT) from oleaginous yeast Candida troplicalis SY005. Besides the increase in size of lipid bodies, total lipid titer by the transformed Colletotrichum (lipid content ∼73% DCW) was found to be ∼1.7-fold more than the wild type (lipid content ∼38% DCW) due to functional activity of the CtDGAT2b transgene when grown under standard condition of growth without imposition of any nutrient-stress. Analysis of lipid fractionation revealed that the neutral lipid titer in transformants increased up to 1.8-, 1.6- and 1.5-fold compared to the wild type when grown under standard, nitrogen stress and phosphorus stress conditions, respectively. Lipid titer of transformed cells was further increased to 1.7-fold following model-based optimization of culture conditions. Taken together, ∼2.9-fold higher lipid titer was achieved in Colletotrichum fungus due to overexpression of a rate-limiting crucial enzyme of lipid biosynthesis coupled with prediction-based bioprocess optimization. PMID:25375973

  5. Effects of dicer-like endoribonucleases 2 and 4 on infection of Arabidopsis thaliana by cucumber mosaic virus and a mutant virus lacking the 2b counter-defence protein gene.

    PubMed

    Ziebell, Heiko; Carr, John P

    2009-09-01

    In tobacco and Nicotiana benthamiana, limited cross-protection against cucumber mosaic virus strain Fny (Fny-CMV) was provided by prior inoculation with a deletion mutant lacking the 2b silencing-suppressor protein gene (Fny-CMVDelta2b). Cross-protection by Fny-CMVDelta2b did not result from induction of systemic RNA silencing. We investigated whether protection occurs through induction of localized RNA silencing by using Arabidopsis thaliana plants harbouring mutations in genes encoding the dicer-like (DCL) endoribonucleases 2, 3 and 4 involved in antiviral silencing. In wild-type A. thaliana (Col-0) plants, Fny-CMVDelta2b was symptomless and cross-protected against Fny-CMV infection. Cross-protection by Fny-CMVDelta2b against Fny-CMV infection was not abolished in dcl2, dcl3 or dcl4 mutant plants and was strongest in dcl2/4 double mutants, although in these plants and in dcl4 mutants, Fny-CMVDelta2b replicated to high levels and induced strong symptoms. The results suggest that Fny-CMVDelta2b/Fny-CMV cross-protection is not completely dependent on RNA silencing and also involves competition between these viruses. PMID:19474248

  6. Gonadotrophin subunit and GnRH receptor gene expression in the pars distalis of the equine pituitary.

    PubMed

    Townsend, Julie; Westcott, Karen; Tortonese, Domingo J

    2009-02-01

    In the horse, pronounced changes in fertility occur annually in response to photoperiod. However, the mechanisms regulating gonadotrophin synthesis and release in this species remain unclear. Here, we investigated the expression of gonadotrophin subunits and GnRH receptor (GnRH-R) mRNA in the pituitary glands of Thoroughbred horses during the breeding (BS) and non-breeding (NBS) season. Seasonal effects on the prevalence of gonadotrophs in the pars distalis were also examined. GnRH-R and common alpha-, LHbeta- and FSHbeta-subunit mRNA contents were determined by Northern analysis and the prevalence of LH-gonadotrophs assessed by immunohistochemistry in pituitaries from sexually active females (mares) in the BS, and sexually inactive mares in the NBS. These variables were then measured in castrated male horses (geldings). In mares, pituitary content of FSHbeta mRNA was significantly higher in the NBS (P<0.01). Conversely, the content of common alpha-subunit mRNA was significantly higher during the BS (P<0.05). In contrast, GnRH-R and LHbeta mRNA abundance were unaffected by season. Interestingly, whereas no seasonal effects were apparent on the number of LH-gonadotrophs/field, the proportion of LH cells (in relation to all other cells) was higher in BS than NBS animals (P<0.05); this resulted from an increased number of non-gonadotroph cells during the NBS (P<0.05). In geldings, no significant seasonal effects were detected for any of the variables investigated (P>0.05). These results reveal robust seasonal effects on common alpha-subunit and FSHbeta gene expression in the pituitary of the mare, in the absence of detectable changes in the content of LHbeta or GnRH-R mRNA. PMID:19114046

  7. Multi site polyadenylation and transcriptional response to stress of a vacuolar type H+-ATPase subunit A gene in Arabidopsis thaliana

    PubMed Central

    Magnotta, Scot M; Gogarten, Johann Peter

    2002-01-01

    Background Vacuolar type H+-ATPases play a critical role in the maintenance of vacuolar homeostasis in plant cells. V-ATPases are also involved in plants' defense against environmental stress. This research examined the expression and regulation of the catalytic subunit of the vacuolar type H+-ATPase in Arabidopsis thaliana and the effect of environmental stress on multiple transcripts generated by this gene. Results Evidence suggests that subunit A of the vacuolar type H+-ATPase is encoded by a single gene in Arabidopsis thaliana. Genome blot analysis showed no indication of a second subunit A gene being present. The single gene identified was shown by whole RNA blot analysis to be transcribed in all organs of the plant. Subunit A was shown by sequencing the 3' end of multiple cDNA clones to exhibit multi site polyadenylation. Four different poly (A) tail attachment sites were revealed. Experiments were performed to determine the response of transcript levels for subunit A to environmental stress. A PCR based strategy was devised to amplify the four different transcripts from the subunit A gene. Conclusions Amplification of cDNA generated from seedlings exposed to cold, salt stress, and etiolation showed that transcript levels for subunit A of the vacuolar type H+-ATPase in Arabidopsis were responsive to stress conditions. Cold and salt stress resulted in a 2–4 fold increase in all four subunit A transcripts evaluated. Etiolation resulted in a slight increase in transcript levels. All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation. PMID:11985780

  8. Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial

    PubMed Central

    Alton, Eric W F W; Armstrong, David K; Ashby, Deborah; Bayfield, Katie J; Bilton, Diana; Bloomfield, Emily V; Boyd, A Christopher; Brand, June; Buchan, Ruaridh; Calcedo, Roberto; Carvelli, Paula; Chan, Mario; Cheng, Seng H; Collie, D David S; Cunningham, Steve; Davidson, Heather E; Davies, Gwyneth; Davies, Jane C; Davies, Lee A; Dewar, Maria H; Doherty, Ann; Donovan, Jackie; Dwyer, Natalie S; Elgmati, Hala I; Featherstone, Rosanna F; Gavino, Jemyr; Gea-Sorli, Sabrina; Geddes, Duncan M; Gibson, James S R; Gill, Deborah R; Greening, Andrew P; Griesenbach, Uta; Hansell, David M; Harman, Katharine; Higgins, Tracy E; Hodges, Samantha L; Hyde, Stephen C; Hyndman, Laura; Innes, J Alastair; Jacob, Joseph; Jones, Nancy; Keogh, Brian F; Limberis, Maria P; Lloyd-Evans, Paul; Maclean, Alan W; Manvell, Michelle C; McCormick, Dominique; McGovern, Michael; McLachlan, Gerry; Meng, Cuixiang; Montero, M Angeles; Milligan, Hazel; Moyce, Laura J; Murray, Gordon D; Nicholson, Andrew G; Osadolor, Tina; Parra-Leiton, Javier; Porteous, David J; Pringle, Ian A; Punch, Emma K; Pytel, Kamila M; Quittner, Alexandra L; Rivellini, Gina; Saunders, Clare J; Scheule, Ronald K; Sheard, Sarah; Simmonds, Nicholas J; Smith, Keith; Smith, Stephen N; Soussi, Najwa; Soussi, Samia; Spearing, Emma J; Stevenson, Barbara J; Sumner-Jones, Stephanie G; Turkkila, Minna; Ureta, Rosa P; Waller, Michael D; Wasowicz, Marguerite Y; Wilson, James M; Wolstenholme-Hogg, Paul

    2015-01-01

    Summary Background Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis. Methods We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50–90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene–liposome complex or 0·9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. Randomisation was stratified by % predicted FEV1 (<70 vs ≥70%), age (<18 vs ≥18 years), inclusion in the mechanistic substudy, and dosing site (London or Edinburgh). Participants and investigators were masked to treatment allocation. The primary endpoint was the relative change in % predicted FEV1. The primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01621867. Findings Between June 12, 2012, and June 24, 2013, we randomly assigned 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 116 (83%) patients comprised the per-protocol population. We noted a significant, albeit modest, treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3·7%, 95% CI 0·1–7·3; p=0·046). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. We recorded no significant difference in treatment-attributable adverse events between groups. Interpretation Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of

  9. Structural Characterization and Evolutionary Relationship of High-Molecular-Weight Glutenin Subunit Genes in Roegneria nakaii and Roegneria alashanica.

    PubMed

    Zhang, Lujun; Li, Zhixin; Fan, Renchun; Wei, Bo; Zhang, Xiangqi

    2016-01-01

    The Roegneria of Triticeae is a large genus including about 130 allopolyploid species. Little is known about its high-molecular-weight glutenin subunits (HMW-GSs). Here, we reported six novel HMW-GS genes from R. nakaii and R. alashanica. Sequencing indicated that Rny1, Rny3, and Ray1 possessed intact open reading frames (ORFs), whereas Rny2, Rny4, and Ray2 harbored in-frame stop codons. All of the six genes possessed a similar primary structure to known HMW-GS, while showing some unique characteristics. Their coding regions were significantly shorter than Glu-1 genes in wheat. The amino acid sequences revealed that all of the six genes were intermediate towards the y-type. The phylogenetic analysis showed that the HMW-GSs from species with St, StY, or StH genome(s) clustered in an independent clade, varying from the typical x- and y-type clusters. Thus, the Glu-1 locus in R. nakaii and R. alashanica is a very primitive glutenin locus across evolution. The six genes were phylogenetically split into two groups clustered to different clades, respectively, each of the two clades included the HMW-GSs from species with St (diploid and tetraploid species), StY, and StH genomes. Hence, it is concluded that the six Roegneria HMW-GS genes are from two St genomes undergoing slight differentiation. PMID:27447615

  10. Structural Characterization and Evolutionary Relationship of High-Molecular-Weight Glutenin Subunit Genes in Roegneria nakaii and Roegneria alashanica

    PubMed Central

    Zhang, Lujun; Li, Zhixin; Fan, Renchun; Wei, Bo; Zhang, Xiangqi

    2016-01-01

    The Roegneria of Triticeae is a large genus including about 130 allopolyploid species. Little is known about its high-molecular-weight glutenin subunits (HMW-GSs). Here, we reported six novel HMW-GS genes from R. nakaii and R. alashanica. Sequencing indicated that Rny1, Rny3, and Ray1 possessed intact open reading frames (ORFs), whereas Rny2, Rny4, and Ray2 harbored in-frame stop codons. All of the six genes possessed a similar primary structure to known HMW-GS, while showing some unique characteristics. Their coding regions were significantly shorter than Glu-1 genes in wheat. The amino acid sequences revealed that all of the six genes were intermediate towards the y-type. The phylogenetic analysis showed that the HMW-GSs from species with St, StY, or StH genome(s) clustered in an independent clade, varying from the typical x- and y-type clusters. Thus, the Glu-1 locus in R. nakaii and R. alashanica is a very primitive glutenin locus across evolution. The six genes were phylogenetically split into two groups clustered to different clades, respectively, each of the two clades included the HMW-GSs from species with St (diploid and tetraploid species), StY, and StH genomes. Hence, it is concluded that the six Roegneria HMW-GS genes are from two St genomes undergoing slight differentiation. PMID:27447615

  11. Isolation and sequencing of a putative promoter region of the murine G protein beta 1 subunit (GNB1) gene.

    PubMed

    Kitanaka, Junichi; Kitanaka, Nobue; Takemura, Motohiko; Wang, Xiao-Bing; Hembree, Cambria M; Goodman, Nancy L; Uhl, George R

    2002-02-01

    The expression of the heterotrimeric GTP-binding protein beta 1 subunit gene (GNB1) is regulated by psychostimulants such as cocaine and amphetamines. Since the up-regulation appears to be one of the candidate processes of sensitization, it is necessary to elucidate the cellular and molecular mechanism of the GNB1 gene regulation for a better understanding the establishment of sensitization. In the present study, we describe the isolation and nucleotide sequence analysis of the GNB1 gene promoter region. We have isolated approximately 10 kb of the 5'-flanking region of the mouse of GNB1 gene and found potential elements involved in putative transcriptional control of the GNB1, such as AP1, AP2, Sp1, cyclic AMP response element, and nuclear factor kappa B recognition sites, within the sequences 0.3 kb upstream from the putative transcription start site. This region was highly rich in G + C content, but lacked TATA or CATT boxes. Comparing the nucleotide sequence of the cDNA clone with the human genome databases using the BLAST program a region containing putative exon 1 and promoter of the human GNB1 gene in chromosome 1 was found. The cloning and sequence analysis of an extensive portion of the 5'-flanking regulatory region of the GNB1 gene provides new insights into the factors involved in the regulation by psychostimulants of GNB1 expression. PMID:12180136

  12. LHX3 Interacts with Inhibitor of Histone Acetyltransferase Complex Subunits LANP and TAF-1β to Modulate Pituitary Gene Regulation

    PubMed Central

    Witzmann, Frank A.; Rhodes, Simon J.

    2013-01-01

    LIM-homeodomain 3 (LHX3) is a transcription factor required for mammalian pituitary gland and nervous system development. Human patients and animal models with LHX3 gene mutations present with severe pediatric syndromes that feature hormone deficiencies and symptoms associated with nervous system dysfunction. The carboxyl terminus of the LHX3 protein is required for pituitary gene regulation, but the mechanism by which this domain operates is unknown. In order to better understand LHX3-dependent pituitary hormone gene transcription, we used biochemical and mass spectrometry approaches to identify and characterize proteins that interact with the LHX3 carboxyl terminus. This approach identified the LANP/pp32 and TAF-1β/SET proteins, which are components of the inhibitor of histone acetyltransferase (INHAT) multi-subunit complex that serves as a multifunctional repressor to inhibit histone acetylation and modulate chromatin structure. The protein domains of LANP and TAF-1β that interact with LHX3 were mapped using biochemical techniques. Chromatin immunoprecipitation experiments demonstrated that LANP and TAF-1β are associated with LHX3 target genes in pituitary cells, and experimental alterations of LANP and TAF-1β levels affected LHX3-mediated pituitary gene regulation. Together, these data suggest that transcriptional regulation of pituitary genes by LHX3 involves regulated interactions with the INHAT complex. PMID:23861948

  13. Regulation of glutathione S-transferase Ya subunit gene expression: identification of a unique xenobiotic-responsive element controlling inducible expression by planar aromatic compounds.

    PubMed Central

    Rushmore, T H; King, R G; Paulson, K E; Pickett, C B

    1990-01-01

    We have identified a region in the 5' flanking sequence of the glutathione S-transferase (RX:glutathione R-transferase, EC 2.5.1.18) Ya subunit gene that contains a unique xenobiotic-responsive element (XRE). The regulatory region spans nucleotides -722 to -682 of the 5' flanking sequence and is responsible for part of the basal level as well as inducible expression of the Ya subunit gene by planar aromatic compounds such as beta-naphthoflavone (beta-NF) and 3-methyl-cholanthrene. The DNA sequence of this region (beta-NF-responsive element) is distinct from the DNA sequence of the XRE found in the cytochrome P-450 IA1 gene. In addition to the region containing the beta-NF-responsive element, two other regulatory regions of the Ya subunit gene have been identified. One region spans nucleotides -867 to -857 and has a DNA sequence with identity to the hepatocyte nuclear factor 1 recognition motif found in several liver-specific genes. The second region spans nucleotides -908 to -899 and contains a DNA sequence with identity to the XRE found in the cytochrome P-450 IA1 gene. The XRE sequence also contributes to part of the responsiveness of the Ya subunit gene to planar aromatic compounds. Our data suggest that regulation of gene expression by planar aromatic compounds can be mediated by a DNA sequence that is distinct from the XRE sequence. Images PMID:2160079

  14. Interferon alpha2b gene delivery using adenoviral vector causes inhibition of tumor growth in xenograft models from a variety of cancers.

    PubMed

    Iqbal Ahmed, C M; Johnson, D E; Demers, G W; Engler, H; Howe, J A; Wills, K N; Wen, S F; Shinoda, J; Beltran, J; Nodelman, M; Machemer, T; Maneval, D C; Nagabhushan, T L; Sugarman, B J

    2001-10-01

    A recombinant adenovirus expressing human interferon alpha2b driven by the cytomegalovirus promoter, IACB, was shown to produce and secrete biologically active protein in vitro and in vivo. Intravenous administration of IACB in Buffalo rats resulted in circulating levels of biologically active human interferon at 70,000 international units/mL for up to 15 days. Distribution of interferon protein after IACB administration was different from that seen with the subcutaneous delivery of interferon protein. Higher levels of interferon protein were observed in liver and spleen after IACB delivery compared to protein delivery. The antitumor efficacy of IACB, as measured by suppression of tumor growth, was tested in athymic nude mice bearing established human tumor xenografts from different types of human cancer. Subcutaneous tumors most responsive to the intratumoral administration of IACB ranked as U87MG (glioblastoma) and K562 (chronic myelogenous leukemia), followed by Hep 3B (hepatocellular carcinoma) and LN229 cells (glioblastoma). Intravenous administration of IACB in animals bearing U87MG or Hep 3B xenografts was also effective in suppressing tumor growth, although to a lesser extent than the intratumoral administration. IACB was also tested in a metastatic model in beige/SCID mice generated with H69 (small cell lung carcinoma) cells and was found to prolong survival in tumor-bearing animals. This suggested that interferon gene delivery can be effective in suppressing tumor growth in a wide variety of cells. PMID:11687902

  15. Physical linkage of the human growth hormone gene cluster and the skeletal muscle sodium channel {alpha}-subunit gene (SCN4A) on chromosome 17

    SciTech Connect

    Bennani-Baiti, I.M.; Jones, B.K.; Liebhaber, S.A.; Cooke, N.E.

    1995-10-10

    The human growth hormone (GH) locus, a cluster of five genes, spans 47 kb on chromosome 17q22-q24. The skeletal muscle sodium channel {alpha}-subunit locus (SCN4A), a 32.5-kb gene, has previously been mapped to 17q23.1-q25.3. We demonstrate that both the GH gene cluster and the SCN4A gene colocalize to a single 525-kb yeast artificial chromosome (YAC) containing DNA derived from human chromosome 17. Restriction maps of two cosmids encompassing the 5{prime} terminus of the GH locus and including up to 40 kb of 5{prime}-flanking sequences demonstrate a perfect 20-kb overlap with previously published maps of the SCN4A gene. A 720-bp DNA segment, encompassing sequences 32.3 to 31.6 kb 5{prime} to GH, was sequenced and found to be identical to exon 14 of SCN4A. These data demonstrate that the SCN4A gene and the entire GH gene cluster are contained within 100 kb on chromosome 17 and are separated by only 21.5 kb. Remarkably, this physical linkage between GH and SCN4A also reveals that multiple elements critical to tissue-specific transcriptional activation of the GH gene lie within the SCN4A gene. 48 refs., 5 figs.

  16. Cloning of Genes Coding for the Three Subunits of Thiocyanate Hydrolase of Thiobacillus thioparus THI 115 and Their Evolutionary Relationships to Nitrile Hydratase

    PubMed Central

    Katayama, Yoko; Matsushita, Yasuhiko; Kaneko, Miyuki; Kondo, Mai; Mizuno, Tadayoshi; Nyunoya, Hiroshi

    1998-01-01

    Thiocyanate hydrolase is a newly found enzyme from Thiobacillus thioparus THI 115 that converts thiocyanate to carbonyl sulfide and ammonia (Y. Katayama, Y. Narahara, Y. Inoue, F. Amano, T. Kanagawa, and H. Kuraishi, J. Biol. Chem. 267:9170–9175, 1992). We have cloned and sequenced the scn genes that encode the three subunits of the enzyme. The scnB, scnA, and scnC genes, arrayed in this order, contained open reading frames encoding sequences of 157, 126, and 243 amino acid residues, respectively, for the β, α, and γ subunits, respectively. Each open reading frame was preceded by a typical Shine-Dalgarno sequence. The deduced amino-terminal peptide sequences for the three subunits were in fair agreement with the chemically determined sequences. The protein molecular mass calculated for each subunit was compatible with that determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From a computer analysis, thiocyanate hydrolase showed significant homologies to bacterial nitrile hydratases known to convert nitrile to the corresponding amide, which is further hydrolyzed by amidase to form acid and ammonia. The two enzymes were homologous over regions corresponding to almost the entire coding regions of the genes: the β and α subunits of thiocyanate hydrolase were homologous to the amino- and carboxyl-terminal halves of the β subunit of nitrile hydratase, and the γ subunit of thiocyanate hydrolase was homologous to the α subunit of nitrile hydratase. Comparisons of the catalytic properties of the two homologous enzymes support the model for the reaction steps of thiocyanate hydrolase that was previously presented on the basis of biochemical analyses. PMID:9573140

  17. The loss of histone H3 lysine 9 acetylation due to dSAGA-specific dAda2b mutation influences the expression of only a small subset of genes

    PubMed Central

    Zsindely, Nóra; Pankotai, Tibor; Újfaludi, Zsuzsanna; Lakatos, Dániel; Komonyi, Orbán; Bodai, László; Tora, László; Boros, Imre M.

    2009-01-01

    In Drosophila, the dADA2b-containing dSAGA complex is involved in histone H3 lysine 9 and 14 acetylation. Curiously, although the lysine 9- and 14-acetylated histone H3 levels are drastically reduced in dAda2b mutants, these animals survive until a late developmental stage. To study the molecular consequences of the loss of histone H3 lysine 9 and 14 acetylation, we compared the total messenger ribonucleic acid (mRNA) profiles of wild type and dAda2b mutant animals at two developmental stages. Global gene expression profiling indicates that the loss of dSAGA-specific H3 lysine 9 and 14 acetylation results in the expression change (up- or down-regulation) of a rather small subset of genes and does not cause a general transcription de-regulation. Among the genes up-regulated in dAda2b mutants, particularly high numbers are those which play roles in antimicrobial defense mechanisms. Results of chromatin immunoprecipitation experiments indicate that in dAda2b mutants, the lysine 9-acetylated histone H3 levels are decreased both at dSAGA up- and down-regulated genes. In contrast to that, in the promoters of dSAGA-independent ribosomal protein genes a high level of histone H3K9ac is maintained in dAda2b mutants. Our data suggest that by acetylating H3 at lysine 9, dSAGA modifies Pol II accessibility to specific promoters differently. PMID:19740772

  18. Mapping of the gene for the p60 subunit of the human chromatin assembly factor (CAF1A) to the Down syndrome region of chromosome 21

    SciTech Connect

    Blouin, J.L.; Gos, A.; Morris, M.A.; Antonarakis, S.E.

    1996-04-15

    Exon trapping was used to clone portions of genes from the Down syndrome critical region (DSCR) of human chromosome 21. One trapped sequence showed complete homology with nucleotide sequence U20980 (GenBank), which corresponds to the gene for the p60 subunit of the human chromatin assembly factor-1 (CAF1A). We mapped this gene to human chromosome 21 by fluorescence in situ hybridization, by the use of somatic cell hybrids, and by hybridization to chromosome 21-specific YACs and cosmids. The CAF1A gene localizes to YACs 745H11 and 230E8 of the Chumakov et al. YAC contig, within the DSCR on 21q22. This CAF1A, which belongs to the WD-motif family of genes and interacts with other polypeptide subunits to promote assembly of histones to replicating DNA, may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome. 22 refs., 1 fig.

  19. Cloning of the cDNAs for the small subunits of bovine and human DNA polymerase {delta} and chromosomal location of the human gene (POLD2)

    SciTech Connect

    Zhang, Jian; Tan, Cheng-Keat; Downey, K.M.

    1995-09-01

    cDNAs encoding the small subunit of bovine and human DNA polymerase {delta} have been cloned and sequenced. The predicted polypeptides, 50,885 and 51,289 Daltons, respectively, are 94% identical, similar to the catalytic subunits. The high degree of conservation of the polypeptides suggests an essential function for the small subunit in the heterodimeric core enzyme. Although the catalytic subunit of DNA polymerase 5 shares significant homology with those of the herpes virus family of DNA polymerases, the small subunit of mammalian DNA polymerase 6 is not homologous to the small subunit of either herpes simplex virus type 1 DNA polymerase (UL42 protein) or the Epstein-Barr virus DNA polymerase (BMRF1 protein). Searches of the protein databases failed to detect significant homology with any protein sequenced thus far. PCR analysis of DNA from a panel of human-hamster hybrid cell lines localized the gene (POLD2) for the small subunit of DNA polymerase 5 to human chromosome 7. 45 refs., 2 figs., 2 tabs.

  20. Gene Expression Analysis of the 26S Proteasome Subunit PSMB4 Reveals Significant Upregulation, Different Expression and Association with Proliferation in Human Pulmonary Neuroendocrine Tumours

    PubMed Central

    Mairinger, Fabian Dominik; Walter, Robert Fred Henry; Theegarten, Dirk; Hager, Thomas; Vollbrecht, Claudia; Christoph, Daniel Christian; Worm, Karl; Ting, Saskia; Werner, Robert; Stamatis, Georgios; Mairinger, Thomas; Baba, Hideo; Zarogoulidis, Konstantinos; Huang, Haidong; Li, Qiang; Tsakiridis, Kosmas; Zarogoulidis, Paul; Schmid, Kurt Werner; Wohlschlaeger, Jeremias

    2014-01-01

    Background: Proteasomal subunit PSMB4 was suggested to be a survival gene in an animal model of hepatocellular carcinoma and in glioblastoma cell lines. In pulmonary adenocarcinoma, a high expression of these genes was found to be associated with poor differentiation and survival. This study investigates the gene expression levels of 26S proteasome subunits in human pulmonary neuroendocrine tumours including typical (TC) and atypical (AC) carcinoid tumours as well as small cell (SCLC) and large cell (LCNEC) neuroendocrine carcinomas. Material and methods: Gene expression levels of proteasomal subunits (PSMA1, PSMA5, PSMB4, PSMB5 and PSMD1) were investigated in 80 neuroendocrine pulmonary tumours (each 20 TC, AC, LCNLC and SCLC) and compared to controls. mRNA levels were determined by using TaqMan assays. Immunohistochemistry on tissue microarrays (TMA) was performed to determine the expression of ki67, cleaved caspase 3 and PSMB4. Results: All proteasomal subunit gene expressions were significantly upregulated in TC, AC, SCLC and LCNEC compared to controls. PSMB4 mRNA is differently expressed between all neuroendocrine tumour subtypes demonstrating the highest expression and greatest range in LCNEC (p=0.043), and is significantly associated with proliferative activity (p=0.039). Conclusion: In line with other 26S proteasomal subunits PSMB4 is significantly increased, but differently expressed between pulmonary neuroendocrine tumours and is associated with the proliferative activity. Unlike in pulmonary adenocarcinomas, no association with biological behaviour was observed, suggesting that increased proteasomal subunit gene expression is a common and probably early event in the tumorigenesis of pulmonary neuroendocrine tumours regardless of their differentiation. PMID:25157275

  1. Localization of an alpha-amanitin resistance mutation in the gene encoding the largest subunit of mouse RNA polymerase II.

    PubMed Central

    Bartolomei, M S; Corden, J L

    1987-01-01

    RNA polymerase II is inhibited by the mushroom toxin alpha-amanitin. A mouse BALB/c 3T3 cell line was selected for resistance to alpha-amanitin and characterized in detail. This cell line, designated A21, was heterozygous, possessing both amanitin-sensitive and -resistant forms of RNA polymerase II; the mutant form was 500 times more resistant to alpha-amanitin than the sensitive form. By using the wild-type mouse RNA polymerase II largest subunit (RPII215) gene (J.A. Ahearn, M.S. Bartolomei, M. L. West, and J. L. Corden, submitted for publication) as the probe, RPII215 genes were isolated from an A21 genomic DNA library. The mutant allele was identified by its ability to transfer amanitin resistance in a transfection assay. Genomic reconstructions between mutant and wild-type alleles localized the mutation to a 450-base-pair fragment that included parts of exons 14 and 15. This fragment was sequenced and compared with the wild-type sequence; a single AT-to-GC transition was detected at nucleotide 6819, corresponding to an asparagine-to-aspartate substitution at amino acid 793 of the predicted protein sequence. Knowledge of the position of the A21 mutation should facilitate the study of the mechanism of alpha-amanitin resistance. Furthermore, the A21 gene will be useful for studying the phenotype of site-directed mutations in the RPII215 gene. Images PMID:3821724

  2. Modeling RNA polymerase competition: the effect of σ-subunit knockout and heat shock on gene transcription level

    PubMed Central

    2011-01-01

    Background Modeling of a complex biological process can explain the results of experimental studies and help predict its characteristics. Among such processes is transcription in the presence of competing RNA polymerases. This process involves RNA polymerases collision followed by transcription termination. Results A mathematical and computer simulation model is developed to describe the competition of RNA polymerases during genes transcription on complementary DNA strands. E.g., in the barley Hordeum vulgare the polymerase competition occurs in the locus containing plastome genes psbA, rpl23, rpl2 and four bacterial type promoters. In heat shock experiments on isolated chloroplasts, a twofold decrease of psbA transcripts and even larger increase of rpl23-rpl2 transcripts were observed, which is well reproduced in the model. The model predictions are in good agreement with virtually all relevant experimental data (knockout, heat shock, chromatogram data, etc.). The model allows to hypothesize a mechanism of cell response to knockout and heat shock, as well as a mechanism of gene expression regulation in presence of RNA polymerase competition. The model is implemented for multiprocessor platforms with MPI and supported on Linux and MS Windows. The source code written in C++ is available under the GNU General Public License from the laboratory website. A user-friendly GUI version is also provided at http://lab6.iitp.ru/en/rivals. Conclusions The developed model is in good agreement with virtually all relevant experimental data. The model can be applied to estimate intensities of binding of the holoenzyme and phage type RNA polymerase to their promoters using data on gene transcription levels, as well as to predict characteristics of RNA polymerases and the transcription process that are difficult to measure directly, e.g., the intensity (frequency) of holoenzyme binding to the promoter in correlation to its nucleotide composition and the type of σ-subunit, the

  3. Multi-target parallel processing approach for gene-to-structure determination of the influenza polymerase PB2 subunit.

