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Sample records for 1c subunit cacna1c

  1. CACNA1C hypermethylation is associated with bipolar disorder.

    PubMed

    Starnawska, A; Demontis, D; Pen, A; Hedemand, A; Nielsen, A L; Staunstrup, N H; Grove, J; Als, T D; Jarram, A; O'Brien, N L; Mors, O; McQuillin, A; Børglum, A D; Nyegaard, M

    2016-01-01

    The CACNA1C gene, encoding a subunit of the L-type voltage-gated calcium channel is one of the best-supported susceptibility genes for bipolar disorder (BD). Genome-wide association studies have identified a cluster of non-coding single-nucleotide polymorphisms (SNPs) in intron 3 to be highly associated with BD and schizophrenia. The mechanism by which these SNPs confer risk of BD appears to be through an altered regulation of CACNA1C expression. The role of CACNA1C DNA methylation in BD has not yet been addressed. The aim of this study was to investigate if CACNA1C DNA methylation is altered in BD. First, the methylation status of five CpG islands (CGIs) across CACNA1C in blood from BD subjects (n=40) and healthy controls (n=38) was determined. Four islands were almost completely methylated or completely unmethylated, while one island (CGI 3) in intron 3 displayed intermediate methylation levels. In the main analysis, the methylation status of CGI 3 was analyzed in a larger sample of BD subjects (n=582) and control individuals (n=319). Out of six CpG sites that were investigated, five sites showed significant hypermethylation in cases (lowest P=1.16 × 10(-7) for CpG35). Nearby SNPs were found to influence the methylation level, and we identified rs2238056 in intron 3 as the strongest methylation quantitative trait locus (P=2.6 × 10(-7)) for CpG35. In addition, we found an increased methylation in females, and no difference between bipolar I and II. In conclusion, we find that CACNA1C methylation is associated with BD and suggest that the regulatory effect of the non-coding risk variants involves a shift in DNA methylation. PMID:27271857

  2. CACNA1C hypermethylation is associated with bipolar disorder

    PubMed Central

    Starnawska, A; Demontis, D; Pen, A; Hedemand, A; Nielsen, A L; Staunstrup, N H; Grove, J; Als, T D; Jarram, A; O'Brien, N L; Mors, O; McQuillin, A; Børglum, A D; Nyegaard, M

    2016-01-01

    The CACNA1C gene, encoding a subunit of the L-type voltage-gated calcium channel is one of the best-supported susceptibility genes for bipolar disorder (BD). Genome-wide association studies have identified a cluster of non-coding single-nucleotide polymorphisms (SNPs) in intron 3 to be highly associated with BD and schizophrenia. The mechanism by which these SNPs confer risk of BD appears to be through an altered regulation of CACNA1C expression. The role of CACNA1C DNA methylation in BD has not yet been addressed. The aim of this study was to investigate if CACNA1C DNA methylation is altered in BD. First, the methylation status of five CpG islands (CGIs) across CACNA1C in blood from BD subjects (n=40) and healthy controls (n=38) was determined. Four islands were almost completely methylated or completely unmethylated, while one island (CGI 3) in intron 3 displayed intermediate methylation levels. In the main analysis, the methylation status of CGI 3 was analyzed in a larger sample of BD subjects (n=582) and control individuals (n=319). Out of six CpG sites that were investigated, five sites showed significant hypermethylation in cases (lowest P=1.16 × 10−7 for CpG35). Nearby SNPs were found to influence the methylation level, and we identified rs2238056 in intron 3 as the strongest methylation quantitative trait locus (P=2.6 × 10−7) for CpG35. In addition, we found an increased methylation in females, and no difference between bipolar I and II. In conclusion, we find that CACNA1C methylation is associated with BD and suggest that the regulatory effect of the non-coding risk variants involves a shift in DNA methylation. PMID:27271857

  3. CLOCK-BMAL1 regulate the cardiac L-type calcium channel subunit CACNA1C through PI3K-Akt signaling pathway.

    PubMed

    Chen, Yanhong; Zhu, Didi; Yuan, Jiamin; Han, Zhonglin; Wang, Yao; Qian, Zhiyong; Hou, Xiaofeng; Wu, Tingting; Zou, Jiangang

    2016-09-01

    The heterodimerized transcription factors CLOCK-BMAL1 regulate the cardiomyocyte circadian rhythms. The L-type calcium currents play important role in the cardiac electrogenesis and arrhythmogenesis. Whether and how the CLOCK-BMAL1 regulate the cardiac L-type calcium channels are yet to be determined. The functions of the L-type calcium channels were evaluated with patch clamping techniques. Recombinant adenoviruses of CLOCK and BMAL1 were used in the expression experiments. We reported that the expressions and functions of CACNA1C (the α-subunit of the L-type calcium channels) showed circadian rhythms, with the peak at zeitgeber time 3 (ZT3). The endocardial action potential durations 90 (APD90) were correspondingly longer at ZT3. The protein levels of the phosphorylated Akt at threonine 308 (pAkt T308) also showed circadian rhythms. Overexpressions of CLOCK-BMAL1 significantly reduced the levels of CACNA1C while increasing the levels of pAkt T308 and pik3r1. Furthermore, the inhibitory effects of CLOCK-BMAL1 on CACNA1C could be abolished by the Akt inhibitor MK2206 or the PDK1 inhibitor GSK2334470. Collectively, our findings suggested that the expressions of the cardiac CACNA1C were under the CLOCK-BMAL1 regulation, probably through the PI3K-Akt signal pathway. PMID:27376484

  4. CACNA1C risk variant affects reward responsiveness in healthy individuals.

    PubMed

    Lancaster, T M; Heerey, E A; Mantripragada, K; Linden, D E J

    2014-01-01

    The variant at rs1006737 in the L-type voltage-gated calcium channel (alpha 1c subunit) CACNA1C gene is reliably associated with both bipolar disorder and schizophrenia. We investigated whether this risk variant affects reward responsiveness because reward processing is one of the central cognitive-motivational domains implicated in both disorders. In a sample of 164 young, healthy individuals, we show a dose-dependent response, where the rs1006737 risk genotype was associated with blunted reward responsiveness, whereas discriminability did not significantly differ between genotype groups. This finding suggests that the CACNA1C risk locus may have a role in neural pathways that facilitate value representation for rewarding stimuli. Impaired reward processing may be a transdiagnostic phenotype of variation in CACNA1C that could contribute to anhedonia and other clinical features common to both affective and psychotic disorders. PMID:25290268

  5. Functional Characterization of Schizophrenia-Associated Variation in CACNA1C

    PubMed Central

    Eckart, Nicole; Song, Qifeng; Yang, Rebecca; Wang, Ruihua; Zhu, Heng; McCallion, Andrew S.; Avramopoulos, Dimitrios

    2016-01-01

    Calcium channel subunits, including CACNA1C, have been associated with multiple psychiatric disorders. Specifically, genome wide association studies (GWAS) have repeatedly identified the single nucleotide polymorphism (SNP) rs1006737 in intron 3 of CACNA1C to be strongly associated with schizophrenia and bipolar disorder. Here, we show that rs1006737 marks a quantitative trait locus for CACNA1C transcript levels. We test 16 SNPs in high linkage disequilibrium with rs1007637 and find one, rs4765905, consistently showing allele-dependent regulatory function in reporter assays. We find allele-specific protein binding for 13 SNPs including rs4765905. Using protein microarrays, we identify several proteins binding ≥3 SNPs, but not control sequences, suggesting possible functional interactions and combinatorial haplotype effects. Finally, using circular chromatin conformation capture, we show interaction of the disease-associated region including the 16 SNPs with the CACNA1C promoter and other potential regulatory regions. Our results elucidate the pathogenic relevance of one of the best-supported risk loci for schizophrenia and bipolar disorder. PMID:27276213

  6. Functional Characterization of Schizophrenia-Associated Variation in CACNA1C.

    PubMed

    Eckart, Nicole; Song, Qifeng; Yang, Rebecca; Wang, Ruihua; Zhu, Heng; McCallion, Andrew S; Avramopoulos, Dimitrios

    2016-01-01

    Calcium channel subunits, including CACNA1C, have been associated with multiple psychiatric disorders. Specifically, genome wide association studies (GWAS) have repeatedly identified the single nucleotide polymorphism (SNP) rs1006737 in intron 3 of CACNA1C to be strongly associated with schizophrenia and bipolar disorder. Here, we show that rs1006737 marks a quantitative trait locus for CACNA1C transcript levels. We test 16 SNPs in high linkage disequilibrium with rs1007637 and find one, rs4765905, consistently showing allele-dependent regulatory function in reporter assays. We find allele-specific protein binding for 13 SNPs including rs4765905. Using protein microarrays, we identify several proteins binding ≥3 SNPs, but not control sequences, suggesting possible functional interactions and combinatorial haplotype effects. Finally, using circular chromatin conformation capture, we show interaction of the disease-associated region including the 16 SNPs with the CACNA1C promoter and other potential regulatory regions. Our results elucidate the pathogenic relevance of one of the best-supported risk loci for schizophrenia and bipolar disorder. PMID:27276213

  7. Cacna1c (Cav1.2) Modulates Electroencephalographic Rhythm and Rapid Eye Movement Sleep Recovery

    PubMed Central

    Kumar, Deependra; Dedic, Nina; Flachskamm, Cornelia; Voulé, Stephanie; Deussing, Jan M.; Kimura, Mayumi

    2015-01-01

    Study Objectives: The CACNA1C gene encodes the alpha 1C1C) subunit of the Cav1.2 voltage-dependent L-type calcium channel (LTCC). Some of the other voltage-dependent calcium channels, e.g., P-/Q-type, Cav2.1; N-type, Cav2.2; E-/R-type, Cav2.3; and T-type, Cav3.3 have been implicated in sleep modulation. However, the contribution of LTCCs to sleep remains largely unknown. Based on recent genome-wide association studies, CACNA1C emerged as one of potential candidate genes associated with both sleep and psychiatric disorders. Indeed, most patients with mental illnesses have sleep problems and vice versa. Design: To investigate an impact of Cav1.2 on sleep-wake behavior and electroencephalogram (EEG) activity, polysomnography was performed in heterozygous Cacna1c (HET) knockout mice and their wild-type (WT) littermates under baseline and challenging conditions (acute sleep deprivation and restraint stress). Measurements and Results: HET mice displayed significantly lower EEG spectral power than WT mice across high frequency ranges (beta to gamma) during wake and rapid eye movement (REM) sleep. Although HET mice spent slightly more time asleep in the dark period, daily amounts of sleep did not differ between the two genotypes. However, recovery sleep after exposure to both types of challenging stress conditions differed markedly; HET mice exhibited reduced REM sleep recovery responses compared to WT mice. Conclusions: These results suggest the involvement of Cacna1c (Cav1.2) in fast electroencephalogram oscillations and REM sleep regulatory processes. Lower spectral gamma activity, slightly increased sleep demands, and altered REM sleep responses found in heterozygous Cacna1c knockout mice may rather resemble a sleep phenotype observed in schizophrenia patients. Citation: Kumar D, Dedic N, FLachskamm C, Voulé S, Deussing JM, Kimura M. Cacna1c (Cav1.2) modulates electroencephalographic rhythm and rapid eye movement sleep recovery. SLEEP 2015;38(9):1371–1380. PMID

  8. The Neuropsychiatric Disease-Associated Gene cacna1c Mediates Survival of Young Hippocampal Neurons.

    PubMed

    Lee, Anni S; De Jesús-Cortés, Héctor; Kabir, Zeeba D; Knobbe, Whitney; Orr, Madeline; Burgdorf, Caitlin; Huntington, Paula; McDaniel, Latisha; Britt, Jeremiah K; Hoffmann, Franz; Brat, Daniel J; Rajadhyaksha, Anjali M; Pieper, Andrew A

    2016-01-01

    Genetic variations in CACNA1C, which encodes the Cav1.2 subunit of L-type calcium channels (LTCCs), are associated with multiple forms of neuropsychiatric disease that manifest high anxiety in patients. In parallel, mice harboring forebrain-specific conditional knockout of cacna1c (forebrain-Cav1.2 cKO) display unusually high anxiety-like behavior. LTCCs in general, including the Cav1.3 subunit, have been shown to mediate differentiation of neural precursor cells (NPCs). However, it has not previously been determined whether Cav1.2 affects postnatal hippocampal neurogenesis in vivo. Here, we show that forebrain-Cav1.2 cKO mice exhibit enhanced cell death of young hippocampal neurons, with no change in NPC proliferation, hippocampal size, dentate gyrus thickness, or corticosterone levels compared with wild-type littermates. These mice also exhibit deficits in brain levels of brain-derived neurotrophic factor (BDNF), and Cre recombinase-mediated knockdown of adult hippocampal Cav1.2 recapitulates the deficit in young hippocampal neurons survival. Treatment of forebrain-Cav1.2 cKO mice with the neuroprotective agent P7C3-A20 restored the net magnitude of postnatal hippocampal neurogenesis to wild-type levels without ameliorating their deficit in BDNF expression. The role of Cav1.2 in young hippocampal neurons survival may provide new approaches for understanding and treating neuropsychiatric disease associated with aberrations in CACNA1C. Visual Abstract. PMID:27066530

  9. The Neuropsychiatric Disease-Associated Gene cacna1c Mediates Survival of Young Hippocampal Neurons123

    PubMed Central

    Lee, Anni S.; Kabir, Zeeba D.; Knobbe, Whitney; Orr, Madeline; Burgdorf, Caitlin; Huntington, Paula; McDaniel, Latisha; Britt, Jeremiah K.; Hoffmann, Franz; Brat, Daniel J.; Rajadhyaksha, Anjali M.

    2016-01-01

    Genetic variations in CACNA1C, which encodes the Cav1.2 subunit of L-type calcium channels (LTCCs), are associated with multiple forms of neuropsychiatric disease that manifest high anxiety in patients. In parallel, mice harboring forebrain-specific conditional knockout of cacna1c (forebrain-Cav1.2 cKO) display unusually high anxiety-like behavior. LTCCs in general, including the Cav1.3 subunit, have been shown to mediate differentiation of neural precursor cells (NPCs). However, it has not previously been determined whether Cav1.2 affects postnatal hippocampal neurogenesis in vivo. Here, we show that forebrain-Cav1.2 cKO mice exhibit enhanced cell death of young hippocampal neurons, with no change in NPC proliferation, hippocampal size, dentate gyrus thickness, or corticosterone levels compared with wild-type littermates. These mice also exhibit deficits in brain levels of brain-derived neurotrophic factor (BDNF), and Cre recombinase-mediated knockdown of adult hippocampal Cav1.2 recapitulates the deficit in young hippocampal neurons survival. Treatment of forebrain-Cav1.2 cKO mice with the neuroprotective agent P7C3-A20 restored the net magnitude of postnatal hippocampal neurogenesis to wild-type levels without ameliorating their deficit in BDNF expression. The role of Cav1.2 in young hippocampal neurons survival may provide new approaches for understanding and treating neuropsychiatric disease associated with aberrations in CACNA1C. Visual Abstract PMID:27066530

  10. Functional implications of a psychiatric risk variant within CACNA1C in induced human neurons

    PubMed Central

    Yoshimizu, Takao; Pan, Jen Q.; Mungenast, Alison E.; Madison, Jon M.; Su, Susan; Ketterman, Josh; Ongur, Dost; McPhie, Donna; Cohen, Bruce; Perlis, Roy; Tsai, Li-Huei

    2014-01-01

    Psychiatric disorders have clear heritable risk. Several large-scale genome-wide association studies have revealed a strong association between susceptibility for psychiatric disorders, including bipolar disease, schizophrenia, and major depression, and a haplotype located in an intronic region of the L-type voltage gated calcium channel (VGCC) subunit gene CACNA1C (peak associated SNP rs1006737), making it one of the most replicable and consistent associations in psychiatric genetics. In the current study, we used induced human neurons to reveal a functional phenotype associated with this psychiatric risk variant. We generated induced human neurons, or iN cells, from more than 20 individuals harboring homozygous risk genotypes, heterozygous, or homozygous non-risk genotypes at the rs1006737 locus. Using these iNs, we performed electrophysiology and quantitative PCR experiments that demonstrated increased L-type VGCC current density as well as increased mRNA expression of CACNA1C in induced neurons homozygous for the risk genotype, compared to non-risk genotypes. These studies demonstrate that the risk genotype at rs1006737 is associated with significant functional alterations in human induced neurons, and may direct future efforts at developing novel therapeutics for the treatment of psychiatric disease. PMID:25403839

  11. Functional implications of a psychiatric risk variant within CACNA1C in induced human neurons.

    PubMed

    Yoshimizu, T; Pan, J Q; Mungenast, A E; Madison, J M; Su, S; Ketterman, J; Ongur, D; McPhie, D; Cohen, B; Perlis, R; Tsai, L-H

    2015-02-01

    Psychiatric disorders have clear heritable risk. Several large-scale genome-wide association studies have revealed a strong association between susceptibility for psychiatric disorders, including bipolar disease, schizophrenia and major depression, and a haplotype located in an intronic region of the L-type voltage-gated calcium channel (VGCC) subunit gene CACNA1C (peak associated SNP rs1006737), making it one of the most replicable and consistent associations in psychiatric genetics. In the current study, we used induced human neurons to reveal a functional phenotype associated with this psychiatric risk variant. We generated induced human neurons, or iN cells, from more than 20 individuals harboring homozygous risk genotypes, heterozygous or homozygous non-risk genotypes at the rs1006737 locus. Using these iNs, we performed electrophysiology and quantitative PCR experiments that demonstrated increased L-type VGCC current density as well as increased mRNA expression of CACNA1C in iNs homozygous for the risk genotype, compared with non-risk genotypes. These studies demonstrate that the risk genotype at rs1006737 is associated with significant functional alterations in human iNs, and may direct future efforts at developing novel therapeutics for the treatment of psychiatric disease. PMID:25403839

  12. CACNA1C risk variant affects facial emotion recognition in healthy individuals

    PubMed Central

    Nieratschker, Vanessa; Brückmann, Christof; Plewnia, Christian

    2015-01-01

    Recognition and correct interpretation of facial emotion is essential for social interaction and communication. Previous studies have shown that impairments in this cognitive domain are common features of several psychiatric disorders. Recent association studies identified CACNA1C as one of the most promising genetic risk factors for psychiatric disorders and previous evidence suggests that the most replicated risk variant in CACNA1C (rs1006737) is affecting emotion recognition and processing. However, studies investigating the influence of rs1006737 on this intermediate phenotype in healthy subjects at the behavioral level are largely missing to date. Here, we applied the “Reading the Mind in the Eyes” test, a facial emotion recognition paradigm in a cohort of 92 healthy individuals to address this question. Whereas accuracy was not affected by genotype, CACNA1C rs1006737 risk-allele carries (AA/AG) showed significantly slower mean response times compared to individuals homozygous for the G-allele, indicating that healthy risk-allele carriers require more information to correctly identify a facial emotion. Our study is the first to provide evidence for an impairing behavioral effect of the CACNA1C risk variant rs1006737 on facial emotion recognition in healthy individuals and adds to the growing number of studies pointing towards CACNA1C as affecting intermediate phenotypes of psychiatric disorders. PMID:26611642

  13. CACNA1C risk variant affects facial emotion recognition in healthy individuals.

    PubMed

    Nieratschker, Vanessa; Brückmann, Christof; Plewnia, Christian

    2015-01-01

    Recognition and correct interpretation of facial emotion is essential for social interaction and communication. Previous studies have shown that impairments in this cognitive domain are common features of several psychiatric disorders. Recent association studies identified CACNA1C as one of the most promising genetic risk factors for psychiatric disorders and previous evidence suggests that the most replicated risk variant in CACNA1C (rs1006737) is affecting emotion recognition and processing. However, studies investigating the influence of rs1006737 on this intermediate phenotype in healthy subjects at the behavioral level are largely missing to date. Here, we applied the "Reading the Mind in the Eyes" test, a facial emotion recognition paradigm in a cohort of 92 healthy individuals to address this question. Whereas accuracy was not affected by genotype, CACNA1C rs1006737 risk-allele carries (AA/AG) showed significantly slower mean response times compared to individuals homozygous for the G-allele, indicating that healthy risk-allele carriers require more information to correctly identify a facial emotion. Our study is the first to provide evidence for an impairing behavioral effect of the CACNA1C risk variant rs1006737 on facial emotion recognition in healthy individuals and adds to the growing number of studies pointing towards CACNA1C as affecting intermediate phenotypes of psychiatric disorders. PMID:26611642

  14. Association of CACNA1C Variants with Bipolar Disorder in the Korean Population

    PubMed Central

    Kim, Soojin; Cho, Chul-Hyun; Geum, Dongho

    2016-01-01

    Objective Previous studies have suggested an association between CACNA1C and susceptibility of bipolar disorder. In this study, we examined the association of CACNA1C variants with bipolar disorder in the Korean population. Methods We selected 2 CACNA1C single nucleotide polymorphisms (SNPs), namely, rs723672 and rs1051375, based on their functions and minor allele frequencies described in previous studies. Genotypes of these 2 SNPs were analyzed by extracting DNA from blood samples collected from 287 patients with bipolar disorder and 340 healthy controls. Results Genotype frequencies of both rs723672 and rs1051375 SNPs were significantly different in patients and controls (p=0.0462 and 1.732E-14, respectively). Dominant, recessive, and allele models showed significant differences between patients and controls with respect to the rs1051375 SNP (p=1.72E-11, 4.17E-10, 4.95E-16, respectively). Conclusion Our results suggested that CACNA1C SNPs rs723672 and rs1051375 were associated with bipolar disorder in the Korean population. In addition, our results highlighted the importance of CACNA1C in determining susceptibility to bipolar disorder. PMID:27482248

  15. Analysis of ANK3 and CACNA1C variants identified in bipolar disorder whole genome sequence data

    PubMed Central

    Fiorentino, Alessia; O'Brien, Niamh Louise; Locke, Devin Paul; McQuillin, Andrew; Jarram, Alexandra; Anjorin, Adebayo; Kandaswamy, Radhika; Curtis, David; Blizard, Robert Alan; Gurling, Hugh Malcolm Douglas

    2014-01-01

    Objectives Genetic markers in the genes encoding ankyrin 3 (ANK3) and the α-calcium channel subunit (CACNA1C) are associated with bipolar disorder (BP). The associated variants in the CACNA1C gene are mainly within intron 3 of the gene. ANK3 BP-associated variants are in two distinct clusters at the ends of the gene, indicating disease allele heterogeneity. Methods In order to screen both coding and non-coding regions to identify potential aetiological variants, we used whole-genome sequencing in 99 BP cases. Variants with markedly different allele frequencies in the BP samples and the 1,000 genomes project European data were genotyped in 1,510 BP cases and 1,095 controls. Results We found that the CACNA1C intron 3 variant, rs79398153, potentially affecting an ENCyclopedia of DNA Elements (ENCODE)-defined region, showed an association with BP (p = 0.015). We also found the ANK3 BP-associated variant rs139972937, responsible for an asparagine to serine change (p = 0.042). However, a previous study had not found support for an association between rs139972937 and BP. The variants at ANK3 and CACNA1C previously known to be associated with BP were not in linkage disequilibrium with either of the two variants that we identified and these are therefore independent of the previous haplotypes implicated by genome-wide association. Conclusions Sequencing in additional BP samples is needed to find the molecular pathology that explains the previous association findings. If changes similar to those we have found can be shown to have an effect on the expression and function of ANK3 and CACNA1C, they might help to explain the so-called ‘missing heritability’ of BP. PMID:24716743

  16. Common variants in CACNA1C and MDD susceptibility: A comprehensive meta-analysis.

    PubMed

    Rao, Shuquan; Yao, Yao; Zheng, Chuan; Ryan, Joanne; Mao, Canquan; Zhang, Fuquan; Meyre, David; Xu, Qi

    2016-09-01

    Major depressive disorder (MDD) is one of the most common psychiatric disorders with a relatively high heritability (35-40%). Though rs1006737 in the CACNA1C gene showed significant association with MDD in a British large-scale candidate association study, most of the replication analyses with relatively small sample size reported negative association. Moreover, this locus has never been identified in previous genome-wide association studies (GWAS) for MDD. Here, we conducted a comprehensive meta-analysis of the association between CACNA1C variants and MDD risk by combining all published data. Genetic data from one European GWAS and five individual follow-up studies, which include up to 12,629 patients of MDD and 28,653 controls, that is, the largest sample size on CACNA1C to date, were collected. Rs1006737 showed significant association with MDD in the fixed-effect model (Z = 2.56, P = 0.011, OR = 1.08, 95%CI = 1.04-1.12) and the association remained after reanalyzing the data according to ethnicity. We additionally analyzed other 25 SNPs, genotyped in only one replication study, across the CACNA1C locus, and found that two SNPs, rs4765905 (P = 0.041, OR = 1.05, 95%CI 1.00-1.09) and rs4765937 (P = 0.025, OR = 1.05, 95%CI 1.01-1.09) showed nominal association with MDD, while rs2239073 (P = 0.002, OR = 1.07, 95%CI 1.02-1.11) exhibited significant association with MDD, which survived from multiple corrections. Our study provides support for positive association between CACNA1C and MDD; however, the current data suggest the necessity of replication analyses in a larger-scale sample. © 2016 Wiley Periodicals, Inc. PMID:27260792

  17. Novel Timothy Syndrome Mutation Leading to Increase in CACNA1C Window Current

    PubMed Central

    Boczek, Nicole J.; Miller, Erin M.; Ye, Dan; Nesterenko, Vlad V.; Tester, David J.; Antzelevitch, Charles; Czosek, Richard J.; Ackerman, Michael J.; Ware, Stephanie M.

    2016-01-01

    Background Timothy syndrome (TS) is a rare multi-system genetic disorder characterized by a myriad of abnormalities including QT prolongation, syndactyly, and neurological symptoms. The predominant genetic causes are recurrent de novo missense mutations in exon 8/8A of the CACNA1C-encoded L-type calcium channel, however some cases remain genetically elusive. Objective To identify the genetic cause of TS in a case that did not harbor a CACNA1C mutation in exon 8/8A, and was negative for all other plausible genetic substrates. Methods Utilization of diagnostic exome sequencing to identify the genetic substrate responsible for our case of TS. The identified mutation was characterized using whole cell patch-clamp technique and the results of these analyses were modeled using a modified Luo-Rudy dynamic model to determine the effects on the cardiac action potential. Results Whole exome sequencing revealed a novel CACNA1C mutation, p.Ile1166Thr, in a young male with diagnosed TS. Functional electrophysiological analysis identified a novel mechanism of TS-mediated disease, with an overall loss of current density and a gain-of-function shift in activation, leading to an increased window current. Modeling studies of this variant predicted prolongation of the action potential, as well as the development of spontaneous early afterdepolarizations. Conclusion Through expanded whole exome sequencing, we have identified a novel genetic substrate for TS, p.Ile1166Thr-CACNA1C. Electrophysiological experiments combined with modeling studies have identified a novel TS mechanism through increased window current. Therefore, expanded genetic testing in cases of TS to the entire CACNA1C coding region, if initial targeted testing is negative, may be warranted. PMID:25260352

  18. CACNA1C genotype explains interindividual differences in amygdala volume among patients with schizophrenia.

    PubMed

    Wolf, Claudia; Mohr, Holger; Schneider-Axmann, Thomas; Reif, Andreas; Wobrock, Thomas; Scherk, Harald; Kraft, Susanne; Schmitt, Andrea; Falkai, Peter; Gruber, Oliver

    2014-03-01

    Affective deficits are one common denominator of schizophrenia (SZ), bipolar disorder (BD) and obsessive compulsive disorder (OCD) with the amygdala indicated as one of the major structures involved in emotion regulation. Previous findings of differences in amygdala volume between healthy controls and patients with SZ, BD or OCD diverge with respect to the affected hemisphere, size and direction of the effect. Variability in the CACNA1C gene has been linked to BD, SZ as well as structural and functional variation in the amygdala in healthy people and patients with BD. We were interested to investigate whether amygdala volumes differ between hemispheres, diagnostic or genotype groups, and whether any interactive effects exist. We combined genotyping of SNP rs1006737 in CACNA1C with structural MRI measurements of relative gray matter (GM) amygdala volume in patients with SZ, BD or OCD as well as healthy controls (N Total = 72). The CACNA1C genotype showed a significant effect on relative GM amygdala volume in patients with SZ. There was a significant left versus right relative GM amygdala volume decrease in patients with SZ or BD. The effects of hemisphere and diagnosis (controls vs. patients with SZ) on relative GM amygdala volume were genotype specific. Our data suggest that the CACNA1C genotype may account for some heterogeneity in the effects of hemisphere and diagnosis on amygdala volume when comparing patients with SZ and controls and point to disturbed Ca(2+)-signaling as a plausible mechanism contributing to the pathology in patients with SZ. PMID:23880959

  19. Calcium Signaling Pathway Genes RUNX2 and CACNA1C Are Associated With Calcific Aortic Valve Disease

    PubMed Central

    Guauque-Olarte, Sandra; Messika-Zeitoun, David; Droit, Arnaud; Lamontagne, Maxime; Tremblay-Marchand, Joël; Lavoie-Charland, Emilie; Gaudreault, Nathalie; Arsenault, Benoit J.; Dubé, Marie-Pierre; Tardif, Jean-Claude; Body, Simon C.; Seidman, Jonathan G.; Boileau, Catherine; Mathieu, Patrick; Pibarot, Philippe; Bossé, Yohan

    2016-01-01

    Background Calcific aortic valve stenosis (AS) is a life-threatening disease with no medical therapy. The genetic architecture of AS remains elusive. This study combines genome-wide association studies, gene expression, and expression quantitative trait loci mapping in human valve tissues to identify susceptibility genes of AS. Methods and Results A meta-analysis was performed combining the results of 2 genome-wide association studies in 474 and 486 cases from Quebec City (Canada) and Paris (France), respectively. Corresponding controls consisted of 2988 and 1864 individuals with European ancestry from the database of genotypes and phenotypes. mRNA expression levels were evaluated in 9 calcified and 8 normal aortic valves by RNA sequencing. The results were integrated with valve expression quantitative trait loci data obtained from 22 AS patients. Twenty-five single-nucleotide polymorphisms had P<5×10−6 in the genome-wide association studies meta-analysis. The calcium signaling pathway was the top gene set enriched for genes mapped to moderately AS-associated single-nucleotide polymorphisms. Genes in this pathway were found differentially expressed in valves with and without AS. Two single-nucleotide polymorphisms located in RUNX2 (runt-related transcription factor 2), encoding an osteogenic transcription factor, demonstrated some association with AS (genome-wide association studies P=5.33×10−5). The mRNA expression levels of RUNX2 were upregulated in calcified valves and associated with eQTL-SNPs. CACNA1C encoding a subunit of a voltage-dependent calcium channel was upregulated in calcified valves. The eQTL-SNP with the most significant association with AS located in CACNA1C was associated with higher expression of the gene. Conclusions This integrative genomic study confirmed the role of RUNX2 as a potential driver of AS and identified a new AS susceptibility gene, CACNA1C, belonging to the calcium signaling pathway. PMID:26553695

  20. The impact of CACNA1C allelic variation on regional gray matter volume in Chinese population.

    PubMed

    Huang, Liang; Mo, Yin; Sun, Xuejin; Yu, Hualin; Li, Hao; Wu, Lichuan; Li, Ming

    2016-04-01

    The SNP rs1006737 in CACNA1C gene has been significantly associated with psychiatric disorders (e.g., schizophrenia and bipolar disorder) in European populations. In Han Chinese, rs1006737 is also strongly associated with schizophrenia, although the effects of the psychosis risk SNP on related brain functions and structures in this population remain unclear. Here, we examined the association of rs1006737 with gray matter volume in a sample of 278 healthy Han Chinese. A whole-brain voxel-based morphometry (VBM) analysis revealed a significant association in the region around right superior occipital gyrus (family-wise error corrected, P = 0.023). Our data provides initial evidence for the involvement of this psychosis genetic risk locus in brain structure variations in Chinese population, and calls for further investigations. PMID:26756527

  1. Underexpression of CACNA1C Caused by Overexpression of microRNA-29a Underlies the Pathogenesis of Atrial Fibrillation

    PubMed Central

    Zhao, Yujie; Yuan, Yiqiang; Qiu, Chunguang

    2016-01-01

    Background The objective of this study was to investigate the molecular mechanism of atrial fibrillation (AF), as well as the negative regulatory relationship between miR-29a-3p and CACNA1C. Material/Methods We searched the online miRNA database (www.mirdb.org) and identified the miR-29a-3p binding sequence within the 3′-UTR of the target gene, and then conducted luciferase assay to verify it. The cells were transfected with miR-29a-3p and ICa,L was determined in those cells. Results We validated CACNA1C to be the direct target gene of miR-29a-3p. We also established the negative regulatory relationship between miR-29a-3p and CACNA1C via studying the relative luciferase activity. We also conducted real-time PCR and Western blot analysis to study the mRNA and protein expression level of CACNA1C among different groups of cells treated with scramble control, 30nM miR-29a-3p mimics, and 60nM miR-29a-3p mimics, indicating a negative regulatory relationship between miR-29a-3p and CACNA1C. We next analyzed whether miR-29a-3p transfection in cardiomyocytes produced the effects on the ICa,L induced by electrical remodeling, and found a tonic inhibition of IBa by endogenous miR-29a-3p in atrial myocytes. Conclusions We validated the negative regulation between miR-29a-3p and CACNA1C, and found that miR-29a-3p might a potential therapeutic target in the treatment of AF. PMID:27341015

  2. DNA variants in CACNA1C modify Parkinson disease risk only when vitamin D level is deficient

    PubMed Central

    Maldonado, Lizmarie; Beecham, Gary W.; Martin, Eden R.; Evatt, Marian L.; Ritchie, James C.; Haines, Jonathan L.; Zabetian, Cyrus P.; Payami, Haydeh; Pericak-Vance, Margaret A.; Vance, Jeffery M.

    2016-01-01

    Objective: To evaluate the association between the genetic variants in CACNA1C, which encodes the α1 subunit of the L-type voltage-sensitive calcium channel (LVSCC) and Parkinson disease (PD) while accounting for interactions with vitamin D concentration. Methods: Two independent case-control data sets (478 cases and 431 controls; 482 cases and 412 controls) were used. Joint effects of single nucleotide polymorphisms (SNPs) and SNP-vitamin D interaction were analyzed by comparing models containing vitamin D deficiency, SNP genotypes, SNP-vitamin D interaction, and covariates to a restricted model with only vitamin D deficiency and covariates. Meta-analysis was used to combine the joint effects in the 2 data sets. Analysis was stratified by vitamin D deficiency to demonstrate the pattern of SNP-vitamin D interaction. Results: Vitamin D deficiency was associated with PD in both data sets (odds ratio [OR] = 1.9–2.7, p ≤ 0.009). SNP rs34621387 demonstrated a significant joint effect (meta-analysis, p = 7.5 × 10−5; Bonferroni corrected, p = 0.02). The G allele at rs34621387 is associated with PD in vitamin D-deficient individuals in both data sets (OR = 2.0–2.1, confidence interval = 1.3–3.5, p = 0.002) but is not associated with PD in vitamin D–nondeficient individuals (p > 0.8 in both data sets). Conclusions: Previous studies suggest that vitamin D deficiency is associated with PD and sustained opening of LVSCC contributes to the selective vulnerability of dopaminergic neurons in PD. Our data demonstrate that the association between genetic variations in CACNA1C and PD depends on vitamin D deficiency, providing one potential mechanism underlying the association between vitamin D deficiency and PD. PMID:27123490

  3. Sex-dependent modulation of age-related cognitive decline by the L-type calcium channel gene Cacna1c (Cav 1.2).

    PubMed

    Zanos, Panos; Bhat, Shambhu; Terrillion, Chantelle E; Smith, Robert J; Tonelli, Leonardo H; Gould, Todd D

    2015-10-01

    Increased calcium influx through L-type voltage-gated calcium channels has been implicated in the neuronal dysfunction underlying age-related memory declines. The present study aimed to test the specific role of Cacna1c (which encodes Cav 1.2) in modulating age-related memory dysfunction. Short-term, spatial and contextual/emotional memory was evaluated in young and aged, wild-type as well as mice with one functional copy of Cacna1c (haploinsufficient), using the novel object recognition, Y-maze and passive avoidance tasks, respectively. Hippocampal expression of Cacna1c mRNA was measured by quantitative polymerase chain reaction. Ageing was associated with object recognition and contextual/emotional memory deficits, and a significant increase in hippocampal Cacna1c mRNA expression. Cacna1c haploinsufficiency was associated with decreased Cacna1c mRNA expression in both young and old animals. However, haploinsufficient mice did not manifest an age-related increase in expression of this gene. Behaviourally, Cacna1c haploinsufficiency prevented object recognition deficits during ageing in both male and female mice. A significant correlation between higher Cacna1c levels and decreased object recognition performance was observed in both sexes. Also, a sex-dependent protective role of decreased Cacna1c levels in contextual/emotional memory loss has been observed, specifically in male mice. These data provide evidence for an association between increased hippocampal Cacna1c expression and age-related cognitive decline. Additionally, they indicate an interaction between the Cacna1c gene and sex in the modulation of age-related contextual memory declines. PMID:25989111

  4. Long QT, Syndactyly, Joint Contractures, Stroke and Novel CACNA1C Mutation: Expanding the Spectrum of Timothy Syndrome

    PubMed Central

    Gillis, Jane; Burashnikov, Elena; Antzelevitch, Charles; Blaser, Susan; Gross, Gil; Turner, Lesley; Babul-Hirji, Riyana; Chitayat, David

    2011-01-01

    Timothy syndrome (TS) is described as an autosomal dominant condition with the constellation of features including prolonged QT interval, hand and foot abnormalities and mental retardation or autism. Splawski et al. [2004] previously described two phenotypes associated with TS distinguished by two unique and different mutations within the CACNA1C gene. We report on a newborn who presented with prolonged QT interval and associated polymorphic ventricular tachycardia, dysmorphic facial features, syndactyly of the hands and feet and joint contractures, suggestive of TS. He developed a stroke, subsequent intractable seizures and was found to have cortical blindness and later profound developmental delay. Initial targeted mutation analysis did not identify either of the previously described TS associated mutations; however, full gene sequencing detected a novel CACNA1C gene mutation (p.Ala1473Gly). The clinical and genetic findings in our case expand both the clinical and molecular knowledge of TS. PMID:22106044

  5. The bipolar disorder risk allele at CACNA1C also confers risk of recurrent major depression and of schizophrenia

    PubMed Central

    Green, E K; Grozeva, D; Jones, I; Jones, L; Kirov, G; Caesar, S; Gordon-Smith, K; Fraser, C; Forty, L; Russell, E; Hamshere, M L; Moskvina, V; Nikolov, I; Farmer, A; McGuffin, P; Holmans, P A; Owen, M J; O'Donovan, M C; Craddock, N

    2010-01-01

    Molecular genetic analysis offers opportunities to advance our understanding of the nosological relationship between psychiatric diagnostic categories in general, and the mood and psychotic disorders in particular. Strong evidence (P=7.0 × 10−7) of association at the polymorphism rs1006737 (within CACNA1C, the gene encoding the α-1C subunit of the L-type voltage-gated calcium channel) with the risk of bipolar disorder (BD) has recently been reported in a meta-analysis of three genome-wide association studies of BD, including our BD sample (N=1868) studied within the Wellcome Trust Case Control Consortium. Here, we have used our UK case samples of recurrent major depression (N=1196) and schizophrenia (N=479) and UK non-psychiatric comparison groups (N=15316) to examine the spectrum of phenotypic effect of the bipolar risk allele at rs1006737. We found that the risk allele conferred increased risk for schizophrenia (P=0.034) and recurrent major depression (P=0.013) with similar effect sizes to those previously observed in BD (allelic odds ratio ∼1.15). Our findings are evidence of some degree of overlap in the biological underpinnings of susceptibility to mental illness across the clinical spectrum of mood and psychotic disorders, and show that at least some loci can have a relatively general effect on susceptibility to diagnostic categories, as currently defined. Our findings will contribute to a better understanding of the pathogenesis of major psychiatric illness, and such knowledge should be useful in providing an etiological rationale for shaping psychiatric nosology, which is currently reliant entirely on descriptive clinical data. PMID:19621016

  6. Pro-arrhythmogenic effects of CACNA1C G1911R mutation in human ventricular tachycardia: insights from cardiac multi-scale models.

    PubMed

    Bai, Jieyun; Wang, Kuanquan; Li, Qince; Yuan, Yongfeng; Zhang, Henggui

    2016-01-01

    Mutations in the CACNA1C gene are associated with ventricular tachycardia (VT). Although the CACNA1C mutations were well identified in patients with cardiac arrhythmias, mechanisms by which cardiac arrhythmias are generated in such genetic mutation conditions remain unclear. In this study, we identified a novel mechanism of VT resulted from enhanced repolarization dispersion which is a key factor for arrhythmias in the CACNA1C G1911R mutation using multi-scale computational models of the human ventricle. The increased calcium influx in the mutation prolonged action potential duration (APD), produced steepened action potential duration restitution (APDR) curves as well as augmented membrane potential differences among different cell types during repolarization, increasing transmural dispersion of repolarization (DOR) and the spatial and temporal heterogeneity of cardiac electrical activities. Consequentially, the vulnerability to unidirectional conduction block in response to a premature stimulus increased at tissue level in the G1911R mutation. The increased functional repolarization dispersion anchored reentrant excitation waves in tissue and organ models, facilitating the initiation and maintenance of VT due to less meandering rotor tip. Thus, the increased repolarization dispersion caused by the G1911R mutation is a primary factor that may primarily contribute to the genesis of cardiac arrhythmias in Timothy Syndrome. PMID:27502440

  7. Pro-arrhythmogenic effects of CACNA1C G1911R mutation in human ventricular tachycardia: insights from cardiac multi-scale models

    PubMed Central

    Bai, Jieyun; Wang, Kuanquan; Li, Qince; Yuan, Yongfeng; Zhang, Henggui

    2016-01-01

    Mutations in the CACNA1C gene are associated with ventricular tachycardia (VT). Although the CACNA1C mutations were well identified in patients with cardiac arrhythmias, mechanisms by which cardiac arrhythmias are generated in such genetic mutation conditions remain unclear. In this study, we identified a novel mechanism of VT resulted from enhanced repolarization dispersion which is a key factor for arrhythmias in the CACNA1C G1911R mutation using multi-scale computational models of the human ventricle. The increased calcium influx in the mutation prolonged action potential duration (APD), produced steepened action potential duration restitution (APDR) curves as well as augmented membrane potential differences among different cell types during repolarization, increasing transmural dispersion of repolarization (DOR) and the spatial and temporal heterogeneity of cardiac electrical activities. Consequentially, the vulnerability to unidirectional conduction block in response to a premature stimulus increased at tissue level in the G1911R mutation. The increased functional repolarization dispersion anchored reentrant excitation waves in tissue and organ models, facilitating the initiation and maintenance of VT due to less meandering rotor tip. Thus, the increased repolarization dispersion caused by the G1911R mutation is a primary factor that may primarily contribute to the genesis of cardiac arrhythmias in Timothy Syndrome. PMID:27502440

  8. Further evidence for genetic association of CACNA1C and schizophrenia: new risk loci in a Han Chinese population and a meta-analysis.

    PubMed

    Zheng, Fanfan; Zhang, Yanling; Xie, Wuxiang; Li, Wenqiang; Jin, Chao; Mi, Weifeng; Wang, Fang; Ma, Wenbin; Ma, Cuicui; Yang, Yongfeng; Du, Bo; Li, Keqing; Liu, Chenxing; Wang, Lifang; Lu, Tianlan; Zhang, Hongyan; Wang, Yun; Lu, Lin; Lv, Luxian; Zhang, Dai; Yue, Weihua

    2014-01-01

    CACNA1C (12p13.3) has been implicated as a susceptibility gene for schizophrenia by several replicated genome wide association studies. While these results have been consistent among studies in European populations, the findings in East Asian populations have varied. To test whether CACNA1C is a risk gene for schizophrenia, we conducted a case-control study in 5897 schizophrenic patients and 6323 healthy control subjects selected from Han Chinese population. Our study replicated the positive associations of rs1006737 (P=0.0108, OR=1.16, 95% CI: 1.03-1.29) and rs1024582 (P=0.0062, OR=1.18, 95% CI: 1.05-1.33), and identified a novel risk locus, rs2007044 (P=0.0053, OR=1.08, 95% CI: 1.02-1.14). A meta-analysis of rs1006737 combining our study and previous studies was conducted in a total of 8222 schizophrenia cases and 24,661 healthy controls. In the meta-analysis, the association between rs1006737 and schizophrenia remained significant (OR=1.14, 95% CI: 1.07-1.22, P=0.0001). Stratified analysis showed no heterogeneity between East Asian and European ancestries (χ(2)[1]=0.07, P=0.795), and the difference in pooled ORs between ancestries was not significant (Z=0.25, P=0.801). Our results provide further support for associations of rs1006737 and rs1024582 with schizophrenia, identify a new risk locus rs2007044 in a Han Chinese population, and further establish CACNA1C as an important susceptibility gene for the disease across world populations. PMID:24355530

  9. A common risk variant in CACNA1C supports a sex-dependent effect on longitudinal functioning and functional recovery from episodes of schizophrenia-spectrum but not bipolar disorder.

    PubMed

    Heilbronner, Urs; Malzahn, Dörthe; Strohmaier, Jana; Maier, Sandra; Frank, Josef; Treutlein, Jens; Mühleisen, Thomas W; Forstner, Andreas J; Witt, Stephanie H; Cichon, Sven; Falkai, Peter; Nöthen, Markus M; Rietschel, Marcella; Schulze, Thomas G

    2015-12-01

    Sex is a powerful modulator of disease susceptibility, course and outcome. The gene CACNA1C is among the best replicated vulnerability genes of bipolar disorder and schizophrenia. The aim of the present study was to investigate whether sex and a variant in CACNA1C (rs10774035 as a proxy for the well-acknowledged risk variant rs1006737) influence psychosocial adaptation in a large German patient sample with schizophrenia-spectrum (n=297) and bipolar (n=516) disorders. We analyzed Global Assessment of Functioning (GAF) scores, retrospectively collected for different time points during disease course. We investigated whether CACNA1C sex-dependently modulates longitudinal GAF scores and recovery from episodes of psychiatric disturbance in the above mentioned disorders. Psychosocial recovery was measured as difference score between the current GAF score (assessing the last remission) and the worst GAF score ever during an illness episode. Covariate- adjusted association analyses revealed a sex × rs10774035 genotype interaction on longitudinal GAF and recovery from illness episodes only in schizophrenia-spectrum but not in bipolar disorders. In schizophrenia-spectrum affected males, rs10774035 minor allele (T) carriers had higher GAF scores at three time points (premorbid, worst ever, current). In contrast, females carrying rs10774035 minor alleles had impaired recovery from schizophrenia-spectrum episodes. These results encourage further investigations of gene × sex interactions and longitudinal quantitative phenotypes to unravel the rich variety of behavioral consequences of genetic individuality. PMID:26475575

  10. Novel Regulation of the Synthesis of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Subunit GluA1 by Carnitine Palmitoyltransferase 1C (CPT1C) in the Hippocampus.

    PubMed

    Fadó, Rut; Soto, David; Miñano-Molina, Alfredo J; Pozo, Macarena; Carrasco, Patricia; Yefimenko, Natalia; Rodríguez-Álvarez, José; Casals, Núria

    2015-10-16

    The regulation of AMPA-type receptor (AMPAR) abundance in the postsynaptic membrane is an important mechanism involved in learning and memory formation. Recent data suggest that one of the constituents of the AMPAR complex is carnitine palmitoyltransferase 1C (CPT1C), a brain-specific isoform located in the endoplasmic reticulum of neurons. Previous results had demonstrated that CPT1C deficiency disrupted spine maturation in hippocampal neurons and impaired spatial learning, but the role of CPT1C in AMPAR physiology had remained mostly unknown. In the present study, we show that CPT1C binds GluA1 and GluA2 and that the three proteins have the same expression profile during neuronal maturation. Moreover, in hippocampal neurons of CPT1C KO mice, AMPAR-mediated miniature excitatory postsynaptic currents and synaptic levels of AMPAR subunits GluA1 and GluA2 are significantly reduced. We show that AMPAR expression is dependent on CPT1C levels because total protein levels of GluA1 and GluA2 are decreased in CPT1C KO neurons and are increased in CPT1C-overexpressing neurons, whereas other synaptic proteins remain unaltered. Notably, mRNA levels of AMPARs remained unchanged in those cultures, indicating that CPT1C is post-transcriptionally involved. We demonstrate that CPT1C is directly involved in the de novo synthesis of GluA1 and not in protein degradation. Moreover, in CPT1C KO cultured neurons, GluA1 synthesis after chemical long term depression was clearly diminished, and brain-derived neurotrophic factor treatment was unable to phosphorylate the mammalian target of rapamycin (mTOR) and stimulate GluA1 protein synthesis. These data newly identify CPT1C as a regulator of AMPAR translation efficiency and therefore also synaptic function in the hippocampus. PMID:26338711

  11. Identification of subunits a, b, and c1 from Acetobacterium woodii Na+-F1F0-ATPase. Subunits c1, c2, AND c3 constitute a mixed c-oligomer.

    PubMed

    Aufurth, S; Schägger, H; Müller, V

    2000-10-27

    The Na(+)-F(1)F(0)-ATPase operon of Acetobacterium woodii was recently shown to contain, among eleven atp genes, those genes that encode subunit a and b, a gene encoding a 16-kDa proteolipid (subunit c(1)), and two genes encoding 8-kDa proteolipids (subunits c(2) and c(3)). Because subunits a, b, and c(1) were not found in previous enzyme preparations, we re-determined the subunit composition of the enzyme. The genes were overproduced, and specific antibodies were raised. Western blots revealed that subunits a, b, and c(1) are produced and localized in the cytoplasmic membrane. Membrane protein complexes were solubilized by dodecylmaltoside and separated by blue native-polyacrylamide gel electrophoresis, and the ATPase subunits were resolved by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analyses revealed the presence of subunits a, c(2), c(3), b, delta, alpha, gamma, beta, and epsilon. Biochemical and immunological analyses revealed that subunits c(1), c(2), and c(3) are all part of the c-oligomer, the first of a F(1)F(0)-ATPase that contains 8- and 16-kDa proteolipids. PMID:10913149

  12. The Roles of RNA Polymerase I and III Subunits Polr1c and Polr1d in Craniofacial Development and in Zebrafish Models of Treacher Collins Syndrome.

    PubMed

    Noack Watt, Kristin E; Achilleos, Annita; Neben, Cynthia L; Merrill, Amy E; Trainor, Paul A

    2016-07-01

    Ribosome biogenesis is a global process required for growth and proliferation of all cells, yet perturbation of ribosome biogenesis during human development often leads to tissue-specific defects termed ribosomopathies. Transcription of the ribosomal RNAs (rRNAs) by RNA polymerases (Pol) I and III, is considered a rate limiting step of ribosome biogenesis and mutations in the genes coding for RNA Pol I and III subunits, POLR1C and POLR1D cause Treacher Collins syndrome, a rare congenital craniofacial disorder. Our understanding of the functions of individual RNA polymerase subunits, however, remains poor. We discovered that polr1c and polr1d are dynamically expressed during zebrafish embryonic development, particularly in craniofacial tissues. Consistent with this pattern of activity, polr1c and polr1d homozygous mutant zebrafish exhibit cartilage hypoplasia and cranioskeletal anomalies characteristic of humans with Treacher Collins syndrome. Mechanistically, we discovered that polr1c and polr1d loss-of-function results in deficient ribosome biogenesis, Tp53-dependent neuroepithelial cell death and a deficiency of migrating neural crest cells, which are the primary progenitors of the craniofacial skeleton. More importantly, we show that genetic inhibition of tp53 can suppress neuroepithelial cell death and ameliorate the skeletal anomalies in polr1c and polr1d mutants, providing a potential avenue to prevent the pathogenesis of Treacher Collins syndrome. Our work therefore has uncovered tissue-specific roles for polr1c and polr1d in rRNA transcription, ribosome biogenesis, and neural crest and craniofacial development during embryogenesis. Furthermore, we have established polr1c and polr1d mutant zebrafish as models of Treacher Collins syndrome together with a unifying mechanism underlying its pathogenesis and possible prevention. PMID:27448281

  13. The Roles of RNA Polymerase I and III Subunits Polr1c and Polr1d in Craniofacial Development and in Zebrafish Models of Treacher Collins Syndrome

    PubMed Central

    Achilleos, Annita; Neben, Cynthia L.; Merrill, Amy E.; Trainor, Paul A.

    2016-01-01

    Ribosome biogenesis is a global process required for growth and proliferation of all cells, yet perturbation of ribosome biogenesis during human development often leads to tissue-specific defects termed ribosomopathies. Transcription of the ribosomal RNAs (rRNAs) by RNA polymerases (Pol) I and III, is considered a rate limiting step of ribosome biogenesis and mutations in the genes coding for RNA Pol I and III subunits, POLR1C and POLR1D cause Treacher Collins syndrome, a rare congenital craniofacial disorder. Our understanding of the functions of individual RNA polymerase subunits, however, remains poor. We discovered that polr1c and polr1d are dynamically expressed during zebrafish embryonic development, particularly in craniofacial tissues. Consistent with this pattern of activity, polr1c and polr1d homozygous mutant zebrafish exhibit cartilage hypoplasia and cranioskeletal anomalies characteristic of humans with Treacher Collins syndrome. Mechanistically, we discovered that polr1c and polr1d loss-of-function results in deficient ribosome biogenesis, Tp53-dependent neuroepithelial cell death and a deficiency of migrating neural crest cells, which are the primary progenitors of the craniofacial skeleton. More importantly, we show that genetic inhibition of tp53 can suppress neuroepithelial cell death and ameliorate the skeletal anomalies in polr1c and polr1d mutants, providing a potential avenue to prevent the pathogenesis of Treacher Collins syndrome. Our work therefore has uncovered tissue-specific roles for polr1c and polr1d in rRNA transcription, ribosome biogenesis, and neural crest and craniofacial development during embryogenesis. Furthermore, we have established polr1c and polr1d mutant zebrafish as models of Treacher Collins syndrome together with a unifying mechanism underlying its pathogenesis and possible prevention. PMID:27448281

  14. Direct and remote modulation of L-channels in chromaffin cells: distinct actions on alpha1C and alpha1D subunits?

    PubMed

    Baldelli, Pietro; Hernández-Guijo, Jesus Miguel; Carabelli, Valentina; Novara, Monica; Cesetti, Tiziana; Andrés-Mateos, Eva; Montiel, Carmen; Carbone, Emilio

    2004-02-01

    Understanding precisely the functioning of voltage-gated Ca2+ channels and their modulation by signaling molecules will help clarifying the Ca(2+)-dependent mechanisms controlling exocytosis in chromaffin cells. In recent years, we have learned more about the various pathways through which Ca2+ channels can be up- or down-modulated by hormones and neurotransmitters and how these changes may condition chromaffin cell activity and catecolamine release. Recently, the attention has been focused on the modulation of L-channels (CaV 1), which represent the major Ca2+ current component in rat and human chromaffin cells. L-channels are effectively inhibited by the released content of secretory granules or by applying mixtures of exogenous ATP, opioids, and adrenaline through the activation of receptor-coupled G proteins. This unusual inhibition persists in a wide range of potentials and results from a direct (membrane-delimited) interaction of G protein subunits with the L-channels co-localized in membrane microareas. Inhibition of L-channels can be reversed when the cAMP/PKA pathway is activated by membrane permeable cAMP analog or when cells are exposed to isoprenaline (remote action), suggesting the existence of parallel and opposite effects on L-channel gating by distinctly activated membrane autoreceptors. Here, the authors review the molecular components underlying these two opposing signaling pathways and present new evidence supporting the presence of two L-channel types in rat chromaffin cells (alpha1C and alpha1D), which open new interesting issues concerning Ca(2+)-channel modulation. In light of recent findings on the regulation of exocytosis by Ca(2+)-channel modulation, the authors explore the possible role of L-channels in the autocontrol of catecholamine release. PMID:15034224

  15. Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development1[C][W][OA

    PubMed Central

    Steiner, Sebastian; Schröter, Yvonne; Pfalz, Jeannette; Pfannschmidt, Thomas

    2011-01-01

    The major RNA polymerase activity in mature chloroplasts is a multisubunit, Escherichia coli-like protein complex called PEP (for plastid-encoded RNA polymerase). Its subunit structure has been extensively investigated by biochemical means. Beside the “prokaryotic” subunits encoded by the plastome-located RNA polymerase genes, a number of additional nucleus-encoded subunits of eukaryotic origin have been identified in the PEP complex. These subunits appear to provide additional functions and regulation modes necessary to adapt transcription to the varying functional situations in chloroplasts. However, despite the enormous progress in genomic data and mass spectrometry techniques, it is still under debate which of these subunits belong to the core complex of PEP and which ones represent rather transient or peripheral components. Here, we present a catalog of true PEP subunits that is based on comparative analyses from biochemical purifications, protein mass spectrometry, and phenotypic analyses. We regard reproducibly identified protein subunits of the basic PEP complex as essential when the corresponding knockout mutants reveal an albino or pale-green phenotype. Our study provides a clearly defined subunit catalog of the basic PEP complex, generating the basis for a better understanding of chloroplast transcription regulation. In addition, the data support a model that links PEP complex assembly and chloroplast buildup during early seedling development in vascular plants. PMID:21949211

  16. Ca(2+)-dependent inactivation of a cloned cardiac Ca2+ channel alpha 1 subunit (alpha 1C) expressed in Xenopus oocytes.

    PubMed Central

    Neely, A; Olcese, R; Wei, X; Birnbaumer, L; Stefani, E

    1994-01-01

    The alpha 1 subunit of cardiac Ca2+ channel, expressed alone or coexpressed with the corresponding beta subunit in Xenopus laevis oocytes, elicits rapidly inactivating Ca2+ currents. The inactivation has the following properties: 1) It is practically absent in external Ba2+; 2) it increases with Ca2+ current amplitudes; 3) it is faster at more negative potentials for comparable Ca2+ current amplitudes; 4) it is independent of channel density; and 5) it does not require the beta subunit. These findings indicate that the Ca2+ binding site responsible for inactivation is encoded in the alpha 1 subunit and suggest that it is located near the inner channel mouth but outside the membrane electric field. PMID:8075326

  17. Cloning of the gamma-aminobutyric acid (GABA) rho 1 cDNA: a GABA receptor subunit highly expressed in the retina.

    PubMed Central

    Cutting, G R; Lu, L; O'Hara, B F; Kasch, L M; Montrose-Rafizadeh, C; Donovan, D M; Shimada, S; Antonarakis, S E; Guggino, W B; Uhl, G R

    1991-01-01

    Type A gamma-aminobutyric acid (GABAA) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. We have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence in 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABAA subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA rho 1, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family. Images PMID:1849271

  18. Ectopic Expression of Rubisco Subunits in Maize Mesophyll Cells Does Not Overcome Barriers to Cell Type-Specific Accumulation1[C][W][OA

    PubMed Central

    Wostrikoff, Katia; Clark, Aimee; Sato, Shirley; Clemente, Tom; Stern, David

    2012-01-01

    In maize (Zea mays), Rubisco accumulates in bundle sheath but not mesophyll chloroplasts, but the mechanisms that underlie cell type-specific expression are poorly understood. To explore the coordinated expression of the chloroplast rbcL gene, which encodes the Rubisco large subunit (LS), and the two nuclear RBCS genes, which encode the small subunit (SS), RNA interference was used to reduce RBCS expression. This resulted in Rubisco deficiency and was correlated with translational repression of rbcL. Thus, as in C3 plants, LS synthesis depends on the presence of its assembly partner SS. To test the hypothesis that the previously documented transcriptional repression of RBCS in mesophyll cells is responsible for repressing LS synthesis in mesophyll chloroplasts, a ubiquitin promoter-driven RBCS gene was expressed in both bundle sheath and mesophyll cells. This did not lead to Rubisco accumulation in the mesophyll, suggesting that LS synthesis is impeded even in the presence of ectopic SS expression. To attempt to bypass this putative mechanism, a ubiquitin promoter-driven nuclear version of the rbcL gene was created, encoding an epitope-tagged LS that was expressed in the presence or absence of the Ubi-RBCS construct. Both transgenes were robustly expressed, and the tagged LS was readily incorporated into Rubisco complexes. However, neither immunolocalization nor biochemical approaches revealed significant accumulation of Rubisco in mesophyll cells, suggesting a continuing cell type-specific impairment of its assembly or stability. We conclude that additional cell type-specific factors limit Rubisco expression to bundle sheath chloroplasts. PMID:22744982

  19. Interaction between the Rev1 C-terminal Domain and the PolD3 Subunit of Polζ Suggests a Mechanism of Polymerase Exchange upon Rev1/Polζ-Dependent Translesion Synthesis

    PubMed Central

    Pustovalova, Yulia; Magalhães, Mariana T. Q.; D’Souza, Sanjay; Rizzo, Alessandro A.; Korza, George; Walker, Graham C.; Korzhnev, Dmitry M.

    2016-01-01

    Translesion synthesis (TLS) is a mutagenic branch of cellular DNA damage tolerance that enables bypass replication over DNA lesions carried out by specialized low-fidelity DNA polymerases. The replicative bypass of most types of DNA damage is performed in a two-step process of Rev1/Polζ-dependent TLS. In the first step, a Y-family TLS enzyme, typically Polη, Polι or Polκ, inserts a nucleotide across DNA lesion. In the second step, a four-subunit B-family DNA polymerase Polζ (Rev3/Rev7/PolD2/PolD3 complex) extends the distorted DNA primer-template. The coordinated action of error-prone TLS enzymes is regulated through their interactions with the two scaffold proteins, the sliding clamp PCNA and the TLS polymerase Rev1. Rev1 interactions with all other TLS enzymes are mediated by its C-terminal domain (Rev1-CT), which can simultaneously bind the Rev7 subunit of Polζ and Rev1-interacting regions (RIRs) from Polη, Polι or Polκ. In this work, we identified a previously unknown RIR motif in the C-terminal part of PolD3 subunit of Polζ whose interaction with the Rev1-CT is among the tightest mediated by RIR motifs. Three-dimensional structure of the Rev1-CT/PolD3-RIR complex determined by NMR spectroscopy revealed a structural basis for the relatively high affinity of this interaction. The unexpected discovery of PolD3-RIR motif suggests a mechanism of 'inserter' to 'extender' DNA polymerase switch upon Rev1/Polζ-dependent TLS, in which the PolD3-RIR binding to the Rev1-CT (i) helps displace the 'inserter' Polη, Polι or Polκ from its complex with Rev1, and (ii) facilitates assembly of the four-subunit 'extender' Polζ through simultaneous interaction of Rev1-CT with Rev7 and PolD3 subunits. PMID:26982350

  20. Vitis labrusca extract effects on cellular dynamics and redox modulations in a SH-SY5Y neuronal cell model: a similar role to lithium.

    PubMed

    Scola, Gustavo; Laliberte, Victoria Louise Marina; Kim, Helena Kyunghee; Pinguelo, Arsene; Salvador, Mirian; Young, L Trevor; Andreazza, Ana Cristina

    2014-12-01

    Oxidative stress and calcium imbalance are consistently reported in bipolar disorder (BD). Polymorphism of voltage-dependent calcium channel, L type, alpha 1C subunit (CACNA1c), which is responsible for the regulation of calcium influx, was also shown to have a strong association with BD. These alterations can lead to a number of different consequences in the cell including production of reactive species causing oxidative damage to proteins, lipids and DNA. Lithium is the most frequent medication used for the treatment of BD. Despite lithium's effects, long-term use can result in many negative side effects. Therefore, there is an urgent need for the development of drugs that may have similar biological effects as lithium without the negative consequences. Moreover, polyphenols are secondary metabolites of plants that present multi-faceted molecular abilities, such as regulation of cellular responses. Vitis labrusca extract (VLE), a complex mixture of polyphenols obtained from seeds of winery wastes of V. labrusca, was previously characterized by our group. This extract presented powerful antioxidant and neuroprotective properties. Therefore, the ability of VLE to ameliorate the consequences of hydrogen peroxide (H2O2)-induced redox alterations to cell viability, intracellular calcium levels and the relative levels of the calcium channel CACNA1c in comparison to lithium's effects were evaluated using a neuroblastoma cell model. H2O2 treatment increased cell mortality through apoptotic and necrotic pathways leading to an increase in intracellular calcium levels and alterations to relative CACNA1c levels. VLE and lithium were found to similarly ameliorate cell mortality through regulation of the apoptotic/necrotic pathways, decreasing intracellular calcium levels and preventing alterations to the relative levels of CACNA1c. The findings of this study suggest that VLE exhibits protective properties against oxidative stress-induced alterations similar to that of lithium

  1. Reverse-Genetic Analysis of the Two Biotin-Containing Subunit Genes of the Heteromeric Acetyl-Coenzyme A Carboxylase in Arabidopsis Indicates a Unidirectional Functional Redundancy1[C][W][OA

    PubMed Central

    Li, Xu; Ilarslan, Hilal; Brachova, Libuse; Qian, Hui-Rong; Li, Ling; Che, Ping; Wurtele, Eve Syrkin; Nikolau, Basil J.

    2011-01-01

    The heteromeric acetyl-coenzyme A carboxylase catalyzes the first and committed reaction of de novo fatty acid biosynthesis in plastids. This enzyme is composed of four subunits: biotin carboxyl-carrier protein (BCCP), biotin carboxylase, α-carboxyltransferase, and β-carboxyltransferase. With the exception of BCCP, single-copy genes encode these subunits in Arabidopsis (Arabidopsis thaliana). Reverse-genetic approaches were used to individually investigate the physiological significance of the two paralogous BCCP-coding genes, CAC1A (At5g16390, codes for BCCP1) and CAC1B (At5g15530, codes for BCCP2). Transfer DNA insertional alleles that completely eliminate the accumulation of BCCP2 have no perceptible effect on plant growth, development, and fatty acid accumulation. In contrast, transfer DNA insertional null allele of the CAC1A gene is embryo lethal and deleteriously affects pollen development and germination. During seed development the effect of the cac1a null allele first becomes apparent at 3-d after flowering, when the synchronous development of the endosperm and embryo is disrupted. Characterization of CAC1A antisense plants showed that reducing BCCP1 accumulation to 35% of wild-type levels, decreases fatty acid accumulation and severely affects normal vegetative plant growth. Detailed expression analysis by a suite of approaches including in situ RNA hybridization, promoter:reporter transgene expression, and quantitative western blotting reveal that the expression of CAC1B is limited to a subset of the CAC1A-expressing tissues, and CAC1B expression levels are only about one-fifth of CAC1A expression levels. Therefore, a likely explanation for the observed unidirectional redundancy between these two paralogous genes is that whereas the BCCP1 protein can compensate for the lack of BCCP2, the absence of BCCP1 cannot be tolerated as BCCP2 levels are not sufficient to support heteromeric acetyl-coenzyme A carboxylase activity at a level that is required for

  2. A1C test

    MedlinePlus

    HbA1C test; Glycated hemoglobin test; Glycosylated hemoglobin test; Hemoglobin glycosylated test; Glycohemoglobin test ... have recently eaten does not affect the A1C test, so you do not need to fast to ...

  3. A1C

    MedlinePlus

    A1C is a blood test for type 2 diabetes and prediabetes. It measures your average blood glucose, or blood sugar, level over the past 3 ... A1C alone or in combination with other diabetes tests to make a diagnosis. They also use the ...

  4. A1C Test

    MedlinePlus

    ... to minimize the complications caused by chronically elevated glucose levels, such as progressive damage to body organs like the kidneys, eyes, cardiovascular system, and nerves. The A1c test result ...

  5. Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation

    PubMed Central

    He, Fengping; Xu, Xin; Yuan, Shuguo; Tan, Liangqiu; Gao, Lingjun; Ma, Shaochun; Zhang, Shebin; Ma, Zhanzhong; Jiang, Wei; Liu, Fenglian; Chen, Baofeng; Zhang, Beibei; Pang, Jungang; Huang, Xiuyan; Weng, Jiaqiang

    2016-01-01

    Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease. PMID:27488468

  6. Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation

    NASA Astrophysics Data System (ADS)

    He, Fengping; Xu, Xin; Yuan, Shuguo; Tan, Liangqiu; Gao, Lingjun; Ma, Shaochun; Zhang, Shebin; Ma, Zhanzhong; Jiang, Wei; Liu, Fenglian; Chen, Baofeng; Zhang, Beibei; Pang, Jungang; Huang, Xiuyan; Weng, Jiaqiang

    2016-08-01

    Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease.

  7. Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation.

