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Sample records for carboxylase beta gene

  1. A single nucleotide polymorphism within the acetyl-coenzyme A carboxylase beta gene is associated with proteinuria in patients with type 2 diabetes.

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    Shiro Maeda

    2010-02-01

    Full Text Available It has been suggested that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy. A large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs in Japanese patients with type 2 diabetes identified the gene encoding acetyl-coenzyme A carboxylase beta (ACACB as a candidate for a susceptibility to diabetic nephropathy; the landmark SNP was found in the intron 18 of ACACB (rs2268388: intron 18 +4139 C > T, p = 1.4x10(-6, odds ratio = 1.61, 95% confidence interval [CI]: 1.33-1.96. The association of this SNP with diabetic nephropathy was examined in 9 independent studies (4 from Japan including the original study, one Singaporean, one Korean, and two European with type 2 diabetes. One case-control study involving European patients with type 1 diabetes was included. The frequency of the T allele for SNP rs2268388 was consistently higher among patients with type 2 diabetes and proteinuria. A meta-analysis revealed that rs2268388 was significantly associated with proteinuria in Japanese patients with type 2 diabetes (p = 5.35 x 10(-8, odds ratio = 1.61, 95% Cl: 1.35-1.91. Rs2268388 was also associated with type 2 diabetes-associated end-stage renal disease (ESRD in European Americans (p = 6 x 10(-4, odds ratio = 1.61, 95% Cl: 1.22-2.13. Significant association was not detected between this SNP and nephropathy in those with type 1 diabetes. A subsequent in vitro functional analysis revealed that a 29-bp DNA fragment, including rs2268388, had significant enhancer activity in cultured human renal proximal tubular epithelial cells. Fragments corresponding to the disease susceptibility allele (T had higher enhancer activity than those of the major allele. These results suggest that ACACB is a strong candidate for conferring susceptibility for proteinuria in patients with type 2 diabetes.

  2. A single nucleotide polymorphism within the acetyl-coenzyme A carboxylase beta gene is associated with proteinuria in patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Maeda, Shiro; Kobayashi, Masa-aki; Araki, Shin-ichi

    2010-01-01

    It has been suggested that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy. A large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese patients with type 2 diabetes identified the gene encoding acetyl-coenzyme A ca......It has been suggested that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy. A large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese patients with type 2 diabetes identified the gene encoding acetyl...... among patients with type 2 diabetes and proteinuria. A meta-analysis revealed that rs2268388 was significantly associated with proteinuria in Japanese patients with type 2 diabetes (p = 5.35 x 10(-8), odds ratio = 1.61, 95% Cl: 1.35-1.91). Rs2268388 was also associated with type 2 diabetes......-associated end-stage renal disease (ESRD) in European Americans (p = 6 x 10(-4), odds ratio = 1.61, 95% Cl: 1.22-2.13). Significant association was not detected between this SNP and nephropathy in those with type 1 diabetes. A subsequent in vitro functional analysis revealed that a 29-bp DNA fragment, including...

  3. Interaction of C/EBP-beta and NF-Y factors constrains activity levels of the nutritionally controlled promoter IA expressing the acetyl-CoA carboxylase-alpha gene in cattle

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    Shi Xuanming

    2012-06-01

    Full Text Available Abstract Background The enzyme acetyl-CoA carboxylase-alpha (ACC-α is rate limiting for de novo fatty acid synthesis. Among the four promoters expressing the bovine gene, promoter IA (PIA is dominantly active in lipogenic tissues. This promoter is in principal repressed but activated under favorable nutritional conditions. Previous analyses already coarsely delineated the repressive elements on the distal promoter but did not resolve the molecular nature of the repressor. Knowledge about the molecular functioning of this repressor is fundamental to understanding the nutrition mediated regulation of PIA activity. We analyzed here the molecular mechanism calibrating PIA activity. Results We finely mapped the repressor binding sites in reporter gene assays and demonstrate together with Electrophoretic Mobility Shift Assays that nuclear factor-Y (NF-Y and CCAAT/enhancer binding protein-β(C/EBPβ each separately repress PIA activity by binding to their cognate low affinity sites, located on distal elements of the promoter. Simultaneous binding of both factors results in strongest repression. Paradoxically, over expression of NFY factors, but also - and even more so - of C/EBPβ significantly activated the promoter when bound to high affinity sites on the proximal promoter. However, co-transfection experiments revealed that NF-Y may eventually diminish the strong stimulatory effect of C/EBPβ at the proximal PIA in a dose dependent fashion. We validated by chromatin immunoprecipitation, that NF-Y and C/EBP factors may physically interact. Conclusion The proximal promoter segment of PIA appears to be principally in an active state, since even minute concentrations of both, NF-Y and C/EBPβ factors can saturate the high affinity activator sites. Higher factor concentrations will saturate the low affinity repressive sites on the distal promoter resulting in reduced and calibrated promoter activity. Based on measurements of the mRNA concentrations of

  4. AMPK activation represses the human gene promoter of the cardiac isoform of acetyl-CoA carboxylase: Role of nuclear respiratory factor-1

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    Adam, Tasneem; Opie, Lionel H. [Hatter Cardiovascular Research Institute, Faculty of Health Sciences, University of Cape Town, Observatory 7925 (South Africa); Essop, M. Faadiel, E-mail: mfessop@sun.ac.za [Cardio-Metabolic Research Group (CMRG), Department of Physiological Sciences, Stellenbosch University, Stellenbosch 7600 (South Africa)

    2010-07-30

    Research highlights: {yields} AMPK inhibits acetyl-CoA carboxylase beta gene promoter activity. {yields} Nuclear respiratory factor-1 inhibits acetyl-CoA carboxylase beta promoter activity. {yields} AMPK regulates acetyl-CoA carboxylase beta at transcriptional level. -- Abstract: The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC{beta}) produces malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase-1. AMPK inhibits ACC{beta} activity, lowering malonyl-CoA levels and promoting mitochondrial fatty acid {beta}-oxidation. Previously, AMPK increased promoter binding of nuclear respiratory factor-1 (NRF-1), a pivotal transcriptional modulator controlling gene expression of mitochondrial proteins. We therefore hypothesized that NRF-1 inhibits myocardial ACC{beta} promoter activity via AMPK activation. A human ACC{beta} promoter-luciferase construct was transiently transfected into neonatal cardiomyocytes {+-} a NRF-1 expression construct. NRF-1 overexpression decreased ACC{beta} gene promoter activity by 71 {+-} 4.6% (p < 0.001 vs. control). Transfections with 5'-end serial promoter deletions revealed that NRF-1-mediated repression of ACC{beta} was abolished with a pPII{beta}-18/+65-Luc deletion construct. AMPK activation dose-dependently reduced ACC{beta} promoter activity, while NRF-1 addition did not further decrease it. We also investigated NRF-1 inhibition in the presence of upstream stimulatory factor 1 (USF1), a known transactivator of the human ACC{beta} gene promoter. Here NRF-1 blunted USF1-dependent induction of ACC{beta} promoter activity by 58 {+-} 7.5% (p < 0.001 vs. control), reversed with a dominant negative NRF-1 construct. NRF-1 also suppressed endogenous USF1 transcriptional activity by 55 {+-} 6.2% (p < 0.001 vs. control). This study demonstrates that NRF-1 is a novel transcriptional inhibitor of the human ACC{beta} gene promoter in the mammalian heart. Our data extends AMPK regulation of ACC{beta} to the transcriptional level.

  5. Organization and expression of two tandemly oriented genes encoding ribulosebisphosphate carboxylase/oxygenase activase in barley.

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    Rundle, S J; Zielinski, R E

    1991-03-15

    We have isolated and structurally characterized genomic DNA and cDNA sequences encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase) activase from barley (Hordeum vulgare L.). Three Rbu-P2 carboxylase activase (Rca) polypeptides are encoded in the barley genome by two closely linked, tandemly oriented nuclear genes (RcaA and RcaB); cDNAs encoding each of the three Rbu-P2 carboxylase activase polypeptides were isolated from cDNA libraries of barley leaf mRNA. RcaA produces two mRNAs, which encode polypeptides of 42 and 46 kDa, by an alternative splicing mechanism identical to that previously reported for spinach and Arabidopsis Rca genes (Werneke, J.M., Chatfield, J.M., and Ogren, W. L. (1989) Plant Cell 1, 815-825). RcaB is transcribed to produce a single mRNA, which encodes a mature peptide of 42 kDa. Genomic Southern blots indicate that RcaA and RcaB represent the entire Rbu-P2 carboxylase activase gene family in barley. The genes share 80% nucleotide sequence identity, and the 42-kDa polypeptides encoded by RcaA and RcaB share 87% amino acid sequence identity. Coding regions of the two barley Rca genes are separated by 1 kilobase pair of flanking DNA. DNA sequence motifs similar to those thought to control light-regulated gene expression in other nuclear-encoded plastid polypeptide genes are found at the 5' end of both barley Rca genes. Probes specific to three mRNAs were used to determine the relative contribution each species makes to the total Rca mRNA pool.

  6. Genes encoding biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution

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    Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric ACCase that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin-carboxyl-carrier protein and CO2 to form carboxybiotin-carbo...

  7. Multiple inter-kingdom horizontal gene transfers in the evolution of the phosphoenolpyruvate carboxylase gene family.

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    Yingmei Peng

    Full Text Available Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC while the other as bacteria-type pepcase (BTPC because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought.

  8. A Novel Approach to Functional Analysis of the Ribulose Bisphosphate Carboxylase Small Subunit Gene by Agrobacterium-Mediated Gene Silencing

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    Xiao-Fu Zhou; Peng-Da Ma; Ren-Hou Wang; Bo Liu; Xing-Zhi Wang

    2006-01-01

    A novel approach to virus-induced post-transcriptional gene silencing for studying the function of the ribulose bisphosphate carboxylase small subunlt (rbcS) gene was established and optimized using potato virus X vector and Nicotiana benthamiana as experimental material. The analysis of silencing phenomena,transcriptional level, protein expression, and pigment measurement showed that the expression of the rbcS endogenous gene was inactivated by the expression of a 500-bp homologous cDNA fragment carried in the virus vector.

  9. Molecular evolution of urea amidolyase and urea carboxylase in fungi

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    Harris Steven D

    2011-03-01

    Full Text Available Abstract Background Urea amidolyase breaks down urea into ammonia and carbon dioxide in a two-step process, while another enzyme, urease, does this in a one step-process. Urea amidolyase has been found only in some fungal species among eukaryotes. It contains two major domains: the amidase and urea carboxylase domains. A shorter form of urea amidolyase is known as urea carboxylase and has no amidase domain. Eukaryotic urea carboxylase has been found only in several fungal species and green algae. In order to elucidate the evolutionary origin of urea amidolyase and urea carboxylase, we studied the distribution of urea amidolyase, urea carboxylase, as well as other proteins including urease, across kingdoms. Results Among the 64 fungal species we examined, only those in two Ascomycota classes (Sordariomycetes and Saccharomycetes had the urea amidolyase sequences. Urea carboxylase was found in many but not all of the species in the phylum Basidiomycota and in the subphylum Pezizomycotina (phylum Ascomycota. It was completely absent from the class Saccharomycetes (phylum Ascomycota; subphylum Saccharomycotina. Four Sordariomycetes species we examined had both the urea carboxylase and the urea amidolyase sequences. Phylogenetic analysis showed that these two enzymes appeared to have gone through independent evolution since their bacterial origin. The amidase domain and the urea carboxylase domain sequences from fungal urea amidolyases clustered strongly together with the amidase and urea carboxylase sequences, respectively, from a small number of beta- and gammaproteobacteria. On the other hand, fungal urea carboxylase proteins clustered together with another copy of urea carboxylases distributed broadly among bacteria. The urease proteins were found in all the fungal species examined except for those of the subphylum Saccharomycotina. Conclusions We conclude that the urea amidolyase genes currently found only in fungi are the results of a horizontal

  10. Cloning and characterization of cotton heteromeric acetyl-CoA carboxylase genes

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Heteromeric acetyl-coanzyme A(CoA)carboxylese(ACCase)catalyzes the formation of malonyl-CoA from acetyl-CoA.It plays an essential role in fatty acid synthesis in prokaryotes and most of plants.The heteromeric ACCase is composed of four subunits:biotin carboxylase (BC),biotin carboxyl carrier protein (BCCP),and α-and β-subunits of carboxyltransferese(α-andβ-CT).In this study,we cloned five novel genes encoding the subunits of heteromeric ACCese(one BC,BCCP and β-CT,and two α-CTs) from cotton (Gossypium hirsutum cv.zhongmian 35)by RACE-PCR.The deduced amino acid sequence of these cDNAs shares high similarity with other reported heteromeric ACCese subunits.The phylogenetic analysis indicated that the different subunits of heteromeric ACCase were grouped in a similar pattern.Southern blot analysis revealed the milti-copy patterns of these heteromeric ACCase genes in cotton genome.Semi-quantitative RT-PCR demonstrated that heteromeric ACCese genes were constitutively expressed in all of the cotton tissues,but the transcripts accumulated at a relatively low level in roots.To our knowledge,this is the first report about characterization of the heteromeric ACCase genes in cotton.

  11. Determination of ploidy level and isolation of genes encoding acetyl-CoA carboxylase in Japanese Foxtail (Alopecurus japonicus.

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    Hongle Xu

    Full Text Available Ploidy level is important in biodiversity studies and in developing strategies for isolating important plant genes. Many herbicide-resistant weed species are polyploids, but our understanding of these polyploid weeds is limited. Japanese foxtail, a noxious agricultural grass weed, has evolved herbicide resistance. However, most studies on this weed have ignored the fact that there are multiple copies of target genes. This may complicate the study of resistance mechanisms. Japanese foxtail was found to be a tetraploid by flow cytometer and chromosome counting, two commonly used methods in the determination of ploidy levels. We found that there are two copies of the gene encoding plastidic acetyl-CoA carboxylase (ACCase in Japanese foxtail and all the homologous genes are expressed. Additionally, no difference in ploidy levels or ACCase gene copy numbers was observed between an ACCase-inhibiting herbicide-resistant and a herbicide-sensitive population in this study.

  12. Nuclear-cytoplasmic conflict in pea (Pisum sativum L.) is associated with nuclear and plastidic candidate genes encoding acetyl-CoA carboxylase subunits.

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    Bogdanova, Vera S; Zaytseva, Olga O; Mglinets, Anatoliy V; Shatskaya, Natalia V; Kosterin, Oleg E; Vasiliev, Gennadiy V

    2015-01-01

    In crosses of wild and cultivated peas (Pisum sativum L.), nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized.

  13. Novel Mutations in the PC Gene in Patients with Type B Pyruvate Carboxylase Deficiency

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    Ostergaard, Elsebet; Duno, Morten; Møller, Lisbeth Birk

    2013-01-01

    We have investigated seven patients with the type B form of pyruvate carboxylase (PC) deficiency. Mutation analysis revealed eight mutations, all novel. In a patient with exon skipping on cDNA analysis, we identified a homozygous mutation located in a potential branch point sequence, the first...... possible branch point mutation in PC. Two patients were homozygous for missense mutations (with normal protein amounts on western blot analysis), and two patients were homozygous for nonsense mutations. In addition, a duplication of one base pair was found in a patient who also harboured a splice site...... mutation. Another splice site mutation led to the activation of a cryptic splice site, shown by cDNA analysis.All patients reported until now with at least one missense mutation have had the milder type A form of PC deficiency. We thus report for the first time two patients with homozygous missense...

  14. Transcritption regulation of soybean ribulose-1,5-bisphos-phate carboxylase small sub-unit gene by external factors

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Ribulose-1,5-bisphosphate carboxylase small subunit gene (rbcS) is present with multi-gene family in plant genome. In Glycine max, the rbcS polypeptide (EC4.1.1.39) is encoded by a gene family containing 4-8 members. Three full-length rbcS cDNA clones were isolated and characterized from soybean seedlings, and both of their nucleotide and amino acid sequences showed high similarity. Differential accumulation of the rbcS mRNA was observed among roots, hypocotyls, cotyledons, epicotyls and leaves. The rbcS genes were up-regulated by various external factors such as salicylic acid (SA), salt stress and drought stress. The expression level of rbcS genes after being treated by 2.0 mmol/L SA and 0.4% NaCl, respectively, is 2.5-3.0-fold as high as that of control sample. Moreover, soybean rbcS mRNA was accumulated with diurnal variation but easily influenced by light and low temperature.

  15. 小麦导入磷酸烯醇式丙酮酸羧化酶(PEPCase)基因的初步研究%Preliminary Study on Phosphoenolpyruvate Carboxylase (PEPCase) Gene Introduced into Wheat

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    张彬; 马建军; 贾栋

    2009-01-01

    [Objective] The aim of this study was to introduce Phosphoenolpyruvate Carboxylase (PEPCase) gene into common wheat Linyou 145. [Method] With the material of common wheat Linyou 145, Phosphoenolpyruvate Carboxylase (PEPCase) gene was introduced into wheat embryo callus by the agrobacterium-mediated transformation system, and then analyzed through successive selection with selective medium containg gygromycin to detect the gene at the molecular level. [Result] The hyg-resistant plants were obtained, and GUS histochemical staining showed the leaf of resistant plants was stained dark blue. The target bands appeared in PCR analysis. [Conclusion] Phosphoenolpyruvate Carboxylase (PEPCase) gene has been primarily introduced into the recipient material.

  16. Alterations in barley ribulose-1,5-bisphosphate carboxylase/oxygenase activase gene expression during development and in response to illumination.

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    Rundle, S J; Zielinski, R E

    1991-08-05

    Two genes encode Rbu-P2-carboxylase activase in barley (RcaA and RcaB): RcaA encodes polypeptides of 46 and 42 kDa, which are generated by the alternatively spliced RcaA1 and RcaA2 mRNAs, respectively; RcaB encodes a 42-kDa polypeptide (Rundle, S. J., and Zielinski, R. E. (1991) J. Biol. Chem. 266, 4677-4685). In the cellular differentiation gradient of the first leaf of barley, the three Rca mRNAs accumulate differentially. RcaA1 and A2 mRNAs accumulate predominantly in the mature, most photosynthetically active regions of the leaf in a pattern that parallels accumulation of total Rbu-P2-carboxylase activase protein. However, the kinetics of accumulation of RcaA1 and RcaA2 mRNA differ slightly, indicating that either changes in RcaA pre-mRNA splicing or mRNA turnover occur during development. RcaB mRNA, in contrast, accumulates in the youngest and oldest cell populations at the base and tip of the leaf, respectively. In the mid-region of the leaf, the difference in accumulation between RcaA and RcaB mRNAs is largely attributable to differences in the rates of transcription of the two Rca genes. In this region of the leaf, the three Rca mRNAs accumulate differentially throughout the course of the diurnal cycle. Steady state levels of the three Rca mRNA species increase in parallel in response to increasing irradiance; these changes were accompanied by increased Rbu-P2-carboxylase activase protein accumulation.