    PubMed

    Armour, Brianna L; Barnes, Steve R; Moen, Spencer O; Smith, Eric; Raymond, Amy C; Fairman, James W; Stewart, Lance J; Staker, Bart L; Begley, Darren W; Edwards, Thomas E; Lorimer, Donald D

    2013-01-01

    Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year (1). Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans (2). Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target. The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design. Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains. PMID:23851357

  4. Intronic Polymorphisms in the CDKN2B-AS1 Gene Are Strongly Associated with the Risk of Myocardial Infarction and Coronary Artery Disease in the Saudi Population

    PubMed Central

    AbdulAzeez, Sayed; Al-Nafie, Awatif N.; Al-Shehri, Abdullah; Borgio, J. Francis; Baranova, Ekaterina V.; Al-Madan, Mohammed S.; Al-Ali, Rudaynah A.; Al-Muhanna, Fahad; Al-Ali, Abdullah; Al-Mansori, Mohammed; Ibrahim, Mohammed Fakhry; Asselbergs, Folkert W.; Keating, Brendan; Koeleman, Bobby P. C.; Al-Ali, Amein K.

    2016-01-01

    Recent genome-wide association studies identified single nucleotide polymorphisms (SNPs) on the chromosome 9p21.3 conferring the risk for CAD (coronary artery disease) in individuals of Caucasian ancestry. We performed a genetic association study to investigate the effect of 12 candidate SNPs within 9p21.3 locus on the risk of CAD in the Saudi population of the Eastern Province of Saudi Arabia. A total of 250 Saudi CAD patients who had experienced an myocardial infarction (MI) and 252 Saudi age-matched healthy controls were genotyped using TaqMan assay. Controls with evidenced lack of CAD provided 90% of statistical power at the type I error rate of 0.05. Five percent of the results were rechecked for quality control using Sanger sequencing, the results of which concurred with the TaqMan genotyping results. Association analysis of 12 SNPs indicated a significant difference in the genotype distribution for four SNPs between cases and controls (rs564398 p = 0.0315, χ2 = 4.6, odds ratio (OD) = 1.5; rs4977574 p = 0.0336, χ2 = 4.5, OD = 1.4; rs2891168 p = 1.85 × 10 − 10, χ2 = 40.6, OD = 2.1 and rs1333042 p = 5.14 × 10 − 9, χ2 = 34.1, OD = 2.2). The study identified three protective haplotypes (TAAG p = 1.00 × 10 − 4; AGTA p = 0.022 and GGGCC p = 0.0175) and a risk haplotype (TGGA p = 2.86 × 10 − 10) for the development of CAD. This study is in line with others that indicated that the SNPs located in the intronic region of the CDKN2B-AS1 gene are associated with CAD. PMID:26999117

  5. Isolation, characterization, and tissue-specific expression of GABA A receptor α1 subunit gene of Carassius auratus gibelio after avermectin treatment.

    PubMed

    Zhao, Yini; Sun, Qi; Hu, Kun; Ruan, Jiming; Yang, Xianle

    2016-02-01

    Carassius auratus gibelio has been widely cultivated in fish farms in China, with avermectin (AVM) being used to prevent parasite infection. Recently, AVM was found to pass through the Carassius auratus gibelio blood-brain barrier (BBB). Although AVM acts mainly through a GABA receptor and specifically the α1 subunit gene, the most common isoform of the GABA A receptor, which is widely expressed in brain neurons and has been studied in other fish, Carassius auratus gibelio GABA A receptor α1 subunit gene cloning, and whether AVM passes through the BBB to induce Carassius auratus gibelio GABA A receptor α1 subunit gene expression have not been studied. The aim of this study was to clone, sequence, and phylogenetically analyze the GABA A receptor α1 subunit gene and to investigate the correlation of its expression with neurotoxicity in brain, liver, and kidney after AVM treatment by quantitative real-time reverse transcription polymerase chain reaction. The α1 subunit gene was 1550 bp in length with an open reading frame of 1380 bp encoding a predicted protein with 459 amino acid residues. The gene contained 128 bp of 5' terminal untranslated region (URT) and 72 bp of 3' terminal UTR. The α1 subunit structural features conformed to the Cys-loop ligand-gated ion channels family, which includes a signal peptide, an extracellular domain at the N-terminal, and four transmembrane domains. The established phylogenetic tree indicated that the α1 subunits of Carassius auratus gibelio and Danio rerio were the most closely related to each other. The α1 subunit was found to be highly expressed in brain and ovary, and the α1 mRNA transcription level increased significantly in brain. Moreover, the higher the concentration of AVM was, the higher the GABA A receptor expression was, indicating that AVM can induce significant neurotoxicity to Carassius auratus gibelio. Therefore, the α1 subunit mRNA expression was positively correlated with the neurotoxicity of AVM in

  6. Isolation and gene expression of yellow grouper ferritin heavy chain subunit after lipopolysaccharide treatment.

    PubMed

    Wang, Li; Wei, Yong

    2012-06-01

    Ferritin is a ubiquitous and conserved iron storage protein that plays a central role in iron metabolism. The ferritin heavy chain subunit (FerH) homolog was isolated from yellow grouper (Epinephelus awoara) spleen using suppression subtractive hybridization and RACE-PCR. The nucleotide sequence of FerH full-length cDNA was 1173 bp and contained an open reading frame of 534 bp, encoding a putative protein of 177 amino acids. The encoded protein shows 78-94% identity with homologs. Based on phylogenetic analysis, yellow grouper FerH is highly conserved throughout evolution and is closer to European seabass than to other species. RT-PCR analysis demonstrated that FerH was widely expressed in various healthy tissues and significantly up-regulated in liver, spleen, and anterior kidney by lipopolysaccharide. The results suggest that yellow grouper FerH may play a role in immune response. PMID:22210544

  7. Multiple Rieske genes in prokaryotes: exchangeable Rieske subunits in the cytochrome bc-complex of Rubrivivax gelatinosus.

    PubMed

    Ouchane, Soufian; Nitschke, Wolfgang; Bianco, Pierre; Vermeglio, André; Astier, Chantal

    2005-07-01

    Bacterial cytochrome bc1-complex encoded by the petABC operon consists of three subunits, the Rieske iron-sulphur protein, the b-type cytochrome, and the c1-type cytochrome. Disruption of the petA gene of Rubrivivax gelatinosus is not lethal under photosynthetic growth conditions. However, deletion of both petA and petB results in a photosynthesis-deficient strain, suggesting the presence of a second gene encoding a Rieske protein and rescuing a functional cytochrome bc1-complex in the PETA1 mutant. The corresponding petA2 gene was identified and the PETA2 mutant could also grow under photosynthetic conditions. The double mutant PETA12, however, was unable to grow photosynthetically. The presence of a photo-induced cyclic electron transfer was tested by monitoring the kinetics of cytochrome photo-oxidation on intact cells; the data confirm the capacity of petA2 to replace petA1 in the bc1-complex to support photosynthesis. Soluble forms of both PetA1 and PetA2 Rieske proteins were purified from Escherichia coli and found to contain correctly inserted [2Fe-2S] clusters. Electron paramagnetic resonance (EPR) spectroscopy and midpoint potential measurements showed typical [2Fe-2S] signals and E(m) values of +275 mV for both Rieske proteins. The high amino acid sequence similarity and the obtained midpoint potential values argue for a functional role of these proteins in the cytochrome bc1-complex. The presence of duplicated Rieske genes is not restricted to R. gelatinosus. Phylogenetic trees of Rieske genes from Rubrivivax and other proteobacteria as well as from cyanobacteria were reconstructed. On the basis of the phylogenetic analyses, differing evolutionary origins of duplicated Rieske genes in proteo- and cyanobacteria are proposed. PMID:15948965

  8. Isolation, characterization, and expression of the gene encoding the beta subunit of the mitochondrial processing peptidase from Blastocladiella emersonii.

    PubMed

    Costa Rocha, C R; Lopes Gomes, S

    1998-08-01

    A 2.3-kb BamHI-KpnI fragment was isolated from a partial genomic library and shown by nucleotide sequence analysis to contain the entire coding region of the gene encoding the beta subunit of the Blastocladiella mitochondrial processing peptidase (beta-MPP). The predicted beta-MPP protein has 465 amino acids and a calculated molecular mass of 50.8 kDa. S1 nuclease protection assays revealed an intron, 209 bp in size, interrupting the coding region between the putative signal sequence and the mature protein. Northern blot analysis showed that beta-MPP mRNA levels decrease significantly during B. emersonii sporulation, reaching basal levels in the zoospore stage. The amount of beta-MPP protein, determined in Western blots, unlike its mRNA, does not vary significantly throughout the fungal life cycle. PMID:9683495

  9. Histone H2B gene cloning, with implication for its function during nuclear shaping in the Chinese mitten crab, Eriocheir sinensis.

    PubMed

    Wu, Jiangli; Mu, Shumei; Guo, Mingshen; Chen, Tingrong; Zhang, Zhaohui; Li, Zhenqiu; Li, Yanqin; Kang, Xianjiang

    2016-01-10

    Spermatogenesis in animals is the process by which male spermatogonia develop into mature spermatozoa. In most taxa, the process involves changes in the basic proteins associated with DNA. Somatic-type histones are partially or totally replaced by transition proteins, which in turn are replaced by protamines producing compact packaging of the genome. Sperm chromatin in the Chinese mitten crab (Eriocheir sinensis) has a noncompacted loosely arranged organization. However, its formation during spermatogenesis is not clear. In this study, a cDNA sequence encoding histone H2B was cloned by polymerase chain reaction amplification, and its recombinant protein was expressed and purified. Protein alignment studies demonstrated that this histone H2B had 80.80%, 95.12%, 80.16%, 91.87%, 81.75%, 77.78% and 99.19% identity with its counterparts in zebrafish, fruit fly, human, prawn, mouse, African clawed frog, and crayfish, respectively. Western blotting indicated that the recombinant protein could be recognized by an anti-H2B antibody and confirmed that histone H2B exists in sperm nuclei. Immunofluorescence demonstrated that histone H2B was present in the nuclei of spermatogonia, spermatocytes, spermatids, and mature spermatozoa. This is the first report that the mature sperm nucleus of E. sinensis contains histone H2B. This work complements a previous study of sperm histones of this species and provides a basis for further study of the noncondensed sperm nuclei of decapod crustaceans. PMID:26343795

  10. Glucagon regulates gluconeogenesis through KAT2B- and WDR5-mediated epigenetic effects

    PubMed Central

    Ravnskjaer, Kim; Hogan, Meghan F.; Lackey, Denise; Tora, Laszlo; Dent, Sharon Y.R.; Olefsky, Jerrold; Montminy, Marc

    2013-01-01

    Circulating pancreatic glucagon is increased during fasting and maintains glucose balance by stimulating hepatic gluconeogenesis. Glucagon triggering of the cAMP pathway upregulates the gluconeogenic program through the phosphorylation of cAMP response element–binding protein (CREB) and the dephosphorylation of the CREB coactivator CRTC2. Hormonal and nutrient signals are also thought to modulate gluconeogenic gene expression by promoting epigenetic changes that facilitate assembly of the transcriptional machinery. However, the nature of these modifications is unclear. Using mouse models and in vitro assays, we show that histone H3 acetylation at Lys 9 (H3K9Ac) was elevated over gluconeogenic genes and contributed to increased hepatic glucose production during fasting and in diabetes. Dephosphorylation of CRTC2 promoted increased H3K9Ac through recruitment of the lysine acetyltransferase 2B (KAT2B) and WD repeat–containing protein 5 (WDR5), a core subunit of histone methyltransferase (HMT) complexes. KAT2B and WDR5 stimulated the gluconeogenic program through a self-reinforcing cycle, whereby increases in H3K9Ac further potentiated CRTC2 occupancy at CREB binding sites. Depletion of KAT2B or WDR5 decreased gluconeogenic gene expression, consequently breaking the cycle. Administration of a small-molecule KAT2B antagonist lowered circulating blood glucose concentrations in insulin resistance, suggesting that this enzyme may be a useful target for diabetes treatment. PMID:24051374

  11. Virus-induced gene silencing of the RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana.

    PubMed

    Nemchinov, Lev G; Boutanaev, Alexander M; Postnikova, Olga A

    2016-01-01

    In eukaryotic cells, RNA polymerase III is highly conserved and transcribes housekeeping genes such as ribosomal 5S rRNA, tRNA and other small RNAs. The RPC5-like subunit is one of the 17 subunits forming RNAPIII and its exact functional roles in the transcription are poorly understood. In this work, we report that virus-induced gene silencing of transcripts encoding a putative RPC5-like subunit of the RNA Polymerase III in a model species Nicotiana benthamiana had pleiotropic effects, including but not limited to severe dwarfing appearance, chlorosis, nearly complete reduction of internodes and abnormal leaf shape. Using transcriptomic analysis, we identified genes and pathways affected by RPC5 silencing and thus presumably related to the cellular roles of the subunit as well as to the downstream cascade of reactions in response to partial loss of RNA Polymerase III function. Our results suggest that silencing of the RPC5L in N. benthamiana disrupted not only functions commonly associated with the core RNA Polymerase III transcripts, but also more diverse cellular processes, including responses to stress. We believe this is the first demonstration that activity of the RPC5 subunit is critical for proper functionality of RNA Polymerase III and normal plant development. PMID:27282827

  12. Virus-induced gene silencing of the RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana

    PubMed Central

    Nemchinov, Lev G.; Boutanaev, Alexander M.; Postnikova, Olga A.

    2016-01-01

    In eukaryotic cells, RNA polymerase III is highly conserved and transcribes housekeeping genes such as ribosomal 5S rRNA, tRNA and other small RNAs. The RPC5-like subunit is one of the 17 subunits forming RNAPIII and its exact functional roles in the transcription are poorly understood. In this work, we report that virus-induced gene silencing of transcripts encoding a putative RPC5-like subunit of the RNA Polymerase III in a model species Nicotiana benthamiana had pleiotropic effects, including but not limited to severe dwarfing appearance, chlorosis, nearly complete reduction of internodes and abnormal leaf shape. Using transcriptomic analysis, we identified genes and pathways affected by RPC5 silencing and thus presumably related to the cellular roles of the subunit as well as to the downstream cascade of reactions in response to partial loss of RNA Polymerase III function. Our results suggest that silencing of the RPC5L in N. benthamiana disrupted not only functions commonly associated with the core RNA Polymerase III transcripts, but also more diverse cellular processes, including responses to stress. We believe this is the first demonstration that activity of the RPC5 subunit is critical for proper functionality of RNA Polymerase III and normal plant development. PMID:27282827

  13. Differential role of PKA catalytic subunits in mediating phenotypes caused by knockout of the Carney complex gene Prkar1a.

    PubMed

    Yin, Zhirong; Pringle, Daphne R; Jones, Georgette N; Kelly, Kimberly M; Kirschner, Lawrence S

    2011-10-01

    The Carney complex is an inherited tumor predisposition caused by activation of the cAMP-dependent protein kinase [protein kinase A (PKA)] resulting from mutation of the PKA-regulatory subunit gene PRKAR1A. Myxomas and tumors in cAMP-responsive tissues are cardinal features of this syndrome, which is unsurprising given the important role played by PKA in modulating cell growth and function. Previous studies demonstrated that cardiac-specific knockout of Prkar1a causes embryonic heart failure and myxomatous degeneration in the heart, whereas limited Schwann cell-specific knockout of the gene causes schwannoma formation. In this study, we sought to determine the role of PKA activation in this phenotype by using genetic means to reduce PKA enzymatic activity. To accomplish this goal, we introduced null alleles of the PKA catalytic subunits Prkaca (Ca) or Prkacb (Cb) into the Prkar1a-cardiac knockout (R1a-CKO) or limited Schwann cell knockout (R1a-TEC3KO) line. Heterozygosity for Prkaca rescued the embryonic lethality of the R1a-CKO, although mice had a shorter than normal lifespan and died from cardiac failure with atrial thrombosis. In contrast, heterozygosity for Prkacb only enabled the mice to survive 1 extra day during embryogenesis. Biochemical analysis indicated that reduction of Ca markedly reduced PKA activity in embryonic hearts, whereas reduction of Cb had minimal effects. In R1a-TEC3KO mice, tumorigenesis was completely suppressed by a heterozygosity for Prkaca, and by more than 80% by heterozygosity for Prkacb. These data suggest that both developmental and tumor phenotypes caused by Prkar1a mutation result from excess PKA activity due to PKA-Ca. PMID:21852354

  14. Cyclic AMP regulation of the human glycoprotein hormone. cap alpha. -subunit gene is mediated by an 18-base-pair element

    SciTech Connect

    Silver, B.J.; Bokar, J.A.; Virgin, J.B.; Vallen, E.A.; Milsted, A.; Nilson, J.H.

    1987-04-01

    cAMP regulates transcription of the gene encoding the ..cap alpha..-subunit of human chorionic gonadotropin (hCG) in the choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, the authors inserted fragments from the 5' flanking region of the ..cap alpha..-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between position -146 and -111. In the absence of cAMP, the ..cap alpha..-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. They localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the ..cap alpha..-subunit gene.

  15. Identification, cDNA Cloning, and Characterization of Luteinizing Hormone Beta Subunit (lhb) Gene in Catla catla.

    PubMed

    Rather, Mohd Ashraf; Bhat, Irfan Ahmad; Sharma, Rupam

    2016-07-01

    Reproductive hormones play a significant role in the gonadal development and gametogenesis process of animals. In the present study luteinizing hormone beta, (lhb) subunit gene was cloned and characterized from the brain of Catla catla. The lhb full-length of cDNA sequence is 629 bp which consists of 43bp 5'-UTR (untranslated region) 447bp, ORF(open reading frame) and 139 bp of 3'-UTR respectively. The coding region of lhb gene encoded a peptide of 148 amino acids. The coding sequence of lhb gene consist of a single N-linked glycosylation site (NET) and 12 cysteine knot residues. Phylogenetic analysis of C. catla Lhβ deduced amino acid sequence showed high similarity with Carassius auratus followed by Gobiocypris rarus. 3D structure Lhβ protein comprises of five β-sheets and six coils/loops. The qPCR results revealed lhb mRNA is mainly expressed in the pituitary, ovary while moderate expression was observed in brain and testis. To best our knowledge, this is the first report on the identification, molecular characterization and structural information regarding luteinizing hormone in Indian major carp. PMID:26980432

  16. Nucleotide sequences of an important functional gene hnRNPA2/B1 from Ailuropoda melanoleuca and Ursus thibetanus mupinensis and its potential value in phylogenetic study.

    PubMed

    Du, Yu-jie; Hou, Yi-ling; Hou, Wan-ru

    2014-01-01

    The cDNA fragments of hnRNPA2/B1 were cloned from the giant panda and black bear using RT-PCR method, which were, respectively, 1029bp and 1026bp in length encoding 343 and 341 amino acids. Analysis indicated the cDNA cloned from the giant panda encoded variant B1 while the cDNA cloned from black bear encoded variant A2. Analyzing the hnRNPA2B1 peptide of the giant panda and black bear, 76 glycine residues and 86 glycine residues were, respectively, found, and moreover, most glycine are concentrated in the latter halves of the hnRNPA2B1 peptides. Functional sites prediction also showed many N-myristoylation sites existed in the glycine-rich domain, which is probably related to the role of telomere maintenance. From base bias and substitution analysis, we can conclude that the ORF of hnRNPA2/B1 biased G while hated C, and transition of the third site did not achieve the level of saturation. Orthology analysis indicated that both the nucleotide sequence and the deduced amino acid sequence showed high identity to other 26 hnRNPA2/B1 sequences from mammals and nonmammals reported. These sequences were used to construct phylogenetic trees employing the NJ method with 1000 bootstrap, and the obtained tree demonstrated similar topology with the classical systematics, which suggested the potential value of hnRNPA2/B1 in phylogenetic analysis. This report will be the first step to the study function of hnRNPA2/B1 in the giant panda and black bear, and will provide a scientific basis to disease surveillance, captive breeding, and conservation of the endangered species. PMID:24588753

  17. Mediator subunit MED1 is a T3-dependent and T3-independent coactivator on the thyrotropin β gene promoter

    SciTech Connect

    Matsui, Keiji; Oda, Kasumi; Mizuta, Shumpei; Ishino, Ruri; Urahama, Norinaga; Hasegawa, Natsumi; Roeder, Robert G.; Ito, Mitsuhiro

    2013-10-11

    Highlights: •MED1 is a bona fide T3-dependent coactivator on TSHB promoter. •Mice with LxxLL-mutant MED1 have attenuated TSHβ mRNA and thyroid hormone levels. •MED1 activates TSHB promoter T3-dependently in cultured cells. •T3-dependent MED1 action is enhanced when SRC1/SRC2 or HDAC2 is downregulated. •MED1 is also a T3-independent GATA2/Pit1 coactivator on TSHB promoter. -- Abstract: The MED1 subunit of the Mediator transcriptional coregulator complex is a nuclear receptor-specific coactivator. A negative feedback mechanism of thyroid-stimulating hormone (TSH, or thyrotropin) expression in the thyrotroph in the presence of triiodothyronine (T3) is employed by liganded thyroid hormone receptor β (TRβ) on the TSHβ gene promoter, where conventional histone-modifying coactivators act as corepressors. We now provide evidence that MED1 is a ligand-dependent positive cofactor on this promoter. TSHβ gene transcription was attenuated in MED1 mutant mice in which the nuclear receptor-binding ability of MED1 was specifically disrupted. MED1 stimulated GATA2- and Pit1-mediated TSHβ gene promoter activity in a ligand-independent manner in cultured cells. MED1 also stimulated transcription from the TSHβ gene promoter in a T3-dependent manner. The transcription was further enhanced when the T3-dependent corepressors SRC1, SRC2, and HDAC2 were downregulated. Hence, MED1 is a T3-dependent and -independent coactivator on the TSHβ gene promoter.

  18. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes

    PubMed Central

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K.; Mayer, Mark L.

    2015-01-01

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species. PMID:26460032

  19. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes.

    PubMed

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K; Mayer, Mark L

    2015-11-01

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species. PMID:26460032

  20. TNF-{alpha} upregulates the A{sub 2B} adenosine receptor gene: The role of NAD(P)H oxidase 4

    SciTech Connect

    St Hilaire, Cynthia; Koupenova, Milka; Carroll, Shannon H.; Smith, Barbara D.; Ravid, Katya

    2008-10-24

    Proliferation of vascular smooth muscle cells (VSMC), oxidative stress, and elevated inflammatory cytokines are some of the components that contribute to plaque formation in the vasculature. The cytokine tumor necrosis factor-alpha (TNF-{alpha}) is released during vascular injury, and contributes to lesion formation also by affecting VSMC proliferation. Recently, an A{sub 2B} adenosine receptor (A{sub 2B}AR) knockout mouse illustrated that this receptor is a tissue protector, in that it inhibits VSMC proliferation and attenuates the inflammatory response following injury, including the release of TNF-{alpha}. Here, we show a regulatory loop by which TNF-{alpha} upregulates the A{sub 2B}AR in VSMC in vitro and in vivo. The effect of this cytokine is mimicked by its known downstream target, NAD(P)H oxidase 4 (Nox4). Nox4 upregulates the A{sub 2B}AR, and Nox inhibitors dampen the effect of TNF-{alpha}. Hence, our study is the first to show that signaling associated with Nox4 is also able to upregulate the tissue protecting A{sub 2B}AR.

  1. Fit 2-B FATHERS.

    ERIC Educational Resources Information Center

    Maiorano, Joseph J.

    2001-01-01

    Fit 2-B FATHERS is a parenting-skills education program for incarcerated adult males. The goals of this program are for participants to have reduced recidivism rates and a reduced risk of their children acquiring criminal records. These goals are accomplished by helping participants become physically, practically, and socially fit for the demands…

  2. The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells

    SciTech Connect

    Watanabe, Kanako; Kanno, Takeshi; Oshima, Tadayuki; Miwa, Hiroto; Tashiro, Chikara; Nishizaki, Tomoyuki

    2008-03-07

    The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-D-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G{sub 1} phase of cell cycling and decreased the proportion in the S/G{sub 2} phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G{sub 1} phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling.

  3. Confirmation of the 2p locus for the mild autosomal recessive lim-girdle muscular dystrophy gene (LGMD2B) in three families allows refinement of the candidate region

    SciTech Connect

    Bashir, R.; Iughetti, P.; Strachan, T.

    1995-05-01

    The mild autosomal recessive limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of muscle diseases. The first gene to be mapped and associated with this phenotype was a locus on 15q geographic isolate. These results have been confirmed in other populations, but it was shown that there is genetic heterogeneity for this form of LGMD. Recently, a second locus has been mapped to chromosome 2p. The confirmation of the mapping of this second locus in LGMD families from different populations is of utmost importance for the positional cloning of this gene (HGMW-approved symbol LGMD2B). In this publication, haplotypes generated from five chromosome 2 markers from all of the known large families linked to chromosome 2p are reported together with the recombinants that show the current most likely location of the LGMD 2B gene. 9 refs., 2 figs., 1 tab.

  4. A cis-active regulatory gene in the mouse: direct demonstration of cis-active control of the rate of enzyme subunit synthesis

    SciTech Connect

    Bernstine, E.G.; Koh, C.

    1980-07-01

    Mouse mitochondrial malic enzyme (L-malate:NADP/sup +/ oxidoreductase (oxaloacetate-decarboxylating), EC1.1.1.40) is a tetrameric protein. Two alleles of the structural gene (Mod-2) are known which code for electrophoretically distinct enzyme subunits: Mod-2/sup a/ and Mod-2/sup b/. A regulatory gene (Mdr-1), closely linked to Mod-2 on chromosome 7, determines the rate of mitochondrial malic enzyme synthesis in brain. Two alleles of Mdr-1 are known: Mdr-1/sup a/ (high activity) and Mdr-1/sup b/ (low activity). By pulse-labeling with (/sup 35/S)methionine, immune precipitation, and isoelectric focusing under dissociating conditions, we have measured the relative rates of synthesis of the two types of enzyme subunit in animals of genotypes Mdr-1/sup a/ Mod-2/sup a//Mdr-1/sup a/ Mod-2/sup b/ and Mdr-1/sup a/ Mod-2/sup a//Mdr-1/sup b/ Mod-2/sup b/. The results show that in the former animals both types of subunit are made at an identical rate, whereas in the latter animals the Mod-2/sup a/ gene product is synthesized at a rate 2.2 times that of the Mod-2/sup b/-coded subunit. Thus we have unambiguously demonstrated that Mdr-1 is cis-active in its control of the expression of the Mod-2 structural gene.

  5. Genes encoding biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric ACCase that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin-carboxyl-carrier protein and CO2 to form carboxybiotin-carbo...

  6. Analysis of the mitochondrial ATP synthase beta-subunit gene in Drosophilidae: structure, transcriptional regulatory features and developmental pattern of expression in Drosophila melanogaster.

    PubMed Central

    Peña, P; Ugalde, C; Calleja, M; Garesse, R

    1995-01-01

    We have cloned and determined the structure of the gene encoding the H(+)-ATP synthase beta subunit in two distantly related Drosophila species, D. melanogaster and D. virilis. The gene contains three exons that are extremely well conserved at the amino acid level, not only in the region encoding the mature protein but also in that encoding the leader peptide. Primer extension analysis indicates that the 5' untranslated region is extremely short, and reveals the presence of multiple initiation sites of transcription in both Drosophila species. The promoters of D. melanogaster and D. virilis H(+)-ATP synthase beta-subunit genes contain a conserved region surrounding the initiation transcription sites. Nucleotide sequence analysis has revealed the absence of canonical TATA and CCAAT boxes and the presence of several putative regulatory elements in both promoter regions, including GAGA, GATA and Ets binding sites. We have analysed the pattern of gene expression during D. melanogaster development. The mRNA is stored in oocytes, and activation of transcription takes place after 10 h of development. The expression of the nuclear-encoded H(+)-ATP synthase beta subunit is strictly coordinated with the expression of subunits 6 and 8 of the same complex that are encoded in the mitochondrial genome. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 8 PMID:8554535

  7. Comparative transcriptional and proteomic profiling of bread wheat cultivar and its derived transgenic line over-expressing a low molecular weight glutenin subunit gene in the endosperm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have carried out a parallel transcriptional and proteomic comparison of seeds from a transformed bread wheat line that over-expresses a transgenic low molecular weight glutenin subunit gene relative to the corresponding non-transformed genotype. Proteomic analyses showed that, during seed develop...

  8. Novel mutation-deletion in the PHOX2B gene of the patient diagnosed with Neuroblastoma, Hirschsprung’s Disease, and Congenital Central Hypoventilation Syndrome (NB-HSCR-CCHS) Cluster

    PubMed Central

    Szymońska, Izabela; Borgenvik, Thore Langfeldt; Karlsvik, Tina Margrethe; Halsen, Anders; Malecki, Bianka Kathryn; Saetre, Sindre Ervik; Jagła, Mateusz; Kruczek, Piotr; Talowska, Anna Madetko; Drabik, Grażyna; Zasada, Magdalena; Malecki, Marek

    2015-01-01

    Introduction Neuroblastoma (NB), Hirschsprung disease (HSCR), Congenital Central Hypoventilation Syndrome (CCHS), clinically referred as the NB-HSCR-CCHS cluster, are genetic disorders linked to mutations in the PHOX2B gene on chromosome 4p12. Specific Aim The specific aim of this project is to define the PHOX2B gene mutations as the genomic basis for the clinical manifestations of the NB-HSCR-CCHS cluster. Patient A one day old male patient presented to the Jagiellonian University Medical College (JUMC), American Children Hospital, neonatal Intensive Care Unit (ICU) due to abdominal distention, vomiting, and severe apneic episodes. With the preliminary diagnosis of the NB-HSCR-CCHS, the blood and tissue samples were acquired from the child, as well as from the child’s parents. All procedures were pursued in accordance with the Declaration of Helsinki, with the patient’s Guardian Informed Consent and the approval from the Institutional Review Board. Genetic/Genomic Methods Karyotyping was analyzed based upon Giemsa banding. The patient’s genomic DNA was extracted from peripheral blood and amplified by polymerase chain reaction. Direct microfluidic Sanger sequencing was performed on the genomic DNA amplicons. These procedures were pursued in addition to the routine clinical examinations and tests. Results G-banding showed the normal 46 XY karyotype. However, genomic sequencing revealed a novel, heterozygous deletion (8 nucleotides: c.699–706, del8) in exon 3 of the PHOX2B gene on chromosome 4. This led to the frame-shift mutation and malfunctioning gene expression product. Conclusion Herein, we report a novel PHOX2B gene mutation in the patient diagnosed with the NB-HSCR-CCHS cluster. The resulting gene expression product may be a contributor to the clinical manifestations of these genetic disorders. It adds to the library of the mutations linked to this syndrome. Consequently, we suggest that screening for the PHOX2B mutations becomes an integral part of

  9. Linkage of TATA-binding protein and proteasome subunit C5 genes in mice and humans reveals synteny conserved between mammals and invertebrates.