    PubMed

    He, Fengping; Xu, Xin; Yuan, Shuguo; Tan, Liangqiu; Gao, Lingjun; Ma, Shaochun; Zhang, Shebin; Ma, Zhanzhong; Jiang, Wei; Liu, Fenglian; Chen, Baofeng; Zhang, Beibei; Pang, Jungang; Huang, Xiuyan; Weng, Jiaqiang

    2016-01-01

    Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease. PMID:27488468

  8. MUC1-C drives MYC in multiple myeloma.

    PubMed

    Tagde, Ashujit; Rajabi, Hasan; Bouillez, Audrey; Alam, Maroof; Gali, Reddy; Bailey, Shannon; Tai, Yu-Tzu; Hideshima, Teru; Anderson, Kenneth; Avigan, David; Kufe, Donald

    2016-05-26

    Multiple myeloma (MM) cell lines and primary tumor cells are addicted to the MYC oncoprotein for survival. Little is known, however, about how MYC expression is upregulated in MM cells. The mucin 1 C-terminal subunit (MUC1-C) is an oncogenic transmembrane protein that is aberrantly expressed in MM cell lines and primary tumor samples. The present studies demonstrate that targeting MUC1-C with silencing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 editing or with the GO-203 inhibitor is associated with downregulation of MYC messenger RNA and protein. The results show that MUC1-C occupies the MYC promoter and thereby activates the MYC gene by a β-catenin/transcription factor 4 (TCF4)-mediated mechanism. In this way, MUC1-C (1) increases β-catenin occupancy on the MYC promoter, (2) forms a complex with β-catenin and TCF4, and, in turn, (3) drives MYC transcription. Analysis of MM cells using quantitative real-time reverse transcription polymerase chain reaction arrays further demonstrated that silencing MUC1-C is associated with downregulation of MYC target genes, including CCND2, hTERT, and GCLC Analysis of microarray data sets further demonstrated that MUC1 levels positively correlate with MYC expression in MM progression and in primary cells from over 800 MM patients. These findings collectively provide convincing evidence that MUC1-C drives MYC expression in MM. PMID:26907633

  9. The action of calcium channel blockers on recombinant L-type calcium channel α1-subunits

    PubMed Central

    Morel, Nicole; Buryi, Vitali; Feron, Olivier; Gomez, Jean-Pierre; Christen, Marie-Odile; Godfraind, Théophile

    1998-01-01

    CHO cells expressing the α1C-a subunit (cardiac isoform) and the α1C-b subunit (vascular isoform) of the voltage-dependent L-type Ca2+ channel were used to investigate whether tissue selectivity of Ca2+ channel blockers could be related to different affinities for α1C isoforms.Inward current evoked by the transfected α1 subunit was recorded by the patch-clamp technique in the whole-cell configuration.Neutral dihydropyridines (nifedipine, nisoldipine, (+)-PN200-110) were more potent inhibitors of α1C-b-subunit than of α1C-a-subunit. This difference was more marked at a holding potential of −100 mV than at −50 mV. SDZ 207-180 (an ionized dihydropyridine) exhibited the same potency on the two isoforms.Pinaverium (ionized non-dihydropyridine derivative) was 2 and 4 fold more potent on α1C-a than on α1C-b subunit at Vh of −100 mV and −50 mV, respectively. Effects of verapamil were identical on the two isoforms at both voltages.[3H]-(+)-PN 200-110 binding experiments showed that neutral dihydropyridines had a higher affinity for the α1C-b than for the α1C-a subunit. SDZ 207-180 had the same affinity for the two isoforms and pinaverium had a higher affinity for the α1C-a subunit than for the α1C-b subunit.These results indicate marked differences among Ca2+ channel blockers in their selectivity for the α1C-a and α1C-b subunits of the Ca2+ channel. PMID:9846638

  10. The action of calcium channel blockers on recombinant L-type calcium channel alpha1-subunits.

    PubMed

    Morel, N; Buryi, V; Feron, O; Gomez, J P; Christen, M O; Godfraind, T

    1998-11-01

    1. CHO cells expressing the alpha(1C-a) subunit (cardiac isoform) and the alpha(1C-b) subunit (vascular isoform) of the voltage-dependent L-type Ca2+ channel were used to investigate whether tissue selectivity of Ca2+ channel blockers could be related to different affinities for alpha1C isoforms. 2. Inward current evoked by the transfected alpha1 subunit was recorded by the patch-clamp technique in the whole-cell configuration. 3. Neutral dihydropyridines (nifedipine, nisoldipine, (+)-PN200-110) were more potent inhibitors of alpha(1C-)b-subunit than of alpha(1C-a)-subunit. This difference was more marked at a holding potential of -100 mV than at -50 mV. SDZ 207-180 (an ionized dihydropyridine) exhibited the same potency on the two isoforms. 4. Pinaverium (ionized non-dihydropyridine derivative) was 2 and 4 fold more potent on alpha(1C-a) than on alpha(1C-b) subunit at Vh of -100 mV and -50 mV, respectively. Effects of verapamil were identical on the two isoforms at both voltages. 5. [3H]-(+)-PN 200-110 binding experiments showed that neutral dihydropyridines had a higher affinity for the alpha(1C-b) than for the alpha(1C-a) subunit. SDZ 207-180 had the same affinity for the two isoforms and pinaverium had a higher affinity for the alpha(1C-a) subunit than for the alpha(1C-b) subunit. 6. These results indicate marked differences among Ca2+ channel blockers in their selectivity for the alpha(1C-a) and alpha(1C-b) subunits of the Ca2+ channel. PMID:9846638

  11. Large-scale genome-wide association analysis of bipolar disorder identifies a new susceptibility locus near ODZ4

    PubMed Central

    Sklar, Pamela; Ripke, Stephan; Scott, Laura J.; Andreassen, Ole A.; Cichon, Sven; Craddock, Nick; Edenberg, Howard J.; Nurnberger, John I.; Rietschel, Marcella; Blackwood, Douglas; Corvin, Aiden; Flickinger, Matthew; Guan, Weihua; Mattingsdal, Morten; Mcquillin, Andrew; Kwan, Phoenix; Wienker, Thomas F.; Daly, Mark; Dudbridge, Frank; Holmans, Peter A.; Lin, Danyu; Burmeister, Margit; Greenwood, Tiffany A.; Hamshere, Marian L.; Muglia, Pierandrea; Smith, Erin N.; Zandi, Peter P.; Nievergelt, Caroline M.; Mckinney, Rebecca; Shilling, Paul D.; Schork, Nicholas J.; Bloss, Cinnamon S.; Foroud, Tatiana; Koller, Daniel L.; Gershon, Elliot S.; Liu, Chunyu; Badner, Judith A.; Scheftner, William A.; Lawson, William B.; Nwulia, Evaristus A.; Hipolito, Maria; Coryell, William; Rice, John P.; Byerley, William; McMahon, Francis J.; Schulze, Thomas G.; Berrettini, Wade; Lohoff, Falk W.; Potash, James B.; Mahon, Pamela B.; Mcinnis, Melvin G.; Zöllner, Sebastian; Zhang, Peng; Craig, David W.; Szelinger, Szabocls; Barrett, Thomas B.; Breuer, René; Meier, Sandra; Strohmaier, Jana; Witt, Stephanie H.; Tozzi, Federica; Farmer, Anne; McGuffin, Peter; Strauss, John; Xu, Wei; Kennedy, James L.; Vincent, John B.; Matthews, Keith; Day, Richard; Ferreira, Manuel D.C.; O'Dushlaine, Colm; Perlis, Roy; Raychaudhuri, Soumya; Ruderfer, Douglas; Hyoun, Phil L.; Smoller, Jordan W.; Li, Jun; Absher, Devin; Thompson, Robert C.; Meng, Fan Guo; Schatzberg, Alan F.; Bunney, William E.; Barchas, Jack D.; Jones, Edward G.; Watson, Stanley J.; Myers, Richard M.; Akil, Huda; Boehnke, Michael; Chambert, Kim; Moran, Jennifer; Scolnick, Ed; Djurovic, Srdjan; Melle, Ingrid; Morken, Gunnar; Gill, Michael; Morris, Derek; Quinn, Emma; Mühleisen, Thomas W.; Degenhardt, Franziska A.; Mattheisen, Manuel; Schumacher, Johannes; Maier, Wolfgang; Steffens, Michael; Propping, Peter; Nöthen, Markus M.; Anjorin, Adebayo; Bass, Nick; Gurling, Hugh; Kandaswamy, Radhika; Lawrence, Jacob; Mcghee, Kevin; Mcintosh, Andrew; Mclean, Alan W.; Muir, Walter J.; Pickard, Benjamin S.; Breen, Gerome; St Clair, David; Caesar, Sian; Gordon-Smith, Katherine; Jones, Lisa; Fraser, Christine; Green, Elaine K.; Grozeva, Detelina; Jones, Ian R.; Kirov, George; Moskvina, Valentina; Nikolov, Ivan; O'Donovan, Michael C.; Owen, Michael J.; Collier, David A.; Elkin, Amanda; Williamson, Richard; Young, Allan H.; Ferrier, I Nicol; Stefansson, Kari; Stefansson, Hreinn; Porgeirsson, Porgeir; Steinberg, Stacy; Gustafsson, Omar; Bergen, Sarah E.; Nimgaonkar, Vishwajit; hultman, Christina; Landén, Mikael; Lichtenstein, Paul; Sullivan, Patrick; Schalling, Martin; Osby, Urban; Backlund, Lena; Frisén, Louise; Langstrom, Niklas; Jamain, Stéphane; Leboyer, Marion; Etain, Bruno; Bellivier, Frank; Petursson, Hannes; Sigur Sson, Engilbert; Müller-Mysok, Bertram; Lucae, Susanne; Schwarz, Markus; Schofield, Peter R.; Martin, Nick; Montgomery, Grant W.; Lathrop, Mark; Oskarsson, Högni; Bauer, Michael; Wright, Adam; Mitchell, Philip B.; Hautzinger, Martin; Reif, Andreas; Kelsoe, John R.; Purcell, Shaun M.

    2011-01-01

    We conducted a combined genome-wide association (GWAS) analysis of 7,481 individuals affected with bipolar disorder and 9,250 control individuals within the Psychiatric Genomewide Association Study Consortium Bipolar Disorder group (PGC-BD). We performed a replication study in which we tested 34 independent SNPs in 4,493 independent bipolar disorder cases and 42,542 independent controls and found strong evidence for replication. In the replication sample, 18 of 34 SNPs had P value < 0.05, and 31 of 34 SNPs had signals with the same direction of effect (P = 3.8 × 10−7). In the combined analysis of all 63,766 subjects (11,974 cases and 51,792 controls), genome-wide significant evidence for association was confirmed for CACNA1C and found for a novel gene ODZ4. In a combined analysis of non-overlapping schizophrenia and bipolar GWAS samples we observed strong evidence for association with SNPs in CACNA1C and in the region of NEK4/ITIH1,3,4. Pathway analysis identified a pathway comprised of subunits of calcium channels enriched in the bipolar disorder association intervals. The strength of the replication data implies that increasing samples sizes in bipolar disorder will confirm many additional loci. PMID:21926972

  12. Rare Mutations of CACNB2 Found in Autism Spectrum Disease-Affected Families Alter Calcium Channel Function

    PubMed Central

    Breitenkamp, Alexandra F. S.; Matthes, Jan; Nass, Robert Daniel; Sinzig, Judith; Lehmkuhl, Gerd; Nürnberg, Peter; Herzig, Stefan

    2014-01-01

    Autism Spectrum Disorders (ASD) are complex neurodevelopmental diseases clinically defined by dysfunction of social interaction. Dysregulation of cellular calcium homeostasis might be involved in ASD pathogenesis, and genes coding for the L-type calcium channel subunits CaV1.2 (CACNA1C) and CaVβ2 (CACNB2) were recently identified as risk loci for psychiatric diseases. Here, we present three rare missense mutations of CACNB2 (G167S, S197F, and F240L) found in ASD-affected families, two of them described here for the first time (G167S and F240L). All these mutations affect highly conserved regions while being absent in a sample of ethnically matched controls. We suggest the mutations to be of physiological relevance since they modulate whole-cell Ba2+ currents through calcium channels when expressed in a recombinant system (HEK-293 cells). Two mutations displayed significantly decelerated time-dependent inactivation as well as increased sensitivity of voltage-dependent inactivation. In contrast, the third mutation (F240L) showed significantly accelerated time-dependent inactivation. By altering the kinetic parameters, the mutations are reminiscent of the CACNA1C mutation causing Timothy Syndrome, a Mendelian disease presenting with ASD. In conclusion, the results of our first-time biophysical characterization of these three rare CACNB2 missense mutations identified in ASD patients support the hypothesis that calcium channel dysfunction may contribute to autism. PMID:24752249

  13. Modulation of the skeletal muscle sodium channel alpha-subunit by the beta 1-subunit.

    PubMed

    Wallner, M; Weigl, L; Meera, P; Lotan, I

    1993-12-28

    Co-expression of cloned sodium channel beta 1-subunit with the rat skeletal muscle-subunit (alpha microI) accelerated the macroscopic current decay, enhanced the current amplitude, shifted the steady state inactivation curve to more negative potentials and decreased the time required for complete recovery from inactivation. Sodium channels expressed from skeletal muscle mRNA showed a similar behaviour to that observed from alpha microI/beta 1, indicating that beta 1 restores 'physiological' behaviour. Northern blot analysis revealed that the Na+ channel beta 1-subunit is present in high abundance (about 0.1%) in rat heart, brain and skeletal muscle, and the hybridization with untranslated region of the 'brain' beta 1 cDNA to skeletal muscle and heart mRNA indicated that the different Na+ channel alpha-subunits in brain, skeletal muscle and heart may share a common beta 1-subunit. PMID:8282123

  14. A1C Test and Diabetes

    MedlinePlus

    ... laboratory tests. How does the A1C relate to estimated average glucose? Estimated average glucose (eAG) is calculated from the A1C. ... levels have the A1C test twice a year. Estimated average glucose (eAG) is calculated from the A1C ...

  15. The roles of AKR1C1 and AKR1C2 in ethyl-3,4-dihydroxybenzoate induced esophageal squamous cell carcinoma cell death

    PubMed Central

    Zhou, Dianrong; Lou, Xiaomin; Xu, Yang; Liu, Siqi; Zhao, Xiaohang

    2016-01-01

    The aldo-keto reductase (AKR) superfamily of enzymes is critical for the detoxification of drugs and toxins in the human body; these enzymes are involved not only in the development of drug resistance in cancer cells but also in the metabolism of polycyclic aromatic hydrocarbons. Here, we demonstrated that AKR1C1/C2 increased the metabolism of ethyl-3,4-dihydroxybenzoate (EDHB) in esophageal squamous cell carcinoma (ESCC) cells. Previous studies have shown that EDHB can effectively induce esophageal cancer cell autophagy and apoptosis, and the AKR1C family represents one set of highly expressed genes after EDHB treatment. To explore the cytotoxic effects of EDHB, esophageal cancer cells with higher (KYSE180) or lower (KYSE510) AKR1C expression levels were evaluated in this study. The proliferation of KYSE180 cells was inhibited more effectively than that of KYSE510 cells by EDHB treatment. Furthermore, the effective subunits of the AKR superfamily, AKR1C1/C2, were quantitatively identified using multiple reaction monitoring (MRM) assays. The sensitivity of esophageal cancer cells to EDHB was significantly attenuated by the siRNA knockdown of AKR1C1/C2. Moreover, the expression of autophagy inducers (Beclin, LC3II and BNIP3) and NDRG1 was significantly elevated in KYSE180 cells, but not in KYSE510 cells, after EDHB treatment. When autophagy was inhibited by 3-methyladenine, KYSE180 cells exhibited an increased sensitivity to EDHB, which may be a metabolic substrate of AKR1C1/C2. These results indicated that ESCC patients with high AKR1C1/C2 expression may be more sensitive to EDHB, and AKR1C1/C2 may facilitate EDHB-induced autophagy and apoptosis, thus providing potential guidance for the chemoprevention of ESCC. PMID:26934124

  16. AMPAR interacting protein CPT1C enhances surface expression of GluA1-containing receptors

    PubMed Central

    Gratacòs-Batlle, Esther; Yefimenko, Natalia; Cascos-García, Helena; Soto, David

    2015-01-01

    AMPARs mediate the vast majority of fast excitatory synaptic transmission in the brain and their biophysical and trafficking properties depend on their subunit composition and on several posttranscriptional and posttranslational modifications. Additionally, in the brain AMPARs associate with auxiliary subunits, which modify the properties of the receptors. Despite the abundance of AMPAR partners, recent proteomic studies have revealed even more interacting proteins that could potentially be involved in AMPAR regulation. Amongst these, carnitine palmitoyltransferase 1C (CPT1C) has been demonstrated to form an integral part of native AMPAR complexes in brain tissue extracts. Thus, we aimed to investigate whether CPT1C might be able to modulate AMPAR function. Firstly, we confirmed that CPT1C is an interacting protein of AMPARs in heterologous expression systems. Secondly, CPT1C enhanced whole-cell currents of GluA1 homomeric and GluA1/GluA2 heteromeric receptors. However, CPT1C does not alter the biophysical properties of AMPARs and co-localization experiments revealed that AMPARs and CPT1C are not associated at the plasma membrane despite a strong level of co-localization at the intracellular level. We established that increased surface GluA1 receptor number was responsible for the enhanced AMPAR mediated currents in the presence of CPT1C. Additionally, we revealed that the palmitoylable residue C585 of GluA1 is important in the enhancement of AMPAR trafficking to the cell surface by CPT1C. Nevertheless, despite its potential as a depalmitoylating enzyme, CPT1C does not affect the palmitoylation state of GluA1. To sum up, this work suggests that CPT1C plays a role as a novel regulator of AMPAR surface expression in neurons. Fine modulation of AMPAR membrane trafficking is fundamental in normal synaptic activity and in plasticity processes and CPT1C is therefore a putative candidate to regulate neuronal AMPAR physiology. PMID:25698923

  17. 7 CFR 1c.102 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 1 2014-01-01 2014-01-01 false Definitions. 1c.102 Section 1c.102 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.102 Definitions. (a) Department or agency head means the head of any federal department or agency and any other officer or employee of any department or agency to whom authority...

  18. 7 CFR 1c.102 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 1 2013-01-01 2013-01-01 false Definitions. 1c.102 Section 1c.102 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.102 Definitions. (a) Department or agency head means the head of any federal department or agency and any other officer or employee of any department or agency to whom authority...

  19. 7 CFR 1c.102 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Definitions. 1c.102 Section 1c.102 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.102 Definitions. (a) Department or agency head means the head of any federal department or agency and any other officer or employee of any department or agency to whom authority...

  20. 7 CFR 1c.124 - Conditions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Conditions. 1c.124 Section 1c.124 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.124 Conditions. With respect to any research project or any class of research projects the department or agency head may impose...

  1. 7 CFR 1c.124 - Conditions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 1 2014-01-01 2014-01-01 false Conditions. 1c.124 Section 1c.124 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.124 Conditions. With respect to any research project or any class of research projects the department or agency head may impose...

  2. 7 CFR 1c.124 - Conditions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 1 2011-01-01 2011-01-01 false Conditions. 1c.124 Section 1c.124 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.124 Conditions. With respect to any research project or any class of research projects the department or agency head may impose...

  3. 7 CFR 1c.124 - Conditions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 1 2012-01-01 2012-01-01 false Conditions. 1c.124 Section 1c.124 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.124 Conditions. With respect to any research project or any class of research projects the department or agency head may impose...

  4. 7 CFR 1c.124 - Conditions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 1 2013-01-01 2013-01-01 false Conditions. 1c.124 Section 1c.124 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.124 Conditions. With respect to any research project or any class of research projects the department or agency head may impose...

  5. 7 CFR 1c.115 - IRB records.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false IRB records. 1c.115 Section 1c.115 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.115 IRB records. (a) An institution, or when appropriate an IRB, shall prepare and maintain adequate documentation of IRB activities, including the following: (1) Copies of...

  6. Led Astray by Hemoglobin A1c

    PubMed Central

    Chen, Jean; Diesburg-Stanwood, Amy; Bodor, Geza; Rasouli, Neda

    2016-01-01

    Hemoglobin A1c (A1c) is used frequently to diagnose and treat diabetes mellitus. Therefore, it is important be aware of factors that may interfere with the accuracy of A1c measurements. This is a case of a rare hemoglobin variant that falsely elevated a nondiabetic patient’s A1c level and led to a misdiagnosis of diabetes. A 67-year-old male presented to endocrine clinic for further management after he was diagnosed with diabetes based on an elevated A1c of 10.7%, which is approximately equivalent to an average blood glucose of 260 mg/dL. Multiple repeat A1c levels remained >10%, but his home fasting and random glucose monitoring ranged from 92 to 130 mg/dL. Hemoglobin electrophoresis and subsequent genetic analysis diagnosed the patient with hemoglobin Wayne, a rare hemoglobin variant. This variant falsely elevates A1c levels when A1c is measured using cation-exchange high-performance liquid chromatography. When the boronate affinity method was applied instead, the patient’s A1c level was actually 4.7%. Though hemoglobin Wayne is clinically silent, this patient was erroneously diagnosed with diabetes and started on an antiglycemic medication. Due to this misdiagnosis, the patient was at risk of escalation in his “diabetes management” and hypoglycemia. Therefore, it is important that providers are aware of factors that may result in hemoglobin A1c inaccuracy including hemoglobin variants. PMID:26848480

  7. 9 CFR 73.1c - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Definitions. 73.1c Section 73.1c Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS SCABIES IN CATTLE §...

  8. 9 CFR 73.1c - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Definitions. 73.1c Section 73.1c Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS SCABIES IN CATTLE §...

  9. 9 CFR 73.1c - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Definitions. 73.1c Section 73.1c Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS SCABIES IN CATTLE §...

  10. 9 CFR 73.1c - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Definitions. 73.1c Section 73.1c Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS SCABIES IN CATTLE §...

  11. 9 CFR 73.1c - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Definitions. 73.1c Section 73.1c Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS SCABIES IN CATTLE §...

  12. MUC1-C oncoprotein promotes FLT3 receptor activation in acute myeloid leukemia cells

    PubMed Central

    Liu, Suiyang; Yin, Li; Stroopinsky, Dina; Rajabi, Hasan; Puissant, Alexandre; Stegmaier, Kimberly; Avigan, David; Kharbanda, Surender; Kufe, Donald

    2014-01-01

    Blasts from approximately one-third of patients with acute myeloid leukemia (AML) harbor activating mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase that confer a poor prognosis. The Mucin 1-C-terminal subunit (MUC1-C) oncoprotein is aberrantly expressed in AML blasts and stem cells; however, there is no known interaction between MUC1-C and FLT3. The present studies demonstrate that MUC1-C associates with wild-type and mutant FLT3 in AML cells. Targeting MUC1-C with the cell-penetrating peptide inhibitor GO-203 disrupts MUC1-C/FLT3 complexes and downregulates FLT3 activation. GO-203 treatment of AML cells was also associated with inhibition of the FLT3 downstream effectors AKT, extracellular signal-regulated kinase, and STAT5. The results further show that AML cells with FLT3-activating mutations and resistant to the FLT3 inhibitor midostaurin/PKC412 are sensitive to GO-203–induced growth arrest and death. Moreover, GO-203 increases sensitivity of mutant FLT3 AML cells to FLT3 inhibitor treatment. These results indicate that MUC1-C contributes to FLT3 activation in AML cells and that targeting MUC1-C inhibits the FLT3 signaling pathway. Our findings support the development of MUC1-C inhibitors alone and in combination with agents that target FLT3 for the treatment of wild-type and mutant FLT3 AML. PMID:24282218

  13. Expression of GluK1c underlies the developmental switch in presynaptic kainate receptor function

    PubMed Central

    Vesikansa, Aino; Sakha, Prasanna; Kuja-Panula, Juha; Molchanova, Svetlana; Rivera, Claudio; Huttunen, Henri J.; Rauvala, Heikki; Taira, Tomi; Lauri, Sari E.

    2012-01-01

    Kainate-type glutamate receptors (KARs) regulate synaptic transmission and neuronal excitability via multiple mechanisms, depending on their subunit composition. Presynaptic KARs tonically depress glutamatergic transmission during restricted period of synapse development; however, the molecular basis behind this effect is unknown. Here, we show that the developmental and cell-type specific expression pattern of a KAR subunit splice variant, GluK1c, corresponds to the immature-type KAR activity in the hippocampus. GluK1c localizes to dendritic contact sites at distal axons, the distal targeting being promoted by heteromerization with the subunit GluK4. Presynaptic expression of GluK1c strongly suppresses glutamatergic transmission in cell-pairs in vitro and mimics the immature-type KAR activity at CA3-CA1 synapses in vivo, at a developmental stage when the endogenous expression is already downregulated. These data support a central role for GluK1c in mediating tonic inhibition of glutamate release and the consequent effects on excitability and activity-dependent fine-tuning of the developing hippocampal circuitry. PMID:22413061

  14. Expression of GluK1c underlies the developmental switch in presynaptic kainate receptor function.

    PubMed

    Vesikansa, Aino; Sakha, Prasanna; Kuja-Panula, Juha; Molchanova, Svetlana; Rivera, Claudio; Huttunen, Henri J; Rauvala, Heikki; Taira, Tomi; Lauri, Sari E

    2012-01-01

    Kainate-type glutamate receptors (KARs) regulate synaptic transmission and neuronal excitability via multiple mechanisms, depending on their subunit composition. Presynaptic KARs tonically depress glutamatergic transmission during restricted period of synapse development; however, the molecular basis behind this effect is unknown. Here, we show that the developmental and cell-type specific expression pattern of a KAR subunit splice variant, GluK1c, corresponds to the immature-type KAR activity in the hippocampus. GluK1c localizes to dendritic contact sites at distal axons, the distal targeting being promoted by heteromerization with the subunit GluK4. Presynaptic expression of GluK1c strongly suppresses glutamatergic transmission in cell-pairs in vitro and mimics the immature-type KAR activity at CA3-CA1 synapses in vivo, at a developmental stage when the endogenous expression is already downregulated. These data support a central role for GluK1c in mediating tonic inhibition of glutamate release and the consequent effects on excitability and activity-dependent fine-tuning of the developing hippocampal circuitry. PMID:22413061

  15. A1C Test and Diabetes

    MedlinePlus

    ... of Diabetes Educators American Diabetes Association JDRF MedlinePlus Diabetes Disease Organizations ​There are many organizations who provide ... KB). Alternate Language URL The A1C Test and Diabetes Page Content On this page: What is the ...

  16. Blood Test: Hemoglobin A1C

    MedlinePlus

    ... the person's average blood sugar levels over that time. Why It's Done Doctors use the hemoglobin A1c test to determine if your child's diabetes management plan needs to be adjusted. Typically the test ...

  17. Minimum variance and variance of outgoing quality limit MDS-1(c1, c2) plans

    NASA Astrophysics Data System (ADS)

    Raju, C.; Vidya, R.

    2016-06-01

    In this article, the outgoing quality (OQ) and total inspection (TI) of multiple deferred state sampling plans MDS-1(c1,c2) are studied. It is assumed that the inspection is rejection rectification. Procedures for designing MDS-1(c1,c2) sampling plans with minimum variance of OQ and TI are developed. A procedure for obtaining a plan for a designated upper limit for the variance of the OQ (VOQL) is outlined.

  18. Characterization of the MUC1-C Cytoplasmic Domain as a Cancer Target

    PubMed Central

    Raina, Deepak; Agarwal, Praveen; Lee, James; Bharti, Ajit; McKnight, C. James; Sharma, Pankaj; Kharbanda, Surender; Kufe, Donald

    2015-01-01

    Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly expressed in diverse human carcinomas and certain hematologic malignancies. The oncogenic MUC1 transmembrane C-terminal subunit (MUC1-C) functions in part by transducing growth and survival signals from cell surface receptors. However, little is known about the structure of the MUC1-C cytoplasmic domain as a potential drug target. Using methods for structural predictions, our results indicate that a highly conserved CQCRRK sequence, which is adjacent to the cell membrane, forms a small pocket that exposes the two cysteine residues for forming disulfide bonds. By contrast, the remainder of the MUC1-C cytoplasmic domain has no apparent structure, consistent with an intrinsically disordered protein. Our studies thus focused on targeting the MUC1 CQCRRK region. The results show that L- and D-amino acid CQCRRK-containing peptides bind directly to the CQC motif. We further show that the D-amino acid peptide, designated GO-203, blocks homodimerization of the MUC1-C cytoplasmic domain in vitro and in transfected cells. Moreover, GO-203 binds directly to endogenous MUC1-C in breast and lung cancer cells. Colocalization studies further demonstrate that GO-203 predominantly binds to MUC1-C at the cell membrane. These findings support the further development of agents that target the MUC1-C cytoplasmic domain CQC motif and thereby MUC1-C function in cancer cells. PMID:26267657

  19. 7 CFR 1c.114 - Cooperative research.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    .... Cooperative research projects are those projects covered by this policy which involve more than one institution. In the conduct of cooperative research projects, each institution is responsible for safeguarding... 7 Agriculture 1 2013-01-01 2013-01-01 false Cooperative research. 1c.114 Section...

  20. 7 CFR 1c.114 - Cooperative research.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    .... Cooperative research projects are those projects covered by this policy which involve more than one institution. In the conduct of cooperative research projects, each institution is responsible for safeguarding... 7 Agriculture 1 2014-01-01 2014-01-01 false Cooperative research. 1c.114 Section...

  1. 7 CFR 1c.114 - Cooperative research.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    .... Cooperative research projects are those projects covered by this policy which involve more than one institution. In the conduct of cooperative research projects, each institution is responsible for safeguarding... 7 Agriculture 1 2010-01-01 2010-01-01 false Cooperative research. 1c.114 Section...

  2. 7 CFR 1c.114 - Cooperative research.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    .... Cooperative research projects are those projects covered by this policy which involve more than one institution. In the conduct of cooperative research projects, each institution is responsible for safeguarding... 7 Agriculture 1 2011-01-01 2011-01-01 false Cooperative research. 1c.114 Section...

  3. 7 CFR 1c.114 - Cooperative research.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    .... Cooperative research projects are those projects covered by this policy which involve more than one institution. In the conduct of cooperative research projects, each institution is responsible for safeguarding... 7 Agriculture 1 2012-01-01 2012-01-01 false Cooperative research. 1c.114 Section...

  4. 7 CFR 1c.102 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.102 Definitions. (a) Department or..., Wage and Hour requirements administered by the Department of Labor). (f) Human subject means a living...) in order for obtaining the information to constitute research involving human subjects. (g) IRB...

  5. 7 CFR 1c.115 - IRB records.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.115 IRB records. (a) An institution..., including the following: (1) Copies of all research proposals reviewed, scientific evaluations, if any, that... members voting for, against, and abstaining; the basis for requiring changes in or disapproving...

  6. 7 CFR 1c.115 - IRB records.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.115 IRB records. (a) An institution..., including the following: (1) Copies of all research proposals reviewed, scientific evaluations, if any, that... members voting for, against, and abstaining; the basis for requiring changes in or disapproving...

  7. Identification of a regulatory subunit of protein phosphatase 1 which mediates blue light signaling for stomatal opening.

    PubMed

    Takemiya, Atsushi; Yamauchi, Shota; Yano, Takayuki; Ariyoshi, Chie; Shimazaki, Ken-ichiro

    2013-01-01

    Protein phosphatase 1 (PP1) is a eukaryotic serine/threonine protein phosphatase comprised of a catalytic subunit (PP1c) and a regulatory subunit that modulates catalytic activity, subcellular localization and substrate specificity. PP1c positively regulates stomatal opening through blue light signaling between phototropins and the plasma membrane H(+)-ATPase in guard cells. However, the regulatory subunit functioning in this process is unknown. We identified Arabidopsis PRSL1 (PP1 regulatory subunit2-like protein1) as a regulatory subunit of PP1c. Tautomycin, a selective inhibitor of PP1c, inhibited blue light responses of stomata in the single mutants phot1 and phot2, supporting the idea that signals from phot1 and phot2 converge on PP1c. We obtained PRSL1 based on the sequence similarity to Vicia faba PRS2, a PP1c-binding protein isolated by a yeast two-hybrid screen. PRSL1 bound to Arabidopsis PP1c through its RVxF motif, a consensus PP1c-binding sequence. Arabidopsis prsl1 mutants were impaired in blue light-dependent stomatal opening, H(+) pumping and phosphorylation of the H(+)-ATPase, but showed normal phototropin activities. PRSL1 complemented the prsl1 phenotype, but not if the protein carried a mutation in the RVxF motif, suggesting that PRSL1 functions through binding PP1c via the RVxF motif. PRSL1 did not affect the catalytic activity of Arabidopsis PP1c but it stimulated the localization of PP1c in the cytoplasm. We conclude that PRSL1 functions as a regulatory subunit of PP1 and regulates blue light signaling in stomata. PMID:22585556

  8. Grumman OV-1C in hangar

    NASA Technical Reports Server (NTRS)

    1983-01-01

    Grumman OV-1C in the hangar used at the time by the Army at Edwards Air Force Base. This OV-1C Mohawk, serial #67-15932, was used in a joint NASA/US Army Aviation Engineering Flight Activity (USAAEFA) program to study a stall-speed warning system in the early 1980s. NASA designed and built an automated stall-speed warning system which presented both airspeed and stall speed to the pilot. Visual indication of impending stall would be displayed to the pilot as a cursor or pointer located on a conventional airspeed indicator. In addition, an aural warning at predetermined stall margins was presented to the pilot through a voice synthesizer. The Mohawk was developed by Grumman Aircraft as a photo observation and electronic reconnaissance aircraft for the US Marines and the US Army. The OV-1 entered production in October 1959 and served the US Army in Europe, Korea, the Viet Nam War, Central and South America, Alaska, and during Desert Shield/Desert Storm in the Middle East. The Mohawk was retired from service in September 1996. 133 OV-1Cs were built, the 'C' designating the model which used an IR (infrared) imaging system to provide reconnaissance.

  9. Grumman OV-1C in flight

    NASA Technical Reports Server (NTRS)

    1983-01-01

    Grumman OV-1C in flight. This OV-1C Mohawk, serial #67-15932, was used in a joint NASA/US Army Aviation Engineering Flight Activity (USAAEFA) program to study a stall-speed warning system in the early 1980s. NASA designed and built an automated stall-speed warning system which presented both airspeed and stall speed to the pilot. Visual indication of impending stall would be displayed to the pilot as a cursor or pointer located on a conventional airspeed indicator. In addition, an aural warning at predetermined stall margins was presented to the pilot through a voice synthesizer. The Mohawk was developed by Grumman Aircraft as a photo observation and reconnaissance aircraft for the US Marines and the US Army. The OV-1 entered production in October 1959 and served the US Army in Europe, Korea, the Viet Nam War, Central and South America, Alaska, and during Desert Shield/Desert Storm in the Middle East. The Mohawk was retired from service in September 1996. 133 OV-1Cs were built, the 'C' designating the model which used an IR (infrared) imaging system to provide reconnaissance.

  10. Candidate gene associations with mood disorder, cognitive vulnerability, and fronto-limbic volumes

    PubMed Central

    Frazier, Thomas W; Youngstrom, Eric A; Frankel, Brian A; Zunta-Soares, Giovana B; Sanches, Marsal; Escamilla, Michael; Nielsen, David A; Soares, Jair C

    2014-01-01

    Background Four of the most consistently replicated variants associated with mood disorder occur in genes important for synaptic function: ANK3 (rs10994336), BDNF (rs6265), CACNA1C (rs1006737), and DGKH (rs1170191). Aims The present study examined associations between these candidates, mood disorder diagnoses, cognition, and fronto-limbic regions implicated in affect regulation. Methods and materials Participants included 128 individuals with bipolar disorder (33% male, Mean age = 38.5), 48 with major depressive disorder (29% male, Mean age = 40.4), and 149 healthy controls (35% male, Mean age = 36.5). Genotypes were determined by 5′-fluorogenic exonuclease assays (TaqMan®). Fronto-limbic volumes were obtained from high resolution brain images using Freesurfer. Chi-square analyses, bivariate correlations, and mediational models examined relationships between genetic variants, mood diagnoses, cognitive measures, and brain volumes. Results Carriers of the minor BDNF and ANK3 alleles showed nonsignificant trends toward protective association in controls relative to mood disorder patients (P = 0.047). CACNA1C minor allele carriers had larger bilateral caudate, insula, globus pallidus, frontal pole, and nucleus accumbens volumes (smallest r = 0.13, P = 0.043), and increased IQ (r = 0.18, P < 0.001). CACNA1C associations with brain volumes and IQ were independent; larger fronto-limbic volumes did not mediate increased IQ. Other candidate variants were not significantly associated with diagnoses, cognition, or fronto-limbic volumes. Discussion and conclusions CACNA1C may be associated with biological systems altered in mood disorder. Increases in fronto-limbic volumes and cognitive ability associated with CACNA1C minor allele genotypes are congruent with findings in healthy samples and may be a marker for increased risk for neuropsychiatric phenotypes. Even larger multimodal studies are needed to quantify the magnitude and specificity of genetic

  11. Dependence on the MUC1-C Oncoprotein in Non-Small Cell Lung Cancer Cells

    PubMed Central

    Raina, Deepak; Kosugi, Michio; Ahmad, Rehan; Panchamoorthy, Govind; Rajabi, Hasan; Alam, Maroof; Shimamura, Takeshi; Shapiro, Geoffrey I.; Supko, Jeffrey; Kharbanda, Surender; Kufe, Donald

    2011-01-01

    Non-small cell lung cancer (NSCLC) cells are often associated with constitutive activation of the phosphatidylinositol 3-kinase (PI3K)->Akt->mTOR pathway. The mucin 1 (MUC1) heterodimeric glycoprotein is aberrantly overexpressed in NSCLC and induces gene signatures that are associated with poor survival of NSCLC patients. The present results demonstrate that the MUC1 C-terminal subunit (MUC1-C) cytoplasmic domain associates with PI3K p85 in NSCLC cells. We show that inhibition of MUC1-C with cell-penetrating peptides blocks this interaction with PI3K p85 and suppresses constitutive phosphorylation of Akt and its downstream effector, mTOR. In concert with these results, treatment of NSCLC cells with the MUC1-C peptide inhibitor, GO-203, was associated with downregulation of PI3K->Akt signaling and inhibition of growth. GO-203 treatment was also associated with increases in reactive oxygen species (ROS) and induction of necrosis by a ROS-dependent mechanism. Moreover, GO-203 treatment of H1975 (EGFR L858R/T790M) and A549 (K-Ras G12S) xenografts growing in nude mice resulted in tumor regressions. These findings indicate that NSCLC cells are dependent on MUC1-C for activation of the PI3K->Akt pathway and for survival. PMID:21421804

  12. 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

  13. Expression of ATP6V1C1 during oral carcinogenesis.

    PubMed

    Oliveira Alves, M G; Carta, Cfl; Padín-Iruegas, M-E; Pérez-Sayáns, M; Suarez-Peñaranda, J M; Issa, J S; García-García, A; Almeida, J D

    2016-05-01

    We investigated the gene and protein expressions of V-type ATPase protein subunit C1 (ATP6V1C1) in cases of oral squamous cell carcinoma (OSCC) and contralateral normal mucosa in smokers, nonsmokers and former smokers. Subjects were separated into five groups of 15: group 1, smokers with OSCC; group 2, normal contralateral mucosa of OSCC patients; group 3, chronic smokers; group 4, former smokers who had stopped smoking 1 year earlier; group 5, individuals who had never smoked. Exfoliative cytology specimens from oral mucosa of smokers, former smokers and nonsmokers showed normal gene and protein expression. We found significantly greater gene expression in the OSCC group than in the nonsmoker groups. No difference in gene expression was observed between normal contralateral mucosa and nonsmoker groups, smoker and nonsmoker groups or former smoker and nonsmoker groups. We observed intense immunostaining for ATP6V1C1 protein in all cases of OSCC and weak or no staining in smoker, former smoker and nonsmoker groups. Significantly greater expression of ATP6V1C1 protein was observed in the OSCC group compared to the other groups, which supports the role of ATP6V1C1 in effecting changes associated with oral cancer. Analysis of the mucosae of chronic smokers, former smokers and the normal contralateral mucosa of patients with OSCC showed unaltered ATP6V1C1 gene and protein expression. Early stages of carcinogenesis, represented by altered epithelium of chronic smokers, had neither gene nor protein alterations as seen in OSCC. Therefore, we infer that the changes in ATP6V1C1 occur during later stages of carcinogenesis. Our preliminary study provides a basis for future studies of using ATP6V1C1 levels for detecting early stage OSCC. PMID:26984774

  14. MUC1-C ACTIVATES THE TAK1 INFLAMMATORY PATHWAY IN COLON CANCER

    PubMed Central

    Takahashi, Hidekazu; Jin, Caining; Rajabi, Hasan; Pitroda, Sean; Alam, Maroof; Ahmad, Rehan; Raina, Deepak; Hasegawa, Masanori; Suzuki, Yozo; Tagde, Ashujit; Bronson, Roderick T.; Weichselbaum, Ralph; Kufe, Donald

    2015-01-01

    The mucin 1 (MUC1) oncoprotein has been linked to the inflammatory response by promoting cytokine-mediated activation of the NF-κB pathway. The TGF-β-activated kinase 1 (TAK1) is an essential effector of proinflammatory NF-κB signaling that also regulates cancer cell survival. The present studies demonstrate that the MUC1-C transmembrane subunit induces TAK1 expression in colon cancer cells. MUC1 also induces TAK1 in a MUC1+/−/IL-10−/− mouse model of colitis and colon tumorigenesis. We show that MUC1-C promotes NF-κB-mediated activation of TAK1 transcription and, in a positive regulatory loop, MUC1-C contributes to TAK1-induced NF-κB signaling. In this way, MUC1-C binds directly to TAK1 and confers the association of TAK1 with TRAF6, which is necessary for TAK1-mediated activation of NF-κB. Targeting MUC1-C thus suppresses the TAK1→NF-κB pathway, downregulates BCL-XL, and in turn sensitizes colon cancer cells to MEK inhibition. Analysis of colon cancer databases further indicates that MUC1, TAK1 and TRAF6 are upregulated in tumors associated with decreased survival and that MUC1-C-induced gene expression patterns predict poor outcomes in patients. These results support a model in which MUC1-C-induced TAK1→NF-κB signaling contributes to intestinal inflammation and colon cancer progression. PMID:25659581

  15. Effect of Cavβ Subunits on Structural Organization of Cav1.2 Calcium Channels

    PubMed Central

    Duong, Son Q.; Thomas, Sam; Harry, Jo Beth; Patel, Chirag; Lao, Qi Zong; Soldatov, Nikolai M.

    2009-01-01

    Background Voltage-gated Cav1.2 calcium channels play a crucial role in Ca2+ signaling. The pore-forming α1C subunit is regulated by accessory Cavβ subunits, cytoplasmic proteins of various size encoded by four different genes (Cavβ1 - β4) and expressed in a tissue-specific manner. Methods and Results Here we investigated the effect of three major Cavβ types, β1b, β2d and β3, on the structure of Cav1.2 in the plasma membrane of live cells. Total internal reflection fluorescence microscopy showed that the tendency of Cav1.2 to form clusters depends on the type of the Cavβ subunit present. The highest density of Cav1.2 clusters in the plasma membrane and the smallest cluster size were observed with neuronal/cardiac β1b present. Cav1.2 channels containing β3, the predominant Cavβ subunit of vascular smooth muscle cells, were organized in a significantly smaller number of larger clusters. The inter- and intramolecular distances between α1C and Cavβ in the plasma membrane of live cells were measured by three-color FRET microscopy. The results confirm that the proximity of Cav1.2 channels in the plasma membrane depends on the Cavβ type. The presence of different Cavβ subunits does not result in significant differences in the intramolecular distance between the termini of α1C, but significantly affects the distance between the termini of neighbor α1C subunits, which varies from 67 Å with β1b to 79 Å with β3. Conclusions Thus, our results show that the structural organization of Cav1.2 channels in the plasma membrane depends on the type of Cavβ subunits present. PMID:19492014

  16. PAF Complex Plays Novel Subunit-Specific Roles in Alternative Cleavage and Polyadenylation

    PubMed Central

    Yang, Yan; Li, Wencheng; Hoque, Mainul; Hou, Liming; Shen, Steven; Tian, Bin; Dynlacht, Brian D.

    2016-01-01

    The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and RNA polymerase II (PolII) elongation. Here, we provide the first genome-wide profiles for the distribution of the entire complex in mammalian cells using chromatin immunoprecipitation and high throughput sequencing. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3’ ends, a profile that sharply contrasted with the yeast complex. Remarkably, we identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription using RNAi coupled with deep sequencing of the 3’ ends of transcripts. Moreover, we found that depletion of Paf1C subunits resulted in the accumulation of PolII over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits led to global loss of histone H2B ubiquitylation, although there was little impact of Paf1C depletion on other histone modifications, including tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and indicate that Paf1C subunits could play roles in controlling transcript length through suppression of PolII accumulation at transcription start site (TSS)-proximal pA sites and regulating pA site choice in 3’UTRs. PMID:26765774

  17. Recessive mutations in POLR1C cause a leukodystrophy by impairing biogenesis of RNA polymerase III

    PubMed Central

    Thiffault, Isabelle; Wolf, Nicole I.; Forget, Diane; Guerrero, Kether; Tran, Luan T.; Choquet, Karine; Lavallée-Adam, Mathieu; Poitras, Christian; Brais, Bernard; Yoon, Grace; Sztriha, Laszlo; Webster, Richard I.; Timmann, Dagmar; van de Warrenburg, Bart P.; Seeger, Jürgen; Zimmermann, Alíz; Máté, Adrienn; Goizet, Cyril; Fung, Eva; van der Knaap, Marjo S.; Fribourg, Sébastien; Vanderver, Adeline; Simons, Cas; Taft, Ryan J.; Yates III, John R.; Coulombe, Benoit; Bernard, Geneviève

    2015-01-01

    A small proportion of 4H (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) or RNA polymerase III (POLR3)-related leukodystrophy cases are negative for mutations in the previously identified causative genes POLR3A and POLR3B. Here we report eight of these cases carrying recessive mutations in POLR1C, a gene encoding a shared POLR1 and POLR3 subunit, also mutated in some Treacher Collins syndrome (TCS) cases. Using shotgun proteomics and ChIP sequencing, we demonstrate that leukodystrophy-causative mutations, but not TCS mutations, in POLR1C impair assembly and nuclear import of POLR3, but not POLR1, leading to decreased binding to POLR3 target genes. This study is the first to show that distinct mutations in a gene coding for a shared subunit of two RNA polymerases lead to selective modification of the enzymes' availability leading to two different clinical conditions and to shed some light on the pathophysiological mechanism of one of the most common hypomyelinating leukodystrophies, POLR3-related leukodystrophy. PMID:26151409

  18. The ribosomal subunit assembly line

    PubMed Central

    Dlakić, Mensur

    2005-01-01

    Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes. PMID:16207363

  19. 7 CFR 1c.116 - General requirements for informed consent.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 1 2011-01-01 2011-01-01 false General requirements for informed consent. 1c.116 Section 1c.116 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.116 General requirements for informed consent. Except as provided elsewhere in this policy, no investigator may involve a human being as a subject...

  20. 7 CFR 1c.112 - Review by institution.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Review by institution. 1c.112 Section 1c.112 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.112 Review by institution... review and approval or disapproval by officials of the institution. However, those officials may...

  1. 7 CFR 1c.116 - General requirements for informed consent.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false General requirements for informed consent. 1c.116 Section 1c.116 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.116 General requirements for informed consent. Except as provided elsewhere in this policy, no investigator may involve a human being as a subject...

  2. 7 CFR 1c.108 - IRB functions and operations.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 1 2012-01-01 2012-01-01 false IRB functions and operations. 1c.108 Section 1c.108 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.108 IRB functions and operations. In order to fulfill the requirements of this policy each IRB shall: (a) Follow written...

  3. 7 CFR 1c.108 - IRB functions and operations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 1 2011-01-01 2011-01-01 false IRB functions and operations. 1c.108 Section 1c.108 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.108 IRB functions and operations. In order to fulfill the requirements of this policy each IRB shall: (a) Follow written...

  4. 7 CFR 1c.108 - IRB functions and operations.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 1 2014-01-01 2014-01-01 false IRB functions and operations. 1c.108 Section 1c.108 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.108 IRB functions and operations. In order to fulfill the requirements of this policy each IRB shall: (a) Follow written...

  5. 7 CFR 1c.108 - IRB functions and operations.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 1 2013-01-01 2013-01-01 false IRB functions and operations. 1c.108 Section 1c.108 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.108 IRB functions and operations. In order to fulfill the requirements of this policy each IRB shall: (a) Follow written...

  6. Assembly of human mitochondrial ATP synthase through two separate intermediates, F1-c-ring and b-e-g complex.

    PubMed

    Fujikawa, Makoto; Sugawara, Kanako; Tanabe, Tsutomu; Yoshida, Masasuke

    2015-09-14

    Mitochondrial ATP synthase is a motor enzyme in which a central shaft rotates in the stator casings fixed with the peripheral stator stalk. When expression of d-subunit, a stator stalk component, was knocked-down, human cells could not form ATP synthase holocomplex and instead accumulated two subcomplexes, one containing a central rotor shaft plus catalytic subunits (F1-c-ring) and the other containing stator stalk components ("b-e-g" complex). F1-c-ring was also formed when expression of mitochondrial DNA-coded a-subunit and A6L was suppressed. Thus, the central rotor shaft and the stator stalk are formed separately and they assemble later. Similar assembly strategy has been known for ATP synthase of yeast and Escherichia coli and could be common to all organisms. PMID:26297831

  7. Apoptotic and autophagic responses to photodynamic therapy in 1c1c7 murine hepatoma cells

    PubMed Central

    Andrzejak, Michelle; Price, Michael

    2011-01-01

    Photodynamic therapy (PDT) is a process that can induce apoptosis, autophagy or both depending on the cell phenotype. Apoptosis is a pathway to cell death while autophagy can protect from photokilling or act as a death pathway. In a previous study, we reported a cytoprotective effect of autophagy in murine leukemia cell lines where both autophagy and apoptosis occur within minutes after irradiation of photosensitized cells. In this study, we examined the effects of mitochondrial photodamage catalyzed by low (≤1 µM) concentrations of the photosensitizing agent termed benzoporphyrin derivative (BPD, Verteporfin) on murine hepatoma 1c1c7 cells. Apoptosis was not observed until several hours after irradiation of photosensitized cells. Autophagy was clearly cytoprotective since PDT efficacy was significantly enhanced in a knockdown sub-line (KD) in which the level of a critical autophagy protein (Atg7) was markedly reduced. This result indicates that autophagy can protect from phototoxicity even when apoptosis is substantially delayed. Much higher concentrations (≥10 µM) of BPD had previously been shown to inhibit autophagosome formation. Phototoxicity studies performed with 10 µM BPD and a proportionally reduced light dose were consistent with the absence of an autophagic process in wild-type (WT) cells under these conditions. PMID:21555918

  8. 7 CFR 1c.109 - IRB review of research.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 1 2011-01-01 2011-01-01 false IRB review of research. 1c.109 Section 1c.109... research. (a) An IRB shall review and have authority to approve, require modifications in (to secure approval), or disapprove all research activities covered by this policy. (b) An IRB shall require...

  9. 7 CFR 1c.117 - Documentation of informed consent.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 1 2013-01-01 2013-01-01 false Documentation of informed consent. 1c.117 Section 1c... Documentation of informed consent. (a) Except as provided in paragraph (c) of this section, informed consent... following: (1) A written consent document that embodies the elements of informed consent required by §...

  10. 7 CFR 1c.117 - Documentation of informed consent.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 1 2012-01-01 2012-01-01 false Documentation of informed consent. 1c.117 Section 1c... Documentation of informed consent. (a) Except as provided in paragraph (c) of this section, informed consent... following: (1) A written consent document that embodies the elements of informed consent required by §...

  11. 7 CFR 1c.117 - Documentation of informed consent.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 1 2011-01-01 2011-01-01 false Documentation of informed consent. 1c.117 Section 1c... Documentation of informed consent. (a) Except as provided in paragraph (c) of this section, informed consent... following: (1) A written consent document that embodies the elements of informed consent required by §...

  12. 7 CFR 1c.117 - Documentation of informed consent.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 1 2014-01-01 2014-01-01 false Documentation of informed consent. 1c.117 Section 1c... Documentation of informed consent. (a) Except as provided in paragraph (c) of this section, informed consent... following: (1) A written consent document that embodies the elements of informed consent required by §...

  13. CDKN1C mutations: two sides of the same coin.

    PubMed

    Eggermann, Thomas; Binder, Gerhard; Brioude, Frédéric; Maher, Eamonn R; Lapunzina, Pablo; Cubellis, Maria Vittoria; Bergadá, Ignacio; Prawitt, Dirk; Begemann, Matthias

    2014-11-01

    Cyclin-dependent kinase (CDK)-inhibitor 1C (CDKN1C) negatively regulates cellular proliferation and it has been shown that loss-of-function mutations in the imprinted CDKN1C gene (11p15.5) are associated with the overgrowth disorder Beckwith-Wiedemann syndrome (BWS). With recent reports of gain-of-function mutations of the PCNA domain of CDKN1C in growth-retarded patients with IMAGe syndrome or Silver-Russell syndrome (SRS), its key role for growth has been confirmed. Thereby, the last gap in the spectrum of molecular alterations in 11p15.5 in growth-retardation and overgrowth syndromes could be closed. Recent functional studies explain the strict association of CDKN1C mutations with clinically opposite phenotypes and thereby contribute to our understanding of the function and regulation of the gene in particular and epigenetic regulation in general. PMID:25262539

  14. Pathogenesis of POLR1C-dependent Type 3 Treacher Collins Syndrome revealed by a zebrafish model.

    PubMed

    Lau, Marco Chi Chung; Kwong, Ernest Man Lok; Lai, Keng Po; Li, Jing-Woei; Ho, Jeff Cheuk Hin; Chan, Ting-Fung; Wong, Chris Kong Chu; Jiang, Yun-Jin; Tse, William Ka Fai

    2016-06-01

    Treacher Collins Syndrome (TCS) is a rare congenital birth disorder (1 in 50,000 live births) characterized by severe craniofacial defects, including the downward slanting palpebral fissures, hypoplasia of the facial bones, and cleft palate (CP). Over 90% of patients with TCS have a mutation in the TCOF1 gene. However, some patients exhibit mutations in two new causative genes, POLR1C and POLR1D, which encode subunits of RNA polymerases I and III, that affect ribosome biogenesis. In this study, we examine the role of POLR1C in TCS using zebrafish as a model system. Our data confirmed that polr1c is highly expressed in the facial region, and dysfunction of this gene by knockdown or knock-out resulted in mis-expression of neural crest cells during early development that leads to TCS phenotype. Next generation sequencing and bioinformatics analysis of the polr1c mutants further demonstrated the up-regulated p53 pathway and predicted skeletal disorders. Lastly, we partially rescued the TCS facial phenotype in the background of p53 mutants, which supported the hypothesis that POLR1C-dependent type 3 TCS is associated with the p53 pathway. PMID:26972049

  15. Targeting MUC1-C is synergistic with bortezomib in downregulating TIGAR and inducing ROS-mediated myeloma cell death

    PubMed Central

    Yin, Li; Kufe, Turner; Avigan, David

    2014-01-01

    The proteosome inhibitor bortezomib (BTZ) induces endoplasmic reticulum and oxidative stress in multiple myeloma (MM) cells. The mucin 1 C-terminal subunit (MUC1-C) oncoprotein is aberrantly expressed in most MM cells, and targeting MUC1-C with GO-203, a cell-penetrating peptide inhibitor of MUC1-C homodimerization, is effective in inducing reactive oxygen species (ROS)-mediated MM cell death. The present results demonstrate that GO-203 and BTZ synergistically downregulate expression of the p53-inducible regulator of glycolysis and apoptosis (TIGAR), which promotes shunting of glucose-6-phosphate into the pentose phosphate pathway to generate reduced glutathione (GSH). In turn, GO-203 blocks BTZ-induced increases in GSH and results in synergistic increases in ROS and MM cell death. The results also demonstrate that GO-203 is effective against BTZ-resistant MM cells. We show that BTZ resistance is associated with BTZ-induced increases in TIGAR and GSH levels, and that GO-203 resensitizes BTZ-resistant cells to BTZ treatment by synergistically downregulating TIGAR and GSH. The GO-203/BTZ combination is thus highly effective in killing BTZ-resistant MM cells. These findings support a model in which targeting MUC1-C is synergistic with BTZ in suppressing TIGAR-mediated regulation of ROS levels and provide an experimental rationale for combining GO-203 with BTZ in certain settings of BTZ resistance. PMID:24632713

  16. Carnitine palmitoyltransferase 1C deficiency causes motor impairment and hypoactivity.

    PubMed

    Carrasco, Patricia; Jacas, Jordi; Sahún, Ignasi; Muley, Helena; Ramírez, Sara; Puisac, Beatriz; Mezquita, Pau; Pié, Juan; Dierssen, Mara; Casals, Núria

    2013-11-01

    Carnitine palmitoyltransferase 1c (CPT1C), a brain-specific protein localized in the endoplasmic reticulum of neurons, is expressed in almost all brain regions, but its only known functions to date are involved in the hypothalamic control of energy homeostasis and in hippocampus-dependent spatial learning. To identify other physiological and behavioral functions of this protein, we performed a battery of neurological tests on Cpt1c-deficient mice. The animals showed intact autonomic and sensory systems, but some motor disturbances were observed. A more detailed study of motor function revealed impaired coordination and gait, severe muscle weakness, and reduced daily locomotor activity. Analysis of motor function in these mice at ages of 6-24 weeks showed that motor disorders were already present in young animals and that impairment increased progressively with age. Analysis of CPT1C expression in different motor brain areas during development revealed that CPT1C levels were low from birth to postnatal day 10 and then rapidly increased peaking at postnatal day 21, which suggests that CPT1C plays a relevant role in motor function during and after weaning. As CPT1C is known to regulate ceramide levels, we measured these biolipids in different motor areas in adult mice. Cerebellar, striatum, and motor cortex extracts from Cpt1c knockout mice showed reduced levels of ceramide and its derivative sphingosine when compared to wild-type animals. Our results indicate that altered ceramide metabolism in motor brain areas induced by Cpt1c deficiency causes progressive motor dysfunction from a young age. PMID:23973755

  17. The Correlation of Hemoglobin A1c to Blood Glucose

    PubMed Central

    Sikaris, Ken

    2009-01-01

    The understanding that hemoglobin A1c (HbA1c) represents the average blood glucose level of patients over the previous 120 days underlies the current management of diabetes. Even in making such a statement, we speak of “average blood glucose” as though “blood glucose” were itself a simple idea. When we consider all the blood glucose forms—arterial versus venous versus capillary, whole blood versus serum versus fluoride-preserved plasma, fasting versus nonfasting—we can start to see that this is not a simple issue. Nevertheless, it seems as though HbA1c correlates to any single glucose measurement. Having more than one measurement and taking those measurements in the preceding month improves the correlation further. In particular, by having glucose measurements that reflect both the relatively lower overnight glucose levels and measurements that reflect the postprandial peaks improves not only our ability to manage diabetes patients, but also our understanding of how HbA1c levels are determined. Modern continuous glucose monitoring (CGM) devices may take thousands of glucose results over a week. Several studies have shown that CGM glucose averages account for the vast proportion of the variation of HbA1c. The ability to relate HbA1c to average glucose may become a popular method for reporting HbA1c, eliminating current concerns regarding differences in HbA1c standardization. Hemoglobin A1c expressed as an average glucose may be more understandable to patients and improve not only their understanding, but also their ability to improve their diabetes management. PMID:20144279

  18. 7 CFR 1c.109 - IRB review of research.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... in accordance with § 1c.117. (d) An IRB shall notify investigators and the institution in writing of... investigator an opportunity to respond in person or in writing. (e) An IRB shall conduct continuing review...

  19. 7 CFR 1c.108 - IRB functions and operations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...) Except when an expedited review procedure is used (see § 1c.110), review proposed research at convened... primary concerns are in nonscientific areas. In order for the research to be approved, it shall...

  20. Involvement of glomerular SREBP-1c in diabetic nephropathy

    SciTech Connect

    Ishigaki, Naomi; Yamamoto, Takashi; Shimizu, Yoshio; Kobayashi, Kazuto; Yatoh, Shigeru; Sone, Hirohito; Takahashi, Akimitsu; Suzuki, Hiroaki; Yamagata, Kunihiro; Yamada, Nobuhiro; Shimano, Hitoshi

    2007-12-21

    The role of glomerular SREBP-1c in diabetic nephropathy was investigated. PEPCK-promoter transgenic mice overexpressing nuclear SREBP-1c exhibited enhancement of proteinuria with mesangial proliferation and matrix accumulation, mimicking diabetic nephropathy, despite the absence of hyperglycemia or hyperlipidemia. Isolated transgenic glomeruli had higher expression of TGF{beta}-1, fibronectin, and SPARC in the absence of marked lipid accumulation. Gene expression of P47phox, p67phox, and PU.1 were also activated, accompanying increased 8-OHdG in urine and kidney, demonstrating that glomerular SREBP-1c could directly cause oxidative stress through induced NADPH oxidase. Similar changes were observed in STZ-treated diabetic mice with activation of endogenous SREBP-1c. Finally, diabetic proteinuria and oxidative stress were ameliorated in SREBP-1-null mice. Adenoviral overexpression of active and dominant-negative SREBP-1c caused consistent reciprocal changes in expression of both profibrotic and oxidative stress genes in MES13 mesangial cells. These data suggest that activation of glomerular SREBP-1c could contribute to emergence and/or progression of diabetic nephropathy.

  1. Carnitine palmitoyltransferase 1C: From cognition to cancer.

    PubMed

    Casals, Núria; Zammit, Victor; Herrero, Laura; Fadó, Rut; Rodríguez-Rodríguez, Rosalía; Serra, Dolors

    2016-01-01

    Carnitine palmitoyltransferase 1 (CPT1) C was the last member of the CPT1 family of genes to be discovered. CPT1A and CPT1B were identified as the gate-keeper enzymes for the entry of long-chain fatty acids (as carnitine esters) into mitochondria and their further oxidation, and they show differences in their kinetics and tissue expression. Although CPT1C exhibits high sequence similarity to CPT1A and CPT1B, it is specifically expressed in neurons (a cell-type that does not use fatty acids as fuel to any major extent), it is localized in the endoplasmic reticulum of cells, and it has minimal CPT1 catalytic activity with l-carnitine and acyl-CoA esters. The lack of an easily measurable biological activity has hampered attempts to elucidate the cellular and physiological role of CPT1C but has not diminished the interest of the biomedical research community in this CPT1 isoform. The observations that CPT1C binds malonyl-CoA and long-chain acyl-CoA suggest that it is a sensor of lipid metabolism in neurons, where it appears to impact ceramide and triacylglycerol (TAG) metabolism. CPT1C global knock-out mice show a wide range of brain disorders, including impaired cognition and spatial learning, motor deficits, and a deregulation in food intake and energy homeostasis. The first disease-causing CPT1C mutation was recently described in humans, with Cpt1c being identified as the gene causing hereditary spastic paraplegia. The putative role of CPT1C in the regulation of complex-lipid metabolism is supported by the observation that it is highly expressed in certain virulent tumor cells, conferring them resistance to glucose- and oxygen-deprivation. Therefore, CPT1C may be a promising target in the treatment of cancer. Here we review the molecular, biochemical, and structural properties of CPT1C and discuss its potential roles in brain function, and cancer. PMID:26708865

  2. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 28 Judicial Administration 2 2012-07-01 2012-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  3. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  4. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 2 2011-07-01 2011-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  5. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 28 Judicial Administration 2 2013-07-01 2013-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  6. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 28 Judicial Administration 2 2014-07-01 2014-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  7. Prenylated chalcones and flavanones as inducers of quinone reductase in mouse Hepa 1c1c7 cells.

    PubMed

    Miranda, C L; Aponso, G L; Stevens, J F; Deinzer, M L; Buhler, D R

    2000-02-28

    The objective of this study was to determine if prenylchalcones (open C-ring flavonoids) and prenylflavanones from hops and beer are inducers of quinone reductase (QR) in the mouse hepatoma Hepa 1c1c7 cell line. All the prenylchalcones and prenylflavanones tested were found to induce QR but not CYP1A1 in this cell line. In contrast, the synthetic chalcone, chalconaringenin, and the flavanone, naringenin, with no prenyl or geranyl groups, were ineffective in inducing QR. The hop chalcones, xanthohumol and dehydrocycloxanthohumol hydrate, also induced QR in the Ah-receptor-defective mutant cell line, Hepa 1c1c7 bp(r)c1. Thus, the prenylflavonoids represent a new class of monofunctional inducers of QR. PMID:10737704

  8. The Association Between A1C and Subclinical Cardiovascular Disease

    PubMed Central

    McNeely, Marguerite J.; McClelland, Robyn L.; Bild, Diane E.; Jacobs, David R.; Tracy, Russell P.; Cushman, Mary; Goff, David C.; Astor, Brad C.; Shea, Steven; Siscovick, David S.

    2009-01-01

    OBJECTIVE To test the hypothesis that A1C is associated with subclinical cardiovascular disease (CVD) in a population without evident diabetes, after adjusting for traditional CVD risk factors and BMI. RESEARCH DESIGN AND METHODS This was a cross-sectional study of 5,121 participants without clinically evident CVD or diabetes (fasting glucose ≥7.0 mmol/l or use of diabetes medication), aged 47–86 years, enrolled in the Multi-Ethnic Study of Atherosclerosis (MESA). Measurements included carotid intimal-medial wall thickness (CIMT) and coronary artery calcification (CAC). Results were adjusted for age, sex, ethnicity, smoking, systolic blood pressure, LDL cholesterol, HDL cholesterol, antihypertensive medication use, lipid-lowering medication use, and BMI. RESULTS Compared with those in the lowest quartile for A1C ([mean ± SD] 5.0 ± 0.2%), participants in the highest quartile (6.0 ± 0.3%) had higher adjusted mean values for common CIMT (0.85 vs. 0.87 mm, P = 0.003) and internal CIMT (1.01 vs. 1.08 mm, P = 0.003). A1C quartile was not associated with prevalence of CAC in the entire cohort (P = 0.27); however, the association was statistically significant in women (adjusted prevalence of CAC in lowest and highest A1C quartiles 37.5 vs. 43.0%, P = 0.01). Among those with some CAC, higher A1C quartile tended to be associated with higher CAC score, but the results were not statistically significant (adjusted P = 0.11). CONCLUSIONS In this multiethnic cohort, there were small, positive associations between A1C, common CIMT, and internal CIMT in the absence of clinically evident diabetes. An association between higher A1C and CAC prevalence was evident only in women. PMID:19549732

  9. Cyclic nucleotide phosphodiesterase-1C (PDE1C) drives cell proliferation, migration and invasion in glioblastoma multiforme cells in vitro.

    PubMed

    Rowther, Farjana B; Wei, Weinbin; Dawson, Timothy P; Ashton, Katherine; Singh, Anushree; Madiesse-Timchou, Mylene P; Thomas, D G T; Darling, John L; Warr, Tracy

    2016-03-01

    Cyclic nucleotides (cAMP & cGMP) are critical intracellular second messengers involved in the transduction of a diverse array of stimuli and their catabolism is mediated by phosphodiesterases (PDEs). We previously detected focal genomic amplification of PDE1C in >90 glioblastoma multiforme (GBM) cells suggesting a potential as a novel therapeutic target in these cells. In this report, we show that genomic gain of PDE1C was associated with increased expression in low passage GBM-derived cell cultures. We demonstrate that PDE1C is essential in driving cell proliferation, migration and invasion in GBM cultures since silencing of this gene significantly mitigates these functions. We also define the mechanistic basis of this functional effect through whole genome expression analysis by identifying down-stream gene effectors of PDE1C which are involved in cell cycle and cell adhesion regulation. In addition, we also demonstrate that Vinpocetine, a general PDE1 inhibitor, can also attenuate proliferation with no effect on invasion/migration. Up-regulation of at least one of this gene set (IL8, CXCL2, FOSB, NFE2L3, SUB1, SORBS2, WNT5A, and MMP1) in TCGA GBM cohorts is associated with worse outcome and PDE1C silencing down-regulated their expression, thus also indicating potential to influence patient survival. Therefore we conclude that proliferation, migration, and invasion of GBM cells could also be regulated downstream of PDE1C. PMID:25620587

  10. Hipk2 and PP1c cooperate to maintain Dvl protein levels required for Wnt signal transduction.

    PubMed

    Shimizu, Nobuyuki; Ishitani, Shizuka; Sato, Atsushi; Shibuya, Hiroshi; Ishitani, Tohru

    2014-09-11

    The phosphoprotein Dishevelled (Dvl) is a common essential component of Wnt/β-catenin and Wnt/planar cell polarity (PCP) signaling pathways. However, the regulation and significance of Dvl phosphorylation are not fully understood. Here, we show that homeodomain-interacting protein kinase 2 (Hipk2) facilitates protein phosphatase 1 catalytic subunit (PP1c)-mediated dephosphorylation of Dvl via its C-terminal domain and that this dephosphorylation blocks ubiquitination and consequent degradation mediated by the E3 ubiquitin ligase Itch, which targets the phosphorylated form of Dvl proteins. Inhibition of Hipk2 or PP1c function reduces Dvl protein levels and suppresses Wnt/β-catenin and Wnt/PCP pathway-dependent events in mammalian cells and zebrafish embryos, suggesting that Hipk2 and PP1c are essential for maintaining Dvl protein levels that are sufficient to activate Wnt signaling. We also show that Wnt-3a, a Wnt/β-catenin ligand, induces dissociation of the Dvl-Hipk2-PP1c complex and Dvl degradation under high-cell-density conditions. This regulation may be a negative feedback mechanism that fine-tunes Wnt/β-catenin signaling. PMID:25159144

  11. Epistatic interaction of genetic depression risk variants in the human subgenual cingulate cortex during memory encoding

    PubMed Central

    Schott, B H; Assmann, A; Schmierer, P; Soch, J; Erk, S; Garbusow, M; Mohnke, S; Pöhland, L; Romanczuk-Seiferth, N; Barman, A; Wüstenberg, T; Haddad, L; Grimm, O; Witt, S; Richter, S; Klein, M; Schütze, H; Mühleisen, T W; Cichon, S; Rietschel, M; Noethen, M M; Tost, H; Gundelfinger, E D; Düzel, E; Heinz, A; Meyer-Lindenberg, A; Seidenbecher, C I; Walter, H

    2014-01-01

    Recent genome-wide association studies have pointed to single-nucleotide polymorphisms (SNPs) in genes encoding the neuronal calcium channel CaV1.2 (CACNA1C; rs1006737) and the presynaptic active zone protein Piccolo (PCLO; rs2522833) as risk factors for affective disorders, particularly major depression. Previous neuroimaging studies of depression-related endophenotypes have highlighted the role of the subgenual cingulate cortex (CG25) in negative mood and depressive psychopathology. Here, we aimed to assess how recently associated PCLO and CACNA1C depression risk alleles jointly affect memory-related CG25 activity as an intermediate phenotype in clinically healthy humans. To investigate the combined effects of rs1006737 and rs2522833 on the CG25 response, we conducted three functional magnetic resonance imaging studies of episodic memory formation in three independent cohorts (N=79, 300, 113). An epistatic interaction of PCLO and CACNA1C risk alleles in CG25 during memory encoding was observed in all groups, with carriers of no risk allele and of both risk alleles showing higher CG25 activation during encoding when compared with carriers of only one risk allele. Moreover, PCLO risk allele carriers showed lower memory performance and reduced encoding-related hippocampal activation. In summary, our results point to region-specific epistatic effects of PCLO and CACNA1C risk variants in CG25, potentially related to episodic memory. Our data further suggest that genetic risk factors on the SNP level do not necessarily have additive effects but may show complex interactions. Such epistatic interactions might contribute to the ‘missing heritability' of complex phenotypes. PMID:24643163

  12. Impact of Ancillary Subunits on Ventricular Repolarization

    PubMed Central

    Abbott, Geoffrey W.; Xu, Xianghua; Roepke, Torsten K.