  17. Cloning and Characterization of a Pyruvate Carboxylase Gene from Penicillium rubens and Overexpression of the Genein the Yeast Yarrowia lipolytica for Enhanced Citric Acid Production.

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    Fu, Ge-Yi; Lu, Yi; Chi, Zhe; Liu, Guang-Lei; Zhao, Shou-Feng; Jiang, Hong; Chi, Zhen-Ming

    2016-02-01

    In this study, a pyruvate carboxylase gene (PYC1) from a marine fungus Penicillium rubens I607 was cloned and characterized. ORF of the gene (accession number: KM397349.1) had 3534 bp encoding 1177 amino acids with a molecular weight of 127.531 kDa and a PI of 6.20. The promoter of the gene was located at -1200 bp and contained a TATAA box, several CAAT boxes and a sequence 5'-SYGGRG-3'. The PYC1 deduced from the gene had no signal peptide, was a homotetramer (α4), and had the four functional domains. After expression of the PYC1 gene from the marine fungus in the marine-derived yeast Yarrowia lipolytica SWJ-1b, the transformant PR32 obtained had much higher specific pyruvate carboxylase activity (0.53 U/mg) than Y. lipolytica SWJ-1b (0.07 U/mg), and the PYC1 gene expression (133.8%) and citric acid production (70.2 g/l) by the transformant PR32 were also greatly enhanced compared to those (100 % and 27.3 g/l) by Y. lipolytica SWJ-1b. When glucose concentration in the medium was 60.0 g/l, citric acid (CA) concentration formed by the transformant PR32 was 36.1 g/l, leading to conversion of 62.1% of glucose into CA. During a 10-l fed-batch fermentation, the final concentration of CA was 111.1 ± 1.3 g/l, the yield was 0.93 g/g, the productivity was 0.46 g/l/h, and only 1.72 g/l reducing sugar was left in the fermented medium within 240 h. HPLC analysis showed that most of the fermentation products were CA. However, minor malic acid and other unknown products also existed in the culture.

  18. Analysis of betaS and betaA genes in a Mexican population with African roots.

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    Magaña, María Teresa; Ongay, Zoyla; Tagle, Juan; Bentura, Gilberto; Cobián, José G; Perea, F Javier; Casas-Castañeda, Maricela; Sánchez-López, Yoaly J; Ibarra, Bertha

    2002-01-01

    To investigate the origin of the beta(A) and beta(S) genes in a Mexican population with African roots and a high frequency of hemoglobin S, we analyzed 467 individuals (288 unrelated) from different towns in the states of Guerrero and Oaxaca in the Costa Chica region. The frequency of the sickle-cell trait was 12.8%, which may represent a public health problem. The frequencies of the beta-haplotypes were determined from 350 nonrelated chromosomes (313 beta(A) and 37 beta(S)). We observed 15 different beta(A) haplotypes, the most common of which were haplotypes 1 (48.9%), 2 (13.4%), and 3 (13.4%). The calculation of pairwise distributions and Nei's genetic distance analysis using 32 worldwide populations showed that the beta(A) genes are more closely related to those of Mexican Mestizos and North Africans. Bantu and Benin haplotypes and haplotype 9 were related to the beta(S) genes, with frequencies of 78.8, 18.2, and 3.0%, respectively. Comparison of these haplotypes with 17 other populations revealed a high similitude with the population of the Central African Republic. These data suggest distinct origins for the beta(A) and beta(S) genes in Mexican individuals from the Costa Chica region.

  19. Phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit genes of bacterioplankton in the East China Sea

    Institute of Scientific and Technical Information of China (English)

    ZENG Yonghui; JIAO Nianzhi; CAI Haiyuan; CHEN Xihan; WEI Chaoling

    2004-01-01

    Phylogenetic diversity of Form I and Form Ⅱ ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit (rbcL) genes in the inshore and offshore areas of the East China Sea were investigated. Two new primer sets were designed for amplifying partial sequences of rbcL genes from Proteobacteria. Four rbcL gene clone libraries were constructed by amplification and cloning of approximately 640~800 bp sequences of bacterioplankton populations.The method of screening library by denaturing gradient gel electrophoresis (DGGE) was introduced. The results show that the diversity of Form I is higher in offshore waters with higher salinity and lower productivity, while that of Form Ⅱ is higher at the inshore station where salinity is lower and productivity is higher. Several clusters of sequences obtained are deeply rooted and show low similarity (60%~78%) to the known rbcL in existing databases.The degree of diversity of rbcL genes is directly related to environmental variables, including temperature, salinity,pH, dissolved oxygen, etc. These results indicate that rbcL gene can be used as an effective indicator for genetic diversity and population variability of bacterioplankton with the ability of carbon dioxide fixation in the sea.

  20. Engineering the α-ketoglutarate overproduction from raw glycerol by overexpression of the genes encoding NADP+-dependent isocitrate dehydrogenase and pyruvate carboxylase in Yarrowia lipolytica.

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    Yovkova, Venelina; Otto, Christina; Aurich, Andreas; Mauersberger, Stephan; Barth, Gerold

    2014-03-01

    To establish and develop a biotechnological process of α-ketoglutaric acid (KGA) production by Yarrowia lipolytica, it is necessary to increase the KGA productivity and to reduce the amounts of by-products, e.g. pyruvic acid (PA) as major by-product and fumarate, malate and succinate as minor by-products. The aim of this study was the improvement of KGA overproduction with Y. lipolytica by a gene dose-dependent overexpression of genes encoding NADP(+)-dependent isocitrate dehydrogenase (IDP1) and pyruvate carboxylase (PYC1) under KGA production conditions from the renewable carbon source raw glycerol. Recombinant Y. lipolytica strains were constructed, which harbour multiple copies of the respective IDP1, PYC1 or IDP1 and PYC1 genes together. We demonstrated that a selective increase in IDP activity in IDP1 multicopy transformants changes the produced amount of KGA. Overexpression of the gene IDP1 in combination with PYC1 had the strongest effect on increasing the amount of secreted KGA. About 19% more KGA compared to strain H355 was produced in bioreactor experiments with raw glycerol as carbon source. The applied cultivation conditions with this strain significantly reduced the main by-product PA and increased the KGA selectivity to more than 95% producing up to 186 g l(-1) KGA. This proved the high potential of this multicopy transformant for developing a biotechnological KGA production process.

  1. Characterization of ribulose-1, 5-bisphosphate carboxylase/oxygenase and transcriptional analysis of its related genes in Saccharina japonica (Laminariales, Phaeophyta)

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    Shao, Zhanru; Liu, Fuli; Li, Qiuying; Yao, Jianting; Duan, Delin

    2014-03-01

    Saccharina japonica is a common macroalga in sublittoral communities of cold seawater environments, and consequently may have highly efficient ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity for carbon assimilation. In our study, we cloned the full-length Rubisco gene from S. japonica ( SJ-rbc). It contained an open reading frame for a large subunit gene ( SJ — rbcL) of 1 467 bp, a small subunit gene ( SJ-rbcS) of 420 bp, and a SJ-rbcL/S intergenic spacer of 269 bp. The deduced peptides of SJ-rbcL and SJ-rbcS were 488 and 139 amino acids with theoretical molecular weights and isoelectric points of 53.97 kDa, 5.81 and 15.84 kDa, 4.71, respectively. After induction with 1 mmol/L isopropyl- β-D-thiogalactopyranoside for 5 h and purification by Ni2+ affinity chromatography, electrophoresis and western blot detection demonstrated successful expression of the 55 kDa SJ-rbcL protein. Real-time quantitative PCR showed that the mRNA levels of SJ-rbcL in gametophytes increased when transferred into normal growth conditions and exhibited diurnal variations: increased expression during the day but suppressed expression at night. This observation implied that Rubisco played a role in normal gametophytic growth and development. In juvenile sporophytes, mRNA levels of SJ-rbcL, carbonic anhydrase, Calvin-Benson-Bassham cycle-related enzyme, and chloroplast light-harvesting protein were remarkably increased under continuous light irradiance. Similarly, expression of these genes was up-regulated under blue light irradiance at 350 μmol/(m2·s). Our results indicate that long-term white light and short-term blue light irradiance enhances juvenile sporophytic growth by synergistic effects of various photosynthetic elements.

  2. Enhanced drought tolerance in transgenic rice over-expressing of maize C4 phosphoenolpyruvate carboxylase gene via NO and Ca(2+).

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    Qian, Baoyun; Li, Xia; Liu, Xiaolong; Chen, Pingbo; Ren, Chengang; Dai, Chuanchao

    2015-03-01

    We determined the effects of endogenous nitric oxide and Ca(2+) on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C4pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) under drought. In this study, seedlings were subjected to PEG 6000 treatments using PC and wild type (WT; Kitaake). The results showed that, compared with WT, PC had higher relative water content (RWC) and net photosynthetic rate (Pn) under drought. During a 2-day re-watering treatment, Pn recovered faster in PC than in WT. Further analyses showed that, under the drought treatment, the amount of endogenous hydrogen peroxide (H2O2) increased in WT mainly via NADPH oxidase. While in PC, the endogenous nitric oxide (NO) content increased via nitrate reductase and nitric oxide synthase on day 2 of the drought treatment and day 1 of the re-watering treatment. After 2 days of drought treatment, PC also showed higher PEPC activity, calcium content, phospholipase D (PLD) activity, C4-pepc and NAC6 transcript levels, and protein kinase activity as compared with PC without treatment. These changes did not occur in WT. Correlation analysis also proved NO associated with these indicators in PC. Based on these results, there was a particular molecular mechanism of drought tolerance in PC. The mechanism is related to the signaling processes via NO and Ca(2+) involving the protein kinase and the transcription factor, resulted in up-regulation of PEPC activity and its gene expression, such as C4pepc. Some genes encode antioxidant system, cu/zn-sod as well, which promote antioxidant system to clear MDA and superoxide anion radical, thereby conferring drought tolerance.

  3. The human thyrotropin beta-subunit gene differs in 5' structure from murine TSH-beta genes.

    Science.gov (United States)

    Guidon, P T; Whitfield, G K; Porti, D; Kourides, I A

    1988-12-01

    The gene encoding the beta-subunit of human thyrotropin (hTSH-beta) was isolated, and its nucleotide sequence was determined. The gene is 4.3 kb in length, consists of three exons and two introns, and is present as a single copy as determined by Southern blot analysis of total genomic DNA. The protein coding portion of the gene, which includes exons 2 and 3, was isolated from a human genomic phage library, while exon 1, which encodes only 5' untranslated mRNA sequence, was isolated from a plasmid library of size-selected genomic DNA fragments. Here we describe the isolation of the 5' untranslated exon of the hTSH-beta subunit and 5'-flanking region. The structure of the hTSH-beta gene is very similar to the previously characterized TSH-beta genes from mouse and rat. The genes from all three species have two distinct promoter regions, but while both promoters are utilized by the murine TSH-beta genes, the human TSH-beta gene apparently utilizes only the proximal promoter for transcription initiation. A striking difference in hTSH-beta gene structure compared to the murine genes is that exon 1 of the human gene is 36 nucleotides. An analysis of the mouse, rat, and human exon 1 and 5'-flanking region shows a high percentage of sequence homology, with the exception of a 9-nucleotide insertion 13 bases 3' from the proximal TATA box found in the human gene but not found in the other two species. We propose that this insertion results in the additional length of human exon 1 compared to the mouse and rat genes. By isolating the promoter region of the hTSH-beta gene, we can begin to identify specific sequences involved in the regulation of hTSH gene expression.

  4. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

    DEFF Research Database (Denmark)

    Martens, Geert A; Jiang, Lei; Hellemans, Karine H;

    2011-01-01

    The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture...... microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators....

  5. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

    DEFF Research Database (Denmark)

    Martens, Geert A; Jiang, Lei; Hellemans, Karine H;

    2011-01-01

    The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators.......The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture...

  6. A preclinical approach for gene therapy of beta-thalassemia.

    Science.gov (United States)

    Breda, Laura; Kleinert, Dorothy A; Casu, Carla; Casula, Laura; Cartegni, Luca; Fibach, Eitan; Mancini, Irene; Giardina, Patricia J; Gambari, Roberto; Rivella, Stefano

    2010-08-01

    Lentiviral-mediated beta-globin gene transfer successfully treated beta-thalassemic mice. Based on this result, clinical trials were initiated. To date, however, no study has investigated the efficacy of gene therapy in relation to the nature of the different beta-globin mutations found in patients. Most mutations can be classified as beta(0) or beta(+), based on the amount of beta-globin protein produced. Therefore, we propose that a screening in vitro is necessary to verify the efficacy of gene transfer prior to treatment of individual patients. We used a two-phase liquid culture system to expand and differentiate erythroid progenitor cells (ErPCs) transduced with lentiviral vectors. We propose the use of this system to test the efficiency of lentiviral vectors carrying the human beta-globin gene, to correct the phenotype of ErPCs from patients preparing for gene therapy. This new approach might have profound implications for designing gene therapy and for understanding the genotype/phenotype variability observed in Cooley's anemia patients.

  7. Inverted repeat of Olisthodiscus luteus chloroplast DNA contains genes for both subunits of ribulose-1,5-bisphosphate carboxylase and the 32,000-dalton QB protein: Phylogenetic implications

    Science.gov (United States)

    Reith, Michael; Cattolico, Rose Ann

    1986-01-01

    The chloroplast DNA of the chromophytic alga Olisthodiscus luteus has been physically mapped with four restriction enzymes. An inverted repeat of 22 kilobase pairs is present in this 150-kilobase-pair plastid genome. The inverted repeat contains the genes for the large and small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and also codes for the 32,000-dalton QB protein. These observations demonstrate that significant differences exist in chloroplast genome structure and organization among major plant taxa. Images PMID:16578794

  8. Beta-thalassemia and beta[A] globin gene haplotypes in Mexican mestizos.

    Science.gov (United States)

    Villalobos-Arámbula, A R; Bustos, R; Casas-Castañeda, M; Gutiérrez, E; Perea, F J; Thein, S L; Ibarra, B

    1997-04-01

    B-globin haplotypes of 20 beta-thalassemia (beta-thal) and 87 beta(A) Mexican mestizo chromosomes were analyzed to ascertain the origin of the beta-thal alleles and the frequencies and distribution of the beta(A) haplotypes among northwestern Mexican mestizos. Sixteen beta-thal chromosomes carried six Mediterranean alleles [five codon 39 C-->T; two IVS1:1 G-->A; two IVS1:5 G-->A; three IVS1:110 G(A; one codon 11 (-T) and three (deltabeta)zero-thal]; the remaining four were linked to three rare alleles (two -28 A-->C and one each: -87 C-->T and initiation codon ATG-->GTG). Among the 87 beta(A) chromosomes, 17 different 5' haplotypes with frequencies for 1, 3, 2 and 5 of 39.0%, 17. 2%, 9.2% and 6.9%, respectively, were observed. The beta-haplotype analysis showed that 13 out of 16 Mediterranean chromosomes could easily be explained by gene migration; however, one codon 39 associated with haplotype 4 (----+ +-), one IVS1:1 with haplotype 1(+----++) and one IVS1:5 G-->A, may represent separate mutational events. Analysis of the rare alleles showed that the -28 A-->C mutation was associated with the commonest beta(A) haplotype in Mexican mestizos, Mediterraneans and the total world population; therefore an independent origin cannot be ruled out. The -87 C-->T and initiation codon ATG-->GTG were found with beta-haplotypes different from the reported ones, suggesting an indigenous origin.

  9. Characterization of beta-R1, a gene that is selectively induced by interferon beta (IFN-beta) compared with IFN-alpha.

    Science.gov (United States)

    Rani, M R; Foster, G R; Leung, S; Leaman, D; Stark, G R; Ransohoff, R M

    1996-09-13

    We report preliminary characterization of a gene designated beta-R1, which is selectively expressed in response to interferon beta (IFN-beta) compared with IFN-alpha. In human astrocytoma cells, beta-R1 was induced to an equivalent extent by 10 IU/mL IFN-beta or 2500 IU/mL IFN-alpha2. To address the mechanism of this differential response, we analyzed induction of the beta-R1 gene in fibrosarcoma cells and derivative mutant cells lacking components required for signaling by type I IFNs. beta-R1 was readily induced by IFN-beta in the parental 2fTGH cell line, but not by recombinant IFN-alpha2, IFN-alpha Con1, or a mixture of IFN-alpha subtypes. IFN-alpha8 induced beta-R1 weakly. beta-R1 was not induced by IFN-beta in mutant cell lines U2A, U3A, U4A, and U6A, which lack, respectively, p48, STAT1, JAK1, and STAT2. U5A cells, which lack the Ifnar 2.2 component of the IFN-alpha and -beta receptor, also failed to express beta-R1. U1A cells are partially responsive to IFN-beta and IFN-alpha8 but lacked beta-R1 expression, indicating that TYK2 protein is essential for induction of this gene. Taken together, these results suggest that the expression of beta-R1 in response to type I IFN requires IFN-stimulated gene factor 3 plus an additional component, which is more efficiently formed on induction by IFN-beta compared with IFN-alpha.

  10. Meiotic recombination in the beta globin gene cluster causing an error in prenatal diagnosis of beta thalassaemia.

    OpenAIRE

    Camaschella, C.; Serra, A.; Saglio, G; Bertero, M T; Mazza, U; Terzoli, S; Brambati, B; Cremonesi, L.; Travi, M; Ferrari, M

    1988-01-01

    In the course of a prenatal diagnosis for beta thalassaemia by linkage analysis of restriction fragment length polymorphisms, a homozygous beta thalassaemia fetus was misdiagnosed as beta thalassaemia trait. Extensive studies of the polymorphic sites within the beta globin gene cluster in all the members of the family resulted in the conclusion that the paternal chromosome 11 of the newborn was different from that expected. Paternity was confirmed by HLA typing and blood group studies. The an...

  11. Analysis of iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage from a Japanese pyrite mine by use of ribulose-1, 5-bisphosphate carboxylase/oxygenase large-subunit gene.