    PubMed

    Trachtulec, Z; Hamvas, R M; Forejt, J; Lehrach, H R; Vincek, V; Klein, J

    1997-08-15

    The TATA-binding protein (TBP) is a factor required for the transcription of all classes of eukaryotic genes. Here, we demonstrate that in the mouse the TBP-encoding gene (Tbp) resides next to the proteasomal subunit C5-encoding gene (Psmb1). The genes are located on mouse chromosome 17 in the t complex within the Hybrid sterility 1 (Hst1) region. We demonstrate that the homologous human genes (TBP AND PSMB1) are tightly linked on the long arm of chromosome 6, in a region syntenic with the proximal part of mouse chromosome 17. The mouse Tbp and Psmb1 and the human TBP and PSMB1 genes are transcribed in the opposite orientation. The TATA-binding protein and proteasomal subunit C5 genes are also linked on chromosome III of Caenorhabditis elegans, and together they are linked to other genes whose homologs map to human chromosome 6 and mouse chromosome 17. In the Drosophila genome, the housekeeping TATA-binding protein gene maps close to two other genes with homologs in the mammalian major histocompatibility complex. There thus exists conserved synteny of unrelated genes between mammals and invertebrates. PMID:9286694

  10. Expression of the Native Cholera Toxin B Subunit Gene and Assembly as Functional Oligomers in Transgenic Tobacco Chloroplasts

    PubMed Central

    Daniell, Henry; Lee, Seung-Bum; Panchal, Tanvi; Wiebe, Peter O.

    2012-01-01

    The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1% of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expresssion levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast-synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast- synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of

  11. Establishment of a continuous culture system for Entamoeba muris and analysis of the small subunit rRNA gene.

    PubMed

    Kobayashi, S; Suzuki, J; Takeuchi, T

    2009-06-01

    We established a culture system for Entamoeba muris (MG-EM-01 strain isolated from a Mongolian gerbil) using a modified Balamuth's egg yolk infusion medium supplemented with 4% adult bovine serum and Bacteroides fragilis cocultured with Escherichia coli. Further, encystation was observed in the culture medium. The morphological characteristics of E. muris are similar to those of Entamoeba coli (E. coli); moreover, the malic isoenzyme electrophoretic band, which shows species-specific electrophoretic mobility, of E. muris had almost the same mobility as that observed with the malic isoenzyme electrophorectic band of E. coli (UZG-EC-01 strain isolated from a gorilla). We determined the small subunit rRNA (SSU-rRNA) gene sequence of the MG-EM-01 strain, and this sequence was observed to show 82.7% homology with that of the UZG-EC-01 strain. Further, the resultant phylogenetic tree for molecular taxonomy based on the SSU-rRNA genes of the 21 strains of the intestinal parasitic amoeba species indicated that the MG-EM-01 strain was most closely related to E. coli. PMID:19585892

  12. Sp1 trans-activates the murine H(+)-K(+)-ATPase alpha(2)-subunit gene.

    PubMed

    Yu, Zhiyuan; Li, Mei; Zhang, Dongyu; Xu, William; Kone, Bruce C

    2009-07-01

    The H(+)-K(+)-ATPase alpha(2) (HKalpha2) gene of the renal collecting duct and distal colon plays a central role in potassium and acid-base homeostasis, yet its transcriptional control remains poorly characterized. We previously demonstrated that the proximal 177 bp of its 5'-flanking region confers basal transcriptional activity in murine inner medullary collecting duct (mIMCD3) cells and that NF-kappaB and CREB-1 bind this region to alter transcription. In the present study, we sought to determine whether the -144/-135 Sp element influences basal HKalpha2 gene transcription in these cells. Electrophoretic mobility shift and supershift assays using probes for -154/-127 revealed Sp1-containing DNA-protein complexes in nuclear extracts of mIMCD3 cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that Sp1, but not Sp3, binds to this promoter region of the HKalpha2 gene in mIMCD3 cells in vivo. HKalpha2 minimal promoter-luciferase constructs with point mutations in the -144/-135 Sp element exhibited much lower activity than the wild-type promoter in transient transfection assays. Overexpression of Sp1, but not Sp3, trans-activated an HKalpha2 proximal promoter-luciferase construct in mIMCD3 cells as well as in SL2 insect cells, which lack Sp factors. Conversely, small interfering RNA knockdown of Sp1 inhibited endogenous HKalpha2 mRNA expression, and binding of Sp1 to chromatin associated with the proximal HKalpha2 promoter without altering the binding or regulatory influence of NF-kappaB p65 or CREB-1 on the proximal HKalpha2 promoter. We conclude that Sp1 plays an important and positive role in controlling basal HKalpha2 gene expression in mIMCD3 cells in vivo and in vitro. PMID:19420113

  13. Characterization of two trpE genes encoding anthranilate synthase {alpha}-subunit in Azospirillum brasilense

    SciTech Connect

    Ge Shimei; Xie Baoen; Chen Sanfeng . E-mail: chensf@cau.edu.cn

    2006-03-10

    The previous report from our laboratory has recently identified a new trpE gene (termed trpE {sub 2}) which exists independently in Azospirillum brasilense Yu62. In this study, amplification of trpE(G) (termed trpE {sub 1}(G) here) confirmed that there are two copies of trpE gene, one trpE being fused into trpG while the other trpE existed independently. This is First report to suggest that two copies of the trpE gene exist in this bacterium. Comparison of the nucleotide sequence demonstrated that putative leader peptide, terminator, and anti-terminator were found upstream of trpE {sub 1}(G) while these sequence features did not exist in front of trpE {sub 2}. The {beta}-galactosidase activity of an A. brasilense strain carrying a trpE {sub 2}-lacZ fusion remained constant at different tryptophan concentrations, but the {beta}-galactosidase activity of the same strain carrying a trpE {sub 1}(G)-lacZ fusion decreased as the tryptophan concentration increased. These data suggest that the expression of trpE {sub 1}(G) is regulated at the transcriptional level by attenuation while trpE {sub 2} is constantly expressed. The anthranilate synthase assays with trpE {sub 1}(G){sup -} and trpE {sub 2} {sup -} mutants demonstrated that TrpE{sub 1}(G) fusion protein is feedback inhibited by tryptophan while TrpE{sub 2} protein is not. We also found that both trpE {sub 1}(G) and trpE {sub 2} gene products were involved in IAA synthesis.

  14. The Expression of Tubb2b Undergoes a Developmental Transition in Murine Cortical Neurons.

    PubMed

    Breuss, Martin; Morandell, Jasmin; Nimpf, Simon; Gstrein, Thomas; Lauwers, Mattias; Hochstoeger, Tobias; Braun, Andreas; Chan, Kelvin; Sánchez Guajardo, Edmundo R; Zhang, Lijuan; Suplata, Marek; Heinze, Katrin G; Elsayad, Kareem; Keays, David A

    2015-10-15

    The development of the mammalian brain requires the generation, migration, and differentiation of neurons, cellular processes that are dependent on a dynamic microtubule cytoskeleton. Mutations in tubulin genes, which encode for the structural subunits of microtubules, cause detrimental neurological disorders known as the tubulinopathies. The disease spectra associated with different tubulin genes are overlapping but distinct, an observation believed to reflect functional specification of this multigene family. Perturbation of the β-tubulin TUBB2B is known to cause polymicrogyria, pachygyria, microcephaly, and axon guidance defects. Here we provide a detailed analysis of the expression pattern of its murine homolog Tubb2b. The generation and characterization of BAC-transgenic eGFP reporter mouse lines has revealed that it is highly expressed in progenitors and postmitotic neurons during cortical development. This contrasts with the 8-week-old cortex, in which Tubb2b expression is restricted to macroglia, and expression is almost completely absent in mature neurons. This developmental transition in neurons is mirrored in the adult hippocampus and the cerebellum but is not a universal feature of Tubb2b; its expression persists in a population of postmitotic neurons in the 8-week-old retina. We propose that the dynamic spatial and temporal expression of Tubb2b reflects specific functional requirements of the microtubule cytoskeleton. PMID:26105993

  15. Five subunit genes of the human muscle nicotinic acetylcholine receptor are mapped to two linkage groups on chromosome 2 and 17

    SciTech Connect

    Lobos, E.A. )

    1993-09-01

    RFLPs were detected in the five subunit genes of the human muscle nicotinic acetylcholine receptor (nAChR) using genomic DNA or cDNA probes from the homologous mouse loci. The RFLPs at the alpha-, beta-, gamma-, delta-, and epsilon-subunit gene loci were analyzed for genetic linkage in 16 families (n = 188). Significant evidence was obtained for close linkage of the [beta]- and [epsilon]-nAChR genes and much greater genetic distance between the [alpha]-nAChR gene and the [gamma]/[delta]-nAChR gene complex. The linkage analysis program CRI-MAP was used to map the positions of the [beta]- and [epsilon]-nAChR genes relative to seven markers on chromosome 17. The results indicate the [beta]- and [epsilon]-nAChR probes to a panel of human-hamster somatic cell hybrids. The [alpha]-, [gamma]-, and [delta]-nAChR genes were placed on a map of 13 chromosome 2 markers. The linkage analysis placed the nAChR genes at two sites on chromosome 2q about equidistant from the marker CRYGP1, with the [alpha]-nAChR gene about 27 cM proximal and the [gamma]/[delta]-nAChR gene complex about 31 cM distal to CRYGP1. 46 refs., 2 figs., 5 tabs.

  16. Protective immunity of rAd5/NR2B vaccine against concomitant aversiveness of spontaneous neuropathic pain following spinal nerve ligation injury

    PubMed Central

    Wang, Gong-Ming; Wang, Xiao-Yan; Liu, Guang-Jie; Cheng, Kun; Wang, Hua; Guo, Shou-Gang

    2015-01-01

    Objective: Peripheral nerve injury elicits an aversive state of spontaneous neuropathic pain, and up to now, the modulation of this concomitant aversive state remains a major therapeutic challenge. NMDA receptor subunits NR2B in the rACC are critically involved in the processing of this aversive state and then a strategy targeted at the NR2B subunit might be promising for modulation of the aversive state. Thus, in the present study, using negative reinforcement animal model to reveal spontaneous pain, we investigated the effect of oral immunization with recombinant adenovirus serotype 5-mediated NR2B gene transfer (rAd5/NR2B) on the modulation of the tonic pain. Material and methods: Following oral administration of the rAd5/NR2B vaccine, NR2B-specific antibodies were induced in serum. And the humoral response was involved in the decreased expression of NR2B protein in the rACC. Results: The present study demonstrated that CPP achieved by spinal administration of clonidine in spinal nerve ligation (SNL) rats revealed the presence of aversive state of spontaneous neuropathic pain. Notably, the humoral autoimmune response blocked the CPP by spinal clonidine, suggesting the relief of the concomitant aversive of spontaneous neuropathic pain in the SNL rats. Conclusion: These data proved the feasibility of oral immunization with rAd5/NR2B for modulation of concomitant aversive of spontaneous neuropathic pain due to peripheral nerve injury. PMID:26309509

  17. Candidate gene association studies of the alpha 4 (CHRNA4) and beta 2 (CHRNB2) neuronal nicotinic acetylcholine receptor subunit genes in Alzheimer's disease.

    PubMed

    Cook, Lynnette J; Ho, Luk W; Taylor, Alison E; Brayne, Carol; Evans, John Grimley; Xuereb, John; Cairns, Nigel J; Pritchard, Antonia; Lemmon, Helen; Mann, David; St Clair, David; Turic, Dragana; Hollingworth, Paul; Moore, Pamela J; Jehu, Luke; Archer, Nicola; Walter, Sarah; Foy, Catherine; Edmondson, Amanda; Powell, John; Lovestone, Simon; Owen, Michael J; Williams, Julie; Lendon, Corinne; Rubinsztein, David C

    2004-03-25

    Consistent deficits in the cholinergic system are evident in Alzheimer's disease (AD) patients, including selective loss of alpha4beta2 nicotinic acetylcholine receptors in the brains of AD patients. Knockout mice for the beta2 subunit have impaired neuronal survival in ageing. Accordingly, we have analysed polymorphisms in the genes that encode the alpha4 and beta2 subunits, CHRNA4 and CHRNB2 respectively, for genetic associations with late-onset AD. A significant association for disease was observed for a non-coding polymorphism in CHRNB2 (odds ratio=0.57, 95% confidence interval=0.35-0.95, P=0.024). Replication analysis was performed in two further sample sets. While these did not individually yield significant results, a significant association remained when all samples were pooled (odds ratio=0.70, 95% confidence interval=0.52-0.95, P=0.019). These data suggest that this variant warrants further examination in large case-control series. PMID:15026168

  18. Cloning and structural analysis of the gene for the regulatory subunit of cAMP-dependent protein kinase in Blastocladiella emersonii.

    PubMed

    Marques, M do V; Gomes, S L

    1992-08-25

    We have isolated and characterized cDNA and genomic DNA clones encoding the regulatory subunit of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. Nucleotide sequence analysis has shown that the predicted protein comprises 403 amino acids with a calculated molecular mass of 44,263 Da and an overall 40% identity to mammalian RII subunits, including a serine in the phosphorylation site, which confirms the Blastocladiella protein as a type II regulatory subunit. The B. emersonii R gene presents two introns, one located in the 5'-noncoding region, whereas the other interrupts the coding region, just after the dimerization domain of the protein. The promoter region does not contain recognizable TATA or CCAAT sequences and is very GC rich, a characteristic shared by mammalian cAMP-dependent protein kinase subunit genes previously analyzed. S1 mapping and primer extension experiments revealed multiple transcription initiation sites. Several sequence motifs were identified in the 5'-flanking region which could be responsible for the regulation of this gene. PMID:1512258

  19. Cloning and characterization of the gene for the catalytic subunit of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii.

    PubMed

    de Oliveira, J C; Borges, A C; Marques, M do V; Gomes, S L

    1994-01-15

    We have isolated and characterized cDNA and genomic DNA clones encoding the catalytic subunit (C) of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. The C-subunit amino acid sequence derived from the nucleotide sequence predicts a basic polypeptide of 424 residues, excluding the initiator methionine, which by amino-terminal sequence analysis has been shown to be absent from the mature protein. The Blastocladiella C presents a 70-amino-acid extension at the amino terminus, when aligned to the mouse C alpha subunit, being one of the largest C subunits already characterized. The B. emersonii C-gene-coding region is interrupted by three introns, ranging in size over 57-69 bp. The positions of the introns are quite different from those found in other species, suggesting a considerable amount of evolutionary drift in the gene structure. The 5'-flanking region lacks recognizable TATA or CCAAT sequences, is remarkably high in GC content (70%), and primer extension experiments indicate that transcription initiates from multiple sites. Several sequence motifs were identified in the promoter region which could be involved in the developmental control of this gene. PMID:8307021

  20. [Cloning, expression and characterization of a gene encoding alpha2 subunit of the proteasome in Schistosoma japonicum].

    PubMed

    Hong, Yang; Han, Hongxiao; Peng, Jinbiao; Li, Ye; Shi, Yaojun; Fu, Zhiqiang; Liu, Jinming; Li, Xiangrui; Lin, Jiaojiao

    2010-04-01

    The 26S proteasome is a proteolytic complex responsible for the degradation of the vast majority of eukaryotic proteins. Regulated proteolysis by the proteasome is thought to influence cell cycle progression, transcriptional control, and other critical cellular processes. A novel Schistosoma japonicum gene (GenBank Accession No. AY813725) proteasome alpha2 subunit (SjPSMA2) was cloned. Sequence analysis revealed that the ORF of SjPSMA2 gene contains 708 nucleotides encoding 235 amino acids, and the molecular weight was estimated to be 25.84 kDa. Real-time PCR analysis showed that this gene expressed in 7 d, 13 d, 18 d, 23 d, 32 d and 42 d schistosoma. The mRNA level of SjPSMA2 was lower in 7 d and 23 d schistosomulum than that in other stages. The SjPSMA2 cDNA fragment was subcloned into an expression vector pET28a(+) and transformed into E. coli BL21 (DE3) cells. After induction with IPTCQ the 30 kDa fusion protein was produced as included bodies. Western-blotting revealed that the fusion protein could be recognized by the rabbit serum anti-Schistosoma japonicum adult worm antigen preparation, and the protein in native could be detected. After immunization of BALB/c mice with the fusion protein, the reduction rates of worm counts and liver egg counts were 12.33% and 35.23%. ELISA results revealed that the vaccinated group showed a significant increase in the level of IgG antibody. This study provided an important basis for investigating the regulation mechanism of the proteasome during the development of Schistosoma japonicum. PMID:20575440

  1. Association of AMPK subunit gene polymorphisms with growth, feed intake, and feed efficiency in meat-type chickens.

    PubMed

    Jin, Sihua; Moujahid, El Mostafa El; Duan, Zhongyi; Zheng, Jiawei; Qu, Lujiang; Xu, Guiyun; Yang, Ning; Chen, Sirui

    2016-07-01

    Investigations on regulatory genes of feed intake will provide a rational scientific basis to improve future selection indices for more efficient chickens. In the present study, we investigated the association of 13 previously reported SNPs in the chicken adenosine monophosphate activated protein kinase (AMPK) subunits PRKAB1, PRKAG2, and PRKAG3 genes with body weight (BW), body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) in two distinct yellow meat-type strains. Six SNPs with a very low minor allele frequency were removed by genotype quality control and data filtering. The experimental population comprised 796 pedigreed males from two strains with different genetic backgrounds, 335 chickens from N202 and 461 chickens from N301. BW at 49 (BW49) and 70 days of age (BW70) and FI (from 49 to 70 days of age) were determined individually. BWG and FCR were computed based on BW and FI in the interval between 49 to 70 days. The results indicated that PRKAB1 SNPs rs14094358 and rs14094362 were significantly associated with BW70, BWG, and FI in the N202 strain, and rs14094361 and rs14094363 were significantly associated with FI and FCR in the N301 strain (P < 0.05). In addition, the PRKAG2 SNP rs14133282 showed significant association with FI in N202, and rs13535812 was significantly associated with BW70 in N202 (P < 0.05). Moreover, the PRKAG3 SNP rs13595570 was significantly associated with BW in N202 (P < 0.05), and significantly associated with FI and FCR in N301 (P < 0.05). Additionally, a two-SNP haplotype comprising rs14094361 and rs14094362 in PRKAB1 was significantly associated with BWG in N202 (P < 0.05). Meanwhile, haplotypes based on two SNPs, rs14133282, and rs13535812, showed significant effects on FI in N202 (P < 0.05). Our findings therefore provide important evidence for association of AMPK subunits polymorphisms with body weight, feed intake, and feed efficiency that may be applied in meat-type chicken breeding programs. PMID

  2. The effects of clobazam treatment in rats on the expression of genes and proteins encoding glucronosyltransferase 1A/2B (UGT1A/2B) and multidrug resistance‐associated protein-2 (MRP2), and development of thyroid follicular cell hypertrophy

    SciTech Connect

    Miyawaki, Izuru Tamura, Akitoshi; Matsumoto, Izumi; Inada, Hiroshi; Kunimatsu, Takeshi; Kimura, Juki; Funabashi, Hitoshi

    2012-12-15

    Clobazam (CLB) is known to increase hepatobiliary thyroxine (T4) clearance in Sprague–Dawley (SD) rats, which results in hypothyroidism followed by thyroid follicular cell hypertrophy. However, the mechanism of the acceleration of T4-clearance has not been fully investigated. In the present study, we tried to clarify the roles of hepatic UDP-glucronosyltransferase (UGT) isoenzymes (UGT1A and UGT2B) and efflux transporter (multidrug resistance–associated protein-2; MRP2) in the CLB-induced acceleration of T4-clearance using two mutant rat strains, UGT1A-deficient mutant (Gunn) and MRP2-deficient mutant (EHBR) rats, especially focusing on thyroid morphology, levels of circulating hormones (T4 and triiodothyronine (T3)) and thyroid-stimulating hormone (TSH), and mRNA or protein expressions of UGTs (Ugt1a1, Ugt1a6, and Ugt2b1/2) and MRP2 (Mrp). CLB induced thyroid morphological changes with increases in TSH in SD and Gunn rats, but not in EHBR rats. T4 was slightly decreased in SD and Gunn rats, and T3 was decreased in Gunn rats, whereas these hormones were maintained in EHBR rats. Hepatic Ugt1a1, Ugt1a6, Ugt2b1/2, and Mrp2 mRNAs were upregulated in SD rats. In Gunn rats, UGT1A mRNAs (Ugt1a1/6) and protein levels were quite low, but UGT2B mRNAs (Ugt2b1/2) and protein were prominently upregulated. In SD and Gunn rats, MRP2 mRNA and protein were upregulated to the same degree. These results suggest that MRP2 is an important contributor in development of the thyroid cellular hypertrophy in CLB-treated rats, and that UGT1A and UGT2B work in concert with MRP2 in the presence of MRP2 function to enable the effective elimination of thyroid hormones. -- Highlights: ► Role of UGT and MRP2 in thyroid pathology was investigated in clobazam-treated rats. ► Clobazam induced thyroid cellular hypertrophy in SD and Gunn rats, but not EHBR rats. ► Hepatic Mrp2 gene and protein were upregulated in SD and Gunn rats, but not EHBR rats. ► Neither serum thyroid hormones (T3/T4

  3. Regulation of Aerobic Energy Metabolism in Podospora anserina by Two Paralogous Genes Encoding Structurally Different c-Subunits of ATP Synthase

    PubMed Central

    Sellem, Carole H.; di Rago, Jean-Paul; Lasserre, Jean-Paul; Ackerman, Sharon H.; Sainsard-Chanet, Annie

    2016-01-01

    Most of the ATP in living cells is produced by an F-type ATP synthase. This enzyme uses the energy of a transmembrane electrochemical proton gradient to synthesize ATP from ADP and inorganic phosphate. Proton movements across the membrane domain (FO) of the ATP synthase drive the rotation of a ring of 8–15 c-subunits, which induces conformational changes in the catalytic part (F1) of the enzyme that ultimately promote ATP synthesis. Two paralogous nuclear genes, called Atp9-5 and Atp9-7, encode structurally different c-subunits in the filamentous fungus Podospora anserina. We have in this study identified differences in the expression pattern for the two genes that correlate with the mitotic activity of cells in vegetative mycelia: Atp9-7 is transcriptionally active in non-proliferating (stationary) cells while Atp9-5 is expressed in the cells at the extremity (apex) of filaments that divide and are responsible for mycelium growth. When active, the Atp9-5 gene sustains a much higher rate of c-subunit synthesis than Atp9-7. We further show that the ATP9-7 and ATP9-5 proteins have antagonist effects on the longevity of P. anserina. Finally, we provide evidence that the ATP9-5 protein sustains a higher rate of mitochondrial ATP synthesis and yield in ATP molecules per electron transferred to oxygen than the c-subunit encoded by Atp9-7. These findings reveal that the c-subunit genes play a key role in the modulation of ATP synthase production and activity along the life cycle of P. anserina. Such a degree of sophistication for regulating aerobic energy metabolism has not been described before. PMID:27442014

  4. Cloning and characterization of Sdga gene encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex in Scoparia dulcis.

    PubMed

    Shite, Masato; Yamamura, Yoshimi; Hayashi, Toshimitsu; Kurosaki, Fumiya

    2008-11-01

    A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein alpha-subunits from various biological sources suggested that, unlike in animal cells, classification of Galpha-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Galpha-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level. PMID:18981590

  5. Boeing XF2B-1 (F2B-1)

    NASA Technical Reports Server (NTRS)

    1931-01-01

    Boeing XF2B-1 (F2B-1): Serving as the prototype for the F2B-1 shipboard fighter, the XF2B-1 differed visually in having a pointed spinner and an unbalanced rudder. Like many aircraft of its day, the Boeing model 69 was powered by a Pratt & Whitney Wasp radial engine.

  6. Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone.

    PubMed Central

    Cyert, M S; Thorner, J

    1992-01-01

    By using an assay specific for detection of calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase, this enzyme was purified approximately 5,000-fold from extracts of the yeast Saccharomyces cerevisiae. Cna1p and Cna2p, the products of two yeast genes encoding the catalytic (A) subunits of calcineurin, were major constituents of the purified fraction. A third prominent component of apparent molecular mass 16 kDa displayed several properties, including ability to bind 45Ca2+, that are characteristic of the regulatory (B) subunit of mammalian calcineurin and was recognized by an antiserum raised against bovine calcineurin. These antibodies were used to isolate the structural gene (CNB1) encoding this protein from a yeast expression library in the vector lambda gt11. The nucleotide sequence of CNB1 predicted a polypeptide similar in length and highly related in amino acid sequence (56% identity) to the mammalian calcineurin B subunit. Like its counterpart in higher cells, yeast Cnb1p was myristoylated at its N terminus. Mutants lacking Cnb1p, or all three calcineurin subunits (Cna1p, Cna2p, and Cnb1p), were viable. Extracts of cnb1 delta mutants contained no detectable calcineurin activity, even though Cna1p and Cna2p were present at normal levels, suggesting that the B subunit is required for full enzymatic activity in vitro. As was observed previously for MATa cna1 cna2 double mutants, MATa cnb1 mutants were defective in their ability to recover from alpha-factor-induced growth arrest. Thus, the B subunit also is required for the function of calcineurin in promoting adaptation of haploid yeast cells to pheromone in vivo. Images PMID:1321337

  7. Analysis of the neurofilament heavy subunit (NFH) gene in familial amyotrophic lateral sclerosis

    SciTech Connect

    Rooke, K.; Rouleau, G.A.; Figlewicz, D.A.

    1994-09-01

    Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset, degenerative disorder of the motor neurons in the cortex, brainstem and spinal cord. Approximately 10% of ALS cases are familial (FALS) and are inherited as an age-dependent autosomal dominant trait. Mutations in the Cu/Zn superoxide dismutase (SOD-1) gene on chromosome 21 have been found in a subset of cases. However, for the remaining FALS cases, the etiology is unknown. The abnormal accumulation of neurofilaments in the cell body and proximal axon of motor neurons is a characteristic pathological finding in ALS. Furthermore, aberrant neuronal swellings that closely resemble those found in ALS have been reported in transgenic mice overexpressing NFH. The C-terminal region of NFH contains a unique functional domain with multiple repeats of the amino acids (Lys-Ser-Pro) (KSP) and forms the side-arms which appear, at the level of electron microscopy, to cross-link neurofilaments. Recently, deletions in the DSP repeat domain have been identified in five ALS patients diagnosed as sporadic cases of the disease. Based on these findings, we propose to analyze all 4 exons of the NFH gene for variation in FALS. DNA from 110 FALS cases has been amplified by the polymerase chain reaction (PCR) and analyzed by single strand conformation polymorphism (SSCP) analysis. Exon 2, exon 3 and the KSP repeat domain (part of exon 4) appear normal in all our FALS individuals under several different SSCP conditions. The analysis of exon 1 and the remainder of exon 4 has yet to be completed.

  8. Imprinting in the schizophrenia candidate gene GABRB2 encoding GABA(A) receptor β(2) subunit.

    PubMed

    Pun, F W; Zhao, C; Lo, W-S; Ng, S-K; Tsang, S-Y; Nimgaonkar, V; Chung, W S; Ungvari, G S; Xue, H

    2011-05-01

    Schizophrenia is a complex genetic disorder, the inheritance pattern of which is likely complicated by epigenetic factors yet to be elucidated. In this study, transmission disequilibrium tests with family trios yielded significant differences between paternal and maternal transmissions of the disease-associated single-nucleotide polymorphism (SNP) rs6556547 and its haplotypes. The minor allele (T) of rs6556547 was paternally undertransmitted to male schizophrenic offsprings, and this parent-of-origin effect strongly suggested that GABRB2 is imprinted. 'Flipping' of allelic expression in heterozygotes of SNP rs2229944 (C/T) in GABRB2 or rs2290732 (G/A) in the neighboring GABRA1 was compatible with imprinting effects on gene expression. Clustering analysis of GABRB2 mRNA expressions suggested that imprinting brought about the observed two-tiered distribution of expression levels in controls with heterozygous genotype at the disease-associated SNP rs1816071 (A/G). The deficit of upper-tiered expressions accounted for the lowered expression levels in the schizophrenic heterozygotes. The occurrence of a two-tiered distribution furnished support for imprinting, and also pointed to the necessity of differentiating between two kinds of heterozygotes of different parental origins in disease association studies on GABRB2. Bisulfite sequencing revealed hypermethylation in the neighborhood of SNP rs1816071, and methylation differences between controls and schizophrenia patients. Notably, the two schizophrenia-associated SNPs rs6556547 and rs1816071 overlapped with a CpG dinucleotide, thereby opening the possibility that CpG methylation status of these sites could have an impact on the risk of schizophrenia. Thus multiple lines of evidence pointed to the occurrence of imprinting in the GABRB2 gene and its possible role in the development of schizophrenia. PMID:20404824

  9. Genome-wide association study of plasma levels of polychlorinated biphenyls disclose an association with the CYP2B6 gene in a population-based sample

    PubMed Central

    Ng, Esther; Salihovic, Samira; Monica Lind, P.; Mahajan, Anubha; Syvänen, Anne-Christine; Axelsson, Tomas; Ingelsson, Erik; Lindgren, Cecilia M.; van Bavel, Bert; Morris, Andrew P.; Lind, Lars

    2015-01-01

    Background Polychlorinated biphenyls (PCBs) are a group of man-made environmental pollutants which accumulate in humans with adverse health effects. To date, very little effort has been devoted to the study of the metabolism of PCBs on a genome-wide level. Objectives Here, we conducted a genome-wide association study (GWAS) to identify genomic regions involved in the metabolism of PCBs. Methods Plasma levels of 16 PCBs ascertained in a cohort of elderly individuals from Sweden (n=1016) were measured using gas chromatography–high resolution mass spectrophotometry (GC-HRMS). DNA samples were genotyped on the Infinium Omni Express bead microarray, and imputed up to reference panels from the 1000 Genomes Project. Association testing was performed in a linear regression framework under an additive model. Results Plasma levels of PCB-99 demonstrated genome-wide significant association with single nucleotide polymorphisms (SNPs) mapping to chromosome 19q13.2. The SNP with the strongest association was rs8109848 (p=3.7×10−13), mapping to an intronic region of CYP2B6. Moreover, when all PCBs were conditioned on PCB-99, further signals were revealed for PCBs -74, -105 and -118, mapping to the same genomic region. The lead SNPs were rs8109848 (p=3.8×10−12) for PCB-118, rs4802104 (p=1.4×10−9) for PCB-74 and rs4803413 (p=2.5×10−9) for PCB-105, all of which map to CYP2B6. Conclusions In our study, we found plasma levels of four lower-chlorinated PCBs to be significantly associated with the genetic region mapping to the CYP2B6 locus. These findings show that CYP2B6 is of importance for the metabolism of PCBs in humans, and may help to identify individuals who may be susceptible to PCB toxicity. PMID:25839716

  10. Suppression of a deletion mutation in the gene encoding essential PBP2b reveals a new lytic transglycosylase involved in peripheral peptidoglycan synthesis in Streptococcus pneumoniae D39.