    2007-01-01

    Voltage-gated potassium (Kv) channels generate the outward K+ ion currents that constitute the primary force in ventricular repolarization. Kv channels comprise tetramers of pore-forming α subunits and, in probably the majority of cases in vivo, ancillary or β subunits that help define the properties of the Kv current generated. Ancillary subunits can be broadly categorized as cytoplasmic or transmembrane, and can modify Kv channel trafficking, conductance, gating, ion selectivity, regulation and pharmacology. Because of their often profound effects on Kv channel function, studies of the molecular correlates of ventricular repolarization must take into account ancillary subunits as well as α subunits. Cytoplasmic ancillary subunits include the Kvβ subunits, which regulate a range of Kv channels and may link channel gating to redox potential; and the KChIPs, which appear most often associated with Kv4 subfamily channels that generate the ventricular Ito current. Transmembrane ancillary subunits include the MinK-related proteins (MiRPs) encoded by KCNE genes, which modulate members of most Kv α subunit subfamilies; and the putative 12-transmembrane domain KCR1 protein which modulates hERG. In some cases, such as the ventricular IKs channel complex, it is well-established that the KCNQ1 α subunit must co-assemble with the MinK (KCNE1) single transmembrane domain ancillary subunit for recapitulation of the characteristic, unusually slowly-activating IKs current. In other cases it is not so clear-cut, and in particular the roles of the other MinK-related proteins (MiRPs 1–4) in regulating cardiac Kv channels such as KCNQ1 and hERG in vivo are under debate. MiRP1 alters hERG function and pharmacology, and inherited MiRP1 mutations are associated with inherited and acquired arrhythmias, but controversy exists over the native role of MiRP1 in regulating hERG (and therefore ventricular IKr) in vivo. Some ancillary subunits may exhibit varied expression to shape

  13. Regulation of Protein Phosphatase 1I by Cdc25C-associated Kinase 1 (C-TAK1) and PFTAIRE Protein Kinase*

    PubMed Central

    Platholi, Jimcy; Federman, Anna; Detert, Julia A.; Heerdt, Paul; Hemmings, Hugh C.

    2014-01-01

    Protein phosphatase 1I (PP-1I) is a major endogenous form of protein phosphatase 1 (PP-1) that consists of the core catalytic subunit PP-1c and the regulatory subunit inhibitor 2 (I-2). Phosphorylation of the Thr-72 residue of I-2 is required for activation of PP-1I. We studied the effects of two protein kinases identified previously in purified brain PP-1I by mass spectrometry, Cdc25C-associated kinase 1 (C-TAK1) and PFTAIRE (PFTK1) kinase, for their ability to regulate PP-1I. Purified C-TAK1 phosphorylated I-2 in reconstituted PP-1I (PP-1c·I-2) on Ser-71, which resulted in partial inhibition of its ATP-dependent phosphatase activity and inhibited subsequent phosphorylation of Thr-72 by the exogenous activating kinase GSK-3. In contrast, purified PFTK1 phosphorylated I-2 at Ser-86, a site known to potentiate Thr-72 phosphorylation and activation of PP-1I phosphatase activity by GSK-3. These findings indicate that brain PP-1I associates with and is regulated by the associated protein kinases C-TAK1 and PFTK1. Multisite phosphorylation of the I-2 regulatory subunit of PP-1I leads to activation or inactivation of PP-1I through bidirectional modulation of Thr-72 phosphorylation, the critical activating residue of I-2. PMID:25028520

  14. Identification of Shigella flexneri Subserotype 1c in Rural Egypt

    PubMed Central

    El-Gendy, Atef; El-Ghorab, Nemat; Lane, Edward M.; Elyazeed, Remon Abu; Carlin, Nils I. A.; Mitry, Mary M.; Kay, Bradford A.; Savarino, Stephen J.; Peruski, Leonard F.

    1999-01-01

    In a population-based study of diarrhea in rural, northern Egypt, 60 Shigella flexneri strains were identified, of which 10 could not be definitively serotyped. Serological analysis with commercial reagents suggested that they were serotype 1, but the strains failed to react with subserotype 1a- or 1b-specific antibodies. All 10 strains reacted with MASF 1c, a monoclonal antibody specific for a provisional S. flexneri subserotype, 1c, first identified in Bangladesh and not previously detected outside of that region. Our results show that S. flexneri subserotype 1c is not unique to Bangladesh and that the inability to detect it may reflect both the limited use of suitable screening methods and the rarity of this subserotype. PMID:9986881

  15. Selecting an A1C Point-of-Care Instrument

    PubMed Central

    Yong, Ee Vonn; Rasinen, Casey

    2015-01-01

    A1C point-of-care (POC) instruments benefit patients with diabetes by facilitating clinician decision making that results in significant glycemic improvements. Three National Glycohemoglobin Standardization Program (NGSP)–certified POC products are available in the United States: the handheld A1CNow (formerly manufactured by Bayer Diabetes Care but now made by Chek Diagnostics) and two bench-top models called the Axis-Shield Afinion Analyzer and the Siemens DCA Vantage. This article compares the three available NGSP-certified POC products in terms of accuracy, precision, ease of use, cost, and additional features. Its goal is to aid health care facilities in conveniently identifying the A1C POC product that best meets their needs. It additionally reviews evidence that supports the continued use of A1C POC instruments in the clinical arena. PMID:26300614

  16. Tracking Diabetes: New York City's A1C Registry

    PubMed Central

    Chamany, Shadi; Silver, Lynn D; Bassett, Mary T; Driver, Cynthia R; Berger, Diana K; Neuhaus, Charlotte E; Kumar, Namrata; Frieden, Thomas R

    2009-01-01

    Context: In December 2005, in characterizing diabetes as an epidemic, the New York City Board of Health mandated the laboratory reporting of hemoglobin A1C laboratory test results. This mandate established the United States’ first population-based registry to track the level of blood sugar control in people with diabetes. But mandatory A1C reporting has provoked debate regarding the role of public health agencies in the control of noncommunicable diseases and, more specifically, both privacy and the doctor-patient relationship. Methods: This article reviews the rationale for adopting the rule requiring the reporting of A1C test results, experience with its implementation, and criticisms raised in the context of the history of public health practice. Findings: For many decades, public health agencies have used identifiable information collected through mandatory laboratory reporting to monitor the population's health and develop programs for the control of communicable and noncommunicable diseases. The registry program sends quarterly patient rosters stratified by A1C level to more than one thousand medical providers, and it also sends letters, on the provider's letterhead whenever possible, to patients at risk of diabetes complications (A1C level >9 percent), advising medical follow-up. The activities of the registry program are similar to those of programs for other reportable conditions and constitute a joint effort between a governmental public health agency and medical providers to improve patients’ health outcomes. Conclusions: Mandatory reporting has proven successful in helping combat other major epidemics. New York City's A1C Registry activities combine both traditional and novel public health approaches to reduce the burden of an epidemic chronic disease, diabetes. Despite criticism that mandatory reporting compromises individuals’ right to privacy without clear benefit, the early feedback has been positive and suggests that the benefits will

  17. MUC1-C Stabilizes MCL-1 in the Oxidative Stress Response of Triple-Negative Breast Cancer Cells to BCL-2 Inhibitors

    PubMed Central

    Hiraki, Masayuki; Suzuki, Yozo; Alam, Maroof; Hinohara, Kunihiko; Hasegawa, Masanori; Jin, Caining; Kharbanda, Surender; Kufe, Donald

    2016-01-01

    Aberrant expression of myeloid cell leukemia-1 (MCL-1) is a major cause of drug resistance in triple-negative breast cancer (TNBC) cells. Mucin 1 (MUC1) is a heterodimeric oncoprotein that is aberrantly overexpressed in most TNBC. The present studies show that targeting the oncogenic MUC1 C-terminal subunit (MUC1-C) in TNBC cells with silencing or pharmacologic inhibition with GO-203 is associated with downregulation of MCL-1 levels. Targeting MUC1-C suppresses the MEK → ERK and PI3K → AKT pathways, and in turn destabilizes MCL-1. The small molecules ABT-737 and ABT-263 target BCL-2, BCL-XL and BCL-w, but not MCL-1. We show that treatment with ABT-737 increases reactive oxygen species and thereby MUC1-C expression. In this way, MUC1-C is upregulated in TNBC cells resistant to ABT-737 or ABT-263. We also demonstrate that MUC1-C is necessary for the resistance-associated increases in MCL-1 levels. Significantly, combining GO-203 with ABT-737 is synergistic in inhibiting survival of parental and drug resistant TNBC cells. These findings indicate that targeting MUC1-C is a potential strategy for reversing MCL-1-mediated resistance in TNBC. PMID:27217294

  18. MUC1-C Oncoprotein Activates ERK→C/EBPβ Signaling and Induction of Aldehyde Dehydrogenase 1A1 in Breast Cancer Cells*

    PubMed Central

    Alam, Maroof; Ahmad, Rehan; Rajabi, Hasan; Kharbanda, Akriti; Kufe, Donald

    2013-01-01

    Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is used as a marker of breast cancer stem cells; however, little is known about the regulation of ALDH1A1 expression. Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed in most human breast cancers. In studies of breast cancer cells stably silenced for MUC1 or overexpressing the oncogenic MUC1-C subunit, we demonstrate that MUC1-C is sufficient for induction of MEK→ERK signaling and that treatment with a MUC1-C inhibitor suppresses ERK activation. In turn, MUC1-C induces ERK-mediated phosphorylation and activation of the CCAAT/enhancer-binding protein β (C/EBPβ) transcription factor. The results further show that MUC1-C and C/EBPβ form a complex on the ALDH1A1 gene promoter and activate ALDH1A1 gene transcription. MUC1-C-induced up-regulation of ALDH1A1 expression is associated with increases in ALDH activity and is detectable in stem-like cells when expanded as mammospheres. These findings demonstrate that MUC1-C (i) activates a previously unrecognized ERK→C/EBPβ→ALDH1A1 pathway, and (ii) promotes the induction of ALDH activity in breast cancer cells. PMID:24043631

  19. MUC1-C Stabilizes MCL-1 in the Oxidative Stress Response of Triple-Negative Breast Cancer Cells to BCL-2 Inhibitors.

    PubMed

    Hiraki, Masayuki; Suzuki, Yozo; Alam, Maroof; Hinohara, Kunihiko; Hasegawa, Masanori; Jin, Caining; Kharbanda, Surender; Kufe, Donald

    2016-01-01

    Aberrant expression of myeloid cell leukemia-1 (MCL-1) is a major cause of drug resistance in triple-negative breast cancer (TNBC) cells. Mucin 1 (MUC1) is a heterodimeric oncoprotein that is aberrantly overexpressed in most TNBC. The present studies show that targeting the oncogenic MUC1 C-terminal subunit (MUC1-C) in TNBC cells with silencing or pharmacologic inhibition with GO-203 is associated with downregulation of MCL-1 levels. Targeting MUC1-C suppresses the MEK → ERK and PI3K → AKT pathways, and in turn destabilizes MCL-1. The small molecules ABT-737 and ABT-263 target BCL-2, BCL-XL and BCL-w, but not MCL-1. We show that treatment with ABT-737 increases reactive oxygen species and thereby MUC1-C expression. In this way, MUC1-C is upregulated in TNBC cells resistant to ABT-737 or ABT-263. We also demonstrate that MUC1-C is necessary for the resistance-associated increases in MCL-1 levels. Significantly, combining GO-203 with ABT-737 is synergistic in inhibiting survival of parental and drug resistant TNBC cells. These findings indicate that targeting MUC1-C is a potential strategy for reversing MCL-1-mediated resistance in TNBC. PMID:27217294

  20. Cleft Lip Repair: The Hybrid Subunit Method.

    PubMed

    Tollefson, Travis T

    2016-04-01

    The unilateral cleft lip repair is one of the most rewarding and challenging of plastic surgery procedures. Surgeons have introduced a variety of straight line, geometric, and rotation-advancement designs, while in practice the majority of North American surgeons have been using hybrids of the rotation-advancement techniques. The anatomic subunit approach was introduced in 2005 by Fisher and has gained popularity, with early adopters of the design touting its simplicity and effectiveness. The objectives of this article are to summarize the basic tenets of respecting the philtral subunit, accurate measurement and planning, and tips for transitioning to this subunit approach. PMID:27097136

  1. 7 CFR 1c.116 - General requirements for informed consent.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 1 2012-01-01 2012-01-01 false General requirements for informed consent. 1c.116... General requirements for informed consent. Except as provided elsewhere in this policy, no investigator... obtained the legally effective informed consent of the subject or the subject's legally...

  2. 7 CFR 1c.109 - IRB review of research.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... information given to subjects as part of informed consent is in accordance with § 1c.116. The IRB may require... welfare of subjects. (c) An IRB shall require documentation of informed consent or may waive documentation... year, and shall have authority to observe or have a third party observe the consent process and...

  3. 7 CFR 1c.116 - General requirements for informed consent.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 1 2014-01-01 2014-01-01 false General requirements for informed consent. 1c.116... General requirements for informed consent. Except as provided elsewhere in this policy, no investigator... obtained the legally effective informed consent of the subject or the subject's legally...

  4. 7 CFR 1c.109 - IRB review of research.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... information given to subjects as part of informed consent is in accordance with § 1c.116. The IRB may require... welfare of subjects. (c) An IRB shall require documentation of informed consent or may waive documentation... year, and shall have authority to observe or have a third party observe the consent process and...

  5. 7 CFR 1c.116 - General requirements for informed consent.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 1 2013-01-01 2013-01-01 false General requirements for informed consent. 1c.116... General requirements for informed consent. Except as provided elsewhere in this policy, no investigator... obtained the legally effective informed consent of the subject or the subject's legally...

  6. 7 CFR 1c.109 - IRB review of research.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... information given to subjects as part of informed consent is in accordance with § 1c.116. The IRB may require... welfare of subjects. (c) An IRB shall require documentation of informed consent or may waive documentation... year, and shall have authority to observe or have a third party observe the consent process and...

  7. Protective effect of Porphyra yezoensis glycoprotein on D-galactosamine‑induced cytotoxicity in Hepa 1c1c7 cells.

    PubMed

    Choi, Jeong-Wook; Kim, Young-Min; Park, Su-Jin; Kim, In-Hye; Nam, Taek-Jeong

    2015-05-01

    The present study aimed to examine the signaling pathways and enzyme activity associated with the protective effect of Porphyra yezoensis glycoprotein (PYGP) on D‑galactosamine (D‑GaIN)‑induced cytotoxicity in Hepa 1c1c7 cells. D‑GaIN is commonly used to induce hepatic injury models in vivo as well as in vitro. PYGP was extracted from Porphyra yezoensis, a red algae distributed along the coasts of Republic of Korea, China and Japan. In the present study, Hepa 1c1c7 cells were pre‑treated with PYGP (20 and 40 µg/ml) for 24 h and then the media was replaced with D‑GaIN (20 mM) and PYGP (20 and 40 µg/ml). The results demonstrated that D‑GaIN induced Hepa 1c1c7 cell death and pretreatment with PYGP was found to attenuate D‑GaIN toxicity. In addition, D‑GaIN decreased the antioxidant activity and increased lipid peroxidation processes; however, pre‑treatment with PYGP reduced the generation of lipid peroxidation products, such as thiobarbituric acid reactive substances, as well as increased the activity of antioxidant enzymes, including superoxide dismutase, catalase and glutathione‑s‑transferase (GST). PYGP was shown to suppress the overexpression of extracellular signal‑regulated kinase, c‑jun N‑terminal kinase and p38 mitogen‑activated protein kinase (MAPK) phosphorylation induced by D‑GaIN. Furthermore, PYGP increased the protein expression of nuclear factor erythroid 2‑related factor 2 (Nrf2), quinine oxidoreductase 1, GST and heme oxygenase 1 protein expression. These results suggested that PYGP had cytoprotective effects against D‑GaIN‑induced cell damage, which may be associated with MAPKs and the Nrf2 signaling pathway. PMID:25626067

  8. The aromatic hydrocarbon receptor modulates the Hepa 1c1c7 cell cycle and differentiated state independently of dioxin.

    PubMed Central

    Ma, Q; Whitlock, J P

    1996-01-01

    The aromatic hydrocarbon receptor (AhR) has been defined and characterized according to its ability to mediate biological responses to exogenous ligands, such as the synthetic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The natural ligand(s) for AhR is unknown, and we know relatively little about AhR function in the absence of TCDD. Here, we have exploited the availability of AhR-defective (AhR-D) mouse hepatoma (Hepa 1c1c7) cells to analyze AhR's effects under conditions in which TCDD is not present. Our results reveal that AhR-D cells exhibit a different morphology, decreased albumin synthesis, and a prolonged doubling time compared with wild-type cells. Introduction of AhR cDNA into AhR-D cells by stable transfection alters these characteristics such that the cells resemble wild-type cells. Conversely, introduction of antisense AhR cDNA into wild-type cells changes their phenotype such that they resemble AhR-D cells. Fluorescence microscopy reveals that AhR-D cells do not exhibit an increased rate of death. Flow cytometric and biochemical analyses imply that the slowed growth rate of AhR-D cells reflects prolongation of G1. Our findings reveal a potential link between AhR and the G1 phase of the Hepa 1c1c7 cell cycle. These effects of AhR occur in the absence of TCDD. We speculate that they represent responses to an endogenous AhR ligand in Hepa 1c1c7 cells. PMID:8628281

  9. Suppression of CYP1A1 expression by naringenin in murine Hepa-1c1c7 cells.

    PubMed

    Kim, Ji Young; Han, Eun Hee; Shin, Dong Weon; Jeong, Tae Cheon; Lee, Eung Seok; Woo, Eun-Rhan; Jeong, Hye Gwang

    2004-08-01

    Naringenin, dietary flavonoid, is antioxidant constituents of many citrus fruits. In the present study, we investigated the effect of naringenin on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible CYP1A1 gene expression in mouse hepatoma Hepa-1c1c7 cells. Naringenin alone did not affect CYP1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity. In contrast, the TCDD-inducible EROD activities were markedly reduced upon concomitant treatment with TCDD and naringenin in a dose dependent manner. TCDD-induced CYP1A1 mRNA level was also markedly suppressed by naringenin. A transient transfection assay using dioxin-response element (DRE)-linked luciferase and electrophoretic mobility shift assay revealed that naringenin reduced transformation of the aryl hydrocarbons receptor(AhR) to a form capable of specifically binding to the DRE sequence in the promoter of the CYP1A1 gene. These results suggest the down regulation of the CYP1A1 gene expression by either naringenin in Hepa-1c1c7 cells might be antagonism of the DRE binding potential of nuclear AhR. PMID:15460448

  10. Suppression of CYP1A1 expression by 4-nonylphenol in murine Hepa-1c1c7 cells.

    PubMed

    Jeong, H G; Kim, J Y; Choi, C Y; You, H J; Hahm, K

    2001-04-10

    This study investigated the effects that 4-nonylphenol (NP) has on CYP1A1 expression in Hepa-1c1c7 cell cultures. NP alone did not affect CYP1A1-specific 7-ethoxyresorufin-O-deethylase (EROD) activity. In contrast, the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible EROD activities were markedly reduced upon concomitant treatment with TCDD and NP in a dose-dependent manner. Treatment with tamoxifen, an anti-estrogen that acts through the estrogen receptor, did not affect the suppressive effects that NP has on TCDD-inducible EROD activity. The TCDD-inducible CYP1A1 mRNA levels were markedly suppressed upon concomitant treatment with TCDD and NP that is consistent with their effects on EROD activity. A transient transfection assay using dioxin-response element (DRE)-linked luciferase and an electrophoretic mobility shift assay revealed that NP reduced the transformation of the aryl hydrocarbon (Ah) receptor to a form capable of binding specifically to the DRE sequence of the CYP1A1 gene promoter. These results suggest that the down-regulation of CYP1A1 gene expression by NP in Hepa-1c1c7 cells might be an antagonism of the DRE-binding potential of the nuclear Ah receptor, but is not mediated through the estradiol receptor. PMID:11248424

  11. Cytokine-mediated induction of metallothionein in Hepa-1c1c7 cells by oleanolic acid.

    PubMed

    Kim, Ji Young; Lee, Kyung Jin; Kim, Dong Hee; Jeong, Tae Cheon; Lee, Eung Seok; Choi, Young Muk; Jeong, Hye Gwang

    2004-12-17

    Oleanolic acid (OA), a pentacyclic triterpene acid, has been reported to possess inducing activity of hepatic metallothionein (MT). However, the mechanism underlying its effects is unknown. This study investigated the effects of OA on the regulation of MT expression in an in vitro model. OA that was added directly to Hepa-1c1c7 cells had no effect on MT induction. However, MT and its mRNA levels increased markedly when the Hepa-1c1c7 cells were cultured with the OA-treated conditioned media from the RAW 264.7 cells. Co-treating the RAW 264.7 cells with OA and pentoxifylline, a TNF-alpha synthesis inhibitor, resulted in a decrease in the effects of OA on the MT induction. In the OA-exposed RAW 264.7 cell cultures, production and mRNA levels of TNF-alpha and IL-6 were increased. However, the MT induction activity was inhibited when antibodies to TNF-alpha and/or IL-6 were added to the OA-treated conditioned media from the RAW 264.7 cells. These results suggest that the up-regulation of MT expression by OA was mediated by the TNF-alpha and IL-6 released from UA-activated macrophages. PMID:15541359

  12. Alcohol dehydrogenase 1C (ADH1C) gene polymorphism and alcoholic liver cirrhosis risk: a meta analysis

    PubMed Central

    He, Lei; Deng, Tao; Luo, He-Sheng

    2015-01-01

    The association between alcohol dehydrogenase 1C (ADH1C) gene polymorphism and alcoholic liver cirrhosis (ALC) has been analyzed in several studies, but results have been conflicting. In this study, a meta-analysis was performed to assess the associations between the ADH1C polymorphism and risk of ALC. Relevant studies were identified using PubMed, Web of Science, CNKI and Wanfang databases up to January 10, 2015. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the association using the fixed or random effect model. A total of 16 case-control studies, including 1375 cases and 1802 controls, were included. Overall, no significant association between the ADH1C polymorphism and ALC risk was found (dominant model: OR=0.87, 95% CI: 0.62-1.23; recessive model: OR=1.30, 95% CI: 0.84-1.99; *1/*2 vs. *1/*1: OR=0.87, 95% CI: 0.63-1.21; *2/*2 vs. *1/*1: OR=1.10, 95% CI: 0.71-1.70). In the subgroup analysis by ethnicity, we observed a significant association in Asian descent (*1/*2 vs. *1/*1: OR=1.63, 95% CI: 1.07-2.49), while a decreased risk was found among Caucasians (dominant model: OR=0.81, 95% CI: 0.66-0.99; *1/*2 vs. *1/*1: OR=0.76, 95% CI: 0.61-0.95). This meta-analysis demonstrated that the ADH1C polymorphism might increase the risk of ALC in Asians, while it may be a protective factor for ALC among Caucasians. PMID:26379912

  13. LQTS gene LOVD database.

    PubMed

    Zhang, Tao; Moss, Arthur; Cong, Peikuan; Pan, Min; Chang, Bingxi; Zheng, Liangrong; Fang, Quan; Zareba, Wojciech; Robinson, Jennifer; Lin, Changsong; Li, Zhongxiang; Wei, Junfang; Zeng, Qiang; Qi, Ming

    2010-11-01

    The Long QT Syndrome (LQTS) is a group of genetically heterogeneous disorders that predisposes young individuals to ventricular arrhythmias and sudden death. LQTS is mainly caused by mutations in genes encoding subunits of cardiac ion channels (KCNQ1, KCNH2,SCN5A, KCNE1, and KCNE2). Many other genes involved in LQTS have been described recently(KCNJ2, AKAP9, ANK2, CACNA1C, SCNA4B, SNTA1, and CAV3). We created an online database(http://www.genomed.org/LOVD/introduction.html) that provides information on variants in LQTS-associated genes. As of February 2010, the database contains 1738 unique variants in 12 genes. A total of 950 variants are considered pathogenic, 265 are possible pathogenic, 131 are unknown/unclassified, and 292 have no known pathogenicity. In addition to these mutations collected from published literature, we also submitted information on gene variants, including one possible novel pathogenic mutation in the KCNH2 splice site found in ten Chinese families with documented arrhythmias. The remote user is able to search the data and is encouraged to submit new mutations into the database. The LQTS database will become a powerful tool for both researchers and clinicians. PMID:20809527

  14. Rare variants in neuronal excitability genes influence risk for bipolar disorder

    PubMed Central

    Ament, Seth A.; Szelinger, Szabolcs; Glusman, Gustavo; Ashworth, Justin; Hou, Liping; Akula, Nirmala; Shekhtman, Tatyana; Badner, Judith A.; Brunkow, Mary E.; Mauldin, Denise E.; Stittrich, Anna-Barbara; Rouleau, Katherine; Detera-Wadleigh, Sevilla D.; Nurnberger, John I.; Gershon, Elliot S.; Schork, Nicholas; Price, Nathan D.; Gelinas, Richard; Hood, Leroy; Craig, David; McMahon, Francis J.; Kelsoe, John R.; Roach, Jared C.; Kelsoe, John R.; Greenwood, Tiffany A.; Nievergelt, Caroline M.; Shilling, Paul D.; Shekhtman, Tatyana; Roach, Jared C.; Ament, Seth A.; Hood, Leroy; Nurnberger, John I.; Edenberg, Howard J.; Foroud, Tatiana; Koller, Daniel L.; Gershon, Elliot S.; Badner, Judith A.; Liu, Chunyu; Scheftner, William A.; Lawson, William B.; Coryell, William; Potash, James B.; Rice, John; Byerley, William; McMahon, Francis J.; Hou, Liping; Berrettini, Wade H.; Zandi, Peter P.; McInnis, Melvin G.; Craig, David W.; Szelinger, Szabolcs; Schulze, Thomas G.; Schork, Nicholas J.; Quarless, Danjuma

    2015-01-01

    We sequenced the genomes of 200 individuals from 41 families multiply affected with bipolar disorder (BD) to identify contributions of rare variants to genetic risk. We initially focused on 3,087 candidate genes with known synaptic functions or prior evidence from genome-wide association studies. BD pedigrees had an increased burden of rare variants in genes encoding neuronal ion channels, including subunits of GABAA receptors and voltage-gated calcium channels. Four uncommon coding and regulatory variants also showed significant association, including a missense variant in GABRA6. Targeted sequencing of 26 of these candidate genes in an additional 3,014 cases and 1,717 controls confirmed rare variant associations in ANK3, CACNA1B, CACNA1C, CACNA1D, CACNG2, CAMK2A, and NGF. Variants in promoters and 5′ and 3′ UTRs contributed more strongly than coding variants to risk for BD, both in pedigrees and in the case-control cohort. The genes and pathways identified in this study regulate diverse aspects of neuronal excitability. We conclude that rare variants in neuronal excitability genes contribute to risk for BD. PMID:25730879

  15. Cloning, expression, and preliminary structural characterization of RTN-1C

    SciTech Connect

    Fazi, Barbara; Melino, Sonia; Sano, Federica Di; Cicero, Daniel O.; Piacentini, Mauro . E-mail: mauro.piacentini@uniroma2.it; Paci, Maurizio

    2006-04-14

    Reticulons (RTNs) are endoplasmic reticulum-associated proteins widely distributed in plants, yeast, and animals. They are characterized by unique N-terminal parts and a common 200 amino acid C-terminal domain containing two long hydrophobic sequences. Despite their implication in many cellular processes, their molecular structure and function are still largely unknown. In this study, the reticulon family member RTN-1C has been expressed and purified in Escherichia coli and its molecular structure has been analysed by fluorescence and CD spectroscopy in different detergents in order to obtain a good solubility and a relative stability. The isotopically enriched protein has been also produced to perform structural studies by NMR spectroscopy. The preliminary results obtained showed that RTN-1C protein possesses helical transmembrane segments when a membrane-like environment is produced by detergents. Moreover, fluorescence experiments indicated the exposure of tryptophan side chains as predicted by structure prediction programs. We also produced the isotopically labelled protein and the procedure adopted allowed us to plan future NMR studies to investigate the biochemical behaviour of reticulon-1C and of its peptides spanning out from the membrane.

  16. MUC1-C nuclear localization drives invasiveness of renal cancer cells through a sheddase/gamma secretase dependent pathway.

    PubMed

    Bouillez, Audrey; Gnemmi, Viviane; Gaudelot, Kelly; Hémon, Brigitte; Ringot, Bélinda; Pottier, Nicolas; Glowacki, François; Butruille, Caroline; Cauffiez, Christelle; Hamdane, Malika; Sergeant, Nicolas; Van Seuningen, Isabelle; Leroy, Xavier; Aubert, Sébastien; Perrais, Michaël

    2014-02-15

    MUC1 is a membrane-anchored mucin and its cytoplasmic tail (CT) can interact with many signaling pathways and act as a co-transcription factor to activate genes involved in tumor progression and metastasis. MUC1 is overexpressed in renal cell carcinoma with correlation to prognosis and has been implicated in the hypoxic pathway, the main renal carcinogenetic pathway. In this context, we assessed the effects of MUC1 overexpression on renal cancer cells properties. Using shRNA strategy and/or different MUC1 constructs, we found that MUC1-extracellular domain and MUC1-CT are involved in increase of migration, cell viability, resistance to anoikis and in decrease of cell aggregation in cancer cells. Invasiveness depends only on MUC1-CT. Then, by using siRNA strategy and/or pharmacological inhibitors or peptides, we showed that sheddases ADAM10, ADAM17 and gamma-secretase are necessary for MUC1 C-terminal subunit (MUC1-C) nuclear location and in increase of invasion property. Finally, MUC1 overexpression increases ADAM10/17 protein expression suggesting a positive regulatory loop. In conclusion, we report that MUC1 acts in renal cancer progression and MUC1-C nuclear localization drives invasiveness of cancer cells through a sheddase/gamma secretase dependent pathway. MUC1 appears as a therapeutic target by blocking MUC1 cleavage or nuclear translocation by using pharmacological approach and peptide strategies. PMID:24504508

  17. MUC1-C nuclear localization drives invasiveness of renal cancer cells through a sheddase/gamma secretase dependent pathway

    PubMed Central

    Gaudelot, Kelly; Hémon, Brigitte; Ringot, Bélinda; Pottier, Nicolas; Glowacki, François; Butruille, Caroline; Cauffiez, Christelle; Hamdane, Malika; Sergeant, Nicolas; Seuningen, Isabelle Van; Leroy, Xavier

    2014-01-01

    MUC1 is a membrane-anchored mucin and its cytoplasmic tail (CT) can interact with many signaling pathways and act as a co-transcription factor to activate genes involved in tumor progression and metastasis. MUC1 is overexpressed in renal cell carcinoma with correlation to prognosis and has been implicated in the hypoxic pathway, the main renal carcinogenetic pathway. In this context, we assessed the effects of MUC1 overexpression on renal cancer cells properties. Using shRNA strategy and/or different MUC1 constructs, we found that MUC1-extracellular domain and MUC1-CT are involved in increase of migration, cell viability, resistance to anoikis and in decrease of cell aggregation in cancer cells. Invasiveness depends only on MUC1-CT. Then, by using siRNA strategy and/or pharmacological inhibitors or peptides, we showed that sheddases ADAM10, ADAM17 and gamma-secretase are necessary for MUC1 C-terminal subunit (MUC1-C) nuclear location and in increase of invasion property. Finally, MUC1 overexpression increases ADAM10/17 protein expression suggesting a positive regulatory loop. In conclusion, we report that MUC1 acts in renal cancer progression and MUC1-C nuclear localization drives invasiveness of cancer cells through a sheddase/gamma secretase dependent pathway. MUC1 appears as a therapeutic target by blocking MUC1 cleavage or nuclear translocation by using pharmacological approach and peptide strategies. PMID:24504508

  18. New Determinant for the CaVβ2 Subunit Modulation of the CaV1.2 Calcium Channel*

    PubMed Central

    Lao, Qi Zong; Kobrinsky, Evgeny; Harry, Jo Beth; Ravindran, Arippa; Soldatov, Nikolai M.

    2008-01-01

    Cavβ subunits support voltage gating of Cav1.2 calcium channels and play important role in excitation-contraction coupling. The common central membrane-associated guanylate kinase (MAGUK) region of Cavβ binds to the α-interaction domain (AID) and the IQ motif of the pore-forming α1C subunit, but these two interactions do not explain why the cardiac Cavβ2 subunit splice variants differentially modulate inactivation of Ca2+ currents (ICa). Previously we described β2Δg, a functionally active splice variant of human Cavβ2 lacking MAGUK. By deletion analysis of β2Δg, we have now identified a 41-amino acid C-terminal essential determinant (β2CED) that stimulates ICa in the absence of Cavβ subunits and conveys a +20-mV shift in the peak of the ICa-voltage relationship. The β2CED is targeted by α1C to the plasma membrane, forms a complex with α1C but does not bind to AID. Electrophysiology and binding studies point to the calmodulin-interacting LA/IQ region in the α1C subunit C terminus as a functionally relevant β2CED binding site. The β2CED interacts with LA/IQ in a Ca2+- and calmodulin-independent manner and need LA, but not IQ, to activate the channel. Deletion/mutation analyses indicated that each of the three Cavβ2/α1C interactions is sufficient to support ICa. However, β2CED does not support Ca2+-dependent inactivation, suggesting that interactions of MAGUK with AID and IQ are crucial for Ca2+-induced inactivation. The β2CED is conserved only in Cavβ2 subunits. Thus, β2CED constitutes a previously unknown integrative part of the multifactorial mechanism of Cavβ2-subunit differential modulation of the Cav1.2 calcium channel that in β2Δg occurs without MAGUK. PMID:18411278

  19. Differential regulation of polysome mRNA levels in mouse Hepa-1C1C7 cells exposed to dioxin.

    PubMed

    Thornley, Jessica A; Trask, Heidi W; Ridley, Christian J A; Korc, Murray; Gui, Jiang; Ringelberg, Carol S; Wang, Sinny; Tomlinson, Craig R

    2011-10-01

    The environmental agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) causes a multitude of human illnesses. In order to more fully understand the underlying biology of TCDD toxicity, we tested the hypothesis that new candidate genes could be identified using polysome RNA from TCDD-treated mouse Hepa-1c1c7 cells. We found that (i) differentially expressed whole cell and cytoplasm RNA levels are both poor predictors of polysome RNA levels; (ii) for a majority of RNAs, differential RNA levels are regulated independently in the nucleus, cytoplasm, and polysomes; (iii) for the remaining polysome RNAs, levels are regulated via several different mechanisms, including a "tagging" of mRNAs in the nucleus for immediate polysome entry; and (iv) most importantly, a gene list derived from differentially expressed polysome RNA generated new genes and cell pathways potentially related to TCDD biology. PMID:21570461

  20. Effects of rutaecarpine on hydrogen peroxide-induced apoptosis in murine hepa-1c1c7 cells.

    PubMed

    Lee, Sung-Jin; Ahn, Hyunjin; Nam, Kung-Woo; Kim, Kyeong Ho; Mar, Woongchon

    2012-09-01

    The aim of this study was to investigate the inhibitory effects of rutaecarpine on DNA strand breaks and apoptosis induced by hydrogen peroxide (H2O2) in murine Hepa-1c1c7 cells. Oxidative DNA damage was estimated by nuclear condensation assessment, fluorescence-activated cell sorting analysis, and Comet assay. Rutaecarpine inhibited cell death induced by 500 μM H2O2, as assessed by 4',6-diamidino-2-phenylindole (DAPI) staining. Treatment with rutaecarpine reduced the number of DNA strand breaks induced by H2O2, as assessed by DAPI staining and Comet assay, and increased quinone reductase, phosphatidylinositol 3-kinase, and pAkt protein levels, as assessed by western blotting. PMID:24009839

  1. Induction of quinone reductase activity by stilbene analogs in mouse Hepa 1c1c7 cells.

    PubMed

    Heo, Y H; Kim, S; Park, J E; Jeong, L S; Lee, S K

    2001-12-01

    Based on the potential cancer chemopreventive activity of resveratrol, a trihydroxystilbene with the induction of quinone reductase activity, this study was designed to determine if stilbene-related compounds were inducers of phase II detoxifying metabolic enzyme quinone reductase (QR) in the mouse hepatoma Hepa 1c1c7 cells. Among the thirteen compounds tested, several compounds including 3,4,5,3',5'-pentamethoxy-trans-stilbene were found to potentially induce QR activity in this cell line. In addition, substitution with 3-thiofurane ring instead of phenyl ring in the stilbene skeleton also exhibited potential induction of QR activity. This result will give primary information to design the potential inducers of QR activity in the stilbene analogs. PMID:11794542

  2. Effects of Rutaecarpine on Hydrogen Peroxide-Induced Apoptosis in Murine Hepa-1c1c7 Cells

    PubMed Central

    Lee, Sung-Jin; Ahn, Hyunjin; Nam, Kung-Woo; Kim, Kyeong Ho; Mar, Woongchon

    2012-01-01

    The aim of this study was to investigate the inhibitory effects of rutaecarpine on DNA strand breaks and apoptosis induced by hydrogen peroxide (H2O2) in murine Hepa-1c1c7 cells. Oxidative DNA damage was estimated by nuclear condensation assessment, fluorescence-activated cell sorting analysis, and Comet assay. Rutaecarpine inhibited cell death induced by 500 μM H2O2, as assessed by 4',6-diamidino-2-phenylindole (DAPI) staining. Treatment with rutaecarpine reduced the number of DNA strand breaks induced by H2O2, as assessed by DAPI staining and Comet assay, and increased quinone reductase, phosphatidylinositol 3-kinase, and pAkt protein levels, as assessed by western blotting. PMID:24009839

  3. DIFFERENTIAL REGULATION OF POLYSOME mRNA LEVELS IN MOUSE HEPA-1C1C7 CELLS EXPOSED TO DIOXIN

    PubMed Central

    Thornley, Jessica A.; Trask, Heidi W.; Ridley, Christian J. A.; Korc, Murray; Gui, Jiang; Ringelberg, Carol S.; Wang, Sinny; Tomlinson, Craig R.

    2011-01-01

    The environmental agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) causes a multitude of human illnesses. In order to more fully understand the underlying biology of TCDD toxicity, we tested the hypothesis that new candidate genes could be identified using polysome RNA from TCDD-treated mouse Hepa-1c1c7 cells. We found that (i) differentially expressed whole cell and cytoplasm RNA levels are both poor predictors of polysome RNA levels; (ii) for a majority of RNAs, differential RNA levels are regulated independently in the nucleus, cytoplasm, and polysomes; (iii) for the remaining polysome RNAs, levels are regulated via several different mechanisms, including a “tagging” of mRNAs in the nucleus for immediate polysome entry; and (iv) most importantly, a gene list derived from differentially expressed polysome RNA generated new genes and cell pathways potentially related to TCDD biology. PMID:21570461

  4. Induction of quinone reductase activity by psoralidin isolated from Psoralea corylifolia in mouse hepa 1c1c7 cells.

    PubMed

    Lee, Sung-Jin; Nam, Kung-Woo; Mar, Woongchon

    2009-07-01

    Quinone reductase (QR) is a protective phase II enzyme against mutagens and carcinogens which is inducible by a number of chemical compounds in plants. This study was carried out to investigate effects of the fractions from the seeds of Psoralea corylifolia on the induction of QR with Hepa 1c1c7 murine hepatoma cell line. The ethyl acetate-soluble fraction of the methanolic extract from the seeds was found to induce QR and the concentration of 1.5 fold QR induction (1.5 FIC) was 1.2 mug/mL. We obtained as an active compound, psoralidin, isolated from the ethyl acetate-soluble fraction after further sequential fractionation with column chromatography and 1.5 FIC of psoralidin was 0.5 mug/mL. The seeds of Psoralea corylifolia and psoralidin might be a candidate for developing QR inducers. PMID:19641888

  5. Amaranth (Amaranthus hypochondriacus) vicilin subunit structure.

    PubMed

    Quiroga, Alejandra; Martínez, E Nora; Rogniaux, Hélène; Geairon, Audrey; Añón, M Cristina

    2010-12-22

    The 7S-globulin fraction is a minor component of the amaranth storage proteins. The present work provides new information about this protein. The amaranth 7S-globulin or vicilin presented a sedimentation coefficient of 8.6 ± 0.6 S and was composed of main subunits of 66, 52, 38, and 16 kDa. On the basis of mass spectrometry (MS) analysis of tryptic fragments, the 52, 38, and 16 kDa subunits presented sequence homology with sesame vicilin, whereas the 66 kDa subunit showed sequence similarity with a putative vicilin. Several characteristics of the 66 kDa subunit were similar to members of the convicilin family. Results support the hypothesis that the 7S-globulin molecules are composed of subunits coming from at least two gene families with primary products of 66 and 52 kDa, respectively. According to the present information, amaranth vicilin may be classified into the vicilin group that includes pea, broad bean, and sesame vicilins, among others. PMID:21117690

  6. Catechol-O-methyltransferase association with hemoglobin A1c

    PubMed Central

    Hall, Kathryn T.; Jablonski, Kathleen A.; Chen, Ling; Harden, Maegan; Tolkin, Benjamin R.; Kaptchuk, Ted J.; Bray, George A.; Ridker, Paul M.; Florez, Jose C.; Chasman, Daniel I.

    2016-01-01

    Aims Catecholamines have metabolic effects on blood pressure, insulin sensitivity and blood glucose. Genetic variation in catechol-O-methyltransferase (COMT), an enzyme that degrades catecholamines, is associated with cardiometabolic risk factors and incident cardiovascular disease (CVD). Here we examined COMT effects on glycemic function and type 2 diabetes. Methods We tested whether COMT polymorphisms were associated with baseline HbA1c in the Women’s Genome Health Study (WGHS), and Meta-Analyses of Glucose and Insulin-related traits Consortium (MAGIC), and with susceptibility to type 2 diabetes in WGHS, DIAbetes Genetics Replication And Meta-analysis consortium (DIAGRAM), and the Diabetes Prevention Program (DPP). Given evidence that COMT modifies some drug responses, we examined association with type 2 diabetes and randomized metformin and aspirin treatment. Results COMT rs4680 high-activity G-allele was associated with lower HbA1c in WGHS (β = −0.032% [0.012], p = 0.008) and borderline significant in MAGIC (β = −0.006% [0.003], p = 0.07). Combined COMT per val allele effects on type 2 diabetes were significant (OR = 0.98 [0.96–0.998], p = 0.03) in fixed-effects analyses across WGHS, DIAGRAM, and DPP. Similar results were obtained for 2 other COMT SNPs rs4818 and rs4633. In the DPP, the rs4680 val allele was borderline associated with lower diabetes incidence among participants randomized to metformin (HR = 0.81 [0.65–1.00], p = 0.05). Conclusions COMT rs4680 high-activity G-allele was associated with lower HbA1c and modest protection from type 2 diabetes. The directionality of COMT associations was concordant with those previously observed for cardiometabolic risk factors and CVD. PMID:27282867

  7. Subunit architecture of general transcription factor TFIIH.

    PubMed

    Gibbons, Brian J; Brignole, Edward J; Azubel, Maia; Murakami, Kenji; Voss, Neil R; Bushnell, David A; Asturias, Francisco J; Kornberg, Roger D

    2012-02-01

    Structures of complete 10-subunit yeast TFIIH and of a nested set of subcomplexes, containing 5, 6, and 7 subunits, have been determined by electron microscopy (EM) and 3D reconstruction. Consistency among all the structures establishes the location of the "minimal core" subunits (Ssl1, Tfb1, Tfb2, Tfb4, and Tfb5), and additional densities can be specifically attributed to Rad3, Ssl2, and the TFIIK trimer. These results can be further interpreted by placement of previous X-ray structures into the additional densities to give a preliminary picture of the RNA polymerase II preinitiation complex. In this picture, the key catalytic components of TFIIH, the Ssl2 ATPase/helicase and the Kin28 protein kinase are in proximity to their targets, downstream promoter DNA and the RNA polymerase C-terminal domain. PMID:22308316

  8. Heteromeric assembly of P2X subunits

    PubMed Central

    Saul, Anika; Hausmann, Ralf; Kless, Achim; Nicke, Annette

    2013-01-01

    Transcripts and/or proteins of P2X receptor (P2XR) subunits have been found in virtually all mammalian tissues. Generally more than one of the seven known P2X subunits have been identified in a given cell type. Six of the seven cloned P2X subunits can efficiently form functional homotrimeric ion channels in recombinant expression systems. This is in contrast to other ligand-gated ion channel families, such as the Cys-loop or glutamate receptors, where homomeric assemblies seem to represent the exception rather than the rule. P2XR mediated responses recorded from native tissues rarely match exactly the biophysical and pharmacological properties of heterologously expressed homomeric P2XRs. Heterotrimerization of P2X subunits is likely to account for this observed diversity. While the existence of heterotrimeric P2X2/3Rs and their role in physiological processes is well established, the composition of most other P2XR heteromers and/or the interplay between distinct trimeric receptor complexes in native tissues is not clear. After a description of P2XR assembly and the structure of the intersubunit ATP-binding site, this review summarizes the distribution of P2XR subunits in selected mammalian cell types and the biochemically and/or functionally characterized heteromeric P2XRs that have been observed upon heterologous co-expression of P2XR subunits. We further provide examples where the postulated heteromeric P2XRs have been suggested to occur in native tissues and an overview of the currently available pharmacological tools that have been used to discriminate between homo- and heteromeric P2XRs. PMID:24391538

  9. Level-1C Product from AIRS: Principal Component Filtering

    NASA Technical Reports Server (NTRS)

    Manning, Evan M.; Jiang, Yibo; Aumann, Hartmut H.; Elliott, Denis A.; Hannon, Scott

    2012-01-01

    The Atmospheric Infrared Sounder (AIRS), launched on the EOS Aqua spacecraft on May 4, 2002, is a grating spectrometer with 2378 channels in the range 3.7 to 15.4 microns. In a grating spectrometer each individual radiance measurement is largely independent of all others. Most measurements are extremely accurate and have very low noise levels. However, some channels exhibit high noise levels or other anomalous behavior, complicating applications needing radiances throughout a band, such as cross-calibration with other instruments and regression retrieval algorithms. The AIRS Level-1C product is similar to Level-1B but with instrument artifacts removed. This paper focuses on the "cleaning" portion of Level-1C, which identifies bad radiance values within spectra and produces substitute radiances using redundant information from other channels. The substitution is done in two passes, first with a simple combination of values from neighboring channels, then with principal components. After results of the substitution are shown, differences between principal component reconstructed values and observed radiances are used to investigate detailed noise characteristics and spatial misalignment in other channels.

  10. TARGETING THE ONCOGENIC MUC1-C PROTEIN INHIBITS MUTANT EGFR-MEDIATED SIGNALING AND SURVIVAL IN NON-SMALL CELL LUNG CANCER CELLS

    PubMed Central

    Kharbanda, Akriti; Rajabi, Hasan; Jin, Caining; Tchaicha, Jeremy; Kikuchi, Eiki; Wong, Kwok-Kin; Kufe, Donald

    2014-01-01

    Purpose Non-small cell lung cancers (NSCLC) that express the EGF receptor (EGFR) with activating mutations frequently develop resistance to EGFR kinase inhibitors. The mucin 1 (MUC1) heterodimeric protein is aberrantly overexpressed in NSCLC cells and confers a poor prognosis; however, the functional involvement of MUC1 in mutant EGFR signaling is not known. Experimental Design Targeting the oncogenic MUC1 C-terminal subunit (MUC1-C) in NSCLC cells harboring mutant EGFR was studied for effects on signaling, growth, clonogenic survival and tumorigenicity. Results Stable silencing of MUC1-C in H1975/EGFR(L858R/T790M) cells resulted in downregulation of AKT signaling and inhibition of growth, colony formation and tumorigenicity. Similar findings were obtained when MUC1-C was silenced in gefitinib-resistant PC9GR cells expressing EGFR(delE746_A750/T790M). The results further show that expression of a MUC1-C(CQC→AQA) mutant, which blocks MUC1-C homodimerization, suppresses EGFR(T790M), AKT and MEK→ERK activation, colony formation and tumorigenicity. In concert with these results, treatment of H1975 and PC9GR cells with GO-203, a cell-penetrating peptide that blocks MUC1-C homodimerization, resulted in inhibition of EGFR, AKT and MEK→ERK signaling and in loss of survival. Combination studies of GO-203 and afatinib, an irreversible inhibitor of EGFR, further demonstrate that these agents are synergistic in inhibiting growth of NSCLC cells harboring the activating EGFR(T790M) or EGFR(delE746-A750) mutants. Conclusions These findings indicate that targeting MUC1-C inhibits mutant EGFR signaling and survival, and thus represents a potential approach alone and in combination for the treatment of NSCLCs resistant to EGFR kinase inhibitors. PMID:25189483

  11. The MUC1 Extracellular Domain Subunit Is Found in Nuclear Speckles and Associates with Spliceosomes

    PubMed Central

    Kumar, Priyadarsina; Ji, Jennifer W.; Martsching, Lindsay; Douglas, Gordon C.

    2012-01-01

    MUC1 is a large transmembrane glycoprotein and oncogene expressed by epithelial cells and overexpressed and underglycosylated in cancer cells. The MUC1 cytoplasmic subunit (MUC1-C) can translocate to the nucleus and regulate gene expression. It is frequently assumed that the MUC1 extracellular subunit (MUC1-N) does not enter the nucleus. Based on an unexpected observation that MUC1 extracellular domain antibody produced an apparently nucleus-associated staining pattern in trophoblasts, we have tested the hypothesis that MUC1-N is expressed inside the nucleus. Three different antibodies were used to identify MUC1-N in normal epithelial cells and tissues as well as in several cancer cell lines. The results of immunofluorescence and confocal microscopy analyses as well as subcellular fractionation, Western blotting, and siRNA/shRNA studies, confirm that MUC1-N is found within nuclei of all cell types examined. More detailed examination of its intranuclear distribution using a proximity ligation assay, subcellular fractionation, and immunoprecipitation suggests that MUC1-N is located in nuclear speckles (interchromatin granule clusters) and closely associates with the spliceosome protein U2AF65. Nuclear localization of MUC1-N was abolished when cells were treated with RNase A and nuclear localization was altered when cells were incubated with the transcription inhibitor 5,6-dichloro-1-b-d-ribofuranosylbenzimidazole (DRB). While MUC1-N predominantly associated with speckles, MUC1-C was present in the nuclear matrix, nucleoli, and the nuclear periphery. In some nuclei, confocal microscopic analysis suggest that MUC1-C staining is located close to, but only partially overlaps, MUC1-N in speckles. However, only MUC1-N was found in isolated speckles by Western blotting. Also, MUC1-C and MUC1-N distributed differently during mitosis. These results suggest that MUC1-N translocates to the nucleus where it is expressed in nuclear speckles and that MUC1-N and MUC1-C have

  12. Oxidant regulated inter-subunit disulfide bond formation between ASIC1a subunits

    PubMed Central

    Zha, Xiang-ming; Wang, Runping; Collier, Dan M.; Snyder, Peter M.; Wemmie, John A.; Welsh, Michael J.

    2009-01-01

    The acid-sensing ion channel-1a (ASIC1a) is composed of 3 subunits and is activated by a decrease in extracellular pH. It plays an important role in diseases associated with a reduced pH and production of oxidants. Previous work showed that oxidants reduce ASIC1a currents. However, the effects on channel structure and composition are unknown. We found that ASIC1a formed inter-subunit disulfide bonds and the oxidant H2O2 increased this link between subunits. Cys-495 in the ASIC1a C terminus was particularly important for inter-subunit disulfide bond formation, although other C-terminal cysteines contributed. Inter-subunit disulfide bonds also produced some ASIC1a complexes larger than trimers. Inter-subunit disulfide bond formation reduced the proportion of ASIC1a located on the cell surface and contributed to the H2O2-induced decrease in H+-gated current. These results indicate that channel function is controlled by disulfide bond formation between intracellular residues on distinct ASIC1a subunits. They also suggest a mechanism by which the redox state can dynamically regulate membrane protein activity by forming intracellular bridges. PMID:19218436

  13. Targeting Atp6v1c1 Prevents Inflammation and Bone Erosion Caused by Periodontitis and Reveals Its Critical Function in Osteoimmunology

    PubMed Central

    Wang, Lin; Lu, Yun; Li, Qian; Zhu, Zheng; Shao, Jian-Zhong; Chen, Wei

    2015-01-01

    Periodontal disease (Periodontitis) is a serious disease that affects a majority of adult Americans and is associated with other systemic diseases, including diabetes, rheumatoid arthritis, and other inflammatory diseases. While great efforts have been devoted toward understanding the pathogenesis of periodontitis, there remains a pressing need for developing potent therapeutic strategies for targeting this pervasive and destructive disease. In this study, we utilized novel adeno-associated virus (AAV)-mediated Atp6v1c1 knockdown gene therapy to treat bone erosion and inflammatory caused by periodontitis in mouse model. Atp6v1c1 is a subunit of the V-ATPase complex and regulator of the assembly of the V0 and V1 domains of the V-ATPase complex. We demonstrated previously that Atp6v1c1 has an essential function in osteoclast mediated bone resorption. We hypothesized that Atp6v1c1 may be an ideal target to prevent the bone erosion and inflammation caused by periodontitis. To test the hypothesis, we employed AAV RNAi knockdown of Atp6v1c1 gene expression to prevent bone erosion and gingival inflammation simultaneously. We found that lesion-specific injection of AAV-shRNA-Atp6v1c1 into the periodontal disease lesions protected against bone erosion (>85%) and gingival inflammation caused by P. gingivalis W50 infection. AAV-mediated Atp6v1c1 knockdown dramatically reduced osteoclast numbers and inhibited the infiltration of dendritic cells and macrophages in the bacteria-induced inflammatory lesions in periodontitis. Silencing of Atp6v1c1 expression also prevented the expressions of osteoclast-related genes and pro-inflammatory cytokine genes. Our data suggests that AAV-shRNA-Atp6v1c1 treatment can significantly attenuate the bone erosion and inflammation caused by periodontitis, indicating the dual function of AAV-shRNA-Atp6v1c1 as an inhibitor of bone erosion mediated by osteoclasts, and as an inhibitor of inflammation through down-regulation of pro

  14. A1c Variability Can Predict Coronary Artery Disease in Patients with Type 2 Diabetes with Mean A1c Levels Greater than 7

    PubMed Central

    Lee, Eun Ju; Kim, You Jeong; Kim, Tae Nyun; Kim, Tae Ik; Lee, Won Kee; Park, Jeong Hyun; Rhee, Byoung Doo

    2013-01-01

    Background Recent studies suggested that the association of acute glucose variability and diabetic complications was not consistent, and that A1c variability representing long term glucose fluctuation may be related to coronary atherosclerosis in patients with type 1 diabetes. In this study, we attempt to determine whether or not A1c variability can predict coronary artery disease (CAD) in patients with type 2 diabetes. Methods We reviewed data of patients with type 2 diabetes who had undergone coronary angiography (CAG) and had been followed up with for 5 years. The intrapersonal standard deviation (SD) of serially-measured A1c levels adjusted by the different number of assessments among patients (adj-A1c-SD) was considered to be a measure of the variability of A1c. Results Among the 269 patients, 121 of them had type 2 diabetes with CAD. In patients with A1c ≥7%, the mean A1c levels and A1c levels at the time of CAG among the three groups were significantly different. The ratio of patients with CAD was the highest in the high adj-A1c-SD group and the lowest in the low adj-A1c-SD group (P=0.017). In multiple regression analysis, adj-A1c-SD was an independent predictor for CAD in subjects with A1c ≥7% (odds ratio, 2.140; P=0.036). Conclusion Patients with higher A1c variability for several years showed higher mean A1c levels. A1c variability can be an independent predictor for CAD as seen in angiographs of patients with type 2 diabetes with mean A1c levels over 7%. PMID:24396666

  15. A polymorphic motif in the small subunit of ADP-glucose pyrophosphorylase modulates interactions between the small and large subunits.

    PubMed

    Cross, Joanna M; Clancy, Maureen; Shaw, Janine R; Boehlein, Susan K; Greene, Thomas W; Schmidt, Robert R; Okita, Thomas W; Hannah, L Curtis

    2005-02-01

    The heterotetrameric, allosterically regulated enzyme, adenosine-5'-diphosphoglucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch synthesis. Despite vast differences in allosteric properties and a long evolutionary separation, heterotetramers of potato small subunit and maize large subunit have activity comparable to either parent in an Escherichia coli expression system. In contrast, co-expression of maize small subunit with the potato large subunit produces little activity as judged by in vivo activity stain. To pinpoint the region responsible for differential activity, we expressed chimeric maize/potato small subunits in E. coli. This identified a 55-amino acid motif of the potato small subunit that is critical for glycogen production when expressed with the potato large subunit. Potato and maize small subunit sequences differ at five amino acids in this motif. Replacement experiments revealed that at least four amino acids of maize origin were required to reduce staining. An AGPase composed of a chimeric potato small subunit containing the 55-amino acid maize motif with the potato large subunit exhibited substantially less affinity for the substrates, glucose-1-phosphate and ATP and an increased Ka for the activator, 3-phosphoglyceric acid. Placement of the potato motif into the maize small subunit restored glycogen synthesis with the potato large subunit. Hence, a small polymorphic motif within the small subunit influences both catalytic and allosteric properties by modulating subunit interactions. PMID:15686515

  16. Step 1: C3 Flight Demo Data Analysis Plan

    NASA Technical Reports Server (NTRS)

    2005-01-01

    The Data Analysis Plan (DAP) describes the data analysis that the C3 Work Package (WP) will perform in support of the Access 5 Step 1 C3 flight demonstration objectives as well as the processes that will be used by the Flight IPT to gather and distribute the data collected to satisfy those objectives. In addition to C3 requirements, this document will encompass some Human Systems Interface (HSI) requirements in performing the C3 flight demonstrations. The C3 DAP will be used as the primary interface requirements document between the C3 Work Package and Flight Test organizations (Flight IPT and Non-Access 5 Flight Programs). In addition to providing data requirements for Access 5 flight test (piggyback technology demonstration flights, dedicated C3 technology demonstration flights, and Airspace Operations Demonstration flights), the C3 DAP will be used to request flight data from Non- Access 5 flight programs for C3 related data products

  17. Regulation of persistent Na current by interactions between beta subunits of voltage-gated Na channels.

    PubMed

    Aman, Teresa K; Grieco-Calub, Tina M; Chen, Chunling; Rusconi, Raffaella; Slat, Emily A; Isom, Lori L; Raman, Indira M

    2009-02-18

    The beta subunits of voltage-gated Na channels (Scnxb) regulate the gating of pore-forming alpha subunits, as well as their trafficking and localization. In heterologous expression systems, beta1, beta2, and beta3 subunits influence inactivation and persistent current in different ways. To test how the beta4 protein regulates Na channel gating, we transfected beta4 into HEK (human embryonic kidney) cells stably expressing Na(V)1.1. Unlike a free peptide with a sequence from the beta4 cytoplasmic domain, the full-length beta4 protein did not block open channels. Instead, beta4 expression favored open states by shifting activation curves negative, decreasing the slope of the inactivation curve, and increasing the percentage of noninactivating current. Consequently, persistent current tripled in amplitude. Expression of beta1 or chimeric subunits including the beta1 extracellular domain, however, favored inactivation. Coexpressing Na(V)1.1 and beta4 with beta1 produced tiny persistent currents, indicating that beta1 overcomes the effects of beta4 in heterotrimeric channels. In contrast, beta1(C121W), which contains an extracellular epilepsy-associated mutation, did not counteract the destabilization of inactivation by beta4 and also required unusually large depolarizations for channel opening. In cultured hippocampal neurons transfected with beta4, persistent current was slightly but significantly increased. Moreover, in beta4-expressing neurons from Scn1b and Scn1b/Scn2b null mice, entry into inactivated states was slowed. These data suggest that beta1 and beta4 have antagonistic roles, the former favoring inactivation, and the latter favoring activation. Because increased Na channel availability may facilitate action potential firing, these results suggest a mechanism for seizure susceptibility of both mice and humans with disrupted beta1 subunits. PMID:19228957

  18. Regulation of persistent Na current by interactions between β subunits of voltage-gated Na channels

    PubMed Central

    Aman, Teresa K.; Grieco-Calub, Tina M.; Chen, Chunling; Rusconi, Raffaella; Slat, Emily A.; Isom, Lori L.; Raman, Indira M.

    2009-01-01

    The β subunits of voltage-gated Na channels (Scnxb) regulate the gating of pore-forming α subunits, as well as their trafficking and localization. In heterologous expression systems, β1, β2, and β3 subunits influence inactivation and persistent current in different ways. To test how the β4 protein regulates Na channel gating, we transfected β4 into HEK cells stably expressing NaV1.1. Unlike a free peptide with a sequence from the β4 cytoplasmic domain, the full-length β4 protein did not block open channels. Instead, β4 expression favored open states by shifting activation curves negative, decreasing the slope of the inactivation curve, and increasing the percentage of non-inactivating current. Consequently, persistent current tripled in amplitude. Expression of β1 or chimeric subunits including the β1 extracellular domain, however, favored inactivation. Co-expressing NaV1.1 and β4 with β1 produced tiny persistent currents, indicating that β1 overcomes the effects of β4 in heterotrimeric channels. In contrast, β1C121W, which contains an extracellular epilepsy-associated mutation, did not counteract the destabilization of inactivation by β4, and also required unusually large depolarizations for channel opening. In cultured hippocampal neurons transfected with β4, persistent current was slightly but significantly increased. Moreover, in β4-expressing neurons from Scn1b and Scn1b/Scn2b null mice, entry into inactivated states was slowed. These data suggest that β1 and β4 have antagonistic roles, the former favoring inactivation and the latter favoring activation. Because increased Na channel availability may facilitate action potential firing, these results suggest a mechanism for seizure susceptibility of both mice and humans with disrupted β1 subunits. PMID:19228957

  19. Conserved Residues in the N Terminus of Lipin-1 Are Required for Binding to Protein Phosphatase-1c, Nuclear Translocation, and Phosphatidate Phosphatase Activity*

    PubMed Central

    Kok, Bernard P. C.; Skene-Arnold, Tamara D.; Ling, Ji; Benesch, Matthew G. K.; Dewald, Jay; Harris, Thurl E.; Holmes, Charles F. B.; Brindley, David N.

    2014-01-01

    Lipin-1 is a phosphatidate phosphatase in glycerolipid biosynthesis and signal transduction. It also serves as a transcriptional co-regulator to control lipid metabolism and adipogenesis. These functions are controlled partly by its subcellular distribution. Hyperphosphorylated lipin-1 remains sequestered in the cytosol, whereas hypophosphorylated lipin-1 translocates to the endoplasmic reticulum and nucleus. The serine/threonine protein phosphatase-1 catalytic subunit (PP-1c) is a major protein dephosphorylation enzyme. Its activity is controlled by interactions with different regulatory proteins, many of which contain conserved RVXF binding motifs. We found that lipin-1 binds to PP-1cγ through a similar HVRF binding motif. This interaction depends on Mg2+ or Mn2+ and is competitively inhibited by (R/H)VXF-containing peptides. Mutating the HVRF motif in the highly conserved N terminus of lipin-1 greatly decreases PP-1cγ interaction. Moreover, mutations of other residues in the N terminus of lipin-1 also modulate PP-1cγ binding. PP-1cγ binds poorly to a phosphomimetic mutant of lipin-1 and binds well to the non-phosphorylatable lipin-1 mutant. This indicates that lipin-1 is dephosphorylated before PP-1cγ binds to its HVRF motif. Importantly, mutating the HVRF motif also abrogates the nuclear translocation and phosphatidate phosphatase activity of lipin-1. In conclusion, we provide novel evidence of the importance of the lipin-1 N-terminal domain for its catalytic activity, nuclear localization, and binding to PP-1cγ. PMID:24558042

  20. The NMDA Receptor NR1 C1 Region Bound to Calmodulin: Structural Insights into Functional Differences between Homologous Domains

    SciTech Connect

    Ataman, Zeynep Akyol; Gakhar, Lokesh; Sorensen, Brenda R.; Hell, Johannes W.; Shea, Madeline A.

    2008-09-17

    Calmodulin (CaM) regulates tetrameric N-methyl-D-aspartate receptors (NMDARs) by binding tightly to the C0 and C1 regions of its NR1 subunit. A crystal structure (2HQW; 1.96 {angstrom}) of calcium-saturated CaM bound to NR1C1 (peptide spanning 875-898) showed that NR1 S890, whose phosphorylation regulates membrane localization, was solvent protected, whereas the endoplasmic reticulum retention motif was solvent exposed. NR1 F880 filled the CaM C-domain pocket, whereas T886 was closest to the N-domain pocket. This 1-7 pattern was most similar to that in the CaM-MARCKS complex. Comparison of CaM-ligand wrap-around conformations identified a core tetrad of CaM C-domain residues (FLMM{sub C}) that contacted all ligands consistently. An identical tetrad of N-domain residues (FLMM{sub N}) made variable sets of contacts with ligands. This CaM-NR1C1 structure provides a foundation for designing mutants to test the role of CaM in NR1 trafficking as well as insights into how the homologous CaM domains have different roles in molecular recognition.

  1. 50 CFR Table 1c to Part 679 - Product Type Codes

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 13 2013-10-01 2013-10-01 false Product Type Codes 1c Table 1c to Part 679 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES OF THE EXCLUSIVE ECONOMIC ZONE OFF ALASKA Pt. 679, Table 1c Table 1c to Part 679—Product...

  2. Selective AKR1C3 inhibitors do not recapitulate the anti-leukaemic activities of the pan-AKR1C inhibitor medroxyprogesterone acetate

    PubMed Central

    Khanim, F; Davies, N; Veliça, P; Hayden, R; Ride, J; Pararasa, C; Chong, M G; Gunther, U; Veerapen, N; Winn, P; Farmer, R; Trivier, E; Rigoreau, L; Drayson, M; Bunce, C

    2014-01-01

    Background: We and others have identified the aldo-keto reductase AKR1C3 as a potential drug target in prostate cancer, breast cancer and leukaemia. As a consequence, significant effort is being invested in the development of AKR1C3-selective inhibitors. Methods: We report the screening of an in-house drug library to identify known drugs that selectively inhibit AKR1C3 over the closely related isoforms AKR1C1, 1C2 and 1C4. This screen initially identified tetracycline as a potential AKR1C3-selective inhibitor. However, mass spectrometry and nuclear magnetic resonance studies identified that the active agent was a novel breakdown product (4-methyl(de-dimethylamine)-tetracycline (4-MDDT)). Results: We demonstrate that, although 4-MDDT enters AML cells and inhibits their AKR1C3 activity, it does not recapitulate the anti-leukaemic actions of the pan-AKR1C inhibitor medroxyprogesterone acetate (MPA). Screens of the NCI diversity set and an independently curated small-molecule library identified several additional AKR1C3-selective inhibitors, none of which had the expected anti-leukaemic activity. However, a pan AKR1C, also identified in the NCI diversity set faithfully recapitulated the actions of MPA. Conclusions: In summary, we have identified a novel tetracycline-derived product that provides an excellent lead structure with proven drug-like qualities for the development of AKR1C3 inhibitors. However, our findings suggest that, at least in leukaemia, selective inhibition of AKR1C3 is insufficient to elicit an anticancer effect and that multiple AKR1C inhibition may be required. PMID:24569460

  3. SMOS L1C and L2 Validation in Australia

    NASA Technical Reports Server (NTRS)

    Rudiger, Christoph; Walker, Jeffrey P.; Kerr, Yann H.; Mialon, Arnaud; Merlin, Olivier; Kim, Edward J.