    Science.gov (United States)

    Kamimura, Kazuo; Okabayashi, Ai; Kikumoto, Mei; Manchur, Mohammed Abul; Wakai, Satoshi; Kanao, Tadayoshi

    2010-03-01

    Iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage (ARD) from a pyrite mine in Yanahara, Okayama prefecture, Japan, were analyzed using the gene (cbbL) encoding the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO). Analyses of partial sequences of cbbL genes from Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidithiobacillus caldus strains revealed the diversity in their cbbL gene sequences. In contrast to the presence of two copies of form I cbbL genes (cbbL1 and cbbL2) in A. ferrooxidans genome, A. thiooxidans and A. caldus had a single copy of form I cbbL gene in their genomes. A phylogenetic analysis based on deduced amino acid sequences from cbbL genes detected in the ARD treatment plant and their close relatives revealed that 89% of the total clones were affiliated with A. ferrooxidans. Clones loosely affiliated with the cbbL from A. thiooxidans NB1-3 or Thiobacillus denitrificans was also detected in the treatment plant. cbbL gene sequences of iron- or sulfur-oxidizing bacteria isolated from the ARD and the ARD treatment plant were not detected in the cbbL libraries from the treatment plant, suggesting the low frequencies of isolates in the samples.

  12. The organization of the gamma-delta-beta gene complex in normal and thalassemia cells.

    Science.gov (United States)

    Bank, A; Mears, J G; Ramirez, F; Burns, A L; Spence, S; Feldenzer, J; Baird, M

    1980-01-01

    Restriction enzyme digestion analysis and direct human globin gene cloning have permitted analysis of the physical arrangement of nucleotide sequences within and surrounding the human globin genes. With these methods it has been shown that the linear arrangement 5' to 3' of the globin genes is G gamma-A gamma-delta-beta. The G gamma and A gamma genes are separated by about 3.5 kilobases (kb), while the A gamma and delta genes are 15 kb apart, and the delta and beta 6.5 kb apart. Each of these genes contains a large intervening sequence (IVS) of approximately 1 kb in precisely the same position between condons 104 and 105. In addition, each of these genes has a small IVS between codons 30 and 31. In homozygous delta beta thalassemia DNA, there is deletion of all of the normal delta and beta gene fragments. However, a new fragment 4.2 kb in size containing the 5' end of the delta globin gene is retained. Retention of this fragment in delta beta thalassemia, but not in HPFH is consistent with a role for sequences in this region for limiting gamma globin gene expression. Studies to date suggest that the beta + and beta 0 thalassemias will be due to a heterogeneous group of DNA defects affecting either beta globin gene transcription or beta mRNA processing. In most cases of beta + and beta 0 thalassemia DNA analyzed, there is no detectable deletion of beta or delta genes. In three India beta 0 patients, deletion of the 3' end of the beta gene has been found. Analysis of cloned beta globin genes from a patient with beta + thalasseia shows differences from normal in the fragments generated by restriction enzymes which cut frequently. Whether these differences are responsible for the defect in thalassemia or are polymorphisms unrelated to thalassemia remains to be determined.

  13. The role of EKLF in human beta-globin gene competition.

    Science.gov (United States)

    Wijgerde, M; Gribnau, J; Trimborn, T; Nuez, B; Philipsen, S; Grosveld, F; Fraser, P

    1996-11-15

    We have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human beta-globin genes in compound EKLF knockout/human beta-locus transgenic mice. EKLF affects only the adult mouse beta-globin genes in homozygous knockout mice; heterozygous mice are unaffected. Here we show that EKLF knockout mice express the human epsilon and gamma-globin genes normally in embryonic red cells. However, fetal liver erythropoiesis, which is marked by a period of gamma- and beta-gene competition in which the genes are alternately transcribed, exhibits an altered ratio of gamma- to beta-gene transcription. EKLF heterozygous fetal livers display a decrease in the number of transcriptionally active beta genes with a reciprocal increase in the number of transcriptionally active gamma genes. beta-Gene transcription is absent in homozygous knockout fetuses with coincident changes in chromatin structure at the beta promoter. There is a further increase in the number of transcriptionally active gamma genes and accompanying gamma gene promoter chromatin alterations. These results indicate that EKLF plays a major role in gamma- and beta-gene competition and suggest that EKLF is important in stabilizing the interaction between the Locus Control Region and the beta-globin gene. In addition, these findings provide further evidence that developmental modulation of globin gene expression within individual cells is accomplished by altering the frequency and/or duration of transcriptional periods of a gene rather than changing the rate of transcription.

  14. Gene expression of beta carotene genes in transgenic biofortified cassava

    OpenAIRE

    Telengech, P. K.; Maling’a, J. N.; Nyende, A. B.; Gichuki, S. T.; Wanjala, B. W.

    2014-01-01

    Cassava is an important food for millions of people around the world. However, cassava is deficient in protein, iron, zinc, pro-vitamin A and vitamin E. Cassava biofortified with pro-vitamin A can help reduce Vitamin A Deficiency among the undernourished communities that rely upon it for sustenance. BioCassava Plus project has developed transgenic cassava that expresses beta carotene in roots using root specific patatin promoter. This study aimed at confirming expression of nptII, crtB and DX...

  15. Gene expression analysis of interferon-beta treatment in multiple sclerosis

    DEFF Research Database (Denmark)

    Sellebjerg, F.; Datta, P.; Larsen, J.;

    2008-01-01

    by treatment with IFN-beta. We use DNA microarrays to study gene expression in 10 multiple sclerosis (MS) patients who began de novo treatment with IFN-beta. After the first injection of IFN-beta, the expression of 74 out of 3428 genes changed at least two-fold and statistically significantly (after Bonferroni...... correction). In contrast, we observed no persisting effects of IFN-beta on gene expression. Among the most strongly induced genes was MXA, which has been used in previous biomarker studies in MS. In addition, the study identified the induction of LGALS9 and TCIR1G, involved in negative regulation of T helper......Treatment with interferon-beta (IFN-beta) induces the expression of hundreds of genes in blood mononuclear cells, and the expression of several genes has been proposed as a marker of the effect of treatment with IFN-beta. However, to date no molecules have been identified that are stably induced...

  16. Molecular analysis of beta-globin gene mutations among Thai beta-thalassemia children: results from a single center study

    Directory of Open Access Journals (Sweden)

    Boonyawat B

    2014-12-01

    Full Text Available Boonchai Boonyawat,1 Chalinee Monsereenusorn,2 Chanchai Traivaree2 1Division of Genetics, Department of Pediatrics, Phramongkutklao Hospital and College of Medicine, Bangkok, Thailand; 2Division of Hematology/Oncology, Department of Pediatrics, Phramongkutklao Hospital and College of Medicine, Bangkok, Thailand Background: Beta-thalassemia is one of the most common genetic disorders in Thailand. Clinical phenotype ranges from silent carrier to clinically manifested conditions including severe beta-thalassemia major and mild beta-thalassemia intermedia. Objective: This study aimed to characterize the spectrum of beta-globin gene mutations in pediatric patients who were followed-up in Phramongkutklao Hospital. Patients and methods: Eighty unrelated beta-thalassemia patients were enrolled in this study including 57 with beta-thalassemia/hemoglobin E, eight with homozygous beta-thalassemia, and 15 with heterozygous beta-thalassemia. Mutation analysis was performed by multiplex amplification refractory mutation system (M-ARMS, direct DNA sequencing of beta-globin gene, and gap polymerase chain reaction for 3.4 kb deletion detection, respectively. Results: A total of 13 different beta-thalassemia mutations were identified among 88 alleles. The most common mutation was codon 41/42 (-TCTT (37.5%, followed by codon 17 (A>T (26.1%, IVS-I-5 (G>C (8%, IVS-II-654 (C>T (6.8%, IVS-I-1 (G>T (4.5%, and codon 71/72 (+A (2.3%, and all these six common mutations (85.2% were detected by M-ARMS. Six uncommon mutations (10.2% were identified by DNA sequencing including 4.5% for codon 35 (C>A and 1.1% initiation codon mutation (ATG>AGG, codon 15 (G>A, codon 19 (A>G, codon 27/28 (+C, and codon 123/124/125 (-ACCCCACC, respectively. The 3.4 kb deletion was detected at 4.5%. The most common genotype of beta-thalassemia major patients was codon 41/42 (-TCTT/codon 26 (G>A or betaE accounting for 40%. Conclusion: All of the beta-thalassemia alleles have been characterized by

  17. Identification and expression of a soybean nodule-enhanced PEP-carboxylase kinase gene (NE-PpcK) that shows striking up-/down-regulation in vivo.

    Science.gov (United States)

    Xu, Wenxin; Zhou, You; Chollet, Raymond

    2003-05-01

    Various isoforms of plant phosphoenolpyruvate carboxylase (PEPC (Ppc)) are controlled post-translationally by an intricate interaction between allosteric regulation and reversible protein phosphorylation. In leaves and root nodules of legumes, these changes in PEPC phosphorylation state are governed primarily by PEPC-kinase (PpcK), a novel, 'minimal but functional' Ser/Thr kinase. To date, this plant-specific kinase has been investigated in molecular terms exclusively in non-leguminous plants, such as Crassulacean-acid-metabolism (CAM) species and Arabidopsis. As an important extension of our earlier biochemical studies on this dedicated kinase and PEPC phosphorylation in soybean (Glycine max) nodules, we now report the molecular cloning of the first legume PpcK from a soybean nodule cDNA library, which encodes a functional, 31.0 kDa PpcK polypeptide. Besides displaying organ, developmental, and spatial expression properties that are strikingly up-regulated in mature nodules, the expression pattern of this transcript is distinct from that of a second soybean PpcK isogene (GmPpcK). The steady-state abundance of this former, nodule-enhanced transcript (NE-PpcK) is markedly influenced by photosynthate supply from the shoots. This latter up-/down-regulation of NE-PpcK transcript level occurs in vivo in concert with the corresponding changes in the nodule PpcK activity, the phosphorylation-state of PEPC, and the abundance of a previously identified, nodule-enhanced transcript (GmPEPC7) that encodes the target enzyme (NE-Ppc). Furthermore, genomic Southern analysis and inspection of the public database indicate that there are at least three distinct PpcK and Ppc isogenes in soybean. Collectively, these and recent findings with Arabidopsis implicate the existence of multiple PpcK-Ppc'expression-partners' in plants, exemplified by NE-PpcK and NE-Ppc in the soybean nodule.

  18. Design of retrovirus vectors for transfer and expression of the human. beta. -globin gene

    Energy Technology Data Exchange (ETDEWEB)

    Miller, A.D.; Bender, M.A.; Harris, E.A.S.; Kaleko, M.; Gelinas, R.E.

    1988-11-01

    Regulated expression of the human ..beta..-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective ..beta..-globin genes. The authors found regions that interfered with virus production within intron 2 of the ..beta..-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for ..beta..-globin expression. Inclusion of ..beta..-globin introns necessitates an antisense orientation of the gene within the retrovirus vector. However, they found no effect of the antisense ..beta..-globin transcription on virus production. A region downstream of the ..beta..-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10/sup 6/ CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human ..beta..-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human ..beta..-globin gene in hematopoietic cells (CFU-S cells) in mice.

  19. Regulation of laminin beta2 chain gene expression in human cancer cell lines

    DEFF Research Database (Denmark)

    Durkin, M E; Nielsen, F C; Loechel, F;

    2001-01-01

    The laminin beta2 chain is a basement membrane component expressed in a tissue- and developmental stage-specific manner. In this report we have examined the transcriptional and post-transcriptional regulation of the human laminin beta2 chain in human tumor cell lines. Both the A204 rhabdomyosarcoma...... and clone A colon carcinoma cells express the laminin beta2 chain mRNA, but only the A204 cells secrete laminin heterotrimers containing the beta2 chain. Segments of the beta2 chain gene promoter region were cloned into luciferase reporter vectors, and their ability to stimulate transcription was tested...... of the human laminin beta2 chain gene generates two isoforms of the 5' untranslated region of the beta2 chain mRNA. The translational efficiencies of the two laminin beta2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell beta2 chain m...

  20. Phylogeny of 16S rRNA, ribulose 1,5-bisphosphate carboxylase/oxygenase, and adenosine 5'-phosphosulfate reductase genes from gamma- and alphaproteobacterial symbionts in gutless marine worms (oligochaeta) from Bermuda and the Bahamas.

    Science.gov (United States)

    Blazejak, Anna; Kuever, Jan; Erséus, Christer; Amann, Rudolf; Dubilier, Nicole

    2006-08-01

    Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluorescence in situ hybridization. As in all other gutless oligochaetes examined to date, I. leukodermatus and I. makropetalos harbor large, oval bacteria identified as Gamma 1 symbionts. The presence of genes coding for ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) and adenosine 5'-phosphosulfate reductase (aprA) supports earlier studies indicating that these symbionts are chemoautotrophic sulfur oxidizers. Alphaproteobacteria, previously identified only in the gutless oligochaete Olavius loisae from the southwest Pacific Ocean, coexist with the Gamma 1 symbionts in both I. leukodermatus and I. makropetalos, with the former harboring four and the latter two alphaproteobacterial phylotypes. The presence of these symbionts in hosts from such geographically distant oceans as the Atlantic and Pacific suggests that symbioses with alphaproteobacterial symbionts may be widespread in gutless oligochaetes. The high phylogenetic diversity of bacterial endosymbionts in two species of the genus Inanidrilus, previously known only from members of the genus Olavius, shows that the stable coexistence of multiple symbionts is a common feature in gutless oligochaetes.

  1. Phylogeny of 16S rRNA, Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase, and Adenosine 5′-Phosphosulfate Reductase Genes from Gamma- and Alphaproteobacterial Symbionts in Gutless Marine Worms (Oligochaeta) from Bermuda and the Bahamas

    Science.gov (United States)

    Blazejak, Anna; Kuever, Jan; Erséus, Christer; Amann, Rudolf; Dubilier, Nicole

    2006-01-01

    Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluorescence in situ hybridization. As in all other gutless oligochaetes examined to date, I. leukodermatus and I. makropetalos harbor large, oval bacteria identified as Gamma 1 symbionts. The presence of genes coding for ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) and adenosine 5′-phosphosulfate reductase (aprA) supports earlier studies indicating that these symbionts are chemoautotrophic sulfur oxidizers. Alphaproteobacteria, previously identified only in the gutless oligochaete Olavius loisae from the southwest Pacific Ocean, coexist with the Gamma 1 symbionts in both I. leukodermatus and I. makropetalos, with the former harboring four and the latter two alphaproteobacterial phylotypes. The presence of these symbionts in hosts from such geographically distant oceans as the Atlantic and Pacific suggests that symbioses with alphaproteobacterial symbionts may be widespread in gutless oligochaetes. The high phylogenetic diversity of bacterial endosymbionts in two species of the genus Inanidrilus, previously known only from members of the genus Olavius, shows that the stable coexistence of multiple symbionts is a common feature in gutless oligochaetes. PMID:16885306

  2. Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls

    Science.gov (United States)

    Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T. V.; Alyethodi, Rafeeque R.; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B.

    2015-12-01

    Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44 °C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly ( P P < 0.01) with HSP70, representing that the change in the expression pattern of these genes is positive and synergistic. These may provide a foundation for understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in cattle.

  3. Detection of Rare Beta Globin Gene Mutations in Northwestern Iran

    Directory of Open Access Journals (Sweden)

    M Haghi

    2007-04-01

    Full Text Available Introduction: Recent molecular studies on Iranian β-thalassemia genes revealed the presence of eight common mutations associated with thalassemia. Although these mutations are frequent, there are other rare and unknown mutations that can create large problems in designing preventive programs. We detected and explained the common mutations in north-western Iran previously and detection of the rare and unknown mutations could be useful in diagnosis and design of future preventive programs. Methods: In this study, 5ml peripheral blood from 20 Azari- β-thalassemia patients whose mutation was not revealed in the previous study was collected and DNA extraction was done by isopropanol and proteinase k method. Initially, samples were examined for the rare mutations: Codon6, Codon16, Codon41/42, Codon36/37, -88 and Codon22 by ARMS – PCR techniques and then the unknown cases were directly sequenced. Results: According to our results, Codon15(TGG-TGA, Codon16(-C, Codon36/37(-T, IVSII-848(C-A, IVSII-745(C-G, -28(A-C( and Codon25/26(+T were recognized and added to the spectrom of beta globin gene mutations in Azerbaijan and Iran. Also, we detected four SNP sites: 5’UTR+20(C-T, Codon2 (CAC-CAT , IVSII-16(C-G and IVSII-666(T-C in β-thalassemia genes. Conclusion: Our results could be useful for developing molecular screening plans and help prenatal diagnosis of beta thalassemia in Azerbaijan , Iran and other neighboring countries.

  4. mRNA expression of iron regulatory genes in beta-thalassemia intermedia and beta-thalassemia major mouse models.

    Science.gov (United States)

    Weizer-Stern, Orly; Adamsky, Konstantin; Amariglio, Ninette; Rachmilewitz, Eliezer; Breda, Laura; Rivella, Stefano; Rechavi, Gideon

    2006-07-01

    beta-Thalassemia is an inherited anemia in which synthesis of the hemoglobin beta-chain is decreased. The excess unmatched alpha-globin chains accumulate in the growing erythroid precursors, causing their premature death (ineffective erythropoiesis). Clinical features of beta-thalassemia include variably severe anemia and iron accumulation due to increased intestinal iron absorption. The most anemic patients require regular blood transfusions, which exacerbate their iron overload and result in damage to vital organs. The hepatic peptide hepcidin, a key regulator of iron metabolism in mammals, was recently found to be low in the urine of beta-thalassemia patients, compared with healthy controls, despite their iron overload. In our work, we measured by RQ-PCR the liver mRNA expression of hepcidin and other iron regulatory genes in beta-thalassemia major mouse model (C57Bl/6 Hbb(th3/th3)), and compared it with beta-thalassemia intermedia mouse model (C57Bl/6 Hbb(th3/+)) and control mice. We found decreased expression of hepcidin and TfR2 and increased expression of TfR1 and NGAL in the beta-thalassemia mouse models, compared with the control mice. Significant down-regulation of hepcidin expression in beta-thalassemia major, despite iron overload, might explain the increased iron absorption typically observed in thalassemia.