    PubMed

    Tsui, Ho-Ching Tiffany; Zheng, Jiaqi J; Magallon, Ariel N; Ryan, John D; Yunck, Rachel; Rued, Britta E; Bernhardt, Thomas G; Winkler, Malcolm E

    2016-06-01

    In ellipsoid-shaped ovococcus bacteria, such as the pathogen Streptococcus pneumoniae (pneumococcus), side-wall (peripheral) peptidoglycan (PG) synthesis emanates from midcells and is catalyzed by the essential class B penicillin-binding protein PBP2b transpeptidase (TP). We report that mutations that inactivate the pneumococcal YceG-domain protein, Spd_1346 (renamed MltG), remove the requirement for PBP2b. ΔmltG mutants in unencapsulated strains accumulate inactivation mutations of class A PBP1a, which possesses TP and transglycosylase (TG) activities. The 'synthetic viable' genetic relationship between Δpbp1a and ΔmltG mutations extends to essential ΔmreCD and ΔrodZ mutations that misregulate peripheral PG synthesis. Remarkably, the single MltG(Y488D) change suppresses the requirement for PBP2b, MreCD, RodZ and RodA. Structural modeling and comparisons, catalytic-site changes and an interspecies chimera indicate that pneumococcal MltG is the functional homologue of the recently reported MltG endo-lytic transglycosylase of Escherichia coli. Depletion of pneumococcal MltG or mltG(Y488D) increases sphericity of cells, and MltG localizes with peripheral PG synthesis proteins during division. Finally, growth of Δpbp1a ΔmltG or mltG(Y488D) mutants depends on induction of expression of the WalRK TCS regulon of PG hydrolases. These results fit a model in which MltG releases anchored PG glycan strands synthesized by PBP1a for crosslinking by a PBP2b:RodA complex in peripheral PG synthesis. PMID:26933838

  11. Assignment of the gene for the core protein II (UQCRC2) subunit of the mitochondrial cytochrome bc[sub 1] complex to human chromosome 16p12

    SciTech Connect

    Duncan, A.M.V. Kingston General Hospital ); Ozawa, Takayuki; Suzuki, Hiroshi ); Rozen, R. Montreal Children's Hospital )

    1993-11-01

    The mammalian cytochrome be[sub 1] complex (complex III) of the mitochondrial respiratory chain catalyzes electron transfer from ubiquinol to cytochrome c. The complex consists of 10-11 subunits: Core proteins I and II, cytochromes b and c[sub 1], the Rieske iron-sulfur protein, the ubiquinone-binding protein, the hinge protein, and 3-4 subunits of low molecular weight. Cytochrome b is encoded by the mitochondrial genome; the other subunits are encoded by nuclear genes. Both the human cytochrome c[sub 1] and the human ubiquinone-binding protein subunits have been assigned to chromosome 8 by somatic cell hybrid mapping. In this study, the authors used in situ hybridization to map core protein II. In situ hybridization to BrdU-synchronized peripheral blood lymphocytes was performed using the method of Harper and Saunders. Chromosomes were stained with a modified fluorescence, 0.25% Wright's stain procedure. The positions of silver grains directly over or touching well-banded metaphase chromosomes were mapped to an ISCN idiogram. The analysis of the distribution of 200 silver grams following in situ hybridization revealed a significant clustering of grains in the p12 region of chromosome 16. The assignment of the core II subunit to human chromosome 16p12 confirms that it is encoded by the nuclear, rather than the mitochondrial, genome. The identification of a single strong hybridization signal is indicative of one locus with no pseudogenes. 6 refs., 1 fig.

  12. Isolation and properties of Drosophila melanogaster ferritin--molecular cloning of a cDNA that encodes one subunit, and localization of the gene on the third chromosome.

    PubMed

    Charlesworth, A; Georgieva, T; Gospodov, I; Law, J H; Dunkov, B C; Ralcheva, N; Barillas-Mury, C; Ralchev, K; Kafatos, F C

    1997-07-15

    Ferritin was purified from iron-fed Drosophila melanogaster extracts by centrifugation in a gradient of potassium bromide. On polyacrylamide gel electrophoresis, the product showed two protein bands corresponding to the ferritin monomer and dimer. Electrophoresis following dissociation with SDS and 2-mercaptoethanol revealed three strong bands of approximately 25, 26, and 28 kDa. N-terminal amino acid sequences were identical for the 25-kDa and 26-kDa subunits, but different for the 28-kDa subunit. Conserved ferritin PCR primers were used to amplify a 360-bp cDNA product, which was used to isolate a clone from a D. melanogaster cDNA library that contained the complete coding sequence for a ferritin subunit. Additional 5' sequence obtained by the RACE method revealed the presence of a putative iron regulatory element. The PCR product was also used to locate the position of the ferritin subunit gene at region 99F on the right arm of the third chromosome. The deduced amino acid sequence of the D. melanogaster ferritin subunit contained a signal sequence and resembled most closely ferritin of the mosquito Aedes aegypti. The evolution of ferritin sequences is discussed. PMID:9266686

  13. Chronic SSRI stimulation of astrocytic 5-HT2B receptors change multiple gene expressions/editings and metabolism of glutamate, glucose and glycogen: a potential paradigm shift

    PubMed Central

    Hertz, Leif; Rothman, Douglas L.; Li, Baoman; Peng, Liang

    2015-01-01

    It is firmly believed that the mechanism of action of SSRIs in major depression is to inhibit the serotonin transporter, SERT, and increase extracellular concentration of serotonin. However, this undisputed observation does not prove that SERT inhibition is the mechanism, let alone the only mechanism, by which SSRI’s exert their therapeutic effects. It has recently been demonstrated that 5-HT2B receptor stimulation is needed for the antidepressant effect of fluoxetine in vivo. The ability of all five currently used SSRIs to stimulate the 5-HT2B receptor equipotentially in cultured astrocytes has been known for several years, and increasing evidence has shown the importance of astrocytes and astrocyte-neuronal interactions for neuroplasticity and complex brain activity. This paper reviews acute and chronic effects of 5-HT2B receptor stimulation in cultured astrocytes and in astrocytes freshly isolated from brains of mice treated with fluoxetine for 14 days together with effects of anti-depressant therapy on turnover of glutamate and GABA and metabolism of glucose and glycogen. It is suggested that these events are causally related to the mechanism of action of SSRIs and of interest for development of newer antidepressant drugs. PMID:25750618

  14. An atypical 12q24.31 microdeletion implicates six genes including a histone demethylase KDM2B and a histone methyltransferase SETD1B in syndromic intellectual disability.

    PubMed

    Labonne, Jonathan D J; Lee, Kang-Han; Iwase, Shigeki; Kong, Il-Keun; Diamond, Michael P; Layman, Lawrence C; Kim, Cheol-Hee; Kim, Hyung-Goo

    2016-07-01

    Microdeletion syndromes are frequent causes of neuropsychiatric disorders leading to intellectual disability as well as autistic features accompanied by epilepsy and craniofacial anomalies. From comparative deletion mapping of the smallest microdeletion to date at 12q24.31, found in a patient with overlapping clinical features of 12q24.31 microdeletion syndrome, we narrowed the putative critical region to 445 kb containing seven genes, one microRNA, and one non-coding RNA. Zebrafish in situ hybridization and comprehensive transcript analysis of annotated genes in the panels of human organ and brain suggest that these are all candidates for neurological phenotypes excluding the gene HPD. This is also corroborated by synteny analysis revealing the conservation of the order of these six candidate genes between humans and zebrafish. Among them, we propose histone demethylase KDM2B and histone methyltransferase SETD1B as the two most plausible candidate genes involved in intellectual disability, autism, epilepsy, and craniofacial anomalies. These two chromatin modifiers located approximately 224 kb apart were both commonly deleted in six patients, while two additional patients had either KDM2B or SETD1B deleted. The four additional candidate genes (ORAI1, MORN3, TMEM120B, RHOF), a microRNA MIR548AQ, and a non-coding RNA LINC01089 are localized between KDM2B and SETD1B. The 12q24.31 microdeletion syndrome with syndromic intellectual disability extends the growing list of microdeletion syndromes and underscores the causative roles of chromatin modifiers in cognitive and craniofacial development. PMID:27106595

  15. Mutations in Two Genes Encoding Different Subunits of a Receptor Signaling Complex Result in an Identical Disease Phenotype

    PubMed Central

    Paloneva, Juha; Manninen, Tuula; Christman, Grant; Hovanes, Karine; Mandelin, Jami; Adolfsson, Rolf; Bianchin, Marino; Bird, Thomas; Miranda, Roxana; Salmaggi, Andrea; Tranebjærg, Lisbeth; Konttinen, Yrjö; Peltonen, Leena

    2002-01-01

    Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), also known as “Nasu-Hakola disease,” is a globally distributed recessively inherited disease leading to death during the 5th decade of life and is characterized by early-onset progressive dementia and bone cysts. Elsewhere, we have identified PLOSL mutations in TYROBP (DAP12), which codes for a membrane receptor component in natural-killer and myeloid cells, and also have identified genetic heterogeneity in PLOSL, with some patients carrying no mutations in TYROBP. Here we complete the molecular pathology of PLOSL by identifying TREM2 as the second PLOSL gene. TREM2 forms a receptor signaling complex with TYROBP and triggers activation of the immune responses in macrophages and dendritic cells. Patients with PLOSL have no defects in cell-mediated immunity, suggesting a remarkable capacity of the human immune system to compensate for the inactive TYROBP-mediated activation pathway. Our data imply that the TYROBP-mediated signaling pathway plays a significant role in human brain and bone tissue and provide an interesting example of how mutations in two different subunits of a multisubunit receptor complex result in an identical human disease phenotype. PMID:12080485

  16. Redescription of Rhizodomus tagatzi (Ciliophora: Spirotrichea: Tintinnida), based on morphology and small subunit ribosomal RNA gene sequence.

    PubMed

    Saccà, Alessandro; Strüder-Kypke, Michaela C; Lynn, Denis H

    2012-01-01

    Herein, we redescribe a tintinnid ciliate that is most commonly known as Tintinnopsis corniger Hada, 1964; but it has been described several times with different names, specifically Tintinnopsis nudicauda Paulmier, 1997 and Rhizodomus tagatzi Strelkow & Wirketis, 1950. Neotype material was collected from the water column of the coastal saline Lake Faro, a meromictic basin connected to the Straits of Messina, Central Mediterranean. The Lake Faro population is characterized by a hyaline or sparsely agglomerated lorica, which made it possible to observe in detail the basal layer structure, usually concealed by abundant incrusting particles. Along with an improved description of the lorica, we provide novel information, such as the general zooid morphology, the ciliary pattern, and the small subunit rRNA (SSU rRNA) gene sequence. Our phylogenetic analysis, based on the SSU rRNA, groups this species with Tintinnopsis radix, while the first taxonomic study designated it as R. tagatzi, introducing a new genus due to peculiarities in lorica morphology. We conclude that the species should be known as R. tagatzi, the senior synonym for the species. However, we do not transfer any other species to this genus, despite strong molecular similarities. Although it is obvious that the genus Tintinnopsis is in need of a thorough revision, current molecular and cytological information for this genus is too sparse, and the type species has not yet been redescribed with modern methods. PMID:22452414

  17. Prevalent Ciliate Symbiosis on Copepods: High Genetic Diversity and Wide Distribution Detected Using Small Subunit Ribosomal RNA Gene

    PubMed Central

    Guo, Zhiling; Liu, Sheng; Hu, Simin; Li, Tao; Huang, Yousong; Liu, Guangxing; Zhang, Huan; Lin, Senjie

    2012-01-01

    Toward understanding the genetic diversity and distribution of copepod-associated symbiotic ciliates and the evolutionary relationships with their hosts in the marine environment, we developed a small subunit ribosomal RNA gene (18S rDNA)-based molecular method and investigated the genetic diversity and genotype distribution of the symbiotic ciliates on copepods. Of the 10 copepod species representing six families collected from six locations of Pacific and Atlantic Oceans, 9 were found to harbor ciliate symbionts. Phylogenetic analysis of the 391 ciliate 18S rDNA sequences obtained revealed seven groups (ribogroups), six (containing 99% of all the sequences) belonging to subclass Apostomatida, the other clustered with peritrich ciliate Vorticella gracilis. Among the Apostomatida groups, Group III were essentially identical to Vampyrophrya pelagica, and the other five groups represented the undocumented ciliates that were close to Vampyrophrya/Gymnodinioides/Hyalophysa. Group VI ciliates were found in all copepod species but one (Calanus sinicus), and were most abundant among all ciliate sequences obtained, indicating that they are the dominant symbiotic ciliates universally associated with copepods. In contrast, some ciliate sequences were found only in some of the copepods examined, suggesting the host selectivity and geographic differentiation of ciliates, which requires further verification by more extensive sampling. Our results reveal the wide occurrence and high genetic diversity of symbiotic ciliates on marine copepods and highlight the need to systematically investigate the host- and geography-based genetic differentiation and ecological roles of these ciliates globally. PMID:23024768

  18. Molecular characterization of Echinococcus granulosus from Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene.

    PubMed

    Sánchez, Elizabeth; Cáceres, Omar; Náquira, César; Garcia, David; Patiño, Gladys; Silvia, Herrera; Volotão, Aline C; Fernandes, Octavio

    2010-09-01

    Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE) in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10), have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44), sheep (41) and humans (14) from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4). The G1 genotype is considered the most widespread and infectious form of E. granulosus worldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru. PMID:20944997

  19. Departure from neutrality at the mitochondrial NADH dehydrogenase subunit 2 gene in humans, but not in chimpanzees.

    PubMed

    Wise, C A; Sraml, M; Easteal, S

    1998-01-01

    To test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution, nucleotide sequences were determined for the 1041 bp of the NADH dehydrogenase subunit 2 (ND2) gene in 20 geographically diverse humans and 20 common chimpanzees. Contingency tests of neutrality were performed using four mutational categories for the ND2 molecule: synonymous and nonsynonymous mutations in the transmembrane regions, and synonymous and nonsynonymous mutations in the surface regions. The following three topological mutational categories were also used: intraspecific tips, intraspecific interiors, and interspecific fixed differences. The analyses reveal a significantly greater number of nonsynonymous polymorphisms within human transmembrane regions than expected based on interspecific comparisons, and they are inconsistent with a neutral equilibrium model. This pattern of excess nonsynonymous polymorphism is not seen within chimpanzees. Statistical tests of neutrality, such as TAJIMA's D test, and the D and F tests proposed by FU and LI, indicate an excess of low frequency polymorphisms in the human data, but not in the chimpanzee data. This is consistent with recent directional selection, a population bottleneck or background selection of slightly deleterious mutations in human mtDNA samples. The analyses further support the idea that mitochondrial genome evolution is governed by selective forces that have the potential to affect its use as a "neutral" marker in evolutionary and population genetic studies. PMID:9475751

  20. Natural alleles of a proteasome α2 subunit gene contribute to thermotolerance and adaptation of African rice.

    PubMed

    Li, Xin-Min; Chao, Dai-Yin; Wu, Yuan; Huang, Xuehui; Chen, Ke; Cui, Long-Gang; Su, Lei; Ye, Wang-Wei; Chen, Hao; Chen, Hua-Chang; Dong, Nai-Qian; Guo, Tao; Shi, Min; Feng, Qi; Zhang, Peng; Han, Bin; Shan, Jun-Xiang; Gao, Ji-Ping; Lin, Hong-Xuan

    2015-07-01

    Global warming threatens many aspects of human life, for example, by reducing crop yields. Breeding heat-tolerant crops using genes conferring thermotolerance is a fundamental way to help deal with this challenge. Here we identify a major quantitative trait locus (QTL) for thermotolerance in African rice (Oryza glaberrima), Thermo-tolerance 1 (TT1), which encodes an α2 subunit of the 26S proteasome involved in the degradation of ubiquitinated proteins. Ubiquitylome analysis indicated that OgTT1 protects cells from heat stress through more efficient elimination of cytotoxic denatured proteins and more effective maintenance of heat-response processes than achieved with OsTT1. Variation in TT1 has been selected for on the basis of climatic temperature and has had an important role in local adaptation during rice evolution. In addition, we found that overexpression of OgTT1 was associated with markedly enhanced thermotolerance in rice, Arabidopsis and Festuca elata. This discovery may lead to an increase in crop security in the face of the ongoing threat of global warming. PMID:25985140

  1. Mitochondrial cytochrome c oxidase subunit 1 (cox1) gene sequence of Spirocerca lupi (Nematoda, Spirurida): avenues for potential implications.

    PubMed

    Traversa, Donato; Costanzo, Francesca; Iorio, Raffaella; Aroch, Itamar; Lavy, Eran

    2007-05-31

    Canine spirocercosis is a life-threatening parasitosis caused by Spirocerca lupi (Nematoda, Spirurida) that is presently emerging in several countries. This study characterised an informative region within the mitochondrial (mtDNA) gene encoding for the cytochrome c oxidase subunit 1 (cox1) of S. lupi by Polymerase Chain Reaction (PCR)-coupled sequencing. Specimens from five different countries in Europe, Asia and Africa were examined and two different sequence variants of cox1 (i.e. haplotypes) were determined, displaying nucleotidic variation at 6 of 689 positions. All of these positions were invariable among all the parasite individuals from Europe (haplotype 1) and among the African and Asian individuals (haplotype 2), but differed between Europe and Asia/Africa. The S. lupi cox1 sequences were consistent with those of other common Spirurida previously reported at both nucleotidic and phylogenetic levels. This study provides molecular information essential for identification of the nematode, irrespective of its life cycle stage. Crucial implications for the specific molecular diagnosis of clinical spirocercosis and investigation of the evolution, population genetics, ecology and epidemiology of S. lupi are discussed. PMID:17428608

  2. Multiple genes, including a member of the AAA family, are essential for degradation of unassembled subunit 2 of cytochrome c oxidase in yeast mitochondria.

    PubMed

    Nakai, T; Yasuhara, T; Fujiki, Y; Ohashi, A

    1995-08-01

    Cytochrome c oxidase consists of three mitochondrion- and several nucleus-encoded subunits. We previously found that in a mutant of Saccharomyces cerevisiae lacking nucleus-encoded subunit 4 of this enzyme (CoxIV), subunits 2 and 3 (CoxII and CoxIII), both encoded by the mitochondrial DNA, were unstable and rapidly degraded in mitochondria, presumably because the subunits cannot assemble normally. To analyze the molecular machinery involved in this proteolytic pathway, we obtained four mutants defective in the degradation of unassembled CoxII (osd mutants) by screening CoxIV-deficient cells for the accumulation of CoxII. All of the mutants were recessive and were classified into three different complementation groups. Tetrad analyses revealed that the phenotype of each mutant was caused by a single nuclear mutation. These results suggest strongly that at least three nuclear genes (the OSD genes) are required for this degradation system. Interestingly, degradation of CoxIII was not affected in the mutants, implying that the two subunits are degraded by distinct pathways. We also cloned the OSD1 gene by complementation of the temperature sensitivity of osd1-1 mutants with a COXIV+ genetic background on a nonfermentable glycerol medium. We found it to encode a member of a family (the AAA family) of putative ATPases, which proved to be identical to recently described YME1 and YTA11. Immunological analyses revealed that Osd1 protein is localized to the mitochondrial inner membrane. Disruption of the predicted ATP-binding cassette by site-directed mutagenesis eliminated biological activities, thereby underscoring the importance of ATP for function. PMID:7623837

  3. Multiple genes, including a member of the AAA family, are essential for degradation of unassembled subunit 2 of cytochrome c oxidase in yeast mitochondria.

    PubMed Central

    Nakai, T; Yasuhara, T; Fujiki, Y; Ohashi, A

    1995-01-01

    Cytochrome c oxidase consists of three mitochondrion- and several nucleus-encoded subunits. We previously found that in a mutant of Saccharomyces cerevisiae lacking nucleus-encoded subunit 4 of this enzyme (CoxIV), subunits 2 and 3 (CoxII and CoxIII), both encoded by the mitochondrial DNA, were unstable and rapidly degraded in mitochondria, presumably because the subunits cannot assemble normally. To analyze the molecular machinery involved in this proteolytic pathway, we obtained four mutants defective in the degradation of unassembled CoxII (osd mutants) by screening CoxIV-deficient cells for the accumulation of CoxII. All of the mutants were recessive and were classified into three different complementation groups. Tetrad analyses revealed that the phenotype of each mutant was caused by a single nuclear mutation. These results suggest strongly that at least three nuclear genes (the OSD genes) are required for this degradation system. Interestingly, degradation of CoxIII was not affected in the mutants, implying that the two subunits are degraded by distinct pathways. We also cloned the OSD1 gene by complementation of the temperature sensitivity of osd1-1 mutants with a COXIV+ genetic background on a nonfermentable glycerol medium. We found it to encode a member of a family (the AAA family) of putative ATPases, which proved to be identical to recently described YME1 and YTA11. Immunological analyses revealed that Osd1 protein is localized to the mitochondrial inner membrane. Disruption of the predicted ATP-binding cassette by site-directed mutagenesis eliminated biological activities, thereby underscoring the importance of ATP for function. PMID:7623837

  4. The genes encoding the delta subunits of dinitrogenases 2 and 3 are required for mo-independent diazotrophic growth by Azotobacter vinelandii.

    PubMed

    Waugh, S I; Paulsen, D M; Mylona, P V; Maynard, R H; Premakumar, R; Bishop, P E

    1995-03-01

    vnfG and anfG encode the delta subunits of alternative nitrogenases 2 and 3 in Azotobacter vinelandii, respectively. As a first step towards elucidating the role of these subunits, diazotrophic growth and acetylene reduction studies were conducted on mutants containing alterations in the genes encoding these subunits. Mutants containing a stop codon (C36stop) or an in-frame deletion in anfG were unable to grow in N-free, Mo-deficient medium (Anf-). Mutants in which cysteine 36 of AnfG (a residue conserved between VnfG and AnfG) was changed to Ala or Ser were Anf+. Thus, this conserved cysteine is not essential for the function of AnfG in dinitrogenase 3. A mutant with a stop codon in vnfG (C17stop) grew after a lag of 25 h in N-free, Mo-deficient medium containing V2O5. However, a Nif- Anf- strain with this mutation was unable to grow under these conditions. This shows that the vnfG gene product is required for nitrogenase 2-dependent growth. Strains with mutations in vnfG and anfG reduced acetylene to different degrees. This indicates that the delta subunits are not required for acetylene reduction by nitrogenases 2 and 3. PMID:7883707

  5. Neuronal-type alpha-bungarotoxin receptors and the alpha 5-nicotinic receptor subunit gene are expressed in neuronal and nonneuronal human cell lines.

    PubMed Central

    Chini, B; Clementi, F; Hukovic, N; Sher, E

    1992-01-01

    alpha-Bungarotoxin (alpha Bgtx) is a toxin known to interact with muscle nicotinic receptors and with some neuronal nicotinic receptors. We show that alpha Bgtx binding sites are also expressed in nonmuscle and nonneuronal human cells, including small cell lung carcinoma and several epithelial cell lines. These receptors are immunologically related to the alpha Bgtx receptors of unknown function described in the nervous system and in the IMR32 neuroblastoma cell line and are distinct from muscle nicotinic receptors. We have also cloned from IMR32 cells the human alpha 5-nicotinic receptor subunit, which is supposed to participate in the formation of alpha Bgtx receptors. Transcripts corresponding to the alpha 5-subunit gene were found not only in neuroblastoma cells but also in all the cell lines expressing alpha Bgtx receptors, with the exception of the TE671 cell line, whose nicotinic receptor subunits are of the muscle type. We conclude that both alpha Bgtx receptors and the alpha 5-nicotinic subunit gene are not neuron-specific, as previously thought, but are expressed in a number of human cell lines of various origin. Images PMID:1542648

  6. An SNP in Exon 11 of Chicken 5'-AMP-Activated Protein Kinase Gamma 3 Subunit Gene was Associated with Meat Water Holding Capacity.

    PubMed

    Yang, Yunzhou; Xiong, Dan; Yao, Ling; Zhao, Chunjiang

    2016-01-01

    The 5'-Adenosine-monophosphate -activated protein kinase plays a key role in regulating cellular energy homeostasis, and it was reported that nucleotide variants in the genes coding the protein were associated with meat quality. In the present study, genetic variations in the exons of gamma non-catalytic subunit genes of the protein kinase were screened among 284 White Plymouth Rock chickens from 7 families with denaturing gradient gel electrophoresis, and their meat quality traits including drip loss and cooked meat rate which reflect water holding capacity and serum biochemical indices were also measured, and the association between the genotypes and the traits was analyzed with a SAS GLM procedure. Our results showed that there were three G/A nucleotide variants including one in exon 6 of the gamma subunit 2 gene and two in exon 11 of the gamma subunit 3 gene, which resulted in amino acid substitutions V150I, V315 M, and A337 T, respectively. And locus V315 M was associated with water holding capacity significantly (P < 0.05). The studied polymorphic locus has the potential to be used as a genetic marker for poultry breeding work. PMID:26597656

  7. Repeated ketamine administration alters N-methyl-D-aspartic acid receptor subunit gene expression: Implication of genetic vulnerability for ketamine abuse and ketamine psychosis in humans

    PubMed Central

    Xu, Ke; Lipsky, Robert H

    2015-01-01

    For more than 40 years following its approval by the Food and Drug Administration (FDA) as an anesthetic, ketamine, a non-competitive N-methyl-D-aspartic acid (NMDA) receptor antagonist, has been used as a tool of psychiatric research. As a psychedelic drug, ketamine induces psychotic symptoms, cognitive impairment, and mood elevation, which resemble some symptoms of schizophrenia. Recreational use of ketamine has been increasing in recent years. However, little is known of the underlying molecular mechanisms responsible for ketamine-associated psychosis. Recent animal studies have shown that repeated ketamine administration significantly increases NMDA receptor subunit gene expression, in particular subunit 1 (NR1 or GluN1) levels. This results in neurodegeneration, supporting a potential mechanism where up-regulation of NMDA receptors could produce cognitive deficits in chronic ketamine abuse patients. In other studies, NMDA receptor gene variants are associated with addictive behavior. Here, we focus on the roles of NMDA receptor gene subunits in ketamine abuse and ketamine psychosis and propose that full sequencing of NMDA receptor genes may help explain individual vulnerability to ketamine abuse and ketamine-associated psychosis. PMID:25245072

  8. Crystal structure of eukaryotic translation initiation factor 2B.

    PubMed

    Kashiwagi, Kazuhiro; Takahashi, Mari; Nishimoto, Madoka; Hiyama, Takuya B; Higo, Toshiaki; Umehara, Takashi; Sakamoto, Kensaku; Ito, Takuhiro; Yokoyama, Shigeyuki

    2016-03-01

    Eukaryotic cells restrict protein synthesis under various stress conditions, by inhibiting the eukaryotic translation initiation factor 2B (eIF2B). eIF2B is the guanine nucleotide exchange factor for eIF2, a heterotrimeric G protein consisting of α-, β- and γ-subunits. eIF2B exchanges GDP for GTP on the γ-subunit of eIF2 (eIF2γ), and is inhibited by stress-induced phosphorylation of eIF2α. eIF2B is a heterodecameric complex of two copies each of the α-, β-, γ-, δ- and ε-subunits; its α-, β- and δ-subunits constitute the regulatory subcomplex, while the γ- and ε-subunits form the catalytic subcomplex. The three-dimensional structure of the entire eIF2B complex has not been determined. Here we present the crystal structure of Schizosaccharomyces pombe eIF2B with an unprecedented subunit arrangement, in which the α2β2δ2 hexameric regulatory subcomplex binds two γε dimeric catalytic subcomplexes on its opposite sides. A structure-based in vitro analysis by a surface-scanning site-directed photo-cross-linking method identified the eIF2α-binding and eIF2γ-binding interfaces, located far apart on the regulatory and catalytic subcomplexes, respectively. The eIF2γ-binding interface is located close to the conserved 'NF motif', which is important for nucleotide exchange. A structural model was constructed for the complex of eIF2B with phosphorylated eIF2α, which binds to eIF2B more strongly than the unphosphorylated form. These results indicate that the eIF2α phosphorylation generates the 'nonproductive' eIF2-eIF2B complex, which prevents nucleotide exchange on eIF2γ, and thus provide a structural framework for the eIF2B-mediated mechanism of stress-induced translational control. PMID:26901872

  9. The search for mutations in the gene for the beta subunit of the cGMP phosphodiesterase (PDEB) in patients with autosomal recessive retinitis pigmentosa

    SciTech Connect

    Riess, O.; Weber, B.; Hayden, M.R. ); Noerremoelle, A. ); Musarella, M.A. )

    1992-10-01

    The finding of a mutation in the beta subunit of the cyclic GMP (cGMP) phosphodiesterase gene causing retinal degeneration in mice (the Pdeb gene) prompted a search for disease-causing mutations in the human phosphodiesterase gene (PDEB gene) in patients with retinitis pigmentosa. All 22 exons including 196 bp of the 5[prime] region of the PDEB gene have been assessed for mutations by using single-strand conformational polymorphism analysis in 14 patients from 13 unrelated families with autosomal recessive retinitis pigmentosa (ARRP). No disease-causing mutations were found in this group of affected individuals of seven different ancestries. However, a frequent intronic and two exonic polymorphisms (Leu[sup 489][yields]Gln and Gly[sup 842][yields]Gly) were identified. Segregation analysis using these polymorphic sites excludes linkage of ARRP to the PDEB gene in a family with two affected children. 43 refs., 3 figs., 2 tabs.