    2012-01-01

    Extensive airborne field campaigns (Australian Airborne Cal/val Experiments for SMOS - AACES) were undertaken during the 2010 summer and winter seasons of the southern hemisphere. The purpose of those campaigns was the validation of the Level 1c (brightness temperature) and Level 2 (soil moisture) products of the ESA-led Soil Moisture and Ocean Salinity (SMOS) mission. As SMOS is the first satellite to globally map L-band (1.4GHz) emissions from the Earth?s surface, and the first 2-dimensional interferometric microwave radiometer used for Earth observation, large scale and long-term validation campaigns have been conducted world-wide, of which AACES is the most extensive. AACES combined large scale medium-resolution airborne L-band and spectral observations, along with high-resolution in-situ measurements of soil moisture across a 50,000km2 area of the Murrumbidgee River catchment, located in south-eastern Australia. This paper presents a qualitative assessment of the SMOS brightness temperature and soil moisture products.

  4. Black hole collapse in the 1 /c expansion

    NASA Astrophysics Data System (ADS)

    Anous, Tarek; Hartman, Thomas; Rovai, Antonin; Sonner, Julian

    2016-07-01

    We present a first-principles CFT calculation corresponding to the spherical collapse of a shell of matter in three dimensional quantum gravity. In field theory terms, we describe the equilibration process, from early times to thermalization, of a CFT following a sudden injection of energy at time t = 0. By formulating a continuum version of Zamolodchikov's monodromy method to calculate conformal blocks at large central charge c, we give a framework to compute a general class of probe observables in the collapse state, incorporating the full backreaction of matter fields on the dual geometry. This is illustrated by calculating a scalar field two-point function at time-like separation and the time-dependent entanglement entropy of an interval, both showing thermalization at late times. The results are in perfect agreement with previous gravity calculations in the AdS3-Vaidya geometry. Information loss appears in the CFT as an explicit violation of unitarity in the 1 /c expansion, restored by nonperturbative corrections.

  5. PKA regulatory subunit expression in tooth development.

    PubMed

    de Sousa, Sílvia Ferreira; Kawasaki, Katsushige; Kawasaki, Maiko; Volponi, Ana Angelova; Gomez, Ricardo Santiago; Gomes, Carolina Cavaliéri; Sharpe, Paul T; Ohazama, Atsushi

    2014-05-01

    Protein kinase A (PKA) plays critical roles in many biological processes including cell proliferation, cell differentiation, cellular metabolism and gene regulation. Mutation in PKA regulatory subunit, PRKAR1A has previously been identified in odontogenic myxomas, but it is unclear whether PKA is involved in tooth development. The aim of the present study was to assess the expression of alpha isoforms of PKA regulatory subunit (Prkar1a and Prkar2a) in mouse and human odontogenesis by in situ hybridization. PRKAR1A and PRKAR2A mRNA transcription was further confirmed in a human deciduous germ by qRT-PCR. Mouse Prkar1a and human PRKAR2A exhibited a dynamic spatio-temporal expression in tooth development, whereas neither human PRKAR1A nor mouse Prkar2a showed their expression in odontogenesis. These isoforms thus showed different expression pattern between human and mouse tooth germs. PMID:24755349

  6. Recent Advances in Subunit Vaccine Carriers

    PubMed Central

    Vartak, Abhishek; Sucheck, Steven J.

    2016-01-01

    The lower immunogenicity of synthetic subunit antigens, compared to live attenuated vaccines, is being addressed with improved vaccine carriers. Recent reports indicate that the physio-chemical properties of these carriers can be altered to achieve optimal antigen presentation, endosomal escape, particle bio-distribution, and cellular trafficking. The carriers can be modified with various antigens and ligands for dendritic cells targeting. They can also be modified with adjuvants, either covalently or entrapped in the matrix, to improve cellular and humoral immune responses against the antigen. As a result, these multi-functional carrier systems are being explored for use in active immunotherapy against cancer and infectious diseases. Advancing technology, improved analytical methods, and use of computational methodology have also contributed to the development of subunit vaccine carriers. This review details recent breakthroughs in the design of nano-particulate vaccine carriers, including liposomes, polymeric nanoparticles, and inorganic nanoparticles. PMID:27104575

  7. Recent Advances in Subunit Vaccine Carriers.

    PubMed

    Vartak, Abhishek; Sucheck, Steven J

    2016-01-01

    The lower immunogenicity of synthetic subunit antigens, compared to live attenuated vaccines, is being addressed with improved vaccine carriers. Recent reports indicate that the physio-chemical properties of these carriers can be altered to achieve optimal antigen presentation, endosomal escape, particle bio-distribution, and cellular trafficking. The carriers can be modified with various antigens and ligands for dendritic cells targeting. They can also be modified with adjuvants, either covalently or entrapped in the matrix, to improve cellular and humoral immune responses against the antigen. As a result, these multi-functional carrier systems are being explored for use in active immunotherapy against cancer and infectious diseases. Advancing technology, improved analytical methods, and use of computational methodology have also contributed to the development of subunit vaccine carriers. This review details recent breakthroughs in the design of nano-particulate vaccine carriers, including liposomes, polymeric nanoparticles, and inorganic nanoparticles. PMID:27104575

  8. The Catalytic Subunit of Protein Phosphatase 1 Gamma Regulates Thrombin-Induced Murine Platelet αIIbβ3 Function

    PubMed Central

    Gushiken, Francisca C.; Hyojeong, Han; Pradhan, Subhashree; Langlois, Kimberly W.; Alrehani, Nawaf; Cruz, Miguel A.; Rumbaut, Rolando E.; Vijayan, K. Vinod

    2009-01-01

    Background Hemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin αIIbβ3. Integrin activation, in turn is mediated by cellular signaling via protein kinases and protein phosphatases. Although the catalytic subunit of protein phosphatase 1 (PP1c) interacts with αIIbβ3, the role of PP1c in platelet reactivity is unclear. Methodology/Principal Findings Using γ isoform of PP1c deficient mice (PP1cγ−/−), we show that the platelets have moderately decreased soluble fibrinogen binding and aggregation to low concentrations of thrombin or protease-activated receptor 4 (PAR4)-activating peptide but not to adenosine diphosphate (ADP), collagen or collagen-related peptide (CRP). Thrombin-stimulated PP1cγ−/− platelets showed decreased αIIbβ3 activation despite comparable levels of αIIbβ3, PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin αIIbβ3 signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cγ−/− platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cγ−/− mice. Phosphorylation of glycogen synthase kinase (GSK3)β-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cγ−/− platelets by an AKT independent mechanism. Inhibition of GSK3β partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cγ−/− platelets. Conclusions/Significance These studies illustrate a role for PP1cγ in maintaining GSK3β-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation. PMID:20016849

  9. Glycogen synthase kinase-3-mediated phosphorylation of serine 73 targets sterol response element binding protein-1c (SREBP-1c) for proteasomal degradation.

    PubMed

    Dong, Qingming; Giorgianni, Francesco; Beranova-Giorgianni, Sarka; Deng, Xiong; O'Meally, Robert N; Bridges, Dave; Park, Edwards A; Cole, Robert N; Elam, Marshall B; Raghow, Rajendra

    2016-01-01

    Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates genes involved in the de novo lipid synthesis and glycolysis pathways. The structure, turnover and transactivation potential of SREBP-1c are regulated by macronutrients and hormones via a cascade of signalling kinases. Using MS, we have identified serine 73 as a novel glycogen synthase kinase-3 (GSK-3) phosphorylation site in the rat SREBP-1c purified from McA-RH7777 hepatoma cells. Our site-specific mutagenesis strategy revealed that the turnover of SREBP-1c, containing wild type, phospho-null (serine to alanine) or phospho-mimetic (serine to aspartic acid) substitutions, was differentially regulated. We show that the S73D mutant of pSREBP-1c, that mimicked a state of constitutive phosphorylation, dissociated from the SREBP-1c-SCAP complex more readily and underwent GSK-3-dependent proteasomal degradation via SCF(Fbw7) ubiquitin ligase pathway. Pharmacologic inhibition of GSK-3 or knockdown of GSK-3 by siRNA prevented accelerated degradation of SREBP-1c. As demonstrated by MS, SREBP-1c was phosphorylated in vitro by GSK-3β at serine 73. Phosphorylation of serine 73 also occurs in the intact liver. We propose that GSK-3-mediated phosphorylation of serine 73 in the rat SREBP-1c and its concomitant destabilization represents a novel mechanism involved in the inhibition of de novo lipid synthesis in the liver. PMID:26589965

  10. CD1c-Expression by Monocytes – Implications for the Use of Commercial CD1c+ Dendritic Cell Isolation Kits

    PubMed Central

    Schrøder, Martine; Melum, Guro Reinholt; Landsverk, Ole J. B.; Bujko, Anna; Yaqub, Sheraz; Gran, Einar; Aamodt, Henrik; Bækkevold, Espen S.; Jahnsen, Frode L.

    2016-01-01

    Conventional dendritic cells (cDCs) comprise a heterogeneous population of cells that are important regulators of immunity and homeostasis. CD1c+ cDCs are present in human blood and tissues, and found to efficiently activate naïve CD4+ T cells. While CD1c is thought to specifically identify this subset of human cDCs, we show here that also classical and intermediate monocytes express CD1c. Accordingly, the commercial CD1c (BDCA-1)+ Dendritic Cell Isolation Kit isolates two distinct cell populations from blood: CD1c+CD14− cDCs and CD1c+CD14+ monocytes. CD1c+ cDCs and CD1c+ monocytes exhibited strikingly different properties, including their differential regulation of surface marker expression, their levels of cytokine production, and their ability to stimulate naïve CD4+ T cells. These results demonstrate that a commercial CD1c (BDCA-1)+ Dendritic Cell Isolation Kit isolates two functionally different cell populations, which has important implications for the interpretation of previously generated data using this kit to characterize CD1c+ cDCs. PMID:27311059

  11. Staggering of subunits in NMDAR channels.

    PubMed Central

    Sobolevsky, Alexander I; Rooney, LeeAnn; Wollmuth, Lonnie P

    2002-01-01

    Functional N-methyl-D-aspartate receptors (NMDARs) are heteromultimers formed by NR1 and NR2 subunits. The M3 segment, as contributed by NR1, forms the core of the extracellular vestibule, including binding sites for channel blockers, and represents a critical molecular link between ligand binding and channel opening. Taking advantage of the substituted cysteine accessibility method along with channel block and multivalent coordination, we studied the contribution of the M3 segment in NR2C to the extracellular vestibule. We find that the M3 segment in NR2C, like that in NR1, contributes to the core of the extracellular vestibule. However, the M3 segments from the two subunits are staggered relative to each other in the vertical axis of the channel. Compared to NR1, homologous positions in NR2C, including those in the highly conserved SYTANLAAF motif, are located about four amino acids more externally. The staggering of subunits may represent a key structural feature underlying the distinct functional properties of NMDARs. PMID:12496098

  12. Na Channel β Subunits: Overachievers of the Ion Channel Family.

    PubMed

    Brackenbury, William J; Isom, Lori L

    2011-01-01

    Voltage-gated Na(+) channels (VGSCs) in mammals contain a pore-forming α subunit and one or more β subunits. There are five mammalian β subunits in total: β1, β1B, β2, β3, and β4, encoded by four genes: SCN1B-SCN4B. With the exception of the SCN1B splice variant, β1B, the β subunits are type I topology transmembrane proteins. In contrast, β1B lacks a transmembrane domain and is a secreted protein. A growing body of work shows that VGSC β subunits are multifunctional. While they do not form the ion channel pore, β subunits alter gating, voltage-dependence, and kinetics of VGSCα subunits and thus regulate cellular excitability in vivo. In addition to their roles in channel modulation, β subunits are members of the immunoglobulin superfamily of cell adhesion molecules and regulate cell adhesion and migration. β subunits are also substrates for sequential proteolytic cleavage by secretases. An example of the multifunctional nature of β subunits is β1, encoded by SCN1B, that plays a critical role in neuronal migration and pathfinding during brain development, and whose function is dependent on Na(+) current and γ-secretase activity. Functional deletion of SCN1B results in Dravet Syndrome, a severe and intractable pediatric epileptic encephalopathy. β subunits are emerging as key players in a wide variety of physiopathologies, including epilepsy, cardiac arrhythmia, multiple sclerosis, Huntington's disease, neuropsychiatric disorders, neuropathic and inflammatory pain, and cancer. β subunits mediate multiple signaling pathways on different timescales, regulating electrical excitability, adhesion, migration, pathfinding, and transcription. Importantly, some β subunit functions may operate independently of α subunits. Thus, β subunits perform critical roles during development and disease. As such, they may prove useful in disease diagnosis and therapy. PMID:22007171

  13. Cardiac phenotype induced by a dysfunctional α1C transgene

    PubMed Central

    Lao, Qi Zong; Ravindran, Arippa; Herbert, Ron; Canuto, Holly C

    2011-01-01

    Based on stable integration of recombinant DNA into a host genome, transgenic technology has become an important genetic engineering methodology. An organism whose genetic characteristics have been altered by the insertion of foreign DNA is supposed to exhibit a new phenotype associated with the function of the transgene. However, successful insertion may not be sufficient to achieve specific modification of function. In this study we describe a strain of transgenic mouse, G7-882, generated by incorporation into the mouse genome of human Cav1.2 α1C cDNA deprived of 3′-UTR to exclude transcription. We found that, in response to chronic infusion of isoproterenol, G7-882 develops dilated cardiomyopathy, a misleading “transgenic artifact” compatible with the expected function of the incorporated “correct” transgene. Specifically, using magnetic resonance imaging (MRI), we found that chronic β-adrenergic stimulation of G7-882 mice caused left ventricular hypertrophy and aggravated development of dilated cardiomyopathy, although no significant changes in the kinetics, density and voltage dependence of the calcium current were observed in G7-882 cardiomyocytes as compared to cells from wild type mice. This result illustrates the possibility that even when a functional transgene is expressed, an observed change in phenotype may be due to the artifact of “incidental incorporation” leading to misleading conclusions. To exclude this possibility and thus provide a robust tool for exploring biological function, the new transgenic phenotype must be replicated in several independently generated transgenic strains. PMID:21224729

  14. Identification of primary HIV-1C infection in Botswana.

    PubMed

    Novitsky, V; Woldegabriel, E; Wester, C; McDonald, E; Rossenkhan, R; Ketunuti, M; Makhema, J; Seage, G R; Essex, M

    2008-08-01

    Methods for identification of primary HIV infections seem increasingly important to understand pathogenesis, and to prevent transmission, which is particularly efficient during acute infection. Most current algorithms for HIV testing are based on detection of HIV antibodies and are unable to identify early infections before seroconversion. The efficiency of prospective cohorts, which is a standard approach for identifying primary HIV-1 infection, depends on a variety of epidemiological and cultural factors including HIV incidence and stigma and, not surprisingly, varies significantly in different geographical areas. We report a voluntary counseling and testing (VCT)-based approach to identifying primary HIV-1C infection that was developed as part of a primary HIV-1 subtype C infection study in Botswana. The referral strategy was based on: (1) collaboration with VCT centers at city clinics operated by the Ministry of Health; (2) partnering with the busiest non-government VCT center; (3) educating healthcare workers and the community about primary HIV infection; and (4) pairing with diverse VCT providers, including NGOs and private-sector organizations. Acute HIV-1 infections were defined by a negative HIV-1 serology combined with a positive HIV-1 RT-PCR test. Recent HIV-1 infections were identified by detuned EIA testing according to the classic STARTH algorithm. The VCT-based referral strategy resulted in the successful identification of 57 cases of acute and early HIV infection. A referral strategy of expanded VCT with viral RNA (Ribonucleic acid) testing to a national program in Botswana may be a promising approach for identification of primary HIV infections on a countrywide level. The program should offer VCT with viral RNA testing to the general public, facilitate proper counseling and risk reduction, and allow initiation of early HAART, and may reduce new viral transmissions. PMID:18608056

  15. Regulation of Human Cytosolic Sulfotransferases 1C2 and 1C3 by Nuclear Signaling Pathways in LS180 Colorectal Adenocarcinoma Cells

    PubMed Central

    Rondini, Elizabeth A.; Fang, Hailin; Runge-Morris, Melissa

    2014-01-01

    Cytosolic sulfotransferases (SULTs) catalyze the sulfate conjugation of a myriad of endogenous and xenobiotic substrates. Among the 13 human SULTs, little is known regarding regulation of the SULT1C subfamily. We evaluated the effects of a panel of transcription factor activators on levels of SULT1C mRNA (1C2 and 1C3) and protein (1C2) in LS180 colorectal adenocarcinoma cells. Treatment with 3-[3-[N-(2-chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy]phenylacetic acid hydrochloride [GW3965, liver X receptor (LXR) activator], 3-(2,6-dichlorophenyl)-4-(3′-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole [GW4064, farnesoid X receptor (FXR)], or rifampicin [pregnane X receptor (PXR)] moderately (≤2-fold) increased both SULT1C2 and SULT1C3 mRNA levels. 1α,25-Dihydroxyvitamin D3 [1,25(OH)2D3, vitamin D receptor (VDR)] selectively upregulated SULT1C2, whereas ciprofibrate [peroxisome proliferator-activated receptor α (PPARα)], rosiglitazone (PPARγ), and 2,3,7,8-tetrachlorodibenzo-p-dioxin [aryl hydrocarbon receptor (AhR)] selectively increased SULT1C3 mRNA levels. SULT1C2 protein content was strongly increased by 1,25(OH)2D3 treatment and moderately increased by GW3965, GW4064, and rifampicin. To evaluate SULT1C2 transcriptional regulation, treatment effects were determined on reporter activity from transfected constructs containing ∼10 kb of the SULT1C2 gene. Treatment with GW3965, GW4064, or 1,25(OH)2D3 increased reporter activity ∼2-, 5-, and 5.5-fold, respectively, from a construct containing mostly intron 1 of the SULT1C2 gene. Expression of AhR, LXRα, LXRβ, PPARα, PPARγ, PXR, and VDR was confirmed in LS180 cells using quantitative reverse-transcription polymerase chain reaction; however, FXR expression was negligible, suggesting that GW4064 increased SULT1C expression through an FXR-independent mechanism. Collectively, our findings are the first to characterize the regulation of human SULT1C2 and SULT1C3 expression by

  16. How subunit coupling produces the γ-subunit rotary motion in F1-ATPase

    PubMed Central

    Pu, Jingzhi; Karplus, Martin

    2008-01-01

    FoF1-ATP synthase manufactures the energy “currency,” ATP, of living cells. The soluble F1 portion, called F1-ATPase, can act as a rotary motor, with ATP binding, hydrolysis, and product release, inducing a torque on the γ-subunit. A coarse-grained plastic network model is used to show at a residue level of detail how the conformational changes of the catalytic β-subunits act on the γ-subunit through repulsive van der Waals interactions to generate a torque that drives unidirectional rotation, as observed experimentally. The simulations suggest that the calculated 85° substep rotation is driven primarily by ATP binding and that the subsequent 35° substep rotation is produced by product release from one β-subunit and a concomitant binding pocket expansion of another β-subunit. The results of the simulation agree with single-molecule experiments [see, for example, Adachi K, et al. (2007) Cell 130:309–321] and support a tri-site rotary mechanism for F1-ATPase under physiological condition. PMID:18216260

  17. Studies on chromatin. II. Isolation and characterization of chromatin subunits.

    PubMed Central

    Bakayev, V V; Melnickov, A A; Osicka, V D; Varshausky, A J

    1975-01-01

    Earlier findings /1-10/ bearing on a subunit organization of chromatin were confirmed and in some points detailed. Besides this, a large-scale isolation of chromatin subunits; their protein composition, electron microscopic appearance and CsCl banding pattern are described. Although the purified chromatin subunit contains all five histones, the relative content of histone H1 i in it is two times lower than that in the original chromatin. tit is shown that a mild digestion of chromatin with staphylococcal nuclease produced not only separate chromatin subunits and their "oligomers' but also deoxyribonucleoprotein particles which sediment more slowly than subunits. It appears that these particles and subunits are produced from different initial structures in the chromatin. Finally, a crystallization of the purified chromatin subunit as a cetyltrimethyl ammonium salt is described. Images PMID:1178523

  18. Glycogen synthase kinase-3-mediated phosphorylation of serine 73 targets sterol response element binding protein-1c (SREBP-1c) for proteasomal degradation

    PubMed Central

    Dong, Qingming; Giorgianni, Francesco; Beranova-Giorgianni, Sarka; Deng, Xiong; O'Meally, Robert N.; Bridges, Dave; Park, Edwards A.; Cole, Robert N.; Elam, Marshall B.; Raghow, Rajendra

    2015-01-01

    Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates genes involved in the de novo lipid synthesis and glycolysis pathways. The structure, turnover and transactivation potential of SREBP-1c are regulated by macronutrients and hormones via a cascade of signalling kinases. Using MS, we have identified serine 73 as a novel glycogen synthase kinase-3 (GSK-3) phosphorylation site in the rat SREBP-1c purified from McA-RH7777 hepatoma cells. Our site-specific mutagenesis strategy revealed that the turnover of SREBP-1c, containing wild type, phospho-null (serine to alanine) or phospho-mimetic (serine to aspartic acid) substitutions, was differentially regulated. We show that the S73D mutant of pSREBP-1c, that mimicked a state of constitutive phosphorylation, dissociated from the SREBP-1c–SCAP complex more readily and underwent GSK-3-dependent proteasomal degradation via SCFFbw7 ubiquitin ligase pathway. Pharmacologic inhibition of GSK-3 or knockdown of GSK-3 by siRNA prevented accelerated degradation of SREBP-1c. As demonstrated by MS, SREBP-1c was phosphorylated in vitro by GSK-3β at serine 73. Phosphorylation of serine 73 also occurs in the intact liver. We propose that GSK-3-mediated phosphorylation of serine 73 in the rat SREBP-1c and its concomitant destabilization represents a novel mechanism involved in the inhibition of de novo lipid synthesis in the liver. PMID:26589965

  19. MYOSIN PHOSPHATASE TARGETING SUBUNIT1 REGULATES MITOSIS BY ANTAGONIZING POLO-LIKE KINASE1

    PubMed Central

    Yamashiro, S.; Yamakita, Y.; Totsukawa, G.; Goto, H.; Kaibuchi, K.; Ito, M.; Hartshorne, D.; Matsumura, F.

    2009-01-01

    SUMMARY Myosin phosphatase targeting subunit1 (MYPT1) binds to the catalytic subunit of protein phosphatase1 (PP1C). This binding is believed to target PP1C to specific substrates including myosin II, thus controlling cellular contractility. Surprisingly, we found that during mitosis mammalian MYPT1 binds to polo-like kinase1 (PLK1). MYPT1 is phosphorylated during mitosis by proline-directed kinases including cdc2, which generates the binding motif for the polo box domain of PLK1. Depletion of PLK1 by small interfering RNAs is known to results in loss of γ-tubulin recruitment to the centrosomes, blocking centrosome maturation, leading to mitotic arrest. We found that co-depletion of MYPT1 and PLK1 reinstates γ-tubulin at the centrosomes, rescuing the mitotic arrest. MYPT1 depletion increases phosphorylation of PLK1 at its activating site (Thr210) in vivo, explaining, at least in part, the rescue phenotype by co-depletion. Taken together, our results identify a previously unrecognized role for MYPT1 in regulating mitosis by antagonizing PLK1. PMID:18477460

  20. Inherent conformational flexibility of F1-ATPase α-subunit.

    PubMed

    Hahn-Herrera, Otto; Salcedo, Guillermo; Barril, Xavier; García-Hernández, Enrique

    2016-09-01

    The core of F1-ATPase consists of three catalytic (β) and three noncatalytic (α) subunits, forming a hexameric ring in alternating positions. A wealth of experimental and theoretical data has provided a detailed picture of the complex role played by catalytic subunits. Although major conformational changes have only been seen in β-subunits, it is clear that α-subunits have to respond to these changes in order to be able to transmit information during the rotary mechanism. However, the conformational behavior of α-subunits has not been explored in detail. Here, we have combined unbiased molecular dynamics (MD) simulations and calorimetrically measured thermodynamic signatures to investigate the conformational flexibility of isolated α-subunits, as a step toward deepening our understanding of its function inside the α3β3 ring. The simulations indicate that the open-to-closed conformational transition of the α-subunit is essentially barrierless, which is ideal to accompany and transmit the movement of the catalytic subunits. Calorimetric measurements of the recombinant α-subunit from Geobacillus kaustophilus indicate that the isolated subunit undergoes no significant conformational changes upon nucleotide binding. Simulations confirm that the nucleotide-free and nucleotide-bound subunits show average conformations similar to that observed in the F1 crystal structure, but they reveal an increased conformational flexibility of the isolated α-subunit upon MgATP binding, which might explain the evolutionary conserved capacity of α-subunits to recognize nucleotides with considerable strength. Furthermore, we elucidate the different dependencies that α- and β-subunits show on Mg(II) for recognizing ATP. PMID:27137408

  1. Cytokine induced changes in proteasome subunit composition are concentration dependent.

    PubMed

    Stohwasser, R; Kloetzel, P M

    1996-09-01

    In eukaryotes, 20S proteasome subunit composition is controlled by the cytokine interferon-gamma (IFN-gamma). IFN-gamma induces the synthesis of the beta-subunits LMP2, LMP7 and MECL-1, which in consequence replace their constitutive subunit homologs delta, MB1 and MC14/Z in the 20S complex. By pulse labeling mouse RMA cells and immunoprecipitation of proteasome complexes with the antibody MP3, we have analysed the effect of different IFN-gamma concentrations on proteasomal subunit composition. Our experiments show that IFN-gamma concentrations as low as 5 U/ml induce subunit substitutions and that overall proteasomal subunit composition is dependent on the cytokine concentration used. An IFN-gamma concentration of 50 U/ml is sufficient for complete replacement of subunit delta by LMP2. In contrast, IFN-gamma treatment never induces a complete replacement of subunit MC14 by MECL-1. These subunits are present at an approximate 1:1 molar ratio, suggesting that both subunits coexist in the same 20S proteasome complex. Furthermore, different regulatory mechanisms have to be postulated for the synthesis and incorporation of the three IFN-gamma inducible proteasome subunits. Both IFN-gamma as well as IL-2 also seem to influence the modification state of the alpha subunit C8. Since the subunit composition is dependent on the cytokine concentration used and strongly influences the proteolytic properties of the 20S proteasome complex, our experiments represent a caveat for experiments in which IFN-gamma dependent proteasomal enzyme characteristics have been analysed without monitoring the subunit composition. PMID:9067255

  2. Cloning and characterization of GABAA α subunits and GABAB subunits in Xenopus laevis during development

    PubMed Central

    Kaeser, Gwendolyn E.; Rabe, Brian A.; Saha, Margaret S.

    2011-01-01

    Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult nervous system, acts via two classes of receptors, the ionotropic GABAA and metabotropic GABAB receptors. During the development of the nervous system GABA acts in a depolarizing, excitatory manner and plays an important role in various neural developmental processes including cell proliferation, migration, synapse formation and activity-dependent differentiation. Here we describe the spatial and temporal expression patterns of the GABAA and GABAB receptors during early development of Xenopus laevis. Using in situ hybridization and qRT-PCR, GABAA α2 was detected as a maternal mRNA. All other α-subunits were first detected by tailbud through hatching stages. Expression of the various subunits was seen in the brain, spinal cord, cranial ganglia, olfactory epithelium, pineal, and pituitary gland. Each receptor subunit showed a distinctive, unique expression pattern suggesting these receptors have specific functions and are regulated in a precise spatial and temporal manner. PMID:21384470

  3. PKA catalytic subunit mutations in adrenocortical Cushing's adenoma impair association with the regulatory subunit.

    PubMed

    Calebiro, Davide; Hannawacker, Annette; Lyga, Sandra; Bathon, Kerstin; Zabel, Ulrike; Ronchi, Cristina; Beuschlein, Felix; Reincke, Martin; Lorenz, Kristina; Allolio, Bruno; Kisker, Caroline; Fassnacht, Martin; Lohse, Martin J

    2014-01-01

    We recently identified a high prevalence of mutations affecting the catalytic (Cα) subunit of protein kinase A (PKA) in cortisol-secreting adrenocortical adenomas. The two identified mutations (Leu206Arg and Leu199_Cys200insTrp) are associated with increased PKA catalytic activity, but the underlying mechanisms are highly controversial. Here we utilize a combination of biochemical and optical assays, including fluorescence resonance energy transfer in living cells, to analyze the consequences of the two mutations with respect to the formation of the PKA holoenzyme and its regulation by cAMP. Our results indicate that neither mutant can form a stable PKA complex, due to the location of the mutations at the interface between the catalytic and the regulatory subunits. We conclude that the two mutations cause high basal catalytic activity and lack of regulation by cAMP through interference of complex formation between the regulatory and the catalytic subunits of PKA. PMID:25477193

  4. Changes in expression of splice cassettes of NMDA receptor GluN1 subunits within the frontal lobe and memory in mice during aging.

    PubMed

    Das, Siba R; Magnusson, Kathy R

    2011-09-12

    Age-related decline in memory has been associated with changes in mRNA and protein expression of different NMDA receptor subunits. The NMDA receptor GluN1 subunit appears to be necessary and sufficient for receptor function. There is evidence that the mRNA expressions of some splice forms of the subunit are influenced by aging and/or behavioral testing experience in old mice. The present study explored the relationships between behavioral testing experience and protein expression of different GluN1 subunit isoforms in the prefrontal/frontal cortex of the brain during aging. Aged C57BL/6 mice with behavioral testing experience showed declines in performance in both spatial working and reference memory tasks. Protein expression of GluN1 C-terminal cassettes C2 and C2', but not the C1 or N1 cassettes, was observed to decline with increasing age, regardless of experience. In middle-age animals, higher expressions of the GluN1 subunit and C2' cassette proteins were associated with good reference memory on initial search. Aged animals with a higher protein expression of GluN1 subunits containing C1 cassettes and the whole population of GluN1 subunits exhibited a closer proximity to the former platform location within the final phase of probe trials. However, the old mice with high expression of the C1 cassette did not show an accurate search during this phase. The old mice with lower expression of the C1 cassette protein more closely mimicked the performances of the young and middle-aged mice. These results indicate that there was heterogeneity in the effect of aging on the expression of the GluN1 subunits containing different splice cassettes. It also suggests that the GluN1 subunit might be most important for good reference memory during middle age, but this relationship may not be maintained into old age. PMID:21443909

  5. Docosahexaenoic acid inhibits proteolytic processing of sterol regulatory element-binding protein-1c (SREBP-1c) via activation of AMP-activated kinase.

    PubMed

    Deng, Xiong; Dong, Qingming; Bridges, Dave; Raghow, Rajendra; Park, Edwards A; Elam, Marshall B

    2015-12-01

    In hyperinsulinemic states including obesity and T2DM, overproduction of fatty acid and triglyceride contributes to steatosis of the liver, hyperlipidemia and hepatic insulin resistance. This effect is mediated in part by the transcriptional regulator sterol responsive element binding protein-1c (SREBP-1c), which stimulates the expression of genes involved in hepatic fatty acid and triglyceride synthesis. SREBP-1c is up regulated by insulin both via increased transcription of nascent full-length SREBP-1c and by enhanced proteolytic processing of the endoplasmic reticulum (ER)-bound precursor to yield the transcriptionally active n-terminal form, nSREBP-1c. Polyunsaturated fatty acids of marine origin (n-3 PUFA) prevent induction of SREBP-1c by insulin thereby reducing plasma and hepatic triglycerides. Despite widespread use of n-3 PUFA supplements to reduce triglycerides in clinical practice, the exact mechanisms underlying their hypotriglyceridemic effect remain elusive. Here we demonstrate that the n-3 PUFA docosahexaenoic acid (DHA; 22:5 n-3) reduces nSREBP-1c by inhibiting regulated intramembrane proteolysis (RIP) of the nascent SREBP-1c. We further show that this effect of DHA is mediated both via activation of AMP-activated protein kinase (AMPK) and by inhibition of mechanistic target of rapamycin complex 1 (mTORC1). The inhibitory effect of AMPK on SREBP-1c processing is linked to phosphorylation of serine 365 of SREBP-1c in the rat. We have defined a novel regulatory mechanism by which n-3 PUFA inhibit induction of SREBP-1c by insulin. These findings identify AMPK as an important negative regulator of hepatic lipid synthesis and as a potential therapeutic target for hyperlipidemia in obesity and T2DM. PMID:26327595

  6. [Nose surgical anatomy in six aesthetic subunits].

    PubMed

    Chaput, B; Lauwers, F; Lopez, R; Saboye, J; André, A; Grolleau, J-L; Chavoin, J-P

    2013-04-01

    The nose is a complex entity, combining aesthetic and functional roles. Descriptive anatomy is a fundamental science that it can be difficult to relate directly to our daily surgical activity. Reasoning in terms of aesthetic subunits to decide on his actions appeared to us so obvious. The aim of this paper is to resume the anatomical bases relevant to our daily practice in order to fully apprehend the restorative or cosmetic procedures. We discuss the limits of the systematization of these principles in nasal oncology. PMID:22699003

  7. MspA Nanopores from Subunit Dimers

    PubMed Central

    Pavlenok, Mikhail; Derrington, Ian M.; Gundlach, Jens H.; Niederweis, Michael

    2012-01-01

    Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA. PMID

  8. MspA nanopores from subunit dimers.

    PubMed

    Pavlenok, Mikhail; Derrington, Ian M; Gundlach, Jens H; Niederweis, Michael

    2012-01-01

    Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA. PMID

  9. Analysis of the phosphofructokinase subunits and isoenzymes in human tissues.

    PubMed Central

    Dunaway, G A; Kasten, T P; Sebo, T; Trapp, R

    1988-01-01

    The 6-phosphofructo-1-kinase (PFK) subunits and isoenzymes were studied in human muscle, heart, brain, liver, platelets, fibroblasts, erythrocytes, placenta and umbilical cord. In each tissue, the subunit types in the native isoenzymes were characterized by immunological titration with subunit-specific antibodies and by column chromatography on QAE (quaternary aminoethyl)-Sephadex. Further, the subunits of the partially purified native isoenzymes were resolved by SDS/polyacrylamide-gel electrophoresis, identified by immunoblotting, and quantified by scanning gel densitometry of silver-stained gels and immunoblots. Depending on the type of tissue, one to three subunits were detected. The Mr values of the L, M and C subunits regardless of tissue were 76,700 +/- 1400, 82,500 +/- 1640 and 86,500 +/- 1620. Of the tissues studied, only the muscle PFK isoenzymes exhibited one subunit, which was the M-type subunit. Of the other tissues studied, the PFK isoenzymes contained various amounts of all three subunits. Considering the properties of the native PFK isoenzymes, it is clear that, in human tissues, they are not simply various combinations of two or three homotetrameric isoenzymes, but complex mixtures of homotetramers and heterotetramers. The kinetic/regulatory properties of the various isoenzyme pools were found to be dependent on subunit composition. Images Fig. 1. PMID:2970843

  10. Aldo-keto Reductase 1C3 (AKR1C3) is overexpressed in skin squamous cell carcinoma (SCC) and affects SCC growth via prostaglandin metabolism

    PubMed Central

    Mantel, Alon; Carpenter-Mendini, Amanda; VanBuskirk, JoAnne; Pentland, Alice P.

    2014-01-01

    Aldo-keto reductase 1C3 (AKR1C3) is an enzyme involved in metabolizing prostaglandins (PGs) and sex hormones. It metabolizes PGD2 to 9α11β-PGF2, diverting the spontaneous conversion of PGD2 to the PPARγ agonist, 15-Deoxy-Delta-12,14-prostaglandin J2 (15d-PGJ2). AKR1C3 is overexpressed in various malignancies, suggesting a tumor promoting function. This work investigates AKR1C3 expression in human non-melanoma skin cancers, revealing overexpression in squamous cell carcinoma (SCC). Effects of AKR1C3 overexpression were then evaluated using 3 SCC cell lines. AKR1C3 was detected in all SCC cell lines and its expression was upregulated in response to its substrate, PGD2. Although attenuating AKR1C3 expression in SCC cells by siRNA did not affect growth, treatment with PGD2 and its dehydration metabolite, 15d-PGJ2, decreased SCC proliferation in a PPARγ-dependent manner. In addition, treatment with the PPARγ agonist pioglitazone profoundly inhibited SCC proliferation. Finally, we generated an SCC cell line that stably overexpressed AKR1C3 (SCC-AKR1C3). SCC-AKR1C3 metabolized PGD2 to 9α11β-PGF2 12 fold faster than the parent cell line and was protected from the anti-proliferative effect mediated by PGD2. This work suggests that PGD2 and its metabolite 15d-PGJ2 attenuate SCC proliferation in a PPARγ-dependent manner, therefore activation of PPARγ by agonists such as Pioglitazone may benefit those at high risk of SCC. PMID:24917395

  11. Knowledge of A1c Predicts Diabetes Self-Management and A1c Level among Chinese Patients with Type 2 Diabetes.

    PubMed

    Yang, Shengnan; Kong, Weimin; Hsue, Cunyi; Fish, Anne F; Chen, Yufeng; Guo, Xiaohui; Lou, Qingqing; Anderson, Robert

    2016-01-01

    This study was to identify current A1c understanding status among Chinese patients with type 2 diabetes, assess if knowledge of A1c affects their diabetes self-management and their glycemic control and recognize the factors influencing knowledge of A1c among patients with type 2 diabetes. A multi-center, cross-sectional survey was conducted between April and July 2010 in 50 medical centers in the Mainland China. Participants were recruited from inpatients and outpatients who were admitted to or visited those medical centers. The survey included core questions about their demographic characteristics, diabetes self-management behavior, and A1c knowledge. Overall, of 5957 patients, the percentage of patients with good understanding was 25.3%. In the multivariable logistic regression model, the variables related to the knowledge of A1c status are presented. We discovered that patients with longer diabetes duration (OR = 1.05; 95%CI = 1.04-1.06) and having received diabetes education (OR = 1.80; 95%CI = 1.49-2.17) were overrepresented in the good understanding of A1c group. In addition, compared to no education level, higher education level was statistically associated with good understanding of A1c (P<0.001). The percentage of patients with good understanding varied from region to region (P<0.001), with Eastern being highest (OR = 1.54; 95%CI = 1.32-1.80), followed by Central (OR = 1.25; 95%CI = 1.02-1.53), when referring to Western. Only a minority of patients with type 2 diabetes in China understood their A1c value. The patients who had a good understanding of their A1c demonstrated significantly better diabetes self-management behavior and had lower A1c levels than those who did not. PMID:26959422

  12. 7 CFR 1c.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... involvement of human subjects. 1c.118 Section 1c.118 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types of applications for grants, cooperative agreements, or contracts...

  13. 7 CFR 1c.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... involvement of human subjects. 1c.118 Section 1c.118 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types of applications for grants, cooperative agreements, or contracts...

  14. 7 CFR 1c.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... involvement of human subjects. 1c.118 Section 1c.118 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types of applications for grants, cooperative agreements, or contracts...

  15. Cholesteryl esters stabilize human CD1c conformations for recognition by self-reactive T cells

    PubMed Central

    Mansour, Salah; Tocheva, Anna S.; Cave-Ayland, Chris; Machelett, Moritz M.; Sander, Barbara; Lissin, Nikolai M.; Molloy, Peter E.; Baird, Mark S.; Stübs, Gunthard; Schröder, Nicolas W. J.; Schumann, Ralf R.; Rademann, Jörg; Postle, Anthony D.; Jakobsen, Bent K.; Marshall, Ben G.; Gosain, Rajendra; Elkington, Paul T.; Elliott, Tim; Skylaris, Chris-Kriton; Essex, Jonathan W.; Tews, Ivo; Gadola, Stephan D.

    2016-01-01

    Cluster of differentiation 1c (CD1c)-dependent self-reactive T cells are abundant in human blood, but self-antigens presented by CD1c to the T-cell receptors of these cells are poorly understood. Here we present a crystal structure of CD1c determined at 2.4 Å revealing an extended ligand binding potential of the antigen groove and a substantially different conformation compared with known CD1c structures. Computational simulations exploring different occupancy states of the groove reenacted these different CD1c conformations and suggested cholesteryl esters (CE) and acylated steryl glycosides (ASG) as new ligand classes for CD1c. Confirming this, we show that binding of CE and ASG to CD1c enables the binding of human CD1c self-reactive T-cell receptors. Hence, human CD1c adopts different conformations dependent on ligand occupancy of its groove, with CE and ASG stabilizing CD1c conformations that provide a footprint for binding of CD1c self-reactive T-cell receptors. PMID:26884207

  16. 7 CFR 1c.119 - Research undertaken without the intention of involving human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... human subjects. 1c.119 Section 1c.119 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.119 Research undertaken without the intention of involving human subjects. In the event research is undertaken without the intention of involving human subjects, but it is later...

  17. 7 CFR 1c.119 - Research undertaken without the intention of involving human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... to involve human subjects in the research, the research shall first be reviewed and approved by an... human subjects. 1c.119 Section 1c.119 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.119 Research undertaken without the intention of involving human subjects. In...

  18. 18 CFR 1c.2 - Prohibition of electric energy market manipulation.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... energy market manipulation. 1c.2 Section 1c.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES PROHIBITION OF ENERGY MARKET MANIPULATION § 1c.2 Prohibition of electric energy market manipulation. (a) It shall be unlawful for any entity, directly...

  19. 18 CFR 1c.2 - Prohibition of electric energy market manipulation.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... energy market manipulation. 1c.2 Section 1c.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES PROHIBITION OF ENERGY MARKET MANIPULATION § 1c.2 Prohibition of electric energy market manipulation. (a) It shall be unlawful for any entity, directly...

  20. 18 CFR 1c.2 - Prohibition of electric energy market manipulation.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... energy market manipulation. 1c.2 Section 1c.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES PROHIBITION OF ENERGY MARKET MANIPULATION § 1c.2 Prohibition of electric energy market manipulation. (a) It shall be unlawful for any entity, directly...

  1. 7 CFR 1c.119 - Research undertaken without the intention of involving human subjects.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... to involve human subjects in the research, the research shall first be reviewed and approved by an... human subjects. 1c.119 Section 1c.119 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.119 Research undertaken without the intention of involving human subjects. In...

  2. 7 CFR 1c.119 - Research undertaken without the intention of involving human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... to involve human subjects in the research, the research shall first be reviewed and approved by an... human subjects. 1c.119 Section 1c.119 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.119 Research undertaken without the intention of involving human subjects. In...

  3. 50 CFR Table 1c to Part 679 - Product Tyoe Codes

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 9 2010-10-01 2010-10-01 false Product Tyoe Codes 1c Table 1c to Part 679 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION... Table 1c to Part 679—Product Tyoe Codes Description Code Ancillary product.A product, such as...

  4. 7 CFR 1c.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... involvement of human subjects. 1c.118 Section 1c.118 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.118 Applications and proposals lacking definite plans for involvement of... projects in which human subjects' involvement will depend upon completion of instruments, prior...

  5. 7 CFR 1c.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... involvement of human subjects. 1c.118 Section 1c.118 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.118 Applications and proposals lacking definite plans for involvement of... projects in which human subjects' involvement will depend upon completion of instruments, prior...

  6. 7 CFR 1c.123 - Early termination of research support: Evaluation of applications and proposals.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 1 2012-01-01 2012-01-01 false Early termination of research support: Evaluation of applications and proposals. 1c.123 Section 1c.123 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.123 Early termination of research support: Evaluation of applications...

  7. 7 CFR 1c.119 - Research undertaken without the intention of involving human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... human subjects. 1c.119 Section 1c.119 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.119 Research undertaken without the intention of involving human subjects. In the event research is undertaken without the intention of involving human subjects, but it is later...

  8. Subunit Arrangement and Function in NMDA Receptors

    SciTech Connect

    Furukawa,H.; Singh, S.; Mancusso, R.; Gouaux, E.

    2005-01-01

    Excitatory neurotransmission mediated by NMDA (N-methyl-D-aspartate) receptors is fundamental to the physiology of the mammalian central nervous system. These receptors are heteromeric ion channels that for activation require binding of glycine and glutamate to the NR1 and NR2 subunits, respectively. NMDA receptor function is characterized by slow channel opening and deactivation, and the resulting influx of cations initiates signal transduction cascades that are crucial to higher functions including learning and memory. Here we report crystal structures of the ligand-binding core of NR2A with glutamate and that of the NR1-NR2A heterodimer with glutamate and glycine. The NR2A-glutamate complex defines the determinants of glutamate and NMDA recognition, and the NR1-NR2A heterodimer suggests a mechanism for ligand-induced ion channel opening. Analysis of the heterodimer interface, together with biochemical and electrophysiological experiments, confirms that the NR1-NR2A heterodimer is the functional unit in tetrameric NMDA receptors and that tyrosine 535 of NR1, located in the subunit interface, modulates the rate of ion channel deactivation.

  9. TSH Receptor Cleavage Into Subunits and Shedding of the A-Subunit; A Molecular and Clinical Perspective.

    PubMed

    Rapoport, Basil; McLachlan, Sandra M

    2016-04-01

    The TSH receptor (TSHR) on the surface of thyrocytes is unique among the glycoprotein hormone receptors in comprising two subunits: an extracellular A-subunit, and a largely transmembrane and cytosolic B-subunit. Unlike its ligand TSH, whose subunits are encoded by two genes, the TSHR is expressed as a single polypeptide that subsequently undergoes intramolecular cleavage into disulfide-linked subunits. Cleavage is associated with removal of a C-peptide region, a mechanism similar in some respects to insulin cleavage into disulfide linked A- and B-subunits with loss of a C-peptide region. The potential pathophysiological importance of TSHR cleavage into A- and B-subunits is that some A-subunits are shed from the cell surface. Considerable experimental evidence supports the concept that A-subunit shedding in genetically susceptible individuals is a factor contributing to the induction and/or affinity maturation of pathogenic thyroid-stimulating autoantibodies, the direct cause of Graves' disease. The noncleaving gonadotropin receptors are not associated with autoantibodies that induce a "Graves' disease of the gonads." We also review herein current information on the location of the cleavage sites, the enzyme(s) responsible for cleavage, the mechanism by which A-subunits are shed, and the effects of cleavage on receptor signaling. PMID:26799472

  10. Sodium channel β subunits: emerging targets in channelopathies

    PubMed Central

    O’Malley, Heather A.; Isom, Lori L.

    2016-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Originally called “auxiliary,” we now know that β subunit proteins are multifunctional signaling molecules that play roles in both excitable and non-excitable cell types, and with or without the pore-forming α subunit present. β subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding β subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. While VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target. PMID:25668026

  11. Sodium channel β subunits: emerging targets in channelopathies.

    PubMed

    O'Malley, Heather A; Isom, Lori L

    2015-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Although β subunits were originally termed auxiliary, we now know that they are multifunctional signaling molecules that play roles in both excitable and nonexcitable cell types and with or without the pore-forming α subunit present. β subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding β subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. Although VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target. PMID:25668026

  12. Haemoglobin J-Baltimore can be detected by HbA1c electropherogram but with underestimated HbA1c value

    PubMed Central

    Brunel, Valéry; Lahary, Agnčs; Chagraoui, Abdeslam; Thuillez, Christian

    2016-01-01

    Glycated haemoglobin (HbA1c) is considered the gold standard for assessing diabetes compensation and treatment. In addition, fortuitous detection of haemoglobin variants during HbA1c measurement is not rare. Recently, two publications reported different conclusions on accuracy of HbA1c value using capillary electrophoresis method in presence of haemoglobin J-Baltimore (HbJ).
Here we describe the fortuitous detection of unknown HbJ using capillary electrophoresis for measurement of HbA1c. A patient followed for gestational diabetes in our laboratory presented unknown haemoglobin on Capillarys 2 Flex Piercing analyser which was identified as HbJ. HbJ is not associated with haematological abnormalities. High Performance Liquid Chromatography methods are known to possibly underestimate HbA1c value in the presence of this variant. This variant and its glycated form are clearly distinguished on electropherogram but HbJ was responsible for underestimating the true area of HbA1c.
Capillary electrophoresis is a good method for detecting HbJ but does not seem suitable for evaluation of HbA1C value in patients in presence of HbJ variant. PMID:27346969

  13. Quantifying the cooperative subunit action in a multimeric membrane receptor

    PubMed Central

    Wongsamitkul, Nisa; Nache, Vasilica; Eick, Thomas; Hummert, Sabine; Schulz, Eckhard; Schmauder, Ralf; Schirmeyer, Jana; Zimmer, Thomas; Benndorf, Klaus

    2016-01-01

    In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels. PMID:26858151

  14. Phenotypic variation of erythrocyte linker histone H1.c in a pheasant (Phasianus colchicus L.) population.

    PubMed

    Kowalski, Andrzej; Pa Yga, Jan; Górnicka-Michalska, Ewa; Bernacki, Zenon; Adamski, Marek

    2010-07-01

    Our goal was to characterize a phenotypic variation of the pheasant erythrocyte linker histone subtype H1.c. By using two-dimensional polyacrylamide gel electrophoresis three histone H1.c phenotypes were identified. The differently migrating allelic variants H1.c1 and H1.c2 formed either two homozygous phenotypes, c1 and c2, or a single heterozygous phenotype, c1c2. In the pheasant population screened, birds with phenotype c2 were the most common (frequency 0.761) while individuals with phenotype c1 were rare (frequency 0.043). PMID:21637419

  15. Inter- and Intra-Subunit Butanol/Isoflurane Sites of Action in the Human Glycine Receptor

    PubMed Central

    McCracken, Mandy L.; Gorini, Giorgio; McCracken, Lindsay M.; Mayfield, R. Dayne; Harris, R. Adron; Trudell, James R.

    2016-01-01

    Glycine receptors (GlyRs) mediate inhibitory neurotransmission and are targets for alcohols and anesthetics in brain. GlyR transmembrane (TM) domains contain critical residues for alcohol/anesthetic action: amino acid A288 in TM3 forms crosslinks with TM1 (I229) in the adjacent subunit as well as TM2 (S267) and TM4 (Y406, W407, I409, Y410) in the same subunit. We hypothesized that these residues may participate in intra-subunit and inter-subunit sites of alcohol/anesthetic action. The following double and triple mutants of GLRA1 cDNA (encoding human glycine receptor alpha 1 subunit) were injected into Xenopus laevis oocytes: I229C/A288C, I229C/A288C/C290S, A288C/Y406C, A288C/W407C, A288C/I409C, and A288C/Y410C along with the corresponding single mutants and wild-type GLRA1. Butanol (22 mM) or isoflurane (0.6 mM) potentiation of GlyR-mediated currents before and after application of the cysteine crosslinking agent HgCl2 (10 μM) was measured using two-electrode voltage clamp electrophysiology. Crosslinking nearly abolished butanol and isoflurane potentiation in the I229C/A288C and I229C/A288C/C290S mutants but had no effect in single mutants or wild-type. Crosslinking also inhibited butanol and isoflurane potentiation in the TM3-4 mutants (A288C/Y406C, A288C/W407C, A288C/I409C, A288C/Y410C) with no effect in single mutants or wild-type. We extracted proteins from oocytes expressing I229C/288C, A288C/Y410C, or wild-type GlyRs, used mass spectrometry to verify their expression and possible inter-subunit dimerization, plus immunoblotting to investigate the biochemical features of proposed crosslinks. Wild-type GlyR subunits measured about 50 kDa; after crosslinking, the dimeric/monomeric 100:50 kDa band ratio was significantly increased in I229C/288C but not A288C/Y410C mutants or wild-type, providing support for TM1-3 inter-subunit and TM3-4 intra-subunit crosslinking. A GlyR homology model based on the GluCl template provides further evidence for a multi-site model

  16. Inter- and Intra-Subunit Butanol/Isoflurane Sites of Action in the Human Glycine Receptor.

    PubMed

    McCracken, Mandy L; Gorini, Giorgio; McCracken, Lindsay M; Mayfield, R Dayne; Harris, R Adron; Trudell, James R

    2016-01-01

    Glycine receptors (GlyRs) mediate inhibitory neurotransmission and are targets for alcohols and anesthetics in brain. GlyR transmembrane (TM) domains contain critical residues for alcohol/anesthetic action: amino acid A288 in TM3 forms crosslinks with TM1 (I229) in the adjacent subunit as well as TM2 (S267) and TM4 (Y406, W407, I409, Y410) in the same subunit. We hypothesized that these residues may participate in intra-subunit and inter-subunit sites of alcohol/anesthetic action. The following double and triple mutants of GLRA1 cDNA (encoding human glycine receptor alpha 1 subunit) were injected into Xenopus laevis oocytes: I229C/A288C, I229C/A288C/C290S, A288C/Y406C, A288C/W407C, A288C/I409C, and A288C/Y410C along with the corresponding single mutants and wild-type GLRA1. Butanol (22 mM) or isoflurane (0.6 mM) potentiation of GlyR-mediated currents before and after application of the cysteine crosslinking agent HgCl2 (10 μM) was measured using two-electrode voltage clamp electrophysiology. Crosslinking nearly abolished butanol and isoflurane potentiation in the I229C/A288C and I229C/A288C/C290S mutants but had no effect in single mutants or wild-type. Crosslinking also inhibited butanol and isoflurane potentiation in the TM3-4 mutants (A288C/Y406C, A288C/W407C, A288C/I409C, A288C/Y410C) with no effect in single mutants or wild-type. We extracted proteins from oocytes expressing I229C/288C, A288C/Y410C, or wild-type GlyRs, used mass spectrometry to verify their expression and possible inter-subunit dimerization, plus immunoblotting to investigate the biochemical features of proposed crosslinks. Wild-type GlyR subunits measured about 50 kDa; after crosslinking, the dimeric/monomeric 100:50 kDa band ratio was significantly increased in I229C/288C but not A288C/Y410C mutants or wild-type, providing support for TM1-3 inter-subunit and TM3-4 intra-subunit crosslinking. A GlyR homology model based on the GluCl template provides further evidence for a multi-site model

  17. Effects of benzyl isothiocyanate and its N-acetylcysteine conjugate on induction of detoxification enzymes in hepa1c1c7 mouse hepatoma cells.

    PubMed

    Hwang, Eun-Sun

    2014-12-01

    The induction of detoxification enzymes by benzyl isothiocyanate (BITC) and its synthetic N-acetyl-L-cysteine (NAC) conjugate (NAC-BITC) was examined in Hepa1c1c7 murine hepatoma cells. BITC and NAC-BITC inhibited Hepa1c1c7 cell growth in a dose-dependent manner. Cell growth was 4.5~57.2% lower in Hepa1c1c7 cells treated with 0.1~10 μM BITC than in control-treated Hepa1c1c7 cells. The NAC-BITC treatment had a similar inhibitory pattern on Hepa1c1c7 cell growth; 0.5 μM and 10 μM NAC-BITC decreased cell growth by 13.6% and 47.4%, respectively. Treatment of Hepa1c1c7 cells with 0.1~2.0 μM BITC also elicited a dose-response effect on the induction of quinone reductase quinone reductase (QR) activity and QR mRNA expression. Treatment with 1 μM and 2 μM BITC caused 1.8- and 2.8-fold inductions of QR mRNA, respectively. By comparison, treatment with 1 μM and 2 μM NAC-BITC caused 1.6- and 1.9-fold inductions of QR mRNA, respectively. Cytochrome P450 (CYP) 1A1 and CYP2E1 induction were lower in 0.1~2 μM BITC-treated cells than in control-treated cells. CYP2E1 activity was 1.2-fold greater in 0.1 μM NAC-BITC-treated cells than in control-treated cells. However, the CYP2E1 activity of cells treated with higher concentrations (i.e., 1~2 μM) of NAC-BITC was similar to the activity of control-treated cells. Considering the potential of isothiocyanatesto prevent cancer, these results provide support for the use of BITC and NAC-BITC conjugates as chemopreventive agents. PMID:25580390

  18. Effects of Benzyl Isothiocyanate and Its N-Acetylcysteine Conjugate on Induction of Detoxification Enzymes in Hepa1c1c7 Mouse Hepatoma Cells

    PubMed Central

    Hwang, Eun-Sun

    2014-01-01

    The induction of detoxification enzymes by benzyl isothiocyanate (BITC) and its synthetic N-acetyl-L-cysteine (NAC) conjugate (NAC-BITC) was examined in Hepa1c1c7 murine hepatoma cells. BITC and NAC-BITC inhibited Hepa1c1c7 cell growth in a dose-dependent manner. Cell growth was 4.5~57.2% lower in Hepa1c1c7 cells treated with 0.1~10 μM BITC than in control-treated Hepa1c1c7 cells. The NAC-BITC treatment had a similar inhibitory pattern on Hepa1c1c7 cell growth; 0.5 μM and 10 μM NAC-BITC decreased cell growth by 13.6% and 47.4%, respectively. Treatment of Hepa1c1c7 cells with 0.1~2.0 μM BITC also elicited a dose-response effect on the induction of quinone reductase quinone reductase (QR) activity and QR mRNA expression. Treatment with 1 μM and 2 μM BITC caused 1.8- and 2.8-fold inductions of QR mRNA, respectively. By comparison, treatment with 1 μM and 2 μM NAC-BITC caused 1.6- and 1.9-fold inductions of QR mRNA, respectively. Cytochrome P450 (CYP) 1A1 and CYP2E1 induction were lower in 0.1~2 μM BITC-treated cells than in control-treated cells. CYP2E1 activity was 1.2-fold greater in 0.1 μM NAC-BITC-treated cells than in control-treated cells. However, the CYP2E1 activity of cells treated with higher concentrations (i.e., 1~2 μM) of NAC-BITC was similar to the activity of control-treated cells. Considering the potential of isothiocyanatesto prevent cancer, these results provide support for the use of BITC and NAC-BITC conjugates as chemopreventive agents. PMID:25580390

  19. Reovirus polypeptide sigma 3 and N-terminal myristoylation of polypeptide mu 1 are required for site-specific cleavage to mu 1C in transfected cells.

    PubMed Central

    Tillotson, L; Shatkin, A J

    1992-01-01

    N-myristoylated viral polypeptide mu 1 was produced in COS cells transfected with a transient expression vector containing a DNA copy of the reovirus M2 gene. The mu 1 product was specifically cleaved to polypeptide mu 1C in cells that were cotransfected with the reovirus S4 gene and that expressed polypeptide sigma 3. Studies with site-specific mutants of the M2 gene demonstrated that conversion of mu 1 to mu 1C was dependent on myristoylation and the presence of the proteolytic cleavage sequence asparagine 42-proline 43 in mu 1, as well as on the presence of polypeptide sigma 3. The mu 1C product and polypeptide sigma 3 formed complexes that were immunoprecipitated by sigma 3-directed antibody, and a myristoylation-negative M2 double mutant, G2A-N42T, yielded mu 1 that did not undergo cleavage to mu 1C or bind sigma 3. However, the N42T single mutant did form immunoprecipitable complexes with sigma 3, indicating that binding can occur in the absence of cleavage. Polypeptide sigma 3 alternatively can bind double-stranded RNA and in COS cells stimulates translation of reporter chloramphenicol acetyltransferase mRNA translation, presumably by blocking double-stranded RNA-mediated activation of the eukaryotic initiation factor 2 alpha subunit kinase which inhibits the initiation of protein synthesis. Consistent with these observations and with the formation of mu 1C-sigma 3 complexes, coexpression of M2 with S4 DNA prevented the translational stimulatory effect of polypeptide sigma 3. Images PMID:1548757

  20. Carnitine palmitoyltransferase-1c gain-of-function in the brain results in postnatal microencephaly.

    PubMed

    Reamy, Amanda A; Wolfgang, Michael J

    2011-08-01

    Carnitine palmitoyltransferase-1c (CPT1c) is a newly identified and poorly understood brain-specific CPT1 homologue. Here, we have generated a new animal model that allows the conditional expression of CPT1c in a tissue specific and/or temporal manner via Cre-lox mediated recombination. Brain-specific, exogenous expression of CPT1c was achieved by crossing transgenic CPT1c mice to Nestin-Cre mice. The resulting double transgenic mice (CPT1c-TgN) displayed severe growth retardation in the postnatal period with a stunted development at 2 weeks of age. CPT1c-TgN mice had a greater than 2.3-fold reduction in brain weight. Even with this degree of microencephaly, CPT1c-TgN mice were viable and fertile and exhibited normal post-weaning growth. When fed a high fat diet CPT1c-TgN mice were protected from weight gain and the difference in body weight between CPT1c-TgN and control mice was further exaggerated. Conversely, low fat, high carbohydrate feeding partially reversed the body weight defects in CPT1c-TgN mice. Analysis of total brain lipids of low fat fed mice revealed a depletion of total very long chain fatty acids in adult CPT1c-TgN mice which was not evident in high fat fed CPT1c-TgN mice. These data show that CPT1c can elicit profound effects on brain physiology and total fatty acid profiles, which can be modulated by the nutritional composition of the diet. PMID:21592121

  1. Whole-genome Association Study of Bipolar Disorder

    PubMed Central

    Sklar, P; Smoller, JW; Fan, J; Ferreira, MAR; Perlis, RH; Chambert, K; Nimgaonkar, VL; McQueen, MB; Faraone, SV; Kirby, A; de Bakker, PIW; Ogdie, MN; Thase, ME; Sachs, GS; Todd-Brown, K; Gabriel, SB; Sougnez, C; Gates, C; Blumenstiel, B; Defelice, M; Ardlie, KG; Franklin, J; Muir, WJ; McGhee, KA; MacIntyre, DM; McLean, A; VanBeck, M; McQuillin, A; Bass, NJ; Robinson, M; Lawrence, J; Anjorin, A; Curtis, D; Scolnick, EM; Daly, MJ; Blackwood, DH; Gurling, HM; Purcell, SM

    2013-01-01

    We performed a genome wide association scan in 1,461 patients with bipolar 1 disorder and 2,008 controls drawn from the Systematic Treatment Enhancement Program for Bipolar Disorder (STEP-BD) and University College London sample collections with successful genotyping for 372,193 SNPs. Our strongest single SNP results are found in myosin5B (MYO5B; p=1.66 × 10−7) and tetraspanin-8 (TSPAN8; p=6.11 × 10−7). Haplotype analysis further supported single SNP results highlighting MYO5B, TSPAN8 and the epidermal growth factor receptor (MYO5B; p=2.04 × 10−8, TSPAN8; p=7.57 × 10−7 and EGFR; p=8.36 × 10−8). For replication, we genotyped 304 SNPs in a family-based NIMH sample (n=409 trios) and a University of Edinburgh case-control sample (n=365 cases, 351 controls) which do not provide independent replication after correction for multiple testing. A comparison of our strongest associations with the genome-wide scan of 1,868 patients with bipolar disorder and 2,938 controls completed as part of the Wellcome Trust Case-Control Consortium (1) indicates concordant signals for SNPs within the voltage-dependent calcium channel, L-type, alpha 1C subunit (CACNA1C) gene, but no other single SNP associations are highly significant in both studies. Given the heritability of bipolar disorder, the lack of agreement between studies emphasizes that susceptibility alleles are likely to be modest in effect size and require even larger samples for detection. PMID:18317468

  2. Compilation of small ribosomal subunit RNA structures.

    PubMed Central

    Neefs, J M; Van de Peer, Y; De Rijk, P; Chapelle, S; De Wachter, R

    1993-01-01

    The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk. PMID:8332525

  3. The synthetic retinoid AGN 193109 but not retinoic acid elevates CYP1A1 levels in mouse embryos and Hepa-1c1c7 cells.

    PubMed

    Soprano, D R; Gambone, C J; Sheikh, S N; Gabriel, J L; Chandraratna, R A; Soprano, K J; Kochhar, D M

    2001-07-15

    The synthetic retinoid AGN 193109 is a potent pan retinoic acid receptor (RAR) antagonist. Treatment of pregnant mice with a single oral 1 mg/kg dose of this antagonist on day 8 postcoitum results in severe craniofacial (median cleft face or frontonasal deficiency) and eye malformations in virtually all exposed fetuses. Using differential display analysis, we have determined that CYP1A1 mRNA levels are elevated in mouse embryos 6 h following treatment with AGN 193109. Similarly, an elevation in CYP1A1 mRNA levels, protein levels, and aryl hydrocarbon hydoxylase activity occurs in Hepa-1c1c7 cells, with the maximal elevation observed when the cells were treated with 10(-5) M AGN 193109 for 4 to 8 h. Elevation in CYP1A1 mRNA levels in mouse embryos and Hepa-1c1c7 cells does not occur upon treatment with the natural retinoid, all-trans-retinoic acid. Finally, elevation in CYP1A1 mRNA levels was not observed when mutant Hepa-1c1c7 cells, which are defective in either the aryl hydrocarbon receptor (AhR) or aryl hydrocarbon receptor nuclear translocator (ARNT), were treated with AGN 193109. This suggests that the AhR/ARNT pathway and not the RAR/RXR pathway is mediating the elevation of CYP1A1 mRNA levels by AGN 193109, at least in the Hepa-1c1c7 cells. This is the first example of a retinoid that displays the abililty to regulate both the RAR/RXR and AhR/ARNT transcriptional regulatory pathways. PMID:11446831

  4. Dynamic regulation of β1 subunit trafficking controls vascular contractility

    PubMed Central

    Leo, M. Dennis; Bannister, John P.; Narayanan, Damodaran; Nair, Anitha; Grubbs, Jordan E.; Gabrick, Kyle S.; Boop, Frederick A.; Jaggar, Jonathan H.

    2014-01-01

    Ion channels composed of pore-forming and auxiliary subunits control physiological functions in virtually all cell types. A conventional view is that channels assemble with their auxiliary subunits before anterograde plasma membrane trafficking of the protein complex. Whether the multisubunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and, potentially, rapid modulation to control activity and cellular excitability is unclear. Arterial smooth muscle cells (myocytes) express large-conductance Ca2+-activated potassium (BK) channel α and auxiliary β1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BKα subunits are primarily (∼95%) plasma membrane-localized in human and rat arterial myocytes. In contrast, only a small fraction (∼10%) of total β1 subunits are located at the cell surface. Immunofluorescence resonance energy transfer microscopy demonstrated that intracellular β1 subunits are stored within Rab11A-postive recycling endosomes. Nitric oxide (NO), acting via cGMP-dependent protein kinase, and cAMP-dependent pathways stimulated rapid (≤1 min) anterograde trafficking of β1 subunit-containing recycling endosomes, which increased surface β1 almost threefold. These β1 subunits associated with surface-resident BKα proteins, elevating channel Ca2+ sensitivity and activity. Our data also show that rapid β1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid β1 subunit surface trafficking controls functional BK channel activity in arterial myocytes and vascular contractility. Conceivably, regulated auxiliary subunit trafficking may control ion channel activity in a wide variety of cell types. PMID:24464482

  5. Carnitine palmitoyltransferase 1C promotes cell survival and tumor growth under conditions of metabolic stress.

    PubMed

    Zaugg, Kathrin; Yao, Yi; Reilly, Patrick T; Kannan, Karuppiah; Kiarash, Reza; Mason, Jacqueline; Huang, Ping; Sawyer, Suzanne K; Fuerth, Benjamin; Faubert, Brandon; Kalliomäki, Tuula; Elia, Andrew; Luo, Xunyi; Nadeem, Vincent; Bungard, David; Yalavarthi, Sireesha; Growney, Joseph D; Wakeham, Andrew; Moolani, Yasmin; Silvester, Jennifer; Ten, Annick You; Bakker, Walbert; Tsuchihara, Katsuya; Berger, Shelley L; Hill, Richard P; Jones, Russell G; Tsao, Ming; Robinson, Murray O; Thompson, Craig B; Pan, Guohua; Mak, Tak W

    2011-05-15

    Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors. PMID:21576264

  6. Carnitine palmitoyltransferase 1C promotes cell survival and tumor growth under conditions of metabolic stress

    PubMed Central

    Zaugg, Kathrin; Yao, Yi; Reilly, Patrick T.; Kannan, Karuppiah; Kiarash, Reza; Mason, Jacqueline; Huang, Ping; Sawyer, Suzanne K.; Fuerth, Benjamin; Faubert, Brandon; Kalliomäki, Tuula; Elia, Andrew; Luo, Xunyi; Nadeem, Vincent; Bungard, David; Yalavarthi, Sireesha; Growney, Joseph D.; Wakeham, Andrew; Moolani, Yasmin; Silvester, Jennifer; Ten, Annick You; Bakker, Walbert; Tsuchihara, Katsuya; Berger, Shelley L.; Hill, Richard P.; Jones, Russell G.; Tsao, Ming; Robinson, Murray O.; Thompson, Craig B.; Pan, Guohua; Mak, Tak W.