  5. The biosynthetic gene cluster for the beta-lactam carbapenem thienamycin in Streptomyces cattleya.

    Science.gov (United States)

    Núñez, Luz Elena; Méndez, Carmen; Braña, Alfredo F; Blanco, Gloria; Salas, José A

    2003-04-01

    beta-lactam ring formation in carbapenem and clavam biosynthesis proceeds through an alternative mechanism to the biosynthetic pathway of classic beta-lactam antibiotics. This involves the participation of a beta-lactam synthetase. Using available information from beta-lactam synthetases, we generated a probe for the isolation of the thienamycin cluster from Streptomyces cattleya. Genes homologous to carbapenem and clavulanic acid biosynthetic genes have been identified. They would participate in early steps of thienamycin biosynthesis leading to the formation of the beta-lactam ring. Other genes necessary for the biosynthesis of thienamycin have also been identified in the cluster (methyltransferases, cysteinyl transferases, oxidoreductases, hydroxylase, etc.) together with two regulatory genes, genes involved in exportation and/or resistance, and a quorum sensing system. Involvement of the cluster in thienamycin biosynthesis was demonstrated by insertional inactivation of several genes generating thienamycin nonproducing mutants.

  6. Total beta-globin gene deletion has high frequency in Filipinos

    Energy Technology Data Exchange (ETDEWEB)

    Patrick, N.; Miyakawa, F.; Hunt, J.A. [Univ. of Hawaii, Honolulu, HI (United States)] [and others

    1994-09-01

    The distribution of {beta}-thalassemia [{beta}{sup Th}] mutations is unique to each ethnic group. Most mutations affect one or a few bases; large deletions have been rare. Among families screened in Hawaii, [{beta}{sup Th}] heterozygotes were diagnosed by microcytosis, absence of abnormal hemoglobins on isoelectric focusing, and raised Hb A{sub 2} by chromatography. Gene frequency for {beta}{sup Th} was 0.02 in Filipinos. In Filipinos, polymerase chain reaction [PCR] with denaturing gradient gel electrophoresis for {beta}{sup Th} mutations detected a mutation in only 6 of 42 {beta}{sup Th} heterozygotes; an IVS2-666 C/T polymorphism showed non-heterozygosity in 37 and heterozygosity in only 5 of these {beta}{sup Th} heterozygotes. One {beta}{sup Th}/{beta}{sup Th} major patient and his mother had no mutation detected by allele-specific oligomer hybridization; PCR failed to amplify any DNA from his {beta}-globin gene. After a total {beta}-globin gene deletion [{beta}{sup Del}] was found in a Filipino family in Ontario, specific PCR amplification for {beta}{sup Del} detected this in 43 of 53 {beta}{sup Th} Filipino samples tested; the above {beta}{sup Th}/{beta}{sup Th} patient was a ({beta}{sup Del}/{beta}{sup Del}) homozygote. The {beta}{sup Del} may account for over 60% of all {beta}{sup Th} alleles in Filipinos; this is the highest proportion of a deletion {beta}{sup Th} mutation reported from any population. Most but not all {beta}{sup Del} heterozygotes had high Hb F [5.13 {plus_minus} 3.94 mean {plus_minus} 1 s.d.] compared to the codon 41/42 four base deletion common in Chinese [2.30 {plus_minus} 0.86], or to {beta}{sup Th} heterozygotes with normal {alpha}-globin genes [2.23 {plus_minus} 0.80].

  7. Intestinal lactase as an autologous beta-galactosidase reporter gene for in vivo gene expression studies.

    Science.gov (United States)

    Salehi, Siamak; Eckley, Lorna; Sawyer, Greta J; Zhang, Xiaohong; Dong, Xuebin; Freund, Jean-Noel; Fabre, John W

    2009-01-01

    Intestinal lactase has potential as an autologous beta-galactosidase reporter gene for long-term gene expression studies in vivo, using chromogenic, luminescent, and fluorogenic substrates developed for Escherichia coli beta-galactosidase. In normal rat tissues, reactivity with a chromogenic fucopyranoside (X-Fuc, the preferred substrate of lactase) was present only at the lumenal surface of small intestine epithelial cells. Full-length lactase (domains I-IV), mature lactase (domains III and IV), and a cytosolic form of mature lactase (domains III and IV, without the signal sequence or transmembrane region) were evaluated. Transfection of HuH-7 cells in vitro, and hydrodynamic gene delivery to the liver in vivo, resulted in excellent gene expression. The full-length and mature (homodimeric, membrane-bound) forms reacted strongly with X-Fuc but not with the corresponding galactopyranoside (X-Gal). However, the presumptively monomeric cytosolic lactase unexpectedly reacted equally well with both substrates. The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was cleaved by cytosolic lactase, but not by full-length or mature lactase. Full-length lactase, when expressed ectopically in hepatocytes in vivo, localized exclusively to the bile canalicular membrane. Intestinal lactase is highly homologous in mice, rats, and humans and has considerable potential for evaluating long-term gene expression in experimental animals and the clinic.

  8. Origin and ascendancy of a chimeric fusion gene: the beta/delta-globin gene of paenungulate mammals.

    Science.gov (United States)

    Opazo, Juan C; Sloan, Angela M; Campbell, Kevin L; Storz, Jay F

    2009-07-01

    The delta-globin gene (HBD) of eutherian mammals exhibits a propensity for recombinational exchange with the closely linked beta-globin gene (HBB) and has been independently converted by the HBB gene in multiple lineages. Here we report the presence of a chimeric beta/delta fusion gene in the African elephant (Loxodonta africana) that was created by unequal crossing-over between misaligned HBD and HBB paralogs. The recombinant chromosome that harbors the beta/delta fusion gene in elephants is structurally similar to the "anti-Lepore" duplication mutant of humans (the reciprocal exchange product of the hemoglobin Lepore deletion mutant). However, the situation in the African elephant is unique in that the chimeric beta/delta fusion gene supplanted the parental HBB gene and is therefore solely responsible for synthesizing the beta-chain subunits of adult hemoglobin. A phylogenetic survey of beta-like globin genes in afrotherian and xenarthran mammals revealed that the origin of the chimeric beta/delta fusion gene and the concomitant inactivation of the HBB gene predated the radiation of "Paenungulata," a clade of afrotherian mammals that includes three orders: Proboscidea (elephants), Sirenia (dugongs and manatees), and Hyracoidea (hyraxes). The reduced fitness of the human Hb Lepore deletion mutant helps to explain why independently derived beta/delta fusion genes (which occur on an anti-Lepore chromosome) have been fixed in a number of mammalian lineages, whereas the reciprocal delta/beta fusion gene (which occurs on a Lepore chromosome) has yet to be documented in any nonhuman mammal. This illustrates how the evolutionary fates of chimeric fusion genes can be strongly influenced by their recombinational mode of origin.

  9. Molecular characterization and endosymbiotic localization of the gene encoding D-ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) form II in the deep-sea vestimentiferan trophosome.

    Science.gov (United States)

    Elsaied, Hosam; Kimura, Hiroyuki; Naganuma, Takeshi

    2002-06-01

    To better understand the contribution of micro-organisms to the primary production in the deep-sea gutless tubeworm Lamellibrachia sp., the 16S-rDNA-based phylogenetic data would be complemented by knowledge of the genes that encode the enzymes relevant to chemoautotrophic carbon fixation, such as D-ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO; EC 4.1.1.39). To phylogenetically characterize the autotrophic endosymbiosis within the trophosome of the tubeworm Lamellibrachia sp., bulk trophosomal DNA was extracted and analysed based on the 16S-rRNA- and RuBisCO-encoding genes. The 16S-rRNA- and RuBisCO-encoding genes were amplified by PCR, cloned and sequenced. For the 16S rDNA, a total of 50 clones were randomly selected and analysed directly by sequencing. Only one operational taxonomic unit resulted from the 16S rDNA sequence analysis. This may indicate the occurrence of one endosymbiotic bacterial species within the trophosome of the Lamellibrachia sp. used in this study. Phylogenetic analysis of the 16S rDNA showed that the Lamellibrachia sp. endosymbiont was closely related to the genus Rhodobacter, a member of the alpha-Protebacteria. For the RuBisCO genes, only the form II gene (cbbM) was amplified by PCR. A total of 50 cbbM clones were sequenced, and these were grouped into two operational RuBisCO units (ORUs) based on their deduced amino acid sequences. The cbbM ORUs showed high amino acid identities with those recorded from the ambient sediment bacteria. To confirm the results of sequence analysis, the localization of the symbiont-specific 16S rRNA and cbbM sequences in the Lamellibrachia sp. trophosome was visualized by in situ hybridization (ISH), using specific probes. Two types of cells, coccoid and filamentous, were observed at the peripheries of the trophosome lobules. Both the symbiont-specific 16S rDNA and cbbM probes hybridized at the same sites coincident with the location of the coccoid cells, whereas the filamentous cells showed no

  10. Structure of the human G gamma-A gamma-delta-beta-globin gene locus

    NARCIS (Netherlands)

    Bernards, R.A.; Little, P.F.R.; Annison, F.; Williamson, R.; Flavell, R.A.

    1979-01-01

    We have constructed a physical map of the human G gamma-, A gamma-, delta-, and beta-globin genes. The previously described maps of the fetal and adult beta-like globin genes have been linked to one another by identification of a DNA fragment, generated by BamHI, that contains part of each of the A

  11. Peroxisome proliferator-activated receptor alpha (PPARalpha) protects against oleate-induced INS-1E beta cell dysfunction by preserving carbohydrate metabolism

    DEFF Research Database (Denmark)

    Frigerio, F; Brun, T; Bartley, C;

    2009-01-01

    AIMS/HYPOTHESIS: Pancreatic beta cells chronically exposed to fatty acids may lose specific functions and even undergo apoptosis. Generally, lipotoxicity is triggered by saturated fatty acids, whereas unsaturated fatty acids induce lipodysfunction, the latter being characterised by elevated basal...... enzyme pyruvate carboxylase. PPARalpha overproduction increased both beta-oxidation and fatty acid storage in the form of neutral triacylglycerol, revealing overall induction of lipid metabolism. These observations were substantiated by expression levels of associated genes. CONCLUSIONS...

  12. Molecular analysis of the beta-globin gene cluster in the Niokholo Mandenka population reveals a recent origin of the beta(S) Senegal mutation.

    Science.gov (United States)

    Currat, Mathias; Trabuchet, Guy; Rees, David; Perrin, Pascale; Harding, Rosalind M; Clegg, John B; Langaney, André; Excoffier, Laurent

    2002-01-01

    A large and ethnically well-defined Mandenka sample from eastern Senegal was analyzed for the polymorphism of the beta-globin gene cluster on chromosome 11. Five RFLP sites of the 5' region were investigated in 193 individuals revealing the presence of 10 different haplotypes. The frequency of the sickle-cell anemia causing mutation (beta(S)) in the Mandenka estimated from this sample is 11.7%. This mutation was found strictly associated with the single Senegal haplotype. Approximately 600 bp of the upstream region of the beta-globin gene were sequenced for a subset of 94 chromosomes, showing the presence of four transversions, five transitions, and a composite microsatellite polymorphism. The sequence of 22 beta(S) chromosomes was also identical to the previously defined Senegal haplotype, suggesting that this mutation is very recent. Monte Carlo simulations (allowing for a specific balancing selection model, a logistic growth of the population, and variable initial frequencies of the Senegal haplotype) were used to estimate the age of the beta(S) mutation. Resulting maximum-likelihood estimates are 45-70 generations (1,350-2,100 years) for very different demographic scenarios. Smallest confidence intervals (25-690 generations) are obtained under the hypothesis that the Mandenka population is large (N(e) >5,000) and stationary or that it has undergone a rapid demographic expansion to a current size of >5,000 reproducing individuals, which is quite likely in view of the great diversity found on beta(A) chromosomes.

  13. Expression profiling of genes regulated by TGF-beta: Differential regulation in normal and tumour cells

    Directory of Open Access Journals (Sweden)

    Takahashi Takashi

    2007-04-01

    Full Text Available Abstract Background TGF-beta is one of the key cytokines implicated in various disease processes including cancer. TGF-beta inhibits growth and promotes apoptosis in normal epithelial cells and in contrast, acts as a pro-tumour cytokine by promoting tumour angiogenesis, immune-escape and metastasis. It is not clear if various actions of TGF-beta on normal and tumour cells are due to differential gene regulations. Hence we studied the regulation of gene expression by TGF-beta in normal and cancer cells. Results Using human 19 K cDNA microarrays, we show that 1757 genes are exclusively regulated by TGF-beta in A549 cells in contrast to 733 genes exclusively regulated in HPL1D cells. In addition, 267 genes are commonly regulated in both the cell-lines. Semi-quantitative and real-time qRT-PCR analysis of some genes agrees with the microarray data. In order to identify the signalling pathways that influence TGF-beta mediated gene regulation, we used specific inhibitors of p38 MAP kinase, ERK kinase, JNK kinase and integrin signalling pathways. The data suggest that regulation of majority of the selected genes is dependent on at least one of these pathways and this dependence is cell-type specific. Interestingly, an integrin pathway inhibitor, RGD peptide, significantly affected TGF-beta regulation of Thrombospondin 1 in A549 cells. Conclusion These data suggest major differences with respect to TGF-beta mediated gene regulation in normal and transformed cells and significant role of non-canonical TGF-beta pathways in the regulation of many genes by TGF-beta.

  14. Cis-cotranscription of two beta globin genes during chicken primitive hematopoiesis.

    Directory of Open Access Journals (Sweden)

    Hiroki Nagai

    Full Text Available Chicken beta globin locus contains four genes, two of which, rho and epsilon, are expressed from the earliest stage of primitive hematopoiesis. Here we show that the transcription of these two genes in the nucleus engages in "on/off" phases. During each "on" phase, cotranscription of rho and epsilon in cis is favored. We propose that these two chicken beta globin genes are transcribed not by competing for a transcription initiation complex, but in a cooperative way.

  15. Phylogeny and evolutionary history of Leymus (Triticeae; Poaceae based on a single-copy nuclear gene encoding plastid acetyl-CoA carboxylase

    Directory of Open Access Journals (Sweden)

    Ding Cun-Bang

    2009-10-01

    Full Text Available Abstract Background Single- and low- copy genes are less likely subject to concerted evolution, thus making themselves ideal tools for studying the origin and evolution of polyploid taxa. Leymus is a polyploid genus with a diverse array of morphology, ecology and distribution in Triticeae. The genomic constitution of Leymus was assigned as NsXm, where Ns was presumed to be originated from Psathyrostachys, while Xm represented a genome of unknown origin. In addition, little is known about the evolutionary history of Leymus. Here, we investigate the phylogenetic relationship, genome donor, and evolutionary history of Leymus based on a single-copy nuclear Acc1 gene. Results Two homoeologues of the Acc1 gene were isolated from nearly all the sampled Leymus species using allele-specific primer and were analyzed with those from 35 diploid taxa representing 18 basic genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that (1 Leymus is closely related to Psathyrostachys, Agropyron, and Eremopyrum; (2 Psathyrostachys juncea is an ancestral Ns-genome donor of Leymus species; (3 the Xm genome in Leymus may be originated from an ancestral lineage of Agropyron and Eremopyrum triticeum; (4 the Acc1 sequences of Leymus species from the Qinghai-Tibetan plateau are evolutionarily distinct; (5 North America Leymus species might originate from colonization via the Bering land bridge; (6 Leymus originated about 11-12MYA in Eurasia, and adaptive radiation might have occurred in Leymus during the period of 3.7-4.3 MYA and 1.7-2.1 MYA. Conclusion Leymus species have allopolyploid origin. It is hypothesized that the adaptive radiation of Leymus species might have been triggered by the recent upliftings of the Qinghai-Tibetan plateau and subsequent climatic oscillations. Adaptive radiation may have promoted the rapid speciation, as well as the fixation of unique morphological characters in Leymus. Our results shed new light on our

  16. Tumor-produced, active Interleukin-1 {beta} regulates gene expression in carcinoma-associated fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Dudas, Jozsef, E-mail: Jozsef.Dudas@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Fullar, Alexandra, E-mail: fullarsz@gmail.com [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); 1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Ulloei ut 26, H-1085 Budapest (Hungary); Bitsche, Mario, E-mail: Mario.Bitsche@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Schartinger, Volker, E-mail: Volker.Schartinger@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Kovalszky, Ilona, E-mail: koval@korb1.sote.hu [1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Ulloei ut 26, H-1085 Budapest (Hungary); Sprinzl, Georg Mathias, E-mail: Georg.Sprinzl@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Riechelmann, Herbert, E-mail: Herbert.Riechelmann@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria)

    2011-09-10

    Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial-mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1{beta} (IL1-{beta}) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-{beta} expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-{beta} processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-{beta}. IL1-{beta} signaling was investigated by western blot and immunocytochemistry. IL1-{beta}-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-{beta}, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NF{kappa}B{alpha}. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-{beta} reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-{beta}-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-{beta} in the tumor cells leads to IL1-{beta}-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression. -- Graphical abstract: SCC-25 cells produce active, processed IL1-{beta}. PDL fibroblasts possess receptor for IL1-{beta}, and its expression is increased 4.56-times in the

  17. Activation of the human beta interferon gene by the adenovirus type 12 E1B gene

    Energy Technology Data Exchange (ETDEWEB)

    Shiroki, K.; Toth, M.

    1988-01-01

    The transcription of endogenous beta interferon mRNA was activated in human embryo kidney (HEK) cells infected with adenovirus 12 (Ad12) but was activated only inefficiently or not at all in HEK cells infected with Ad5 and rc-1 (Ad5 dl312 containing the Ad12 E1A region). The analysis with Ad12 mutants showed that Ad12 E1B products, especially the 19K protein, were important for the expression of the endogenous beta interferon gene and Ad12 E1A products were not involved in the expression. The expression of exogeneously transfected pIFN-CAT (a hybrid plasmid having the human beta interferon promoter fused with the CAT gene) was activated in HEK and chicken embryo fibroblast (CEF) cells infected with either Ad12 or Ad5. The analysis of cotransfection of CEF cells with pIFN-CAT and plasmids containing fragments of Ad12 or Ad5 DNA showed that Ad12 or Ad5 E1B (possibly the 19K protein) was and E1A was not involved in the expression of the exogenous pIFN-CAT.