  10. In silico analysis of Brucella abortus Omp2b and in vitro expression of SOmp2b

    PubMed Central

    2016-01-01

    Purpose At present, there is no vaccine available for the prevention of human brucellosis. Brucella outer membrane protein 2b (Omp2b) is a 36 kD porin existed in common Brucella pathogens and it is considered as priority antigen for designing a new subunit vaccine. Materials and Methods In the current study, we aimed to predict and analyze the secondary and tertiary structures of the Brucella abortus Omp2b protein, and to predict T-cell and B-cell epitopes with the help of bioinformatics tools. Subsequently, cloning and expression of the short form of Omp2b (SOmp2b) was performed using pET28a expression vector and Escherichia coli BL21 host, respectively. The recombinant SOmp2b (rSOmp2b) was purified with Ni-NTA column. Results The recombinant protein was successfully expressed in E. coli host and purified under denaturation conditions. The yield of the purified rSOmp2b was estimated by Bradford method and found to be 220 µg/mL of the culture. Conclusion Our results indicate that Omp2b protein has a potential to induce both B-cell– and T-cell–mediated immune responses and it can be evaluated as a new subunit vaccine candidate against brucellosis. PMID:26866027

  11. SACALC2B

    Energy Science and Technology Software Center (ESTSC)

    2007-02-08

    SACALC2B calculates the average solid angle subtended by a rectangular or circular detector window to a coaxial or non-coaxial rectangular, circular or point source, including where the source and detector planes are not parallel. SACALC_CYL calculates the average solid angle subtended by a cylinder to a rectangular or circular source, plane or thick, at any location and orientation. This is needed, for example, in calculating the intrinsic gamma efficiency of a detector such as amore » GM tube. The program also calculates the number of hits on the cylinder side and on each end, and the average path length through the detector volume (assuming no scattering or absorption). Point sources can be modelled by using a circular source of zero radius.« less

  12. Comparative inter-strain sequence analysis of the putative regulatory region of murine psychostimulant-regulated gene GNB1 (G protein beta 1 subunit gene).

    PubMed

    Kitanaka, Nobue; Kitanaka, Junichi; Walther, Donna; Wang, Xiao-Bing; Uhl, George R

    2003-08-01

    We isolated a cDNA clone from a murine genomic library of C57BL/6 strain, carrying 13.8 kb of nucleotides including exon 1 of heterotrimeric GTP-binding protein beta 1 subunit gene (genetic symbol, GNB1) and 10.6 kb of the 5' flanking region. Sequence comparison with GNB1 gene locus from 129Sv strain revealed a 0.2% divergence in a 13.2 kb common region between these two strains. The divergence consisted of eight single nucleotide polymorphisms, three insertions and one deletion, with 129Sv used as the reference. The exon 1 and the putative regulation elements, such as cyclic AMP response element, AP1, AP2, Sp1 and nuclear factor-kappa B recognition sites, were perfectly conserved. The expression of GNB1 mRNA was significantly increased in mouse striatum 2 h after single methamphetamine administration with an approximately 150% expression level compared with the basal level. In contrast, no change in the expression level was observed in the cerebral cortex. After the chronic methamphetamine treatment regimen, the expression level of GNB1 mRNA did not change in any brain regions examined. These results suggest (1) that the 5' flanking nucleotide sequence of GNB1 gene was strictly conserved for its possible contribution to the same change in the expression level between the mouse strains in response to psychostimulants and (2) that the initial process of development of behavioral sensitization appeared to occur parallel to the significant increase in the expression level of GNB1 gene in the mouse striatum. PMID:14631649

  13. The BAF60 subunit of the SWI/SNF chromatin-remodeling complex directly controls the formation of a gene loop at FLOWERING LOCUS C in Arabidopsis.

    PubMed

    Jégu, Teddy; Latrasse, David; Delarue, Marianne; Hirt, Heribert; Domenichini, Séverine; Ariel, Federico; Crespi, Martin; Bergounioux, Catherine; Raynaud, Cécile; Benhamed, Moussa

    2014-02-01

    SWI/SNF complexes mediate ATP-dependent chromatin remodeling to regulate gene expression. Many components of these complexes are evolutionarily conserved, and several subunits of Arabidopsis thaliana SWI/SNF complexes are involved in the control of flowering, a process that depends on the floral repressor FLOWERING LOCUS C (FLC). BAF60 is a SWI/SNF subunit, and in this work, we show that BAF60, via a direct targeting of the floral repressor FLC, induces a change at the high-order chromatin level and represses the photoperiod flowering pathway in Arabidopsis. BAF60 accumulates in the nucleus and controls the formation of the FLC gene loop by modulation of histone density, composition, and posttranslational modification. Physiological analysis of BAF60 RNA interference mutant lines allowed us to propose that this chromatin-remodeling protein creates a repressive chromatin configuration at the FLC locus. PMID:24510722

  14. A gene-specific effect of an internal deletion in the Bdp1 subunit of the RNA polymerase III transcription initiation factor TFIIIB.

    PubMed

    Ishiguro, Akira; Kassavetis, George A

    2003-07-31

    The Saccharomyces cerevisiae RPR1 gene encodes the RNA subunit of its RNase P, which processes RNA polymerase (pol) III primary transcripts. RPR1, which is transcribed by pol III, has been isolated as a multicopy suppressor of a specific small internal deletion (amino acids 253-269) in the Bdp1 subunit of transcription factor TFIIIB, the core pol III transcription factor. The selective effect of this Bdp1 deletion on RPR1 transcription has been analyzed in vitro. It is shown that TFIIIC-dependent assembly of TFIIIB on the RPR1 promoter is specifically sensitive to this Bdp1 deletion, leading to gene-specifically defective single-round and multiple-round transcription. PMID:12885403

  15. Genetic analysis of the Drosophila DNAprim gene. The function of the 60-kd primase subunit of DNA polymerase opposes the fat facets signaling pathway in the developing eye.

    PubMed Central

    Chen, X; Li, Q; Fischer, J A

    2000-01-01

    The Drosophila DNAprim gene encodes the large subunit (60 kD) of DNA primase, the part of DNA polymerase alpha that synthesizes RNA primers during DNA replication. The precise function of the 60-kD subunit is unknown. In a mutagenesis screen for suppressors of the fat facets (faf) mutant eye phenotype, we identified mutations in DNAprim. The faf gene encodes a deubiquitinating enzyme required specifically for patterning the compound eye. The DNA sequences of four DNAprim alleles were determined and these define essential protein domains. We show that while flies lacking DNAprim activity are lethal, flies with reduced DNAprim activity display morphological defects in their eyes, and unlike faf mutants, cell cycle abnormalities in larval eye discs. Mechanisms by which DNA primase levels might influence the faf-dependent cell communication pathway are discussed. PMID:11102374

  16. Inactivation of genes coding for mitochondrial Nd7 and Nd9 complex I subunits in Chlamydomonas reinhardtii. Impact of complex I loss on respiration and energetic metabolism.

    PubMed

    Massoz, Simon; Larosa, Véronique; Plancke, Charlotte; Lapaille, Marie; Bailleul, Benjamin; Pirotte, Dorothée; Radoux, Michèle; Leprince, Pierre; Coosemans, Nadine; Matagne, René F; Remacle, Claire; Cardol, Pierre

    2014-11-01

    In Chlamydomonas, unlike in flowering plants, genes coding for Nd7 (NAD7/49 kDa) and Nd9 (NAD9/30 kDa) core subunits of mitochondrial respiratory-chain complex I are nucleus-encoded. Both genes possess all the features that facilitate their expression and proper import of the polypeptides in mitochondria. By inactivating their expression by RNA interference or insertional mutagenesis, we show that both subunits are required for complex I assembly and activity. Inactivation of complex I impairs the cell growth rate, reduces the respiratory rate, leads to lower intracellular ROS production and lower expression of ROS scavenging enzymes, and is associated to a diminished capacity to concentrate CO2 without compromising photosynthetic capacity. PMID:24316185

  17. Alterations of the tumor suppressor genes CDKN2A (p16(INK4a)), p14(ARF), CDKN2B (p15(INK4b)), and CDKN2C (p18(INK4c)) in atypical and anaplastic meningiomas.

    PubMed

    Boström, J; Meyer-Puttlitz, B; Wolter, M; Blaschke, B; Weber, R G; Lichter, P; Ichimura, K; Collins, V P; Reifenberger, G

    2001-08-01

    We investigated 67 meningothelial tumors (20 benign meningiomas, 34 atypical meningiomas, and 13 anaplastic meningiomas) for losses of genetic information from chromosome arms 1p and 9p, as well as for deletion, mutation, and expression of the tumor suppressor genes CDKN2A (p16(INKa)/MTS1), p14(ARF), CDKN2B (p15(INK4b)/MTS2) (all located at 9p21) and CDKN2C (1p32). Comparative genomic hybridization and microsatellite analysis showed losses on 1p in 11 anaplastic meningiomas (85%), 23 atypical meningiomas (68%), and 5 benign meningiomas (25%). One atypical meningioma with loss of heterozygosity on 1p carried a somatic CDKN2C mutation (c.202C>T: R68X). Losses on 9p were found in five anaplastic meningiomas (38%), six atypical meningiomas (18%), and one benign meningioma (5%). Six anaplastic meningiomas (46%) and one atypical meningioma (3%) showed homozygous deletions of the CDKN2A, p14(ARF), and CDKN2B genes. Two anaplastic meningiomas carried somatic point mutations in CDKN2A (c.262G>T: E88X and c.262G>A: E88K) and p14(ARF) (c.305G>T: G102V and c.305G>A: G102E). One anaplastic meningioma, three atypical meningiomas, and one benign meningioma without a demonstrated homozygous deletion or mutation of CDKN2A, p14(ARF), or CDKN2B lacked detectable transcripts from at least one of these genes. Hypermethylation of CDKN2A, p14(ARF), and CDKN2B could be demonstrated in one of these cases. Taken together, our results indicate that CDKN2C is rarely altered in meningiomas. However, the majority of anaplastic meningiomas either show homozygous deletions of CDKN2A, p14(ARF), and CDKN2B, mutations in CDKN2A and p14(ARF), or lack of expression of one or more of these genes. Thus, inactivation of the G(1)/S-phase cell-cycle checkpoint is an important aberration in anaplastic meningiomas. PMID:11485924

  18. Retention of NMDA receptor NR2 subunits in the lumen of endoplasmic reticulum in targeted NR1 knockout mice

    PubMed Central

    Fukaya, Masahiro; Kato, Akira; Lovett, Chanel; Tonegawa, Susumu; Watanabe, Masahiko

    2003-01-01

    Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system, and the N-methyl-d-aspartate-selective glutamate receptor (NR) consisting of the NR1 subunit and an NR2 or NR3 subunit plays crucial roles in synaptic transmission, plasticity, and learning and memory. By using a knockout mouse strain, in which the NR1 gene deletion is primarily targeted to the CA1 pyramidal cells of the hippocampus, we investigated the in vivo effect of the loss of the NR1 subunit on the cellular expression and intracellular distribution of the NR2 subunits. The NR1 gene deletion had no apparent effect on the levels of NR2A or NR2B mRNA but led to severe reductions of NR2A and NR2B protein in dendrites of CA1 pyramidal cells. This reduced dendritic distribution of the NR2 subunits accompanied their robust accumulation in perikarya, where they were condensed in the lumen of the endoplasmic reticulum as electron-dense granules. These granules were also observed in CA1 pyramidal cells of the control mice but they were much fewer and contained no detectable levels of the NR2 subunit. The effect of the NR1 knockout on intracellular localization of the NR2 subunits was specific in that no such effect was observed for the GluR1 and PSD-95, two other major postsynaptic proteins. These results suggest that the NR1 subunit plays a crucial role in the release of the NR2 subunit from the endoplasmic reticulum in hippocampal pyramidal cells in vivo, and when the NR1 subunit is unavailable, the NR2 subunits are retained and aggregate into intracisternal granules. PMID:12676993

  19. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene.

    PubMed Central

    Kurtzman, C P; Robnett, C J

    1997-01-01

    Clinically important species of Candida and related organisms were compared for extent of nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region is sufficiently variable to allow reliable separation of all known clinically significant yeast species. Of the 204 described species examined, 21 appeared to be synonyms of previously described organisms. Phylogenetic relationships among the species are presented. PMID:9114410

  20. Acid-Sensing Ion Channel 2a (ASIC2a) Promotes Surface Trafficking of ASIC2b via Heteromeric Assembly

    PubMed Central

    Kweon, Hae-Jin; Kim, Dong-Il; Bae, Yeonju; Park, Jae-Yong; Suh, Byung-Chang

    2016-01-01

    Acid-sensing ion channels (ASICs) are proton-activated cation channels that play important roles as typical proton sensors during pathophysiological conditions and normal synaptic activities. Among the ASIC subunits, ASIC2a and ASIC2b are alternative splicing products from the same gene, ACCN1. It has been shown that ASIC2 isoforms have differential subcellular distribution: ASIC2a targets the cell surface by itself, while ASIC2b resides in the ER. However, the underlying mechanism for this differential subcellular localization remained to be further elucidated. By constructing ASIC2 chimeras, we found that the first transmembrane (TM1) domain and the proximal post-TM1 domain (17 amino acids) of ASIC2a are critical for membrane targeting of the proteins. We also observed that replacement of corresponding residues in ASIC2b by those of ASIC2a conferred proton-sensitivity as well as surface expression to ASIC2b. We finally confirmed that ASIC2b is delivered to the cell surface from the ER by forming heteromers with ASIC2a, and that the N-terminal region of ASIC2a is additionally required for the ASIC2a-dependent membrane targeting of ASIC2b. Together, our study supports an important role of ASIC2a in membrane targeting of ASIC2b. PMID:27477936

  1. Acid-Sensing Ion Channel 2a (ASIC2a) Promotes Surface Trafficking of ASIC2b via Heteromeric Assembly.

    PubMed

    Kweon, Hae-Jin; Kim, Dong-Il; Bae, Yeonju; Park, Jae-Yong; Suh, Byung-Chang

    2016-01-01

    Acid-sensing ion channels (ASICs) are proton-activated cation channels that play important roles as typical proton sensors during pathophysiological conditions and normal synaptic activities. Among the ASIC subunits, ASIC2a and ASIC2b are alternative splicing products from the same gene, ACCN1. It has been shown that ASIC2 isoforms have differential subcellular distribution: ASIC2a targets the cell surface by itself, while ASIC2b resides in the ER. However, the underlying mechanism for this differential subcellular localization remained to be further elucidated. By constructing ASIC2 chimeras, we found that the first transmembrane (TM1) domain and the proximal post-TM1 domain (17 amino acids) of ASIC2a are critical for membrane targeting of the proteins. We also observed that replacement of corresponding residues in ASIC2b by those of ASIC2a conferred proton-sensitivity as well as surface expression to ASIC2b. We finally confirmed that ASIC2b is delivered to the cell surface from the ER by forming heteromers with ASIC2a, and that the N-terminal region of ASIC2a is additionally required for the ASIC2a-dependent membrane targeting of ASIC2b. Together, our study supports an important role of ASIC2a in membrane targeting of ASIC2b. PMID:27477936

  2. Protein factors in Blastocladiella emersonii cell extracts recognize similar sequence elements in the promoters of the genes encoding cAMP-dependent protein kinase subunits.

    PubMed

    de Oliveira, J C; Marques, M V; Gomes, S L

    1997-08-01

    Blastocladiella emersonii contains a single cAMP-dependent protein kinase (PKA), which is similar to the mammalian type II isoforms. Its activity is regulated during development by changes in the levels of the catalytic (C) and regulatory (R) subunits, which occur in parallel with changes in levels of the corresponding mRNAs, suggesting coordinate transcriptional control of the expression of both subunits. Both R and C mRNA levels are low in vegetative cells, rise sharply during sporulation and decrease to basal levels again after germination. To investigate sequence elements common to both Blastocladiella R and C gene promoters, which might be involved in the coordinate regulation of these genes, their 5'-flanking regions were analyzed by gel mobility shift and DNase I footprinting assays. We determined that different DNA-protein complexes are generated when fragments of the R and C gene promoters are incubated with extracts from cells expressing (sporulating cells) or not expressing (vegetative cells) both subunits, and competition experiments suggested that similar protein factors bind to both promoters. DNase I footprinting experiments have indicated that a sequence common to both R and C promoters, and similar to mammalian E-boxes, binds factors present in extracts from vegetative and sporulating cells, whereas sequences flanking the E-boxes in both promoters showed a change in the pattern of DNase I digestion only when the vegetative cell extract was used. This result suggests that the composition of the protein complexes binding to these regions changes during sporulation. PMID:9294034

  3. Association of alpha subunit of GABAA receptor subtype gene polymorphisms with epilepsy susceptibility and drug resistance in north Indian population.

    PubMed

    Kumari, Ritu; Lakhan, Ram; Kalita, J; Misra, U K; Mittal, Balraj

    2010-05-01

    GABA (gamma-amino butyric acid) receptors have always been an inviting target in the etiology and treatment of epilepsy because of its role as a major inhibitory neurotransmitter in the brain. The aim of our study was to find out the possible role of single nucleotide polymorphisms (SNPs) present in GABRA1 IVS11+15 A>G (rs2279020) and GABRG2 588C>T (rs211037) genes in seizure susceptibility and pharmaco-resistance in northern Indian patients with epilepsy. A total of 395 epilepsy patients and 199 control subjects were enrolled for present study. The genotyping was done by PCR-RFLP methods. The GABRA1 IVS11+15 A>G polymorphism conferred high risk for epilepsy susceptibility at genotype 'AG' (P=0.004, OR=1.77, 95% CI=1.20-2.63), 'GG' (P=0.01, OR=1.80, 95% CI=1.15-2.80) and G allele level (P=0.001, OR=1.50, 95% CI=1.16-1.92). Moreover this polymorphism was also associated with multiple drug resistance in patients with epilepsy for homozygous variant 'GG' genotype (P=0.031, OR=1.84, 95% CI=1.05-3.23) and G allele (P=0.020, OR=1.43, 95% CI=1.05-1.95). However GABRG2 588C>T polymorphism was not found to be associated either with epilepsy susceptibility or with drug resistance. Overall results indicate differential role of different subunits of GABA(A) receptor subtypes in epilepsy susceptibility and pharmacotherapy. PMID:20356767

  4. Higher gene expression of CYP1A2, 2B1 and 2D2 in the brain of female compared with male rats.

    PubMed

    Nagai, K; Fukuno, S; Suzuki, H; Konishi, H

    2016-06-01

    Cytochrome P450 (CYP) in the brain plays an essential role in the local metabolism of various compounds, including clinically used drugs, toxins, and endogenous substances. In the present study, we compared the expression profiles of mRNAs for several CYP subtypes in the brain between male and female rats. The expression of CYP1A2, CYP2B1, and CYP2D2 in females was significantly higher than that in males. On the other hand, the expression level of the other CYP subtypes examined in the male brain was similar to that in the female brain. These results strongly suggest that marked gender differences exist in the expression profiles of some CYP subtypes in rat brain. PMID:27455552

  5. Evidence for Association between SH2B1 Gene Variants and Glycated Hemoglobin in Nondiabetic European American Young Adults: The Add Health Study.

    PubMed

    Lange, Leslie A; Graff, Mariaelisa; Lange, Ethan M; Young, Kristin L; Richardson, Andrea S; Mohlke, Karen L; North, Kari E; Harris, Kathleen M; Gordon-Larsen, Penny

    2016-09-01

    Glycated hemoglobin (HbA1c) is used to classify glycaemia and type 2 diabetes (T2D). Body mass index (BMI) is a predictor of HbA1c levels and T2D. We tested 43 established BMI and obesity loci for association with HbA1c in a nationally representative multiethnic sample of young adults from the National Longitudinal Study of Adolescent to Adult Health [Add Health: age 24-34 years; n = 5641 European Americans (EA); 1740 African Americans (AA); 1444 Hispanic Americans (HA)] without T2D, using two levels of covariate adjustment (Model 1: age, sex, smoking, and geographic region; Model 2: Model 1 covariates plus BMI). Bonferroni adjustment was made for 43 SNPs and we considered P < 0.0011 statistically significant. Means (SD) for HbA1c were 5.4% (0.3) in EA, 5.7% (0.4) in AA, and 5.5% (0.3) in HA. We observed significant evidence for association with HbA1c for two variants near SH2B1 in EA (rs4788102, P = 2.2 × 10(-4) ; rs7359397, P = 9.8 × 10(-4) ) for Model 1. Both results were attenuated after adjustment for BMI (rs4788102, P = 1.7 × 10(-3) ; rs7359397, P = 4.6 × 10(-3) ). No variant reached Bonferroni-corrected significance in AA or HA. These results suggest that SH2B1 polymorphisms are associated with HbA1c, largely independent of BMI, in EA young adults. PMID:27530450

  6. Neuron-specific specificity protein 4 (Sp4) bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons

    PubMed Central

    Johar, Kaid; Priya, Anusha; Dhar, Shilpa; Liu, Qiuli; Wong-Riley, Margaret T. T.

    2013-01-01

    Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically-regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1, that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. PMID:24032355

  7. Transcriptional regulation of a gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in soybean tissue is linked to the phytochrome response.

    PubMed Central

    Berry-Lowe, S L; Meagher, R B

    1985-01-01

    The effects of white light, far-red light, and darkness on the transcription of a soybean ribulose-1,5-biphosphate carboxylase small subunit gene, SRS1, were investigated. RNA was labeled with [alpha-32P]UTP in nuclei isolated from plants grown under different conditions of light and darkness and used to probe Southern blots and dot blots. The levels of small subunit mRNA synthesis were normalized to ribosomal RNA synthesis. We demonstrate that the SRS1 gene is transcribed at a rate 16- to 32-fold higher in plants grown in the light than in those grown in darkness. Transcription of the small subunit increased dramatically when plants grown in darkness were given 30 min to 6 h of light and then leveled off after 24 to 48 h of exposure. When light-grown seedlings were exposed to greater than 2 h of darkness, a gradual decrease in transcription was detected. This decrease in transcription reached basal dark-grown levels after 48 h of exposure to darkness. The increase in transcription in etiolated seedlings treated with white light for 15 min could be reduced to basal levels if the treatment was followed by treatment with far-red light for 15 min. In addition, transcription in ligh-grown seedlings was reduced to basal levels when plants were exposed to far-red light for 15 min. The transcription of this ribulose-1,5-biphosphate carboxylase small subunit gene is strongly positively regulated by white light, is negatively regulated by far-red light, and exhibits a classic phytochrome-linked response. Images PMID:3837851

  8. Human locus coeruleus neurons express the GABA(A) receptor gamma2 subunit gene and produce benzodiazepine binding.

    PubMed

    Hellsten, Kati S; Sinkkonen, Saku T; Hyde, Thomas M; Kleinman, Joel E; Särkioja, Terttu; Maksimow, Anu; Uusi-Oukari, Mikko; Korpi, Esa R

    2010-06-21

    Noradrenergic neurons of the locus coeruleus project throughout the cerebral cortex and multiple subcortical structures. Alterations in the locus coeruleus firing are associated with vigilance states and with fear and anxiety disorders. Brain ionotropic type A receptors for gamma-aminobutyric acid (GABA) serve as targets for anxiolytic and sedative drugs, and play an essential regulatory role in the locus coeruleus. GABA(A) receptors are composed of a variable array of subunits forming heteropentameric chloride channels with different pharmacological properties. The gamma2 subunit is essential for the formation of the binding site for benzodiazepines, allosteric modulators of GABA(A) receptors that are clinically often used as sedatives/hypnotics and anxiolytics. There are contradictory reports in regard to the gamma2 subunit's expression and participation in the functional GABA(A) receptors in the mammalian locus coeruleus. We report here that the gamma2 subunit is transcribed and participates in the assembly of functional GABA(A) receptors in the tyrosine hydroxylase-positive neuromelanin-containing neurons within postmortem human locus coeruleus as demonstrated by in situ hybridization with specific gamma2 subunit oligonucleotides and autoradiographic assay for flumazenil-sensitive [(3)H]Ro 15-4513 binding to benzodiazepine sites. These sites were also sensitive to the alpha1 subunit-preferring agonist zolpidem. Our data suggest a species difference in the expression profiles of the alpha1 and gamma2 subunits in the locus coeruleus, with the sedation-related benzodiazepine sites being more important in man than rodents. This may explain the repeated failures in the transition of novel drugs with a promising neuropharmacological profile in rodents to human clinical usage, due to intolerable sedative effects. PMID:20417252

  9. The acquired radioresistance in HeLa cells under conditions mimicking hypoxia was attenuated by a decreased expression of HIF subunit genes induced by RNA interference

    SciTech Connect

    Doi, Nobutaka; Ogawa, Ryohei; Cui, Zheng-Guo; Morii, Akihiro; Watanabe, Akihiko; Kanayama, Shinji; Yoneda, Yuko; Kondo, Takashi

    2015-05-01

    The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment. - Highlights: • Incubation with CoCl{sub 2} confers radioresistance to HeLa cells. • Both HIF-1α and HIF-2α are involved in the acquisition of radioresistance. • An shRNA to a homology region of HIF-1α and HIF-2α suppressed the radioresistance. • The shRNA decreased cells with stem cell markers and a stem cell phenotype.

  10. Potential role for concurrent abnormalities of the cyclin D1, p16CDKN2 and p15CDKN2B genes in certain B cell non-Hodgkin's lymphomas. Functional studies in a cell line (Granta 519).

    PubMed

    Jadayel, D M; Lukas, J; Nacheva, E; Bartkova, J; Stranks, G; De Schouwer, P J; Lens, D; Bartek, J; Dyer, M J; Kruger, A R; Catovsky, D

    1997-01-01

    Abnormalities of several cell-cycle regulatory genes including cyclin D1, p16CDKN2 and p15CDKN2B have been described in B cell non-Hodgkin's lymphoma (B-NHL). We describe a new B-NHL cell line (Granta 519), with concurrent abnormalities of the cyclin D1, pl6CDKN2 and pl5CDKN2B genes. An independent clinical case of mantle cell NHL (Mc-NHL) with concomitant overexpression of cyclin D1, and deletion of the p16CDKN2 gene was also identified, suggesting that this combination of oncogenic aberration is a pathophysiologic contribution to a subset of NHL cases. More in-depth functional studies of this concept were facilitated by the availability of the cell line Granta 519 which was derived from a case of high-grade NHL and has a mature B cell immunophenotype. Cytogenetic analysis identified translocation t(11;14)(q13;q32) and complex rearrangements involving chromosomes 9p22, 13p21, 17pl1, and 18q21. Molecular analysis identified overexpression of cyclin D1 mRNA and biallelic deletion of the p16CDKN2 and p15CDKN2B genes. To elucidate the effect of these genetic abnormalities on the G1 control of Granta 519 cells, the level and function of the major components of the cyclinD/retinoblastoma (RB) pathway were investigated. Cyclin D1 was dominant among the D-type cyclins, formed abundant complexes with cyclin-dependent kinase (Cdk) Cdk4 rather than Cdk6, and the immunoprecipitated cyclin D1/Cdk4 holoenzyme was active as a pRB kinase. Electroporation of wild-type pl6CDKN2 arrested the Granta 519 cells in G1, consistent with the p16CDKN2 loss as a biologically relevant event during multistep evolution of the tumor, and with the expression of functional pRB. Direct cooperation of these distinct abnormalities to cell-cycle, deregulation in NHL cells was suggested by G1 acceleration upon inducible overexpression of cyclin D1 in a control breast cancer cell line lacking p16CDKN2, an effect which could be prevented by ectopic expression of p16CDKN2. Taken together, these data

  11. An unusual insertion/deletion in the gene encoding the. beta. -subunit of propionyl-CoA carboxylase is a frequent mutation in Caucasian propionic acidemia

    SciTech Connect

    Tahara, T.; Kraus, J.P.; Rosenberg, L.E. )

    1990-02-01

    Propionic acidemia is an inherited disorder of organic acid metabolism that is caused by deficiency of propionly-CoA carboxylase. Affected patients fall into two complementation groups, pccA and pccBC (subgroups B, C, and BC), resulting from deficiency of the nonidentical {alpha} and {beta} subunits of PCC, respectively. The authors have detected an unusual insertion/deletion in the DNA of patients from the pccBC and pccC subgroups that replaces 14 nucleotides in the coding sequence of the {beta} subunit with 12 nucleotides unrelated to this region of the gene. Among 14 unrelated Caucasian patients in the pccBc complementation group, this unique mutation was found in 8 of 28 mutant alleles examined. Mutant allele-specific oligonucleotide hybridization to amplified genomic DNAs revealed that the inserted 12 nucleotides do not originate in an {approx}1000-bp region around the mutation. In the course of the investigation, they identified another mutation in the same exon: a 3-bp in-frame deletion that eliminates one of two isoleucine codons immediately preceding the Msp I site. Two unrelated patients were compound heterozygotes for this single-codon deletion and for the insertion/deletion described above. They conclude that either there is a propensity for the PCC {beta}-subunit gene to undergo mutations of this sort at this position or, more likely, the mutations in all of the involved Caucasian patients have a common origin in preceding generations.

  12. Selective expression of a dominant-negative type Iα PKA regulatory subunit in striatal medium spiny neurons impairs gene expression and leads to reduced feeding and locomotor activity.

    PubMed

    Yang, Linghai; Gilbert, Merle L; Zheng, Ruimao; McKnight, G Stanley

    2014-04-01

    Striatal medium spiny neurons (MSNs) mediate many of the physiological effects of dopamine, including the regulation of feeding and motor behaviors. Dopaminergic inputs from the midbrain modulate MSN excitability through pathways that involve cAMP and protein kinase A (PKA), but the physiological role of specific PKA isoforms in MSN neurons remains poorly understood. One of the major PKA regulatory (R) subunit isoforms expressed in MSNs is RIIβ, which localizes the PKA holoenzyme primarily to dendrites by interaction with AKAP5 and other scaffolding proteins. However, RI (RIα and RIβ) subunits are also expressed in MSNs and the RI holoenzyme has a weaker affinity for most scaffolding proteins and tends to localize in the cell body. We generated mice with selective expression of a dominant-negative RI subunit (RIαB) in striatal MSNs and show that this dominant-negative RIαB localizes to the cytoplasm and specifically inhibits type I PKA activity in the striatum. These mice are normal at birth; however, soon after weaning they exhibit growth retardation and the adult mice are hypophagic, lean, and resistant to high-fat diet-induced hyperphagia and obesity. The RIαB-expressing mice also exhibit decreased locomotor activity and decreased dopamine-regulated CREB phosphorylation and c-fos gene expression in the striatum. Our results demonstrate a critical role for cytoplasmic RI-PKA holoenzyme in gene regulation and the overall physiological function of MSNs. PMID:24695708

  13. Molecular cloning and characterization of the gonadotropin subunits GPα, FSHβ, and LHβ genes in the stinging catfish Heteropneustes fossilis: phylogeny, seasonal expression and pituitary localization.