    2011-01-01

    Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors. PMID:21576264

  7. E4BP4 is an insulin-induced stabilizer of nuclear SREBP-1c and promotes SREBP-1c-mediated lipogenesis.

    PubMed

    Tong, Xin; Li, Pei; Zhang, Deqiang; VanDommelen, Kyle; Gupta, Neil; Rui, Liangyou; Omary, M Bishr; Yin, Lei

    2016-07-01

    Upon food intake, insulin stimulates de novo lipogenesis (DNL) in hepatocytes via the AKT-mTORC1-sterol regulatory element-binding protein (SREBP)-1c pathway. How insulin maintains the maximal SREBP-1c activities during the entire feeding state remains elusive. We previously reported that insulin induced b-ZIP transcription factor, E4-binding protein 4 (E4BP4), in hepatocytes. In the current study, we show that insulin injection increases hepatic E4bp4 expression by activating the AKT-mTORC1-SREBP-1c pathway in hepatocytes. E4bp4-deficient hepatocytes not only fail to maintain robust DNL but also become resistant to SREBP-1c-induced lipogenesis. In vivo, acute depletion of E4bp4 in the liver by adenoviral shRNA reduces the expression of lipogenic enzymes and results in reduced levels of serum triglycerides and cholesterol during the postprandial phase. In hepatocytes, E4BP4 interacts with nuclear SREBP-1c to preserve its acetylation, and subsequently protects it from ubiquitination-dependent degradation. In conclusion, the current studies uncover a novel positive feedback pathway mediated by E4BP4 to augment SREBP-1c-mediated DNL in the liver during the fed state. PMID:27252523

  8. Comparative microarray analysis of basal gene expression in mouse Hepa-1c1c7 wild-type and mutant cell lines.

    PubMed

    Fong, C J; Burgoon, L D; Zacharewski, T R

    2005-08-01

    Hepa-1c1c7 wild-type and benzo[a]pyrene-resistant derived mutant cell lines have been used to elucidate pathways and mechanisms involving the aryl hydrocarbon receptor (AhR). However, there has been little focus on other biological processes which may differ between the isolated lines. In this study, mouse cDNA microarrays representing 4858 genes were used to examine differences in basal gene expression between mouse Hepa-1c1c7 wild-type and c1 (truncated Cyp1a1 protein), c4 (AhR nuclear translocator, ARNT, deficient), and c12 (low AhR levels) mutant cell lines. Surprisingly, c1 mutants exhibited the greatest number of gene expression changes compared to wild-type cells, followed by c4 and c12 lines, respectively. Differences in basal gene expression were consistent with cell line specific variations in morphology, mitochondrial activity, and proliferation rate. MTT and direct cell count assays indicate both c4 and c12 mutants exhibit increased proliferative activity when compared to wild-type cells, while the c1 mutants exhibited decreased activity. This study further characterizes Hepa-1c1c7 wild-type and mutant cells and identifies significant differences in biological processes that should be considered when conducting comparative mechanistic studies with these lines. PMID:15888666

  9. Cytokine-mediated suppression of cytochrome P450 1A1 in Hepa-1c1c7 cells by pokeweed mitogen.

    PubMed

    Jeong, H G

    2001-02-28

    This study investigated the effects of pokeweed mitogen (PWM) on the regulation of cytochrome P450 (P450) 1A1 expression in an in vitro model, using murine hepatoma cell line Hepa-1c1c7 and murine macrophage cell line RAW 264.7 cell cultures. PWM added directly to Hepa-1c1c7 cells had no effect on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced P450 1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity. However, TCDD-induced EROD activity and P450 1A1 mRNA levels were markedly suppressed when Hepa-1c1c7 cells were cultured with PWM-treated conditioned media from RAW 264.7 in a dose-dependent manner. Concomitant treatment with PWM and pentoxifylline, a TNFalpha synthesis inhibitor, to RAW 264.7 cells decreased the suppressive effects of PWM on TCDD-induced EROD activity. In PWM-exposed RAW 264.7 cell cultures, TNFalpha and IL-6 levels increased in a dose-dependent fashion. When antibodies to TNFalpha or/and IL-6 were added to PWM-treated conditioned media from RAW 264.7, the suppression of EROD activity was inhibited. These results suggested the suppression of P450 1A1 by PWM was mediated exclusively by TNFalpha and IL-6, released from macrophages. PMID:11311574

  10. Ceramide levels regulated by carnitine palmitoyltransferase 1C control dendritic spine maturation and cognition.

    PubMed

    Carrasco, Patricia; Sahún, Ignasi; McDonald, Jerome; Ramírez, Sara; Jacas, Jordi; Gratacós, Esther; Sierra, Adriana Y; Serra, Dolors; Herrero, Laura; Acker-Palmer, Amparo; Hegardt, Fausto G; Dierssen, Mara; Casals, Núria

    2012-06-15

    The brain-specific isoform carnitine palmitoyltransferase 1C (CPT1C) has been implicated in the hypothalamic regulation of food intake and energy homeostasis. Nevertheless, its molecular function is not completely understood, and its role in other brain areas is unknown. We demonstrate that CPT1C is expressed in pyramidal neurons of the hippocampus and is located in the endoplasmic reticulum throughout the neuron, even inside dendritic spines. We used molecular, cellular, and behavioral approaches to determine CPT1C function. First, we analyzed the implication of CPT1C in ceramide metabolism. CPT1C overexpression in primary hippocampal cultured neurons increased ceramide levels, whereas in CPT1C-deficient neurons, ceramide levels were diminished. Correspondingly, CPT1C knock-out (KO) mice showed reduced ceramide levels in the hippocampus. At the cellular level, CPT1C deficiency altered dendritic spine morphology by increasing immature filopodia and reducing mature mushroom and stubby spines. Total protrusion density and spine head area in mature spines were unaffected. Treatment of cultured neurons with exogenous ceramide reverted the KO phenotype, as did ectopic overexpression of CPT1C, indicating that CPT1C regulation of spine maturation is mediated by ceramide. To study the repercussions of the KO phenotype on cognition, we performed the hippocampus-dependent Morris water maze test on mice. Results show that CPT1C deficiency strongly impairs spatial learning. All of these results demonstrate that CPT1C regulates the levels of ceramide in the endoplasmic reticulum of hippocampal neurons, and this is a relevant mechanism for the correct maturation of dendritic spines and for proper spatial learning. PMID:22539351

  11. Ceramide Levels Regulated by Carnitine Palmitoyltransferase 1C Control Dendritic Spine Maturation and Cognition*

    PubMed Central

    Carrasco, Patricia; Sahún, Ignasi; McDonald, Jerome; Ramírez, Sara; Jacas, Jordi; Gratacós, Esther; Sierra, Adriana Y.; Serra, Dolors; Herrero, Laura; Acker-Palmer, Amparo; Hegardt, Fausto G.; Dierssen, Mara; Casals, Núria

    2012-01-01

    The brain-specific isoform carnitine palmitoyltransferase 1C (CPT1C) has been implicated in the hypothalamic regulation of food intake and energy homeostasis. Nevertheless, its molecular function is not completely understood, and its role in other brain areas is unknown. We demonstrate that CPT1C is expressed in pyramidal neurons of the hippocampus and is located in the endoplasmic reticulum throughout the neuron, even inside dendritic spines. We used molecular, cellular, and behavioral approaches to determine CPT1C function. First, we analyzed the implication of CPT1C in ceramide metabolism. CPT1C overexpression in primary hippocampal cultured neurons increased ceramide levels, whereas in CPT1C-deficient neurons, ceramide levels were diminished. Correspondingly, CPT1C knock-out (KO) mice showed reduced ceramide levels in the hippocampus. At the cellular level, CPT1C deficiency altered dendritic spine morphology by increasing immature filopodia and reducing mature mushroom and stubby spines. Total protrusion density and spine head area in mature spines were unaffected. Treatment of cultured neurons with exogenous ceramide reverted the KO phenotype, as did ectopic overexpression of CPT1C, indicating that CPT1C regulation of spine maturation is mediated by ceramide. To study the repercussions of the KO phenotype on cognition, we performed the hippocampus-dependent Morris water maze test on mice. Results show that CPT1C deficiency strongly impairs spatial learning. All of these results demonstrate that CPT1C regulates the levels of ceramide in the endoplasmic reticulum of hippocampal neurons, and this is a relevant mechanism for the correct maturation of dendritic spines and for proper spatial learning. PMID:22539351

  12. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    PubMed Central

    Tandrup Schmidt, Signe; Foged, Camilla; Smith Korsholm, Karen; Rades, Thomas; Christensen, Dennis

    2016-01-01

    The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs) concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI). Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs), which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR

  13. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators.

    PubMed

    Tandrup Schmidt, Signe; Foged, Camilla; Korsholm, Karen Smith; Rades, Thomas; Christensen, Dennis

    2016-01-01

    The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs) concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI). Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs), which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR

  14. The light subunit of system bo,+ is fully functional in the absence of the heavy subunit

    PubMed Central

    Reig, Núria; Chillarón, Josep; Bartoccioni, Paola; Fernández, Esperanza; Bendahan, Annie; Zorzano, Antonio; Kanner, Baruch; Palacín, Manuel; Bertran, Joan

    2002-01-01

    The heteromeric amino acid transporters are composed of a type II glycoprotein and a non-glycosylated polytopic membrane protein. System bo,+ exchanges dibasic for neutral amino acids. It is composed of rBAT and bo,+AT, the latter being the polytopic membrane subunit. Mutations in either of them cause malfunction of the system, leading to cystinuria. bo,+AT-reconstituted systems from HeLa or MDCK cells catalysed transport of arginine that was totally dependent on the presence of one of the bo,+ substrates inside the liposomes. rBAT was essential for the cell surface expression of bo,+AT, but it was not required for reconstituted bo,+AT transport activity. No system bo,+ transport was detected in liposomes derived from cells expressing rBAT alone. The reconstituted bo,+AT showed kinetic asymmetry. Expressing the cystinuria-specific mutant A354T of bo,+AT in HeLa cells together with rBAT resulted in defective arginine uptake in whole cells, which was paralleled by the reconstituted bo,+AT activity. Thus, subunit bo,+AT by itself is sufficient to catalyse transmembrane amino acid exchange. The polytopic subunits may also be the catalytic part in other heteromeric transporters. PMID:12234930

  15. Epitopes from two soybean glycinin subunits antigenic in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Glycinin is a seed storage protein in soybean (Glycine max) that is allergenic in pigs. Glycinin is a hexamer composed of subunits consisting of a basic and acidic portion joined by disulfide bridges. There are 5 glycinin subunits designated Gy1-Gy5. Results: Twenty seven out of 30 pi...

  16. The Development and Institutionalization of Subunit Power in Organizations.

    ERIC Educational Resources Information Center

    Boeker, Warren

    1989-01-01

    Examines the effects of founding events on the evolution of subunit importance in the semiconductor industry from 1958 to 1985. Distributions of power and subunit importance represent not only influences of current conditions, but also vestiges of earlier events, including the institution's founding. Includes 55 references. (MLH)

  17. Proteopedia Entry: The Large Ribosomal Subunit of "Haloarcula Marismortui"

    ERIC Educational Resources Information Center

    Decatur, Wayne A.

    2010-01-01

    This article presents a "Proteopedia" page that shows the refined version of the structure of the "Haloarcula" large ribosomal subunit as solved by the laboratories of Thomas Steitz and Peter Moore. The landmark structure is of great impact as it is the first atomic-resolution structure of the highly conserved ribosomal subunit which harbors…

  18. Estimating the efficiency of benzodiazepines on GABAA receptors comprising γ1 or γ2 subunits

    PubMed Central

    Baburin, I; Khom, S; Timin, E; Hohaus, A; Sieghart, W; Hering, S

    2008-01-01

    Background and purpose: Heterologous expression of α1, β2 and γ2S(γ1) subunits produces a mixed population of GABAA receptors containing α1β2 or α1β2γ2S(γ1) subunits. GABA sensitivity (lower in receptors containing γ1 or γ2S subunits) and the potentiation of GABA-activated chloride currents (IGABA) by benzodiazepines (BZDs) are dependent on γ2S(γ1) incorporation. A variable γ subunit incorporation may affect the estimation of IGABA potentiation by BZDs. We propose an approach for estimation of BZD efficiency that accounts for mixed population of α1β2 and α1β2γ2S(γ1) receptors. Experimental approach: We investigated the relation between GABA sensitivity (EC50) and BZD modulation by analysing triazolam-, clotiazepam- and midazolam-induced potentiation of IGABA in Xenopus oocytes under two-microelectrode voltage clamp. Key results: Plotting EC50 versus BZD-induced shifts of GABA concentration-response curves (ΔEC50(BZD)) of oocytes injected with different amounts of α1, β2 and γ2S(γ1) cRNA (1:1:1–1:1:10) revealed a linear regression between γ2S(γ1)-mediated reduction of GABA sensitivity (EC50) and ΔEC50(BZD). The slope factors of the regression were always higher for oocytes expressing α1β2γ1 subunit receptors (1.8±0.1 (triazolam), 1.6±0.1 (clotiazepam), 2.3±0.2 (midazolam)) than for oocytes expressing α1β2γ2S receptors (1.4±0.1 (triazolam), 1.4±0.1 (clotiazepam), 1.3±0.1 (midazolam)). Mutant GABAA receptors (α1β2-R207Cγ2S) with lower GABA sensitivity showed higher drug efficiencies (slope factors=1.1±0.1 (triazolam), 1.1±0.1 (clotiazepam), 1.2±0.1 (midazolam)). Conclusions and implications: Regression analysis enabled the estimation of BZD efficiency when variable mixtures of α1β2 and α1β2γ2S(γ1) receptors are expressed and provided new insights into the γ2S(γ1) dependency of BZD action. PMID:18604239

  19. 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

  20. The Glucose Measurement Industry and Hemoglobin A1c: An Opportunity for Creative Destruction.

    PubMed

    Cembrowski, George

    2016-01-01

    The MyStar Extra self-monitoring blood glucose (SMBG) system provides moving estimates of the patient's hemoglobin A1c (HbA1c). There is a treasure trove of highly accurate glucose data available from highly accurate SMBG, CGM and FGM along with highly accurate HPLC HbA1c. If Nathan's criteria are used to select subjects whose glucoses can be correlated to the HbA1c, then algorithms can be developed for robustly transforming glucose into HbA1c. These algorithms can then be implemented in any SMBG or with the CGM and FGM software. This calculated HbA1c would even be accurate with Nathan's excluded population thus reducing the use of fructosamine and glycated protein. Finally, the developer of these new algorithms is advised to use a specific approach for testing her algorithm. PMID:26481643

  1. Geranyl diphosphate synthase large subunit, and methods of use

    DOEpatents

    Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

    2001-10-16

    A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

  2. Modulation of Kv4.3 current by accessory subunits.

    PubMed

    Deschênes, Isabelle; Tomaselli, Gordon F

    2002-09-25

    Kv4.3 encodes the pore-forming subunit of the cardiac transient outward potassium current (I(to)). hKv4.3-encoded current does not fully replicate cardiac I(to), suggesting a functionally significant role for accessory subunits. KChIP2 associates with Kv4.3 and modifies hKv4.3-encoded currents but does not replicate native I(to). We examined the effect of several ancillary subunits expressed in the heart on hKv4.3-encoded currents. Remarkably, the ancillary subunits Kvbeta(3), minK, MiRP-1, the Na channel beta(1) and KChIP2 increased the density and modified the gating of hKv4.3 current. hKv4.3 promiscuously assembles with ancillary subunits in vitro, functionally modifying the encoded currents; however, the physiological significance is uncertain. PMID:12297301

  3. [Evaluation of D10 hemoglobin testing system for hemoglobin A1C assay].

    PubMed

    Marzullo, C; Minery, M

    2008-01-01

    Bio-Rad D10 hemoglobin testing system with rack loader for hemoglobinA1C assay was evaluated. Analytical qualities were satisfactory. Imprecision was good (within-run cv was 0,5% for 4,5% of HBA(1C), 0,63% for 7,4% of HBA1C, 0,46% for 11,1% of HBA1C, between-run cv was 1,16% for 4,7% of HBA1C, 1,01% for 7,6% of HBA1C, 1,04% for 11,2% of HBA1C). Results were very well correlated with those obtained on Bio-Rad Variant II (r = 0,998). Bland and Altman graph showed good agreement between the two methods for HbA1C under 15%. The measuring range was up to 18,3% of HBA1C. There was no specimen related carry over. Triglycerides under 5,5 mmol/L and bilirubin under 734 mumol/L did not interfere. Carbamylation of HBA1C did not interfere for urea concentration under 14 mmol/L. Practicability was very good. Detection of common hemoglobin variants (HbS, C, D, E, O) is available. Fast and easy switching between short and long program allows to perform HBA1C determination for patients with hemoglobin variants. So, D10 is an interesting and easy to use small HPLC automate witch offers accurate HBA1C quantification certified by NGSP. PMID:18227011

  4. RASSF1C modulates the expression of a stem cell renewal gene, PIWIL1

    PubMed Central

    2012-01-01

    Background RASSF1A and RASSF1C are two major isoforms encoded by the Ras association domain family 1 (RASSF1) gene through alternative promoter selection and mRNA splicing. RASSF1A is a well established tumor suppressor gene. Unlike RASSF1A, RASSF1C appears to have growth promoting actions in lung cancer. In this article, we report on the identification of novel RASSF1C target genes in non small cell lung cancer (NSCLC). Methods Over-expression and siRNA techniques were used to alter RASSF1C expression in human lung cancer cells, and Affymetrix-microarray study was conducted using NCI-H1299 cells over-expressing RASSF1C to identify RASSF1C target genes. Results The microarray study intriguingly shows that RASSF1C modulates the expression of a number of genes that are involved in cancer development, cell growth and proliferation, cell death, and cell cycle. We have validated the expression of some target genes using qRT-PCR. We demonstrate that RASSF1C over-expression increases, and silencing of RASSF1C decreases, the expression of PIWIL1 gene in NSCLC cells using qRT-PCR, immunostaining, and Western blot analysis. We also show that RASSF1C over-expression induces phosphorylation of ERK1/2 in lung cancer cells, and inhibition of the MEK-ERK1/2 pathway suppresses the expression of PIWIL1 gene expression, suggesting that RASSF1C may exert its activities on some target genes such as PIWIL1 through the activation of the MEK-ERK1/2 pathway. Also, PIWIL1 expression is elevated in lung cancer cell lines compared to normal lung epithelial cells. Conclusions Taken together, our findings provide significant data to propose a model for investigating the role of RASSF1C/PIWIL1 proteins in initiation and progression of lung cancer. PMID:22591718

  5. 7 CFR 1c.101 - To what does this policy apply?

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false To what does this policy apply? 1c.101 Section 1c.101 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.101 To what does this policy apply? (a) Except as provided in paragraph (b) of this section, this policy applies to all research involving human subjects...

  6. Methods, units and quality requirements for the analysis of haemoglobin A1c in diabetes mellitus.

    PubMed

    Penttilä, Ilkka; Penttilä, Karri; Holm, Päivi; Laitinen, Harri; Ranta, Päivi; Törrönen, Jukka; Rauramaa, Rainer

    2016-06-26

    The formation of glycohemoglobin, especially the hemoglobin A1c (HbA1c) fraction, occurs when glucose becomes coupled with the amino acid valine in the β-chain of Hb; this reaction is dependent on the plasma concentration of glucose. Since the early 1970s it has been known that diabetics display higher values OF HbA1C because they have elevated blood glucose concentrations. Thus HbA1c has acquired a very important role in the treatment and diagnosis of diabetes mellitus. After the introduction of the first quantitative measurement OF HbA1C, numerous methods for glycohemoglobin have been introduced with different assay principles: From a simple mini-column technique to the very accurate automated high-pressure chromatography and lastly to many automated immunochemical or enzymatic assays. In early days, the results of the quality control reports for HbA1c varied extensively between laboratories, therefore in United States and Canada working groups (WG) of the Diabetes Controls and Complications Trial (DCCT) were set up to standardize the HbA1c assays against the DCCT/National Glycohemoglobin Standardization Program reference method based on liquid chromatography. In the 1990s, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) appointed a new WG to plan a reference preparation and method for the HBA1c measurement. When the reference procedures were established, in 2004 IFCC recommended that all manufacturers for equipment used in HbA1c assays should calibrate their methods to their proposals. This led to an improvement in the coefficient of variation (CV%) associated with the assay. In this review, we describe the glycation of Hb, methods, standardization of the HbA1c assays, analytical problems, problems with the units in which HbA1c values are expressed, reference values, quality control aspects, target requirements for HbA1c, and the relationship of the plasma glucose values to HbA1c concentrations. We also note that the acceptance

  7. The brain-specific carnitine palmitoyltransferase-1c regulates energy homeostasis

    PubMed Central

    Wolfgang, Michael J.; Kurama, Takeshi; Dai, Yun; Suwa, Akira; Asaumi, Makoto; Matsumoto, Shun-ichiro; Cha, Seung Hun; Shimokawa, Teruhiko; Lane, M. Daniel

    2006-01-01

    Fatty acid synthesis in the central nervous system is implicated in the control of food intake and energy expenditure. An intermediate in this pathway, malonyl-CoA, mediates these effects. Malonyl-CoA is an established inhibitor of carnitine palmitoyltransferase-1 (CPT1), an outer mitochondrial membrane enzyme that controls entry of fatty acids into mitochondria and, thereby, fatty acid oxidation. CPT1c, a brain-specific enzyme with high sequence similarity to CPT1a (liver) and CPT1b (muscle) was recently discovered. All three CPTs bind malonyl-CoA, and CPT1a and CPT1b catalyze acyl transfer from various fatty acyl-CoAs to carnitine, whereas CPT1c does not. These findings suggest that CPT1c has a unique function or activation mechanism. We produced a targeted mouse knockout (KO) of CPT1c to investigate its role in energy homeostasis. CPT1c KO mice have lower body weight and food intake, which is consistent with a role as an energy-sensing malonyl-CoA target. Paradoxically, CPT1c KO mice fed a high-fat diet are more susceptible to obesity, suggesting that CPT1c is protective against the effects of fat feeding. CPT1c KO mice also exhibit decreased rates of fatty acid oxidation, which may contribute to their increased susceptibility to diet-induced obesity. These findings indicate that CPT1c is necessary for the regulation of energy homeostasis. PMID:16651524

  8. The Rab6-regulated KIF1C kinesin motor domain contributes to Golgi organization

    PubMed Central

    Lee, Peter L; Ohlson, Maikke B; Pfeffer, Suzanne R

    2015-01-01

    Most kinesins transport cargoes bound to their C-termini and use N-terminal motor domains to move along microtubules. We report here a novel function for KIF1C: it transports Rab6A-vesicles and can influence Golgi complex organization. These activities correlate with KIF1C's capacity to bind the Golgi protein Rab6A directly, both via its motor domain and C-terminus. Rab6A binding to the motor domain inhibits microtubule interaction in vitro and in cells, decreasing the amount of motile KIF1C. KIF1C depletion slows protein delivery to the cell surface, interferes with vesicle motility, and triggers Golgi fragmentation. KIF1C can protect Golgi membranes from fragmentation in cells lacking an intact microtubule network. Rescue of fragmentation requires sequences that enable KIF1C to bind Rab6A at both ends, but not KIF1C motor function. Rab6A binding to KIF1C's motor domain represents an entirely new mode of regulation for a kinesin motor, and likely has important consequences for KIF1C's cellular functions. DOI: http://dx.doi.org/10.7554/eLife.06029.001 PMID:25821985

  9. Methods, units and quality requirements for the analysis of haemoglobin A1c in diabetes mellitus

    PubMed Central

    Penttilä, Ilkka; Penttilä, Karri; Holm, Päivi; Laitinen, Harri; Ranta, Päivi; Törrönen, Jukka; Rauramaa, Rainer

    2016-01-01

    The formation of glycohemoglobin, especially the hemoglobin A1c (HbA1c) fraction, occurs when glucose becomes coupled with the amino acid valine in the β-chain of Hb; this reaction is dependent on the plasma concentration of glucose. Since the early 1970s it has been known that diabetics display higher values OF HbA1C because they have elevated blood glucose concentrations. Thus HbA1c has acquired a very important role in the treatment and diagnosis of diabetes mellitus. After the introduction of the first quantitative measurement OF HbA1C, numerous methods for glycohemoglobin have been introduced with different assay principles: From a simple mini-column technique to the very accurate automated high-pressure chromatography and lastly to many automated immunochemical or enzymatic assays. In early days, the results of the quality control reports for HbA1c varied extensively between laboratories, therefore in United States and Canada working groups (WG) of the Diabetes Controls and Complications Trial (DCCT) were set up to standardize the HbA1c assays against the DCCT/National Glycohemoglobin Standardization Program reference method based on liquid chromatography. In the 1990s, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) appointed a new WG to plan a reference preparation and method for the HBA1c measurement. When the reference procedures were established, in 2004 IFCC recommended that all manufacturers for equipment used in HbA1c assays should calibrate their methods to their proposals. This led to an improvement in the coefficient of variation (CV%) associated with the assay. In this review, we describe the glycation of Hb, methods, standardization of the HbA1c assays, analytical problems, problems with the units in which HbA1c values are expressed, reference values, quality control aspects, target requirements for HbA1c, and the relationship of the plasma glucose values to HbA1c concentrations. We also note that the acceptance

  10. Method for the detection of a polypeptide subunit in the presence of a quaternary protein containing the subunit

    SciTech Connect

    Wands, J.R.; Ozturk, M.; Bellet, D.

    1990-06-12

    This patent describes a method for the determination of a free protein subunit of hCG in a sample containing intact quaternary hCG. It comprises: contacting the sample with a first monoclonal antibody which is bound to a carrier, wherein the first monoclonal antibody binds epitopic determinants bindable only on the free protein subunit; incubating the components for a period of time and under conditions sufficient to form an immune complex between the free protein subunit, the first monoclonal antibody, and the carrier; separating the carrier from the sample; adding to the carrier a detectably labeled second monoclonal antibody, wherein the second monoclonal antibody binds epitopic determinants bindable on both the free protein subunit and the intact quaternary hCG; separating the carrier from the liquid phase; and determining the detectably labeled second monoclonal antibody in the carrier or in the liquid phase, which is a measure of the amount of the free protein subunit in the sample.

  11. Gel-based chemical cross-linking analysis of 20S proteasome subunit-subunit interactions in breast cancer.

    PubMed

    Song, Hai; Xiong, Hua; Che, Jing; Xi, Qing-Song; Huang, Liu; Xiong, Hui-Hua; Zhang, Peng

    2016-08-01

    The ubiquitin-proteasome system plays a pivotal role in breast tumorigenesis by controlling transcription factors, thus promoting cell cycle growth, and degradation of tumor suppressor proteins. However, breast cancer patients have failed to benefit from proteasome inhibitor treatment partially due to proteasome heterogeneity, which is poorly understood in malignant breast neoplasm. Chemical crosslinking is an increasingly important tool for mapping protein three-dimensional structures and proteinprotein interactions. In the present study, two cross-linkers, bis (sulfosuccinimidyl) suberate (BS(3)) and its water-insoluble analog disuccinimidyl suberate (DSS), were used to map the subunit-subunit interactions in 20S proteasome core particle (CP) from MDA-MB-231 cells. Different types of gel electrophoresis technologies were used. In combination with chemical cross-linking and mass spectrometry, we applied these gel electrophoresis technologies to the study of the noncovalent interactions among 20S proteasome subunits. Firstly, the CP subunit isoforms were profiled. Subsequently, using native/SDSPAGE, it was observed that 0.5 mmol/L BS(3) was a relatively optimal cross-linking concentration for CP subunit-subunit interaction study. 2-DE analysis of the cross-linked CP revealed that α1 might preinteract with α2, and α3 might pre-interact with α4. Moreover, there were different subtypes of α1α2 and α3α4 due to proteasome heterogeneity. There was no significant difference in cross-linking pattern for CP subunits between BS(3) and DSS. Taken together, the gel-based characterization in combination with chemical cross-linking could serve as a tool for the study of subunit interactions within a multi-subunit protein complex. The heterogeneity of 20S proteasome subunit observed in breast cancer cells may provide some key information for proteasome inhibition strategy. PMID:27465334

  12. Stargazin is an AMPA receptor auxiliary subunit.

    PubMed

    Vandenberghe, Wim; Nicoll, Roger A; Bredt, David S

    2005-01-11

    AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors mediate fast excitatory synaptic transmission in brain and underlie aspects of synaptic plasticity. Numerous AMPA receptor-binding proteins have been implicated in AMPA receptor trafficking and anchoring. However, the relative contributions of these proteins to the composition of native AMPA receptor complexes in brain remain uncertain. Here, we use blue native gel electrophoresis to analyze the composition of native AMPA receptor complexes in cerebellar extracts. We identify two receptor populations: a functional form that contains the transmembrane AMPA receptor-regulatory protein stargazin and an apo-form that lacks stargazin. Limited proteolysis confirms assembly of stargazin with a large proportion of native AMPA receptors. In contrast, other AMPA receptor-interacting proteins, such as synapse-associated protein 97, glutamate receptor-interacting protein 1, protein kinase Calpha binding protein, N-ethylmaleimide-sensitive fusion protein, AP2, and protein 4.1N, do not show significant association with AMPA receptor complexes on native gels. These data identify stargazin as an auxiliary subunit for a neurotransmitter-gated ion channel. PMID:15630087

  13. Down-regulation of murine Cyp1a-1 in mouse hepatoma Hepa-1c1c7 cells by bisphenol A.

    PubMed

    Jeong, H G; Kimand, J Y; Choi, C Y

    2000-11-01

    Cultured mouse hepatoma Hepa-1c1c7 cells were treated with either bisphenol A or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or in combination to assess the role of bisphenol A in the process of Cyp1a-1 induction. Treatment of Hepa-1c1c7 cultures with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) induced Cyp1a-1, as determined by analysis of 7-ethoxyresorufin O-deethylase (EROD) activities. Bisphenol A alone did not affect the activity of Cyp1a-1-specific EROD; in contrast, TCDD-induced EROD activities were markedly reduced in the concomitant treatment of TCDD and bisphenol A in a dose-dependent manner. Treatment with tamoxifen, an antiestrogen that acts through the estrogen receptor, did not affect the suppressive effects of bisphenol A on TCDD-induced EROD activity. TCDD-induced Cyp1a-1 mRNA levels were markedly suppressed in the concomitant treatment of TCDD and bisphenol A consistent with their effects on EROD activity. Transient transfection assay using dioxin-response element (DRE)-linked luciferase revealed that bisphenol A reduced transformation of the aryl hydrocarbons (Ah) receptor to a form capable of specifically binding to the DRE sequence in the promoter of the Cyp1a-1 gene. These results suggest the down-regulation of the Cyp1a-1 gene expression by bisphenol A in Hepa-1c1c7 cells might be antagonism of the DRE binding potential of nuclear Ah receptor but not mediated through estradiol receptor. PMID:11061999

  14. Apoptosis in murine hepatoma hepa 1c1c7 wild-type, C12, and C4 cells mediated by bilirubin.

    PubMed

    Seubert, John M; Darmon, Alison J; El-Kadi, Ayman O S; D'Souza, Sudhir J A; Bend, John R

    2002-08-01

    Elevated serum and tissue bilirubin concentrations that occur in pathological conditions such as cholestasis, jaundice, and other liver diseases are known to stimulate cytotoxic responses. In preliminary studies, we noted that bilirubin seemed to cause apoptosis in murine hepatoma Hepa 1c1c7 wild-type (WT) cells. Consequently, we investigated apoptosis caused by bilirubin in WT, mutant C12 [aryl hydrocarbon receptor (AHR)-deficient], and C4 (AHR nuclear translocator-deficient) Hepa 1c1c7 cells. Three independent measures of apoptosis were used to quantify the effects of exogenous bilirubin (0, 1, 10, 25, 50, or 100 microM). Caspase-3 activity and cytochrome c release from mitochondria increased at 3 h post-treatment, before increased caspase-8 activity at 6 h, and nuclear condensation by 24 h after treatment with bilirubin. No differences in whole-cell lipid peroxidation were observed between the cell types; however, intracellular reactive oxygen species (ROS) production was greater in WT cells than C12 or C4 cells 3 h after bilirubin exposure. Pretreatment of cells for 1 h with 1 or 10 microM alpha-naphthoflavone, an AHR antagonist, before bilirubin exposure resulted in decreased caspase-3 activity at 6 h and nuclear condensation at 24 h in WT cells. These results indicate that bilirubin, a potential AHR ligand, causes apoptosis in murine Hepa 1c1c7 WT cells by a mechanism(s) partially involving the AHR, disruption of membrane integrity, and increased intracellular ROS production. PMID:12130676

  15. Autocatalytic processing of m-AAA protease subunits in mitochondria.

    PubMed

    Koppen, Mirko; Bonn, Florian; Ehses, Sarah; Langer, Thomas

    2009-10-01

    m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individual subunits is only poorly understood. Here, we have examined the processing of nuclear-encoded m-AAA protease subunits upon import into mitochondria and demonstrate autocatalytic processing of Afg3l1 and Afg3l2. The mitochondrial processing peptidase MPP generates an intermediate form of Afg3l2 that is matured autocatalytically. Afg3l1 or Afg3l2 are also required for maturation of newly imported paraplegin subunits after their cleavage by MPP. Our results establish that mammalian m-AAA proteases can act as processing enzymes in vivo and reveal overlapping activities of Afg3l1 and Afg3l2. These findings might be of relevance for the pathogenesis of neurodegenerative disorders associated with mutations in different m-AAA protease subunits. PMID:19656850

  16. Both subunits of ADP-glucose pyrophosphorylase are regulatory.

    PubMed

    Cross, Joanna M; Clancy, Maureen; Shaw, Janine R; Greene, Thomas W; Schmidt, Robert R; Okita, Thomas W; Hannah, L Curtis

    2004-05-01

    The allosteric enzyme ADP-Glc pyrophosphorylase (AGPase) catalyzes the synthesis of ADP-Glc, a rate-limiting step in starch synthesis. Plant AGPases are heterotetramers, most of which are activated by 3-phosphoglyceric acid (3-PGA) and inhibited by phosphate. The objectives of these studies were to test a hypothesis concerning the relative roles of the two subunits and to identify regions in the subunits important in allosteric regulation. We exploited an Escherichia coli expression system and mosaic AGPases composed of potato (Solanum tuberosum) tuber and maize (Zea mays) endosperm subunit fragments to pursue this objective. Whereas potato and maize subunits have long been separated by speciation and evolution, they are sufficiently similar to form active mosaic enzymes. Potato tuber and maize endosperm AGPases exhibit radically different allosteric properties. Hence, comparing the kinetic properties of the mosaics to those of the maize endosperm and potato tuber AGPases has enabled us to identify regions important in regulation. The data herein conclusively show that both subunits are involved in the allosteric regulation of AGPase. Alterations in the small subunit condition drastically different allosteric properties. In addition, extent of 3-PGA activation and extent of 3-PGA affinity were found to be separate entities, mapping to different regions in both subunits. PMID:15122037

  17. RNA polymerase II subunit composition, stoichiometry, and phosphorylation.

    PubMed Central

    Kolodziej, P A; Woychik, N; Liao, S M; Young, R A

    1990-01-01

    RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme. Images PMID:2183013

  18. Prokaryotic and eukaryotic RNA polymerases have homologous core subunits.

    PubMed Central

    Sweetser, D; Nonet, M; Young, R A

    1987-01-01

    Eukaryotic RNA polymerases are complex aggregates whose component subunits are functionally ill-defined. The gene that encodes the 140,000-dalton subunit of Saccharomyces cerevisiae RNA polymerase II was isolated and studied in detail to obtain clues to the protein's function. This gene, RPB2, exists in a single copy in the haploid genome. Disruption of the gene is lethal to the yeast cell. RPB2 encodes a protein of 138,750 daltons, which contains sequences implicated in binding purine nucleotides and zinc ions and exhibits striking sequence homology with the beta subunit of Escherichia coli RNA polymerase. These observations suggest that the yeast and the E. coli subunit have similar roles in RNA synthesis, as the beta subunit contains binding sites for nucleotide substrates and a portion of the catalytic site for RNA synthesis. The subunit homologies reported here, and those observed previously with the largest RNA polymerase subunit, indicate that components of the prokaryotic RNA polymerase "core" enzyme have counterparts in eukaryotic RNA polymerases. PMID:3547406

  19. The role of aldo-keto reductase 1C3 (AKR1C3)-mediated prostaglandin D2 (PGD2) metabolism in keloids.

    PubMed

    Mantel, Alon; Newsome, Austin; Thekkudan, Theresa; Frazier, Robert; Katdare, Meena

    2016-01-01

    Keloids are progressively expanding scars, mostly prevalent in individuals of African descent. Previous data identified increased mast cell number and activation state in keloids suggesting a role in disease progression. The major eicosanoid secreted by mast cells is prostaglandin D2 (PGD2), a relatively unstable pro-inflammatory mediator which can be spontaneously converted to 15-deoxy-(Delta12,14)-prostaglandin J2(15d-PGJ2) or enzymatically metabolized to 9α,11β-PGF2 by aldo-keto reductase 1C3 (AKR1C3). In this work, we investigated the possible role of PGD2 and its metabolites in keloids using CRL1762 keloid fibroblasts (KF) and immunohistochemical staining. Our data suggested approximately 3-fold increase of tryptase-positive mast cell count in keloids compared with normal skin. Furthermore, AKR1C3 was overexpressed in the fibrotic area of keloids while relatively weak staining detected in normal skin. Metabolism of PGD2 to 9α,11β-PGF2 by both, KF and normal fibroblasts, was dependent on AKR1C3 as this reaction was attenuated in the presence of the AKR1C3 inhibitor, 2'-hydroxyflavanone, or in cells with decreased AKR1C3 expression. 15d-PGJ2, but not the other tested PGs, inhibited KF proliferation, attenuated KF-mediated collagen gel contraction and increased caspase-3 activation. In addition, treatment with 15d-PGJ2 activated P38-MAPK, induced reactive oxygen species and upregulated superoxide dismutase-1 (SOD-1). Finally, inhibition of P38-MAPK further augmented 15d-PGJ2-induced caspase-3 cleavage and attenuated its effect on SOD-1 transcription. This work suggests that localized dual inhibition of AKR1C3 and P38-MAPK may inhibit keloid progression. Inhibiting AKR1C3 activity may generate oxidative environment due to redirection of PGD2 metabolism towards 15d-PGJ2 while inhibition of P38-MAPK will sensitize keloid cells to ROS-induced apoptosis. PMID:26308156

  20. Apolipoprotein A1/C3/A5 haplotypes and serum lipid levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The association of single nucleotide polymorphisms (SNPs) in the apolipoprotein (Apo) A1/C3/A4/A5 gene cluster and serum lipid profiles is inconsistent. The present study was undertaken to detect the association between the ApoA1/C3/A5 gene polymorphisms and their haplotypes with serum lipid levels ...

  1. 18 CFR 2.1c - Policy statement on consultation with Indian tribes in Commission proceedings.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Policy statement on consultation with Indian tribes in Commission proceedings. 2.1c Section 2.1c Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES GENERAL POLICY AND INTERPRETATIONS Statements of...

  2. 50 CFR Table 1c to Part 679 - Product Type Codes

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 50 Wildlife and Fisheries 13 2012-10-01 2012-10-01 false Product Type Codes 1c Table 1c to Part 679 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES OF THE EXCLUSIVE ECONOMIC ZONE OFF ALASKA Pt....

  3. 50 CFR Table 1c to Part 679 - Product Type Codes

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 50 Wildlife and Fisheries 11 2011-10-01 2011-10-01 false Product Type Codes 1c Table 1c to Part 679 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES OF THE EXCLUSIVE ECONOMIC ZONE OFF ALASKA Pt....

  4. CD1c tetramers detect ex vivo T cell responses to processed phosphomycoketide antigens

    PubMed Central

    Ly, Dalam; Kasmar, Anne G.; Cheng, Tan-Yun; de Jong, Annemieke; Huang, Shouxiong; Roy, Sobhan; Bhatt, Apoorva; van Summeren, Ruben P.; Altman, John D.; Jacobs, William R.; Adams, Erin J.; Minnaard, Adriaan J.; Porcelli, Steven A.

    2013-01-01

    CD1c is expressed with high density on human dendritic cells (DCs) and B cells, yet its antigen presentation functions are the least well understood among CD1 family members. Using a CD1c-reactive T cell line (DN6) to complete an organism-wide survey of M. tuberculosis lipids, we identified C32 phosphomycoketide (PM) as a previously unknown molecule and a CD1c-presented antigen. CD1c binding and presentation of mycoketide antigens absolutely required the unusual, mycobacteria-specific lipid branching patterns introduced by polyketide synthase 12 (pks12). Unexpectedly, one TCR responded to diversely glycosylated and unglycosylated forms of mycoketide when presented by DCs and B cells. Yet cell-free systems showed that recognition was mediated only by the deglycosylated phosphoantigen. These studies identify antigen processing of a natural bacterial antigen in the human CD1c system, indicating that cells act on glycolipids to generate a highly simplified neoepitope composed of a sugar-free phosphate anion. Using knowledge of this processed antigen, we generated human CD1c tetramers, and demonstrate that CD1c–PM complexes stain T cell receptors (TCRs), providing direct evidence for a ternary interaction among CD1c-lipid-TCR. Furthermore, PM-loaded CD1c tetramers detect fresh human T cells from peripheral blood, demonstrating a polyclonal response to PM antigens in humans ex vivo. PMID:23530121

  5. 18 CFR 1c.2 - Prohibition of electric energy market manipulation.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Prohibition of electric energy market manipulation. 1c.2 Section 1c.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES PROHIBITION OF ENERGY MARKET MANIPULATION §...

  6. 18 CFR 1c.2 - Prohibition of electric energy market manipulation.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Prohibition of electric energy market manipulation. 1c.2 Section 1c.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES PROHIBITION OF ENERGY MARKET MANIPULATION §...

  7. Admissible ŝl (2|1; C) k characters and parafermions

    NASA Astrophysics Data System (ADS)

    Hayes, M.; Taormina, A.

    1998-10-01

    The branching functions of a particular subclass of characters of the affine superalgebra ŝl(2|1;( C) k into characters of the subalgebra ŝl(2|1;( C) k are calculated for fractional levels k = /1 u - 1, u ɛ N. They involve rational torus A u(u-1)andZu-1 parafermion characters.

  8. Frequent Monitoring of A1C During Pregnancy as a Treatment Tool to Guide Therapy

    PubMed Central

    Jovanovič, Lois; Savas, Hatice; Mehta, Manish; Trujillo, Angelina; Pettitt, David J.

    2011-01-01

    OBJECTIVE No guidelines for A1C measurement exist for women with gestational diabetes mellitus (GDM). The aim of this study was to document the rate of A1C decline in women with GDM. RESEARCH DESIGN AND METHODS Women with GDM in the Santa Barbara County Endocrine Clinic are managed with a carbohydrate-restricted diet and self-monitored blood glucose before and 1-h postprandial. Insulin is started if the preprandial glucose concentration is ≥90 mg/dl and/or a 1-h postprandial glucose concentration is ≥120 mg/dl. Capillary A1C was tested weekly using the DCA2000+ analyzer. RESULTS Twenty-four women with GDM (aged 29.0 ± 7.3 years) with initial A1C ≥7.0% were recruited. Baseline A1C was 8.8 ± 1.8%. Mean A1C decline was 0.47% per week (range 0.10–1.15%); the maximum was 4.3% in 4 weeks. CONCLUSIONS This study documents rapid decline in A1C during pregnancy and the utility of weekly A1C to guide therapy. PMID:20921215

  9. Insulin induction of SREBP-1c in rodent liver requires LXRα-C/EBPβ complex

    PubMed Central

    Tian, Jing; Goldstein, Joseph L.; Brown, Michael S.

    2016-01-01

    Insulin increases lipid synthesis in liver by activating transcription of the gene encoding sterol regulatory element-binding protein-1c (SREBP-1c). SREBP-1c activates the transcription of all genes necessary for fatty acid synthesis. Insulin induction of SREBP-1c requires LXRα, a nuclear receptor. Transcription of SREBP-1c also requires transcription factor C/EBPβ, but a connection between LXRα and C/EBPβ has not been made. Here we show that LXRα and C/EBPβ form a complex that can be immunoprecipitated from rat liver nuclei. Chromatin immunoprecipitation assays showed that the LXRα-C/EBPβ complex binds to the SREBP-1c promoter in a region that contains two binding sites for LXRα and is known to be required for insulin induction. Knockdown of C/EBPβ in fresh rat hepatocytes or mouse livers in vivo reduces the ability of insulin to increase SREBP-1c mRNA. The LXRα-C/EBPβ complex is bound to the SREBP-1c promoter in the absence or presence of insulin, indicating that insulin acts not by increasing the formation of this complex, but rather by activating it. PMID:27382175

  10. 18 CFR 2.1c - Policy statement on consultation with Indian tribes in Commission proceedings.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... (3) Is filed with the Secretary of the Commission. See generally 18 CFR 2.19. Statements of General... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Policy statement on consultation with Indian tribes in Commission proceedings. 2.1c Section 2.1c Conservation of Power and...

  11. 18 CFR 2.1c - Policy statement on consultation with Indian tribes in Commission proceedings.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Policy statement on consultation with Indian tribes in Commission proceedings. 2.1c Section 2.1c Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES GENERAL POLICY AND INTERPRETATIONS Statements of...

  12. 18 CFR 2.1c - Policy statement on consultation with Indian tribes in Commission proceedings.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... (3) Is filed with the Secretary of the Commission. See generally 18 CFR 2.19. Statements of General... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Policy statement on consultation with Indian tribes in Commission proceedings. 2.1c Section 2.1c Conservation of Power and...

  13. Diabetes mellitus, hemoglobin A1C, and the incidence of total joint arthroplasty infection.

    PubMed

    Iorio, Richard; Williams, Kelly M; Marcantonio, Andrew J; Specht, Lawrence M; Tilzey, John F; Healy, William L

    2012-05-01

    Patients with diabetes have a higher incidence of infection after total joint arthroplasty (TJA) than patients without diabetes. Hemoglobin A1c (HbA1c) levels are a marker for blood glucose control in diabetic patients. A total of 3468 patients underwent 4241 primary or revision total hip arthroplasty or total knee arthroplasty at one institution. Hemoglobin A1c levels were examined to evaluate if there was a correlation between the control of HbA1c and infection after TJA. There were a total of 46 infections (28 deep and 18 superficial [9 cellulitis and 9 operative abscesses]). Twelve (3.43%) occurred in diabetic patients (n = 350; 8.3%) and 34 (0.87%) in nondiabetic patients (n = 3891; 91.7%) (P < .001). There were 9 deep (2.6%) infections in diabetic patients and 19 (0.49%) in nondiabetic patients. In noninfected, diabetic patients, HbA1c level ranged from 4.7% to 15.1% (mean, 6.92%). In infected diabetic patients, HbA1c level ranged from 5.1% to 11.7% (mean, 7.2%) (P < .445). The average HbA1c level in patients with diabetes was 6.93%. Diabetic patients have a significantly higher risk for infection after TJA. Hemoglobin A1c levels are not reliable for predicting the risk of infection after TJA. PMID:22054905

  14. AKR1C3 as a target in castrate resistant prostate cancer.

    PubMed

    Adeniji, Adegoke O; Chen, Mo; Penning, Trevor M

    2013-09-01

    Aberrant androgen receptor (AR) activation is the major driver of castrate resistant prostate cancer (CRPC). CRPC is ultimately fatal and more therapeutic agents are needed to treat this disease. Compounds that target the androgen axis by inhibiting androgen biosynthesis and or AR signaling are potential candidates for use in CRPC treatment and are currently being pursued aggressively. Aldo-keto reductase 1C3 (AKR1C3) plays a pivotal role in androgen biosynthesis within the prostate. It catalyzes the 17-ketoreduction of weak androgen precursors to give testosterone and 5α-dihydrotestosterone. AKR1C3 expression and activity has been implicated in the development of CRPC, making it a rational target. Selective inhibition of AKR1C3 will be important, however, due to the presence of closely related isoforms, AKR1C1 and AKR1C2 that are also involved in androgen inactivation. We examine the evidence that supports the vital role of AKR1C3 in CRPC and recent developments in the discovery of potent and selective AKR1C3 inhibitors. This article is part of a Special Issue entitled 'CSR 2013'. PMID:23748150

  15. The role of hemoglobin A1c in the assessment of diabetes and cardiovascular risk.

    PubMed

    Sandler, Courtney Nagel; McDonnell, Marie E

    2016-05-01

    Hemoglobin A1c (HbA1c) is a widely used tool for diagnosing, screening, and managing patients with diabetes; however, proper application and interpretation of the HbA1c test is crucial to master for accurate assessment of patients. It also has become the standard test in population-based studies for evaluating the relationship between glycemic control and cardiovascular risk. Results from large clinical trials support the modern perspective that the HbA1c target should be personalized according to the risks and benefits of glycemic control. This likely is most important in patients with diabetes and elevated cardiovascular risk in whom achieving low HbA1c levels early in the natural history may be the most beneficial. PMID:27176682

  16. A novel self-lipid antigen targets human T cells against CD1c+ leukemias

    PubMed Central

    Lepore, Marco; de Lalla, Claudia; Gundimeda, S. Ramanjaneyulu; Gsellinger, Heiko; Consonni, Michela; Garavaglia, Claudio; Sansano, Sebastiano; Piccolo, Francesco; Scelfo, Andrea; Häussinger, Daniel; Montagna, Daniela; Locatelli, Franco; Bonini, Chiara; Bondanza, Attilio; Forcina, Alessandra; Li, Zhiyuan; Ni, Guanghui; Ciceri, Fabio; Jenö, Paul; Xia, Chengfeng

    2014-01-01

    T cells that recognize self-lipids presented by CD1c are frequent in the peripheral blood of healthy individuals and kill transformed hematopoietic cells, but little is known about their antigen specificity and potential antileukemia effects. We report that CD1c self-reactive T cells recognize a novel class of self-lipids, identified as methyl-lysophosphatidic acids (mLPAs), which are accumulated in leukemia cells. Primary acute myeloid and B cell acute leukemia blasts express CD1 molecules. mLPA-specific T cells efficiently kill CD1c+ acute leukemia cells, poorly recognize nontransformed CD1c-expressing cells, and protect immunodeficient mice against CD1c+ human leukemia cells. The identification of immunogenic self-lipid antigens accumulated in leukemia cells and the observed leukemia control by lipid-specific T cells in vivo provide a new conceptual framework for leukemia immune surveillance and possible immunotherapy. PMID:24935257

  17. Mutations in GABAA receptor subunits associated with genetic epilepsies.

    PubMed

    Macdonald, Robert L; Kang, Jing-Qiong; Gallagher, Martin J

    2010-06-01

    Mutations in inhibitory GABAA receptor subunit genes (GABRA1, GABRB3, GABRG2 and GABRD) have been associated with genetic epilepsy syndromes including childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME), pure febrile seizures (FS), generalized epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome (DS)/severe myoclonic epilepsy in infancy (SMEI). These mutations are found in both translated and untranslated gene regions and have been shown to affect the GABAA receptors by altering receptor function and/or by impairing receptor biogenesis by multiple mechanisms including reducing subunit mRNA transcription or stability, impairing subunit folding, stability, or oligomerization and by inhibiting receptor trafficking. PMID:20308251

  18. MUC1-C confers EMT and KRAS independence in mutant KRAS lung cancer cells

    PubMed Central

    Kharbanda, Akriti; Rajabi, Hasan; Jin, Caining; Alam, Maroof; Wong, Kwok-Kin; Kufe, Donald

    2014-01-01

    Non-small cell lung cancers (NSCLCs) that harbor an oncogenic KRAS mutation are often associated with resistance to targeted therapies. The MUC1-C transmembrane protein is aberrantly overexpressed in NSCLCs and confers a poor outcome; however, the functional role for MUC1-C in mutant KRAS NSCLC cells has remained unclear. The present studies demonstrate that silencing MUC1-C in A549/KRAS(G12S) and H460/KRAS(Q61H) NSCLC cells is associated with downregulation of AKT signaling and inhibition of growth. Overexpression of a MUC1-C(CQC→AQA) mutant, which inhibits MUC1-C homodimerization and function, suppressed both AKT and MEK activation. Moreover, treatment with GO-203, an inhibitor of MUC1-C homodimerization, blocked AKT and MEK signaling and decreased cell survival. The results further demonstrate that targeting MUC1-C suppresses expression of the ZEB1 transcriptional repressor by an AKT-mediated mechanism, and in turn induces miR-200c. In concert with these effects on the ZEB1/miR-200c regulatory loop, targeting MUC1-C was associated with reversal of the epithelial-mesenchymal transition (EMT) and inhibition of self-renewal capacity. Loss of MUC1-C function also attenuated KRAS independence and inhibited growth of KRAS mutant NSCLC cells as tumors in mice. These findings support a model in which targeting MUC1-C inhibits mutant KRAS signaling in NSCLC cells and thereby reverses the EMT phenotype and decreases self-renewal. PMID:25245423

  19. A 90-Day Dietary Toxicity Study of Genetically Modified Rice T1C-1 Expressing Cry1C Protein in Sprague Dawley Rats

    PubMed Central

    Tang, Xueming; Han, Fangting; Zhao, Kai; Xu, Yan; Wu, Xiao; Wang, Jinbin; Jiang, Lingxi; Shi, Wei

    2012-01-01

    In a 90-day study, Sprague Dawley rats were fed transgenic T1C-1 rice expressing Cry1C protein and were compared with rats fed non-transgenic parental rice Minghui 63 and rats fed a basal diet. No adverse effects on animal behavior or weight gain were observed during the study. Blood samples were collected and analyzed, and standard hematological and biochemical parameters were compared. A few of these parameters were found to be significantly different, but were within the normal reference intervals for rats of this breed and age, and were thus not considered to be treatment-related. Following sacrifice, a large number of organs were weighed, and macroscopic and histopathological examinations were performed with no changes reported. The aim of this study was to use a known animal model to determine the safety of the genetically modified (GM) rice T1C-1. The results showed no adverse or toxic effects due to T1C-1 rice when tested in this 90-day study. PMID:23300690

  20. Separation and conductimetric detection of C1-C7 aliphatic monocarboxylic acids and C1-C7 aliphatic monoamines on unfunctionized polymethacrylate resin columns.

    PubMed

    Ohta, Kazutoku; Towata, Atsuya; Ohashi, Masayoshi; Takeuchi, Toyohide

    2004-06-11

    The application of unfunctionized polymethacrylate resin (TSKgel G3000PWXL) as a stationary phase in liquid chromatography with conductimetric detection for C1-C7 aliphatic monocarboxylic acids (formic acid, acetic acid, propionic acid, butyric acid, isovaleric acid, valeric acid, 3,3-dimethylbutyric acid, 4-methylvaleric acid, hexanoic acid, 2-methylhexanoic acid, 5-methylhexanoic acid and heptanoic acid) and C1-C7 aliphatic monoamines (methylamine, ethylamine, propylamine, isobutylamine, butylamine, isoamylamine, amylamine, 1,3-dimethylbutylamine, hexylamine, 2-heptylamine and heptylamine) was attempted with C8 aliphatic monocarboxylic acids (2-propylvaleric acid, 2-ethylhexanoic acid, 2-methylheptanoic acid and octanoic acid) and C8 aliphatic monoamines (1,5-dimethylhexylamine, 2-ethylhexylamine, 1-methylheptylamine and octylamine) as eluents, respectively. Using 1 mM 2-methylheptanoic acid at pH 4.0 as the eluent, excellent separation and relatively high sensitive detection for these C1-C7 carboxylic acids were achieved on a TSKgel G3000PWXL column (150 mm x 6 mm i.d.) in 60 min. Using 2 mM octylamine at pH 11.0 as the eluent, excellent separation and relatively high sensitive detection for these C1-C7 amines were also achieved on the TSKgel G3000PWXL column in 60 min. PMID:15250420

  1. Differences in TCDD-elicited gene expression profiles in human HepG2, mouse Hepa1c1c7 and rat H4IIE hepatoma cells

    PubMed Central

    2011-01-01

    Background 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that elicits a broad spectrum of toxic effects in a species-specific manner. Current risk assessment practices routinely extrapolate results from in vivo and in vitro rodent models to assess human risk. In order to further investigate the species-specific responses elicited by TCDD, temporal gene expression responses in human HepG2, mouse Hepa1c1c7 and rat H4IIE cells were compared. Results Microarray analysis identified a core set of conserved gene expression responses across species consistent with the role of AhR in mediating adaptive metabolic responses. However, significant species-specific as well as species-divergent responses were identified. Computational analysis of the regulatory regions of species-specific and -divergent responses suggests that dioxin response elements (DREs) are involved. These results are consistent with in vivo rat vs. mouse species-specific differential gene expression, and more comprehensive comparative DRE searches. Conclusions Comparative analysis of human HepG2, mouse Hepa1c1c7 and rat H4IIE TCDD-elicited gene expression responses is consistent with in vivo rat-mouse comparative gene expression studies, and more comprehensive comparative DRE searches, suggesting that AhR-mediated gene expression is species-specific. PMID:21496263

  2. Calculation of the phase diagrams of ferrimagnetic alloys A cB 1- c and application to transition metal-rare-earth Fe cGd 1- c and Fe cTb 1- c materials

    NASA Astrophysics Data System (ADS)

    Fresneau, M.; Virlouvet, A.; Khater, A.

    1999-07-01

    A theoretical model is presented for the calculation of the magnetic properties of generalised spin ferrimagnetic random substitutional alloys A cB 1- c with antiferromagnetic coupling between the A and B spin species. In particular, we study in an effective field method the phase diagrams of these systems as a function of the alloy concentration c, for given magnetic exchange constants. The model is applied with no adjustable parameters to the transition metal-rare-earth Fe cGd 1- c and Fe cTb 1- c alloys, using the appropriate spins for the Fe, Gd and Tb ions. We report a coherent and an overall qualitative agreement between theory and experiment in the complete range of concentrations 1⩾ c⩾0, for the first time to our knowledge, and determine consequently for these materials a set of ionic exchange constants that are invariant with respect to the alloy concentration. To our knowledge this is the first time that approximate but seemingly reliable exchange constants for the two alloys have been derived.

  3. Electrophysiological and morphological maturation of murine fetal cardiomyocytes during electrical stimulation in vitro.

    PubMed

    Baumgartner, Sven; Halbach, Marcel; Krausgrill, Benjamin; Maass, Martina; Srinivasan, Sureshkumar Perumal; Sahito, Raja Ghazanfar Ali; Peinkofer, Gabriel; Nguemo, Filomain; Müller-Ehmsen, Jochen; Hescheler, Jürgen

    2015-01-01

    The aim of this study was to investigate whether continuous electrical stimulation affects electrophysiological properties and cell morphology of fetal cardiomyocytes (FCMs) in culture. Fetal cardiomyocytes at day 14.5 post coitum were harvested from murine hearts and electrically stimulated for 6 days in culture using a custom-made stimulation chamber. Subsequently, action potentials of FCM were recorded with glass microelectrodes. Immunostainings of α-Actinin, connexin 43, and vinculin were performed. Expression of ion channel subunits Kcnd2, Slc8a1, Cacna1, Kcnh2, and Kcnb1 was analyzed by quantitative reverse-transcriptase polymerase chain reaction. Action potential duration to 50% and 90% repolarization (APD50 and APD90) of electrically stimulated FCMs were significantly decreased when compared to nonstimulated control FCM. Alignment of cells was significantly higher in stimulated FCM when compared to control FCM. The expression of connexin 43 was significantly increased in stimulated FCM when compared to control FCM. The ratio between cell length and cell width of the stimulated FCM was significantly higher than in control FCM. Kcnh2 and Kcnd2 were upregulated in stimulated FCM when compared to control FCM. Expression of Slc8a1, Cacna1c, and Kcnb1 was not different in stimulated and control FCMs. The decrease in APD50 observed after electrical stimulation of FCM in vitro corresponds to the electrophysiological maturation of FCM in vivo. Expression levels of ion channels suggest that some important but not all aspects of the complex process of electrophysiological maturation are promoted by electrical stimulation. Parallel alignment, increased connexin 43 expression, and elongation of FCM are signs of a morphological maturation induced by electrical stimulation. PMID:24917562

  4. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; Chapelle, S; De Wachter, R

    1994-01-01

    A database on large ribosomal subunit RNA is made available. It contains 258 sequences. It provides sequence, alignment and secondary structure information in computer-readable formats. Files can be obtained using ftp. PMID:7524023

  5. A process yields large quantities of pure ribosome subunits

    NASA Technical Reports Server (NTRS)

    Friedman, M.; Lu, P.; Rich, A.

    1972-01-01

    Development of process for in-vitro protein synthesis from living cells followed by dissociation of ribosomes into subunits is discussed. Process depends on dialysis or use of chelating agents. Operation of process and advantages over previous methods are outlined.

  6. Histone demethylase JARID1C inactivation triggers genomic instability in sporadic renal cancer

    PubMed Central

    Rondinelli, Beatrice; Rosano, Dalia; Antonini, Elena; Frenquelli, Michela; Montanini, Laura; Huang, DaChuan; Segalla, Simona; Yoshihara, Kosuke; Amin, Samir B.; Lazarevic, Dejan; The, Bin Tean; Verhaak, Roel G.W.; Futreal, P. Andrew; Di Croce, Luciano; Chin, Lynda; Cittaro, Davide; Tonon, Giovanni

    2015-01-01

    Mutations in genes encoding chromatin-remodeling proteins are often identified in a variety of cancers. For example, the histone demethylase JARID1C is frequently inactivated in patients with clear cell renal cell carcinoma (ccRCC); however, it is largely unknown how JARID1C dysfunction promotes cancer. Here, we determined that JARID1C binds broadly to chromatin domains characterized by the trimethylation of lysine 9 (H3K9me3), which is a histone mark enriched in heterochromatin. Moreover, we found that JARID1C localizes on heterochromatin, is required for heterochromatin replication, and forms a complex with established players of heterochromatin assembly, including SUV39H1 and HP1α, as well as with proteins not previously associated with heterochromatin assembly, such as the cullin 4 (CUL4) complex adaptor protein DDB1. Transcription on heterochromatin is tightly suppressed to safeguard the genome, and in ccRCC cells, JARID1C inactivation led to the unrestrained expression of heterochromatic noncoding RNAs (ncRNAs) that in turn triggered genomic instability. Moreover, ccRCC patients harboring JARID1C mutations exhibited aberrant ncRNA expression and increased genomic rearrangements compared with ccRCC patients with tumors endowed with other genetic lesions. Together, these data suggest that inactivation of JARID1C in renal cancer leads to heterochromatin disruption, genomic rearrangement, and aggressive ccRCCs. Moreover, our results shed light on a mechanism that underlies genomic instability in sporadic cancers. PMID:26551685

  7. Interference of the Hope Hemoglobin With Hemoglobin A1c Results.

    PubMed

    Chakraborty, Sutirtha; Chanda, Dalia; Gain, Mithun; Krishnan, Prasad

    2015-01-01

    Hemoglobin A1c (HbA1c) is now considered to be the marker of choice in diagnosis and management of diabetes mellitus, based on the results of certain landmark clinical trials. Herein, we report the case of a 52-year-old ethnic Southeast Asian Indian man with impaired glucose tolerance whose glycated hemoglobin (ie, HbA1c) levels, as measured via Bio-Rad D10 high-performance liquid chromatography (HPLC) and Roche Tina-quant immunoassay were 47.8% and 44.0%, respectively. No variant hemoglobin (Hb) peak was observed via the D10 chromatogram. We assayed the patient specimen on the Sebia MINICAP capillary electrophoresis platform; the HbA1c level was 6.8%, with a large variant Hb peak of 42.0%. This finding suggested the possible presence of the heterozygous Hb Hope, which can result in spuriously elevated HbA1c results on HPLC and turbidimetric immunoassays. Although the capillary electrophoresis system was able to identify the variant, the A1c results should not be considered accurate due to overlapping of the variant and adult Hb peaks on the electrophoretogram reading. Hb Hope is usually clinically silent but can present such analytical challenges. Through this case study, we critically discuss the limitations of various HbA1c assay methods, highlighting the fact that laboratory professionals need to be aware of occurrences of Hb Hope, to help ensure patient safety. PMID:26199262

  8. The lipid kinase PIP5K1C regulates pain signaling and sensitization

    PubMed Central

    Wright, Brittany D.; Loo, Lipin; Street, Sarah E.; Ma, Anqi; Taylor-Blake, Bonnie; Stashko, Michael A.; Jin, Jian; Janzen, William P.; Frye, Stephen V.; Zylka, Mark J.