  18. Successful treatment of murine beta-thalassemia intermedia by transfer of the human beta-globin gene.

    Science.gov (United States)

    May, Chad; Rivella, Stefano; Chadburn, Amy; Sadelain, Michel

    2002-03-15

    The beta-thalassemias are caused by more than 200 mutations that reduce or abolish beta-globin production. The severity of the resulting anemia can lead to lifelong transfusion dependency. A genetic treatment based on globin gene transfer would require that transgene expression be erythroid specific, elevated, and sustained over time. We report here that long-term synthesis of chimeric hemoglobin (mualpha(2):hubeta(A)(2)) could be achieved in mice with beta-thalassemia intermedia following engraftment with bone marrow cells transduced with a lentiviral vector encoding the human beta-globin gene. In the absence of any posttransduction selection, the treated chimeras exhibit durably increased hemoglobin levels without diminution over 40 weeks. Ineffective erythropoiesis and extramedullary hematopoiesis (EMH) regress, as reflected by normalization of spleen size, architecture, hematopoietic colony formation, and disappearance of liver EMH. These findings establish that a sustained increase of 3 to 4 g/dL hemoglobin is sufficient to correct ineffective erythropoiesis. Hepatic iron accumulation is markedly decreased in 1-year-old chimeras, indicating persistent protection from secondary organ damage. These results demonstrate for the first time that viral-mediated globin gene transfer in hematopoietic stem cells effectively treats a severe hemoglobin disorder.

  19. Structure and expression of the mouse beta-hexosaminidase genes, Hexa and Hexb.

    Science.gov (United States)

    Yamanaka, S; Johnson, O N; Norflus, F; Boles, D J; Proia, R L

    1994-06-01

    Two genes, HEXA and HEXB, encode the alpha- and beta-subunits, respectively, of human beta-hexosaminidase. In the mouse, the corresponding genes are termed Hexa and Hexb. The subunits dimerize to yield three isozymes, beta-hexosaminidase A (alpha beta), B (beta beta), and S (alpha alpha), that have the capacity to degrade a variety of substrates containing beta-linked N-acetylglucosamine and N-acetylgalactosamine residues. Mutations in the HEXA or HEXB gene resulting in a beta-hexosaminidase deficiency cause Tay-Sachs or Sandhoff disease, respectively. As a prelude to the creation of mouse models of these lysosomal storage diseases, we have characterized the molecular biology of the mouse beta-hexosaminidase system. Protein sequences derived from the cloned Hexa and Hexb cDNAs were 55% identical to each other and were also very similar to the cognate human sequences: 84% sequence identity with human HEXA and 75% with HEXB. The mouse hexosaminidase subunits, when expressed in HeLa cells from the cDNAs, displayed specificity toward synthetic substrates similar to the human subunits. The Hexa and Hexb genes were 25 and 22 kb in length, respectively. Each gene was divided into 14 exons, with the positions of introns precisely matching those of the corresponding human genes. The 5' flanking regions of the mouse genes demonstrated promoter activity as ascertained by their ability to drive chloramphenicol acetyltransferase gene expression in transfected NIH 3T3 cells. The sequences of these regulatory regions were G+C-rich in the 200 bp upstream of the respective initiator ATGs. Several putative promoter elements were present, including Sp1, AP2, CAAT, and TATA motifs.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. The transforming growth factor-beta receptor genes and the risk of intracranial aneurysms

    NARCIS (Netherlands)

    Ruigrok, Ynte M.; Baas, Annette F.; Medic, Jelena; Wijmenga, Cisca; Rinkel, Gabriel J. E.

    2012-01-01

    Background Mutations in the receptor genes of the transforming growth factor beta pathway, TGFBR1 and TGFBR2, cause syndromes with thoracic aortic aneurysms, while genetic variants in TGFBR1 and TGFBR2 are associated with abdominal aortic aneurysms. The transforming growth factor-beta pathway may be

  1. Molecular cloning and characterization of mutant and wild-type human. beta. -actin genes

    Energy Technology Data Exchange (ETDEWEB)

    Leavitt, J.; Gunning, P.; Porreca, P.; Ng, S.Y.; Lin, C.H.; Kedes, L.

    1984-10-01

    There are more than 20 ..beta..-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional ..beta..-actin genes, they used the new method of B. Seed for selecting genomic clones by homologous recombination. A derivative of the ..pi..VX miniplasmid, ..pi..AN7..beta..1, was constructed by insertion of the 600-base-pair 3' untranslated region of the ..beta..-actin mRNA expressed in human fibroblasts. Five clones containing ..beta..-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete ..beta..-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then use to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant ..beta..-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived closed verified the identity of the ..beta..-actin gene expressed in human fibroblasts.

  2. The beta-globin gene in Sardinian delta beta 0-thalassemia carries a C----T nonsense mutation at codon 39.

    OpenAIRE

    Guida, S; Giglioni, B; Comi, P; Ottolenghi, S; Camaschella, C.; Saglio, G

    1984-01-01

    Sardinian delta beta 0-thalassemia is an inherited syndrome characterized by the inactivity of the beta-globin gene and the persistent activity of the fetal gamma-globin genes, particularly the A gamma-globin gene. Previous mapping studies with restriction enzymes failed to show any abnormality in the non-alpha globin gene cluster. We have now examined the possibility that this syndrome might result from a single rather than two different defects. Restriction enzyme polymorphisms linked to th...

  3. Molecular cloning and characterization of the human beta-like globin gene cluster.

    Science.gov (United States)

    Fritsch, E F; Lawn, R M; Maniatis, T

    1980-04-01

    The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.

  4. Effects of ultrasound on Transforming Growth Factor-beta genes in bone cells

    Directory of Open Access Journals (Sweden)

    J Harle

    2005-12-01

    Full Text Available Therapeutic ultrasound (US is a widely used form of biophysical stimulation that is increasingly applied to promote fracture healing. Transforming growth factor-beta (TGF-beta, which is encoded by three related but different genes, is known to play a major part in bone growth and repair. However, the effects of US on the expression of the TGF-beta genes and the physical acoustic mechanisms involved in initiating changes in gene expression in vitro, are not yet known. The present study demonstrates that US had a differential effect on these TGF-beta isoforms in a human osteoblast cell line, with the highest dose eliciting the most pronounced up-regulation of both TGF-beta1 and TGF-beta3 at 1 hour after treatment and thereafter declining. In contrast, US had no effect on TGF-beta2 expression. Fluid streaming rather than thermal effects or cavitation was found to be the most likely explanation for the gene responses observed in vitro.

  5. Expression of bacterial poly(3-hydroxybutyrate) synthesis genes in hairy roots of sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Menzel, G; Harloff, H-J; Jung, C

    2003-01-01

    Three genes from Ralstonia eutropha necessary for poly(3-hydroxybutyrate) (PHB) synthesis were introduced into the hairy roots of sugar beet. Transformation of a vector construct harbouring the PHB genes, each fused to the coding region of the pea ribulose-bisphosphate carboxylase plastid targeting sequence, resulted in 20 transgenic hairy-root clones, producing up to 55 mg high molecular PHB/g dry weight, as identified by gas chromatography, gel permeation chromatography and HPLC. Accumulation of PHB polymer in sugar beet root leucoplasts was confirmed by transmission electron microscopy. Thus, for the first time, plastidic PHB production was demonstrated for roots of a carbohydrate-storing crop plant.

  6. Beta Thalassemia

    Science.gov (United States)

    Beta thalassemia is found in people of Mediterranean, Middle Eastern, African, South Asian (Indian, Pakistani, etc.), Southeast Asian and Chinese descent. 1 Beta Thalassemia ßß Normal beta globin genes found on chromosomes ...

  7. Cloning and sequencing of the beta-glucosidase gene from Acetobacter xylinum ATCC 23769.

    Science.gov (United States)

    Tajima, K; Nakajima, K; Yamashita, H; Shiba, T; Munekata, M; Takai, M

    2001-12-31

    The beta-glucosidase gene (bglxA) was cloned from the genomic DNA of Acetobacter xylinum ATCC 23769 and its nucleotide sequence (2200 bp) was determined. This bglxA gene was present downstream of the cellulose synthase operon and coded for a polypeptide of molecular mass 79 kDa. The overexpression of the beta-glucosidase in A. xylinum caused a tenfold increase in activity compared to the wild-type strain. In addition, the action pattern of the enzyme was identified as G3ase activity. The deduced amino acid sequence of the bglxA gene showed 72.3%, 49.6%, and 45.1% identity with the beta-glucosidases from A. xylinum subsp. sucrofermentans, Cellvibrio gilvus, and Mycobacterium tuberculosis, respectively. Based on amino acid sequence similarities, the beta-glucosidase (BglxA) was assigned to family 3 of the glycosyl hydrolases.

  8. Elucidation of IL-1/TGF-beta interactions in mouse chondrocyte cell line by genome-wide gene expression

    DEFF Research Database (Denmark)

    Takahashi, N; Rieneck, K; van der Kraan, P M;

    2005-01-01

    To elucidate the antagonism between interleukin-1 (IL-1) and transforming growth factor-beta (TGF-beta) at the gene expression level, as IL-1 and TGF-beta are postulated to be critical mediators of cartilage degeneration/protection in rheumatic diseases.......To elucidate the antagonism between interleukin-1 (IL-1) and transforming growth factor-beta (TGF-beta) at the gene expression level, as IL-1 and TGF-beta are postulated to be critical mediators of cartilage degeneration/protection in rheumatic diseases....

  9. Arrested rearrangement of TCR V[beta] genes in thymocytes from children with x-linked severe combined immunodeficiency disease

    Energy Technology Data Exchange (ETDEWEB)

    Sleasman, J.W.; Harville, T.O.; White, G.B.; Barrett, D.J. (Univ. of Florida College of Medicine, Gainsville, FL (United States)); George, J.F. (Univ. of Alabama, Birmingham, AL (United States)); Goodenow, M.M. (Univ. of Florida College of Medicine, Gainsville, FL (United States) Univ. of Alabama, Birmingham, AL (United States))

    1994-07-01

    Human X-linked severe combined immunodeficiency disease (SCID) is an immunodeficiency disorder in which T cell development is arrested in the thymic cortex. B lymphocytes in children with X-linked SCID seem to differentiate normally. X-linked SCID is associated with a mutation in the gene that encodes the IL-2R [gamma]-chain. Because TCR-[beta] gene recombination is a pivotal initial event in T lymphocyte onteogeny within the thymus, the authors hypothesized that a failure to express normal IL-2R[gamma] could lead to impaired TCR-[beta] gene recombination in early thymic development. PCR was used to determine the status of TCR-[beta] gene-segment rearrangements in thymic DNA that had been obtained from children with X-linked SCID. The initial step in TCR-[beta] gene rearrangement, that of D[beta] to J[beta] recombination, was readily detected in all thymus samples from children with X-linked SCID; in contrast, V[beta] to DJ[beta] gene rearrangements were undetectable in the same samples. Both D[beta] to J[beta] and V[beta] to DJ[beta] TCR genes were rearranged in the thymic tissues obtained from immunologically normal children. The authors conclude that TCR[beta]-chain gene rearrangement is arrested in children with X-linked SCID. The results suggest a causative relationship between the failure of TCR [beta]-chain gene arrangements to proceed beyond DJ[beta] rearrangements and the production of a nonfunctional IL-2R [gamma]-chain. 45 refs., 3 figs.

  10. Structure of the gene for human. beta. /sub 2/-adrenergic receptor: expression and promoter characterization

    Energy Technology Data Exchange (ETDEWEB)

    Emorine, L.J.; Marullo, S.; Delavier-Klutchko, C.; Kaveri, S.V.; Durieu-Trautmann, O.; Strosberg, A.D.

    1987-10-01

    The genomic gene coding for the human ..beta../sub 2/-adrenergic receptor (..beta../sub 2/AR) from A431 epidermoid cells has been isolated. Transfection of the gene into eukaryotic cells restores a fully active receptor/GTP-binding protein/adenylate cyclase complex with ..beta../sub 2/AR properties. Southern blot analyses with ..beta../sub 2/AR-specific probes show that a single ..beta../sub 2/AR gene is common to various human tissues and that its flanking sequences are highly conserved among humans and between man and rabbit, mouse, and hamster. Functional significance of these regions is supported by the presence of a promoter region (including mRNA cap sites, two TATA boxes, a CAAT box, and three G + C-rich regions that resemble binding sites for transcription factor Sp1) 200-300 base pairs 5' to the translation initiation codon. In the 3' flanking region, sequences homologous to glucocorticoid-response elements might be responsible for the increased expression of the ..beta../sub 2/AR gene observed after treatment of the transfected cells with hydrocortisone. In addition, 5' to the promoter region, an open reading frame encodes a 251-residue polypeptide that displays striking homologies with protein kinases and other nucleotide-binding proteins.

  11. IFN-beta gene deletion leads to augmented and chronic demyelinating experimental autoimmune encephalomyelitis

    DEFF Research Database (Denmark)

    Teige, Ingrid; Treschow, Alexandra; Teige, Anna;

    2003-01-01

    Since the basic mechanisms behind the beneficial effects of IFN-beta in multiple sclerosis (MS) patients are still obscure, here we have investigated the effects of IFN-beta gene disruption on the commonly used animal model for MS, experimental autoimmune encephalomyelitis (EAE). We show that IFN......-beta knockout (KO) mice are more susceptible to EAE than their wild-type (wt) littermates; they develop more severe and chronic neurological symptoms with more extensive CNS inflammation and demyelination. However, there was no discrepancy observed between wt and KO mice regarding the capacity of T cells...... to proliferate or produce IFN-gamma in response to recall Ag. Consequently, we addressed the effect of IFN-beta on encephalitogenic T cell development and the disease initiation phase by passive transfer of autoreactive T cells from KO or wt littermates to both groups of mice. Interestingly, IFN-beta KO mice...

  12. Genes and mechanisms involved in beta-amyloid generation and Alzheimer's disease.

    Science.gov (United States)

    Steiner, H; Capell, A; Leimer, U; Haass, C

    1999-01-01

    Alzheimer's disease is characterized by the invariable accumulation of senile plaques that are predominantly composed of amyloid beta-peptide (Abeta). Abeta is generated by proteolytic processing of the beta-amyloid precursor protein (betaAPP) involving the combined action of beta- and gamma-secretase. Cleavage within the Abeta domain by alpha-secretase prevents Abeta generation. In some very rare cases of familial AD (FAD), mutations have been identified within the betaAPP gene. These mutations are located close to or at the cleavage sites of the secretases and pathologically effect betaAPP processing by increasing Abeta production, specifically its highly amyloidogenic 42 amino acid variant (Abeta42). Most of the mutations associated with FAD have been identified in the two presenilin (PS) genes, particularly the PS1 gene. Like the mutations identified within the betaAPP gene, mutations in PS1 and PS2 cause the increased generation of Abeta42. PS1 has been shown to be functionally involved in Notch signaling, a key process in cellular differentation, and in betaAPP processing. A gene knock out of PS1 in mice leads to an embryonic lethal phenotype similar to that of mice lacking Notch. In addition, absence of PS1 results in reduced gamma-secretase cleavage and leads to an accumulation of betaAPP C-terminal fragments and decreased amounts of Abeta. Recent work may suggest that PS1 could be the gamma-secretase itself, exhibiting the properties of a novel aspartyl protease. Mutagenesis of either of two highly conserved intramembraneous aspartate residues of PS1 leads to reduced Abeta production as observed in the PS1 knockout. A corresponding mutation in PS2 interfered with betaAPP processing and Notch signaling suggesting a functional redundancy of both presenilins. In this issue, some of the recent work on the molecular mechanisms involved in Alzheimer's disease (AD) as well as novel diagnostic approaches and risk factors for AD will be discussed. In the first

  13. Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase {beta}-subunit gene family

    Energy Technology Data Exchange (ETDEWEB)

    Pestov, Nikolay B. [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997 (Russian Federation); Zhao, Hao [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Basrur, Venkatesha [Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109 (United States); Modyanov, Nikolai N., E-mail: nikolai.modyanov@utoledo.edu [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States)

    2011-09-09

    Highlights: {yields} Structural properties of BetaM and Na,K-ATPase {beta}-subunits are sharply different. {yields} BetaM protein is concentrated in nuclear membrane of skeletal myocytes. {yields} BetaM does not associate with a Na,K-ATPase {alpha}-subunit in skeletal muscle. {yields} Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. {yields} BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a {beta}-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase {beta}-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was

  14. Beta-globin gene cluster haplotypes and alpha-thalassemia in sickle cell disease patients from Trinidad.

    Science.gov (United States)

    Jones-Lecointe, Altheia; Smith, Erskine; Romana, Marc; Gilbert, Marie-Georges; Charles, Waveney P; Saint-Martin, Christian; Kéclard, Lisiane

    2008-01-01

    In this study, we have determined the frequency of beta(S) haplotypes in 163 sickle cell disease patients from Trinidad. The alpha(3.7) globin gene deletion status was also studied with an observed gene frequency of 0.17. Among the 283 beta(S) chromosomes analyzed, the Benin haplotype was the most prevalent (61.8%) followed by Bantu (17.3%), Senegal (8.5%), Cameroon (3.5%), and Arab-Indian (3.2%), while 5.7% of them were atypical. This beta(S) haplotypes distribution differed from those previously described in other Caribbean islands (Jamaica, Guadeloupe, and Cuba), in agreement with the known involvement of the major colonial powers (Spain, France, and Great Britain) in the slave trade in Trinidad and documented an Indian origin of the beta(S) gene.

  15. Transformation of frog embryos with a rabbit beta-globin gene.

    OpenAIRE

    Rusconi, S; Schaffner, W

    1981-01-01

    In order to study the fate and possible expression of foreign DNA during embryogenesis of the frog Xenopus laevis, we have injected a rabbit beta-globin gene into fertilized Xenopus eggs. Frog embryo DNA was extracted at various stages of development, fractionated by agarose gel electrophoresis, transferred to nitrocellulose filters, and hybridized to labeled beta-globin recombinant plasmid DNA. It was found that the injected DNA replicated extrachromosomally, reaching, at gastrula stage, a l...

  16. The Survey of Genes Encoding Beta-Lactamases, in Escherichia Coli Resistant to Beta-Lactam and Non-Beta-Lactam Antibiotics

    Directory of Open Access Journals (Sweden)

    Fereshteh Shahcheraghi

    2010-01-01

    Full Text Available Resistance to the new generation of cephalosporins which is mediated by Extended-Spectrum beta-lactamases (ESBLs has been found amongEscherichia coli isolates throughout the world. These resistance genes and their producers, the micro-organisms carrying beta-lactamases, are responsible for serious clinical and therapeutic problems among inpatients and it is necessary to pay more attention to detection of ESBLs producing organisms.Materiasl and MethodsCollectively 260 isolates of E. coli were obtained from 6 hospitals in Tehran (Iran during April-2006 to April-2007. The antibiotic susceptibility patterns of isolates were determined by disk diffusion method. phenotypic confirmatory test (PCT was carried out for screening of ESBLs. Microbroth dilution assay was used to determine the minimum inhibitory concentration (MIC of ceftazidime. Isolates showing MIC≥2 μg/ml were subjected to polymerase chain reaction (PCR targeting blaTEM, blaSHV, blaCTX and blaPER genes. ResultsThe PCT showed that 48.08% of isolates are ESBL producers (125 of 260. The majority of cefotaxime resistant (90.8% and ceftazidime resistant (92.5% isolates were ESBL producers. The obtained results by PCR revealed that 5.77% (n=15 of 260 and 24.23 (n=63 of isolates can produce SHV and TEM type enzymes respectively. blaCTX was detected in 20.38% of isolates (n=53 and none of them could produce blaPER type beta-lactamases. ConclusionThe results of our study showed that the ESBL genes have high prevalence among clinical isolates of E. coli. Such high dissemination of ESBLs is a serious problem for public health and therefore, it's necessary to seek a program for monitoring ESBLs in hospitals.