    PubMed

    Acharjee, Arup; Chaube, Radha; Joy, Keerikkattil Paily

    2015-10-01

    Gonadotropins are heterodimeric glycoproteins secreted by the pituitary, and consist of a common glycoprotein hormone alpha (GPα) and the function-specific follicle-stimulating hormone beta subunit (FSHβ) or luteinizing hormone beta subunit (LHβ). In the present study, the subunit protein genes were cloned and characterized from the pituitary of the catfish Heteropneustes fossilis. Full-length cDNAs of GPα, FSHβ, and LHβ are 511 base pairs (bp), 659 bp and 660 bp long, and encode 92, 108, and 112 aminoacids long mature proteins, respectively. GPα has 10 cysteines with 2 N-linked glycosylation sites while LHβ contains 12 cysteines with a single N-linked glycosylation site. In contrast, FSHβ has 13 cysteines, 1 additional over the conserved 12 cysteines of other vertebrates, and a single glycosylation site between Cys 3 and Cys 4. Phylogenetic analyses of the deduced proteins confirm their homology and relationships with the respective gonadotropin subunit proteins of gnathostome vertebrates. Tissue expression analysis by semi-quantitative RT-PCR shows that GPα mRNA is expressed only in the pituitary while both FSHβ and LHβ mRNA are expressed in extra-pituitary sites. The subunit mRNAs show both seasonal and sex dimorphic variations especially in the expression of FSHβ and LHβ transcripts. In the sexually quiescent phase, the transcript expression is low while in the recrudescent phase, the expressions are differential, high, and varied with regard to sex and reproductive phase. In situ hybridization of the mRNAs gave positive signals in gonadotropes in the pars distalis of the pituitary, which exhibited seasonal variation in staining intensity and numbers. PMID:26205349

  14. Biochemical and functional properties of distinct nicotinic acetylcholine receptors in the superior cervical ganglion of mice with targeted deletions of nAChR subunit genes.

    PubMed

    David, Reinhard; Ciuraszkiewicz, Anna; Simeone, Xenia; Orr-Urtreger, Avi; Papke, Roger L; McIntosh, J M; Huck, Sigismund; Scholze, Petra

    2010-03-01

    Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in ganglia of the autonomic nervous system. Here, we determined the subunit composition of hetero-pentameric nAChRs in the mouse superior cervical ganglion (SCG), the function of distinct receptors (obtained by deletions of nAChR subunit genes) and mechanisms at the level of nAChRs that might compensate for the loss of subunits. As shown by immunoprecipitation and Western blots, wild-type (WT) mice expressed: alpha 3 beta 4 (55%), alpha 3 beta 4 alpha 5 (24%) and alpha 3 beta 4 beta 2 (21%) nAChRs. nAChRs in beta 4 knockout (KO) mice were reduced to < 15% of controls and no longer contained the alpha 5 subunit. Compound action potentials, recorded from the postganglionic (internal carotid) nerve and induced by preganglionic nerve stimulation, did not differ between alpha 5 beta 4 KO and WT mice, suggesting that the reduced number of receptors in the KO mice did not impair transganglionic transmission. Deletions of alpha 5 or beta2 did not affect the overall number of receptors and we found no evidence that the two subunits substitute for each other. In addition, dual KOs allowed us to study the functional properties of distinct alpha 3 beta4 and alpha 3 beta 2 receptors that have previously only been investigated in heterologous expression systems. The two receptors strikingly differed in the decay of macroscopic currents, the efficacy of cytisine, and their responses to the alpha-conotoxins AuIB and MII. Our data, based on biochemical and functional experiments and several mouse KO models, clarify and significantly extend previous observations on the function of nAChRs in heterologous systems and the SCG. PMID:20377613

  15. The organization of the gene for the functionally dominant alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit GluR-B.

    PubMed

    Köhler, M; Kornau, H C; Seeburg, P H

    1994-07-01

    The murine gene encoding the GluR-B subunit of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors was characterized with respect to exon-intron organization, transcriptional start site, alternatively spliced transcripts, and adenosine to guanosine substitutions between gene and complementary DNA sequence. The GluR-B gene spans > 90 kilobase pairs and harbors 17 exons. Transcription appears to initiate approximately 430 nucleotides upstream of the translational start codon, with no intron in the 5'-untranslated region of the gene. Four alternatively spliced mRNAs are generated from the primary GluR-B transcript, two containing the modules Flip and Flop, and another two with alternate C-terminal coding sequence. The major GluR-B mRNAs in murine brain, 4 and 6 kilobase differ in the length of their 3'-untranslated region. PMID:7545935

  16. The human NFKB3 gene encoding the p65 subunit of transcription factor NF-[sub K]B is located on chromosome 11q12

    SciTech Connect

    Deloukas, P.; Loon, A.P.G.M. van ); Dauwerse, J.G.; Ommen, G.J.B. van )

    1994-02-01

    A YAC clone that contains the human gene NFKB3, encoding the p65 subunit of transcription factor nuclear factor [sub K]B (NF-[sub K]B), was isolated. The YAC contains the entire NFKB3 gene, which is smaller than 15 kb and present in a single copy in the genome. Fluorescence in situ hybridization with metaphase chromosomes showed two different chromosomal locations (11q12 and Xp11.4) for sequences present in the YAC. The NFKB3 gene was assigned to chromosome 11q12 by PCR analysis of a panel of relevant hybrid cell lines. Thus, no linkage exists between NFKB3 and genes encoding other known members of the NF-[sub K]B family. 20 refs., 2 figs.

  17. The Mediator Complex MED15 Subunit Mediates Activation of Downstream Lipid-Related Genes by the WRINKLED1 Transcription Factor1[OPEN

    PubMed Central

    Kim, Mi Jung

    2016-01-01

    The Mediator complex is known to be a master coordinator of transcription by RNA polymerase II, and this complex is recruited by transcription factors (TFs) to target promoters for gene activation or repression. The plant-specific TF WRINKLED1 (WRI1) activates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. However, no Mediator subunit has yet been identified that mediates WRI1 transcriptional activity. Promoter-β-glucuronidase fusion experiments showed that MEDIATOR15 (MED15) is expressed in the same cells in the embryo as WRI1. We found that the Arabidopsis (Arabidopsis thaliana) MED15 subunit of the Mediator complex interacts directly with WRI1 in the nucleus. Overexpression of MED15 or WRI1 increased transcript levels of WRI1 target genes involved in glycolysis and fatty acid biosynthesis; these genes were down-regulated in wild-type or WRI1-overexpressing plants by silencing of MED15. However, overexpression of MED15 in the wri1 mutant also increased transcript levels of WRI1 target genes, suggesting that MED15 also may act with other TFs to activate downstream lipid-related genes. Chromatin immunoprecipitation assays confirmed the association of MED15 with six WRI1 target gene promoters. Additionally, silencing of MED15 resulted in reduced fatty acid content in seedlings and mature seeds, whereas MED15 overexpression increased fatty acid content in both developmental stages. Similar results were found in wri1 mutant and WRI1 overexpression lines. Together, our results indicate that the WRI1/MED15 complex transcriptionally regulates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. PMID:27246098

  18. GRIN2B Mutations in West Syndrome and Intellectual Disability with Focal Epilepsy

    PubMed Central

    Lemke, Johannes R; Hendrickx, Rik; Geider, Kirsten; Laube, Bodo; Schwake, Michael; Harvey, Robert J; James, Victoria M; Pepler, Alex; Steiner, Isabelle; Hörtnagel, Konstanze; Neidhardt, John; Ruf, Susanne; Wolff, Markus; Bartholdi, Deborah; Caraballo, Roberto; Platzer, Konrad; Suls, Arvid; De Jonghe, Peter; Biskup, Saskia; Weckhuysen, Sarah

    2014-01-01

    Objective To identify novel epilepsy genes using a panel approach and describe the functional consequences of mutations. Methods Using a panel approach, we screened 357 patients comprising a vast spectrum of epileptic disorders for defects in genes known to contribute to epilepsy and/or intellectual disability (ID). After detection of mutations in a novel epilepsy gene, we investigated functional effects in Xenopus laevis oocytes and screened a follow-up cohort. Results We revealed de novo mutations in GRIN2B encoding the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor in 2 individuals with West syndrome and severe developmental delay as well as 1 individual with ID and focal epilepsy. The patient with ID and focal epilepsy had a missense mutation in the extracellular glutamate-binding domain (p.Arg540His), whereas both West syndrome patients carried missense mutations within the NR2B ion channel-forming re-entrant loop (p.Asn615Ile, p.Val618Gly). Subsequent screening of 47 patients with unexplained infantile spasms did not reveal additional de novo mutations, but detected a carrier of a novel inherited GRIN2B splice site variant in close proximity (c.2011-5_2011-4delTC). Mutations p.Asn615Ile and p.Val618Gly cause a significantly reduced Mg2+ block and higher Ca2+ permeability, leading to a dramatically increased Ca2+ influx, whereas p.Arg540His caused less severe disturbance of channel function, corresponding to the milder patient phenotype. Interpretation We identified GRIN2B gain-of-function mutations as a cause of West syndrome with severe developmental delay as well as of ID with childhood onset focal epilepsy. Severely disturbed channel function corresponded to severe clinical phenotypes, underlining the important role of facilitated NMDA receptor signaling in epileptogenesis. PMID:24272827

  19. PCR-based isolation and identification of full-length low-molecular-weight glutenin subunit genes in bread wheat (Triticum aestivum L.).

    PubMed

    Zhang, Xiaofei; Liu, Dongcheng; Jiang, Wei; Guo, Xiaoli; Yang, Wenlong; Sun, Jiazhu; Ling, Hongqing; Zhang, Aimin

    2011-12-01

    Low-molecular-weight glutenin subunits (LMW-GSs) are encoded by a multi-gene family and are essential for determining the quality of wheat flour products, such as bread and noodles. However, the exact role or contribution of individual LMW-GS genes to wheat quality remains unclear. This is, at least in part, due to the difficulty in characterizing complete sequences of all LMW-GS gene family members in bread wheat. To identify full-length LMW-GS genes, a polymerase chain reaction (PCR)-based method was established, consisting of newly designed conserved primers and the previously developed LMW-GS gene molecular marker system. Using the PCR-based method, 17 LMW-GS genes were identified and characterized in Xiaoyan 54, of which 12 contained full-length sequences. Sequence alignments showed that 13 LMW-GS genes were identical to those found in Xiaoyan 54 using the genomic DNA library screening, and the other four full-length LMW-GS genes were first isolated from Xiaoyan 54. In Chinese Spring, 16 unique LMW-GS genes were isolated, and 13 of them contained full-length coding sequences. Additionally, 16 and 17 LMW-GS genes in Dongnong 101 and Lvhan 328 (chosen from the micro-core collections of Chinese germplasm), respectively, were also identified. Sequence alignments revealed that at least 15 LMW-GS genes were common in the four wheat varieties, and allelic variants of each gene shared high sequence identities (>95%) but exhibited length polymorphism in repetitive regions. This study provides a PCR-based method for efficiently identifying LMW-GS genes in bread wheat, which will improve the characterization of complex members of the LMW-GS gene family and facilitate the understanding of their contributions to wheat quality. PMID:21830110

  20. Low molecular weight glutenin subunit gene Glu-B3h confers superior dough strength and breadmaking quality in wheat (Triticum aestivum L.)

    PubMed Central

    Wang, Yaping; Zhen, Shoumin; Luo, Nana; Han, Caixia; Lu, Xiaobing; Li, Xiaohui; Xia, Xianchun; He, Zhonghu; Yan, Yueming

    2016-01-01

    Low molecular weight glutenin subunit is one of the important quality elements in wheat (Triticum aestivum L.). Although considerable allelic variation has been identified, the functional properties of individual alleles at Glu-3 loci are less studied. In this work, we performed the first comprehensive study on the molecular characteristics and functional properties of the Glu-B3h gene using the wheat cultivar CB037B and its Glu-B3 deletion line CB037C. The results showed that the Glu-B3h deletion had no significant effects on plant morphological or yield traits, but resulted in a clear reduction in protein body number and size and main quality parameters, including inferior mixing property, dough strength, loaf volume, and score. Molecular characterization showed that the Glu-B3h gene consists of 1179 bp, and its encoded B-subunit has a longer repetitive domain and an increased number of α-helices, as well as higher expression, which could contribute to superior flour quality. The SNP-based allele-specific PCR markers designed for the Glu-B3h gene were developed and validated with bread wheat holding various alleles at Glu-B3 locus, which could effectively distinguish the Glu-B3h gene from others at the Glu-B3 locus, and have potential applications for wheat quality improvement through marker-assisted selection. PMID:27273251

  1. Class IA PI3Kinase Regulatory Subunit, p85α, Mediates Mast Cell Development through Regulation of Growth and Survival Related Genes

    PubMed Central

    Krishnan, Subha; Mali, Raghuveer Singh; Koehler, Karl R.; Vemula, Sasidhar; Chatterjee, Anindya; Ghosh, Joydeep; Ramdas, Baskar; Ma, Peilin; Hashino, Eri; Kapur, Reuben

    2012-01-01

    Stem cell factor (SCF) mediated KIT receptor activation plays a pivotal role in mast cell growth, maturation and survival. However, the signaling events downstream from KIT are poorly understood. Mast cells express multiple regulatory subunits of class 1A PI3Kinase (PI3K) including p85α, p85β, p50α, and p55α. While it is known that PI3K plays an essential role in mast cells; the precise mechanism by which these regulatory subunits impact specific mast cell functions including growth, survival and cycling are not known. We show that loss of p85α impairs the growth, survival and cycling of mast cell progenitors (MCp). To delineate the molecular mechanism (s) by which p85α regulates mast cell growth, survival and cycling, we performed microarray analyses to compare the gene expression profile of MCps derived from WT and p85α-deficient mice in response to SCF stimulation. We identified 151 unique genes exhibiting altered expression in p85α-deficient cells in response to SCF stimulation compared to WT cells. Functional categorization based on DAVID bioinformatics tool and Ingenuity Pathway Analysis (IPA) software relates the altered genes due to lack of p85α to transcription, cell cycle, cell survival, cell adhesion, cell differentiation, and signal transduction. Our results suggest that p85α is involved in mast cell development through regulation of expression of growth, survival and cell cycle related genes. PMID:22238586

  2. The porcine gene TBP10 encodes a protein homologous to the human tat-binding protein/26S protease subunit family.

    PubMed

    Leeb, T; Rettenberger, G; Breech, J; Hameister, H; Brenig, B

    1996-03-01

    We have cloned a porcine gene, designated TBP1O, that belongs to the Tat-binding protein/26S protease subunit family. The genomic structure of the porcine TBP1O gene was analyzed after isolation of three overlapping genomic phage lambda clones. The TBP10 gene harbors 12 exons spanning 4.5 kb of chromosomal DNA. The TBP1O gene was assigned to Chromosome (Chr) 12 by fluorescence in situ hybridization (FISH) on metaphase chromosomes. The chromosomal location was confirmed by PCR analysis of a porcine-rodent hybrid cell panel. The TBP1O protein is encoded by a 1221 nucleotide cDNA and has a molecular mass of 45.6 kDa. The predicted amino acid sequence has highest similarity to the human and bovine p45 subunit of the 26S protease and the human transcription factor TRIP1. Further similarities were detected to the slime mold protein DdTBP1O and the Schizosaccharomyces pombe and Saccharomyces cerevisiae protein SUG1. Like DdTBP1O and other members of the protein family, the porcine TBP1O harbors a leucine zipper motif in the N-terminal region and a domain characteristics of ATP-dependent proteases in the C-terminal region. PMID:8833236

  3. Differential Expression of SWI/SNF Chromatin Remodeler Subunits Brahma and Brahma-Related Gene During Drug-Induced Liver Injury and Regeneration in Mouse Model.

    PubMed

    Sinha, Sonal; Verma, Sudhir; Chaturvedi, Madan M

    2016-08-01

    The chromatin remodeling activity of mammalian SWI/SNF complex is carried out by either Brahma (BRM) or Brahma-related gene (BRG-1). The BRG-1 regulates genes involved in cell proliferation, whereas BRM is associated with cell differentiation, and arrest of cell growth. Global modifications of histones and expression of genes of chromatin-remodeling subunits have not been studied in in vivo model systems. In the present study, we investigate epigenetic modifications of histones and the expression of genes in thioacetamide (TAA)-induced liver injury and regeneration in a mouse model. In the present study, we report that hepatocyte proliferation and H3S10 phosphorylation occur during 60 to 72 h post TAA treatment in mice. Furthermore, there was change in the H3K9 acetylation and H3K9 trimethylation pattern with respect to liver injury and regeneration phase. Looking into the expression pattern of Brg-1 and Brm, it is evident that they contribute substantially to the process of liver regeneration. The SWI/SNF remodeler might contain BRG-1 as its ATPase subunit during injury phase. Whereas, BRM-associated SWI/SNF remodeler might probably be predominant during decline of injury phase and initiation of regeneration phase. Furthermore, during the regeneration phase, BRG-1-containing remodeler again predominates. Considering all these observations, the present study depicts an interplay between chromatin interacting machineries in different phases of thioacetamide-induced liver injury and regeneration. PMID:27097303

  4. Low molecular weight glutenin subunit gene Glu-B3h confers superior dough strength and breadmaking quality in wheat (Triticum aestivum L.).

    PubMed

    Wang, Yaping; Zhen, Shoumin; Luo, Nana; Han, Caixia; Lu, Xiaobing; Li, Xiaohui; Xia, Xianchun; He, Zhonghu; Yan, Yueming

    2016-01-01

    Low molecular weight glutenin subunit is one of the important quality elements in wheat (Triticum aestivum L.). Although considerable allelic variation has been identified, the functional properties of individual alleles at Glu-3 loci are less studied. In this work, we performed the first comprehensive study on the molecular characteristics and functional properties of the Glu-B3h gene using the wheat cultivar CB037B and its Glu-B3 deletion line CB037C. The results showed that the Glu-B3h deletion had no significant effects on plant morphological or yield traits, but resulted in a clear reduction in protein body number and size and main quality parameters, including inferior mixing property, dough strength, loaf volume, and score. Molecular characterization showed that the Glu-B3h gene consists of 1179 bp, and its encoded B-subunit has a longer repetitive domain and an increased number of α-helices, as well as higher expression, which could contribute to superior flour quality. The SNP-based allele-specific PCR markers designed for the Glu-B3h gene were developed and validated with bread wheat holding various alleles at Glu-B3 locus, which could effectively distinguish the Glu-B3h gene from others at the Glu-B3 locus, and have potential applications for wheat quality improvement through marker-assisted selection. PMID:27273251

  5. The Chlamydomonas Dhc1 gene encodes a dynein heavy chain subunit required for assembly of the I1 inner arm complex.

    PubMed Central

    Myster, S H; Knott, J A; O'Toole, E; Porter, M E

    1997-01-01

    Multiple members of the dynein heavy chain (Dhc) gene family have been recovered in several organisms, but the relationships between these sequences and the Dhc isoforms that they encode are largely unknown. To identify Dhc loci and determine the specific functions of the individual Dhc isoforms, we have screened a collection of motility mutants generated by insertional mutagenesis in Chlamydomonas. In this report, we characterize one strain, pf9-3, in which the insertion event was accompanied by a deletion of approximately 13 kb of genomic DNA within the transcription unit of the Dhc1 gene. Northern blot analysis confirms that pf9-3 is a null mutation. Biochemical and structural studies of isolated axonemes demonstrate that the pf9-3 mutant fails to assemble the I1 inner arm complex, a two-headed dynein isoform composed of two Dhcs (1 alpha and 1 beta) and three intermediate chains. To determine if the Dhc1 gene product corresponds to one of the Dhcs of the I1 complex, antibodies were generated against a Dhc1-specific peptide sequence. Immunoblot analysis reveals that the Dhc1 gene encodes the 1 alpha Dhc subunit. These studies thus, identify the first inner arm Dhc locus to be described in any organism and further demonstrate that the 1 alpha Dhc subunit plays an essential role in the assembly of the I1 inner arm complex. Images PMID:9247642

  6. Inactivation of Genes Encoding Subunits of the Peripheral and Membrane Arms of Neurospora Mitochondrial Complex I and Effects on Enzyme Assembly

    PubMed Central

    Duarte, M.; Sousa, R.; Videira, A.

    1995-01-01

    We have isolated and characterized the nuclear genes encoding the 12.3-kD subunit of the membrane arm and the 29.9-kD subunit of the peripheral arm of complex I from Neurospora crassa. The former gene was known to be located in linkage group I and the latter is now assigned to linkage group IV of the fungal genome. The genes were separately transformed into different N. crassa strains and transformants with duplicated DNA sequences were isolated. Selected transformants were then mated with other strains to generate repeat-induced point mutations in both copies of the genes present in the nucleus of the parental transformant. From the progeny of the crosses, we were then able to recover two individual mutants lacking the 12.3- and 29.9-kD proteins in their mitochondria, mutants nuo12.3 and nuo29.9, respectively. Several other subunits of complex I are present in the mutant organelles, although with altered stoichiometries as compared with those in the wild-type strain. Based on the analysis of Triton-solubilized mitochondrial complexes in sucrose gradients, neither mutant is able to fully assemble complex I. Our results indicate that mutant nuo12.3 separately assembles the peripheral arm and most of the membrane arm of the enzyme. Mutant nuo29.9 seems to accumulate the membrane arm of complex I and being devoid of the peripheral part. This implicates the 29.9-kD protein in an early step of complex I assembly. PMID:7768434

  7. An analysis of glucocorticoid receptor-mediated gene expression in BEAS-2B human airway epithelial cells identifies distinct, ligand-directed, transcription profiles with implications for asthma therapeutics

    PubMed Central

    Joshi, T; Johnson, M; Newton, R; Giembycz, M

    2015-01-01

    Background and Purpose International asthma guidelines recommend that inhaled glucocorticoids be used as a monotherapy in all patients with mild to moderate disease because of their ability to suppress airways inflammation. Current evidence suggests that the therapeutic benefit of glucocorticoids is due to the transactivation and transrepression of anti-inflammatory and pro-inflammatory genes respectively. However, the extent to which clinically relevant glucocorticoids are equivalent in their ability to modulate gene expression is unclear. Experimental Approach A pharmacodynamics investigation of glucocorticoid receptor (GR)-mediated gene transactivation in BEAS-2B human airway epithelial cells was performed using a glucocorticoid response element luciferase reporter coupled with an analysis of glucocorticoid-inducible genes encoding proteins with anti-inflammatory and adverse-effect potential. Key Results Using transactivation as a functionally relevant output, a given glucocorticoid displayed a unique, gene expression ‘fingerprint’ where intrinsic efficacy and GR density were essential determinants. We showed that depending on the gene selected for analysis, a given glucocorticoid can behave as an antagonist, partial agonist, full agonist or even ‘super agonist’. In the likely event that different, tissue-dependent gene expression profiles are reproduced in vivo, then the anti-inflammatory and adverse-effect potential of many glucocorticoids currently available as asthma therapeutics may not be equivalent. Conclusions and Implications The generation of gene expression ‘fingerprints’ in target and off-target human tissues could assist the rational design of GR agonists with improved therapeutic ratios. This approach could identify compounds that are useful in the management of severe asthma and other inflammatory disorders where systemic exposure is desirable. PMID:25393397

  8. Flour Quality and Related Molecular Characterization of High Molecular Weight Glutenin Subunit Genes from Wild Emmer Wheat Accession TD-256.

    PubMed

    Zhang, Da-Le; He, Ting-Ting; Liang, Hui-Hui; Huang, Lu-Yu; Su, Ya-Zhong; Li, Yu-Ge; Li, Suo-Ping

    2016-06-22

    To clarify the effect of high molecular weight glutenin subunit (HMW-GS) from wild emmer wheat on flour quality, which has the same mobility as that from common wheat, the composition and molecular characterization of HMW-GS from wild emmer wheat accession TD-256, as well as its flour quality, were intensively analyzed. It is found that the mobilities of Glu-A1 and Glu-B1 subunits from TD-256 are consistent with those of bread wheat cv. 'XiaoYan 6'. Nevertheless, dough rheological properties of TD-256 reveal its poor flour quality. In the aspect of molecular structure from HMW-GS, only two conserved cysteine residues can be observed in the deduced protein sequence of 1Bx14* from TD-256, while most Glu-1Bx contain four conserved cysteine residues. In addition, as can be predicted from secondary structure, the quantity both of α-helixes and their amino acid residues of the subunits from TD-256 is fewer than those of common wheat. Though low molecular weight glutenin subunit (LMW-GS) and gliadin can also greatly influence flour quality, the protein structure of the HMW-GS revealed in this work can partly explain the poor flour quality of wild emmer accession TD-256. PMID:27243935

  9. Stably Expressed D Genome-derived HMW Glutenin Subunit Genes Transformed Into Different Durum Wheat Genotypes Change Dough Mixing Properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The glutenin subunits 1Dx5 and 1Dy10 are encoded by chromosome 1D and associated with higher dough strength in hexaploid bread wheats. In order to study the effects of their expression in different durum wheat genotypes, four cultivars commonly grown in the Mediterranean area were co-transformed, vi...

  10. Cloning and characterization of homeologous cellulose synthase catalytic subunit 2 genes from allotetraploid cotton (Gossypium hirsutum L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cellulose synthase catalytic subunits (CesAs) are the catalytic sites within a multisubunit complex for cellulose biosynthesis in plants. CesAs have been extensively studied in diploid plants, but are not well characterized in polyploid plants. Gossypium hirsutum is an allotetraploid cotton specie...

  11. The human gene (CSNK2A1) coding for the casein kinase II subunit [alpha] is located on chromosome 20 and contains tandemly arranged Alu repeats

    SciTech Connect

    Wirkner, U.; Lichter, P.; Pyerin, W. ); Voss, H.; Ansorge, W. )

    1994-01-15

    The authors have isolated and characterized an 18.9-kb genomic clone representing a central portion of the human casein kinase II (CKII) subunit [alpha] gene (CSNK2A1). Using the whole clone as a probe, the gene was localized on chromosome 20p13. The clone contains eight exons whose sequences comprise bases 102 to 824 of the coding region of the human CKII[alpha]. The exon/intron splice junctions conform to the gt/ag rule. Three of the nine introns are located at positions corresponding to those in the CKII[alpha] gene of the nematode Caenorhabditis elegans. The introns contain eight complete and eight incomplete Alu repeats. Some of the Alu sequences are arranged in tandems of two or three, which seem to originate from insertions of younger Alu sequences into the poly(A) region of previously integrated Alu sequences, as indicated by flanking direct repeats. 50 refs., 5 figs., 1 tab.

  12. Isolation and characterization of temperature-sensitive mutations in RPA190, the gene encoding the largest subunit of RNA polymerase I from Saccharomyces cerevisiae.

    PubMed Central

    Wittekind, M; Dodd, J; Vu, L; Kolb, J M; Buhler, J M; Sentenac, A; Nomura, M

    1988-01-01

    The isolation and characterization of temperature-sensitive mutations in RNA polymerase I from Saccharomyces cerevisiae are described. A plasmid carrying RPA190, the gene encoding the largest subunit of the enzyme, was subjected to in vitro mutagenesis with hydroxylamine. Using a plasmid shuffle screening system, five different plasmids were isolated which conferred a temperature-sensitive phenotype in haploid yeast strains carrying the disrupted chromosomal RPA190 gene. These temperature-sensitive alleles were transferred to the chromosomal RPA190 locus for mapping and physiology experiments. Accumulation of RNA was found to be defective in all mutant strains at the nonpermissive temperature. In addition, analysis of pulse-labeled RNA from two mutant strains at 37 degrees C showed that the transcription of rRNA genes was decreased, while that of 5S RNA was relatively unaffected. RNA polymerase I was partially purified from several of the mutant strains grown at the nonpermissive temperature and was shown to be deficient when assayed in vitro. Fine-structure mapping and sequencing of the mutant alleles demonstrated that all five mutations were unique. The rpa190-1 and rpa190-5 mutations are tightly clustered in region I (S.S. Broyles and B. Moss, Proc. Natl. Acad. Sci. USA 83:3141-3145, 1986), the putative zinc-binding region that is common to all eucaryotic RNA polymerase large subunits. The rpa190-3 mutation is located between regions III and IV, and a strain carrying it behaves as a mutant that is defective in the synthesis of the enzyme. This mutation lies within a previously unidentified segment of highly conserved amino acid sequence homology that is shared among the largest subunits of eucaryotic nuclear RNA polymerases. Another temperature-sensitive mutation, rpa190-2, creates a UGA nonsense codon. Images PMID:3054507

  13. The PI3K regulatory subunit gene PIK3R1 is under direct control of androgens and repressed in prostate cancer cells

    PubMed Central

    Munkley, Jennifer; Livermore, Karen E.; McClurg, Urszula L.; Kalna, Gabriela; Knight, Bridget; McCullagh, Paul; McGrath, John; Crundwell, Malcolm; Leung, Hing Y.; Robson, Craig N.; Harries, Lorna W.; Rajan, Prabhakar; Elliott, David J.

    2015-01-01

    Androgen receptor (AR) signalling and the PI3K pathway mediate survival signals in prostate cancer, and have been shown to regulate each other by reciprocal negative feedback, such that inhibition of one activates the other. Understanding the reciprocal regulation of these pathways is important for disease management as tumour cells can adapt and survive when either single pathway is inhibited pharmacologically. We recently carried out genome-wide exon-specific profiling of prostate cancer cells to identify novel androgen-regulated transcriptional events. Here we interrogated this dataset for novel androgen-regulated genes associated with the PI3K pathway. We find that the PI3K regulatory subunits PIK3R1 (p85α) and PIK3R3 (p55γ) are direct targets of the AR which are rapidly repressed by androgens in LNCaP cells. Further characterisation revealed that the PIK3CA p110α catalytic subunit is also indirectly regulated by androgens at the protein level. We show that PIK3R1 mRNA is significantly under-expressed in prostate cancer (PCa) tissue, and provide data to suggest a context-dependent regulatory mechanism whereby repression of the p85α protein by the AR results in destabilisation of the PI3K p110α catalytic subunit and downstream PI3K pathway inhibition that functionally affects the properties of prostate cancer cells. PMID:26501081

  14. Nonsense mutation in the phosphofructokinase muscle subunit gene associated with retention of intron 10 in one of the isolated transcripts in Ashkenazi Jewish patients with Tarui disease.