    2014-01-01

    SUMMARY Numerous pain-producing (pronociceptive) receptors signal via phosphatidylinositol 4,5- bisphosphate (PIP2) hydrolysis. However, it is currently unknown which lipid kinases generate PIP2 in nociceptive dorsal root ganglia (DRG) neurons and if these kinases regulate pronociceptive receptor signaling. Here, we found that phosphatidylinositol 4-phosphate 5 kinase type 1C (PIP5K1C) is expressed at higher levels than any other PIP5K and, based on experiments with Pip5k1c+/− mice, generates at least half of all PIP2 in DRG neurons. Additionally, Pip5k1c haploinsufficiency reduces pronociceptive receptor signaling and TRPV1 sensitization in DRG neurons as well as thermal and mechanical hypersensitivity in mouse models of chronic pain. We identified a novel small molecule inhibitor of PIP5K1C (UNC3230) in a high-throughput screen. UNC3230 lowered PIP2 levels in DRG neurons and attenuated hypersensitivity when administered intrathecally or into the hindpaw. Our studies reveal that PIP5K1C regulates PIP2- dependent nociceptive signaling and suggest that PIP5K1C is a novel therapeutic target for chronic pain. PMID:24853942

  9. Myo1c binding to submembrane actin mediates insulin-induced tethering of GLUT4 vesicles

    PubMed Central

    Boguslavsky, Shlomit; Chiu, Tim; Foley, Kevin P.; Osorio-Fuentealba, Cesar; Antonescu, Costin N.; Bayer, K. Ulrich; Bilan, Philip J.; Klip, Amira

    2012-01-01

    GLUT4-containing vesicles cycle between the plasma membrane and intracellular compartments. Insulin promotes GLUT4 exocytosis by regulating GLUT4 vesicle arrival at the cell periphery and its subsequent tethering, docking, and fusion with the plasma membrane. The molecular machinery involved in GLUT4 vesicle tethering is unknown. We show here that Myo1c, an actin-based motor protein that associates with membranes and actin filaments, is required for insulin-induced vesicle tethering in muscle cells. Myo1c was found to associate with both mobile and tethered GLUT4 vesicles and to be required for vesicle capture in the total internal reflection fluorescence (TIRF) zone beneath the plasma membrane. Myo1c knockdown or overexpression of an actin binding–deficient Myo1c mutant abolished insulin-induced vesicle immobilization, increased GLUT4 vesicle velocity in the TIRF zone, and prevented their externalization. Conversely, Myo1c overexpression immobilized GLUT4 vesicles in the TIRF zone and promoted insulin-induced GLUT4 exposure to the extracellular milieu. Myo1c also contributed to insulin-dependent actin filament remodeling. Thus we propose that interaction of vesicular Myo1c with cortical actin filaments is required for insulin-mediated tethering of GLUT4 vesicles and for efficient GLUT4 surface delivery in muscle cells. PMID:22918957

  10. Myo1c binding to submembrane actin mediates insulin-induced tethering of GLUT4 vesicles.

    PubMed

    Boguslavsky, Shlomit; Chiu, Tim; Foley, Kevin P; Osorio-Fuentealba, Cesar; Antonescu, Costin N; Bayer, K Ulrich; Bilan, Philip J; Klip, Amira

    2012-10-01

    GLUT4-containing vesicles cycle between the plasma membrane and intracellular compartments. Insulin promotes GLUT4 exocytosis by regulating GLUT4 vesicle arrival at the cell periphery and its subsequent tethering, docking, and fusion with the plasma membrane. The molecular machinery involved in GLUT4 vesicle tethering is unknown. We show here that Myo1c, an actin-based motor protein that associates with membranes and actin filaments, is required for insulin-induced vesicle tethering in muscle cells. Myo1c was found to associate with both mobile and tethered GLUT4 vesicles and to be required for vesicle capture in the total internal reflection fluorescence (TIRF) zone beneath the plasma membrane. Myo1c knockdown or overexpression of an actin binding-deficient Myo1c mutant abolished insulin-induced vesicle immobilization, increased GLUT4 vesicle velocity in the TIRF zone, and prevented their externalization. Conversely, Myo1c overexpression immobilized GLUT4 vesicles in the TIRF zone and promoted insulin-induced GLUT4 exposure to the extracellular milieu. Myo1c also contributed to insulin-dependent actin filament remodeling. Thus we propose that interaction of vesicular Myo1c with cortical actin filaments is required for insulin-mediated tethering of GLUT4 vesicles and for efficient GLUT4 surface delivery in muscle cells. PMID:22918957

  11. A Phytophthora infestans RXLR effector targets plant PP1c isoforms that promote late blight disease.

    PubMed

    Boevink, Petra C; Wang, Xiaodan; McLellan, Hazel; He, Qin; Naqvi, Shaista; Armstrong, Miles R; Zhang, Wei; Hein, Ingo; Gilroy, Eleanor M; Tian, Zhendong; Birch, Paul R J

    2016-01-01

    Plant pathogens deliver effectors to alter host processes. Knowledge of how effectors target and manipulate host proteins is critical to understand crop disease. Here, we show that in planta expression of the RXLR effector Pi04314 enhances leaf colonization by Phytophthora infestans via activity in the host nucleus and attenuates induction of jasmonic and salicylic acid-responsive genes. Pi04314 interacts with three host protein phosphatase 1 catalytic (PP1c) isoforms, causing their re-localization from the nucleolus to the nucleoplasm. Re-localization of PP1c-1 also occurs during infection and is dependent on an R/KVxF motif in the effector. Silencing the PP1c isoforms or overexpression of a phosphatase-dead PP1c-1 mutant attenuates infection, demonstrating that host PP1c activity is required for disease. Moreover, expression of PP1c-1mut abolishes enhanced leaf colonization mediated by in planta Pi04314 expression. We argue that PP1c isoforms are susceptibility factors forming holoenzymes with Pi04314 to promote late blight disease. PMID:26822079

  12. Mutation of SIMPLE in Charcot-Marie-Tooth 1C alters production of exosomes.

    PubMed

    Zhu, Hong; Guariglia, Sara; Yu, Raymond Y L; Li, Wenjing; Brancho, Deborah; Peinado, Hector; Lyden, David; Salzer, James; Bennett, Craig; Chow, Chi-Wing

    2013-06-01

    Charcot-Marie-Tooth (CMT) disease is an inherited neurological disorder. Mutations in the small integral membrane protein of the lysosome/late endosome (SIMPLE) account for the rare autosomal-dominant demyelination in CMT1C patients. Understanding the molecular basis of CMT1C pathogenesis is impeded, in part, by perplexity about the role of SIMPLE, which is expressed in multiple cell types. Here we show that SIMPLE resides within the intraluminal vesicles of multivesicular bodies (MVBs) and inside exosomes, which are nanovesicles secreted extracellularly. Targeting of SIMPLE to exosomes is modulated by positive and negative regulatory motifs. We also find that expression of SIMPLE increases the number of exosomes and secretion of exosome proteins. We engineer a point mutation on the SIMPLE allele and generate a physiological mouse model that expresses CMT1C-mutated SIMPLE at the endogenous level. We find that CMT1C mouse primary embryonic fibroblasts show decreased number of exosomes and reduced secretion of exosome proteins, in part due to improper formation of MVBs. CMT1C patient B cells and CMT1C mouse primary Schwann cells show similar defects. Together the data indicate that SIMPLE regulates the production of exosomes by modulating the formation of MVBs. Dysregulated endosomal trafficking and changes in the landscape of exosome-mediated intercellular communications may place an overwhelming burden on the nervous system and account for CMT1C molecular pathogenesis. PMID:23576546

  13. Evolutionary diversification of DYX1C1 transcripts via an HERV-H LTR integration event.

    PubMed

    Kim, Yun-Ji; Huh, Jae-Won; Kim, Dae-Soo; Han, Kyudong; Kim, Hwan-Mook; Kim, Heui-Soo

    2011-01-01

    DYX1C1 is a candidate gene for developmental dyslexia and has three alternative pre-mRNA spliced forms in the human genome. One of the transcripts contains an HERV-H LTR that could affect the expression level of DYX1C1. We speculate that the HERV-H LTR integrated into the DYX1C1 locus in the catarrhine lineage after its divergence from the platyrrhine lineage. Reverse transcription-PCR of the HERV-H LTR-related transcript produced four alternative forms from several human tissues. All of alternative forms were also identified in various rhesus macaque tissues. Through sequencing analysis of various primate DNA samples, we found that a part of the HERV-H LTR sequence was duplicated within the DYX1C1 exon 9 only in catarrhines. However, the duplication event did not cause frameshift mutation of the DYX1C1 transcript. Taken together, this HERV-H LTR insertion into DYX1C1 has contributed to transcript diversification of DYX1C1 during primate evolution. PMID:22214596

  14. Gene delivery of Homer1c rescues spatial learning in a rodent model of cognitive aging.

    PubMed

    Gerstein, Hilary; Lindstrom, Mary J; Burger, Corinna

    2013-08-01

    Homer1c has been shown to play a role in learning and memory. Overexpression of Homer1c in the hippocampus can improve memory in normal rats and can also rescue spatial learning deficits in Homer1 knockout mice. In a previous study, we found that Homer1c mRNA is upregulated after a spatial learning paradigm in aged rats that successfully learn the task, when compared to aged rats that are learning-impaired (AI). This study was designed to validate the role of Homer1c in successful cognitive aging. In this article, we report that gene delivery of Homer1c into the hippocampus of aged learning-impaired rats significantly improves individual performance on an object location memory task. The learning ability of these rats on the Morris Water Maze was also superior to that of AI control rats. In summary, using 2 independent spatial memory tasks, we demonstrate that Homer1c is sufficient to improve the spatial learning deficits in a rodent model of cognitive aging. These results point to Homer1c as a potential therapeutic target for improving age-related cognitive impairment. PMID:23523268

  15. Optimal Hemoglobin A1c Levels for Screening of Diabetes and Prediabetes in the Japanese Population

    PubMed Central

    Shimodaira, Masanori; Okaniwa, Shinji; Hanyu, Norinao; Nakayama, Tomohiro

    2015-01-01

    The aim of this study was to evaluate the utility of hemoglobin A1c (HbA1c) to identify individuals with diabetes and prediabetes in the Japanese population. A total of 1372 individuals without known diabetes were selected for this study. A 75 g oral glucose tolerance test (OGTT) was used to diagnose diabetes and prediabetes. The ability of HbA1c to detect diabetes and prediabetes was investigated using receiver operating characteristic (ROC) analysis. The kappa (κ) coefficient was used to test the agreement between HbA1c categorization and OGTT-based diagnosis. ROC analysis demonstrated that HbA1c was a good test to identify diabetes and prediabetes, with areas under the curve of 0.918 and 0.714, respectively. Optimal HbA1c cutoffs for diagnosing diabetes and prediabetes were 6.0% (sensitivity 83.7%, specificity 87.6%) and 5.7% (sensitivity 60.6%, specificity 72.1%), respectively, although the cutoff for prediabetes showed low accuracy (67.6%) and a high false-negative rate (39.4%). Agreement between HbA1c categorization and OGTT-based diagnosis was low in diabetes (κ = 0.399) and prediabetes (κ = 0.324). In Japanese subjects, the HbA1c cutoff of 6.0% had appropriate sensitivity and specificity for diabetes screening, whereas the cutoff of 5.7% had modest sensitivity and specificity in identifying prediabetes. Thus, HbA1c may be inadequate as a screening tool for prediabetes. PMID:26114121

  16. The Role of Homer1c in Metabotropic Glutamate Receptor-dependent Long-Term Potentiation

    PubMed Central

    O’Riordan, Kenneth; Gerstein, Hilary; Hullinger, Rikki; Burger, Corinna

    2016-01-01

    Group I metabotropic glutamate receptors (mGluR1/5) play a role in synaptic plasticity and they demonstrate direct interactions with the neuronal Homer1c protein. We have previously shown that Homer1c can restore the plasticity deficits in Homer1 knockout mice (H1-KO). Here, we investigated the role of Homer1c in mGluR-dependent synaptic plasticity in wild-type mice, H1-KO, and H1-KO mice overexpressing Homer1c (KO+H1c). We used a form of plasticity induced by activation of mGluR1/5 that transforms short-term potentiaion (STP) induced by a subthreshold theta burst stimulation into long-term potentiation (LTP). We have shown that although acute hippocampal slices from wild-type animals can induce LTP using this stimulation protocol, H1-KO only show STP. Gene delivery of Homer1c into the hippocampus of H1-KO mice rescued LTP to wild-type levels. This form of synaptic plasticity was dependent on mGluR5 but not mGluR1 activation both in wild-type mice and in KO+H1c. mGluR1/5-dependent LTP was blocked with inhibitors of the MEK-ERK and PI3K-mTOR pathways in KO+H1c mice. Moreover, blocking Homer1c–mGluR5 interactions prevented the maintenance of LTP in acute hippocampal slices from KO+H1c. These data indicate that Homer1c–mGluR5 interactions are necessary for mGluR-dependent LTP, and that mGluR1/5-dependent LTP involves PI3K and ERK activation. PMID:24167026

  17. Relationship Between A1C and Fasting Plasma Glucose in Dysglycemia or Type 2 Diabetes

    PubMed Central

    Ramachandran, Ambady; Riddle, Matthew C.; Kabali, Conrad; Gerstein, Hertzel C.

    2012-01-01

    OBJECTIVE A1C measurement has advantages over measures of plasma glucose. Few studies have evaluated the A1C–fasting plasma glucose (FPG) relationship and whether oral antidiabetes drugs (OADs) and ethnic or geographic variations affect the relationship. Baseline A1C and FPG data from the Outcome Reduction with Initial Glargine Intervention (ORIGIN) trial participants were analyzed to 1) elucidate the relationship between A1C and FPG in people with moderate dysglycemia (A1C 5.6–9.0% [38–75 mmol/mol]) and additional risk factors for cardiovascular disease, 2) determine whether this relationship is altered by use of an OAD, and 3) study whether geographic and ethnic differences exist. RESEARCH DESIGN AND METHODS Analysis was performed of 12,527 participants with dysglycemia or early type 2 diabetes recruited in North America, South America, Europe, Australia, and Asia who comprised white, Latin American, Asian, black, and other ethnicities. The A1C-FPG relationships were analyzed using cubic B spline curves in all participants and in subgroups not using an OAD or using an OAD and comprising persons of different ethnic or geographic origin. RESULTS A strong relationship between FPG in the range of 5.6–9.0 mmol/L and the corresponding A1C was seen across different geographic regions and ethnic groups. A smaller increase in A1C per unit increase in FPG occurred for persons taking an OAD versus those not taking an OAD. CONCLUSIONS The strong relationship between A1C and FPG in moderate dysglycemia is not significantly affected by ethnic or geographic differences. Use of an OAD alters the relationship and should be considered when interpreting A1C level. PMID:22323416

  18. Fasting blood glucose and HbA1c in children with ADHD.

    PubMed

    Lindblad, Frank; Eickhoff, Malin; Forslund, Anders H; Isaksson, Johan; Gustafsson, Jan

    2015-04-30

    Reports of hypocortisolism and overweight in pediatric ADHD motivate an investigation of blood glucose regulation in this group. Fasting blood glucose and HbA1c were investigated in 10 children (10-15 years) with ADHD and 22 comparisons. Fasting blood glucose was similar in both groups. HbA1c values were higher in the ADHD-group. BMI-SDS was also higher in the ADHD-group but did not predict HbA1c. The results suggest an association between ADHD and an altered blood glucose homeostasis. PMID:25747679

  19. Effect of Long-Term Periodontal Care on Hemoglobin A1c in Type 2 Diabetes.

    PubMed

    Merchant, A T; Georgantopoulos, P; Howe, C J; Virani, S S; Morales, D A; Haddock, K S

    2016-04-01

    This was a prospective cohort study evaluating 126,805 individuals with diabetes and periodontal disease receiving care at all Veterans Administration medical centers and clinics in the United States from 2005 through 2012. The exposures were periodontal treatment at baseline (PT0) and at follow-up (PT2). The outcomes were change in HbA1c following initial treatment (ΔHbA1c1) and follow-up treatment (ΔHbA1c2), and diabetes control was defined as HbA1c at <7% and <9% following initial and follow-up treatment, respectively. Marginal structural models were used to account for potential confounding and selection bias. The objective was to evaluate the impact of long-term treatment of periodontal disease on glycemic control among individuals with type 2 diabetes. Participants were 64 y old on average, 97% were men, and 71% were white. At baseline, the average diabetes duration was 4 y, 12% of participants were receiving insulin, and 60% had HbA1c <7%. After an average 1.7 y of follow-up, the mean HbA1c increased from 7.03% to 7.21%. About 29.4% of participants attended their periodontal maintenance visit following baseline. Periodontal treatment at baseline and follow-up reduced HbA1c by -0.02% and -0.074%, respectively. Treatment at follow-up increased the likelihood of individuals achieving diabetes control by 5% and 3% at the HbA1c <7% and HbA1c <9% thresholds, respectively, and was observed even among never smokers. HbA1c reduction after periodontal treatment at follow-up was greater (ΔHbA1c2 = -0.25%) among individuals with higher baseline HbA1c. Long-term periodontal care provided in a clinical setting improved long-term glycemic control among individuals with type 2 diabetes and periodontal disease. PMID:26701348

  20. Primary structure of the ovine pituitary follitropin beta-subunit.

    PubMed Central

    Sairam, M R; Seidah, N G; Chrétien, M

    1981-01-01

    The complete amino acids sequence of the ovine pituitary follitropin beta-subunit was established by studying the tryptic, chymotryptic and thermolytic peptides. The N-terminal sequence of the subunit was confirmed by subjecting the oxidated protein to Edman degradation in an automated sequenator. Automated Edman degradation of the reduced and alkylated (with iodo [14C]acetamide) beta-subunit indicated that most of the molecules used in the sequence studies had lost the N-terminal serine residue. This also confirmed the location of the first five half-cystine residues in the sequence. The proposed structure shows the presence of 111 amino acid residues with the two oligosaccharide moieties linked to asparagine residues located at positions 6 and 23. Heterogeneity occurs at both the termini of the polypeptide chain. Comparison of the sequence of beta-subunit of the ovine hormone with that proposed for human follitropin beta-subunit shows the absence of any deletions in the middle of the peptide chain. Of the 13 replacements, 11 residues can be explained on the basis of a single base change in the codon. The single tryptophan residue of the follitropin occupies an identical position in all the four species that have been studied. The region corresponding to residues 63-105 of the ovine beta-subunit is highly conserved in all the species. PMID:6798969

  1. Transcriptional regulators of Na,K-ATPase subunits

    PubMed Central

    Li, Zhiqin; Langhans, Sigrid A.

    2015-01-01

    The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic α-subunit, the β-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids, and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits has been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease. PMID:26579519

  2. A new look at sodium channel β subunits.

    PubMed

    Namadurai, Sivakumar; Yereddi, Nikitha R; Cusdin, Fiona S; Huang, Christopher L H; Chirgadze, Dimitri Y; Jackson, Antony P

    2015-01-01

    Voltage-gated sodium (Nav) channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are a major focus of research in neurobiology, structural biology, membrane biology and pharmacology. Mutations in Nav channels are implicated in a wide variety of inherited pathologies, including cardiac conduction diseases, myotonic conditions, epilepsy and chronic pain syndromes. Drugs active against Nav channels are used as local anaesthetics, anti-arrhythmics, analgesics and anti-convulsants. The Nav channels are composed of a pore-forming α subunit and associated β subunits. The β subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules. They modulate multiple aspects of Nav channel behaviour and play critical roles in controlling neuronal excitability. The recently published atomic resolution structures of the human β3 and β4 subunit Ig domains open a new chapter in the study of these molecules. In particular, the discovery that β3 subunits form trimers suggests that Nav channel oligomerization may contribute to the functional properties of some β subunits. PMID:25567098

  3. A new look at sodium channel β subunits

    PubMed Central

    Namadurai, Sivakumar; Yereddi, Nikitha R.; Cusdin, Fiona S.; Huang, Christopher L.-H.; Chirgadze, Dimitri Y.; Jackson, Antony P.

    2015-01-01

    Voltage-gated sodium (Nav) channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are a major focus of research in neurobiology, structural biology, membrane biology and pharmacology. Mutations in Nav channels are implicated in a wide variety of inherited pathologies, including cardiac conduction diseases, myotonic conditions, epilepsy and chronic pain syndromes. Drugs active against Nav channels are used as local anaesthetics, anti-arrhythmics, analgesics and anti-convulsants. The Nav channels are composed of a pore-forming α subunit and associated β subunits. The β subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules. They modulate multiple aspects of Nav channel behaviour and play critical roles in controlling neuronal excitability. The recently published atomic resolution structures of the human β3 and β4 subunit Ig domains open a new chapter in the study of these molecules. In particular, the discovery that β3 subunits form trimers suggests that Nav channel oligomerization may contribute to the functional properties of some β subunits. PMID:25567098

  4. 7 CFR 1c.111 - Criteria for IRB approval of research.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    .... (4) Informed consent will be sought from each prospective subject or the subject's legally authorized representative, in accordance with, and to the extent required by § 1c.116. (5) Informed consent will...

  5. 7 CFR 1c.111 - Criteria for IRB approval of research.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    .... (4) Informed consent will be sought from each prospective subject or the subject's legally authorized representative, in accordance with, and to the extent required by § 1c.116. (5) Informed consent will...

  6. 7 CFR 1c.111 - Criteria for IRB approval of research.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    .... (4) Informed consent will be sought from each prospective subject or the subject's legally authorized representative, in accordance with, and to the extent required by § 1c.116. (5) Informed consent will...

  7. 98. Catalog HHistory 1, C.C.C., 19 Tree Planting, Negative No. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    98. Catalog H-History 1, C.C.C., 19 Tree Planting, Negative No. P 474c (Photographer and date unknown) TRANSPLANTING TREE. - Skyline Drive, From Front Royal, VA to Rockfish Gap, VA , Luray, Page County, VA

  8. The Clinical Significance of HbA1c in Operable Chronic Thromboembolic Pulmonary Hypertension

    PubMed Central

    Richter, Manuel Jonas; Milger, Katrin; Haase, Sarah; Sommer, Natascha; Tello, Khodr; Seeger, Werner; Mayer, Eckhard; Wiedenroth, Christoph Benjamin; Grimminger, Friedrich; George, Wolfgang; Ghofrani, Hossein Ardeschir; Guth, Stefan; Gall, Henning

    2016-01-01

    Background Glycosylated hemoglobin A1c (HbA1c) has been proposed as an independent predictor of long-term prognosis in pulmonary arterial hypertension. However, the clinical relevance of HbA1c in patients with operable chronic thromboembolic pulmonary hypertension (CTEPH) remains unknown. The aim of the present study was to investigate the clinical significance of HbA1c as a biomarker in CTEPH. Methods Prospectively, 102 patients underwent pulmonary endarterectomy (PEA) in our national referral center between March 2013 and March 2014, of which after exclusion 45 patients were analyzed. HbA1c- levels, hemodynamic and exercise parameters were analyzed prior and one-year post-PEA. Results 45 patients (BMI: 27.3 ± 6.0 kg/m2; age: 62.7 ± 12.3 years) with a mean pulmonary arterial pressure (mPAP) of 43.6 ± 9.4 mmHg, a pulmonary vascular resistance (PVR) of 712.1 ± 520.4 dyn*s/cm5, a cardiac index (CI) of 2.4 ± 0.5 l/min/m2 and a mean HbA1c-level of 39.8 ± 5.6 mmol/mol were included. One-year post-PEA pulmonary hemodynamic and functional status significantly improved in our cohort. Baseline HbA1c-levels were significantly associated with CI, right atrial pressure, peak oxygen uptake and the change of 6-minute walking distance using linear regression analysis. However, using logistic regression analysis baseline HbA1c-levels were not significantly associated with residual post-PEA PH. Conclusions This is the first prospective study to describe an association of HbA1c-levels with pulmonary hemodynamics and exercise capacity in operable CTEPH patients. Our preliminary results indicate that in these patients impaired glucose metabolism as assessed by HbA1c is of clinical significance. However, HbA1c failed as a predictor of the hemodynamic outcome one-year post-PEA. PMID:27031508

  9. Epigenetic Characterization of CDKN1C in Placenta Samples from Non-syndromic Intrauterine Growth Restriction.

    PubMed

    López-Abad, Miriam; Iglesias-Platas, Isabel; Monk, David

    2016-01-01

    The cyclin-dependent kinase (CDK)-inhibitor 1C (CDKN1C) gene is expressed from the maternal allele and is located within the centromeric imprinted domain at chromosome 11p15. It is a negative regulator of proliferation, with loss-of-function mutations associated with the overgrowth disorder Beckwith-Wiedemann syndrome. Recently, gain-of-function mutations within the PCNA domain have been described in two disorders characterized by growth failure, namely IMAGe (intra-uterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital abnormalities) syndrome and Silver-Russell syndrome (SRS). Over-expression of CDKN1C by maternally inherited microduplications also results in SRS, suggesting that in addition to activating mutations this gene may regulate growth by changes in dosage. To determine if CDKN1C is involved in non-syndromic IUGR we compared the expression and DNA methylation levels in a large cohort of placental biopsies from IUGR and uneventful pregnancies. We observe higher levels of expression of CDKN1C in IUGR placentas compared to those of controls. All placenta biopsies heterozygous for the PAPA repeat sequence in exon 2 showed appropriate monoallelic expression and no mutations in the PCNA domain were observed. The expression profile was independent of both genetic or methylation variation in the minimal CDKN1C promoter interval and of methylation of the cis-acting maternally methylated region associated with the neighboring KCNQ1OT1 non-coding RNA. Chromatin immunoprecipitation revealed binding sites for CTCF within the unmethylated CDKN1C gene body CpG island and putative enhancer regions, associated with the canonical enhancer histone signature, H3K4me1 and H3K27ac, located ∼58 and 360 kb away. Using 3C-PCR we identify constitutive higher-order chromatin loops that occur between one of these putative enhancer regions and CDKN1C in human placenta tissues, which we propose facilitates expression. PMID:27200075

  10. Epigenetic Characterization of CDKN1C in Placenta Samples from Non-syndromic Intrauterine Growth Restriction

    PubMed Central

    López-Abad, Miriam; Iglesias-Platas, Isabel; Monk, David

    2016-01-01

    The cyclin-dependent kinase (CDK)-inhibitor 1C (CDKN1C) gene is expressed from the maternal allele and is located within the centromeric imprinted domain at chromosome 11p15. It is a negative regulator of proliferation, with loss-of-function mutations associated with the overgrowth disorder Beckwith–Wiedemann syndrome. Recently, gain-of-function mutations within the PCNA domain have been described in two disorders characterized by growth failure, namely IMAGe (intra-uterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital abnormalities) syndrome and Silver–Russell syndrome (SRS). Over-expression of CDKN1C by maternally inherited microduplications also results in SRS, suggesting that in addition to activating mutations this gene may regulate growth by changes in dosage. To determine if CDKN1C is involved in non-syndromic IUGR we compared the expression and DNA methylation levels in a large cohort of placental biopsies from IUGR and uneventful pregnancies. We observe higher levels of expression of CDKN1C in IUGR placentas compared to those of controls. All placenta biopsies heterozygous for the PAPA repeat sequence in exon 2 showed appropriate monoallelic expression and no mutations in the PCNA domain were observed. The expression profile was independent of both genetic or methylation variation in the minimal CDKN1C promoter interval and of methylation of the cis-acting maternally methylated region associated with the neighboring KCNQ1OT1 non-coding RNA. Chromatin immunoprecipitation revealed binding sites for CTCF within the unmethylated CDKN1C gene body CpG island and putative enhancer regions, associated with the canonical enhancer histone signature, H3K4me1 and H3K27ac, located ∼58 and 360 kb away. Using 3C-PCR we identify constitutive higher-order chromatin loops that occur between one of these putative enhancer regions and CDKN1C in human placenta tissues, which we propose facilitates expression. PMID:27200075

  11. Mutations in JMJD1C are involved in Rett syndrome and intellectual disability

    PubMed Central

    Sáez, Mauricio A.; Fernández-Rodríguez, Juana; Moutinho, Catia; Sanchez-Mut, Jose V.; Gomez, Antonio; Vidal, Enrique; Petazzi, Paolo; Szczesna, Karolina; Lopez-Serra, Paula; Lucariello, Mario; Lorden, Patricia; Delgado-Morales, Raul; de la Caridad, Olga J.; Huertas, Dori; Gelpí, Josep L.; Orozco, Modesto; López-Doriga, Adriana; Milà, Montserrat; Perez-Jurado, Luís A.; Pineda, Mercedes; Armstrong, Judith; Lázaro, Conxi; Esteller, Manel

    2016-01-01

    Purpose: Autism spectrum disorders are associated with defects in social response and communication that often occur in the context of intellectual disability. Rett syndrome is one example in which epilepsy, motor impairment, and motor disturbance may co-occur. Mutations in histone demethylases are known to occur in several of these syndromes. Herein, we aimed to identify whether mutations in the candidate histone demethylase JMJD1C (jumonji domain containing 1C) are implicated in these disorders. Genet Med 18 1, 378–385. Methods: We performed the mutational and functional analysis of JMJD1C in 215 cases of autism spectrum disorders, intellectual disability, and Rett syndrome without a known genetic defect. Genet Med 18 1, 378–385. Results: We found seven JMJD1C variants that were not present in any control sample (~ 6,000) and caused an amino acid change involving a different functional group. From these, two de novo JMJD1C germline mutations were identified in a case of Rett syndrome and in a patient with intellectual disability. The functional study of the JMJD1C mutant Rett syndrome patient demonstrated that the altered protein had abnormal subcellular localization, diminished activity to demethylate the DNA damage-response protein MDC1, and reduced binding to MECP2. We confirmed that JMJD1C protein is widely expressed in brain regions and that its depletion compromises dendritic activity. Genet Med 18 1, 378–385. Conclusions: Our findings indicate that mutations in JMJD1C contribute to the development of Rett syndrome and intellectual disability. Genet Med 18 1, 378–385. PMID:26181491

  12. Novel mutations in the USH1C gene in Usher syndrome patients

    PubMed Central

    Aparisi, María José; García-García, Gema; Jaijo, Teresa; Rodrigo, Regina; Graziano, Claudio; Seri, Marco; Simsek, Tulay; Simsek, Enver; Bernal, Sara; Baiget, Montserrat; Pérez-Garrigues, Herminio; Millán, José María

    2010-01-01

    Purpose Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by severe-profound sensorineural hearing loss, retinitis pigmentosa, and vestibular areflexia. To date, five USH1 genes have been identified. One of these genes is Usher syndrome 1C (USH1C), which encodes a protein, harmonin, containing PDZ domains. The aim of the present work was the mutation screening of the USH1C gene in a cohort of 33 Usher syndrome patients, to identify the genetic cause of the disease and to determine the relative involvement of this gene in USH1 pathogenesis in the Spanish population. Methods Thirty-three patients were screened for mutations in the USH1C gene by direct sequencing. Some had already been screened for mutations in the other known USH1 genes (myosin VIIA [MYO7A], cadherin-related 23 [CDH23], protocadherin-related 15 [PCDH15], and Usher syndrome 1G [USH1G]), but no mutation was found. Results Two novel mutations were found in the USH1C gene: a non-sense mutation (p.C224X) and a frame-shift mutation (p.D124TfsX7). These mutations were found in a homozygous state in two unrelated USH1 patients. Conclusions In the present study, we detected two novel pathogenic mutations in the USH1C gene. Our results suggest that mutations in USH1C are responsible for 1.5% of USH1 disease in patients of Spanish origin (considering the total cohort of 65 Spanish USH1 patients since 2005), indicating that USH1C is a rare form of USH in this population. PMID:21203349

  13. Characters of admissible representations of the affine superalgebra ŝl(2|1; C) k

    NASA Astrophysics Data System (ADS)

    Bowcock, P.; Hayes, M.; Taormina, A.

    1998-02-01

    We calculate characters and supercharacters for irreducible, admissible representations of the affine superalgebra ŝl(2|1; C) k in both the Ramond and Neveu-Schwarz sectors and discuss their modular properties in the special case of level k = - {1}/{2}. We also show that the non-degenerate integrable ŝl(2|1; C) k characters coincide with some N = 4 superconformal characters.

  14. Baicalin induces NAD(P)H:quinone reductase through the transactivation of AP-1 and NF-kappaB in Hepa 1c1c7 cells.

    PubMed

    Park, H J; Lee, Y W; Lee, S K

    2004-12-01

    Baicalin (5,6,7-trihydroxyflavone-7-O-D-glucuronic acid, BA) is a flavone isolated from Scutellariae radix. In our previous report BA was a major active principle of NAD(P)H:quinone reductase (QR) induction mediated by Scutellariae radix extract and the induction was related to the transcriptional activation of the QR gene in Hepa 1c1c7 cells. The primary aim of the present study was to determine the molecular mechanism of QR gene expression by baicalin. The antioxidant or electrophile response element (ARE/EpRE) found at the 5'-flanking region of phase II genes may play an important role in mediating their induction by xenobiotics, including chemopreventive agents. In accordance, to study the molecular mechanisms of QR gene expression by BA, electrophoretic mobility shift assay (EMSA), using nuclear extracts of treated and untreated cells against ARE, activator protein-1 (AP-1) or nuclear factor-kappaB (NF-kappaB) binding sites, showed that BA increased the binding levels of the parameters in a dose-dependent manner. Further, Hepa 1c1c7 cells were transiently transfected with a plasmid containing three copies of the AP-1- or NF-kappaB-binding site linked to a chloramphenicol acetyltransferase (CAT) reporter gene. Using the CAT reporter gene assay, a dose-dependent transactivation of AP-1- or NF-kappaB-mediated CAT expression was observed with the treatment of BA. These results clearly indicate that BA induces the QR gene expression and activity by transactivation of AP-1 and NF-kappaB, and thus BA may be considered as a potential cancer chemopreventive agent with the induction of phase II detoxification enzyme. PMID:15548947

  15. Significance of HbA1c Test in Diagnosis and Prognosis of Diabetic Patients

    PubMed Central

    Sherwani, Shariq I.; Khan, Haseeb A.; Ekhzaimy, Aishah; Masood, Afshan; Sakharkar, Meena K.

    2016-01-01

    Diabetes is a global endemic with rapidly increasing prevalence in both developing and developed countries. The American Diabetes Association has recommended glycated hemoglobin (HbA1c) as a possible substitute to fasting blood glucose for diagnosis of diabetes. HbA1c is an important indicator of long-term glycemic control with the ability to reflect the cumulative glycemic history of the preceding two to three months. HbA1c not only provides a reliable measure of chronic hyperglycemia but also correlates well with the risk of long-term diabetes complications. Elevated HbA1c has also been regarded as an independent risk factor for coronary heart disease and stroke in subjects with or without diabetes. The valuable information provided by a single HbA1c test has rendered it as a reliable biomarker for the diagnosis and prognosis of diabetes. This review highlights the role of HbA1c in diagnosis and prognosis of diabetes patients. PMID:27398023

  16. What is the Role of HbA1c in Diabetic Hemodialysis Patients?

    PubMed

    Coelho, Silvia

    2016-01-01

    The definition of a good glycemic control in patients with diabetes mellitus on hemodialysis is far from settled. In the general population, hemoglobin A1c is highly correlated with the average glycemia of the last 8-12 weeks. However, in hemodialysis patients, the correlation of hbA1c with glycemia is weaker as it also reflects changes in hemoglobin characteristics and red blood cells half-life. As expected, studies show that the association between HbA1c and outcomes in these patients differ from the general population. Therefore, the value of HbA1c in the treatment of hemodialysis patients has been questioned. Guidelines are generally cautious in their recommendations about possible targets of HbA1c in this population. Indeed, the risk of not treating hyperglycemia should be weighed against the particularly high risk of precipitating hypoglycemia in dialysis patients. In this review, a critical analysis of the current role of HbA1c in the care of hemodialysis patients is presented. PMID:26138753

  17. A Phytophthora infestans RXLR effector targets plant PP1c isoforms that promote late blight disease

    PubMed Central

    Boevink, Petra C.; Wang, Xiaodan; McLellan, Hazel; He, Qin; Naqvi, Shaista; Armstrong, Miles R.; Zhang, Wei; Hein, Ingo; Gilroy, Eleanor M.; Tian, Zhendong; Birch, Paul R. J.

    2016-01-01

    Plant pathogens deliver effectors to alter host processes. Knowledge of how effectors target and manipulate host proteins is critical to understand crop disease. Here, we show that in planta expression of the RXLR effector Pi04314 enhances leaf colonization by Phytophthora infestans via activity in the host nucleus and attenuates induction of jasmonic and salicylic acid-responsive genes. Pi04314 interacts with three host protein phosphatase 1 catalytic (PP1c) isoforms, causing their re-localization from the nucleolus to the nucleoplasm. Re-localization of PP1c-1 also occurs during infection and is dependent on an R/KVxF motif in the effector. Silencing the PP1c isoforms or overexpression of a phosphatase-dead PP1c-1 mutant attenuates infection, demonstrating that host PP1c activity is required for disease. Moreover, expression of PP1c–1mut abolishes enhanced leaf colonization mediated by in planta Pi04314 expression. We argue that PP1c isoforms are susceptibility factors forming holoenzymes with Pi04314 to promote late blight disease. PMID:26822079

  18. Significance of HbA1c Test in Diagnosis and Prognosis of Diabetic Patients.

    PubMed

    Sherwani, Shariq I; Khan, Haseeb A; Ekhzaimy, Aishah; Masood, Afshan; Sakharkar, Meena K

    2016-01-01

    Diabetes is a global endemic with rapidly increasing prevalence in both developing and developed countries. The American Diabetes Association has recommended glycated hemoglobin (HbA1c) as a possible substitute to fasting blood glucose for diagnosis of diabetes. HbA1c is an important indicator of long-term glycemic control with the ability to reflect the cumulative glycemic history of the preceding two to three months. HbA1c not only provides a reliable measure of chronic hyperglycemia but also correlates well with the risk of long-term diabetes complications. Elevated HbA1c has also been regarded as an independent risk factor for coronary heart disease and stroke in subjects with or without diabetes. The valuable information provided by a single HbA1c test has rendered it as a reliable biomarker for the diagnosis and prognosis of diabetes. This review highlights the role of HbA1c in diagnosis and prognosis of diabetes patients. PMID:27398023

  19. A bioinformatic and computational study of myosin phosphatase subunit diversity

    PubMed Central

    Dippold, Rachael P.

    2014-01-01

    Variability in myosin phosphatase (MP) subunits may provide specificity in signaling pathways that regulate muscle tone. We utilized public databases and computational algorithms to investigate the phylogenetic diversity of MP regulatory (PPP1R12A-C) and inhibitory (PPP1R14A-D) subunits. The comparison of exonic coding sequences and expression data confirmed or refuted the existence of isoforms and their tissue-specific expression in different model organisms. The comparison of intronic and exonic sequences identified potential expressional regulatory elements. As examples, smooth muscle MP regulatory subunit (PPP1R12A) is highly conserved through evolution. Its alternative exon E24 is present in fish through mammals with two invariant features: 1) a reading frame shift generating a premature termination codon and 2) a hexanucleotide sequence adjacent to the 3′ splice site hypothesized to be a novel suppressor of exon splicing. A characteristic of the striated muscle MP regulatory subunit (PPP1R12B) locus is numerous and phylogenetically variable transcriptional start sites. In fish this locus only codes for the small (M21) subunit, suggesting the primordial function of this gene. Inhibitory subunits show little intragenic variability; their diversity is thought to have arisen by expansion and tissue-specific expression of different gene family members. We demonstrate differences in the regulatory landscape between smooth muscle enriched (PPP1R14A) and more ubiquitously expressed (PPP1R14B) family members and identify deeply conserved intronic sequence and predicted transcriptional cis-regulatory elements. This bioinformatic and computational study has uncovered a number of attributes of MP subunits that supports selection of ideal model organisms and testing of hypotheses regarding their physiological significance and regulated expression. PMID:24898838

  20. Functional diversity of complex I subunits in Candida albicans mitochondria.

    PubMed

    Li, Dongmei; She, Xiaodong; Calderone, Richard

    2016-02-01

    Our interest in the mitochondria of Candida albicans has progressed to the identification of several proteins that are critical to complex I (CI) activity. We speculated that there should be major functional differences at the protein level between mammalian and fungal mitochondria CI. In our pursuit of this idea, we were helped by published data of CI subunit proteins from a broad diversity of species that included two subunit proteins that are not found in mammals. These subunit proteins have been designated as Nuo1p and Nuo2p (NADH-ubiquinone oxidoreductases). Since functional assignments of both C. albicans proteins were unknown, other than having a putative NADH-oxidoreductase activity, we constructed knock-out strains that could be compared to parental cells. The relevance of our research relates to the critical roles of both proteins in cell biology and pathogenesis and their absence in mammals. These features suggest they may be exploited in antifungal drug discovery. Initially, we characterized Goa1p that apparently regulates CI activity but is not a CI subunit protein. We have used the goa1∆ for comparisons to Nuo1p and Nuo2p. We have demonstrated the critical role of these proteins in maintaining CI activities, virulence, and prolonging life span. More recently, transcriptional profiling of the three mutants and an ndh51∆ (protein is a highly conserved CI subunit) has revealed that there are overlapping yet also different functional assignments that suggest subunit specificity. The differences and similarities of each are described below along with our hypotheses to explain these data. Our conclusion and perspective is that the C. albicans CI subunit proteins are highly conserved except for two that define non-mammalian functions. PMID:26373419

  1. Cytochrome c oxidase: Evolution of control via nuclear subunit addition☆

    PubMed Central

    Pierron, Denis; Wildman, Derek E.; Hüttemann, Maik; Markondapatnaikuni, Gopi Chand; Aras, Siddhesh; Grossman, Lawrence I.

    2014-01-01

    According to theory, present eukaryotic cells originated from a beneficial association between two free-living cells. Due to this endosymbiotic event the pre-eukaryotic cell gained access to oxidative phosphorylation (OXPHOS), which produces more than 15 times as much ATP as glycolysis. Because cellular ATP needs fluctuate and OXPHOS both requires and produces entities that can be toxic for eukaryotic cells such as ROS or NADH, we propose that the success of endosymbiosis has largely depended on the regulation of endosymbiont OXPHOS. Several studies have presented cytochrome c oxidase as a key regulator of OXPHOS; for example, COX is the only complex of mammalian OXPHOS with known tissue-specific isoforms of nuclear encoded subunits. We here discuss current knowledge about the origin of nuclear encoded subunits and the appearance of different isozymes promoted by tissue and cellular environments such as hypoxia. We also review evidence for recent selective pressure acting on COX among vertebrates, particularly in primate lineages, and discuss the unique pattern of co-evolution between the nuclear and mitochondrial genomes. Finally, even though the addition of nuclear encoded subunits was a major event in eukaryotic COX evolution, this does not lead to emergence of a more efficient COX, as might be expected from an anthropocentric point of view, for the “higher” organism possessing large brains and muscles. The main function of these subunits appears to be “only” to control the activity of the mitochondrial subunits. We propose that this control function is an as yet underappreciated key point of evolution. Moreover, the importance of regulating energy supply may have caused the addition of subunits encoded by the nucleus in a process comparable to a “domestication scenario” such that the host tends to control more and more tightly the ancestral activity of COX performed by the mtDNA encoded subunits. This article is part of a Special Issue entitled

  2. Cytochrome c oxidase: evolution of control via nuclear subunit addition.

    PubMed

    Pierron, Denis; Wildman, Derek E; Hüttemann, Maik; Markondapatnaikuni, Gopi Chand; Aras, Siddhesh; Grossman, Lawrence I

    2012-04-01

    According to theory, present eukaryotic cells originated from a beneficial association between two free-living cells. Due to this endosymbiotic event the pre-eukaryotic cell gained access to oxidative phosphorylation (OXPHOS), which produces more than 15 times as much ATP as glycolysis. Because cellular ATP needs fluctuate and OXPHOS both requires and produces entities that can be toxic for eukaryotic cells such as ROS or NADH, we propose that the success of endosymbiosis has largely depended on the regulation of endosymbiont OXPHOS. Several studies have presented cytochrome c oxidase as a key regulator of OXPHOS; for example, COX is the only complex of mammalian OXPHOS with known tissue-specific isoforms of nuclear encoded subunits. We here discuss current knowledge about the origin of nuclear encoded subunits and the appearance of different isozymes promoted by tissue and cellular environments such as hypoxia. We also review evidence for recent selective pressure acting on COX among vertebrates, particularly in primate lineages, and discuss the unique pattern of co-evolution between the nuclear and mitochondrial genomes. Finally, even though the addition of nuclear encoded subunits was a major event in eukaryotic COX evolution, this does not lead to emergence of a more efficient COX, as might be expected from an anthropocentric point of view, for the "higher" organism possessing large brains and muscles. The main function of these subunits appears to be "only" to control the activity of the mitochondrial subunits. We propose that this control function is an as yet under appreciated key point of evolution. Moreover, the importance of regulating energy supply may have caused the addition of subunits encoded by the nucleus in a process comparable to a "domestication scenario" such that the host tends to control more and more tightly the ancestral activity of COX performed by the mtDNA encoded subunits. PMID:21802404

  3. Neue biosensorische Prinzipien für die Hämoglobin-A1c Bestimmung

    NASA Astrophysics Data System (ADS)

    Stöllner, Daniela

    2002-06-01

    Hämoglobin-A1c (HbA1c) ist ein Hämoglobin (Hb)-Subtypus, der durch nicht-enzymatische Glykierung des N-terminalen Valinrestes der Hämoglobin-beta-Kette entsteht. Das gemessene Verhältnis von HbA1c zum Gesamt-Hämoglobin (5-20 % bei Diabetikern) repräsentiert den Mittelwert der Blutglucosekonzentration über einen zweimonatigen Zeitraum und stellt zur Beurteilung der diabetischen Stoffwechsellage eine Ergänzung zur Akutkontrolle der Glukosekonzentration dar. Ziel der vorliegenden Arbeit war es, einen amperometrischen Biosensor für die Bestimmung des medizinisch relevanten Parameters HbA1c zu entwickeln. Durch Selektion geeigneter Bioerkennungselemente und deren Immobilisierung unter Erhalt der Bindungsfunktion für die Zielmoleküle Hämoglobin bzw. HbA1c wurden spezifische, hochaffine und regenerationsstabile Sensoroberflächen geschaffen. Für die Entwicklung des HbA1c-Biosensors wurden zwei Konzepte - Enzymsensor und Immunosensor - miteinander verglichen. Die enzymatische Umsetzung von HbA1c erfolgte mit der Fructosylamin Oxidase (FAO) aus Pichia pastoris N 1-1 unter Freisetzung von H2O2, welches sowohl optisch über eine Indikatorreaktion als auch elektrochemisch nach Einschluss der FAO in PVA-SbQ und Fixierung des Immobilisats vor einer H2O2-Elektrode nachgewiesen wurde. Die Kalibration des Enzymsensors mit der HbA1c-Modellsubstanz Fructosyl-Valin ergab Nachweisgrenzen, die ausserhalb des physiologisch relevanten HbA1c-Konzentrationsbereich lagen. Aus der Umsetzung von glykierten Peptiden mit einer nicht HbA1c analogen Aminosäurensequenz, z.B. Fructosyl-Valin-Glycin wurde zudem eine geringe HbA1c-Spezifität abgeleitet. Für den Immunosensor wurden zwei heterogene Immunoassay-Formate unter Verwendung von hochaffinen und spezifischen Antikörpern in Kombination mit Glucose Oxidase (GOD) als Markerenzym zum Nachweis von HbA1c untersucht. Beim indirekt-kompetitiven Immunoassay wurde anstelle des kompletten HbA1c-Moleküls das glykierte Pentapeptid

  4. Challenges in HbA1c Analysis and Reporting in Patients with Variant Hemoglobins.

    PubMed

    Sultana, T A; Sheme, Z A; Sultana, G S; Sultana, B; Mishu, F A; Khan, N Z; Sarkar, B C; Muttalib, M A; Khan, S A; Choudhury, S; Mahtab, H

    2016-04-01

    Hemoglobin A1c (HbA(1)c) is a well-established indicator of mean glycemia. The presence of genetic variants of hemoglobin can profoundly affect the accuracy of HbA(1)c measurements. Variants of hemoglobin especially Hemoglobin E (HbE) is prevalent in South East Asia including Bangladesh. The objective of our study is to compare the HbA(1)c values measured on high performance liquid chromatography (HPLC) and Turbidimetric Inhibition Immunoassay (TINIA) in diabetic patients with variant hemoglobins including HbE. A total of 7595 diabetic patients receiving treatment at BIRDEM General Hospital were analyzed for HbA(1)c results within a period of two months from December 2013 to January 2014. Seventy two cases out of 7595 (0.95%) had either undetectable or below normal HbA(1)c levels (males-33 and females-39; ratio = 0.82:1) by HPLC method. In 34(0.45%) cases, HbA(1)c value was undetectable by HPLC method but was in the reportable range by TINIA method. In the other 38 (0.55%) cases, HbA(1)c levels were below the reportable range (<4%) by HPLC method but were in the normal or higher range by TINIA method. TINIA method did not agree with HPLC method on Bland Altman plot in the 38 cases with below normal HbA(1)c levels, [Mean bias -5.2(-9.3 to 1.0), 95% CI] but agreed very well [mean bias -0.21 (-0.84 to 0.42), y=1.1037+0.776X; r(2)=0.30, p<0.01] in controls. In control group mean MCV was 83.80±7.48 and in study group was 73.65±10.44. Alkaline electrophoresis confirmed the variant hemoglobin to be HbE. The fasting blood sugar levels of all the 72 cases correlated strongly with TINIA method (r(2) =0.75, p<0.0001) but not with HPLC (r = 0.24, p=0.13). In our regions where populations have a high prevalence of Hb variant, proper knowledge of hemoglobin variants which affect the measurements HbA(1)c level is essential. MCV of 80fl or below may serve as a rough guide to select samples that require analysis by TINIA method. Moreover, HPLC may be a convenient and inexpensive

  5. HbA1c Test as a Tool in the Diagnosis of Gestational Diabetes Mellitus

    PubMed Central

    Renz, Paula Breitenbach; Cavagnolli, Gabriela; Weinert, Letícia Schwerz; Silveiro, Sandra Pinho; Camargo, Joíza Lins

    2015-01-01

    Aims Gestational diabetes mellitus (GDM) is a prevalent and potentially serious condition which may put both mothers and neonates at risk. The current recommendation for diagnosis is the oral glucose tolerance test (OGTT). This study aimed to determine the usefulness of HbA1c test as a diagnostic tool for GDM as compared to the traditional criteria based on the OGTT. Methods This was a diagnostic test accuracy study. We performed OGTT and HbA1c test in women attending prenatal visits at a tertiary hospital. GDM was defined according to WHO1999 or ADA/WHO 2013 criteria. ROC curve was used to evaluate the diagnostic performance of HbA1c. Sensitivity, specificity and likelihood ratios for different HbA1c cut-off points were calculated. Results Of the 262 women in the third trimester of gestation enrolled in the study, 86 (33%) were diagnosed with GDM. Only five of these women presented HbA1c ≥48 mmol/mol (6.5%). This cut-off point presented 100% specificity but very low sensitivity (7%). Based on ROC curve, and considering OGTT as the reference criterion, HbA1c ≥40 mmol/mol (5.8%) showed adequate specificity in diagnosing GDM (94.9%) but low sensitivity (26.4%). Unlike, HbA1c values of 31 mmol/mol (5.0%) presented adequate sensitivity (89.7%) but low specificity (32.6%) to detect GDM. For women with HbA1c ≥40 mmol/mol (5.8%), the positive and negative likelihood ratios were 5.14 (95%CI 2.49–10.63) and 0.78 (0.68–0.88), respectively. The post-test probability of GDM was about 40%, representing a 4.0-fold increase in the mean pre-test probability. This cut-off point could eliminate the need for the unpleasant and laborious OGTT tests in almost one third of cases, as 38% of patients with GDM may be diagnosable by HbA1c test alone. Conclusions Our results show that combined HbA1c and OGTT measurements may be useful in diagnosing GDM. PMID:26292213

  6. The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

    PubMed Central

    Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

    2012-01-01

    The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

  7. Intracrine androgens and AKR1C3 activation confer resistance to enzalutamide in prostate cancer

    PubMed Central

    Liu, Chengfei; Lou, Wei; Zhu, Yezi; Yang, Joy C.; Nadiminty, Nagalakshmi; Gaikwad, Nilesh W.; Evans, Christopher P.; Gao, Allen C.

    2015-01-01

    The introduction of enzalutamide and abiraterone has led to improvement in the treatment of metastatic castration-resistant prostate cancer (mCRPC). However, acquired resistance to enzalutamide and abiraterone therapies frequently develops within a short period in many patients. In the present study, we developed enzalutamide resistant prostate cancer cells in an effort to understand the mechanisms of resistance. Global gene expression analysis showed that steroid biosynthesis pathway is activated in enzalutamide resistant prostate cancer cells. One of the crucial steroidogenic enzymes, AKR1C3, was significantly elevated in enzalutamide resistant cells. In addition, AKR1C3 is highly expressed in metastatic and recurrent prostate cancer and in enzalutamide resistant prostate xenograft tumors. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis of the steroid metabolites revealed that androgen precursors such as cholesterol, DHEA and progesterone, as well as androgens are highly up regulated in enzalutamide resistant prostate cancer cells compared to the parental cells. Knock down of AKR1C3 expression by shRNA or inhibition of AKR1C3 enzymatic activity by indomethacin resensitized enzalutamide resistant prostate cancer cells to enzalutamide treatment both in vitro and in vivo. In contrast, overexpression of AKR1C3 confers resistance to enzalutamide. Furthermore, the combination of indomethacin and enzalutamide resulted in significant inhibition of enzalutamide-resistant tumor growth. These results suggest that AKR1C3 activation is a critical resistance mechanism associated with enzalutamide resistance, targeting intracrine androgens and AKR1C3 will overcome enzalutamide resistance and improve survival of advanced prostate cancer patients. PMID:25649766

  8. Intracrine Androgens and AKR1C3 Activation Confer Resistance to Enzalutamide in Prostate Cancer.

    PubMed

    Liu, Chengfei; Lou, Wei; Zhu, Yezi; Yang, Joy C; Nadiminty, Nagalakshmi; Gaikwad, Nilesh W; Evans, Christopher P; Gao, Allen C

    2015-04-01

    The introduction of enzalutamide and abiraterone has led to improvement in the treatment of metastatic castration-resistant prostate cancer. However, acquired resistance to enzalutamide and abiraterone therapies frequently develops within a short period in many patients. In the present study, we developed enzalutamide-resistant prostate cancer cells in an effort to understand the mechanisms of resistance. Global gene-expression analysis showed that the steroid biosynthesis pathway is activated in enzalutamide-resistant prostate cancer cells. One of the crucial steroidogenic enzymes, AKR1C3, was significantly elevated in enzalutamide-resistant cells. In addition, AKR1C3 is highly expressed in metastatic and recurrent prostate cancer and in enzalutamide-resistant prostate xenograft tumors. LC/MS analysis of the steroid metabolites revealed that androgen precursors such as cholesterol, DHEA and progesterone, as well as androgens are highly upregulated in enzalutamide-resistant prostate cancer cells compared to the parental cells. Knockdown of AKR1C3 expression by shRNA or inhibition of AKR1C3 enzymatic activity by indomethacin resensitized enzalutamide-resistant prostate cancer cells to enzalutamide treatment both in vitro and in vivo. In contrast, overexpression of AKR1C3 confers resistance to enzalutamide. Furthermore, the combination of indomethacin and enzalutamide resulted in significant inhibition of enzalutamide-resistant tumor growth. These results suggest that AKR1C3 activation is a critical resistance mechanism associated with enzalutamide resistance; targeting intracrine androgens and AKR1C3 will overcome enzalutamide resistance and improve survival of advanced prostate cancer patients. PMID:25649766

  9. Characterization, solubilization and partial purification of serotonin 5-HT1C receptors

    SciTech Connect

    Yagaloff, K.A.

    1986-01-01

    /sup 125/I-Lysergic acid diethylamide (/sup 125/I-LSD) binds with high affinity to a unique serotonergic site on rat choroid plexus. These sites were localized to choroid plexus epithelial cells using a novel high resolution autoradiographic technique. In membrane preparations, the serotonergic site density was 3100 fmol/mg protein, which is 10 fold higher than the density of any other serotonergic site in brain homogenates. The pharmacology of this site, termed the 5-HT1c site, does not match that of 5-Ht1a, 5-HT1b or 5HT2 serotonergic sites. 5-Ht1c sites were solubilized from pig choroid plexus using the zwitterionic detergent, CHAPS. High affinity labelling of the solubilized site was obtained using the serotonergic radioligand, N1-methyl-2-(/sup 125/I)lysergic acid diethylamide (/sup 125/I-MIL). Choroid plexus tumors obtained from transgenic mice were examined for the presence of serotonin 5-HT1c receptors. /sup 125/I-LSD binding to choroid plexus tumors displays a pharmacological profile that matches the properties of 5-HT1c receptors in normal choroid plexus. The tumor exhibits the highest site density of serotonin receptors (6600 fmol/mg protein) found in any tissue. /sup 125/I-LSD autoradiography of brain sections from transgenic mice shows high levels of specific labelling over the tumor. The affinities of various indolealkyl, phenlakyl and beta-carboline derivatives for the serotonin 5-HT1c receptor were measured in pig choroid plexus using /sup 125/I-MIL. Serotonin precursors and metabolites were all very weak inhibitors of specific /sup 125/I-MIL binding. Structure-affinity relationships were determined for a number of indolealkylamine analogues. Only serotonin is present in cerebrospinal fluid at concentrations near its 5-HT1c inhibition constant, suggesting that serotonin is the natural 5-HT1c agonist.

  10. Progress towards development of a cholera subunit vaccine.

    PubMed

    Taylor, Ronald K; Kirn, Thomas J; Bose, Niranjan; Stonehouse, Emily; Tripathi, Shital A; Kovác, Pavol; Wade, William F

    2004-07-01

    Cholera, an enteric disease that can reach pandemic proportions, remains a world-wide problem that is positioned to increase in incidence as changes in global climate or armed conflict spawn the conditions that enhance transmission to humans and, thus, precipitate epidemic cholera. An effective subunit cholera vaccine that can provide protective immunity with one parenteral immunization would be a major advantage over the existing oral vaccines that can require two doses for optimal protection. The existing vaccines are clearly effective in some settings, but are less so in others, especially with respect to specific groups such as young (2-5 years) children. In our efforts to develop a cholera subunit vaccine, we focused on two Vibrio cholerae antigens, LPS (lipopolysaccharide) and TCP (toxin co-regulated pilus), that are known to induce protective antibodies in animal models and, in the case of anti-LPS antibodies, to be associated with clinical protection of V. cholerae exposed or vaccinated individuals. This review discusses the current cholera vaccines and compares the advantages of a cholera subunit vaccine to that of the whole cell vaccines. We discuss the possible subunit antigens and prospective targeted use of a subunit cholera vaccine. PMID:17191897

  11. Neue biosensorische Prinzipien für die Hämoglobin-A1c Bestimmung

    NASA Astrophysics Data System (ADS)

    Stöllner, Daniela

    2002-06-01

    Hämoglobin-A1c (HbA1c) ist ein Hämoglobin (Hb)-Subtypus, der durch nicht-enzymatische Glykierung des N-terminalen Valinrestes der Hämoglobin-beta-Kette entsteht. Das gemessene Verhältnis von HbA1c zum Gesamt-Hämoglobin (5-20 % bei Diabetikern) repräsentiert den Mittelwert der Blutglucosekonzentration über einen zweimonatigen Zeitraum und stellt zur Beurteilung der diabetischen Stoffwechsellage eine Ergänzung zur Akutkontrolle der Glukosekonzentration dar. Ziel der vorliegenden Arbeit war es, einen amperometrischen Biosensor für die Bestimmung des medizinisch relevanten Parameters HbA1c zu entwickeln. Durch Selektion geeigneter Bioerkennungselemente und deren Immobilisierung unter Erhalt der Bindungsfunktion für die Zielmoleküle Hämoglobin bzw. HbA1c wurden spezifische, hochaffine und regenerationsstabile Sensoroberflächen geschaffen. Für die Entwicklung des HbA1c-Biosensors wurden zwei Konzepte - Enzymsensor und Immunosensor - miteinander verglichen. Die enzymatische Umsetzung von HbA1c erfolgte mit der Fructosylamin Oxidase (FAO) aus Pichia pastoris N 1-1 unter Freisetzung von H2O2, welches sowohl optisch über eine Indikatorreaktion als auch elektrochemisch nach Einschluss der FAO in PVA-SbQ und Fixierung des Immobilisats vor einer H2O2-Elektrode nachgewiesen wurde. Die Kalibration des Enzymsensors mit der HbA1c-Modellsubstanz Fructosyl-Valin ergab Nachweisgrenzen, die ausserhalb des physiologisch relevanten HbA1c-Konzentrationsbereich lagen. Aus der Umsetzung von glykierten Peptiden mit einer nicht HbA1c analogen Aminosäurensequenz, z.B. Fructosyl-Valin-Glycin wurde zudem eine geringe HbA1c-Spezifität abgeleitet. Für den Immunosensor wurden zwei heterogene Immunoassay-Formate unter Verwendung von hochaffinen und spezifischen Antikörpern in Kombination mit Glucose Oxidase (GOD) als Markerenzym zum Nachweis von HbA1c untersucht. Beim indirekt-kompetitiven Immunoassay wurde anstelle des kompletten HbA1c-Moleküls das glykierte Pentapeptid

  12. A Novel Pentapeptide Targeting Integrin β3-Subunit Inhibits Platelet Aggregation and Its Application in Rat for Thrombosis Prevention

    PubMed Central

    Qu, Qingrong; Liu, Yamin; Yan, Xuejiao; Fan, Xiaobo; Liu, Naifeng; Wu, Guoqiu

    2016-01-01

    Background: Antiplatelet therapy plays a pivotal role in the prevention and treatment of thrombotic diseases. We reported the screening of P1C as a novel integrin-binding peptide from the C-terminal of connective tissue growth factor. Primary study indicated that P1C has potential against platelet aggregation. Objectives: We aimed to find the shortest active unit from the P1C fragments and explore its in vivo and in vitro activities. Methods: A series of truncated P1C fragments was prepared and screened for antiplatelet activity. The most active fragment was evaluated using coagulation assays. Flow cytometry and confocal microscopy were used to determine the interaction between the peptide and the integrin. The in vivo potential was further explored using two types of rat models. Results: From a series of truncated P1C forms, a so-called P1Cm peptide of 5-amino acids, namely, IRTPK was screened out as the shortest active unit with superior activity. Coagulation experiments and an in vivo toxicity assay demonstrated that P1Cm is safe in vivo and inhibits ADP- and TH-induced human platelet aggregation in vitro in a concentration-dependent manner. Furthermore, it has limited effect on the coagulation parameters. Flow cytometry and confocal microscopy experiments consistently indicated that the peptide specifically binds the β3-subunit of integrin on platelets. Further experiments using rat models of artery-vein shunt and carotid arterial thrombosis illustrated that P1Cm can effectively prevent thrombosis formation. Conclusion: P1Cm may be a new, promising antithrombotic alternative to currently available antiplatelet treatments. PMID:27014063

  13. Ammonia oxidation is not required for growth of Group 1.1c soil Thaumarchaeota.

    PubMed

    Weber, Eva B; Lehtovirta-Morley, Laura E; Prosser, James I; Gubry-Rangin, Cécile

    2015-03-01

    Thaumarchaeota are among the most abundant organisms on Earth and are ubiquitous. Within this phylum, all cultivated representatives of Group 1.1a and Group 1.1b Thaumarchaeota are ammonia oxidizers, and play a key role in the nitrogen cycle. While Group 1.1c is phylogenetically closely related to the ammonia-oxidizing Thaumarchaeota and is abundant in acidic forest soils, nothing is known about its physiology or ecosystem function. The goal of this study was to perform in situ physiological characterization of Group 1.1c Thaumarchaeota by determining conditions that favour their growth in soil. Several acidic grassland, birch and pine tree forest soils were sampled and those with the highest Group 1.1c 16S rRNA gene abundance were incubated in microcosms to determine optimal growth temperature, ammonia oxidation and growth on several organic compounds. Growth of Group 1.1c Thaumarchaeota, assessed by qPCR of Group 1.1c 16S rRNA genes, occurred in soil, optimally at 30°C, but was not associated with ammonia oxidation and the functional gene amoA could not be detected. Growth was also stimulated by addition of organic nitrogen compounds (glutamate and casamino acids) but not when supplemented with organic carbon alone. This is the first evidence for non-ammonia oxidation associated growth of Thaumarchaeota in soil. PMID:25764563

  14. Low HbA1c and Increased Mortality Risk-is Frailty a Confounding Factor?

    PubMed Central

    Abdelhafiz, Ahmed H; Sinclair, Alan J

    2015-01-01

    Diabetes mellitus is increasingly becoming an older person disease due to the increased survival and aging of the population. Previous studies which showed benefits of tight glycemic control and a linear relationship between HbA1c and mortality have largely included younger patients newly diagnosed with diabetes and with less comorbidities. Recent studies, which included older population with diabetes, have shown a U-shaped relationship of increased mortality associated with low HbA1c. The mechanism of such relationship is unclear. There was no direct causal link between low HbA1c and mortality. It appears that malnutrition, inflammation and functional decline are characteristics shared by the populations that showed increased mortality and low HbA1c. In these studies functional status, disability or frailty was not routinely measured. Therefore, although adjustment for comorbidities was made there may be a residual confounding by unmeasured factors such as frailty. Thus, frailty or decline in functional reserve may be the main confounding factor explaining the relationship between increased mortality risk and low HbA1c. PMID:26236548

  15. Dyslexia and DYX1C1: deficits in reading and spelling associated with a missense mutation.

    PubMed

    Bates, T C; Lind, P A; Luciano, M; Montgomery, G W; Martin, N G; Wright, M J

    2010-12-01

    The status of DYX1C1 (C15q21.3) as a susceptibility gene for dyslexia is unclear. We report the association of this gene with reading and spelling ability in a sample of adolescent twins and their siblings. Family-based association analyses were carried out on 13 single-nucleotide polymorphisms (SNPs) in DYX1C1, typed in 790 families with up to 5 offspring and tested on 6 validated measures of lexical processing (irregular word) and grapheme-phoneme decoding (pseudo-word) reading- and spelling-based measures of dyslexia, as well as a short-term memory measure. Significant association was observed at the misssense mutation rs17819126 for all reading measures and for spelling of lexical processing words, and at rs3743204 for both irregular and nonword reading. Verbal short-term memory was associated with rs685935. Support for association was not found at rs3743205 and rs61761345 as previously reported by Taipale et al., but these SNPs had very low (0.002 for rs3743205) minor allele frequencies in this sample. These results suggest that DYX1C1 influences reading and spelling ability with additional effects on short-term information storage or rehearsal. Missense mutation rs17819126 is a potential functional basis for the association of DYX1C1 with dyslexia. PMID:19901951

  16. Human autoantibodies specific for the α1A calcium channel subunit reduce both P-type and Q-type calcium currents in cerebellar neurons

    PubMed Central

    Pinto, Ashwin; Gillard, Samantha; Moss, Fraser; Whyte, Kathryn; Brust, Paul; Williams, Mark; Stauderman, Ken; Harpold, Michael; Lang, Bethan; Newsom-Davis, John; Bleakman, David; Lodge, David; Boot, John

    1998-01-01

    The pharmacological properties of voltage-dependent calcium channel (VDCC) subtypes appear mainly to be determined by the α1 pore-forming subunit but, whether P-and Q-type VDCCs are encoded by the same α1 gene presently is unresolved. To investigate this, we used IgG antibodies to presynaptic VDCCs at motor nerve terminals that underlie muscle weakness in the autoimmune Lambert–Eaton myasthenic syndrome (LEMS). We first studied their action on changes in intracellular free Ca2+ concentration [Ca2+]i in human embryonic kidney (HEK293) cell lines expressing different combinations of human recombinant VDCC subunits. Incubation for 18 h with LEMS IgG (2 mg/ml) caused a significant dose-dependent reduction in the K+-stimulated [Ca2+]i increase in the α1A cell line but not in the α1B, α1C, α1D, and α1E cell lines, establishing the α1A subunit as the target for these autoantibodies. Exploiting this specificity, we incubated cultured rat cerebellar neurones with LEMS IgG and observed a reduction in P-type current in Purkinje cells and both P- and Q-type currents in granule cells. These data are consistent with the hypothesis that the α1A gene encodes for the pore-forming subunit of both P-type and Q-type VDCCs. PMID:9653186

  17. Dengue vaccine: an update on recombinant subunit strategies.

    PubMed

    Martin, J; Hermida, L

    2016-03-01

    Dengue is an increasing public health problem worldwide, with the four serotypes of the virus infecting over 390 million people annually. There is no specific treatment or antiviral drug for dengue, and prevention is largely limited to controlling the mosquito vectors or disrupting the human-vector contact. Despite the considerable progress made in recent years, an effective vaccine against the virus is not yet available. The development of a dengue vaccine has been hampered by many unique challenges, including the need to ensure the absence of vaccine-induced enhanced severity of disease. Recombinant protein subunit vaccines offer a safer alternative to other vaccine approaches. Several subunit vaccine candidates are presently under development, based on different structural and non-structural proteins of the virus. Novel adjuvants or immunopotentiating strategies are also being tested to improve their immunogenicity. This review summarizes the current status and development trends of subunit dengue vaccines. PMID:26982462

  18. Functional biosynthesis of an allophycocyan beta subunit in Escherichia coli.

    PubMed

    Ge, Baosheng; Sun, Haixiang; Feng, Yang; Yang, Jinying; Qin, Song

    2009-03-01

    Allophycocyanin is a phycobiliprotein with various biological and pharmacological properties. An expression vector was constructed using CpeS as the bilin lyase for the allophycocyanin beta subunit, resulting in overexpression of a fluorescent allophycocyanin beta-subunit in Escherichia coli. A high-density cell culture was developed using a continuous feeding strategy. After 16 h of culture, the dry cell density reached 21.4 g l(-1), the expression of the allophycocyanin beta-subunit was 0.86 g l(-1) broth, and the relative chromoprotein yield was 81.4%. The recombinant protein showed spectral features similar to native allophycocyanin, which provide an efficient methodology for large-scale production of this valuable fluorescent protein. PMID:19269586

  19. Cholera Toxin B: One Subunit with Many Pharmaceutical Applications

    PubMed Central

    Baldauf, Keegan J.; Royal, Joshua M.; Hamorsky, Krystal Teasley; Matoba, Nobuyuki

    2015-01-01

    Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT), which consists of two subunits: the A subunit (CTA) and the B subunit (CTB). CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction. PMID:25802972

  20. 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

  1. Stress and A1c Among People with Diabetes Across the Lifespan.

    PubMed

    Hilliard, Marisa E; Yi-Frazier, Joyce P; Hessler, Danielle; Butler, Ashley M; Anderson, Barbara J; Jaser, Sarah

    2016-08-01

    Stress is known to negatively affect health and is a potentially serious barrier to diabetes-related health outcomes. This paper synthesizes what is known about stress and glycemic control among people with type 1 and type 2 diabetes across the lifespan. Chronic stress-especially in relation to living with diabetes-was most strongly associated with A1c, particularly among subgroups that face disproportionate stress, such as minority groups or adolescents/young adults. Mechanisms of the stress-A1c association include physiological, psychological, behavioral, and environmental links. Understanding the dimensions of stress as they relate to health in diabetes can be of significant clinical importance, and interventions targeting mechanisms that either exacerbate or buffer stress have reported modest improvements in A1c. PMID:27287017

  2. Fast Adaptation in Vestibular Hair Cells Requires Myosin-1c Activity

    PubMed Central

    Stauffer, Eric A.; Scarborough, John D.; Hirono, Moritoshi; Miller, Emilie D.; Shah, Kavita; Mercer, John A.; Holt, Jeffrey R.; Gillespie, Peter G.