  17. Promoters for the human beta-hexosaminidase genes, HEXA and HEXB.

    Science.gov (United States)

    Norflus, F; Yamanaka, S; Proia, R L

    1996-02-01

    Human lysosomal beta-hexosaminidases are encoded by two genes, HEXA and HEXB, specifying an alpha- and a beta-subunit, respectively. The subunits dimerize to form beta-hexosaminidase A (alpha beta), beta-hexosaminidase B (beta beta), and beta-hexosaminidase S (alpha alpha). This enzyme system has the capacity to degrade a variety of cellular substrates: oligosaccharides, glycosaminoglycans, and glycolipids containing beta-linked N-acetylglucosaminyl or N-galactosaminyl residues. Mutations in either the HEXA gene or HEXB gene lead to an accumulation of GM2 ganglioside in neurons, resulting in the severe neurodegenerative disorders termed the GM2 gangliosidoses. To identify the DNA elements responsible for hexosaminidase expression, we ligated the 5'-flanking sequences of both the human and mouse hexosaminidase genes to a chloramphenicol acetyltransferase (CAT) gene. The resulting plasmids were transfected into NIH-3T3 cells and CAT activity was determined as a measure of promoter strength. By 5' deletion analysis, it was found that essential sequences for HEXA expression resided within a 40-bp region between 100 bp and 60 bp upstream of the ATG initiation codon. This area contained two potential estrogen response element half-sites as well as potential binding sites for transcription factors NF-E1 and AP-2. Similarly, important HEXB promoter sequences were localized to a 60-bp region between 150 bp and 90 bp upstream of the ATG codon. By performing scanning mutagenesis on a 60-bp region within the 150-bp HEXB construct, we defined an essential promoter element of 12 bp that contained two potential AP-1 sites. The mouse Hexa and Hexb 5'-flanking sequences were found to contain regions similar in sequence, location, and activity to the essential promoter elements defined in the cognate human genes. No sequence similarity was found, however, between 5'-flanking regions of the HEXA and HEXB genes. These essential promoter elements represent potential sites for HEXA and

  18. A mutation of the beta-globin gene initiation codon, ATG-->AAG, found in a French Caucasian man.

    Science.gov (United States)

    Lacan, Philippe; Aubry, Martine; Couprie, Nicole; Francina, Alain

    2005-01-01

    A new mutation of the beta-globin gene initiation codon, ATG-->AAG (Met-->Tyr), is reported in a man originating from the southeast of France. Typical hematological findings of beta-thalassemia (thal) trait were found. We emphasize the importance of characterizing uncommon beta-thal mutations for genetic counseling.

  19. Elucidation of IL-1/TGF-beta interactions in mouse chondrocyte cell line by genome-wide gene expression

    DEFF Research Database (Denmark)

    Takahashi, N; Rieneck, K; van der Kraan, P M;

    2005-01-01

    To elucidate the antagonism between interleukin-1 (IL-1) and transforming growth factor-beta (TGF-beta) at the gene expression level, as IL-1 and TGF-beta are postulated to be critical mediators of cartilage degeneration/protection in rheumatic diseases....

  20. Identification and molecular characterization of four new large deletions in the beta-globin gene cluster.

    Science.gov (United States)

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Couprie, Nicole; Francina, Alain

    2009-01-01

    Despite the fact that mutations in the human beta-globin gene cluster are essentially point mutations, a significant number of large deletions have also been described. We present here four new large deletions in the beta-globin gene cluster that have been identified on patients displaying an atypical hemoglobin phenotype (high HbF) at routine analysis. The first deletion, which spreads over 2.0 kb, removes the entire beta-globin gene, including its promoter, and is associated with a typical beta-thal minor phenotype. The three other deletions are larger (19.7 to 23.9 kb) and remove both the delta and beta-globin genes. Phenotypically, they look like an HPFH-deletion as they are associated with normal hematological parameters. The precise localization of their 5' and 3' breakpoints gives new insights about the differences between HPFH and (deltabeta)(0)-thalassemia at the molecular level. The importance of detection of these deletions in prenatal diagnosis and newborn screening of hemoglobinopathies is also discussed.

  1. Alpha beta T-cell development is not affected by inversion of TCR beta gene enhancer sequences: polar enhancement of gene expression regardless of enhancer orientation.

    Science.gov (United States)

    Huang, Fang; Cabaud, Olivier; Verthuy, Christophe; Hueber, Anne-Odile; Ferrier, Pierre

    2003-08-01

    V(D)J recombination and expression of the T-cell receptor beta (TCRbeta) gene are required for the development of the alphabeta T lymphocyte lineage. These processes depend on a transcriptional enhancer (Ebeta) which acts preferentially on adjacent upstream sequences, and has little impact on the 5' distal and 3' proximal regions of the TCRbeta locus. Using knock-in mice, we show that alphabeta T-cell differentiation and TCRbeta gene recombination and expression are not sensitive to the orientation of Ebeta sequences. We discuss the implication of these results regarding the mode of enhancer function at this locus during T lymphocyte development.

  2. Estrogen Receptor beta 2 Induces Hypoxia Signature of Gene Expression by Stabilizing HIF-1 alpha in Prostate Cancer

    OpenAIRE

    Prasenjit Dey; Velazquez-Villegas, Laura A.; Michelle Faria; Anthony Turner; Philp Jonsson; Paul Webb; Cecilia Williams; Jan-Åke Gustafsson; Ström, Anders M.

    2015-01-01

    The estrogen receptor (ER) beta variant ER beta 2 is expressed in aggressive castration-resistant prostate cancer and has been shown to correlate with decreased overall survival. Genome-wide expression analysis after ER beta 2 expression in prostate cancer cells revealed that hypoxia was an overrepresented theme. Here we show that ER beta 2 interacts with and stabilizes HIF-1 alpha protein in normoxia, thereby inducing a hypoxic gene expression signature. HIF-1 alpha is known to stimulate met...

  3. Metabolic analysis of Escherichia coli in the presence and absence of the carboxylating enzymes phosphoenolpyruvate carboxylase and pyruvate carboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Gokarn, R.R.; Eiteman, M.A.; Altman, E.

    2000-05-01

    Fermentation patterns of Escherichia coli with and without the phosphoenolpyruvate carboxylase (PPC) and pyruvate carboxylase (PYC) enzymes were compared under anaerobic conditions with glucose as a carbon source. Time profiles of glucose and fermentation product concentrations were determined and used to calculate metabolic fluxes through central carbon pathways during exponential cell growth. The presence of the Rhizobium etli pyc gene in E. coli (JCL1242/pTrc99A-pyc) restored the succinate producing ability of E. coli ppc null mutants (JCL1242), with PYC competing favorably with both pyruvate formate lyase and lactate dehydrogenase. Succinate formation was slightly greater by JCL1242/pTrc99a-pyc than by cells which overproduced PPC(JCL1242/pPC201, ppc{sup +}), even though PPC activity in cell extracts of JCL1242/pPC201 (ppc{sup +}) was 40-fold greater than PYC activity in extracts of JCL1242/pTrc99a-pyc. Flux calculations indicate that during anaerobic metabolism the pyc{sup +} strain had a 34% greater specific glucose consumption rate, a 37% greater specific rate of ATP formation, and a 6% greater specific growth rate compared to the ppc{sup +} strain. In light of the important position of pyruvate at the juncture of NADH-generating pathways and NADH-dissimilating branches, the results show that when PPC or PYC is expressed, the metabolic network adapts by altering the flux to lactate and the molar ratio of ethanol to acetate formation.

  4. Comparative modeling and molecular dynamics suggest high carboxylase activity of the Cyanobium sp. CACIAM14 RbcL protein.

    Science.gov (United States)

    Siqueira, Andrei Santos; Lima, Alex Ranieri Jerônimo; Dall'Agnol, Leonardo Teixeira; de Azevedo, Juliana Simão Nina; da Silva Gonçalves Vianez, João Lídio; Gonçalves, Evonnildo Costa

    2016-03-01

    Rubisco catalyzes the first step reaction in the carbon fixation pathway, bonding atmospheric CO2/O2 to ribulose 1,5-bisphosphate; it is therefore considered one of the most important enzymes in the biosphere. Genetic modifications to increase the carboxylase activity of rubisco are a subject of great interest to agronomy and biotechnology, since this could increase the productivity of biomass in plants, algae and cyanobacteria and give better yields in crops and biofuel production. Thus, the aim of this study was to characterize in silico the catalytic domain of the rubisco large subunit (rbcL gene) of Cyanobium sp. CACIAM14, and identify target sites to improve enzyme affinity for ribulose 1,5-bisphosphate. A three-dimensional model was built using MODELLER 9.14, molecular dynamics was used to generate a 100 ns trajectory by AMBER12, and the binding free energy was calculated using MM-PBSA, MM-GBSA and SIE methods with alanine scanning. The model obtained showed characteristics of form-I rubisco, with 15 beta sheets and 19 alpha helices, and maintained the highly conserved catalytic site encompassing residues Lys175, Lys177, Lys201, Asp203, and Glu204. The binding free energy of the enzyme-substrate complexation of Cyanobium sp. CACIAM14 showed values around -10 kcal mol(-1) using the SIE method. The most important residues for the interaction with ribulose 1,5-bisphosphate were Arg295 followed by Lys334. The generated model was successfully validated, remaining stable during the whole simulation, and demonstrated characteristics of enzymes with high carboxylase activity. The binding analysis revealed candidates for directed mutagenesis sites to improve rubisco's affinity.

  5. Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

    Directory of Open Access Journals (Sweden)

    Cooper John A

    2000-07-01

    Full Text Available Abstract Background Capping protein (CP, a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform. Results We isolated genomic clones corresponding to the β subunit of mouse CP and identified its chromosomal location by interspecies backcross mapping. Conclusions The CPβ gene (Cappb1 mapped to Chromosome 4 between Cdc42 and D4Mit312. Three mouse mutations, snubnose, curly tail, and cribriform degeneration, map in the vicinity of the β gene.

  6. Cell and Gene Therapy for the Beta-Thalassemias: Advances and Prospects.

    Science.gov (United States)

    Mansilla-Soto, Jorge; Riviere, Isabelle; Boulad, Farid; Sadelain, Michel

    2016-04-01

    The beta-thalassemias are inherited anemias caused by mutations that severely reduce or abolish expression of the beta-globin gene. Like sickle cell disease, a related beta-globin gene disorder, they are ideal candidates for performing a genetic correction in patient hematopoietic stem cells (HSCs). The most advanced approach utilizes complex lentiviral vectors encoding the human β-globin gene, as first reported by May et al. in 2000. Considerable progress toward the clinical implementation of this approach has been made in the past five years, based on effective CD34+ cell mobilization and improved lentiviral vector manufacturing. Four trials have been initiated in the United States and Europe. Of 16 evaluable subjects, 6 have achieved transfusion independence. One of them developed a durable clonal expansion, which regressed after several years without transformation. Although globin lentiviral vectors have so far proven to be safe, this occurrence suggests that powerful insulators with robust enhancer-blocking activity will further enhance this approach. The combined discovery of Bcl11a-mediated γ-globin gene silencing and advances in gene editing are the foundations for another gene therapy approach, which aims to reactivate fetal hemoglobin (HbF) production. Its clinical translation will hinge on the safety and efficiency of gene targeting in true HSCs and the induction of sufficient levels of HbF to achieve transfusion independence. Altogether, the progress achieved over the past 15 years bodes well for finding a genetic cure for severe globin disorders in the next decade.

  7. Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and beta-lactamase and alginate production

    DEFF Research Database (Denmark)

    Bagge, Niels; Schuster, Martin; Hentzer, Morten

    2004-01-01

    . The treatment often includes beta-lactam antibiotics. How these antibiotics influence gene expression in the surviving biofilm population of P. aeruginosa is not clear. Thus, we used the microarray technology to study the effects of subinhibitory concentrations of a beta-lactam antibiotic, imipenem, on gene...

  8. Nonblack patients with sickle cell disease have African. beta. sup s gene cluster haplotypes

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, Z.R.; Powars, D.R.; Williams, W.D. (Univ. of Southern California School of Medicine, Los Angeles (USA)); Kinney, T.R. (Duke Univ., Durham, NC (USA)); Schroeder, W.A. (California Institute of Technology, Pasadena (USA))

    1989-05-26

    Of 18 nonblack patients with sickle cell disease, 14 had sickle cell anemia, 2 had hemoglobin SC disease, and 2 had hemoglobin S-{beta}{sup o}-thalassemia. The {beta}{sup s} gene cluster haplotypes that were determined in 7 patients were of African origin and were identified as Central African Republic, Central African Republic minor II, Benin, and Senegal. The haplotype Central African Republic minor II was present on the {beta}{sup o}-thalassemia chromosome in 2 patients. None of 10 patients whose {alpha}-gene status was determined had {alpha}-thalassemia-2. These data strongly support the concept that the {beta}{sup s} gene on chromosome 11 of these individuals is of African origin and that the {alpha}-gene locus on chromosome 16 is of white or native American origin. The clinical severity of the disease in these nonblack patients is appropriate to their haplotype without {alpha}-thalassemia-2 and is comparable with that of black patients. All persons with congenital hemolytic anemia should be examined for the presence of sickle cell disease regardless of physical appearance or ethnic background.

  9. A gene duplication led to specialized gamma-aminobutyrate and beta-alanine aminotransferase in yeast

    DEFF Research Database (Denmark)

    Andersen, Gorm; Andersen, Birgit; Dobritzsch, D.

    2007-01-01

    In humans, beta-alanine (BAL) and the neurotransmitter gamma-aminobutyrate (GABA) are transaminated by a single aminotransferase enzyme. Apparently, yeast originally also had a single enzyme, but the corresponding gene was duplicated in the Saccharomyces kluyveri lineage. SkUGA1 encodes a homologue...

  10. Structure of the gene encoding the murine protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1995-01-01

    The mouse protein kinase CK2 beta subunit gene (Csnk2b) is composed of seven exons contained within 7874 bp. The exon and intron lengths extend from 76 to 321 and 111 to 1272 bp, respectively. The lengths of the murine coding exons correspond exactly to the lengths of the exons in the human CK2...

  11. Organ specificity of beta-carotene induced lung gene-expression changes in Bcmo 1-/- mice

    NARCIS (Netherlands)

    Helden, Y.G.J.; Godschalk, R.W.L.; Schooten, van F.J.; Keijer, J.

    2013-01-01

    Scope - Whole genome transcriptome analysis of male and female beta-carotene 15,15'-monooxygenase knockout (Bcmo1-/-) and Bcmo1+/+ (wild-type) mice with or without 14 wk of BC supplementation was done. We previously showed that only 1.8% of the genes regulated by BC in lung were also regulated in li

  12. Using the NCBI Genome Databases to Compare the Genes for Human & Chimpanzee Beta Hemoglobin

    Science.gov (United States)

    Offner, Susan

    2010-01-01

    The beta hemoglobin protein is identical in humans and chimpanzees. In this tutorial, students see that even though the proteins are identical, the genes that code for them are not. There are many more differences in the introns than in the exons, which indicates that coding regions of DNA are more highly conserved than non-coding regions.

  13. Origin and spread of beta-globin gene mutations in India, Africa, and Mediterranea: analysis of the 5' flanking and intragenic sequences of beta S and beta C genes.

    Science.gov (United States)

    Trabuchet, G; Elion, J; Baudot, G; Pagnier, J; Bouhass, R; Nigon, V M; Labie, D; Krishnamoorthy, R

    1991-06-01

    Nucleotide polymorphisms of both the 5' flanking and intragenic regions of the human beta-globin gene were investigated by directly sequencing genomic DNA after amplification by the polymerase chain reaction in 47 subjects homozygous for the beta S or the beta C mutation. The sickle-cell mutation was found in the context of five different haplotypes defined by eight nucleotide substitutions and various structures of a region of the simple repeated sequence (AT) chi Ty. All subjects from the same geographic origin bear an identical chromosomal structure, defining the Senegal-, Bantu-, Benin-, Cameroon-, and Indian-type chromosomes. These results strengthen our previous conclusions about the multiple occurrence of the sickle-cell mutation. The Benin-type chromosome was also found among Algerian and Sicilian sickle-cell patients, whereas the Indian-type chromosome was observed in two geographically distant tribes, illustrating the spread of these sickle-cell genes. We also found that the intragenic sequence polymorphisms (frameworks) are not always in linkage disequilibrium with the BamH I polymorphism downstream from the beta-globin gene, as had been previously observed. Finally, we present a tentative phylogenetic tree of the different alleles at this locus. Some polymorphisms of this sequence might be contemporary with our last common ancestor, the great apes, that is, about 4-6 millions years old.

  14. Isolation and functional characterization of a lycopene beta-cyclase gene that controls fruit colour of papaya (Carica papaya L.).

    Science.gov (United States)

    Devitt, Luke C; Fanning, Kent; Dietzgen, Ralf G; Holton, Timothy A

    2010-01-01

    The colour of papaya fruit flesh is determined largely by the presence of carotenoid pigments. Red-fleshed papaya fruit contain lycopene, whilst this pigment is absent from yellow-fleshed fruit. The conversion of lycopene (red) to beta-carotene (yellow) is catalysed by lycopene beta-cyclase. This present study describes the cloning and functional characterization of two different genes encoding lycopene beta-cyclases (lcy-beta1 and lcy-beta2) from red (Tainung) and yellow (Hybrid 1B) papaya cultivars. A mutation in the lcy-beta2 gene, which inactivates enzyme activity, controls lycopene production in fruit and is responsible for the difference in carotenoid production between red and yellow-fleshed papaya fruit. The expression level of both lcy-beta1 and lcy-beta2 genes is similar and low in leaves, but lcy-beta2 expression increases markedly in ripe fruit. Isolation of the lcy-beta2 gene from papaya, that is preferentially expressed in fruit and is correlated with fruit colour, will facilitate marker-assisted breeding for fruit colour in papaya and should create possibilities for metabolic engineering of carotenoid production in papaya fruit to alter both colour and nutritional properties.