    PubMed Central

    Vasconcelos, O; Sivakumar, K; Dalakas, M C; Quezado, M; Nagle, J; Leon-Monzon, M; Dubnick, M; Gajdusek, D C; Goldfarb, L G

    1995-01-01

    Mutations in the human phosphofructokinase muscle subunit gene (PFKM) are known to cause myopathy classified as glycogenosis type VII (Tarui disease). Previously described molecular defects include base substitutions altering encoded amino acids or resulting in abnormal splicing. We report a mutation resulting in phosphofructokinase deficiency in three patients from an Ashkenazi Jewish family. Using a reverse transcription PCR assay, PFKM subunit transcripts differing by length were detected in skeletal muscle tissue of all three affected subjects. In the longer transcript, an insertion of 252 nucleotides totally homologous to the structure of the 10th intron of the PFKM gene was found separating exon 10 from exon 11. In addition, two single base transitions were identified by direct sequencing: [exon 6; codon 95; CGA (Arg) to TGA (stop)] and [exon 7; codon 172; ACC (Thr) to ACT (Thr)] in either transcript. Single-stranded conformational polymorphism and restriction enzyme analyses confirmed the presence of these point substitutions in genomic DNA and strongly suggested homozygosity for the pathogenic allele. The nonsense mutation at codon 95 appeared solely responsible for the phenotype in these patients, further expanding genetic heterogeneity of Tarui disease. Transcripts with and without intron 10 arising from identical mutant alleles probably resulted from differential pre-mRNA processing and may represent a novel message from the PFKM gene. Images Fig. 2 Fig. 4 Fig. 5 PMID:7479776

  15. Mutations in GAS8, a Gene Encoding a Nexin-Dynein Regulatory Complex Subunit, Cause Primary Ciliary Dyskinesia with Axonemal Disorganization.

    PubMed

    Jeanson, Ludovic; Thomas, Lucie; Copin, Bruno; Coste, André; Sermet-Gaudelus, Isabelle; Dastot-Le Moal, Florence; Duquesnoy, Philippe; Montantin, Guy; Collot, Nathalie; Tissier, Sylvie; Papon, Jean-François; Clement, Annick; Louis, Bruno; Escudier, Estelle; Amselem, Serge; Legendre, Marie

    2016-08-01

    Primary ciliary dyskinesia (PCD) is an autosomal recessive disease characterized by chronic respiratory infections of the upper and lower airways, hypofertility, and, in approximately half of the cases, situs inversus. This complex phenotype results from defects in motile cilia and sperm flagella. Among the numerous genes involved in PCD, very few-including CCDC39 and CCDC40-carry mutations that lead to a disorganization of ciliary axonemes with microtubule misalignment. Focusing on this particular phenotype, we identified bi-allelic loss-of-function mutations in GAS8, a gene that encodes a subunit of the nexin-dynein regulatory complex (N-DRC) orthologous to DRC4 of the flagellated alga Chlamydomonas reinhardtii. Unlike the majority of PCD patients, individuals with GAS8 mutations have motile cilia, which, as documented by high-speed videomicroscopy, display a subtle beating pattern defect characterized by slightly reduced bending amplitude. Immunofluorescence studies performed on patients' respiratory cilia revealed that GAS8 is not required for the proper expression of CCDC39 and CCDC40. Rather, mutations in GAS8 affect the subcellular localization of another N-DRC subunit called DRC3. Overall, this study, which identifies GAS8 as a PCD gene, unveils the key importance of the corresponding protein in N-DRC integrity and in the proper alignment of axonemal microtubules in humans. PMID:27120127

  16. Elevated breast cancer risk in irradiated BALB/c mice associates with unique functional polymorphism of the Prkdc (DNA-dependent protein kinase catalytic subunit) gene

    NASA Technical Reports Server (NTRS)

    Yu, Y.; Okayasu, R.; Weil, M. M.; Silver, A.; McCarthy, M.; Zabriskie, R.; Long, S.; Cox, R.; Ullrich, R. L.

    2001-01-01

    Female BALB/c mice are unusually radiosensitive and more susceptible than C57BL/6 and other tested inbred mice to ionizing radiation (IR)-induced mammary tumors. This breast cancer susceptibility is correlated with elevated susceptibility for mammary cell transformation and genomic instability following irradiation. In this study, we report the identification of two BALB/c strain-specific polymorphisms in the coding region of Prkdc, the gene encoding the DNA-dependent protein kinase catalytic subunit, which is known to be involved in DNA double-stranded break repair and post-IR signal transduction. First, we identified an A --> G transition at base 11530 resulting in a Met --> Val conversion at codon 3844 (M3844V) in the phosphatidylinositol 3-kinase domain upstream of the scid mutation (Y4046X). Second, we identified a C --> T transition at base 6418 resulting in an Arg --> Cys conversion at codon 2140 (R2140C) downstream of the putative leucine zipper domain. This unique PrkdcBALB variant gene is shown to be associated with decreased DNA-dependent protein kinase catalytic subunit activity and with increased susceptibility to IR-induced genomic instability in primary mammary epithelial cells. The data provide the first evidence that naturally arising allelic variation in a mouse DNA damage response gene may associate with IR response and breast cancer risk.

  17. Nuclear-Cytoplasmic Conflict in Pea (Pisum sativum L.) Is Associated with Nuclear and Plastidic Candidate Genes Encoding Acetyl-CoA Carboxylase Subunits

    PubMed Central

    Bogdanova, Vera S.; Zaytseva, Olga O.; Mglinets, Anatoliy V.; Shatskaya, Natalia V.; Kosterin, Oleg E.; Vasiliev, Gennadiy V.

    2015-01-01

    In crosses of wild and cultivated peas (Pisum sativum L.), nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized. PMID:25789472

  18. The Mitochondrial Cytochrome Oxidase Subunit I Gene Occurs on a Minichromosome with Extensive Heteroplasmy in Two Species of Chewing Lice, Geomydoecus aurei and Thomomydoecus minor.

    PubMed

    Pietan, Lucas L; Spradling, Theresa A; Demastes, James W

    2016-01-01

    In animals, mitochondrial DNA (mtDNA) typically occurs as a single circular chromosome with 13 protein-coding genes and 22 tRNA genes. The various species of lice examined previously, however, have shown mitochondrial genome rearrangements with a range of chromosome sizes and numbers. Our research demonstrates that the mitochondrial genomes of two species of chewing lice found on pocket gophers, Geomydoecus aurei and Thomomydoecus minor, are fragmented with the 1,536 base-pair (bp) cytochrome-oxidase subunit I (cox1) gene occurring as the only protein-coding gene on a 1,916-1,964 bp minicircular chromosome in the two species, respectively. The cox1 gene of T. minor begins with an atypical start codon, while that of G. aurei does not. Components of the non-protein coding sequence of G. aurei and T. minor include a tRNA (isoleucine) gene, inverted repeat sequences consistent with origins of replication, and an additional non-coding region that is smaller than the non-coding sequence of other lice with such fragmented mitochondrial genomes. Sequences of cox1 minichromosome clones for each species reveal extensive length and sequence heteroplasmy in both coding and noncoding regions. The highly variable non-gene regions of G. aurei and T. minor have little sequence similarity with one another except for a 19-bp region of phylogenetically conserved sequence with unknown function. PMID:27589589

  19. Histone H2B Monoubiquitination Mediated by HISTONE MONOUBIQUITINATION1 and HISTONE MONOUBIQUITINATION2 Is Involved in Anther Development by Regulating Tapetum Degradation-Related Genes in Rice1[OPEN

    PubMed Central

    Cao, Hong; Li, Xiaoying; Wang, Zhi; Ding, Meng; Sun, Yongzhen; Dong, Fengqin; Chen, Fengying; Liu, Li’an; Doughty, James; Li, Yong; Liu, Yong-Xiu

    2015-01-01

    Histone H2B monoubiquitination (H2Bub1) is an important regulatory mechanism in eukaryotic gene transcription and is essential for normal plant development. However, the function of H2Bub1 in reproductive development remains elusive. Here, we report rice (Oryza sativa) HISTONE MONOUBIQUITINATION1 (OsHUB1) and OsHUB2, the homologs of Arabidopsis (Arabidopsis thaliana) HUB1 and HUB2 proteins, which function as E3 ligases in H2Bub1, are involved in late anther development in rice. oshub mutants exhibit abnormal tapetum development and aborted pollen in postmeiotic anthers. Knockout of OsHUB1 or OsHUB2 results in the loss of H2Bub1 and a reduction in the levels of dimethylated lysine-4 on histone 3 (H3K4me2). Anther transcriptome analysis revealed that several key tapetum degradation-related genes including OsC4, rice Cysteine Protease1 (OsCP1), and Undeveloped Tapetum1 (UDT1) were down-regulated in the mutants. Further, chromatin immunoprecipitation assays demonstrate that H2Bub1 directly targets OsC4, OsCP1, and UDT1 genes, and enrichment of H2Bub1 and H3K4me2 in the targets is consistent to some degree. Our studies suggest that histone H2B monoubiquitination, mediated by OsHUB1 and OsHUB2, is an important epigenetic modification that in concert with H3K4me2, modulates transcriptional regulation of anther development in rice. PMID:26143250

  20. A study of single nucleotide polymorphisms of GRIN2B in schizophrenia from Chinese Han population.

    PubMed

    Guo, Zhenming; Niu, Weibo; Bi, Yan; Zhang, Rui; Ren, Decheng; Hu, Jiaxin; Huang, Xiaoye; Wu, Xi; Cao, Yanfei; Yang, Fengping; Wang, Lu; Li, Weidong; Li, Xingwang; Xu, Yifeng; He, Lin; Yu, Tao; He, Guang

    2016-09-01

    Schizophrenia is a severe and complex mental disorder with high heritability. There is evidence that mutations in the gene of Nmethyl-d-aspartate-type glutamate receptors (NMDAR) are associated with schizophrenia. GRIN2B encodes a subunit of NMDARs, and has been identified as a candidate gene for many psychiatric disorders, especially schizophrenia. In this study, we investigated whether single nucleotide polymorphisms (SNPs) in GRIN2B were associated with schizophrenia. Four SNPs (rs890, rs1806191, rs219872, rs172677) were genotyped in 752 schizophrenic patients and 846 healthy controls of the Chinese Han population. Our results indicate differences in allele and genotype frequencies of rs890 between case and control. These results were assessed by adapting different genetic models (codominant, dominant, recessive, overdominant, log-additive models). After controlling for confounding factors including sex and age, rs890 remained associated with schizophrenia. In addition, rs890 and rs1806191 were found to form a haplotype associated with schizophrenia. In summary, our results indicate that the GRIN2B SNP rs890 might be associated with schizophrenia in the Chinese Han population. PMID:27453061

  1. Role of the Cyclic AMP Response Element Binding Complex and Activation of Mitogen-Activated Protein Kinases in Synergistic Activation of the Glycoprotein Hormone α Subunit Gene by Epidermal Growth Factor and Forskolin

    PubMed Central

    Roberson, Mark S.; Ban, Makiko; Zhang, Tong; Mulvaney, Jennifer M.

    2000-01-01

    The aim of these studies was to elucidate a role for epidermal growth factor (EGF) signaling in the transcriptional regulation of the glycoprotein hormone α subunit gene, a subunit of chorionic gonadotropin. Studies examined the effects of EGF and the adenylate cyclase activator forskolin on the expression of a transfected α subunit reporter gene in a human choriocarcinoma cell line (JEG3). At maximal doses, administration of EGF resulted in a 50% increase in a subunit reporter activity; forskolin administration induced a fivefold activation; the combined actions of EGF and forskolin resulted in synergistic activation (greater than eightfold) of the α subunit reporter. Mutagenesis studies revealed that the cyclic AMP response elements (CRE) were required and sufficient to mediate EGF-forskolin-induced synergistic activation. The combined actions of EGF and forskolin resulted in potentiated activation of extracellular signal-regulated kinase (ERK) enzyme activity compared with EGF alone. Specific blockade of ERK activation was sufficient to block EGF-forskolin-induced synergistic activation of the α subunit reporter. Pretreatment of JEG3 cells with a p38 mitogen-activated protein kinase inhibitor did not influence activation of the α reporter. However, overexpression of c-Jun N-terminal kinase (JNK)-interacting protein 1 as a dominant interfering molecule abolished the synergistic effects of EGF and forskolin on the α subunit reporter. CRE binding studies suggested that the CRE complex consisted of CRE binding protein and EGF-ERK-dependent recruitment of c-Jun–c-Fos (AP-1) to the CRE. A dominant negative form of c-Fos (A-Fos) that specifically disrupts c-Jun–c-Fos DNA binding inhibited synergistic activation of the α subunit. Thus, synergistic activation of the α subunit gene induced by EGF-forskolin requires the ERK and JNK cascades and the recruitment of AP-1 to the CRE binding complex. PMID:10779323

  2. Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase {beta}-subunit gene family

    SciTech Connect

    Pestov, Nikolay B.; Zhao, Hao; Basrur, Venkatesha; Modyanov, Nikolai N.

    2011-09-09

    Highlights: {yields} Structural properties of BetaM and Na,K-ATPase {beta}-subunits are sharply different. {yields} BetaM protein is concentrated in nuclear membrane of skeletal myocytes. {yields} BetaM does not associate with a Na,K-ATPase {alpha}-subunit in skeletal muscle. {yields} Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. {yields} BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a {beta}-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase {beta}-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was

  3. Glutamate cysteine ligase modifier (GCLM) subunit gene is not associated with methamphetamine-use disorder or schizophrenia in the Japanese population.

    PubMed

    Kishi, Taro; Ikeda, Masashi; Kitajima, Tsuyoshi; Yamanouchi, Yoshio; Kinoshita, Yoko; Kawashima, Kunihiro; Inada, Toshiya; Harano, Mutsuo; Komiyama, Tokutaro; Hori, Toru; Yamada, Mitsuhiko; Iyo, Masaomi; Sora, Ichiro; Sekine, Yoshimoto; Ozaki, Norio; Ujike, Hiroshi; Iwata, Nakao

    2008-10-01

    A recent study showed a significant association between schizophrenia in European samples and the glutamate cysteine ligase modifier (GCLM) subunit gene, which is the key glutathione (GSH)-synthesizing enzyme. Since the symptoms of methamphetamine (METH)-induced psychosis are similar to those of schizophrenia, the GCLM gene is thought to be a good candidate gene for METH-use disorder or related disorders. To evaluate the association between the GCLM gene and METH-use disorder and schizophrenia, we conducted a case-control study of Japanese subjects (METH-use disorder, 185 cases; schizophrenia, 742 cases; and controls, 819). Four SNPs (2 SNPs from an original report and JSNP database, and 2 "tagging SNPs" from HapMap database) in the GCLM gene were examined in this association analysis; one SNP showed an association with both METH-use disorder and METH-induced psychosis. After Bonferroni's correction for multiple testing, however, this significance disappeared. No significant association was found with schizophrenia. Our findings suggest that a common genetic variation in the GCLM gene might not contribute to the risk of METH-use disorder and schizophrenia in the Japanese population. PMID:18991850

  4. Loss of the gene for the alpha subunit of ATP synthase (ATP5A1) from the W chromosome in the African grey parrot (Psittacus erithacus).

    PubMed

    de Kloet, S R

    2001-08-01

    This study describes the results of an analysis using Southern blotting, the polymerase chain reaction, and sequencing which shows that the African grey parrot (Psittacus erithacus) lacks the W-chromosomal gene for the alpha subunit of mitochondrial ATP synthase (ATP5A1W). Additional evidence shows that in other psittacines a fragment of the ATP5A1W gene contains five times as many nonsynonymous nucleotide replacements as the homologous fragment of the Z gene. Therefore, whereas in these other psittacines the corresponding ATP5A1Z protein fragment is highly conserved and varies by only a few, moderately conservative amino acid substitutions, the homologous ATP5A1W fragments contain a considerable number of, sometimes highly nonconservative, amino acid replacements. In one of these species, the ringneck parakeet (Psittacula krameri), the ATP5A1W gene is present in an inactive form because of the presence of a nonsense codon. Other changes, possibly leading to an inactive ATP5A1W gene product, involve the substitution of arginine residues by cysteine in the ATP5A1W protein of the mitred conure (Aratinga mitrata) and the blue and gold macaw (Ara ararauna). The data suggest also that although the divergence of the psittacine ATP5A1W and ATP5A1Z genes preceded the origin of the psittacidae, this divergence occurred independently of a similar process in the myna (Gracula religiosa), the outgroup used in this study. PMID:11479684

  5. Genetic Interactions between Reg1/Hex2 and Glc7, the Gene Encoding the Protein Phosphatase Type 1 Catalytic Subunit in Saccharomyces Cerevisiae

    PubMed Central

    Huang, D.; Chun, K. T.; Goebl, M. G.; Roach, P. J.

    1996-01-01

    Mutations in GLC7, the gene encoding the type 1 protein phosphatase catalytic subunit, cause a variety of abberrant phenotypes in yeast, such as impaired glycogen synthesis and relief of glucose repression of the expression of some genes. Loss of function of the REG1/HEX2 gene, necessary for glucose repression of several genes, was found to suppress the glycogen-deficient phenotype of the glc7-1 allele. Deletion of REG1 in a wild-type background led to overaccumulation of glycogen as well as slow growth and an enlarged cell size. However, loss of REG1 did not suppress other phenotypes associated with GLC7 mutations, such as inability to sporulate or, in cells bearing the glc7(Y-170) allele, lack of growth at 14°. The effect of REG1 deletion on glycogen accumulation is not simply due to derepression of glucose-repressed genes, although it does require the presence of SNF1, which encodes a protein kinase essential for expression of glucose-repressed genes and for glycogen accumulation. We propose that REG1 has a role in controlling glycogen accumulation. PMID:8722767

  6. Mapping of the {beta}{sub 2} subunit gene (GABRB2) to microdissected human chromosome 5q34-q35 defines a gene cluster for the most abundant GABA{sub A} receptor isoform

    SciTech Connect

    Russek, S.J.; Farb, D.H. |

    1994-10-01

    The {gamma}-aminobutyric acid receptor (GABA{sub A}R) is a multisubunit Cl{sup -} channel that mediates most fast inhibitory synaptic transmission in the central nervous system. Molecular evolution has given rise to many genetic variants of GABA{sub A}R subunits, including {alpha}{sub 1-6}, {beta}{sub 1-4}, {gamma}{sub 1-4}, {sigma}, and {rho}{sub 1-2}, suggesting that an enormous number of combinations of subunits are possible. Here we report that the {beta}{sub 2} gene is located on chromosome 5q34-q35, defining a cluster comprising {alpha}{sub 1}, {beta}{sub 2}, and {gamma}{sub 2} genes that together code for the most abundant GABA{sub A}R isoform. The fact that intron position is conserved in the {beta}{sub 1-3} genes, taken together with the observation that chromosomes 4 and 15 also contain distinct {alpha}-{beta}-{gamma} gene clusters, strongly suggests that an ancestral {alpha}-{beta}-{gamma} cluster was duplicated and translocated to at least two different chromosomes. This organization of GABA{sub A}R gene clusters may have been preserved as linkage provides a mechanism for facilitating coordinate gene expression. 34 refs., 5 figs., 1 tab.

  7. Long-chain unsaturated fatty acids reduce the transcriptional activity of the rat follicle-stimulating hormone β-subunit gene

    PubMed Central

    MORIYAMA, Ryutaro; YAMAZAKI, Tsubasa; KATO, Takako; KATO, Yukio

    2016-01-01

    Here, we assessed the effects of long-chain fatty acids (LCFAs) and the LCFA receptor agonist GW9508 on the transcription of the gonadotropin subunit genes Cga, Lhb and Fshb because LCFA receptor GPR120 was observed in mouse gonadotropes in our recent study. A transcription assay using LβT2 cells demonstrated that LCFAs, oleic acid, α-linolenic acid, docosahexaenoic acid and palmitate, repressed the expression of Cga, Lhb, and Fshb at concentrations between 50 and 100 µM. On the other hand, treatment with 10 µM unsaturated LCFAs, oleic acid, α-linolenic acid and docosahexaenoic acid, repressed only Fshb expression, while the same dose of a saturated LCFA, palmitate, had no effect on the expression of gonadotropin subunit genes. Furthermore, GW9508 did not affect promoter activity. Next, we examined deletion mutants of the upstream region of Fshb and found that the upstream regulatory region (-2824 to -2343 bp) of Fshb was responsible for the notable repression by 10 µM unsaturated LCFAs. Our results suggest that the upstream region of Fshb is susceptible to unsaturated LCFAs. In addition, unsaturated LCFAs play a role in repressing Fshb expression through the distal -2824 to -2343 bp region, which might be independent of the LCFA receptor GPR120 pathway. PMID:26853521

  8. The RICE MINUTE-LIKE1 (RML1) gene, encoding a ribosomal large subunit protein L3B, regulates leaf morphology and plant architecture in rice

    PubMed Central

    Zheng, Ming; Wang, Yihua; Liu, Xi; Sun, Juan; Wang, Yunlong; Xu, Yang; Lv, Jia; Long, Wuhua; Zhu, Xiaopin; Guo, Xiuping; Jiang, Ling; Wang, Chunming; Wan, Jianmin

    2016-01-01

    Mutations of ribosomal proteins (RPs) are known to cause developmental abnormalities in yeast, mammals, and dicotyledonous plants; however, their effects have not been studied in rice. Here, we identifiy a ribosomal biogenesis mutant, rice minute-like1 (rml1) that displays a minute phenotype as evidenced by retarded growth and defects in the vascular system. We determine that RML1 encodes a ribosome large subunit protein 3B (RPL3B) in rice by means of map-based cloning and genetic complementation. RPL3B is abundantly expressed in all the tissues, whereas RPL3A, another RPL3 gene family member, is expressed at low levels. Notably, the expression level of RPL3A in the rml1 mutant is similar to that in the wild-type, suggesting that RPL3A provides no functional compensation for RPL3B in rml1 plants. Ribosomal profiles show that mutation of RPL3B leads to a significant reduction in free 60S ribosomal subunits and polysomes, indicating a ribosomal insufficiency in the rml1 mutant. Our results demonstrate that the ribosomal protein gene RPL3B is required for maintaining normal leaf morphology and plant architecture in rice through its regulation of ribosome biogenesis. PMID:27241493

  9. ATP25, a New Nuclear Gene of Saccharomyces cerevisiae Required for Expression and Assembly of the Atp9p Subunit of Mitochondrial ATPase

    PubMed Central

    Zeng, Xiaomei; Barros, Mario H.; Shulman, Theodore

    2008-01-01

    We report a new nuclear gene, designated ATP25 (reading frame YMR098C on chromosome XIII), required for expression of Atp9p (subunit 9) of the Saccharomyces cerevisiae mitochondrial proton translocating ATPase. Mutations in ATP25 elicit a deficit of ATP9 mRNA and of its translation product, thereby preventing assembly of functional F0. Unlike Atp9p, the other mitochondrial gene products, including ATPase subunits Atp6p and Atp8p, are synthesized normally in atp25 mutants. Northern analysis of mitochondrial RNAs in an atp25 temperature-sensitive mutant confirmed that Atp25p is required for stability of the ATP9 mRNA. Atp25p is a mitochondrial inner membrane protein with a predicted mass of 70 kDa. The primary translation product of ATP25 is cleaved in vivo after residue 292 to yield a 35-kDa C-terminal polypeptide. The C-terminal half of Atp25p is sufficient to stabilize the ATP9 mRNA and restore synthesis of Atp9p. Growth on respiratory substrates, however, depends on both halves of Atp25p, indicating that the N-terminal half has another function, which we propose to be oligomerization of Atp9p into a proper size ring structure. PMID:18216280

  10. The RICE MINUTE-LIKE1 (RML1) gene, encoding a ribosomal large subunit protein L3B, regulates leaf morphology and plant architecture in rice.

    PubMed

    Zheng, Ming; Wang, Yihua; Liu, Xi; Sun, Juan; Wang, Yunlong; Xu, Yang; Lv, Jia; Long, Wuhua; Zhu, Xiaopin; Guo, Xiuping; Jiang, Ling; Wang, Chunming; Wan, Jianmin

    2016-05-01

    Mutations of ribosomal proteins (RPs) are known to cause developmental abnormalities in yeast, mammals, and dicotyledonous plants; however, their effects have not been studied in rice. Here, we identifiy a ribosomal biogenesis mutant, rice minute-like1 (rml1) that displays a minute phenotype as evidenced by retarded growth and defects in the vascular system. We determine that RML1 encodes a ribosome large subunit protein 3B (RPL3B) in rice by means of map-based cloning and genetic complementation. RPL3B is abundantly expressed in all the tissues, whereas RPL3A, another RPL3 gene family member, is expressed at low levels. Notably, the expression level of RPL3A in the rml1 mutant is similar to that in the wild-type, suggesting that RPL3A provides no functional compensation for RPL3B in rml1 plants. Ribosomal profiles show that mutation of RPL3B leads to a significant reduction in free 60S ribosomal subunits and polysomes, indicating a ribosomal insufficiency in the rml1 mutant. Our results demonstrate that the ribosomal protein gene RPL3B is required for maintaining normal leaf morphology and plant architecture in rice through its regulation of ribosome biogenesis. PMID:27241493

  11. Sequence Variation in the Small-Subunit rRNA Gene of Plasmodium malariae and Prevalence of Isolates with the Variant Sequence in Sichuan, China

    PubMed Central

    Liu, Qing; Zhu, Shenghua; Mizuno, Sahoko; Kimura, Masatsugu; Liu, Peina; Isomura, Shin; Wang, Xingzhen; Kawamoto, Fumihiko

    1998-01-01

    By two PCR-based diagnostic methods, Plasmodium malariae infections have been rediscovered at two foci in the Sichuan province of China, a region where no cases of P. malariae have been officially reported for the last 2 decades. In addition, a variant form of P. malariae which has a deletion of 19 bp and seven substitutions of base pairs in the target sequence of the small-subunit (SSU) rRNA gene was detected with high frequency. Alignment analysis of Plasmodium sp. SSU rRNA gene sequences revealed that the 5′ region of the variant sequence is identical to that of P. vivax or P. knowlesi and its 3′ region is identical to that of P. malariae. The same sequence variations were also found in P. malariae isolates collected along the Thai-Myanmar border, suggesting a wide distribution of this variant form from southern China to Southeast Asia. PMID:9774600

  12. Disease-Associated Mutations in the HSPD1 Gene Encoding the Large Subunit of the Mitochondrial HSP60/HSP10 Chaperonin Complex

    PubMed Central

    Bross, Peter; Fernandez-Guerra, Paula

    2016-01-01

    Heat shock protein 60 (HSP60) forms together with heat shock protein 10 (HSP10) double-barrel chaperonin complexes that are essential for folding to the native state of proteins in the mitochondrial matrix space. Two extremely rare monogenic disorders have been described that are caused by missense mutations in the HSPD1 gene that encodes the HSP60 subunit of the HSP60/HSP10 chaperonin complex. Investigations of the molecular mechanisms underlying these disorders have revealed that different degrees of reduced HSP60 function produce distinct neurological phenotypes. While mutations with deleterious or strong dominant negative effects are not compatible with life, HSPD1 gene variations found in the human population impair HSP60 function and depending on the mechanism and degree of HSP60 dys- and mal-function cause different phenotypes. We here summarize the knowledge on the effects of disturbances of the function of the HSP60/HSP10 chaperonin complex by disease-associated mutations.

  13. Redescriptions of three trachelocercid ciliates (Protista, Ciliophora, Karyorelictea), with notes on their phylogeny based on small subunit rRNA gene sequences.

    PubMed

    Yan, Ying; Xu, Yuan; Yi, Zhenzhen; Warren, Alan

    2013-09-01

    Three trachelocercid ciliates, Kovalevaia sulcata (Kovaleva, 1966) Foissner, 1997, Trachelocerca sagitta (Müller, 1786) Ehrenberg, 1840 and Trachelocerca ditis (Wright, 1982) Foissner, 1996, isolated from two coastal habitats at Qingdao, China, were investigated using live observation and silver impregnation methods. Data on their infraciliature and morphology are supplied. The small subunit rRNA (SSU rRNA) genes of K. sulcata and Trachelocerca sagitta were sequenced for the first time. Phylogenetic analyses based on SSU rRNA gene sequence data indicate that both organisms, and the previously sequenced Trachelocerca ditis, are located within the trachelocercid assemblage and that K. sulcata is sister to an unidentified taxon forming a clade that is basal to the core trachelocercids. PMID:23847285

  14. TFIIIB subunit locations on U6 gene promoter DNA mapped by site-specific protein-DNA photo-cross-linking.

    PubMed

    Kang, Jin Joo; Kang, Yoon Soon; Stumph, William E

    2016-05-01

    RNA polymerase III-transcribed U6 snRNA genes have gene-external promoters that contain TATA boxes. U6 TATA sequences are bound by TFIIIB that in Drosophila contains the three subunits TBP, Brf1, and Bdp1. The overall structure of TFIIIB is still not well understood. We have therefore studied the mode of TFIIIB binding to DNA by site-specific protein-DNA photo-cross-linking. The results indicate that a portion of Brf1 is sandwiched between Bdp1 and TBP upstream of the TATA box. Furthermore, Bdp1 traverses the DNA under the N-terminal stirrup of TBP to interact with the DNA (and very likely Brf1) downstream of the TATA sequence. PMID:27112515

  15. Interaction of factor XIII subunits.

    PubMed

    Katona, Eva; Pénzes, Krisztina; Csapó, Andrea; Fazakas, Ferenc; Udvardy, Miklós L; Bagoly, Zsuzsa; Orosz, Zsuzsanna Z; Muszbek, László

    2014-03-13

    Coagulation factor XIII (FXIII) is a heterotetramer consisting of 2 catalytic A subunits (FXIII-A2) and 2 protective/inhibitory B subunits (FXIII-B2). FXIII-B, a mosaic protein consisting of 10 sushi domains, significantly prolongs the lifespan of catalytic subunits in the circulation and prevents their slow progressive activation in plasmatic conditions. In this study, the biochemistry of the interaction between the 2 FXIII subunits was investigated. Using a surface plasmon resonance technique and an enzyme-linked immunosorbent assay-type binding assay, the equilibrium dissociation constant (Kd) for the interaction was established in the range of 10(-10) M. Based on the measured Kd, it was calculated that in plasma approximately 1% of FXIII-A2 should be in free form. This value was confirmed experimentally by measuring FXIII-A2 in plasma samples immunodepleted of FXIII-A2B2. Free plasma FXIII-A2 is functionally active, and when activated by thrombin and Ca(2+), it can cross-link fibrin. In cerebrospinal fluid and tears with much lower FXIII subunit concentrations, >80% of FXIII-A2 existed in free form. A monoclonal anti-FXIII-B antibody that prevented the interaction between the 2 subunits reacted with the recombinant combined first and second sushi domains of FXIII-B, and its epitope was localized to the peptide spanning positions 96 to 103 in the second sushi domain. PMID:24408323

  16. Diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes in the MgCl2-dominated deep hypersaline anoxic basin discovery.