    2009-01-01

    Summary In sensory hair cells of the inner ear, mechanical amplification of small stimuli requires fast adaptation, the rapid closing of mechanically activated transduction channels. In frog and mouse vestibular hair cells, we found that the rate of fast adaptation depends on both channel opening and stimulus size and that it is modeled well as a release of a mechanical element in series with the transduction apparatus. To determine whether myosin-1c molecules of the adaptation motor are responsible for the release, we introduced the Y61G mutation into the Myo1c locus and generated mice homozygous for this sensitized allele. Measuring transduction and adaptation in the presence of NMB-ADP, an allele-specific inhibitor, we found that the inhibitor not only blocked slow adaptation, as demonstrated previously in transgenic mice, but also inhibited fast adaptation. These results suggest that mechanical activity of myosin-1c is required for fast adaptation in vestibular hair cells. PMID:16102537

  3. Micelle-Based Adjuvants for Subunit Vaccine Delivery

    PubMed Central

    Trimaille, Thomas; Verrier, Bernard

    2015-01-01

    In the development of subunit vaccines with purified or recombinant antigens for cancer and infectious diseases, the design of improved and safe adjuvants able to efficiently target the antigen presenting cells, such as dendritic cells, represents a crucial challenge. Nanoparticle-based antigen delivery systems have been identified as an innovative strategy to improve the efficacy of subunit vaccines. Among them, self-assembled micellar nanoparticles from amphiphilic (macro)molecules have recently emerged as promising candidates. In this short review, we report on the recent research findings highlighting the versatility and potential of such systems in vaccine delivery. PMID:26426060

  4. Functional analysis of Drosophila DNA polymerase ε p58 subunit

    PubMed Central

    Sahashi, Ritsuko; Matsuda, Risa; Suyari, Osamu; Kawai, Mieko; Yoshida, Hideki; Cotterill, Sue; Yamaguchi, Masamitsu

    2013-01-01

    DNA polymerase ε (polε) plays a central role in DNA replication in eukaryotic cells, and has been suggested to the main synthetic polymerase on the leading strand. It is a hetero-tetrameric enzyme, comprising a large catalytic subunit (the A subunit ~250 kDa), a B subunit of ~60 kDa in most species (~80 kDa in budding yeast) and two smaller subunits (each ~20 kDa). In Drosophila, two subunits of polε (dpolε) have been identified. One is the 255 kDa catalytic subunit (dpolεp255), and the other is the 58 kDa subunit (dpolεp58). The functions of the B subunit have been mainly studied in budding yeast and mammalian cell culture, few studies have been performed in the context of an intact multicellular organism and therefore its functions in this context remain poorly understood. To address this we examined the in vivo role of dpolεp58 in Drosophila. A homozygous dpolεp58 mutant is pupal lethal, and the imaginal discs are less developed in the third instar larvae. In the eye discs of this mutant S phases, as measured by BrdU incorporation assays, were significantly reduced. In addition staining with an anti-phospho histone H3 (PH3) antibody, (a marker of M phase), was increased in the posterior region of eye discs, where usually cells stop replicating and start differentiation. These results indicate that dpolεp58 is essential for Drosophila development and plays an important role in progression of S phase in mitotic cell cycles. We also observed that the size of nuclei in salivary gland cells were decreased in dpolεp58 mutant, indicating that dpolεp58 also plays a role in endoreplication. Furthermore we detect a putative functional interaction between dpolε and ORC2 in discs suggesting that polε plays a role in the initiation of DNA replication in Drosophila. PMID:24224125

  5. Advancements in the development of subunit influenza vaccines

    PubMed Central

    Zhang, Naru; Zheng, Bo-Jian; Lu, Lu; Zhou, Yusen; Jiang, Shibo; Du, Lanying

    2014-01-01

    The ongoing threat of influenza epidemics and pandemics has emphasized the importance of developing safe and effective vaccines against infections from divergent influenza viruses. In this review, we first introduce the structure and life cycle of influenza A viruses, describing major influenza A virus-caused pandemics. We then compare different types of influenza vaccines and discuss current advancements in the development of subunit influenza vaccines, particularly those based on nucleoprotein (NP), extracellular domain of matrix protein 2 (M2e) and hemagglutinin (HA) proteins. We also illustrate potential strategies for improving the efficacy of subunit influenza vaccines. PMID:25529753

  6. INTRACELLULAR TARGETING OF THE ONCOGENIC MUC1-C PROTEIN WITH A NOVEL GO-203 NANOPARTICLE FORMULATION

    PubMed Central

    Hasegawa, Masanori; Sinha, Raj Kumar; Kumar, Manoj; Alam, Maroof; Yin, Li; Raina, Deepak; Kharbanda, Akriti; Panchamoorthy, Govind; Gupta, Dikshi; Singh, Harpal; Kharbanda, Surender; Kufe, Donald

    2015-01-01

    Purpose The MUC1-C oncoprotein is an intracellular target that is druggable with cell-penetrating peptide inhibitors. However, development of peptidyl drugs for treating cancer has been a challenge because of unfavorable pharmacokinetic parameters and limited cell penetrating capabilities. Experimental Design Encapsulation of the MUC1-C inhibitor, GO-203, in novel polymeric nanoparticles (NPs) was studied for effects on intracellular targeting of MUC1-C signaling and function. Results Our results show that loading GO-203 into tetrablock polylactic acid (PLA)-polyethylene glycol (PEG)-polypropylene glycol (PPG)-PEG copolymers is achievable and, notably, is enhanced by increasing PEG chain length. Additionally, we found that release of GO-203 from these NPs is controllable over at least 7 days. GO-203/NP treatment of MUC1-C-positive breast and lung cancer cells in vitro was more active with less frequent dosing than that achieved with non-encapsulated GO-203. Moreover, treatment with GO-203/NPs blocked MUC1-C homodimerization, consistent with on-target effects. GO-203/NP treatment was also effective in downregulating TIGAR, disrupting redox balance and inhibiting the self-renewal capacity of cancer cells. Significantly, weekly administration of GO-203/NPs to mice bearing syngeneic or xenograft tumors was associated with regressions that were comparable to those found when dosing on a daily basis with GO-203. Conclusions These findings thus define an effective approach for (i) sustained administration of GO-203 in polymeric PLA-(PEG-PPG-PEG) NPs to target MUC1-C in cancer cells and (ii) the potential delivery of other anti-cancer peptide drugs. PMID:25712682

  7. Sanger Sequencing for BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del Mutation Screen on Pap Smear Cytology Samples

    PubMed Central

    Lee, Sin Hang; Zhou, Shaoxia; Zhou, Tianjun; Hong, Guofan

    2016-01-01

    Three sets of polymerase chain reaction (PCR) primers were designed for heminested PCR amplification of the target DNA fragments in the human genome which include the site of BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del respectively, to prepare the templates for direct Sanger sequencing screen of these three founder mutations. With a robust PCR mixture, crude proteinase K digestate of the fixed cervicovaginal cells in the liquid-based Papanicolaou (Pap) cytology specimens can be used as the sample for target DNA amplification without pre-PCR DNA extraction, purification and quantitation. The post-PCR products can be used directly as the sequencing templates without further purification or quantitation. By simplifying the frontend procedures for template preparation, the cost for screening these three founder mutations can be reduced to about US $200 per test when performed in conjunction with human papillomavirus (HPV) assays now routinely ordered for cervical cancer prevention. With this projected price structure, selective patients in a high-risk population can be tested and each provided with a set of DNA sequencing electropherograms to document the absence or presence of these founder mutations in her genome to help assess inherited susceptibility to breast and ovarian cancer in this era of precision molecular personalized medicine. PMID:26867194

  8. Spin wave ballistic transport properties of [Co1-c Gdc ] ℓ‧ [Co]ℓ [Co1-c Gdc ] ℓ‧ nanojunctions between Co leads

    NASA Astrophysics Data System (ADS)

    Ashokan, V.; Khater, A.; Abou Ghantous, M.; Ghader, D.

    2015-06-01

    The spin wave (SW) ballistic transport properties are investigated for nanojunction systems composed of thin [Co1-c Gdc ] ℓ‧ [Co]ℓ [Co1-c Gdc ] ℓ‧ layered nanostructures between cobalt leads. The nanojunction is considered as a homogeneous random alloy of concentrations c on an hcp crystal lattice. ℓ corresponds to the numbers of the hcp (0001) atomic planes per given layer. The phase field matching theory (PFMT) is used to investigate the spin dynamics of the nanojunction system in the virtual crystal approximation (VCA), valid in particular for submicroscopic SW wavelengths. The model calculations yield the spin modes localized on the nanojunction, normal to its plane, in their propagating and resonant forms. The eigenvectors of these modes are calculated for certain cases to illustrate the spin precession configurations on the nanojunction. The VCA-PFMT approach yields a general model, and is used to calculate the SW ballistic scattering and transport across the nanojunction for spin waves incident from the Co leads onto the nanojunction. The results demonstrate resonance Fano assisted maxima in the SW transmission spectra due to interactions between incident lead spin waves and localized spin resonances on the nanojunction. It is shown that these maxima change with nanojunction thickness and alloy concentration. The spectral transmission results for low frequency SWs are of specific interest, in particular they correspond to submicroscopic wavelengths which present an interest for current research of magnonic devices.

  9. The effect of aromatic hydrocarbon receptor on the phenotype of the Hepa 1c1c7 murine hepatoma cells in the absence of dioxin.

    PubMed

    Wang, Feng; Zhang, Ruixue; Shi, Shengli; Hankinson, Oliver

    2007-01-01

    The aromatic hydrocarbon receptor (AhR) mediates biological responses to certain exogenous ligands, such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and has also been demonstrated to modulate the cell cycle and differentiated state of several cell lines independently of exogenous ligands. In this study, we used DNA microarray analysis to elucidate the profile of genes responsive to the expression of unliganded AhR by re-introducing AhR into an AhR-deficient mouse derivative (c19) of the mouse hepatoma cell line Hepa 1c1c7. 22 gene products were up-regulated and 8 were down-regulated two-fold or more in c19 cells infected with a retroviral vector expressing mouse AhR. Surprisingly, expression of genes involved in cell proliferation or differentiation were not affected by introduction of AhR. AhR also did not restore expression of the albumin gene in c19 cells. Introduction of AhR into c12, a similar AhR-defective mouse hepatoma cell line, also did not restore albumin expression, and furthermore, did not lead to changes in cellular morphology or cell cycle parameters. These observations fail to support the notion that unliganded AhR regulates proliferation and differentiation of liver-derived cells. PMID:19936078

  10. The Effect of Aromatic Hydrocarbon Receptor on the Phenotype of the Hepa 1c1c7 Murine Hepatoma Cells in the Absence of Dioxin

    PubMed Central

    Wang, Feng; Zhang, Ruixue; Shi, Shengli; Hankinson, Oliver

    2007-01-01

    The aromatic hydrocarbon receptor (AhR) mediates biological responses to certain exogenous ligands, such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and has also been demonstrated to modulate the cell cycle and differentiated state of several cell lines independently of exogenous ligands. In this study, we used DNA micorarray analysis to elucidate the profile of genes responsive to the expression of unliganded AhR by re-introducing AhR into an AhR-deficient mouse derivative (c19) of the mouse hepatoma cell line Hepa1c1c7. 22 gene products were up-regulated and 8 were down-regulated two-fold or more in c19 cells infected with a retroviral vector expressing mouse AhR. Surprisingly, expression of genes involved in cell proliferation or differentiation were not affected by introduction of AhR. AhR also did not restore expression of the albumin gene in c19 cells. Introduction of AhR into c12, a similar AhR-defective mouse hepatoma cell line, also did not restore albumin expression, and furthermore, did not lead to changes in cellular morphology or cell cycle parameters. These observations fail to support the notion that unliganded AhR regulates proliferation and differentiation of liver-derived cells. PMID:19936078

  11. Sanger Sequencing for BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del Mutation Screen on Pap Smear Cytology Samples.

    PubMed

    Lee, Sin Hang; Zhou, Shaoxia; Zhou, Tianjun; Hong, Guofan

    2016-01-01

    Three sets of polymerase chain reaction (PCR) primers were designed for heminested PCR amplification of the target DNA fragments in the human genome which include the site of BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del respectively, to prepare the templates for direct Sanger sequencing screen of these three founder mutations. With a robust PCR mixture, crude proteinase K digestate of the fixed cervicovaginal cells in the liquid-based Papanicolaou (Pap) cytology specimens can be used as the sample for target DNA amplification without pre-PCR DNA extraction, purification and quantitation. The post-PCR products can be used directly as the sequencing templates without further purification or quantitation. By simplifying the frontend procedures for template preparation, the cost for screening these three founder mutations can be reduced to about US $200 per test when performed in conjunction with human papillomavirus (HPV) assays now routinely ordered for cervical cancer prevention. With this projected price structure, selective patients in a high-risk population can be tested and each provided with a set of DNA sequencing electropherograms to document the absence or presence of these founder mutations in her genome to help assess inherited susceptibility to breast and ovarian cancer in this era of precision molecular personalized medicine. PMID:26867194

  12. A Review of the Challenge in Measuring Hemoglobin A1c

    PubMed Central

    Weykamp, Cas; Garry John, W.; Mosca, Andrea

    2009-01-01

    The attraction of the simple biochemical concept combined with a clinical requirement for a long-term marker of glycolic control in diabetes has made hemoglobin A1c (HbA1c) one of the most important assays undertaken in the medical laboratory. The diversity in the biochemistry of glycation, clinical requirements, and management demands has resulted in a broad range of methods being developed since HbA1c was described in the late 1960s. A range of analytic principles are used for the measurement of HbA1c. The charge difference between hemoglobin A0 and HbA1c has been widely utilized to separate these two fractions, most notably found these days in ion-exchange high-performance liquid chromatography systems; the difference in molecular structure (affinity chromatography and immunochemical methods) are becoming widely available. Different results found in different laboratories using a variety of HbA1c analyses resulted in the need for standardization, most notably in the United States, Japan, and Sweden. Designated comparison methods are now located in these three countries, but as they are arbitrarily chosen and have differences in specificity, results of these methods and the reference values and action limits of the methods differ and only harmonized HbA1c in specific geographic areas. A reference measurement system within the concept of metrological traceability is now globally accepted as the only valid analytic anchor. However, there is still discussion over the units to be reported. The consensus statement of the International Federation of Clinical Chemistry (IFCC), the American Diabetes Association, the International Diabetes Federation, and the European Association for the Study of Diabetes suggests reporting HbA1c in IFCC units (mmol/mol), National Glycohemoglobin Standardization Program units (%), and estimated average glucose (either in mg/dl or mmol/liter). The implementation of this consensus statement raised new questions, to be answered in a

  13. Endovascular Treatment of a Vertebral Artery Pseudoaneurysm Following Posterior C1-C2 Transarticular Screw Fixation

    SciTech Connect

    Mendez, Jose C. Gonzalez-Llanos, Francisco

    2005-01-15

    We present a case of vertebral artery pseudoaneurysm after a posterior C1-C2 transarticular screw fixation procedure that was effectively treated with endovascular coil occlusion. Vertebral artery pseudoaneurysm complicating posterior C1-C2 transarticular fixation is extremely rare, with only one previous case having been reported previously. Endovascular occlusion is better achieved in the subacute phase of the pseudoaneurysm, when the wall of the pseudoaneurysm has matured and stabilized. Further follow-up angiographies are mandatory in order to confirm that there is no recurrence of the lesion.

  14. 40 CFR Table W-1c to Subpart W of... - Default Average Component Counts For Major Crude Oil Production Equipment

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) MANDATORY GREENHOUSE GAS REPORTING Petroleum and Natural Gas Systems Pt. 98, Subpt. W, Table W-1C Table W-1C to Subpart W of Part...

  15. Podosome-regulating kinesin KIF1C translocates to the cell periphery in a CLASP-dependent manner

    PubMed Central

    Efimova, Nadia; Grimaldi, Ashley; Bachmann, Alice; Frye, Keyada; Zhu, Xiaodong; Feoktistov, Alexander; Straube, Anne; Kaverina, Irina

    2014-01-01

    ABSTRACT The kinesin KIF1C is known to regulate podosomes, actin-rich adhesion structures that remodel the extracellular matrix during physiological processes. Here, we show that KIF1C is a player in the podosome-inducing signaling cascade. Upon induction of podosome formation by protein kinase C (PKC), KIF1C translocation to the cell periphery intensifies and KIF1C accumulates both in the proximity of peripheral microtubules that show enrichment for the plus-tip-associated proteins CLASPs and around podosomes. Importantly, without CLASPs, both KIF1C trafficking and podosome formation are suppressed. Moreover, chimeric mitochondrially targeted CLASP2 recruits KIF1C, suggesting a transient CLASP–KIF1C association. We propose that CLASPs create preferred microtubule tracks for KIF1C to promote podosome induction downstream of PKC. PMID:25344256

  16. Aspects of Subunit Interactions in the Chloroplast ATP Synthase (I. Isolation of a Chloroplast Coupling Factor 1-Subunit III Complex from Spinach Thylakoids).

    PubMed Central

    Wetzel, C. M.; McCarty, R. E.

    1993-01-01

    A chloroplast ATP synthase complex (CF1 [chloroplast-coupling factor 1]-CF0 [membrane-spanning portion of chloroplast ATP synthase]) depleted of all CF0 subunits except subunit III (also known as the proteolipid subunit) was purified to study the interaction between CF1 and subunit III. Subunit III has a putative role in proton translocation across the thylakoid membrane during photophosphorylation; therefore, an accurate model of subunit inter-actions involving subunit III will be valuable for elucidating the mechanism and regulation of energy coupling. Purification of the complex from a crude CF1-CF0 preparation from spinach (Spinacia oleracea) thylakoids was accomplished by detergent treatment during anion-exchange chromatography. Subunit III in the complex was positively identified by amino acid analysis and N-terminal sequencing. The association of subunit III with CF1 was verified by linear sucrose gradient centrifugation, immunoprecipitation, and incorporation of the complex into asolectin liposomes. After incorporation into liposomes, CF1 was removed from the CF1-III complex by ethylenediaminetetracetate treatment. The subunit III-proteoliposomes were competent to rebind purified CF1. These results indicate that subunit III directly interacts with CF1 in spinach thylakoids. PMID:12231815

  17. Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman.

    PubMed

    Hafver, Tandekile Lubelwana; Hodne, Kjetil; Wanichawan, Pimthanya; Aronsen, Jan Magnus; Dalhus, Bjørn; Lunde, Per Kristian; Lunde, Marianne; Martinsen, Marita; Enger, Ulla Helene; Fuller, William; Sjaastad, Ivar; Louch, William Edward; Sejersted, Ole Mathias; Carlson, Cathrine Rein

    2016-02-26

    The sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na(+)/K(+)-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca(2+) binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X5-8Φ1Φ2-X8-9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation. PMID:26668322

  18. The Kinesin KIF1C and Microtubule Plus Ends Regulate Podosome Dynamics in Macrophages

    PubMed Central

    Kopp, Petra; Lammers, Reiner; Aepfelbacher, Martin; Woehlke, Günther; Rudel, Thomas; Machuy, Nikolaus; Steffen, Walter

    2006-01-01

    Microtubules are important for the turnover of podosomes, dynamic, actin-rich adhesions implicated in migration and invasion of monocytic cells. The molecular basis for this functional dependency, however, remained unclear. Here, we show that contact by microtubule plus ends critically influences the cellular fate of podosomes in primary human macrophages. In particular, we identify the kinesin KIF1C, a member of the Kinesin-3 family, as a plus-end–enriched motor that targets regions of podosome turnover. Expression of mutation constructs or small interfering RNA-/short hairpin RNA-based depletion of KIF1C resulted in decreased podosome dynamics and ultimately in podosome deficiency. Importantly, protein interaction studies showed that KIF1C binds to nonmuscle myosin IIA via its PTPD-binding domain, thus providing an interface between the actin and tubulin cytoskeletons, which may facilitate the subcellular targeting of podosomes by microtubules. This is the first report to implicate a kinesin in podosome regulation and also the first to describe a function for KIF1C in human cells. PMID:16554367

  19. 7 CFR 1c.113 - Suspension or termination of IRB approval of research.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Suspension or termination of IRB approval of research... § 1c.113 Suspension or termination of IRB approval of research. An IRB shall have authority to suspend or terminate approval of research that is not being conducted in accordance with the...

  20. The role of myosin 1c and myosin 1b in surfactant exocytosis.

    PubMed

    Kittelberger, Nadine; Breunig, Markus; Martin, René; Knölker, Hans-Joachim; Miklavc, Pika

    2016-04-15

    Actin and actin-associated proteins have a pivotal effect on regulated exocytosis in secretory cells and influence pre-fusion as well as post-fusion stages of exocytosis. Actin polymerization on secretory granules during the post-fusion phase (formation of an actin coat) is especially important in cells with large secretory vesicles or poorly soluble secretions. Alveolar type II (ATII) cells secrete hydrophobic lipo-protein surfactant, which does not easily diffuse from fused vesicles. Previous work showed that compression of actin coat is necessary for surfactant extrusion. Here, we investigate the role of class 1 myosins as possible linkers between actin and membranes during exocytosis. Live-cell microscopy showed translocation of fluorescently labeled myosin 1b and myosin 1c to the secretory vesicle membrane after fusion. Myosin 1c translocation was dependent on its pleckstrin homology domain. Expression of myosin 1b and myosin 1c constructs influenced vesicle compression rate, whereas only the inhibition of myosin 1c reduced exocytosis. These findings suggest that class 1 myosins participate in several stages of ATII cell exocytosis and link actin coats to the secretory vesicle membrane to influence vesicle compression. PMID:26940917

  1. Magnetic properties of (Ho 1-cGd c) Co 2 compounds

    NASA Astrophysics Data System (ADS)

    Tari, A.; Keith, V.; Hwang, J. S.

    1994-02-01

    E.S.R., magnetisation and susceptibility measurements have been carried out on the series (Ho 1-cGd c)Co 2. In compounds with c≤ 0.40 we find evidence of spin reorientation. In the three most dilute compounds a g-value of about four is obtained which increases with increasing Gd content.

  2. Phosphodiesterase 1C is dispensable for rapid response termination of olfactory sensory neurons

    PubMed Central

    Cygnar, Katherine D.; Zhao, Haiqing

    2009-01-01

    In the nose, odorants are detected on the cilia of olfactory sensory neurons (OSNs), where a cAMP-mediated signaling pathway transforms odor stimulation into electrical responses. Phosphodiesterase (PDE) activity in OSN cilia was long thought to account for rapid response termination by degrading odor-induced cAMP. Two PDEs with distinct cellular localization have been found in OSNs: PDE1C in cilia; PDE4A throughout the cell but absent from cilia. We disrupted both genes in mice and performed electroolfactogram analysis. Unexpectedly, eliminating PDE1C did not prolong response termination. Prolonged termination occurred only in mice lacking both PDEs, suggesting that cAMP degradation by PDE1C in cilia is not a rate-limiting factor for response termination in wildtype. Pde1c−/− OSNs instead displayed reduced sensitivity and attenuated adaptation to repeated stimulation, suggesting potential roles for PDE1C in regulating sensitivity and adaptation. These observations provide new perspectives in regulation of olfactory transduction. PMID:19305400

  3. Is hemoglobin A1c level effective in predicting the prognosis of Fournier gangrene?

    PubMed Central

    Sen, Haluk; Bayrak, Omer; Erturhan, Sakip; Borazan, Ersin; Koc, Mustafa Nihat

    2016-01-01

    Objectives: To evaluate the effect of immune failure and/or diabetes mellitus (DM) association on the mortality and morbidity of the Fournier's Gangrene (FG), and interrelatedly, the usability of HbA1c level in the prediction of prognosis. Materials and Methods: The data of 38 patients with the diagnosis of FG were investigated retrospectively. The patients were divided into two groups as patients with DM (Group 1, n = 18) and non-diabetics (Group 2, n = 20). The patients in group 1 were also divided into two subgroups as patients with HbA1c value ≥7 (Group 1a) and HbA1c value <7 (Group 1b). Results: The mean age of all 38 male patients was 66.3 ± 6.4 years. The initial symptoms were scrotal rash and swelling (n = 20, 52.6%), high fever (>38°C) (n = 22, 57.8%), purulent discharge from genital or perineal areas (n = 13, 34.2%), skin bruises (n = 11, 28.9%) and general state disorder in five patients that were admitted from day care center (13.1%). DM, as the most often comorbid disease, was detected in 18 patients (47.3%). Six patients (15.7%) were deceased during the follow-up period. Conclusion: In the present study, the researchers determined that diabetic patients with HbA1c level of 7 or higher had worse prognosis, and increased mortality. PMID:27453658

  4. To the occiput or not? C1-c2 ligamentous laxity in children with down syndrome.

    PubMed

    Siemionow, Kris; Chou, Dean

    2014-10-01

    Study Design Retrospective case review. Objective Atlantoaxial instability with and without basilar invagination poses a considerable challenge in management regarding reduction, surgical approach, decompression, instrumentation choice, and extent of fusion. A variety of strategies have been described to reduce and stabilize cranial settling with basilar invagination. Modern instrumentation options included extension to the occiput, C1-C2 transarticular fixation, and C1 lateral mass-C2 pars among others. Since not all cases of cranial settling are the same, their treatment strategies also differ. Factors such as local vascular anatomy, amount of subluxation, need for distraction, and shape of occipital condyles will dictate level and type of instrumentation. The objective of this study was to outline treatment options and provide a rationale for the surgical plan. Methods Two cases of C1-C2 instability in patients with Down syndrome are described. Case 2 underwent C1-C2 instrumented fusion, whereas case 1 involved posterior instrumented fusion to the occiput. Results Both patients tolerated the procedures well. There were no complications. Minimum follow-up was 1 year. There was no loss of reduction. Solid arthrodesis was achieved in both cases. Conclusion Successful reduction can be achieved with both C1-C2 instrumented fusion as well as O-C instrument fusion. Factors such as local vascular anatomy, amount of subluxation, need for distraction, and shape of occipital condyles will dictate level and type of instrumentation. PMID:25364324

  5. Stereoselective synthesis of the C1-C29 part of amphidinol 3.

    PubMed

    Tsuruda, Takeshi; Ebine, Makoto; Umeda, Aya; Oishi, Tohru

    2015-01-16

    Stereoselective synthesis of the C1-C29 part of amphidinol 3 (AM3) was achieved. The C1-C20 part was assembled from three building blocks via regioselective cross metathesis to form the C4-C5 double bond and addition of an alkenyllithium and a lithium acetylide to two Weinreb amides followed by asymmetric reduction to form the C9-C10 and C14-C15 bonds, respectively. The C21-C29 part was synthesized via successive cross metathesis and oxa-Michael addition sequence to construct the 1,3-diol system at C25 and C27 and Brown asymmetric crotylation to introduce the stereogenic centers at C23 and C24. Coupling of the C1-C20 and C21-C29 parts was achieved by Julia-Kocienski olefination and regio- and stereoselective dihydroxylation of the C20-C21 double bond in the presence of the C4-C5 and C8-C9 double bonds to afford the C1-C29 part of AM3. PMID:25517178

  6. On the causes of compositional order in the Ni sub c Pt sub (1-c) alloys

    SciTech Connect

    Gyorffy, B.L. . H.H. Wills Physics Lab.); Pinski, F.J. . Dept. of Physics); Ginatempo, B. . Ist. di Fisica Teorica); Johnson, D.D. ); Staunton, J.B. . Dept. of Physics); Shelton, W.A.; Stocks, G.M.; Nicholson, D.M.

    1991-01-01

    We review, briefly, the arguments which gave rise to the current controversy concerning the origin of compositional order in Ni{sub c}Pt{sub 1-c} alloys. We note that strain fluctuations play an important role in determining the state of compositional order in this system and outline a theoretical framework that takes account of them. 29 refs., 4 figs.

  7. Residual Endotoxin Contaminations in Recombinant Proteins Are Sufficient to Activate Human CD1c+ Dendritic Cells

    PubMed Central

    Schwarz, Harald; Schmittner, Maria; Duschl, Albert; Horejs-Hoeck, Jutta

    2014-01-01

    Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002–2 ng/ml). We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14. PMID:25478795

  8. Validation of NHB 8060.1C, Test 18 Arc Tracking, September 30, 1991

    NASA Technical Reports Server (NTRS)

    Linley, Larry

    2005-01-01

    A test project was conducted to validate Test 18 of NASA Handbook (NHB) 8060.1C and, if necessary, identify and recommend improvements in the procedures or criteria of the test. The NHB 8060.1C, Test 18 test system was modified to produce better discrimination of test results. Changes, and their effects on test results, in the graphite immersion-depth, test timing sequence, and atmospheric conditions were investigated for the wire-insulation constructions tested. Based on the test results, the graphite immersion-depths (between 0.8 mm and 1.6 mm), the timing sequence, and the change in the test conditions from ambient to three environments common in manned spaceflight did not significantly affect test results. The criteria used in Test 18 of NHB 8060.1C was found to be appropriate for qualifying arc-tracking and arc-propagation characteristics of wire-insulation materials, Using the Test 18 criteria, Kapton and ETFE were considered inappropriate for use, while PTFE was considered appropriate. Recommendations from this test project for Test 18 of NHB 8060.1C include changing the experimental setup and configurational tests and performing qualification testing in air rather than in the three environments common in manned spaceflight.

  9. Spirastrellolide E: Synthesis of an advanced C(1)-C(24) southern hemisphere

    PubMed Central

    Sokolsky, Alexander; Wang, Xiaozhao; Smith, Amos B.

    2014-01-01

    The synthesis of a C(1)-C(24) advanced southern hemisphere fragment towards the total synthesis of spirastrellolide E has been achieved. Highlights of the route include a highly convergent Type I Anion Relay Chemistry (ARC) tactic for fragment assembly, in conjunction with a directed, regioselective gold-catalyzed alkyne functionalization to generate the central unsaturated [6,6]-spiroketal. PMID:26097261

  10. IGF-1 C Domain-Modified Hydrogel Enhances Cell Therapy for AKI.

    PubMed

    Feng, Guowei; Zhang, Jimin; Li, Yang; Nie, Yan; Zhu, Dashuai; Wang, Ran; Liu, Jianfeng; Gao, Jie; Liu, Na; He, Ningning; Du, Wei; Tao, Hongyan; Che, Yongzhe; Xu, Yong; Kong, Deling; Zhao, Qiang; Li, Zongjin

    2016-08-01

    Low cell retention and engraftment after transplantation limit the successful application of stem cell therapy for AKI. Engineered microenvironments consisting of a hydrogel matrix and growth factors have been increasingly successful in controlling stem cell fate by mimicking native stem cell niche components. Here, we synthesized a bioactive hydrogel by immobilizing the C domain peptide of IGF-1 (IGF-1C) on chitosan, and we hypothesized that this hydrogel could provide a favorable niche for adipose-derived mesenchymal stem cells (ADSCs) and thereby enhance cell survival in an AKI model. In vitro studies demonstrated that compared with no hydrogel or chitosan hydrogel only, the chitosan-IGF-1C hydrogel increased cell viability through paracrine effects. In vivo, cotransplantation of the chitosan-IGF-1C hydrogel and ADSCs in ischemic kidneys ameliorated renal function, likely by the observed promotion of stem cell survival and angiogenesis, as visualized by bioluminescence imaging and attenuation of fibrosis. In conclusion, IGF-1C immobilized on a chitosan hydrogel provides an artificial microenvironment for ADSCs and may be a promising therapeutic approach for AKI. PMID:26869006

  11. 7 CFR 1c.111 - Criteria for IRB approval of research.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 1 2011-01-01 2011-01-01 false Criteria for IRB approval of research. 1c.111 Section... Criteria for IRB approval of research. (a) In order to approve research covered by this policy the IRB... minimized: (i) By using procedures which are consistent with sound research design and which do...

  12. 7 CFR 1c.111 - Criteria for IRB approval of research.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Criteria for IRB approval of research. 1c.111 Section... Criteria for IRB approval of research. (a) In order to approve research covered by this policy the IRB... minimized: (i) By using procedures which are consistent with sound research design and which do...

  13. Nur77 modulates hepatic lipid metabolism through suppression of SREBP1c activity

    SciTech Connect

    Pols, Thijs W.H.; Ottenhoff, Roelof; Vos, Mariska; Levels, Johannes H.M.; Quax, Paul H.A.; Meijers, Joost C.M.; Pannekoek, Hans; Groen, Albert K.; Vries, Carlie J.M. de

    2008-02-22

    NR4A nuclear receptors are induced in the liver upon fasting and regulate hepatic gluconeogenesis. Here, we studied the role of nuclear receptor Nur77 (NR4A1) in hepatic lipid metabolism. We generated mice expressing hepatic Nur77 using adenoviral vectors, and demonstrate that these mice exhibit a modulation of the plasma lipid profile and a reduction in hepatic triglyceride. Expression analysis of >25 key genes involved in lipid metabolism revealed that Nur77 inhibits SREBP1c expression. This results in decreased SREBP1c activity as is illustrated by reduced expression of its target genes stearoyl-coA desaturase-1, mitochondrial glycerol-3-phosphate acyltransferase, fatty acid synthase and the LDL receptor, and provides a mechanism for the physiological changes observed in response to Nur77. Expression of LXR target genes Abcg5 and Abcg8 is reduced by Nur77, and may suggest involvement of LXR in the inhibitory action of Nur77 on SREBP1c expression. Taken together, our study demonstrates that Nur77 modulates hepatic lipid metabolism through suppression of SREBP1c activity.

  14. The role of myosin 1c and myosin 1b in surfactant exocytosis

    PubMed Central

    Kittelberger, Nadine; Breunig, Markus; Martin, René; Knölker, Hans-Joachim; Miklavc, Pika

    2016-01-01

    ABSTRACT Actin and actin-associated proteins have a pivotal effect on regulated exocytosis in secretory cells and influence pre-fusion as well as post-fusion stages of exocytosis. Actin polymerization on secretory granules during the post-fusion phase (formation of an actin coat) is especially important in cells with large secretory vesicles or poorly soluble secretions. Alveolar type II (ATII) cells secrete hydrophobic lipo-protein surfactant, which does not easily diffuse from fused vesicles. Previous work showed that compression of actin coat is necessary for surfactant extrusion. Here, we investigate the role of class 1 myosins as possible linkers between actin and membranes during exocytosis. Live-cell microscopy showed translocation of fluorescently labeled myosin 1b and myosin 1c to the secretory vesicle membrane after fusion. Myosin 1c translocation was dependent on its pleckstrin homology domain. Expression of myosin 1b and myosin 1c constructs influenced vesicle compression rate, whereas only the inhibition of myosin 1c reduced exocytosis. These findings suggest that class 1 myosins participate in several stages of ATII cell exocytosis and link actin coats to the secretory vesicle membrane to influence vesicle compression. PMID:26940917

  15. 102. Catalog HHistory 1, C.C.C., 34 Landscaping, Negative No. 6040a ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    102. Catalog H-History 1, C.C.C., 34 Landscaping, Negative No. 6040a (Photographer and date unknown) BEAUTIFICATION PROGRAM STARTED AS SOON AS GRADING ALONG THE DRIVE WAS COMPLETED. CCC CAMP 3 SHOWN PLANTING LAUREL. - Skyline Drive, From Front Royal, VA to Rockfish Gap, VA , Luray, Page County, VA

  16. Software selection based on analysis and forecasting methods, practised in 1C

    NASA Astrophysics Data System (ADS)

    Vazhdaev, A. N.; Chernysheva, T. Y.; Lisacheva, E. I.

    2015-09-01

    The research focuses on the problem of a “1C: Enterprise 8” platform inboard mechanisms for data analysis and forecasting. It is important to evaluate and select proper software to develop effective strategies for customer relationship management in terms of sales, as well as implementation and further maintenance of software. Research data allows creating new forecast models to schedule further software distribution.

  17. Close Approach Prediction Analysis of the Earth Science Constellation with the Fengyun-1C Debris

    NASA Technical Reports Server (NTRS)

    Duncan, Matthew; Rand, David K.

    2008-01-01

    Routine satellite operations for the Earth Science Constellation (ESC) include collision risk assessment between members of the constellation and other orbiting space objects. Each day, close approach predictions are generated by a U.S. Department of Defense Joint Space Operations Center Orbital Safety Analyst using the high accuracy Space Object Catalog maintained by the Air Force's 1" Space Control Squadron. Prediction results and other ancillary data such as state vector information are sent to NASAJGoddard Space Flight Center's (GSFC's) Collision Risk Assessment analysis team for review. Collision analysis is performed and the GSFC team works with the ESC member missions to develop risk reduction strategies as necessary. This paper presents various close approach statistics for the ESC. The ESC missions have been affected by debris from the recent anti-satellite test which destroyed the Chinese Fengyun- 1 C satellite. The paper also presents the percentage of close approach events induced by the Fengyun-1C debris, and presents analysis results which predict the future effects on the ESC caused by this event. Specifically, the Fengyun-1C debris is propagated for twenty years using high-performance computing technology and close approach predictions are generated for the ESC. The percent increase in the total number of conjunction events is considered to be an estimate of the collision risk due to the Fengyun-1C break- UP.

  18. Abnormalities in alpha-dystroglycan expression in MDC1C and LGMD2I muscular dystrophies.

    PubMed

    Brown, Susan C; Torelli, Silvia; Brockington, Martin; Yuva, Yeliz; Jimenez, Cecilia; Feng, Lucy; Anderson, Louise; Ugo, Isabella; Kroger, Stephan; Bushby, Kate; Voit, Thomas; Sewry, Caroline; Muntoni, Francesco

    2004-02-01

    We recently identified mutations in the fukutin related protein (FKRP) gene in patients with congenital muscular dystrophy type 1C (MDC1C) and limb girdle muscular dystrophy type 2I (LGMD2I). The sarcolemma of these patients typically displays an immunocytochemical reduction of alpha-dystroglycan. In this report we extend these observations and report a clear correlation between the residual expression of alpha-dystroglycan and the phenotype. Three broad categories were identified. Patients at the severe end of the clinical spectrum (MDC1C) were compound heterozygote between a null allele and a missense mutation or carried two missense mutations and displayed a profound depletion of alpha-dystroglycan. Patients with LGMD with a Duchenne-like severity typically had a moderate reduction in alpha-dystroglycan and were compound heterozygotes between a common C826A (Leu276Ileu) FKRP mutation and either a missense or a nonsense mutation. Individuals with the milder form of LGMD2I were almost invariably homozygous for the Leu276Ile FKRP mutation and showed a variable but subtle alteration in alpha-dystroglycan immunolabeling. Our data therefore suggest a correlation between a reduction in alpha-dystroglycan, the mutation and the clinical phenotype in MDC1C and LGMD2I which supports the hypothesis that dystroglycan plays a central role in the pathogenesis of these disorders. PMID:14742276

  19. Interference with hemoglobin A(1C) determination by the hemoglobin variant Shelby.

    PubMed

    Scuderi, Richard T; Griffin, Terrance L; Mehta, Shruti P; Herold, David A; Fitzgerald, Robert L

    2007-09-01

    Hemoglobin variant carrier status was found in a 46-year-old African American man following detection of a falsely elevated hemoglobin A1c (HbA1c) by ionexchange high-performance liquid chromatography (HPLC, VARIANT A1c, Bio-Rad Laboratories, Hercules, CA). Additional analysis of the hemoglobin variant using the Beta Thal Short program (Bio-Rad) revealed an unknown peak with a retention time of 4.84 minutes and a proportion of 26.3%. No mass shift in alpha-globin or beta-globin proteins was observed by mass spectrometry. DNA sequencing revealed a missense mutation in 1 beta-globin allele corresponding to the hemoglobin Shelby trait. The patient was asymptomatic with a normal hemoglobin value of 13.6 g/dL (136 g/L) but had increased target cells on a peripheral blood smear. An alternative method for HbA1c determination using boronate-affinity HPLC provided a value of 3.9% (0.04; reference range, 4.0%-6.9% [0.04-0.07]), more consistent with the patient's recent blood glucose values in the normal range. PMID:17709318

  20. 7 CFR 1c.123 - Early termination of research support: Evaluation of applications and proposals.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... OF HUMAN SUBJECTS § 1c.123 Early termination of research support: Evaluation of applications and... 7 Agriculture 1 2010-01-01 2010-01-01 false Early termination of research support: Evaluation of... the rights and welfare of human subjects (whether or not the research was subject to...

  1. 7 CFR 1c.123 - Early termination of research support: Evaluation of applications and proposals.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... OF HUMAN SUBJECTS § 1c.123 Early termination of research support: Evaluation of applications and... 7 Agriculture 1 2013-01-01 2013-01-01 false Early termination of research support: Evaluation of... the rights and welfare of human subjects (whether or not the research was subject to...

  2. 7 CFR 1c.123 - Early termination of research support: Evaluation of applications and proposals.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... OF HUMAN SUBJECTS § 1c.123 Early termination of research support: Evaluation of applications and... 7 Agriculture 1 2014-01-01 2014-01-01 false Early termination of research support: Evaluation of... the rights and welfare of human subjects (whether or not the research was subject to...

  3. 7 CFR 1c.123 - Early termination of research support: Evaluation of applications and proposals.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... OF HUMAN SUBJECTS § 1c.123 Early termination of research support: Evaluation of applications and... 7 Agriculture 1 2011-01-01 2011-01-01 false Early termination of research support: Evaluation of... the rights and welfare of human subjects (whether or not the research was subject to...

  4. PERCEIVED WEIGHT DISCRIMINATION AMPLIFIES THE LINK BETWEEN CENTRAL ADIPOSITY AND NONDIABETIC GLYCEMIC CONTROL (HBA1C)

    PubMed Central

    Tsenkova, Vera K.; Carr, Deborah; Schoeller, Dale A.; Ryff, Carol D.

    2010-01-01

    Background While the preclinical development of type 2 diabetes is partly explained by obesity and central adiposity, psychosocial research has shown that chronic stressors such as discrimination have health consequences as well. Purpose We investigated the extent to which the well-established effects of obesity and central adiposity on nondiabetic glycemic control (indexed by HbA1c) were moderated by a targeted psychosocial stressor linked to weight: perceived weight discrimination. Methods Data came from the nondiabetic subsample (n=938) of the Midlife in the United States (MIDUS II) survey. Results Body mass index (BMI), waist-to-hip ratio, and waist circumference were linked to significantly higher HbA1c (p < .001). Multivariate-adjusted models showed that weight discrimination exacerbated the effects of waist-to-hip ratio on HbA1c ( p < .05), such that people who had higher WHR and reported weight discrimination had the highest HbA1c levels. Conclusions Understanding how biological and psychosocial factors interact at nondiabetic levels to increase vulnerability could have important implications for public health and education strategies. Effective strategies may include targeting sources of discrimination, rather than solely targeting health behaviors and practices of overweight and obese persons. PMID:21136227

  5. 7 CFR 1c.110 - Expedited review procedures for certain kinds of research involving no more than minimal risk...

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Expedited review procedures for certain kinds of research involving no more than minimal risk, and for minor changes in approved research. 1c.110 Section 1c.110 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.110 Expedited review procedures for certain kinds...

  6. 7 CFR 1c.103 - Assuring compliance with this policy-research conducted or supported by any Federal Department or...

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 1 2014-01-01 2014-01-01 false Assuring compliance with this policy-research conducted or supported by any Federal Department or Agency. 1c.103 Section 1c.103 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.103 Assuring compliance with this...

  7. 77 FR 2573 - International Product Change-Global Plus 1C and 2C Negotiated Service Agreements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-18

    ... International Product Change--Global Plus 1C and 2C Negotiated Service Agreements AGENCY: Postal Service TM... Regulatory Commission to add Global Plus 1C and 2C Negotiated Service Agreements to the Competitive Products... of United States Postal Service to Add Global Plus 1C and 2C Negotiated Service Agreements to...

  8. GABAB(1) receptor subunit isoforms differentially regulate stress resilience.

    PubMed

    O'Leary, Olivia F; Felice, Daniela; Galimberti, Stefano; Savignac, Hélène M; Bravo, Javier A; Crowley, Tadhg; El Yacoubi, Malika; Vaugeois, Jean-Marie; Gassmann, Martin; Bettler, Bernhard; Dinan, Timothy G; Cryan, John F

    2014-10-21

    Stressful life events increase the susceptibility to developing psychiatric disorders such as depression; however, many individuals are resilient to such negative effects of stress. Determining the neurobiology underlying this resilience is instrumental to the development of novel and more effective treatments for stress-related psychiatric disorders. GABAB receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABAB(2) subunit with either a GABAB(1a) or a GABAB(1b) subunit. Here we show that mice lacking the GABAB(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABAB(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wild-type mice. In addition, increased hippocampal expression of the GABAB(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABAB(1b)(-/-) mice is coupled with increased proliferation and survival of newly born cells in the adult ventral hippocampus and increased stress-induced c-Fos activation in the hippocampus following early-life stress. Taken together, the data suggest that GABAB(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression. PMID:25288769

  9. CMF70 is a subunit of the dynein regulatory complex.

    PubMed

    Kabututu, Zakayi P; Thayer, Michelle; Melehani, Jason H; Hill, Kent L

    2010-10-15

    Flagellar motility drives propulsion of several important pathogens and is essential for human development and physiology. Motility of the eukaryotic flagellum requires coordinate regulation of thousands of dynein motors arrayed along the axoneme, but the proteins underlying dynein regulation are largely unknown. The dynein regulatory complex, DRC, is recognized as a focal point of axonemal dynein regulation, but only a single DRC subunit, trypanin/PF2, is currently known. The component of motile flagella 70 protein, CMF70, is broadly and uniquely conserved among organisms with motile flagella, suggesting a role in axonemal motility. Here we demonstrate that CMF70 is part of the DRC from Trypanosoma brucei. CMF70 is located along the flagellum, co-sediments with trypanin in sucrose gradients and co-immunoprecipitates with trypanin. RNAi knockdown of CMF70 causes motility defects in a wild-type background and suppresses flagellar paralysis in cells with central pair defects, thus meeting the functional definition of a DRC subunit. Trypanin and CMF70 are mutually conserved in at least five of six extant eukaryotic clades, indicating that the DRC was probably present in the last common eukaryotic ancestor. We have identified only the second known subunit of this ubiquitous dynein regulatory system, highlighting the utility of combined genomic and functional analyses for identifying novel subunits of axonemal sub-complexes. PMID:20876659

  10. Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

    PubMed

    McCrea, P D; Popot, J L; Engelman, D M

    1987-12-01

    Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit. PMID:3428268

  11. The Essential Anatomical Subunit Approximation Unilateral Cleft Lip Repair.

    PubMed

    Chong, David K; Swanson, Jordan W

    2016-07-01

    The anatomical subunit approximation cleft lip repair advantageously achieves a balanced lip contour, with the line of repair hidden along seams of aesthetic subunits. Dr. David Fisher's original description of the repair reflects the considerable thought that went into the evolution of his design. As his technique has gained acceptance in the intervening 10 years, the authors note several key principles embodied in it that represent a shift in the cleft lip repair paradigm. The authors believe understanding these principles is important to mastery of the anatomical subunit technique, and facilitate its teaching. First, design a plan that adheres to anatomical subunits and perform measurements precisely. Second, identify and adequately release each cleft tissue layer from the lip and nose to enable restoration of balance. Third, drive surgical approximation through inset of the lateral muscle into the superiorly backcut medial orbicularis muscle, followed by skin closure with inferior triangle interposition above the white roll. In this article, the authors present essential components of the technique, and identify several principles that enable its successful execution. PMID:27348690

  12. Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

    PubMed Central

    Römling, Ute; Galperin, Michael Y.

    2015-01-01

    Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

  13. Insecticidal Pilin Subunit from the Insect Pathogen Xenorhabdus nematophila

    PubMed Central

    Khandelwal, Puneet; Choudhury, Devapriya; Birah, Ajanta; Reddy, M. K.; Gupta, Gorakh Prasad; Banerjee, Nirupama

    2004-01-01

    Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X. nematophila. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X. nematophila isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H. armigera, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium. PMID:15375127

  14. Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions.

    PubMed

    Römling, Ute; Galperin, Michael Y

    2015-09-01

    Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits - which differ among various taxa - affect the enzymatic activity and product yield in vivo by modulating (i) the expression of the biosynthesis apparatus, (ii) the export of the nascent β-D-glucan polymer to the cell surface, and (iii) the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of resulting biofilms, which is particularly important for the interactions of bacteria with higher organisms - leading to rhizosphere colonization and modulating the virulence of cellulose-producing bacterial pathogens inside and outside of host cells. We review the organization of four principal types of cellulose synthase operon found in various bacterial genomes, identify additional bcs genes that encode components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms and in the choice between acute infection and persistence in the host. PMID:26077867

  15. Spectroscopic properties of Carcinus aestuarii hemocyanin and its structural subunits

    NASA Astrophysics Data System (ADS)

    Dolashka-Angelova, Pavlina; Hristova, Rumiyana; Stoeva, Stanka; Voelter, Wolfgang

    1999-12-01

    Hemocyanin (Hc) of Carcinus aestuarii contains three major and one minor electrophoretically separable polypeptide chains which were purified by fast protein liquid chromatography (FPLC) ion exchange chromatography. N-terminal amino acid sequences of four structural subunits (SSs) from C. aestuarii were compared with known N-terminal sequences from other arthropodan hemocyanins. The conformational changes, induced by various treatments, were monitored by far UV, CD and fluorescence spectroscopy. The critical temperatures for the structural subunits, Tc, determined by fluorescence spectroscopy, are in the region of 52-59°C and coincide with the melting temperatures, Tm (49-55°C), determined by CD spectroscopy. The free energy of stabilization in water, Δ GDH 2O , toward guanidinium hydrochloride is about 1.3 times higher for the dodecameric Hc as compared to the isolated subunits and about one time higher for Ca1, comparing with other SSs. The studies reveal that the conformational stability of the native dodecamer towards various denaturants (temperature and guanidinium hydrochloride) indicate that the quaternary structure is stabilized by oligomerization between structural subunits, and the possibility of a structural role of the sugar mojeties cannot be excluded.

  16. The receptor subunits generating NMDA receptor mediated currents in oligodendrocytes

    PubMed Central

    Burzomato, Valeria; Frugier, Guillaume; Pérez-Otaño, Isabel; Kittler, Josef T; Attwell, David

    2010-01-01

    NMDA receptors have been shown to contribute to glutamate-evoked currents in oligodendrocytes. Activation of these receptors damages myelin in ischaemia, in part because they are more weakly blocked by Mg2+ than are most neuronal NMDA receptors. This weak Mg2+ block was suggested to reflect an unusual subunit composition including the NR2C and NR3A subunits. Here we expressed NR1/NR2C and triplet NR1/NR2C/NR3A recombinant receptors in HEK cells and compared their currents with those of NMDA-evoked currents in rat cerebellar oligodendrocytes. NR1/NR2C/3A receptors were less blocked by 2 mm Mg2+ than were NR1/NR2C receptors (the remaining current was 30% and 18%, respectively, of that seen without added Mg2+) and showed less channel noise, suggesting a smaller single channel conductance. NMDA-evoked currents in oligodendrocytes showed a Mg2+ block (to 32%) similar to that observed for NR1/NR2C/NR3A and significantly different from that for NR1/NR2C receptors. Co-immunoprecipitation revealed interactions between NR1, NR2C and NR3A subunits in a purified myelin preparation from rat brain. These data are consistent with NMDA-evoked currents in oligodendrocytes reflecting the activation of receptors containing NR1, NR2C and NR3A subunits. PMID:20660562

  17. Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

    PubMed Central

    McCrea, P D; Popot, J L; Engelman, D M

    1987-01-01

    Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit. PMID:3428268

  18. Two orthogonal cleavages separate subunit RNAs in mouse ribosome biogenesis

    PubMed Central

    Wang, Minshi; Anikin, Leonid; Pestov, Dimitri G.

    2014-01-01

    Ribosome biogenesis is a dynamic multistep process, many features of which are still incompletely documented. Here, we show that changes in this pathway can be captured and annotated by means of a graphic set of pre-rRNA ratios, a technique we call Ratio Analysis of Multiple Precursors (RAMP). We find that knocking down a ribosome synthesis factor produces a characteristic RAMP profile that exhibits consistency across a range of depletion levels. This facilitates the inference of affected steps and simplifies comparative analysis. We applied RAMP to examine how endonucleolytic cleavages of the mouse pre-rRNA transcript in the internal transcribed spacer 1 (ITS1) are affected by depletion of factors required for maturation of the small ribosomal subunit (Rcl1, Fcf1/Utp24, Utp23) and the large subunit (Pes1, Nog1). The data suggest that completion of early maturation in a subunit triggers its release from the common pre-rRNA transcript by stimulating cleavage at the proximal site in ITS1. We also find that splitting of pre-rRNA in the 3′ region of ITS1 is prevalent in adult mouse tissues and quiescent cells, as it is in human cells. We propose a model for subunit separation during mammalian ribosome synthesis and discuss its implications for understanding pre-rRNA processing pathways. PMID:25190460

  19. Emergence of ion channel modal gating from independent subunit kinetics.

    PubMed

    Bicknell, Brendan A; Goodhill, Geoffrey J

    2016-09-01

    Many ion channels exhibit a slow stochastic switching between distinct modes of gating activity. This feature of channel behavior has pronounced implications for the dynamics of ionic currents and the signaling pathways that they regulate. A canonical example is the inositol 1,4,5-trisphosphate receptor (IP3R) channel, whose regulation of intracellular Ca(2+) concentration is essential for numerous cellular processes. However, the underlying biophysical mechanisms that give rise to modal gating in this and most other channels remain unknown. Although ion channels are composed of protein subunits, previous mathematical models of modal gating are coarse grained at the level of whole-channel states, limiting further dialogue between theory and experiment. Here we propose an origin for modal gating, by modeling the kinetics of ligand binding and conformational change in the IP3R at the subunit level. We find good agreement with experimental data over a wide range of ligand concentrations, accounting for equilibrium channel properties, transient responses to changing ligand conditions, and modal gating statistics. We show how this can be understood within a simple analytical framework and confirm our results with stochastic simulations. The model assumes that channel subunits are independent, demonstrating that cooperative binding or concerted conformational changes are not required for modal gating. Moreover, the model embodies a generally applicable principle: If a timescale separation exists in the kinetics of individual subunits, then modal gating can arise as an emergent property of channel behavior. PMID:27551100

  20. Abnormal subcellular localization of GABAA receptor subunits in schizophrenia brain.

    PubMed

    Mueller, T M; Remedies, C E; Haroutunian, V; Meador-Woodruff, J H

    2015-01-01

    Inhibitory neurotransmission is primarily mediated by γ-aminobutyric acid (GABA) activating synaptic GABA type A receptors (GABA(A)R). In schizophrenia, presynaptic GABAergic signaling deficits are among the most replicated findings; however, postsynaptic GABAergic deficits are less well characterized. Our lab has previously demonstrated that although there is no difference in total protein expression of the α1-6, β1-3 or γ2 GABA(A)R subunits in the superior temporal gyrus (STG) in schizophrenia, the α1, β1 and β2 GABA(A)R subunits are abnormally N-glycosylated. N-glycosylation is a posttranslational modification that has important functional roles in protein folding, multimer assembly and forward trafficking. To investigate the impact that altered N-glycosylation has on the assembly and trafficking of GABA(A)Rs in schizophrenia, this study used western blot analysis to measure the expression of α1, α2, β1, β2 and γ2 GABA(A)R subunits in subcellular fractions enriched for endoplasmic reticulum (ER) and synapses (SYN) from STG of schizophrenia (N = 16) and comparison (N = 14) subjects and found evidence of abnormal localization of the β1 and β2 GABA(A)R subunits and subunit isoforms in schizophrenia. The β2 subunit is expressed as three isoforms at 52 kDa (β2(52 kDa)), 50 kDa (β2(50 kDa)) and 48 kDa (β2(48 kDa)). In the ER, we found increased total β2 GABA(A)R subunit (β2(ALL)) expression driven by increased β2(50 kDa), a decreased ratio of β(248 kDa):β2(ALL) and an increased ratio of β2(50 kDa):β2(48 kDa). Decreased ratios of β1:β2(ALL) and β1:β2(50 kDa) in both the ER and SYN fractions and an increased ratio of β2(52 kDa):β(248 kDa) at the synapse were also identified in schizophrenia. Taken together, these findings provide evidence that alterations of N-glycosylation may contribute to GABAergic signaling deficits in schizophrenia by disrupting the assembly and trafficking of GABA(A)Rs. PMID:26241350

  1. Abnormal subcellular localization of GABAA receptor subunits in schizophrenia brain

    PubMed Central

    Mueller, T M; Remedies, C E; Haroutunian, V; Meador-Woodruff, J H

    2015-01-01

    Inhibitory neurotransmission is primarily mediated by γ-aminobutyric acid (GABA) activating synaptic GABA type A receptors (GABAAR). In schizophrenia, presynaptic GABAergic signaling deficits are among the most replicated findings; however, postsynaptic GABAergic deficits are less well characterized. Our lab has previously demonstrated that although there is no difference in total protein expression of the α1–6, β1–3 or γ2 GABAAR subunits in the superior temporal gyrus (STG) in schizophrenia, the α1, β1 and β2 GABAAR subunits are abnormally N-glycosylated. N-glycosylation is a posttranslational modification that has important functional roles in protein folding, multimer assembly and forward trafficking. To investigate the impact that altered N-glycosylation has on the assembly and trafficking of GABAARs in schizophrenia, this study used western blot analysis to measure the expression of α1, α2, β1, β2 and γ2 GABAAR subunits in subcellular fractions enriched for endoplasmic reticulum (ER) and synapses (SYN) from STG of schizophrenia (N=16) and comparison (N=14) subjects and found evidence of abnormal localization of the β1 and β2 GABAAR subunits and subunit isoforms in schizophrenia. The β2 subunit is expressed as three isoforms at 52 kDa (β252 kDa), 50 kDa (β250 kDa) and 48 kDa (β248 kDa). In the ER, we found increased total β2 GABAAR subunit (β2ALL) expression driven by increased β250 kDa, a decreased ratio of β248 kDa:β2ALL and an increased ratio of β250 kDa:β248 kDa. Decreased ratios of β1:β2ALL and β1:β250 kDa in both the ER and SYN fractions and an increased ratio of β252 kDa:β248 kDa at the synapse were also identified in schizophrenia. Taken together, these findings provide evidence that alterations of N-glycosylation may contribute to GABAergic signaling deficits in schizophrenia by disrupting the assembly and trafficking of GABAARs. PMID:26241350

  2. Succinate dehydrogenase subunit D and succinate dehydrogenase subunit B mutation analysis in canine phaeochromocytoma and paraganglioma.

    PubMed

    Holt, D E; Henthorn, P; Howell, V M; Robinson, B G; Benn, D E

    2014-07-01

    Phaeochromocytomas (PCs) are tumours of the adrenal medulla chromaffin cells. Paragangliomas (PGLs) arise in sympathetic ganglia (previously called extra-adrenal PCs) or in non-chromaffin parasympathetic ganglia cells that are usually non-secretory. Parenchymal cells from these tumours have a common embryological origin from neural crest ectoderm. Several case series of canine PCs and PGLs have been published and a link between the increased incidence of chemoreceptor neoplasia in brachycephalic dog breeds and chronic hypoxia has been postulated. A similar link to hypoxia in man led to the identification of germline heterozygous mutations in the gene encoding succinate dehydrogenase subunit D (SDHD) and subsequently SDHA, SDHB and SDHC in similar tumours. We investigated canine PCs (n = 6) and PGLs (n = 2) for SDHD and SDHB mutations and in one PGL found a somatic SDHD mutation c.365A>G (p.Lys122Arg) in exon 4, which was not present in normal tissue from this brachycephalic dog. Two PCs were heterozygous for both c.365A>G (p.Lys122Arg) mutation and an exon 3 silent variant c.291G>A. We also identified the heterozygous SDHB exon 2 mutation c.113G>A (p.Arg38Gln) in a PC. These results illustrate that genetic mutations may underlie tumourigenesis in canine PCs and PGLs. The spontaneous nature of these canine diseases and possible association of PGLs with hypoxia in brachycephalic breeds may make them an attractive model for studying the corresponding human tumours. PMID:24813157

  3. THE GSTP1 c.313A>G POLYMORPHISM MODULATES THE CARDIORESPIRATORY RESPONSE TO AEROBIC TRAINING.

    PubMed

    Zarebska, A; Jastrzebski, Z; Kaczmarczyk, M; Ficek, K; Maciejewska-Karlowska, A; Sawczuk, M; Leońska-Duniec, A; Krol, P; Cieszczyk, P; Zmijewski, P; Eynon, N

    2014-12-01

    The GSTP1 c.313A>G polymorphism is a candidate to explain some of the individual differences in cardiorespiratory fitness phenotypes' responses to aerobic exercise training. We aim to explore the association between the GSTP1 c.313A>G polymorphism and the response to low-high impact aerobic exercise training. Sixty-six Polish Caucasian women were genotyped for the GSTP1 c.313A>G polymorphism; 62 of them completed 12-week aerobic (50-75% HRmax) exercise training and were measured for selected somatic features (body mass and BMI) and cardiorespiratory fitness indices - maximal oxygen uptake (VO2max, maximum heart rate (HRmax), maximum ventilation (VEmax) and anaerobic threshold (AT) - before and after the training period. Two-factor analysis of variance revealed a main training effect for body mass reduction (p=0.007) and BMI reduction (p=0.013), improvements of absolute and relative VO2max (both p<0.001), and increased VEmax (p=0.005), but not for changes in fat-free mass (FFM) (p=0.162). However, a significant training x GSTP1 c.313A>G interaction was found only for FFM (p=0.042), absolute and relative VO2max (p=0.029 and p=0.026), and VEmax (p=0.005). As the result of training, significantly greater improvements in VO2max, VEmax and FFM were gained by the GG+GA group compared to the AA genotype group. The results support the hypothesis that heterogeneity in individual response to training stimuli is at least in part determined by genetics, and GSTP1 c.313A>G may be considered as one (of what appear to be many) target polymorphisms to influence these changes. PMID:25435667

  4. THE GSTP1 c.313A>G POLYMORPHISM MODULATES THE CARDIORESPIRATORY RESPONSE TO AEROBIC TRAINING

    PubMed Central

    Zarebska, A; Jastrzebski, Z; Kaczmarczyk, M; Ficek, K; Maciejewska-Karlowska, A; Sawczuk, M; Leońska-Duniec, A; Krol, P; Cieszczyk, P; Zmijewski, P

    2014-01-01

    The GSTP1 c.313A>G polymorphism is a candidate to explain some of the individual differences in cardiorespiratory fitness phenotypes’ responses to aerobic exercise training. We aim to explore the association between the GSTP1 c.313A>G polymorphism and the response to low-high impact aerobic exercise training. Sixty-six Polish Caucasian women were genotyped for the GSTP1 c.313A>G polymorphism; 62 of them completed 12-week aerobic (50-75% HRmax) exercise training and were measured for selected somatic features (body mass and BMI) and cardiorespiratory fitness indices – maximal oxygen uptake (VO2max, maximum heart rate (HRmax), maximum ventilation (VEmax) and anaerobic threshold (AT) – before and after the training period. Two-factor analysis of variance revealed a main training effect for body mass reduction (p=0.007) and BMI reduction (p=0.013), improvements of absolute and relative VO2max (both p<0.001), and increased VEmax (p=0.005), but not for changes in fat-free mass (FFM) (p=0.162). However, a significant training x GSTP1 c.313A>G interaction was found only for FFM (p=0.042), absolute and relative VO2max (p=0.029 and p=0.026), and VEmax (p=0.005). As the result of training, significantly greater improvements in VO2max, VEmax and FFM were gained by the GG+GA group compared to the AA genotype group. The results support the hypothesis that heterogeneity in individual response to training stimuli is at least in part determined by genetics, and GSTP1 c.313A>G may be considered as one (of what appear to be many) target polymorphisms to influence these changes. PMID:25435667

  5. Design of Mutant β2 Subunits as Decoy Molecules to Reduce the Expression of Functional Ca2+ Channels in Cardiac Cells

    PubMed Central

    Télémaque, Sabine; Sonkusare, Swapnil; Grain, Terrie; Rhee, Sung W.; Stimers, Joseph R.; Rusch, Nancy J.; Marsh, James D.

    2016-01-01

    Calcium influx through long-lasting (“L-type”) Ca2+ channels (CaV) drives excitation-contraction in the normal heart. Dysregulation of this process contributes to Ca2+ overload, and interventions that reduce expression of the pore-forming α1 subunit may alleviate cytosolic Ca2+ excess. As a molecular approach to disrupt the assembly of CaV1.2 (α1C) channels at the cell membrane, we targeted the Ca2+ channel β2 subunit, an intracellular chaperone that interacts with α1C via its β interaction domain (BID) to promote CaV1.2 channel expression. Recombinant adenovirus expressing either the full β2 subunit (Full-β2) or truncated β2 subunit constructs lacking either the C terminus, N terminus, or both (N-BID, C-BID, and BID, respectively) fused to green fluorescent protein were developed as potential decoys and overexpressed in HL-1 cells. Fluorescence microscopy revealed that the localization of Full-β2 at the surface membrane was associated with increased Ca2+ current mainly attributed to CaV1.2 channels. In contrast, truncated N-BID and C-BID constructs showed punctate intracellular expression, and BID showed a diffuse cytosolic distribution. Total expression of the α1C protein of CaV1.2 channels was similar between groups, but HL-1 cells overexpressing C-BID and BID exhibited reduced Ca2+ current. C-BID and BID also attenuated Ca2+ current associated with another L-type Ca2+ channel, CaV1.3, but they did not reduce transient Ca2+ currents attributed to CaV3 channels. These results suggest that β2 subunit mutants lacking the N terminus may preferentially disrupt the proper localization of L-type Ca2+ channels in the cell membrane. Cardiac-specific delivery of these decoy molecules in vivo may represent a gene-based treatment for pathologies involving Ca2+ overload. PMID:18184831

  6. 1-Nitropyrene (1-NP) induces apoptosis and apparently a non-apoptotic programmed cell death (paraptosis) in Hepa1c1c7 cells

    SciTech Connect

    Asare, Nana Landvik, Nina E.; Lagadic-Gossmann, Dominique; Rissel, Mary; Tekpli, Xavier; Ask, Kjetil; Lag, Marit; Holme, Jorn A.

    2008-07-15

    Mechanistic studies of nitro-PAHs (polycyclic aromatic hydrocarbons) of interest might help elucidate which chemical characteristics are most important in eliciting toxic effects. 1-Nitropyrene (1-NP) is the predominant nitrated PAH emitted in diesel exhaust. 1-NP-exposed Hepa1c1c7 cells exhibited marked changes in cellular morphology, decreased proliferation and different forms of cell death. A dramatic increase in cytoplasmic vacuolization was observed already after 6 h of exposure and the cells started to round up at 12 h. The rate of cell proliferation was markedly reduced at 24 h and apoptotic as well as propidium iodide (PI)-positive cells appeared. Electron microscopic examination revealed that the vacuolization was partly due to mitochondria swelling. The caspase inhibitor Z-VAD-FMK inhibited only the apoptotic cell death and Nec-1 (an inhibitor of necroptosis) exhibited no inhibitory effects on either cell death or vacuolization. In contrast, cycloheximide markedly reduced both the number of apoptotic and PI-positive cells as well as the cytoplasmic vacuolization, suggesting that 1-NP induced paraptotic cell death. All the MAPKs; ERK1/2, p38 and JNK, appear to be involved in the death process since marked activation was observed upon 1-NP exposure, and their inhibitors partly reduced the induced cell death. The ERK1/2 inhibitor PD 98057 completely blocked the induced vacuolization, whereas the other MAPKs inhibitors only had minor effects on this process. These findings suggest that 1-NP may cause apoptosis and paraptosis. In contrast, the corresponding amine (1-aminopyrene) elicited only minor apoptotic and necrotic cell death, and cells with characteristics typical of paraptosis were absent.

  7. 1-Nitropyrene (1-NP) induces apoptosis and apparently a non-apoptotic programmed cell death (paraptosis) in Hepa1c1c7 cells.

    PubMed

    Asare, Nana; Landvik, Nina E; Lagadic-Gossmann, Dominique; Rissel, Mary; Tekpli, Xavier; Ask, Kjetil; Låg, Marit; Holme, Jørn A

    2008-07-15

    Mechanistic studies of nitro-PAHs (polycyclic aromatic hydrocarbons) of interest might help elucidate which chemical characteristics are most important in eliciting toxic effects. 1-Nitropyrene (1-NP) is the predominant nitrated PAH emitted in diesel exhaust. 1-NP-exposed Hepa1c1c7 cells exhibited marked changes in cellular morphology, decreased proliferation and different forms of cell death. A dramatic increase in cytoplasmic vacuolization was observed already after 6 h of exposure and the cells started to round up at 12 h. The rate of cell proliferation was markedly reduced at 24 h and apoptotic as well as propidium iodide (PI)-positive cells appeared. Electron microscopic examination revealed that the vacuolization was partly due to mitochondria swelling. The caspase inhibitor Z-VAD-FMK inhibited only the apoptotic cell death and Nec-1 (an inhibitor of necroptosis) exhibited no inhibitory effects on either cell death or vacuolization. In contrast, cycloheximide markedly reduced both the number of apoptotic and PI-positive cells as well as the cytoplasmic vacuolization, suggesting that 1-NP induced paraptotic cell death. All the MAPKs; ERK1/2, p38 and JNK, appear to be involved in the death process since marked activation was observed upon 1-NP exposure, and their inhibitors partly reduced the induced cell death. The ERK1/2 inhibitor PD 98057 completely blocked the induced vacuolization, whereas the other MAPKs inhibitors only had minor effects on this process. These findings suggest that 1-NP may cause apoptosis and paraptosis. In contrast, the corresponding amine (1-aminopyrene) elicited only minor apoptotic and necrotic cell death, and cells with characteristics typical of paraptosis were absent. PMID:18417179

  8. Benzo[a]pyrene, 3-methylcholanthrene and beta-naphthoflavone induce oxidative stress in hepatoma hepa 1c1c7 Cells by an AHR-dependent pathway.

    PubMed

    Elbekai, Reem H; Korashy, Hesham M; Wills, Kelly; Gharavi, Negar; El-Kadi, Ayman O S

    2004-11-01

    Polycyclic aromatic hydrocarbons have been shown to cause oxidative stress in vitro and in vivo in various animal models but the mechanisms by which these compounds produce oxidative stress are unknown. In the current study we have investigated the role of the aryl hydrocarbon receptor (AHR) in the production of reactive oxygen species (ROS) by its cognate ligands and the consequent effect on cyp1a1 activity, mRNA and protein expressions. For this purpose, Hepa 1c1c7 cells wild-type (WT) and C12 mutant cells, which are AHR-deficient, were incubated with increasing concentrations of the AHR-ligands, benzo[a]pyrene (B[a]P, 0.25-25 microM), 3-methylcholanthrene (3MC, 0.1-10 microM) and beta-naphthoflavone (betaNF, 1-50 microM). The studied AHR-ligands dose-dependently increased lipid peroxidation in WT but not in C12 cells. However, only B[a]P and betaNF, at the highest concentrations tested, significantly increased H2O2 production in WT but not C12 cells. The increase in lipid peroxidation and H2O2 production by AHR-ligands were accompanied by a decrease in the cyp1a1 catalytic activity but not mRNA or protein expressions, which were significantly induced in a dose-dependent manner by all AHR-ligands, suggesting a post-translational mechanism is involved in the decrease of cyp1a1 activity. The AHR-ligand-mediated decrease in cyp1a1 activity was reversed by the antioxidant N-acetylcysteine. Our results show that the AHR-ligands induce oxidative stress by an AHR-dependent pathway. PMID:15621696

  9. Down-regulation of aryl hydrocarbon receptor-regulated genes by tumor necrosis factor-alpha and lipopolysaccharide in murine hepatoma Hepa 1c1c7 cells.