  15. Beta-Adrenergic gene therapy for cardiovascular disease

    Directory of Open Access Journals (Sweden)

    Koch Walter J

    2000-10-01

    Full Text Available Abstract Gene therapy using in vivo recombinant adenovirus-mediated gene transfer is an effective technique that offers great potential to improve existing drug treatments for the complex cardiovascular diseases of heart failure and vascular smooth muscle intimal hyperplasia. Cardiac-specific adenovirus-mediated transfer of the carboxyl-terminus of the β-adrenergic receptor kinase (βARKct, acting as a Gβγ-β-adrenergic receptor kinase (βARK1 inhibitor, improves basal and agonist-induced cardiac performance in both normal and failing rabbit hearts. In addition, βARKct adenovirus infection of vascular smooth muscle is capable of significantly diminishing neointimal proliferation after angioplasty. Therefore, further investigation is warranted to determine whether inhibition of βARK1 activity and sequestration of Gβγ via an adenovirus that encodes the βARKct transgene might be a useful clinical tool for the treatment of cardiovascular pathologies.

  16. Globin gene transfer for treatment of the beta-thalassemias and sickle cell disease.

    Science.gov (United States)

    Sadelain, Michel; Rivella, Stefano; Lisowski, Leszek; Samakoglu, Selda; Rivière, Isabelle

    2004-09-01

    The beta-thalassemias and sickle cell disease are severe congenital anemias that are caused by mutations that alter the production of the beta chain of hemoglobin. Allogeneic hematopoietic stem cell (HSC) transplantation is curative, but this therapeutic option is not available to the majority of patients. The transfer of a functional globin gene in autologous HCSs thus represents a highly attractive alternative treatment. This strategy, simple in principle, raises major challenges in terms of controlling the expression of the globin transgene, which ideally should be erythroid specific, differentiation-stage restricted, elevated, position independent, and sustained over time. Using lentiviral vectors, we have demonstrated that an optimised combination of proximal and distal transcriptional control elements permits lineage-specific, elevated expression of the beta-globin gene, resulting in therapeutic hemoglobin production and correction of anemia in beta-thalassemic mice. Several groups have now confirmed and extended these findings in various mouse models of severe hemoglobinopathies, thus generating enthusiasm for a genetic treatment based on globin gene transfer. Furthermore, globin vectors represent a general paradigm for the regulation of transgene function and the improvement of vector safety by restricting transgene expression to the differentiated progeny within a single lineage, thereby reducing the risk of activating oncogenes in hematopoietic progenitors. Here we review the principles underlying the genesis of regulated vectors for stem cell therapy.

  17. Class II genes of miniature swine. II. Molecular identification and characterization of B (beta) genes from the SLAc haplotype.

    Science.gov (United States)

    Pratt, K; Sachs, D H; Germana, S; el-Gamil, M; Hirsch, F; Gustafsson, K; LeGuern, C

    1990-01-01

    Genomic clones corresponding to class II beta genes of the SLAc haplotype of miniature swine have been isolated and characterized. These genes have been grouped into seven non-overlapping clusters on the basis of restriction mapping. Ordering of exons within each cluster was accomplished by hybridization of Southern blots of restriction fragments with exon-specific probes. The two clusters (clusters 2 and 3) encoding the DRB and DQB genes were identified on the basis of hybridization with locus-specific 3' untranslated cDNA probes. Cluster 4 contained exons of both DOB and DQB genes, the basis for which remains to be determined. The remaining four clusters (1, 5, 6, 7) were identified as containing DP, DR, and DO coding sequences, respectively, on the basis of sequence analysis. The porcine class II region appears very similar to that of man in number and nature of the class II genes identified and in the intron/exon organization of corresponding genes.

  18. Inter-MAR association contributes to transcriptionally active looping events in human beta-globin gene cluster.

    Science.gov (United States)

    Wang, Li; Di, Li-Jun; Lv, Xiang; Zheng, Wei; Xue, Zheng; Guo, Zhi-Chen; Liu, De-Pei; Liang, Chi-Chuan

    2009-01-01

    Matrix attachment regions (MARs) are important in chromatin organization and gene regulation. Although it is known that there are a number of MAR elements in the beta-globin gene cluster, it is unclear that how these MAR elements are involved in regulating beta-globin genes expression. Here, we report the identification of a new MAR element at the LCR (locus control region) of human beta-globin gene cluster and the detection of the inter-MAR association within the beta-globin gene cluster. Also, we demonstrate that SATB1, a protein factor that has been implicated in the formation of network like higher order chromatin structures at some gene loci, takes part in beta-globin specific inter-MAR association through binding the specific MARs. Knocking down of SATB1 obviously reduces the binding of SATB1 to the MARs and diminishes the frequency of the inter-MAR association. As a result, the ACH establishment and the alpha-like globin genes and beta-like globin genes expressions are affected either. In summary, our results suggest that SATB1 is a regulatory factor of hemoglobin genes, especially the early differentiation genes at least through affecting the higher order chromatin structure.

  19. Mx1 and IP-10: biomarkers to measure IFN-beta activity in mice following gene-based delivery.

    Science.gov (United States)

    Petry, Harald; Cashion, Linda; Szymanski, Paul; Ast, Oliver; Orme, Ann; Gross, Cynthia; Bauzon, Maxine; Brooks, Alan; Schaefer, Caralee; Gibson, Heather; Qian, Husheng; Rubanyi, Gabor M; Harkins, Richard N

    2006-10-01

    Recombinant interferon-beta (IFN-beta) protein is used successfully for the treatment of multiple sclerosis (MS). Gene therapy might be an alternative approach to overcome drawbacks occurring with IFN-beta protein therapy. A critical issue in developing a new approach is detection of biologically active IFN-beta in preclinical models. The goal of the present study was to determine if Mx1 and IP-10, which are known to be activated after IFN-beta treatment in humans, can be used as biomarkers in mice. In three in vivo experiments, the correlation between different methods of murine IFN-beta (MuIFN-beta) delivery and biomarker induction was studied: (1) bolus protein delivery by intravenous (i.v.) or intramuscular (i.m.) injection, (2) gene-based delivery of IFN- beta by i.m. injection of plasmid DNA, followed by electroporation, and (3) gene-based delivery of IFN-beta by i.m. injection of adenovirus-associated type 1 (AAV1). Short-term induction of Mx1 mRNA and IP-10 was observed after treatment with bolus MuIFN-beta protein. Long-term induction of both biomarkers was observed after IFN-beta plasmid DNA delivery or when AAV1 was used as the vector. The experiments demonstrate that gene-based delivery provides sustained levels of IFN-beta compared with bolus protein injection and that Mx1 RNA and IP-10 can be used to monitor biologically active circulating plasma MuIFN-beta protein in mice.

  20. Transforming growth factor-beta inhibits aromatase gene transcription in human trophoblast cells via the Smad2 signaling pathway

    Directory of Open Access Journals (Sweden)

    Fu Guodong

    2009-12-01

    Full Text Available Abstract Background Transforming growth factor-beta (TGF-beta is known to exert multiple regulatory functions in the human placenta, including inhibition of estrodial production. We have previously reported that TGF-beta1 decreased aromatase mRNA levels in human trophoblast cells. The objective of this study was to investigate the molecular mechanisms underlying the regulatory effect of TGF-beta1 on aromatase expression. Methods To determine if TGF-beta regulates aromatase gene transcription, several reporter constructs containing different lengths of the placental specific promoter of the human aromatase gene were generated. JEG-3 cells were transiently transfected with a promoter construct and treated with or without TGF-beta1. The promoter activity was measured by luciferase assays. To examine the downstream signaling molecule mediating the effect of TGF-beta on aromatase transcription, cells were transiently transfected with dominant negative mutants of TGF-beta type II (TbetaRII and type I receptor (ALK5 receptors before TGF-beta treatment. Smad2 activation was assessed by measuring phophorylated Smad2 protein levels in cytosolic and nuclear fractions. Smad2 expression was silenced using a siRNA expression construct. Finally, aromatase mRNA half-life was determined by treating cells with actinomycin D together with TGF-beta1 and measuring aromatase mRNA levels at various time points after treatment. Results and Discussion TGF-beta1 inhibited the aromatase promoter activity in a time- and dose-dependent manner. Deletion analysis suggests that the TGF-β1 response element resides between -422 and -117 nucleotides upstream from the transcription start site where a Smad binding element was found. The inhibitory effect of TGF-beta1 was blocked by dominant negative mutants of TbetaRII and ALK5. TGF-beta1 treatment induced Smad2 phosphorylation and translocation into the nucleus. On the other hand, knockdown of Smad2 expression reversed the

  1. Analysis and expression of the alpha-expansin and beta-expansin gene families in maize

    Science.gov (United States)

    Wu, Y.; Meeley, R. B.; Cosgrove, D. J.

    2001-01-01

    Expansins comprise a multigene family of proteins in maize (Zea mays). We isolated and characterized 13 different maize expansin cDNAs, five of which are alpha-expansins and eight of which are beta-expansins. This paper presents an analysis of these 13 expansins, as well as an expression analysis by northern blotting with materials from young and mature maize plants. Some expansins were expressed in restricted regions, such as the beta-expansins ExpB1 (specifically expressed in maize pollen) and ExpB4 (expressed principally in young husks). Other expansins such as alpha-expansin Exp1 and beta-expansin ExpB2 were expressed in several organs. The expression of yet a third group was not detected in the selected organs and tissues. An analysis of expansin sequences from the maize expressed sequence tag collection is also presented. Our results indicate that expansin genes may have general, overlapping expression in some instances, whereas in other cases the expression may be highly specific and limited to a single organ or cell type. In contrast to the situation in Arabidopsis, beta-expansins in maize seem to be more numerous and more highly expressed than are alpha-expansins. The results support the concept that beta-expansins multiplied and evolved special functions in the grasses.

  2. Molecular cloning and functional characterization of beta-N-acetylglucosaminidase genes from Sf9 cells.

    Science.gov (United States)

    Aumiller, Jared J; Hollister, Jason R; Jarvis, Donald L

    2006-06-01

    Sf9, a cell line derived from the lepidopteran insect, Spodoptera frugiperda, is widely used as a host for recombinant glycoprotein expression and purification by baculovirus vectors. Previous studies have shown that this cell line has one or more beta-N-acetylglucosaminidase activities that may be involved in the degradation and/or processing of N-glycoprotein glycans. However, these enzymes and their functions remain poorly characterized. Therefore, the goal of this study was to isolate beta-N-acetylglucosaminidase genes from Sf9 cells, over-express the gene products, and characterize their enzymatic activities. A degenerate PCR approach yielded three Sf9 cDNAs, which appeared to encode two distinct beta-N-acetylglucosaminidases, according to bioinformatic analyses. Baculovirus-mediated expression of these two cDNA products induced membrane-associated beta-N-acetylglucosaminidase activities in Sf9 cells, which cleaved terminal N-acetylglucosamine residues from the alpha-3 and -6 branches of a biantennary N-glycan substrate with acidic pH optima and completely hydrolyzed chitotriose to its constituent N-acetylglucosamine monomers. GFP-tagged forms of both enzymes exhibited punctate cytoplasmic fluorescence, which did not overlap with either lysosomal or Golgi-specific dyes. Together, these results indicated that the two new Sf9 genes identified in this study encode broad-spectrum beta-N-acetylglucosaminidases that appear to have unusual intracellular distributions. Their relative lack of substrate specificity and acidic pH optima are consistent with a functional role for these enzymes in glycoprotein glycan and chitin degradation, but not with a role in N-glycoprotein glycan processing.

  3. Evidence for the association of the S100beta gene with low cognitive performance and dementia in the elderly

    DEFF Research Database (Denmark)

    Lambert, J-C; Ferreira, S; Gussekloo, J

    2007-01-01

    Variations in the S100beta gene may be instrumental in producing a continuum from mild cognitive decline to overt dementia. After screening 25 single nucleotide polymorphisms (SNPs) in S100beta, we observed association of the rs2300403 intron 2 SNP with poorer cognitive function in three...... corresponding mRNA isoform was called S100beta2). S100beta2 expression was increased in AD brain compared with controls, and the rs2300403 SNP was associated with elevated levels of S100beta2 mRNA in AD brains, especially in women. Therefore, this genetic variant in S100beta increases the risk of low cognitive...... performance and dementia, possibly by favouring a splicing event increasing S100beta2 isoform expression in the brain....

  4. Sterol regulation of acetyl coenzyme A carboxylase: a mechanism for coordinate control of cellular lipid.

    OpenAIRE

    Lopez, J.M.; Bennett, M K; Sanchez, H B; Rosenfeld, J M; Osborne, T E

    1996-01-01

    Transcription from the housekeeping promoter for the acetyl coenzyme A carboxylase (ACC) gene, which encodes the rate-controlling enzyme of fatty acid biosynthesis, is shown to be regulated by cellular sterol levels through novel binding sites for the sterol-sensitive sterol regulatory element binding protein (SREBP)-1 transcription factor. The position of the SREBP sites relative to those for the ubiquitous auxiliary transcription factor Sp1 is reminiscent of that previously described for th...

  5. Genetic structures at the origin of acquisition of the beta-lactamase bla KPC gene.

    Science.gov (United States)

    Naas, Thierry; Cuzon, Gaelle; Villegas, Maria-Virginia; Lartigue, Marie-Frédérique; Quinn, John P; Nordmann, Patrice

    2008-04-01

    Genetic structures surrounding the carbapenem-hydrolyzing Ambler class A bla KPC gene were characterized in several KPC-positive Klebsiella pneumoniae and Pseudomonas aeruginosa strains isolated from the United States, Colombia, and Greece. The bla KPC genes were associated in all cases with transposon-related structures. In the K. pneumoniae YC isolate from the United States, the beta-lactamase bla KPC-2 gene was located on a novel Tn3-based transposon, Tn4401. Tn4401 was 10 kb in size, was delimited by two 39-bp imperfect inverted repeat sequences, and harbored, in addition to the beta-lactamase bla KPC-2 gene, a transposase gene, a resolvase gene, and two novel insertion sequences, ISKpn6 and ISKpn7. Tn4401 has been identified in all isolates. However, two isoforms of this transposon were found: Tn4401a was found in K. pneumoniae YC and K. pneumoniae GR from the United States and Greece, respectively, and differed by a 100-bp deletion, located just upstream of the bla KPC-2 gene, compared to the sequence of Tn4401b, which was found in the Colombian isolates. In all isolates tested, Tn4401 was flanked by a 5-bp target site duplication, the signature of a recent transposition event, and was inserted in different open reading frames located on plasmids that varied in size and nature. Tn4401 is likely at the origin of carbapenem-hydrolyzing beta-lactamase KPC mobilization to plasmids and its further insertion into various-sized plasmids identified in nonclonally related K. pneumoniae and P. aeruginosa isolates.

  6. Enantioselective Degradation Mechanism of Beta-Cypermethrin in Soil From the Perspective of Functional Genes.

    Science.gov (United States)

    Yang, Zhong-Hua; Ji, Guo-Dong

    2015-12-01

    The behavior and mechanisms of the enantioselective degradation of beta-cypermethrin were studied in soil. The four isomers were degraded at different rates, and the enantiomer fractions of alpha-cypermethrin and theta-cypermethrin exceeded 0.5. Moreover, 3-phenoxybenzoic acid, phenol, and protocatechuic acid were detected; based on the presence of these metabolites, we predicted the degradation pathway and identified the functional genes that are related to this degradation process. We established quantitative relationships between the data on degradation kinetics and functional genes; we found that the quantitative relationships between different enantiomers differed even under the same conditions, and the genes pobA and pytH played key roles in limiting the degradation rate. Data obtained using path analysis revealed that the same gene had different direct and indirect effects on the degradation of different isomers. A mechanism was successfully proposed to explain the selective degradation of chiral compounds based on the perspective of functional genes.

  7. Conditional beta1-integrin gene deletion in neural crest cells causes severe developmental alterations of the peripheral nervous system

    DEFF Research Database (Denmark)

    Pietri, Thomas; Eder, Olivier; Breau, Marie Anne;

    2004-01-01

    Integrins are transmembrane receptors that are known to interact with the extracellular matrix and to be required for migration, proliferation, differentiation and apoptosis. We have generated mice with a neural crest cell-specific deletion of the beta1-integrin gene to analyse the role of beta1-...

  8. Decreased gene expression of human beta-defensin-1 in the development of squamous cell carcinoma of the oral cavity.

    NARCIS (Netherlands)

    Wenghoefer, M.H.; Pantelis, A.; Dommisch, H.; Reich, R.; Martini, M.; Allam, J.P.; Novak, N.; Berge, S.; Jepsen, S.; Winter, J.

    2008-01-01

    The aim of this study was to investigate the gene expression of human beta-defensin-1, -2, -3 (hBD-1, -2, -3), interleukin-1beta, tumour necrosis factor-alpha and cyclooxygenase-2 in oral squamous cell carcinoma (OSCC) compared to benign and premalignant lesions as well as healthy controls. Biopsies

  9. Isolation of novel human cDNA (hGMF-gamma) homologous to Glia Maturation Factor-beta gene.

    Science.gov (United States)

    Asai, K; Fujita, K; Yamamoto, M; Hotta, T; Morikawa, M; Kokubo, M; Moriyama, A; Kato, T

    1998-03-13

    A novel full-length human cDNA homologous to Glia Maturation Factor-beta (GMF-beta) gene was isolated. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein sequence of 142 amino acid residues. The deduced amino acid sequences of its putative product is highly homologous to human GMF-beta (82% identity) and named for GMF-gamma. Northern blot analysis indicated that a message of 0.9 kb long, but not 4.1 kb of GMF-beta, is predominantly expressed in human lung, heart, and placenta.

  10. Effect of polymorphic variants of GH, Pit-1, and beta-LG genes on milk production of Holstein cows.

    Science.gov (United States)

    Heidari, M; Azari, M A; Hasani, S; Khanahmadi, A; Zerehdaran, S

    2012-04-01

    Effect of polymorphic variants of growth hormone (GH), beta-lactoglobulin (beta-LG), and Pit-1 genes on milk yield was analyzed in a Holstein herd. Genotypes of the cows for these genes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Allele frequencies were 0.884 and 0.116 for L and V variants of GH, 0.170 and 0.830 for A and B variants of Pit-1, and 0.529 and 0.471 for A and B variants of beta-LG, respectively. GLM procedure of SAS software was used to test the effects of these genes on milk yield. Results indicated significant effects of these genes on milk yield (P LG gene, milk yield of animals with AA genotype was more than BB genotype (P LG (AA) were superior compared to heterozygote genotypes, whereas, the heterozygote genotype of Pit-1 gene (AB) was desirable.