    PubMed

    van der Wielen, Paul W J J

    2006-06-01

    Partial sequences of the form I (cbbL) and form II (cbbM) of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit genes were obtained from the brine and interface of the MgCl2-dominated deep hypersaline anoxic basin Discovery. CbbL and cbbM genes were found in both brine and interface of the Discovery Basin but were absent in the overlying seawater. The diversity of both genes in the brine and interface was low, which might caused by the extreme saline conditions in Discovery of approximately 5 M MgCl2. None of the retrieved sequences were closely related to sequences deposited in the GenBank database. A phylogenetic analysis demonstrated that the cbbL sequences were affiliated with a Thiobacillus sp. or with one of the RuBisCO genes from Hydrogenovibrio marinus. The cbbM sequences clustered with thiobacilli or formed a new group with no close relatives. The results implicate that bacteria with the potential for carbon dioxide fixation and chemoautotrophy are present in the Discovery Basin. This is the first report demonstrating that RuBisCO genes are present under hypersaline conditions of 5 M MgCl2. PMID:16734797

  17. Pancreatic Cancer Stage 2B

    MedlinePlus

    ... 2B Description: Stage IIB pancreatic cancer; drawing shows cancer in the pancreas and in nearby lymph nodes. Also shown are the bile duct, pancreatic duct, and duodenum. Stage IIB pancreatic cancer. Cancer has spread to nearby lymph nodes and ...

  18. Different expression of protein kinase A (PKA) regulatory subunits in cortisol-secreting adrenocortical tumors: Relationship with cell proliferation

    SciTech Connect

    Mantovani, G.; Lania, A.G.; Bondioni, S.; Peverelli, E.; Pedroni, C.; Ferrero, S.; Pellegrini, C.; Vicentini, L.; Arnaldi, G.; Bosari, S.; Beck-Peccoz, P.; Spada, A.

    2008-01-01

    The four regulatory subunits (R1A, R1B, R2A, R2B) of protein kinase A (PKA) are differentially expressed in several cancer cell lines and exert distinct roles in growth control. Mutations of the R1A gene have been found in patients with Carney complex and in a minority of sporadic primary pigmented nodular adrenocortical disease (PPNAD). The aim of this study was to evaluate the expression of PKA regulatory subunits in non-PPNAD adrenocortical tumors causing ACTH-independent Cushing's syndrome and to test the impact of differential expression of these subunits on cell growth. Immunohistochemistry demonstrated a defective expression of R2B in all cortisol-secreting adenomas (n = 16) compared with the normal counterpart, while both R1A and R2A were expressed at high levels in the same tissues. Conversely, carcinomas (n = 5) showed high levels of all subunits. Sequencing of R1A and R2B genes revealed a wild type sequence in all tissues. The effect of R1/R2 ratio on proliferation was assessed in mouse adrenocortical Y-1 cells. The R2-selective cAMP analogue 8-Cl-cAMP dose-dependently inhibited Y-1 cell proliferation and induced apoptosis, while the R1-selective cAMP analogue 8-HA-cAMP stimulated cell proliferation. Finally, R2B gene silencing induced up-regulation of R1A protein, associated with an increase in cell proliferation. In conclusion, we propose that a high R1/R2 ratio favors the proliferation of well differentiated and hormone producing adrenocortical cells, while unbalanced expression of these subunits is not required for malignant transformation.

  19. Analysis of the Type IV Fimbrial-Subunit Gene fimA of Xanthomonas hyacinthi: Application in PCR-Mediated Detection of Yellow Disease in Hyacinths

    PubMed Central

    van Doorn, J.; Hollinger, T. C.; Oudega, B.

    2001-01-01

    A sensitive and specific detection method was developed for Xanthomonas hyacinthi; this method was based on amplification of a subsequence of the type IV fimbrial-subunit gene fimA from strain S148. The fimA gene was amplified by PCR with degenerate DNA primers designed by using the N-terminal and C-terminal amino acid sequences of trypsin fragments of FimA. The nucleotide sequence of fimA was determined and compared with the nucleotide sequences coding for the fimbrial subunits in other type IV fimbria-producing bacteria, such as Xanthomonas campestris pv. vesicatoria, Neisseria gonorrhoeae, and Moraxella bovis. In a PCR internal primers JAAN and JARA, designed by using the nucleotide sequences of the variable central and C-terminal region of fimA, amplified a 226-bp DNA fragment in all X. hyacinthi isolates. This PCR was shown to be pathovar specific, as assessed by testing 71 Xanthomonas pathovars and bacterial isolates belonging to other genera, such as Erwinia and Pseudomonas. Southern hybridization experiments performed with the labelled 226-bp DNA amplicon as a probe suggested that there is only one structural type IV fimbrial-gene cluster in X. hyacinthi. Only two Xanthomonas translucens pathovars cross-reacted weakly in PCR. Primers amplifying a subsequence of the fimA gene of X. campestris pv. vesicatoria (T. Ojanen-Reuhs, N. Kalkkinen, B. Westerlund-Wikström, J. van Doorn, K. Haahtela, E.-L. Nurmiaho-Lassila, K. Wengelink, U. Bonas, and T. K. Korhonen, J. Bacteriol. 179: 1280–1290, 1997) were shown to be pathovar specific, indicating that the fimbrial-subunit sequences are more generally applicable in xanthomonads for detection purposes. Under laboratory conditions, approximately 1,000 CFU of X. hyacinthi per ml could be detected. In inoculated leaves of hyacinths the threshold was 5,000 CFU/ml. The results indicated that infected hyacinths with early symptoms could be successfully screened for X. hyacinthi with PCR. PMID:11157222

  20. The dependence receptor UNC5H2/B triggers apoptosis via PP2A-mediated dephosphorylation of DAP kinase.

    PubMed

    Guenebeaud, Céline; Goldschneider, David; Castets, Marie; Guix, Catherine; Chazot, Guillaume; Delloye-Bourgeois, Céline; Eisenberg-Lerner, Avital; Shohat, Galit; Zhang, Mingjie; Laudet, Vincent; Kimchi, Adi; Bernet, Agnès; Mehlen, Patrick

    2010-12-22

    The UNC5H dependence receptors promote apoptosis in the absence of their ligand, netrin-1, and this is important for neuronal and vascular development and for limitation of cancer progression. UNC5H2 (also called UNC5B) triggers cell death through the activation of the serine-threonine protein kinase DAPk. While performing a siRNA screen to identify genes implicated in UNC5H-induced apoptosis, we identified the structural subunit PR65β of the holoenzyme protein phosphatase 2A (PP2A). We show that UNC5H2/B recruits a protein complex that includes PR65β and DAPk and retains PP2A activity. PP2A activity is required for UNC5H2/B-induced apoptosis, since it activates DAPk by triggering its dephosphorylation. Moreover, netrin-1 binding to UNC5H2/B prevents this effect through interaction of the PP2A inhibitor CIP2A to UNC5H2/B. Thus we show here that, in the absence of netrin-1, recruitment of PP2A to UNC5H2/B allows the activation of DAPk via a PP2A-mediated dephosphorylation and that this mechanism is involved in angiogenesis regulation. PMID:21172653

  1. Distinct roles of NR2A and NR2B cytoplasmic tails in long term potentiation

    PubMed Central

    Foster, Kelly A.; McLaughlin, Nathan; Edbauer, Dieter; Phillips, Marnie; Bolton, Andrew; Constantine-Paton, Martha; Sheng, Morgan

    2010-01-01

    NMDA receptors (NMDARs) are critical mediators of activity-dependent synaptic plasticity, but the differential roles of NR2A- versus NR2B-containing NMDARs have been controversial. Here, we investigate the roles of NR2A and NR2B in LTP in organotypic hippocampal slice cultures using RNAi and overexpression, to complement pharmacological approaches. In young slices, when NR2B is the predominant subunit expressed, LTP is blocked by the NR2B-selective antagonist Ro25-6981. As slices mature, and NR2A expression rises, activation of NR2B receptors became no longer necessary for LTP induction. LTP was blocked, however, by RNAi knockdown of NR2B, and this was rescued by coexpression of an RNAi-resistant NR2B (NR2B*) cDNA. Interestingly, a chimeric NR2B subunit in which the C-terminal cytoplasmic tail was replaced by that of NR2A failed to rescue LTP while the reverse chimera, NR2A channel with NR2B tail, was able to restore LTP. Thus expression of NR2B with its intact cytoplasmic tail is required for LTP induction, at an age when channel activity of NR2B-NMDARs is not required for LTP. Overexpression of wildtype NR2A failed to rescue LTP in neurons transfected with NR2B-RNAi construct, despite restoring NMDA-EPSC amplitude to a similar level as NR2B*. Surprisingly, an NR2A construct lacking its entire C-terminal cytoplasmic tail regained its ability to restore LTP. Together these data suggest that the NR2B subunit plays a critical role for LTP, presumably by recruiting relevant molecules important for LTP via its cytoplasmic tail. By contrast, NR2A is not essential for LTP and its cytoplasmic tail seems to carry inhibitory factors for LTP. PMID:20164351

  2. Mutations in exocyst complex subunit SEC6 gene impaired polar auxin transport and PIN protein recycling in Arabidopsis primary root.

    PubMed

    Tan, Xiaoyun; Feng, Yihong; Liu, Yulong; Bao, Yiqun

    2016-09-01

    Polar auxin transport, which is critical for land plant pattern formation and directional growth, is largely depended on asymmetric distribution of PIN proteins at the plasma membrane (PM). Endocytosis and recycling processes play important roles in regulating PIN protein distribution and abundance at the PM. Two subunits (SEC8, EXO70A1) of exocyst, an octameric vesicle-tethering complex, have been reported to be involved in PIN protein recycling in Arabidopsis. However, the function of exocyst complex in PIN protein recycling and polar auxin transport remains incompletely understood. In this study, we utilized two SEC6 down-regulation mutants (PRsec6-1 and PRsec6-2) to investigate the role of exocyst subunit SEC6 in the primary root development, polar auxin transport and PIN proteins recycling. We found that in PRsec6 mutants: 1. Primary root growth was retarded, and lateral root initiation were compromised. 2. Primary roots were sensitive to exogenous auxin 1-napthalene acetic acid (NAA) but not 2,4-dichlorophenoxy (2.4-D). 3. Recycling of PIN1 and PIN2 proteins from the Brefeldin A (BFA) compartment to the PM was delayed. 4. Vesicles accumulated in the primary root tip cells, especially accumulated in the cytosol closed to the PM. These results further demonstrated that the exocyst complex plays an important role in PIN protein recycling and polar auxin transport in Arabidopsis primary root. PMID:27457987

  3. SRC Inhibition Reduces NR2B Surface Expression and Synaptic Plasticity in the Amygdala

    ERIC Educational Resources Information Center

    Sinai, Laleh; Duffy, Steven; Roder, John C.

    2010-01-01

    The Src protein tyrosine kinase plays a central role in the regulation of N-methyl-d-aspartate receptor (NMDAR) activity by regulating NMDAR subunit 2B (NR2B) surface expression. In the amygdala, NMDA-dependent synaptic plasticity resulting from convergent somatosensory and auditory inputs contributes to emotional memory; however, the role of Src…

  4. Expression of the glycoprotein hormone alpha-subunit gene in the placenta requires a functional cyclic AMP response element, whereas a different cis-acting element mediates pituitary-specific expression.

    PubMed Central

    Bokar, J A; Keri, R A; Farmerie, T A; Fenstermaker, R A; Andersen, B; Hamernik, D L; Yun, J; Wagner, T; Nilson, J H

    1989-01-01

    The single-copy gene encoding the alpha subunit of glycoprotein hormones is expressed in the pituitaries of all mammals and in the placentas of only primates and horses. We have systematically analyzed the promoter-regulatory elements of the human and bovine alpha-subunit genes to elucidate the molecular mechanisms underlying their divergent patterns of tissue-specific expression. This analysis entailed the use of transient expression assays in a chorionic gonadotropin-secreting human choriocarcinoma cell line, protein-DNA binding assays, and expression of chimeric forms of human or bovine alpha subunit genes in transgenic mice. From the results, we conclude that placental expression of the human alpha-subunit gene requires a functional cyclic AMP response element (CRE) that is present as a tandem repeat in the promoter-regulatory region. In contrast, the promoter-regulatory region of the bovine alpha-subunit gene, as well as of the rat and mouse genes, was found to contain a single CRE homolog that differed from its human counterpart by a single nucleotide. This difference substantially reduced the binding affinity of the bovine CRE homolog for the nuclear protein that bound to the human alpha CRE and thereby rendered the bovine alpha-subunit promoter inactive in human choriocarcinoma cells. However, conversion of the bovine alpha CRE homolog to an authentic alpha CRE restored activity to the bovine alpha-subunit promoter in choriocarcinoma cells. Similarly, a human but not a bovine alpha transgene was expressed in placenta in transgenic mice. Thus, placenta-specific expression of the human alpha-subunit gene may be the consequence of the recent evolution of a functional CRE. Expression of the human alpha transgene in mouse placenta further suggests that evolution of placenta-specific trans-acting factors preceded the appearance of this element. Finally, in contrast to their divergent patterns of placental expression, both the human and bovine alpha-subunit

  5. A Common Polymorphism of the Human Cardiac Sodium Channel Alpha Subunit (SCN5A) Gene Is Associated with Sudden Cardiac Death in Chronic Ischemic Heart Disease

    PubMed Central

    Marcsa, Boglárka; Dénes, Réka; Vörös, Krisztina; Rácz, Gergely; Sasvári-Székely, Mária; Rónai, Zsolt; Törő, Klára; Keszler, Gergely

    2015-01-01

    Cardiac death remains one of the leading causes of mortality worldwide. Recent research has shed light on pathophysiological mechanisms underlying cardiac death, and several genetic variants in novel candidate genes have been identified as risk factors. However, the vast majority of studies performed so far investigated genetic associations with specific forms of cardiac death only (sudden, arrhythmogenic, ischemic etc.). The aim of the present investigation was to find a genetic marker that can be used as a general, powerful predictor of cardiac death risk. To this end, a case-control association study was performed on a heterogeneous cohort of cardiac death victims (n=360) and age-matched controls (n=300). Five single nucleotide polymorphisms (SNPs) from five candidate genes (beta2 adrenergic receptor, nitric oxide synthase 1 adaptor protein, ryanodine receptor 2, sodium channel type V alpha subunit and transforming growth factor-beta receptor 2) that had previously been shown to associate with certain forms of cardiac death were genotyped using sequence-specific real-time PCR probes. Logistic regression analysis revealed that the CC genotype of the rs11720524 polymorphism in the SCN5A gene encoding a subunit of the cardiac voltage-gated sodium channel occurred more frequently in the highly heterogeneous cardiac death cohort compared to the control population (p=0.019, odds ratio: 1.351). A detailed subgroup analysis uncovered that this effect was due to an association of this variant with cardiac death in chronic ischemic heart disease (p=0.012, odds ratio = 1.455). None of the other investigated polymorphisms showed association with cardiac death in this context. In conclusion, our results shed light on the role of this non-coding polymorphism in cardiac death in ischemic cardiomyopathy. Functional studies are needed to explore the pathophysiological background of this association. PMID:26146998

  6. Dominant Red Coat Color in Holstein Cattle Is Associated with a Missense Mutation in the Coatomer Protein Complex, Subunit Alpha (COPA) Gene

    PubMed Central

    Dorshorst, Ben; Henegar, Corneliu; Liao, Xiaoping; Sällman Almén, Markus; Rubin, Carl-Johan; Ito, Shosuke; Wakamatsu, Kazumasa; Stothard, Paul; Van Doormaal, Brian; Plastow, Graham; Barsh, Gregory S.; Andersson, Leif

    2015-01-01

    Coat color in Holstein dairy cattle is primarily controlled by the melanocortin 1 receptor (MC1R) gene, a central determinant of black (eumelanin) vs. red/brown pheomelanin synthesis across animal species. The major MC1R alleles in Holsteins are Dominant Black (MC1RD) and Recessive Red (MC1Re). A novel form of dominant red coat color was first observed in an animal born in 1980. The mutation underlying this phenotype was named Dominant Red and is epistatic to the constitutively activated MC1RD. Here we show that a missense mutation in the coatomer protein complex, subunit alpha (COPA), a gene with previously no known role in pigmentation synthesis, is completely associated with Dominant Red in Holstein dairy cattle. The mutation results in an arginine to cysteine substitution at an amino acid residue completely conserved across eukaryotes. Despite this high level of conservation we show that both heterozygotes and homozygotes are healthy and viable. Analysis of hair pigment composition shows that the Dominant Red phenotype is similar to the MC1R Recessive Red phenotype, although less effective at reducing eumelanin synthesis. RNA-seq data similarly show that Dominant Red animals achieve predominantly pheomelanin synthesis by downregulating genes normally required for eumelanin synthesis. COPA is a component of the coat protein I seven subunit complex that is involved with retrograde and cis-Golgi intracellular coated vesicle transport of both protein and RNA cargo. This suggests that Dominant Red may be caused by aberrant MC1R protein or mRNA trafficking within the highly compartmentalized melanocyte, mimicking the effect of the Recessive Red loss of function MC1R allele. PMID:26042826

  7. Dominant Red Coat Color in Holstein Cattle Is Associated with a Missense Mutation in the Coatomer Protein Complex, Subunit Alpha (COPA) Gene.

    PubMed

    Dorshorst, Ben; Henegar, Corneliu; Liao, Xiaoping; Sällman Almén, Markus; Rubin, Carl-Johan; Ito, Shosuke; Wakamatsu, Kazumasa; Stothard, Paul; Van Doormaal, Brian; Plastow, Graham; Barsh, Gregory S; Andersson, Leif

    2015-01-01

    Coat color in Holstein dairy cattle is primarily controlled by the melanocortin 1 receptor (MC1R) gene, a central determinant of black (eumelanin) vs. red/brown pheomelanin synthesis across animal species. The major MC1R alleles in Holsteins are Dominant Black (MC1RD) and Recessive Red (MC1Re). A novel form of dominant red coat color was first observed in an animal born in 1980. The mutation underlying this phenotype was named Dominant Red and is epistatic to the constitutively activated MC1RD. Here we show that a missense mutation in the coatomer protein complex, subunit alpha (COPA), a gene with previously no known role in pigmentation synthesis, is completely associated with Dominant Red in Holstein dairy cattle. The mutation results in an arginine to cysteine substitution at an amino acid residue completely conserved across eukaryotes. Despite this high level of conservation we show that both heterozygotes and homozygotes are healthy and viable. Analysis of hair pigment composition shows that the Dominant Red phenotype is similar to the MC1R Recessive Red phenotype, although less effective at reducing eumelanin synthesis. RNA-seq data similarly show that Dominant Red animals achieve predominantly pheomelanin synthesis by downregulating genes normally required for eumelanin synthesis. COPA is a component of the coat protein I seven subunit complex that is involved with retrograde and cis-Golgi intracellular coated vesicle transport of both protein and RNA cargo. This suggests that Dominant Red may be caused by aberrant MC1R protein or mRNA trafficking within the highly compartmentalized melanocyte, mimicking the effect of the Recessive Red loss of function MC1R allele. PMID:26042826

  8. A new genotype of Trypanosoma cruzi associated with bats evidenced by phylogenetic analyses using SSU rDNA, cytochrome b and Histone H2B genes and genotyping based on ITS1 rDNA.

    PubMed

    Marcili, A; Lima, L; Cavazzana, M; Junqueira, A C V; Veludo, H H; Maia Da Silva, F; Campaner, M; Paiva, F; Nunes, V L B; Teixeira, M M G

    2009-05-01

    We characterized 15 Trypanosoma cruzi isolates from bats captured in the Amazon, Central and Southeast Brazilian regions. Phylogenetic relationships among T. cruzi lineages using SSU rDNA, cytochrome b, and Histone H2B genes positioned all Amazonian isolates into T. cruzi I (TCI). However, bat isolates from the other regions, which had been genotyped as T. cruzi II (TC II) by the traditional genotyping method based on mini-exon gene employed in this study, were not nested within any of the previously defined TCII sublineages, constituting a new genotype designated as TCbat. Phylogenetic analyses demonstrated that TCbat indeed belongs to T. cruzi and not to other closely related bat trypanosomes of the subgenus Schizotrypanum, and that although separated by large genetic distances TCbat is closest to lineage TCI. A genotyping method targeting ITS1 rDNA distinguished TCbat from established T. cruzi lineages, and from other Schizotrypanum species. In experimentally infected mice, TCbat lacked virulence and yielded low parasitaemias. Isolates of TCbat presented distinctive morphological features and behaviour in triatomines. To date, TCbat genotype was found only in bats from anthropic environments of Central and Southeast Brazil. Our findings indicate that the complexity of T. cruzi is larger than currently known, and confirmed bats as important reservoirs and potential source of T. cruzi infections to humans. PMID:19368741

  9. Mutations in a gene encoding the. cap alpha. subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling

    SciTech Connect

    Jahng, K.Y.; Ferguson, J.; Reed, S.I.

    1988-06-01

    Mutations which allowed conjugation by Saccharomyces cerevisiae cells lacking a mating pheromone receptor gene were selected. One of the genes defined by such mutations was isolated from a yeast genomic library by complementation of a temperature-sensitive mutation and is identically to the gene GPA1 (also known as SCG1), recently shown to be highly homologous to gene encoding the ..cap alpha.. subunits of mammalian G proteins. Physiological analysis of temperature-sensitive gpal mutations suggests that the encoded G protein is involved in signaling in response to mating pheromones. Mutational disruption of G-protein activity causes cell-cycle arrest in G/sub 1/, deposition of mating-specific cell surface aggultinins, and induction of pheromone-specific mRNa, all of which are responses to pheromone in wild-type cells. In addition, mutants can conjugate without the benefit of mating pheromone or pheromone receptor. A model is presented where the activated G protein has a negative impact on a constitutive signal which normally keeps the pheromone response repressed.

  10. Msx1 homeodomain transcription factor and TATA-binding protein interact to repress the expression of the glycoprotein hormone α subunit gene.

    PubMed

    Park, Ki-Sun; Kim, Kee K; Kim, Kyoon Eon

    Studying the regulatory mechanism of the glycoprotein hormone α subunit (αGSU) gene in thyrotropes is essential for understanding the synthesis of functional thyroid-stimulating hormone (TSH). Here, we investigated the influence of a homeodomain transcription factor Msx1 (Msh homeobox 1) on αGSU expression in thyrotropes. The transient expression of Msx1 inhibited the activity of an αGSU reporter gene, as well as its endogenous mRNA level in thyrotrope-derived αTSH cells. Luciferase reporter assays with serial deletion constructs and a close examination of the sequences revealed that the putative Msx1 binding site (PMS) in the αGSU promoter is not responsible for Msx1-mediated transcriptional repression. We also identified the TATA-box binding protein (TBP) as an interacting protein in thyrotropes. Interaction of TBP with Msx1 attenuates the inhibitory effect of Msx1 on αGSU gene expression in a DNA binding-independent manner. Furthermore, transient transfection studies with mutant Msx1 revealed that the interaction of TBP and Msx1 is critical for Msx1-mediated transcriptional repression of the αGSU. These results suggest that Msx1 functions as a transcriptional repressor of αGSU and that its interaction with TBP is an integral part of the mechanism by which Msx1 regulates the inhibition of αGSU gene expression. PMID:26505791

  11. Quantitative analyses of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large-subunit genes (cbbL) in typical paddy soils.

    PubMed

    Xiao, Ke-Qing; Bao, Peng; Bao, Qiong-Li; Jia, Yan; Huang, Fu-Yi; Su, Jian-Qiang; Zhu, Yong-Guan

    2014-01-01

    The Calvin cycle is known to be the major pathway for CO2 fixation, but our current understanding of its occurrence and importance in paddy soils is poor. In this study, the diversity of three ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes (cbbLG, cbbLR, cbbM) was investigated by clone library, T-RFLP, qPCR, and enzyme assay in five paddy soils in China. The cbbLG sequences revealed a relatively low level of diversity and were mostly related to the sequences of species from Thiobacillus. In contrast, highly diverse cbbLR and cbbM sequences were dispersed on the phylogenetic trees, and most of them were distantly related to known sequences, even forming separate clusters. Abundances of three cbbL genes ranged from 10(6) to 10(9) copies g(-1) soil, and cbbLR outnumbered cbbM and cbbLG in all soil samples, indicating that cbbLR may play a more important role than other two cbbL genes. Soil properties significantly influenced cbbL diversity in five paddy soils, of which clay content, C/N ratio, CEC, pH, and SOC correlated well with variations in microbial composition and abundance. In summary, this study provided a comparison of three cbbL genes, advancing our understanding of their role in carbon sequestration and nutrient turnover in the paddy soil. PMID:24024547

  12. Molecular characterization and expression analysis of the gene coding for the porcine beta(3) integrin subunit (CD61).

    PubMed

    Jiménez-Marín, Angeles; Yubero, Noemí; Esteso, Gloria; Moreno, Angela; de las Mulas, Juana Martín; Morera, Luis; Llanes, Diego; Barbancho, Manuel; Garrido, Juan J

    2008-01-31

    Integrins are heterodimeric cell adhesion molecules with major roles in a variety of biological processes ranging from cell migration to tissue organization, immune and non-immune defense mechanisms and oncogenic transformation. Members of the beta(3) integrin subfamily are composed of a beta(3) subunit (CD61) non-covalently associated with two alpha subunits, alpha(IIb) (CD41) and alpha(v) (CD51), to constitute a group of transmembrane glycoproteins that participate in many physiologically important events. This investigation has focused on the molecular characterization of the cDNA encoding the porcine beta(3) integrin subunit. The deduced 762-amino acid sequence was 93, 92, 91, 89, 79 and 73% homologous to human, dog, rabbit, mouse, chicken and Xenopus laevis CD61 protein, respectively. Porcine CD61 molecule shares many structural features with human CD61, including a region containing a metal ion-dependent adhesion site (MIDAS) folding into an I domain-like structure. Through PCR-SSCP analysis and sequencing, six polymorphic positions were detected in the cDNA sequence of porcine CD61, and their frequencies were observed from a collection of 47 pigs. Expression analysis was done at two different levels: expression of the CD61 mRNA by RT-PCR and localization of the protein by immunohistochemistry. Our results show that CD61 transcripts were detected mainly in platelets and hematopoietic tissues. The immunohistochemical tissue localization of CD61 protein by a specific monoclonal antibody against CD61 recombinant protein showed that CD61 was expressed on vascular and non-vascular smooth muscle, epithelium and myeloid cells, being undetectable in cells of the lymphoid lineage. Furthermore, pulmonary intravascular macrophages (PIM), a subpopulation of macrophages which seem to play an important role in blood clearance, expressed much more CD61 when compared to pulmonary alveolar macrophages (PAM). The knowledge of the structure and distribution of the CD61 provides

  13. Molecular cloning of cDNA for the B beta subunit of Xenopus fibrinogen, the product of a coordinately-regulated gene family.

    PubMed

    Bhattacharya, A; Shepard, A R; Moser, D R; Roberts, L R; Holland, L J

    1991-02-01

    Fibrinogen, the principal blood-clotting protein, is made up of three different subunits synthesized in the liver. In vitro administration of glucocorticoids to liver cells from the frog Xenopus laevis causes a dramatic increase in fibrinogen synthesis. Investigations of molecular mechanisms underlying this hormonal stimulation at the mRNA level require cDNA clones complementary to the mRNAs coding for the three fibrinogen subunits, called A alpha, B beta, and gamma. We describe here the isolation and characterization of cDNA clones for the B beta subunit of Xenopus fibrinogen. cDNA libraries in both plasmid (pBR322) and phage (lambda gt10) cloning vectors were constructed from frog liver mRNA and screened with a rat B beta cDNA. Clones thus isolated hybridized to two Xenopus liver mRNAs 2500 and 1800 bases long, the previously-determined sizes for B beta mRNAs. The identity of the plasmid clone B beta-27 was confirmed by hybridization-selection of complementary mRNA which translated in vitro into the B beta polypeptide, as determined by size and susceptibility to thrombin cleavage. lambda/B beta 10, a clone representing nearly all of the 2500-base B beta mRNA, was isolated from the phage cDNA library. The 3'-end of this clone includes a polyadenylation signal about 20 residues upstream of a stretch of 34 adenosine residues, which probably represents the 3'-poly(A) tail of the messenger RNA. lambda/B beta 10 lacks only 20 nucleotides of full-length B beta mRNA at the 5'-end and there is one major start site of transcription. The 2500-base B beta mRNA has a 700-base extension at the 3'-end that is not present in the 1800-base mRNA. The Xenopus laevis genome contains two or three genes for the B beta fibrinogen subunit. Using the cDNA clone as a probe, B beta mRNA was shown to be induced at least 20-fold by glucocorticoid treatment of purified parenchymal cells of Xenopus liver maintained in primary culture. PMID:2050271

  14. Evaluation of the DNA-dependent RNA polymerase ß-subunit gene (rpoB) for phytoplasma classification and phylogeny

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytoplasmas are classified into 16Sr groups, subgroups, and ‘Candidatus Phytoplasma’ species, largely or entirely based on analysis of 16S rRNA gene sequences. Yet, distinctions among closely related ‘Candidatus Phytoplasma’ species and strains based on 16S rRNA gene sequences alone has limitation...

  15. Characterization of the proteasome ß2 subunit gene and its mutant allele in the tephritid fruit fly pest, Anastrepha suspensa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Conditional lethal release (CLR) is a proposed variation of the sterile insect technique (SIT) for the biological control of pest insects that would result from the release of transgenic insects carrying dominant conditional lethal genes. After mating with pest insects in the field, lethal gene exp...

  16. Development of multiplex PCR assay for authentication of Cornu Cervi Pantotrichum in traditional Chinese medicine based on cytochrome b and C oxidase subunit 1 genes.

    PubMed

    Gao, Lijun; Xia, Wei; Ai, Jinxia; Li, Mingcheng; Yuan, Guanxin; Niu, Jiamu; Fu, Guilian; Zhang, Lihua

    2016-07-01

    This study describes a method for discriminating the true Cervus antlers from its counterfeits using multiplex PCR. Bioinformatics were carried out to design the spec