    PubMed

    Gharavi, Negar; El-Kadi, Ayman O S

    2005-03-01

    Although much is known concerning the effects of inflammation and oxidative stress on the cytochrome P450 1A1 (CYP1A1), little is known about the modulation of other aryl hydrocarbon receptor (AHR)-regulated genes such as glutathione-S-transferase Ya (GST Ya) and NAD(P)H:quinone oxidoreductase (QOR) by inflammation. In the present study, the effect of tumor necrosis factor (TNF)-alpha and lipopolysaccharides (LPS) on the constitutive and inducible expression of the AHR-regulated genes cyp1a1, GST Ya, and QOR was determined in murine hepatoma Hepa 1c1c7 (WT), AHR-deficient (C12), and AHR nuclear translocator protein (ARNT)-deficient (C4) cells. We found that both TNF-alpha and LPS strongly repressed the constitutive expression and the beta-naphthoflavone-mediated induction of cyp1a1, GST Ya, and QOR in WT but not in C12 and C4 cells. The induction of GST Ya and QOR activities and mRNA levels by phenolic antioxidant, tert-butylhydroquinone, through the antioxidant response element was not significantly affected by TNF-alpha or LPS. In addition, a significant increase in reactive oxygen species was observed in WT, C12, and C4 cells treated with TNF-alpha or LPS which was completely prevented by tert-butylhydroquinone. These results show that the down-regulation of AHR-regulated genes by TNF-alpha and LPS is dependent on the presence of both heterodimeric transcription factors, AHR and ARNT. Furthermore, reactive oxygen species may be involved in the down-regulation of AHR-regulated genes. PMID:15627257

  10. Down-regulation of the detoxifying enzyme NAD(P)H:quinone oxidoreductase 1 by vanadium in Hepa 1c1c7 cells.

    PubMed

    Anwar-Mohamed, Anwar; El-Kadi, Ayman O S

    2009-05-01

    Recent data suggest that vanadium (V5+) compounds exert protective effects against chemical-induced carcinogenesis, mainly through modifying various xenobiotic metabolizing enzymes. In fact, we have shown that V5+ down-regulates the expression of Cyp1a1 at the transcriptional level through an ATP-dependent mechanism. However, incongruously, there is increasing evidence that V5+ is found in higher amounts in cancer cells and tissues than in normal cells or tissues. Therefore, the current study aims to address the possible effect of this metal on the regulation of expression of an enzyme that helps maintain endogenous antioxidants used to protect tissues/cells from mutagens, carcinogens, and oxidative stress damage, NAD(P) H:quinone oxidoreductase 1 (Nqo1). In an attempt to examine these effects, Hepa 1c1c7 cells and its AhRdeficient version, c12, were treated with increasing concentrations of V5+ in the presence of two distinct Nqo1 inducers, the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and isothiocyanate sulforaphane (SUL). Our results showed that V5+ inhibits the TCDD- and SUL-mediated induction of Nqo1 at mRNA, protein, and catalytic activity levels. At transcriptional level, V5+ was able to decrease the TCDD- and SUL-induced nuclear accumulation of Nrf2 and the subsequent binding to antioxidant responsive element (ARE) without affecting Nrf2 protein levels. Looking at post-transcriptional level; we found that V5+ did not affect Nqo1 mRNA transcripts turn-over rates. However, at the post-translational level V5+ increased Nqo1 protein half-life. In conclusion, the present study demonstrates that V5+ down-regulates Nqo1 at the transcriptional level, possibly through inhibiting the ATP-dependent activation of Nrf2. PMID:19367690

  11. Induction of cytochrome P450 1A1 gene expression by a vitamin K3 analog in mouse hepatoma Hepa-1c1c7 cells.

    PubMed

    Chun, Y J; Lee, B Y; Yang, S A; Ryu, C K; Kim, M Y

    2001-10-31

    Nine vitamin K3 analogs were compared with respect to the induction of the cytochrome P450 1A1 (CYP1A1) expression in mouse hepatoma Hepa-1c1c7 cells. 6-(4-Diethylamino)phenyl-7-chloro-5,8-quinolinedione (EA4) caused a significant induction of the CYP1A1-mediated ethoxyresorufin O-deethylase activity in a time- and concentration-dependent manner. The induction was accompanied by an increase of the Cyp1a1 mRNA transcription. The transient expression of the mouse Cyp1a1-CAT gene into cells showed that EA4 induced CAT activity. However, the aryl hydrocarbon receptor and its nuclear partner, aryl hydrocarbon receptor nuclear translocator mRNA transcription, were unaffected by the EA4 treatment. When the cells were incubated with EA4 in the presence of 1 nM TCDD, the ethoxyresorufin O-deethylase activity that was induced by TCDD was significantly suppressed by EA4. Inhibition of protein synthesis by cycloheximide strongly enhanced the EA4-dependent Cyp1a1 mRNA expression. Up-regulation of protein kinase C by a 2 h preincubation with phorbol 12-myristate 13-acetate increased the EA4-dependent expression of the Cyp1a1 gene. In human cells, such as HepG2 (human hepatocarcinoma), MCF-7 (human breast adenocarcinoma cell line), and HL-60 (human promyelocytic cell line), the expression of CYP1A1 mRNA was also induced by EA4 treatment. Moreover, CYP1B1 mRNA was increased by EA4 in MCF-7 cells. These results indicate that EA4 modulates CYP1A1 and CYP1B1 expressions by transcriptional activation. Also, protein kinase C may be involved in the induction mechanism of CYP1A1 by EA4. PMID:11710520

  12. Signalling pathways involved in 1-nitropyrene (1-NP)-induced and 3-nitrofluoranthene (3-NF)-induced cell death in Hepa1c1c7 cells.

    PubMed

    Asare, Nana; Tekpli, Xavier; Rissel, Mary; Solhaug, Anita; Landvik, Nina; Lecureur, Valerie; Podechard, Normand; Brunborg, Gunnar; Låg, Marit; Lagadic-Gossmann, Dominique; Holme, Jørn A

    2009-11-01

    We previously reported that 1-nitropyrene (1-NP) and 3-nitrofluoranthene (3-NF) elicited apoptotic cell death as well as non-apoptotic programmed cell deaths (PCDs) with paraptotic and necroptotic characteristics, respectively. In the present study, we have further confirmed and extended these findings. Flow cytometric analyses of 1-NP-exposed/3NF-exposed Hepa1c1c7 cells revealed that caspase-3 was only activated in the subpopulation of cells corresponding to that with classic apoptotic morphology. Immunocytochemical analysis indicated that leucocyte elastase inhibitor-derived DNaseII (LEI/L-DNaseII), apoptosis-inducing factor (AIF) and endonuclease G (EndoG) were more clearly translocated to the nucleus following 3-NF exposure than after 1-NP. These 3-NF-induced changes in AIF and EndoG translocation were reduced by necrostatin-1, an inhibitor of necroptotic cell death. Both compounds lead to accumulation of lipid droplets and induced DNA damage. Activation of checkpoint kinase (CHK) 1 and H2AX, but not ataxia telangiectasia mutated and CHK2, were observed. Furthermore, inhibition of p53 using pifithrin-alpha reduced the cell death induced by both compounds, suggesting a role of DNA damage/CHK1/p53 pathway in the death process. 1-NP-induced cell death was in addition characterized by increased oxidative damage and intracellular accumulation of Ca(2+). These findings further support the notion that 1-NP elicited apoptotic cell death and PCD with paraptotic characteristics, while 3-NF induced apoptosis and a PCD with necroptotic features. PMID:19703935

  13. Heterotrimeric G protein subunit Gγ13 is critical to olfaction

    PubMed Central

    Li, Feng; Ponissery-Saidu, Samsudeen; Yee, Karen; Wang, Hong; Chen, Meng-Ling; Iguchi, Naoko; Zhang, Genhua; Jiang, Ping; Reisert, Johannes; Huang, Liquan

    2013-01-01

    The activation of G-protein-coupled olfactory receptors on the olfactory sensory neurons (OSNs) triggers a signaling cascade, which is mediated by a heterotrimeric G protein consisting of α, β and γ subunits. Although its α subunit, Gαolf, has been identified and well characterized, the identities of its β and γ subunits and their function in olfactory signal transduction, however, have not been well established yet. We and others have found the expression of Gγ13 in the olfactory epithelium, particularly in the cilia of the OSNs. In this study, we generated a conditional gene knockout mouse line to specifically nullify Gγ13 expression in the olfactory marker protein-expressing OSNs. Immunohistochemical and Western blot results showed that Gγ13 subunit was indeed eliminated in the mutant mice’s olfactory epithelium. Intriguingly, Gαolf, β1 subunits, Ric-8B and CEP290 proteins were also absent in the epithelium whereas the presence of the effector enzyme adenylyl cyclase III remained largely unaltered. Electro-olfactogram studies showed that the mutant animals had greatly reduced responses to a battery of odorants including three presumable pheromones. Behavioral tests indicated that the mutant mice had a remarkably reduced ability to perform an odor-guided search task although their motivation and agility seemed normal. Our results indicate that Gαolf exclusively forms a functional heterotrimeric G protein with Gβ1 and Gγ13 in OSNs, mediating olfactory signal transduction. The identification of the olfactory G protein’s βγ moiety has provided a novel approach to understanding the feedback regulation of olfactory signal transduction pathways as well as the control of subcellular structures of OSNs. PMID:23637188

  14. Antibodies to GABAA receptor α1 and γ2 subunits

    PubMed Central

    Pettingill, Philippa; Kramer, Holger B.; Coebergh, Jan Adriaan; Pettingill, Rosie; Maxwell, Susan; Nibber, Anjan; Malaspina, Andrea; Jacob, Anu; Irani, Sarosh R.; Buckley, Camilla; Beeson, David; Lang, Bethan; Waters, Patrick

    2015-01-01

    Objective: To search for antibodies against neuronal cell surface proteins. Methods: Using immunoprecipitation from neuronal cultures and tandem mass spectrometry, we identified antibodies against the α1 subunit of the γ-aminobutyric acid A receptor (GABAAR) in a patient whose immunoglobulin G (IgG) antibodies bound to hippocampal neurons. We searched 2,548 sera for antibodies binding to GABAAR α, β, and γ subunits on live HEK293 cells and identified the class, subclass, and GABAAR subunit specificities of the positive samples. Results: GABAAR-Abs were identified in 40 of 2,046 (2%) referred sera previously found negative for neuronal antibodies, in 5/502 (1%) previously positive for other neuronal surface antibodies, but not in 92 healthy individuals. The antibodies in 40% bound to either the α1 (9/45, 20%) or the γ2 subunits (9/45, 20%) and were of IgG1 (94%) or IgG3 (6%) subclass. The remaining 60% had lower antibody titers (p = 0.0005), which were mainly immunoglobulin M (IgM) (p = 0.0025), and showed no defined subunit specificity. Incubation of primary hippocampal neurons with GABAAR IgG1 sera reduced surface GABAAR membrane expression. The clinical features of 15 patients (GABAAR α1 n = 6, γ2 n = 5, undefined n = 4) included seizures (47%), memory impairment (47%), hallucinations (33%), or anxiety (20%). Most patients had not been given immunotherapies, but one with new-onset treatment-resistant catatonia made substantial improvement after plasma exchange. Conclusions: The GABAAR α1 and γ2 are new targets for antibodies in autoimmune neurologic disease. The full spectrum of clinical features, treatment responses, correlation with antibody specificity, and in particular the role of the IgM antibodies will need to be assessed in future studies. PMID:25636713

  15. Ion channels, channelopathies, and tooth formation.

    PubMed

    Duan, X

    2014-02-01

    The biological functions of ion channels in tooth development vary according to the nature of their gating, the species of ions passing through those gates, the number of gates, localization of channels, tissue expressing the channel, and interactions between cells and microenvironment. Ion channels feature unique and specific ion flux in ameloblasts, odontoblasts, and other tooth-specific cell lineages. Both enamel and dentin have active chemical systems orchestrating a variety of ion exchanges and demineralization and remineralization processes in a stage-dependent manner. An important role for ion channels is to regulate and maintain the calcium and pH homeostasis that are critical for proper enamel and dentin biomineralization. Specific functions of chloride channels, TRPVs, calcium channels, potassium channels, and solute carrier superfamily members in tooth formation have been gradually clarified in recent years. Mutations in these ion channels or transporters often result in disastrous changes in tooth development. The channelopathies of tooth include altered eruption (CLCN7, KCNJ2, TRPV3), root dysplasia (CLCN7, KCNJ2), amelogenesis imperfecta (KCNJ1, CFTR, AE2, CACNA1C, GJA1), dentin dysplasia (CLCN5), small teeth (CACNA1C, GJA1), tooth agenesis (CLCN7), and other impairments. The mechanisms leading to tooth channelopathies are primarily related to pH regulation, calcium homeostasis, or other alterations of the niche for tooth eruption and development. PMID:24076519

  16. The ratio of glycated albumin to hemoglobin A1c measured in IFCC units accurately represents the glycation gap.

    PubMed

    Akatsuka, Junya; Mochizuki, Mie; Musha, Ikuma; Ohtake, Akira; Kobayashi, Kisho; Kikuchi, Toru; Kikuchi, Nobuyuki; Kawamura, Tomoyuki; Urakami, Tatsuhiko; Sugihara, Shigetaka; Hoshino, Tadao; Amemiya, Shin

    2015-01-01

    The glycation gap (G-gap: difference between measured hemoglobin A1c [A1C] and the value predicted by its regression on the fructosamine level) is stable and associated with diabetic complications. Measuring A1C level in International Federation of Clinical Chemistry (IFCC) units (A1C-SI; mmol/mol) and National Glycohemoglobin Standardization Program units (A1C-NGSP; %) and using glycated albumin (GA) level instead of fructosamine level for calculating the G-gap, we investigated whether the G-gap is better represented by GA/A1C ratio if expressed in SI units (GA/A1C-SI ratio) rather than in NGSP units (GA/A1C-% ratio). We examined 749 Japanese children with type 1 diabetes using simultaneous GA and A1C measurements. Of these, 369 patients were examined more than five times to assess the consistency of the G-gap and the GA/A1C ratio within individuals. The relationship of GA/A1C-% ratio to the corresponding A1C-NGSP was stronger than that of GA/A1C-SI ratio to A1C-IFCC. At enrollment, the inverse relationship between the GA/A1C-SI ratio and G-gap was highly significant (R(2) = 0.95) compared with that between the GA/A1C-% ratio and G-gap (R(2) = 0.69). A highly significant inverse relationship was also observed between the mean GA/A1C-SI ratio and the mean G-gaps obtained individually over time (R(2) = 0.95) compared with that using the corresponding A1C-NGSP (R(2) = 0.67). We conclude that the G-gap is better represented by the GA/A1C-SI ratio. We propose the use of mean GA/A1C-SI ratios easily obtained individually over time as reference values in Japanese children with type 1 diabetes (6.75 ± 0.60 [means ± SD]). PMID:25367400

  17. Identification of newly recognized serotype 1c as the most prevalent Shigella flexneri serotype in northern rural Vietnam

    PubMed Central

    STAGG, R. M.; CAM, P. D.; VERMA, N. K.

    2008-01-01

    SUMMARY We investigated the identity of 37 Shigella flexneri strains that had previously been isolated from northern rural Vietnam (Son Tay Province) and described as untypable. Twenty-four isolates reacted with MASF 1c, a monoclonal antibody specific for S. flexneri serotype 1c. A further ten untypable isolates were found to be rough mutants (no longer expressing O-antigen) that were derived from serotype 1c strains. Pulsed-field gel electrophoresis demonstrated that these strains consisted of many different clones, indicating serotype 1c was well established in this region in the late 1990s. Serotype 1c was the most prevalent S. flexneri serotype isolated in the Son Tay Province, accounting for about 40% of S. flexneri isolates. Subsequent isolation of S. flexneri serotype 1c in this region and elsewhere in Vietnam confirmed that serotype 1c is of genuine importance in Vietnam. PMID:17922932

  18. An Analysis of the FY-1C, Iridium 33, and Cosmos 2251 Fragments

    NASA Technical Reports Server (NTRS)

    Liou, J.-C.

    2014-01-01

    The beginning of the year 2013 marks the sixth anniversary of the destruction of the Fengyun-1C (FY-1C) weather satellite as the result of an anti-satellite test conducted by China in January 2007 and the fourth anniversary of the accidental collision between Cosmos 2251 and the operational Iridium 33 in February 2009. These two events represent the worst satellite breakups in history. A total of 5579 fragments have been cataloged by the U.S. Space Surveillance Network (SSN), and almost 5000 of them were still in orbit in January 2013. In addition to these cataloged objects, hundreds of thousands (or more) of fragments down to the millimeter size regime were also generated during the breakups. These fragments are too small to be tracked by the SSN, but are large enough to be a safety concern for human space activities and robotic missions in low Earth orbit (LEO, the region below 2000 km altitude). Like their cataloged siblings, many of them remain in orbit today. These two breakup events dramatically changed the landscape of the orbital debris environment in LEO. The spatial density of the cataloged population in January 2013 is shown as the top blue curve. The combined FY-1C, Iridium 33, and Cosmos 2251 fragments (black curve) account for about 50 percent of the cataloged population below an altitude of 1000 km. They are also responsible for the concentrations at 770 km and 850 km, altitudes at which the collisions occurred. The effects of the FY-1C, Iridium 33, and Cosmos 2251 fragments will continue to be felt for decades to come. For example, approximately half of the generated FY-1C fragments will remain in orbit 20 years from now. In general, the Iridium 33 and Cosmos 2251 fragments will decay faster than the FY-1C fragments because of their lower altitudes. Of the Iridium 33 and Cosmos 2251 fragments, the former have much shorter orbital lifetimes than the latter, because lightweight composite materials were heavily used in the construction of the Iridium

  19. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development

    PubMed Central

    Beier, Anna; Krisp, Christoph; Wolters, Dirk A.

    2016-01-01

    ABSTRACT The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora. Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. PMID:27329756

  20. Relationship of HbA1c variability, absolute changes in HbA1c, and all-cause mortality in type 2 diabetes: a Danish population-based prospective observational study

    PubMed Central

    Skriver, Mette V; Sandbæk, Annelli; Kristensen, Jette K; Støvring, Henrik

    2015-01-01

    Objective We assessed the relationship of mortality with glycated hemoglobin (HbA1c) variability and with absolute change in HbA1c. Design A population-based prospective observational study with a median follow-up time of 6 years. Methods Based on a validated algorithm, 11 205 Danish individuals with type 2 diabetes during 2001–2006 were identified from public data files, with at least three HbA1c measurements: one index measure, one closing measure 22–26 months later, and one measurement in-between. Medium index HbA1c was 7.3%, median age was 63.9 years, and 48% were women. HbA1c variability was defined as the mean absolute residual around the line connecting index value with closing value. Cox proportional hazard models with restricted cubic splines were used, with all-cause mortality as the outcome. Results Variability between 0 and 0.5 HbA1c percentage point was not associated with mortality, but for index HbA1c ≤8% (64 mmol/mol), a variability above 0.5 was associated with increased mortality (HR of 1 HbA1c percentage point variability was 1.3 (95% CI 1.1 to 1.5) for index HbA1c 6.6–7.4%). For index HbA1c≤8%, mortality increased when HbA1c declined, but was stable when HbA1c rose. For index HbA1c>8%, change in HbA1c was associated with mortality, with the lowest mortality for greatest decline (HR=0.9 (95% CI 0.80 to 0.98) for a 2-percentage point decrease). Conclusions For individuals with an index HbA1c below 8%, both high HbA1c variability and a decline in HbA1c were associated with increased mortality. For individuals with index HbA1c above 8%, change in HbA1c was associated with mortality, whereas variability was not. PMID:25664182

  1. Subunit composition and chromophore content of R-phycoerythrin from Porphyra haitanensis

    NASA Astrophysics Data System (ADS)

    Gao, Hong-Feng; Ji, Ming-Hou; Cao, Wen-Da

    1996-03-01

    R-phycoerythrin from Porphyra haitanensis exists in two aggregation states with different molecular weights. A more highly aggregated form, RPE I, was chromatographed on Bio-Rex 70 column with urea solution (pH 3.0) as eluent, and the molecular weights of the 3 subunits (α, β, γ) obtained were determined on SDS-PAGE at 18000, 19200 and 30000, respectively. α subunit carried two phycoerythrobilin (PEB); β subunit, three PEB and one phycourobilin (PUB); γ subunit, one PEB and three PUB chromophores. The molar ratio of α, β, and γ subunits of RPE I was 6: 6: 1, and their subunit composition was confired to be (αβ)6γ on account of the molecular weight of RPE I, 232000. A lower aggregated form, RPE II, contained α and β subunits similar to those of RPE I, but its subunit composition was the (αβ) monomer of RPE.

  2. Voltage-gated Na+ channels: Potential for β subunits as therapeutic targets

    PubMed Central

    Brackenbury, William J.; Isom, Lori L.

    2012-01-01

    Background Voltage gated Na+ channels (VGSCs) contain a pore-forming α subunit and one or more β subunits. VGSCs are involved in a wide variety of pathophysiologies, including epilepsy, cardiac arrhythmia, Multiple Sclerosis, periodic paralysis, migraine, neuropathic and inflammatory pain, Huntington’s disease, and cancer. Increasing evidence implicates the β subunits as key players in these disorders. Objective The purpose of this report is to review the recent literature describing the multifunctional roles of VGSC β subunits in the context of their role(s) in disease. Methods An extensive review of the literature on β subunits was performed. Results/conclusion β subunits are multifunctional. As components of VGSC complexes, β subunits mediate signaling processes regulating electrical excitability, adhesion, migration, pathfinding, and transcription. β subunits may prove useful in disease diagnosis and therapy. PMID:18694383

  3. GABAA receptor beta subunit heterogeneity: functional expression of cloned cDNAs.

    PubMed Central

    Ymer, S; Schofield, P R; Draguhn, A; Werner, P; Köhler, M; Seeburg, P H

    1989-01-01

    Cloned cDNAs encoding two new beta subunits of the rat and bovine GABAA receptor have been isolated using a degenerate oligonucleotide probe based on a highly conserved peptide sequence in the second transmembrane domain of GABAA receptor subunits. The beta 2 and beta 3 subunits share approximately 72% sequence identity with the previously characterized beta 1 polypeptide. Northern analysis showed that both beta 2 and beta 3 mRNAs are more abundant in the brain than beta 1 mRNA. All three beta subunit encoding cDNAs were also identified in a library constructed from adrenal medulla RNA. Each beta subunit, when co-expressed in Xenopus oocytes with an alpha subunit, forms functional GABAA receptors. These results, together with the known alpha subunit heterogeneity, suggest that a variety of related but functionally distinct GABAA receptor subtypes are generated by different subunit combinations. Images PMID:2548852

  4. Anthracycline resistance mediated by reductive metabolism in cancer cells: The role of aldo-keto reductase 1C3

    SciTech Connect

    Hofman, Jakub; Malcekova, Beata; Skarka, Adam; Novotna, Eva; Wsol, Vladimir

    2014-08-01

    Pharmacokinetic drug resistance is a serious obstacle that emerges during cancer chemotherapy. In this study, we investigated the possible role of aldo-keto reductase 1C3 (AKR1C3) in the resistance of cancer cells to anthracyclines. First, the reducing activity of AKR1C3 toward anthracyclines was tested using incubations with a purified recombinant enzyme. Furthermore, the intracellular reduction of daunorubicin and idarubicin was examined by employing the transfection of A549, HeLa, MCF7 and HCT 116 cancer cells with an AKR1C3 encoding vector. To investigate the participation of AKR1C3 in anthracycline resistance, we conducted MTT cytotoxicity assays with these cells, and observed that AKR1C3 significantly contributes to the resistance of cancer cells to daunorubicin and idarubicin, whereas this resistance was reversible by the simultaneous administration of 2′-hydroxyflavanone, a specific AKR1C3 inhibitor. In the final part of our work, we tracked the changes in AKR1C3 expression after anthracycline exposure. Interestingly, a reciprocal correlation between the extent of induction and endogenous levels of AKR1C3 was recorded in particular cell lines. Therefore, we suggest that the induction of AKR1C3 following exposure to daunorubicin and idarubicin, which seems to be dependent on endogenous AKR1C3 expression, eventually might potentiate an intrinsic resistance given by the normal expression of AKR1C3. In conclusion, our data suggest a substantial impact of AKR1C3 on the metabolism of daunorubicin and idarubicin, which affects their pharmacokinetic and pharmacodynamic behavior. In addition, we demonstrate that the reduction of daunorubicin and idarubicin, which is catalyzed by AKR1C3, contributes to the resistance of cancer cells to anthracycline treatment. - Highlights: • Metabolism of anthracyclines by AKR1C3 was studied at enzyme and cellular levels. • Anthracycline resistance mediated by AKR1C3 was demonstrated in cancer cells. • Induction of AKR1C3

  5. Importance of standardization of hemoglobin A1c in the analysis of factors that predict hemoglobin A1c levels in non-diabetic residents of three distinct areas of Japan.

    PubMed

    Takahashi, Y; Noda, M; Tsugane, S; Kimura, S; Akanuma, Y; Kuzuya, T; Ohashi, Y; Kadowaki, T

    2001-08-01

    We performed a statistical analysis to elucidate effects of standardized measurement of hemoglobin A1c (HbA1c) on analysis of factors that affect HbA1c values. Subjects were participants in the Japan Public Health Center-based Prospective Study on Cancer and Cardiovascular Diseases, and a total of 1789 men and 3150 women in three distinct areas who did not have overt diabetes (HbA1c> or =6.1% or prior diagnosis) were analyzed. A different method of HbA1c assay was used in each area: high-performance liquid chromatography in one area and a different immunochemical method in each of the other two areas. Then, calibration of HbA1c was performed using two HbA1c standards (5.5 and 10.5%) provided by the Japan Diabetes Society. Analysis of co-variance was performed separately in men and women. When raw HbA1c data were used as the outcome, 'area', which represents differences in assay systems, lifestyles, etc. had a significant effect on HbA1c levels. When calibrated HbA1c data were used, however, 'area' was no longer a significant factor. In the latter analysis, age and BMI were the principal contributors to HbA1c, and parental history of diabetes had a weak effect in women. Thus, standardization of HbA1c reduced the difference between assay systems, and uncovered two common factors to determine HbA1c levels. PMID:11403857

  6. Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts

    PubMed Central

    Guo, Lin; Hei, Hongya; Tian, Lulu; Peng, Wen; Cai, Hui

    2015-01-01

    Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β3 subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells. PMID:25902045

  7. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

    PubMed Central

    Garbers, C; DeLong, A; Deruére, J; Bernasconi, P; Söll, D

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis. Images PMID:8641277

  8. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Garbers, C.; DeLong, A.; Deruere, J.; Bernasconi, P.; Soll, D.; Evans, M. L. (Principal Investigator)

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

  9. Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

    PubMed

    Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

    1995-06-01

    Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves. PMID:7610168

  10. Posterior C1-C2 calcium pyrophosphate dihydrate crystal deposition disease.

    PubMed

    Ng, Isaac Bing-Yi; Arkun, Knarik; Riesenburger, Ron I

    2016-01-01

    Calcium pyrophosphate dihydrate (CPPD) crystal deposition disease rarely occurs in the posterior aspect of the craniocervical junction (CCJ). To the best of our knowledge, there have been only 2 previously reported cases of patients with posterior CPPD lesions in this region that have led to cervical myelopathy. We report the case of a 70-year-old man presenting with neck pain and cervical myelopathy with multilevel stenosis from C1-C6. The stenosis was worst at C1-C2, secondary to compression by a CPPD lesion posterior to the spinal cord. The patient underwent a C2-C6 laminectomy and fusion with resection of the CPPD lesion. In this report, we discuss the patient and present a novel theory to explain the preponderance of CPPD lesions in the CCJ occurring anteriorly and not posteriorly to the spinal cord. PMID:26976840

  11. Quality of HbA1c Measurement in the Practice: The German Perspective.

    PubMed

    Heinemann, Lutz; Freckmann, Guido

    2015-05-01

    Hemoglobin A1c (HbA1c) measurement has come to be a cornerstone in modern diabetes therapy. However, the methodological aspects of this type of measurement have been given little attention lately due to its position as an established method of choice. Nevertheless, quite a number of issues face practical application, such as clinically relevant differences between different measurement methods--both lab-based and point-of-care (POCT) systems will show better or worse diabetes management results after switching methods; and there are a number of possible reasons that need to be known and observed in practice. The aim of this review is to draw attention to these problems from a German point of view and provide suggestions for appropriate measures to improve the situation. PMID:25691655

  12. Quality of HbA1c Measurement in the Practice

    PubMed Central

    Freckmann, Guido

    2015-01-01

    Hemoglobin A1c (HbA1c) measurement has come to be a cornerstone in modern diabetes therapy. However, the methodological aspects of this type of measurement have been given little attention lately due to its position as an established method of choice. Nevertheless, quite a number of issues face practical application, such as clinically relevant differences between different measurement methods—both lab-based and point-of-care (POCT) systems will show better or worse diabetes management results after switching methods; and there are a number of possible reasons that need to be known and observed in practice. The aim of this review is to draw attention to these problems from a German point of view and provide suggestions for appropriate measures to improve the situation. PMID:25691655

  13. 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.

  14. Polygonumnolides C1-C4; minor dianthrone glycosides from the roots of Polygonum multiflorum Thunb.

    PubMed

    Yang, Jian-Bo; Li, Li; Dai, Zhong; Wu, Yu; Geng, Xing-Chao; Li, Bo; Ma, Shuang-Cheng; Wang, Ai-Guo; Su, Ya-Lun

    2016-09-01

    Four new dianthrone glycosides, named polygonumnolides C1-C4 (1-4), were isolated from the dried roots of Polygonum multiflorum Thunb, together with two known emodin dianthrones (5-6). Their hepatotoxicities were evaluated against L-02 cell lines. Compounds 1-4 showed weak hepatotoxicity against L-02 cell lines with IC50 values of 313.05, 205.20, 294.20, and 207.35 μM, respectively. PMID:27139982

  15. C1-c2 pedicle screw fixation for treatment of old odontoid fractures.

    PubMed

    Qi, Lei; Li, Mu; Zhang, Shuai; Si, Haipeng; Xue, Jingsong

    2015-02-01

    Nonunion and C1-C2 instability of odontoid fractures usually result from delayed diagnosis and inappropriate treatment. However, the available treatment options for odontoid fractures remain controversial. The authors evaluated the effectiveness of internal screw fixation via the C1 and C2 pedicle in cases of old odontoid fractures. This retrospective study included 21 patients with old odontoid fractures (13 men and 8 women; mean age, 46.5 years; range, 24-69 years). Internal screw fixation via the C1 and C2 pedicle was performed in all patients. Fracture reduction and C1-C2 fusion were assessed with imaging. The neck pain visual analog scale score and cervical spinal cord functional Japanese Orthopaedic Association score (for those who had cervical spinal cord injury) were used to evaluate the effectiveness of treatment. Postoperative complications were recorded. Postoperative imaging showed that the C1-C2 dislocation was satisfactorily repositioned in all patients. Bone fusion was observed 1 year after surgery in all patients. No loosening or breaking of internal fixation occurred. The preoperative neck pain visual analog scale score was 5.9±1.5 and improved significantly to 1.8±0.8 after surgery (P<.001). The Japanese Orthopaedic Association score in patients with cervical spinal injury (n=14) was 9.2±1.9 and also significantly improved to 13.8±1.9 at the last follow-up examination (P<.001), with an average improvement rate of 61.0%. No iatrogenic vertebral artery injury or severe spinal cord injury occurred. Screw fixation via the C1 and C2 pedicle was found to be an effective and safe surgical approach for the treatment of old odontoid fractures with C1-C2 dislocation or instability. PMID:25665108

  16. Attention-deficit hyperactivity disorder and the adrenergic receptors alpha 1C and alpha 2C.

    PubMed

    Barr, C L; Wigg, K; Zai, G; Roberts, W; Malone, M; Schachar, R; Tannock, R; Kennedy, J L

    2001-05-01

    The adrenergic system has been hypothesized to be involved in the etiology of attention-deficit hyperactivity disorder (ADHD) based on pharmacological interventions and animal models. Noradrenergic neurons are implicated in the modulation of vigilance, improvement of visual attention, initiation of adaptive response, learning and memory. In this study we tested the genes for two adrenergic receptors, alpha 1C (ADRA1C) located on chromosome 8p11.2, and alpha 2C (ADRA2C) located on chromosome 4p16, as genetic susceptibility factors in ADHD. For the adrenergic receptor alpha 1C we used a C to T polymorphism that results in a change of Cys to Arg at codon 492 for the linkage study. For the adrenergic receptor alpha 2C gene we examined a dinucleotide repeat polymorphism located approximately 6 kb from the gene. We examined these polymorphisms in a sample of 103 families ascertained through an ADHD proband. Using the transmission disequilibrium test, we did not observe biased transmission of any of the alleles of these polymorphisms. We conclude that the alleles at the polymorphisms tested in these two genes are not linked to the ADHD phenotype in this sample of families. PMID:11326305

  17. The Characteristics and Consequences of the Break-up of the Fengyun-1C Spacecraft

    NASA Technical Reports Server (NTRS)

    Johnson, Nicholas L.; Stansbery, Eugene; Liou, Jer-chyi; Horstman, Matt; Stokeley, Christopher; Whitlock, David

    2007-01-01

    The intentional break-up of the Fengyun-1C spacecraft on 11 January 2007 via hypervelocity collision with a ballistic object created the most severe artificial debris cloud in Earth orbit since the beginning of space exploration. More than 900 debris on the order of 10 cm or greater in size have been identified by the U.S. Space Surveillance Network (SSN). The majority of these debris reside in long-lived orbits. The NASA Orbital Debris Program Office has conducted a thorough examination of the nature of the Fengyun-1C debris cloud, using SSN data for larger debris and special Haystack radar observations for smaller debris. These data have been compared with the NASA standard satellite break-up model for collisions, and the results are presented in this paper. The orbital longevity of the debris have also been evaluated for both small and large debris. The consequent long-term spatial density effects on the low Earth orbit (LEO) regime are then described. Finally, collision probabilities between the Fengyun-1C debris cloud and the resident space object population of 1 January 2007 have been calculated. The potential effect on the growth of the near-Earth satellite population is presented.

  18. Baseline characteristics and Latino versus non-Latino contrasts among Bronx A1C study participants

    PubMed Central

    Walker, Elizabeth A.; Silver, Lynn D.; Chamany, Shadi; Schechter, Clyde B.; Gonzalez, Jeffrey S.; Carrasco, Jeidy; Powell, Danielle; Berger, Diana; Basch, Charles E.

    2015-01-01

    We describe the New York City A1c Registry and associations among baseline characteristics of low-income, diverse adults with diabetes enrolled in a telephonic intervention trial. Baseline data were analyzed from 941 participants randomized to a telephonic/print or a print-only intervention to improve glycemic control in the context of an A1c Registry program. Summary statistics for key variables were calculated and we highlight contrasts between Latino and non-Latino participants. There were high proportions of Latino (67.7%) and Black (28.0%) participants from the South Bronx. Mean age was 56.3 years, almost 70.0% were foreign born, and 55.8% preferred Spanish language. Mean A1c was 9.2% and mean BMI 32.1kg/m2. There were significant contrasts between the Latino and non-Latino participants for behavioral and psychosocial variables. A telephonic intervention study was able to randomize a large number of low-income, ethnically diverse, urban participants with poor diabetes control. Latino vs. non-Latino differences at baseline were striking. PMID:24407771

  19. CD1c bypasses lysosomes to present a lipopeptide antigen with 12 amino acids

    PubMed Central

    Young, David C.; De Jong, Annemieke; Vazquez, Jenny; Cheng, Tan-Yun; Talekar, Rahul; Barral, Duarte C.; León, Luis; Brenner, Michael B.; Katz, Joel T.; Riese, Richard; Ruprecht, Ruth M.; O'Connor, Peter B.; Costello, Catherine E.; Porcelli, Steven A.; Briken, Volker

    2009-01-01

    The recent discovery of dideoxymycobactin (DDM) as a ligand for CD1a demonstrates how a nonribosomal lipopeptide antigen is presented to T cells. DDM contains an unusual acylation motif and a peptide sequence present only in mycobacteria, but its discovery raises the possibility that ribosomally produced viral or mammalian proteins that commonly undergo lipidation might also function as antigens. To test this, we measured T cell responses to synthetic acylpeptides that mimic lipoproteins produced by cells and viruses. CD1c presented an N-acyl glycine dodecamer peptide (lipo-12) to human T cells, and the response was specific for the acyl linkage as well as the peptide length and sequence. Thus, CD1c represents the second member of the CD1 family to present lipopeptides. lipo-12 was efficiently recognized when presented by intact cells, and unlike DDM, it was inactivated by proteases and augmented by protease inhibitors. Although lysosomes often promote antigen presentation by CD1, rerouting CD1c to lysosomes by mutating CD1 tail sequences caused reduction in lipo-12 presentation. Thus, although certain antigens require antigen processing in lysosomes, others are destroyed there, providing a hypothesis for the evolutionary conservation of large CD1 families containing isoforms that survey early endosomal pathways. PMID:19468063

  20. ER sheet persistence is coupled to myosin 1c-regulated dynamic actin filament arrays.

    PubMed

    Joensuu, Merja; Belevich, Ilya; Rämö, Olli; Nevzorov, Ilya; Vihinen, Helena; Puhka, Maija; Witkos, Tomasz M; Lowe, Martin; Vartiainen, Maria K; Jokitalo, Eija

    2014-04-01

    The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network. PMID:24523293

  1. Synthesis of Saturated Long Chain Fatty Acids from Sodium Acetate-1-C14 by Mycoplasma1

    PubMed Central

    Pollack, J. D.; Tourtellotte, M. E.

    1967-01-01

    Three strains of Mycoplasma, M. laidlawii A and B, and Mycoplasma sp. A60549, were grown in broth containing sodium acetate-1-C14. The methyl esters of the phospholipid fatty acids of harvested radioactive cells were prepared and identified by comparison of their mobilities to known radioactive fatty acid methyl esters by use of a modified reversed-phase partition-thin layer chromatographic technique. No radioactive methyl oleate or methyl linoleate was detected. Compounds migrating as radioactive methyl myristate, stearate, palmitate, and, with less certainty, laurate and octanoate were detected. The qualitative findings for all three organisms appeared similar. M. laidlawii B synthesized a radioactive substance, presumably a saturated fatty acid detected as the methyl ester derivative, which migrated in a position intermediate to methyl myristate-1-C14 and methyl palmitate-1-C14. This work indicates that M. laidlawii A and B and Mycoplasma sp. A60549 are capable, in a complex medium containing fatty acids, of synthesizing saturated but not unsaturated fatty acids entirely or in part from acetate. Images PMID:6020566

  2. Molecular basis of mycobacterial lipid antigen presentation by CD1c and its recognition by αβ T cells.

    PubMed

    Roy, Sobhan; Ly, Dalam; Li, Nan-Sheng; Altman, John D; Piccirilli, Joseph A; Moody, D Branch; Adams, Erin J

    2014-10-28

    CD1c is a member of the group 1 CD1 family of proteins that are specialized for lipid antigen presentation. Despite high cell surface expression of CD1c on key antigen-presenting cells and the discovery of its mycobacterial lipid antigen presentation capability, the molecular basis of CD1c recognition by T cells is unknown. Here we present a comprehensive functional and molecular analysis of αβ T-cell receptor (TCR) recognition of CD1c presenting mycobacterial phosphomycoketide antigens. Our structure of CD1c with the mycobacterial phosphomycoketide (PM) shows similarities to that of CD1c-mannosyl-β1-phosphomycoketide in that the A' pocket accommodates the mycoketide alkyl chain; however, the phosphate head-group of PM is shifted ∼6 Å in relation to that of mannosyl-β1-PM. We also demonstrate a bona fide interaction between six human TCRs and CD1c-mycoketide complexes, measuring high to moderate affinities. The crystal structure of the DN6 TCR and mutagenic studies reveal a requirement of five complementarity determining region (CDR) loops for CD1c recognition. Furthermore, mutagenesis of CD1c reveals residues in both the α1 and α2 helices involved in TCR recognition, yet not entirely overlapping among the examined TCRs. Unlike patterns for MHC I, no archetypical binding footprint is predicted to be shared by CD1c-reactive TCRs, even when recognizing the same or similar antigens. PMID:25298532

  3. Molecular basis of mycobacterial lipid antigen presentation by CD1c and its recognition by αβ T cells

    PubMed Central

    Roy, Sobhan; Ly, Dalam; Li, Nan-Sheng; Altman, John D.; Piccirilli, Joseph A.; Moody, D. Branch; Adams, Erin J.

    2014-01-01

    CD1c is a member of the group 1 CD1 family of proteins that are specialized for lipid antigen presentation. Despite high cell surface expression of CD1c on key antigen-presenting cells and the discovery of its mycobacterial lipid antigen presentation capability, the molecular basis of CD1c recognition by T cells is unknown. Here we present a comprehensive functional and molecular analysis of αβ T-cell receptor (TCR) recognition of CD1c presenting mycobacterial phosphomycoketide antigens. Our structure of CD1c with the mycobacterial phosphomycoketide (PM) shows similarities to that of CD1c-mannosyl-β1-phosphomycoketide in that the A' pocket accommodates the mycoketide alkyl chain; however, the phosphate head-group of PM is shifted ∼6 Å in relation to that of mannosyl-β1-PM. We also demonstrate a bona fide interaction between six human TCRs and CD1c-mycoketide complexes, measuring high to moderate affinities. The crystal structure of the DN6 TCR and mutagenic studies reveal a requirement of five complementarity determining region (CDR) loops for CD1c recognition. Furthermore, mutagenesis of CD1c reveals residues in both the α1 and α2 helices involved in TCR recognition, yet not entirely overlapping among the examined TCRs. Unlike patterns for MHC I, no archetypical binding footprint is predicted to be shared by CD1c-reactive TCRs, even when recognizing the same or similar antigens. PMID:25298532

  4. Structural Analysis of the Myo1c and Neph1 Complex Provides Insight into the Intracellular Movement of Neph1.

    PubMed

    Arif, Ehtesham; Sharma, Pankaj; Solanki, Ashish; Mallik, Leena; Rathore, Yogendra S; Twal, Waleed O; Nath, Samir K; Gandhi, Darpan; Holzman, Lawrence B; Ostap, E Michael; Ashish; Nihalani, Deepak

    2016-06-01

    The Myo1c motor functions as a cargo transporter supporting various cellular events, including vesicular trafficking, cell migration, and stereociliary movements of hair cells. Although its partial crystal structures were recently described, the structural details of its interaction with cargo proteins remain unknown. This study presents the first structural demonstration of a cargo protein, Neph1, attached to Myo1c, providing novel insights into the role of Myo1c in intracellular movements of this critical slit diaphragm protein. Using small angle X-ray scattering studies, models of predominant solution conformation of unliganded full-length Myo1c and Myo1c bound to Neph1 were constructed. The resulting structures show an extended S-shaped Myo1c with Neph1 attached to its C-terminal tail. Importantly, binding of Neph1 did not induce a significant shape change in Myo1c, indicating this as a spontaneous process or event. Analysis of interaction surfaces led to the identification of a critical residue in Neph1 involved in binding to Myo1c. Indeed, a point mutant from this site abolished interaction between Neph1 and Myo1c when tested in the in vitro and in live-cell binding assays. Live-cell imaging, including fluorescence recovery after photobleaching, provided further support for the role of Myo1c in intracellular vesicular movement of Neph1 and its turnover at the membrane. PMID:27044863

  5. Functional properties of the CaV1.2 calcium channel activated by calmodulin in the absence of alpha2delta subunits.

    PubMed

    Ravindran, Arippa; Kobrinsky, Evgeny; Lao, Qi Zong; Soldatov, Nikolai M

    2009-01-01

    Voltage-activated CaV1.2 calcium channels require association of the pore-forming alpha1C subunit with accessory CaVbeta and alpha2delta subunits. Binding of a single calmodulin (CaM) to alpha1C supports Ca2+-dependent inactivation (CDI). The human CaV1.2 channel is silent in the absence of CaVbeta and/or alpha2delta. Recently, we found that coexpression of exogenous CaM (CaMex) supports plasma membrane targeting, gating facilitation and CDI of the channel in the absence of CaVbeta. Here we discovered that CaMex and its Ca2+-insensitive mutant (CaM1234) rendered active alpha1C/CaVbeta channel in the absence of alpha2delta. Coexpression of CaMex with alpha1C and beta2d in calcium-channel-free COS-1 cells recovered gating of the channel and supported CDI. Voltage-dependence of activation was shifted by approximately +40 mV to depolarization potentials. The calcium current reached maximum at +40 mV (20 mM Ca2+) and exhibited approximately 3 times slower activation and 5 times slower inactivation kinetics compared to the wild-type channel. Furthermore, both CaMex and CaM1234 accelerated recovery from inactivation and induced facilitation of the calcium current by strong depolarization prepulse, the properties absent from the human vascular/neuronal CaV1.2 channel. The data suggest a previously unknown action of CaM that in the presence of CaVbeta; translates into activation of the alpha2delta-deficient calcium channel and alteration of its properties. PMID:19106618

  6. Functional properties of the Cav1.2 calcium channel activated by calmodulin in the absence of α2δ subunits

    PubMed Central

    Ravindran, Arippa; Kobrinsky, Evgeny; Lao, Qi Zong; Soldatov, Nikolai M.

    2009-01-01

    Voltage-activated Cav1.2 calcium channels require association of the pore-forming α1C subunit with accessory Cavβ and α2δ subunits. Binding of a single calmodulin (CaM) to α1C supports Ca2+-dependent inactivation (CDI). The human Cav1.2 channel is silent in the absence of Cavβ and/or α2δ. Recently, we found that coexpression of exogenous CaM (CaMex) supports plasma membrane targeting, gating facilitation and CDI of the channel in the absence of Cavβ. Here we discovered that CaMex and its Ca2+-insensitive mutant (CaM1234) rendered active α1C/Cavβ channel in the absence of α2δ. Coexpression of CaMex with α1C and β2d in calcium-channel-free COS-1 cells recovered gating of the channel and supported CDI. Voltage-dependence of activation was shifted by ≈ +40 mV to depolarization potentials. The calcium current reached maximum at +40 mV (20 mM Ca2+) and exhibited approximately 3 times slower activation and 5 times slower inactivation kinetics compared to the wild-type channel. Furthermore, both CaMex and CaM1234 accelerated recovery from inactivation and induced facilitation of the calcium current by strong depolarization prepulse, the properties absent from the human vascular/neuronal Cav1.2 channel. The data suggest a previously unknown action of CaM that in the presence of Cavβ translates into activation of the α2δ-deficient calcium channel and alteration of its properties. PMID:19106618

  7. Development and Characterization of Diamondback Moth Resistance to Transgenic Broccoli Expressing High Levels of Cry1C

    PubMed Central

    Zhao, Jian-Zhou; Collins, Hilda L.; Tang, Juliet D.; Cao, Jun; Earle, Elizabeth D.; Roush, Richard T.; Herrero, Salvador; Escriche, Baltasar; Ferré, Juan; Shelton, Anthony M.

    2000-01-01

    A field-collected colony of the diamondback moth, Plutella xylostella, had 31-fold resistance to Cry1C protoxin of Bacillus thuringiensis. After 24 generations of selection with Cry1C protoxin and transgenic broccoli expressing a Cry1C protein, the resistance that developed was high enough that neonates of the resistant strain could complete their entire life cycle on transgenic broccoli expressing high levels of Cry1C. After 26 generations of selection, the resistance ratios of this strain to Cry1C protoxin were 12,400- and 63,100-fold, respectively, for the neonates and second instars by a leaf dip assay. The resistance remained stable until generation 38 (G38) under continuous selection but decreased to 235-fold at G38 when selection ceased at G28. The Cry1C resistance in this strain was seen to be inherited as an autosomal and incompletely recessive factor or factors when evaluated using a leaf dip assay and recessive when evaluated using Cry1C transgenic broccoli. Saturable binding of 125I-Cry1C was found with brush border membrane vesicles (BBMV) from both susceptible and Cry1C-resistant strains. Significant differences in Cry1C binding to BBMV from the two strains were detected. BBMV from the resistant strain had about sevenfold-lower affinity for Cry1C and threefold-higher binding site concentration than BBMV from the susceptible strain. The overall Cry1C binding affinity was just 2.5-fold higher for BBMV from the susceptible strain than it was for BBMV from the resistant strain. These results suggest that reduced binding is not the major mechanism of resistance to Cry1C. PMID:10966391

  8. The structure of cucurbitin: subunit symmetry and organization in situ.

    PubMed

    Colman, P M; Suzuki, E; Van Donkelaar, A

    1980-02-01

    The low-resolution (2 nm) subunit symmetry of cucurbitin, the crystalline seed storage globulin of cucurbits, has been determined by X-ray diffraction. The wet crystals belong to the cubic space group F23 and there are 4 molecules per unit cell. The molecules therefore possess point-group symmetry 23 and contain 12 structural units which at this resolution are indistinguishable. On drying, the crystal lattice dimension shrinks from 13.6 nm to 12.4 nm with no apparent change in symmetry. Diffraction patterns of small crystals spun into a pellet, and sections of dry and wet native seed indicate that in situ the protein is organised in microcrystals of the same unit cell and symmetry. Edestin, the crystalline storage globulin from cannabis, and a crystalline globulin from tobacco seed both have the same crystal lattice as cucurbitin and, very likely, the same subunit symmetry. PMID:7358051

  9. Complete subunit architecture of the proteasome regulatory particle

    PubMed Central

    Lander, Gabriel C.; Estrin, Eric; Matyskiela, Mary E.; Bashore, Charlene; Nogales, Eva; Martin, Andreas

    2011-01-01

    The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information strongly restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes poly-ubiquitinated substrates. We used electron microscopy and a newly-developed heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes, and the protein unfolding machinery at subnanometer resolution, outlining the substrate’s path to degradation. Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore. Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination. We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes. PMID:22237024

  10. Characterization of inhibitors of phosphodiesterase 1C on a human cellular system.

    PubMed

    Dunkern, Torsten R; Hatzelmann, Armin

    2007-09-01

    Different inhibitors of the Ca(2+)/calmodulin-stimulated phosphodiesterase 1 family have been described and used for the examination of phosphodiesterase 1 in cellular, organ or animal models. However, the inhibitors described differ in potency and selectivity for the different phosphodiesterase family enzymes, and in part exhibit additional pharmacodynamic actions. In this study, we demonstrate that phosphodiesterase 1C is expressed in the human glioblastoma cell line A172 with regard to mRNA, protein and activity level, and that lower activities of phosphodiesterase 2, phosphodiesterase 3, phosphodiesterase 4 and phosphodiesterase 5 are also present. The identity of the phosphodiesterase 1C activity detected was verified by downregulation of the mRNA and protein through human phosphodiesterase 1C specific small interfering RNA. In addition, the measured K(m) values (cAMP, 1.7 microm; cGMP, 1.3 microm) are characteristic of phosphodiesterase 1C. We demonstrate that treatment with the Ca(2+) ionophore ionomycin increases intracellular Ca(2+) in a concentration-dependent way without affecting cell viability. Under conditions of enhanced intracellular Ca(2+) concentration, a rapid increase in cAMP levels caused by the adenylyl cyclase activator forskolin was abolished, indicating the involvement of Ca(2+)-activated phosphodiesterase 1C. The reduction of forskolin-stimulated cAMP levels was reversed by phosphodiesterase 1 inhibitors in a concentration-dependent way. Using this cellular system, we compared the cellular potency of published phosphodiesterase 1 inhibitors, including 8-methoxymethyl-3-isobutyl-1-methylxanthine, vinpocetine, SCH51866, and two established phosphodiesterase 1 inhibitors developed by Schering-Plough (named compounds 31 and 30). We demonstrate that up to 10 microm 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine had no effect on the reduction of forskolin-stimulated cAMP levels by ionomycin, whereas the more selective and up to 10

  11. Single subunit type of ferritin from visceral mass of Saccostrea cucullata: cloning, expression and cisplatin-subunit analysis.

    PubMed

    Zhu, Bo; Lin, Qing; Ke, Cai-Huan; Huang, He-Qing

    2011-09-01

    Ferritin, the iron storage protein, plays a key role in iron metabolism. Here, we have cloned an inducible ferritin cDNA with 516 bp within the open reading frame fragment from the visceral mass of Saccostrea cucullata. The subunit sequence of the ferritin was predicted to be a polypeptide of 171 amino acids with a molecular weight (MW) of 19.9182 kDa and an isoelectric point of 5.24. The cDNA sequence of S. cucullata ferritin was constructed into a pET-32a expression system for expressing its relative protein efficiently in the Escherichia coli BL21 strain under isopropyl-β-D-thiogalactoside (IPTG) induction. The recombinant ferritin, which was further purified on a Ni-NTA resin column and digested with enterokinase, was detected as a single subunit of approximately MW 20 kDa using both SDS-PAGE and mass spectrometry. S. cucullata ferritin (ScFer) showed 98% identity with Crassostrea gigas ferritin at the amino acid level. The secondary structure and phosphorylation sites of deduced amino acids were predicted with ExPASy proteomics tools and the NetPhos 2.0 server, respectively, and the subunit space structure of recombinant S. cucullata ferritin (rScFer) was built using the molecular operating environmental software system. The results of both in-gel digestion and identification using MALDI-TOF MS/MS showed that the recombinant protein was ScFer. ICP-MS indicated that rScFer subunit can directly bind to cisplatin[cis-Diaminedichloroplatinum(CDDP)], giving approximately 22.9 CDDP/ferritin subunit for forming a novel complex of CDDP-subunit, which suggests that it constructs a nanometer CDDP core-ferritin for developing a new drug of anti-cancer. The results of both the real-time PCR and Western blotting showed that the expression of ScFer mRNA was up-regulated in the oyster under the stress of Cd(2+). In addition, the expression increment of ScFer mRNA under bacterial challenge indicated that ferritin participated in the immune response of S. cucullata. The

  12. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Caers, A; De Rijk, P; De Wachter, R

    1998-01-01

    About 8600 complete or nearly complete sequences are now available from the Antwerp database on small ribosomal subunit RNA. All these sequences are aligned with one another on the basis of the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Literature references, accession numbers and detailed taxonomic information are also compiled. The database can be consulted via the World Wide Web at URL http://rrna.uia.ac.be/ssu/ PMID:9399829

  13. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; De Wachter, R

    1996-01-01

    Our database on large ribosomal subunit RNA contained 334 sequences in July, 1995. All sequences in the database are aligned, taking into account secondary structure. The aligned sequences are provided, together with incorporated secondary structure information, in several computer-readable formats. These data can easily be obtained through the World Wide Web. The files in the database are also available via anonymous ftp. PMID:8594610

  14. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Caers, A; Van de Peer, Y; De Wachter, R

    1998-01-01

    The rRNA WWW Server at URL http://rrna.uia.ac.be/ now provides a database of 496 large subunit ribosomal RNA sequences. All these sequences are aligned, incorporate secondary structure information, and can be obtained in a number of formats. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available and searchable. If necessary, the data on the server can also be obtained by anonymous ftp. PMID:9399830

  15. Instability of toxin A subunit of AB5 toxins in the bacterial periplasm caused by deficiency of their cognate B subunits

    PubMed Central

    Kim, Sang-Hyun; Ryu, Su Hyang; Lee, Sang-Ho; Lee, Yong-Hoon; Lee, Sang-Rae; Huh, Jae-Won; Kim, Sun-Uk; Kim, Ekyune; Kim, Sunghyun; Jon, Sangyong; Bishop, Russell E.; Chang, Kyu-Tae

    2016-01-01

    Shiga toxin (STx) belongs to the AB5 toxin family and is transiently localized in the periplasm before secretion into the extracellular milieu. While producing outer membrane vesicles (OMVs) containing only A subunit of the toxin (STxA), we created specific STx1B- and STx2B-deficient mutants of E. coli O157:H7. Surprisingly, STxA subunit was absent in the OMVs and periplasm of the STxB-deficient mutants. In parallel, the A subunit of heat-labile toxin (LT) of enterotoxigenic E. coli (ETEC) was absent in the periplasm of the LT-B-deficient mutant, suggesting that instability of toxin A subunit in the absence of the B subunit is a common phenomenon in the AB5 bacterial toxins. Moreover, STx2A was barely detectable in the periplasm of E. coli JM109 when stx2A was overexpressed alone, while it was stably present when stxB was co-expressed. Compared with STx2 holotoxin, purified STx2A was degraded rapidly by periplasmic proteases when assessed for in vitro proteolytic susceptibility, suggesting that the B subunit contributes to stability of the toxin A subunit in the periplasm. We propose a novel role for toxin B subunits of AB5 toxins in protection of the A subunit from proteolysis during holotoxin assembly in the periplasm. PMID:21762677

  16. Purification and subunit heterogeneity of pili of Bordetella bronchiseptica.

    PubMed Central

    Lee, S W; Way, A W; Osen, E G

    1986-01-01

    Pili were isolated and purified from Bordetella bronchiseptica. Electron microscopic observations revealed that pili are ubiquitous in this species. The occurrence of pili and flagella appeared to correlate with growth phase and colonial morphology. Pili were about 3 to 4 nm in diameter and morphologically similar to pili isolated from other gram-negative bacteria. Internal core structure was not evident. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified pili showed that up to three different pilus subunit variants could be observed on a single strain, depending on the colonial phase and culture condition. Enzyme immunoassay and immunoblot, however, showed that these subunit variants are serologically related. Mice vaccinated with purified pili were protected against a virulent intraperitoneal challenge of B. bronchiseptica. B. bronchiseptica pili were also found to be similar to Bordetella pertussis pili in morphology and in the molecular size and antigenic structure of pilus subunits. The intact pili of B. bronchiseptica and B. pertussis, however, appeared to have weak serological cross-reactivity. Images PMID:2867974

  17. Ribosomal small subunit domains radiate from a central core.

    PubMed

    Gulen, Burak; Petrov, Anton S; Okafor, C Denise; Vander Wood, Drew; O'Neill, Eric B; Hud, Nicholas V; Williams, Loren Dean

    2016-01-01

    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2'OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit. PMID:26876483

  18. RNA degradation paths in a 12-subunit nuclear exosome complex.

    PubMed

    Makino, Debora Lika; Schuch, Benjamin; Stegmann, Elisabeth; Baumgärtner, Marc; Basquin, Claire; Conti, Elena

    2015-08-01

    The eukaryotic exosome is a conserved RNA-degrading complex that functions in RNA surveillance, turnover and processing. How the same machinery can either completely degrade or precisely trim RNA substrates has long remained unexplained. Here we report the crystal structures of a yeast nuclear exosome containing the 9-subunit core, the 3'-5' RNases Rrp44 and Rrp6, and the obligate Rrp6-binding partner Rrp47 in complex with different RNAs. The combined structural and biochemical data of this 12-subunit complex reveal how a single-stranded RNA can reach the Rrp44 or Rrp6 active sites directly or can bind Rrp6 and be threaded via the central channel towards the distal RNase Rrp44. When a bulky RNA is stalled at the entrance of the channel, Rrp6-Rrp47 swings open. The results suggest how the same molecular machine can coordinate processive degradation and partial trimming in an RNA-dependent manner by a concerted swinging mechanism of the two RNase subunits. PMID:26222026

  19. Ribosomal small subunit domains radiate from a central core

    NASA Astrophysics Data System (ADS)

    Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O'Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

    2016-02-01

    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2‧OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit.

  20. Ribosomal small subunit domains radiate from a central core

    PubMed Central

    Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O’Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

    2016-01-01

    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2′OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit. PMID:26876483

  1. Serotonergic modulation of muscle acetylcholine receptors of different subunit composition.

    PubMed Central

    García-Colunga, J; Miledi, R

    1996-01-01

    Modulation of muscle acetylcholine (AcCho) receptors (AcChoRs) by serotonin [5-hydroxytryptamine (5HT)] and other serotonergic compounds was studied in Xenopus laevis oocytes. Various combinations of alpha, beta, gamma, and delta subunit RNAs were injected into oocytes, and membrane currents elicited by AcCho were recorded under voltage clamp. Judging by the amplitudes of AcCho currents generated, the levels of functional receptor expression were: alpha beta gamma delta > alpha beta delta > alpha beta gamma > alpha gamma delta. The alpha beta gamma delta and alpha beta delta AcChoR Subtypes were strongly blocked by 5HT, whereas the alpha beta gamma receptor was blocked only slightly. The order of blocking potency of AcChoRs by 5HT was: alpha beta delta > alpha beta gamma delta > alpha beta gamma. 5HT receptor antagonists, such as methysergide and spiperone, were even more potent blockers of AcChoRs than 5HT but did not show much subunit selectivity. Blockage of alpha beta gamma delta and alpha beta delta receptors by 5HT was voltage-dependent, and the voltage dependence was abolished when the delta subunit was omitted. These findings may need to be taken into consideration when trying to elucidate the mode of action of many clinically important serotonergic compounds. Images Fig. 3 PMID:8633003

  2. Direct Observation of Subunit Exchange along Mature Vimentin Intermediate Filaments

    PubMed Central

    Nöding, Bernd; Herrmann, Harald; Köster, Sarah

    2014-01-01

    Actin filaments, microtubules, and intermediate filaments (IFs) are central elements of the metazoan cytoskeleton. At the molecular level, the assembly mechanism for actin filaments and microtubules is fundamentally different from that of IFs. The former two types of filaments assemble from globular proteins. By contrast, IFs assemble from tetrameric complexes of extended, half-staggered, and antiparallel oriented coiled-coils. These tetramers laterally associate into unit-length filaments; subsequent longitudinal annealing of unit-length filaments yields mature IFs. In vitro, IFs form open structures without a fixed number of tetramers per cross-section along the filament. Therefore, a central question for the structural biology of IFs is whether individual subunits can dissociate from assembled filaments and rebind at other sites. Using the fluorescently labeled IF-protein vimentin for assembly, we directly observe and quantitatively determine subunit exchange events between filaments as well as with soluble vimentin pools. Thereby we demonstrate that the cross-sectional polymorphism of donor and acceptor filaments plays an important role. We propose that in segments of donor filaments with more than the standard 32 molecules per cross-section, subunits are not as tightly bound and are predisposed to be released from the filament. PMID:25517157

  3. Mutant GABA(A) receptor subunits in genetic (idiopathic) epilepsy.

    PubMed

    Hirose, Shinichi

    2014-01-01

    The γ-aminobutyric acid receptor type A (GABAA receptor) is a ligand-gated chloride channel that mediates major inhibitory functions in the central nervous system. GABAA receptors function mainly as pentamers containing α, β, and either γ or δ subunits. A number of antiepileptic drugs have agonistic effects on GABAA receptors. Hence, dysfunctions of GABAA receptors have been postulated to play important roles in the etiology of epilepsy. In fact, mutations or genetic variations of the genes encoding the α1, α6, β2, β3, γ2, or δ subunits (GABRA1, GABRA6, GABRB2, GABRB3, GABRG2, and GABRD, respectively) have been associated with human epilepsy, both with and without febrile seizures. Epilepsy resulting from mutations is commonly one of following, genetic (idiopathic) generalized epilepsy (e.g., juvenile myoclonic epilepsy), childhood absence epilepsy, genetic epilepsy with febrile seizures, or Dravet syndrome. Recently, mutations of GABRA1, GABRB2, and GABRB3 were associated with infantile spasms and Lennox-Gastaut syndrome. These mutations compromise hyperpolarization through GABAA receptors, which is believed to cause seizures. Interestingly, most of the insufficiencies are not caused by receptor gating abnormalities, but by complex mechanisms, including endoplasmic reticulum (ER)-associated degradation, nonsense-mediated mRNA decay, intracellular trafficking defects, and ER stress. Thus, GABAA receptor subunit mutations are now thought to participate in the pathomechanisms of epilepsy, and an improved understanding of these mutations should facilitate our understanding of epilepsy and the development of new therapies. PMID:25194483

  4. Subunit interactions in yeast transcription/repair factor TFIIH. Requirement for Tfb3 subunit in nucleotide excision repair.

    PubMed

    Feaver, W J; Huang, W; Gileadi, O; Myers, L; Gustafsson, C M; Kornberg, R D; Friedberg, E C

    2000-02-25

    A yeast strain harboring a temperature-sensitive allele of TFB3 (tfb3(ts)), the 38-kDa subunit of the RNA polymerase II transcription/nucleotide excision repair factor TFIIH, was found to be sensitive to ultraviolet (UV) radiation and defective for nucleotide excision repair in vitro. Interestingly, tfb3(ts) failed to grow on medium containing caffeine. A comprehensive pairwise two-hybrid analysis between yeast TFIIH subunits identified novel interactions between Rad3 and Tfb3, Tfb4 and Ssl1, as well as Ssl2 and Tfb2. These interactions have facilitated a more complete model of the structure of TFIIH and the nucleotide excision repairosome. PMID:10681587

  5. On the multiple roles of the voltage gated sodium channel β1 subunit in genetic diseases

    PubMed Central

    Baroni, Debora; Moran, Oscar

    2015-01-01

    Voltage-gated sodium channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are composed of a pore-forming α-subunit and associated β-subunits. The β1-subunit was the first accessory subunit to be cloned. It can be important for controlling cell excitability and modulating multiple aspects of sodium channel physiology. Mutations of β1 are implicated in a wide variety of inherited pathologies, including epilepsy and cardiac conduction diseases. This review summarizes β1-subunit related channelopathies pointing out the current knowledge concerning their genetic background and their underlying molecular mechanisms. PMID:26042039

  6. Comparison of Large Subunits of Type II DNA-dependent RNA Polymerases from Higher Plants.

    PubMed

    Kidd, G H; Link, G; Bogorad, L

    1979-10-01

    Two-dimensional tryptic mapping of (125)I-labeled polypeptides has been employed to compare the large subunits of type II DNA-dependent RNA polymerases from maize, parsley (Petroselinum sativum), and wheat. Maps of the 220 kilodalton (kd) and 140 kd subunits from wheat RNA polymerase II differ from those of the corresponding subunits from parsley enzyme II. The 180 kd subunits from maize and parsley type II enzymes also yield dissimilar tryptic maps. Thus, despite similarities in molecular mass, the large subunits of wheat, parsley, and maize type II RNA polymerases are unique to each individual plant species. PMID:16661032

  7. Screening Baccharin Analogs as Selective Inhibitors Against Type 5 17β-Hydroxysteroid Dehydrogenase (AKR1C3)

    PubMed Central

    Zang, Tianzhu; Verma, Kshitij; Chen, Mo; Jin, Yi; Trippier, Paul C.; Penning, Trevor M.

    2015-01-01

    Aldo-keto reductase 1C3 (AKR1C3), also known as type 5 17β-hydroxysteroid dehydrogenase, is a downstream steroidogenic enzyme and converts androgen precursors to the potent androgen receptor ligands: testosterone and 5α-dihydrotestosterone. Studies have shown that AKR1C3 is involved in the development of castration resistant prostate cancer (CRPC) and that it is a rational drug target for the treatment of CRPC. Baccharin, a component of Brazilian propolis, has been observed to exhibit a high inhibitory potency and selectivity for AKR1C3 over other AKR1C isoforms and is a promising lead compound for developing more potent and selective inhibitors. Here, we report the screening of fifteen baccharin analogs as selective inhibitors against AKR1C3 versus AKR1C2 (type 3 3α-hydroxysteroid dehydrogenase). Among these analogs, the inhibitory activity and selectivity of thirteen compounds were evaluated for the first time. The substitution of the 4-dihydrocinnamoyloxy group of baccharin by an acetate group displayed nanomolar inhibitory potency (IC50: 440 nM) and a 102-fold selectivity over AKR1C2. By contrast, when the cinnamic acid group of baccharin was esterified, there was a dramatic decrease in potency and selectivity for AKR1C3 in comparison to baccharin. Low or sub- micromolar inhibition was observed when the 3-prenyl group of baccharin was removed, and the selectivity over AKR1C2 was low. Although unsubstituted baccharin was still the most potent (IC50: 100 nM) and selective inhibitor for AKR1C3, these data provide structure-activity relationships required for the optimization of new baccharin analogs. They suggest that the carboxylate group on cinnamic acid, the prenyl group, and either retention of 4′-dihydrocinnamoyloxy group or acetate substituent on cinnamic acid are important to maintain the high potency and selectivity for AKR1C3. PMID:25555457

  8. Transcriptional Regulation of Cytosolic Sulfotransferase 1C2 by Vitamin D Receptor in LS180 Human Colorectal Adenocarcinoma Cells.

    PubMed

    Barrett, Kathleen G; Fang, Hailin; Kocarek, Thomas A; Runge-Morris, Melissa

    2016-08-01

    The factors that regulate expression of genes in the 1C family of human cytosolic sulfotransferases (SULT1C) are not well understood. In a recent study evaluating the effects of a panel of transcription factor activators on SULT1C family member expression in LS180 human colorectal adenocarcinoma cells, we found that SULT1C2 expression was significantly increased by 1α,25-dihydroxyvitamin D3 (VitD3) treatment. The objective of our current study was to identify the mechanism responsible for VitD3-mediated activation of SULT1C2 transcription. VitD3 treatment of LS180 cells activated transcription of a transfected luciferase reporter plasmid that contained ∼5 kilobase pairs (kbp) of the SULT1C2 gene, which included 402 nucleotides (nt) of the noncoding exon 1, all of intron 1, and 21 nt of exon 2. Although computational analysis of the VitD3-responsive region of the SULT1C2 gene identified a pregnane X receptor (PXR)-binding site within exon 1, the transfected 5 kbp SULT1C2 reporter was not activated by treatment with rifampicin, a prototypical PXR agonist. However, deletion or mutation of the predicted PXR-binding site abolished VitD3-mediated SULT1C2 transcriptional activation, identifying the site as a functional vitamin D response element (VDRE). We further demonstrated that vitamin D receptor (VDR) can interact directly with the SULT1C2 VDRE sequence using an enzyme-linked immunosorbent assay-based transcription factor binding assay. In conclusion, VitD3-inducible SULT1C2 transcription is mediated through a VDRE in exon 1. These results suggest a role for SULT1C2 in VitD3-regulated physiologic processes in human intestine. PMID:27130351

  9. Hybridization of glutamate aspartate transaminase. Investigation of subunit interaction.

    PubMed

    Boettcher, B; Martinez-Carrion, M

    1975-10-01

    Glutamate aspartate transaminase (EC 2.6.1.1) is a dimeric enzyme with identical subunits with each active site containing pyridoxal 5'-phosphate linked via an