  11. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

    Science.gov (United States)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes.

  12. INS-gene mutations: from genetics and beta cell biology to clinical disease.

    Science.gov (United States)

    Liu, Ming; Sun, Jinhong; Cui, Jinqiu; Chen, Wei; Guo, Huan; Barbetti, Fabrizio; Arvan, Peter

    2015-04-01

    A growing list of insulin gene mutations causing a new form of monogenic diabetes has drawn increasing attention over the past seven years. The mutations have been identified in the untranslated regions of the insulin gene as well as the coding sequence of preproinsulin including within the signal peptide, insulin B-chain, C-peptide, insulin A-chain, and the proteolytic cleavage sites both for signal peptidase and the prohormone convertases. These mutations affect a variety of different steps of insulin biosynthesis in pancreatic beta cells. Importantly, although many of these mutations cause proinsulin misfolding with early onset autosomal dominant diabetes, some of the mutant alleles appear to engage different cellular and molecular mechanisms that underlie beta cell failure and diabetes. In this article, we review the most recent advances in the field and discuss challenges as well as potential strategies to prevent/delay the development and progression of autosomal dominant diabetes caused by INS-gene mutations. It is worth noting that although diabetes caused by INS gene mutations is rare, increasing evidence suggests that defects in the pathway of insulin biosynthesis may also be involved in the progression of more common types of diabetes. Collectively, the (pre)proinsulin mutants provide insightful molecular models to better understand the pathogenesis of all forms of diabetes in which preproinsulin processing defects, proinsulin misfolding, and ER stress are involved.

  13. beta(S)-Globin gene cluster haplotypes in the West Bank of Palestine.

    Science.gov (United States)

    Samarah, Fekri; Ayesh, Suhail; Athanasiou, Miranda; Christakis, John; Vavatsi, Norma

    2009-01-01

    Sickle cell disease is an inherited autosomal recessive disorder of the beta-globin chain. In Palestine it is accompanied by a low level of Hb F (mean 5.14%) and a severe clinical presentation. In this study, 59 Palestinian patients, homozygotes for Hb S were studied for their haplotype background. Eight polymorphic sites in the beta-globin gene cluster were examined. The Benin haplotype was predominant with a frequency of 88.1%, followed by a frequency of 5.1% for the Bantu haplotype. One chromosome was found to carry the Cameroon haplotype (0.85%). Three atypical haplotypes were also found (5.95%). Heterogeneity was observed in Hb F production, ranging between 1.5 and 17.0%, whereas the (G)gamma ratio was homogeneous among all haplotypes with a normal amount of about 41%. Our results are in agreement with previous reports of the Benin haplotype origin in the Mediterranean.

  14. Comprehensive identification of single nucleotide polymorphisms associated with beta-lactam resistance within pneumococcal mosaic genes.

    Directory of Open Access Journals (Sweden)

    Claire Chewapreecha

    2014-08-01

    Full Text Available Traditional genetic association studies are very difficult in bacteria, as the generally limited recombination leads to large linked haplotype blocks, confounding the identification of causative variants. Beta-lactam antibiotic resistance in Streptococcus pneumoniae arises readily as the bacteria can quickly incorporate DNA fragments encompassing variants that make the transformed strains resistant. However, the causative mutations themselves are embedded within larger recombined blocks, and previous studies have only analysed a limited number of isolates, leading to the description of "mosaic genes" as being responsible for resistance. By comparing a large number of genomes of beta-lactam susceptible and non-susceptible strains, the high frequency of recombination should break up these haplotype blocks and allow the use of genetic association approaches to identify individual causative variants. Here, we performed a genome-wide association study to identify single nucleotide polymorphisms (SNPs and indels that could confer beta-lactam non-susceptibility using 3,085 Thai and 616 USA pneumococcal isolates as independent datasets for the variant discovery. The large sample sizes allowed us to narrow the source of beta-lactam non-susceptibility from long recombinant fragments down to much smaller loci comprised of discrete or linked SNPs. While some loci appear to be universal resistance determinants, contributing equally to non-susceptibility for at least two classes of beta-lactam antibiotics, some play a larger role in resistance to particular antibiotics. All of the identified loci have a highly non-uniform distribution in the populations. They are enriched not only in vaccine-targeted, but also non-vaccine-targeted lineages, which may raise clinical concerns. Identification of single nucleotide polymorphisms underlying resistance will be essential for future use of genome sequencing to predict antibiotic sensitivity in clinical microbiology.

  15. Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter

    Science.gov (United States)

    Inducible gene promoters that are specifically activated by pathogen invasion or insect pest attack are needed for effective expression of resistance genes to control plant diseases. In the present study, a promoter from a serine proteinase inhibitor gene (BvSTI) shown to be up-regulated in resist...

  16. Transcriptome analysis of sugar beet root maggot (Tetanops myopaeformis) genes modulated by the Beta vulgaris host.

    Science.gov (United States)

    Li, Haiyan; Smigocki, Ann C

    2016-10-03

    Sugar beet root maggot (SBRM, Tetanops myopaeformis von Röder) is a major but poorly understood insect pest of sugar beet (Beta vulgaris L.). The molecular mechanisms underlying plant defense responses are well documented, however, little information is available about complementary mechanisms for insect adaptive responses to overcome host resistance. To date, no studies have been published on SBRM gene expression profiling. Suppressive subtractive hybridization (SSH) generated more than 300 SBRM ESTs differentially expressed in the interaction of the pest with a moderately resistant (F1016) and a susceptible (F1010) sugar beet line. Blast2GO v. 3.2 search indicated that over 40% of the differentially expressed genes had known functions, primarily driven by fruit fly D. melanogaster genes. Expression patterns of 18 selected EST clones were confirmed by RT-PCR analysis. Gene Ontology (GO) analysis predicted a dominance of metabolic and catalytic genes involved in the interaction of SBRM with its host. SBRM genes functioning during development, regulation, cellular process, signaling and under stress conditions were annotated. SBRM genes that were common or unique in response to resistant or susceptible interactions with the host were identified and their possible roles in insect responses to the host are discussed.

  17. Effect of casein genes - beta-LGB, DGAT1, GH, and LHR - on milk production and milk composition traits in crossbred Holsteins.

    Science.gov (United States)

    Molee, A; Poompramun, C; Mernkrathoke, P

    2015-03-30

    The objectives of this study were to determine the effects of a single gene and composite genotype of the casein gene family, including the beta-lactoglobulin gene (beta-LGB), acyl-CoA: diacylglycerol acyltransferase 1 gene (DGAT1), growth hormone gene (GH), and luteinizing hormone receptor gene (LHR) on milk yield, milk composition, the percentage of fat, protein, solids-not-fat, and total solid in crossbred Holsteins. A total of 231 crossbred Holstein cows were examined for the study. The genotype of the beta-casein gene was analyzed by allele-specific polymerase chain reaction, while the alpha-S1, alpha-S2, kappa-casein, DGAT1, beta-LGB, and GH genes were analyzed using a polymerase chain reaction-restriction fragment length polymorphism assay. The association between genes and milk yield and milk composition was analyzed. Three pairs of genes, for which significant associations were detected, were beta + kappa-casein, DGAT1 + beta-casein, and GH + beta-LGB. In the single-gene model, most loci are significantly associated with traits. A significant association between the composite genotype and the traits was detected in all composite genotypes. GH + beta-LGB appears to be the most suitable variants for improving milk production and percentage of milk protein. Overall, the effects of the composite genotype and single gene were different. A physical or functional relationship between genes is necessary for investigating gene markers.

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

    Directory of Open Access Journals (Sweden)

    Zhong Tao P

    2007-07-01

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

  19. CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster

    DEFF Research Database (Denmark)

    Kalmykova, Alla I; Shevelyov, Yuri Y; Polesskaya, Oksana O

    2002-01-01

    An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta...... and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two...

  20. Beta-globin gene cluster haplotypes in Venezuelan sickle cell patients from the State of Aragua

    OpenAIRE

    Nancy Moreno; Martínez, José A.; Zorella Blanco; Leidys Osorio; Patrick Hackshaw

    2002-01-01

    Seven polymorphic sites in the beta-globin gene cluster were analyzed on a sample of 96 chromosomes of Venezuelan sickle cell patients from the State of Aragua. The Benin haplotype was predominant with a frequency of 0.479, followed by the Bantu haplotype (0.406); a minority of cases with other haplotypes was also identified: atypical Bantu A2 (0.042), Senegal (0.031), atypical Bantu A7 (0.021) and Saudi Arabia/Indian (0.021) haplotypes; however, the Cameroon haplotype was not identified in t...

  1. Gene expression profile of amyloid beta protein-injected mouse model for Alzheimer disease

    Institute of Scientific and Technical Information of China (English)

    Ling-na KONG; Ping-ping ZUO; Liang MU; Yan-yong LIU; Nan YANG

    2005-01-01

    Aim: To investigate the gene expression profile changes in the cerebral cortex of mice injected icv with amyloid beta-protein (Aβ) fragment 25-35 using cDNA microarray. Methods: Balb/c mice were randomly divided into a control group and Aβ-treated group. The Morris water maze test was performed to detect the effect of Aβ-injection on the learning and memory of mice. Atlas Mouse 1.2 Expression Arrays containing 1176 genes were used to investigate the gene expression pattern of each group. Results: The gene expression profiles showed that 19 genes including TBX1, NF-κB, AP-1/c-Jun, cadherin, integrin, erb-B2, and FGFR1 were up-regulated after 2 weeks oficv administration of Aβ; while 12 genes were downregulated, including NGF, glucose phosphate isomerase 1, AT motif binding factor 1, Na+/K+-ATPase, and Akt. Conclusions: The results provide important leads for pursuing a more complete understanding of the molecular events of Aβ-injection into mice with Alzheimer disease.

  2. Beta s gene in Central Iran is in linkage disequilibrium with the Indian-Arab haplotype.

    Science.gov (United States)

    Rahgozar, S; Poorfathollah, A A; Moafi, A R; Old, J M

    2000-11-01

    Sickle cell anemia is not considered to be a significant disease in Iran, although the sickle cell trait is estimated to have a high incidence in the Southern provinces. Since 1977, when the presence of a mild sickle cell anemia was reported in this country, there have been no further investigations published giving precise data on the incidence and origins of the sickle cell mutation in Iran. We report here the finding of patients with the sickle cell trait, sickle cell anemia, and sickle-beta thalassemia in Central Iran. A survey of 300 individuals from a village in Southeast Esfahan revealed an incidence of the sickle cell trait of 8.33%. "Cascade screening" enabled 96 relatives in four surrounding villages to be tested, and the at-risk couples were offered counseling as a "sickle cell control program." The hematological indices and HbF levels of the affected patients were determined. The HbF levels were unusually high, ranging from 18% to 41.4%, and SS patients with the highest levels were asymptomatic. Linkage analysis revealed the betaS gene haplotype in this population to be the Indian-Arab haplotype.

  3. Effect of interleukin-1beta gene functional polymorphism on dorsolateral prefrontal cortex activity in schizophrenic patients.

    Science.gov (United States)

    Papiol, Sergi; Molina, Vicente; Rosa, Araceli; Sanz, Javier; Palomo, Tomás; Fañanás, Lourdes

    2007-12-05

    Hypoactivity of the dorsolateral prefrontal cortex (DLPFC) during cognitive tasks is among the most consistent findings in schizophrenia. The biological factors contributing to this hypofrontality are only partially known. Previous reports have shown the influence of genes mapped to IL-1 cluster (i) in the risk to develop schizophrenia and (ii) on brain morphological abnormalities in these patients. Moreover, Interleukin-1beta (IL-1beta), encoded by IL-1B gene (IL-1 cluster, chromosome 2q13) has a key role in dopaminergic differentiation and dendrite growth in developing cortical neurons. The authors explored the role of a genetic functional polymorphism at IL-1B gene in relation to DLPFC activity. DLPFC (left and right) metabolic activity was measured in a sample of 19 DSM-IV diagnosed schizophrenic patients of Spanish origin using a procedure based on MRI/PET image fusion. During PET studies, subjects performed a contingent Continuous Performance Test aiming to activate DLPFC. Functional promoter polymorphism -511 C/T (rs16944) of IL-1B gene was genotyped in these patients. Those patients who were allele 2 (-511 T) carriers showed a lower metabolic activity in the left DLPFC with respect to patients homozygous for allele 1 (-511 C) (U = 16, z = -2.32, P = 0.02). Our results suggest that hypofrontality reported in some schizophrenic patients might be explained, at least in part, by this functional polymorphism at IL-1B gene. Genetic variants with influence on brain functionality may account for the neurocognitive heterogeneity observed in schizophrenic patients.

  4. Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and beta-lactamase and alginate production

    DEFF Research Database (Denmark)

    Bagge, N.; Schuster, M.; Hentzer, Morten

    2004-01-01

    . As expected, the most strongly induced gene was ampC, which codes for chromosomal beta-lactamase. We also found that genes coding for alginate biosynthesis were induced by exposure to imipenem. Alginate production is correlated to the development of impaired lung function, and P. aeruginosa strains isolated...

  5. Detection of genes mediating beta-lactamase production in isolates of enterobacteria recovered from wild pets in Saudi Arabia

    Directory of Open Access Journals (Sweden)

    Sabry A. Hassan

    2015-12-01

    Full Text Available Aim: To determine the genetic basis and types of beta-lactamase encountered among enterobacterial isolates of wild pets from the animal exhibit. Materials and Methods: A total of 17 beta-lactamase-producing enterobacteria recovered from fecal samples of wild pet animals were analyzed for a selected beta-lactamase gene by polymerase chain reaction. Results: Molecular analysis identified one or more β-lactamase-encoding genes in 14 enterobacterial isolates as a single or gene combination. The most frequent extended-spectrum β-lactamases types were transmission electron microscopy and CTX-M, and the most common AmpC enzymes were CMY-2 and DHA types. Conclusions: The study is the first in Saudi Arabia, have established the presence of β-lactamase-encoding genes in the fecal isolates of wild pets.

  6. Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice.

    Science.gov (United States)

    Itakura, Tomohiro; Kuroki, Aya; Ishibashi, Yasuhiro; Tsuji, Daisuke; Kawashita, Eri; Higashine, Yukari; Sakuraba, Hitoshi; Yamanaka, Shoji; Itoh, Kohji

    2006-08-01

    Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (betabeta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (GlcNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (alphabeta heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine alpha-subunit. However, co-introduction of the HEXA encoding the human alpha-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.

  7. Genomic organization of the human {beta}-catenin gene (CTNNB1)

    Energy Technology Data Exchange (ETDEWEB)

    Nollet, F.; Berx, G.; Molemans, F.; Roy, F. van [Univ. of Ghent (Belgium)

    1996-03-05

    The cytoplasmic {beta}-catenin protein is implicated in signal transduction and associates with both the cell-cell adhesion protein E-cadherin and the tumor suppressor gene product APC. We determined the primary structure of the human {beta}-catenin gene (CTNNB1) by analysis cDNA and genomic clones. The size of the complete gene was determined to be 23.2 kb. Restriction mapping and partial sequence analysis revealed 16 exons. All splice donor and acceptor sites were conformable to the GT/AG rule. The exon size ranged from 61 to 790 bp. Half of the introns were smaller than 550 bp, with the smallest being 84 pb and the longest being 6700 bp. The intron-exon boundaries did not coincide either with conserved sites in the 12 armadillo repeat sequences of {beta}-catenin or with intron-exon boundaries in the armadillo gene of Drosophila. A major site for transcription initiation was identified as an A residue 214 nucleotides upstream of the ATG initiation codon. The resulting transcript is 3362 nucleotides long. Compared to the previously published mRNA sequence, additional residues were identified, 16 at the 5{prime} end and 766 at the 3{prime} end of the mRNA. An alternative splice acceptor site within exon 16 reduced the 3{prime} UTR sequence by 159 bp. Polymerase chain reaction on cDNA from 14 human cell lines demonstrated the general occurrence of both splice variants. The 5{prime}-flanking region is highly GC-rich and lacks a CCAAT box, but contains a TATA box and potential binding sites for several transcription factors, such as NFkB, SP1, AP2, and EGR1. Both a 437-bp fragment and a 6-kb fragment, containing about 4.7 kb of the 5{prime}-flanking region in addition to the noncoding exon 1 and 1 kb of intron 1, showed clear promoter activity when these fragments were linked to a secreted alkaline phosphatase reporter gene and transfected into a mouse epithelial cell line. 53 refs., 5 figs., 2 tabs.

  8. Detection of Amp C genes encoding for beta-lactamases in Escherichia coli and Klebsiella pneumoniae

    Directory of Open Access Journals (Sweden)

    M Shanthi

    2012-01-01

    Full Text Available Purpose : Amp C beta-lactamase are Ambler class C enzymes that confer resistance to extended spectrum cephalosporins and are not inhibited by beta-lactamase inhibitors. Their detection is crucial, since the phenotypic tests are not standardised leading to ambiguity in interpretation of results. This study was done to detect the types of Amp C prevalent in Escherichia coli and Klebsiella pneumoniae by multiplex polymerase chain reaction (PCR. Materials and Methods : Seventy-seven consecutive cefoxitin resistant clinical isolates of E. coli (n = 25 and K. pneumoniae (n = 52 were included in the study. Antibiotic susceptibility testing to various classes of antibiotics was performed by disc diffusion using Clinical Laboratory Standards Institute (CLSI guidelines. Minimum inhibitory concentration (MIC to cefoxitin, imipenem and meropenem were determined by broth microdilution method. Isolates were screened for production of Extended Spectrum Beta-Lactamase (ESBL. Multiplex PCR was performed for the detection of Amp C genes after phenotypic testing (Hodge test and inhibitor based test. Results : Cefoxitin Hodge test was positive in 40 isolates which included 20 E. coli and 20 K. pneumoniae. There was zone enhancement with boronic acid in 55 isolates, of which 36 were K. pneumoniae and 19 were E. coli. Multiplex PCR detected Amp C in 11/25 E. coli and 12/52 K. pneumoniae isolates. The Amp C genes detected were CIT (Amp C origin - Citrobacter freundii, DHA (Dhahran Hospital, Saudi Arabia, ACC (Ambler class C, EBC (Amp C origin - Enterobacter cloacae groups. ESBL was co-produced in 54 isolates. Conclusions : Amp C was detected in 29.87% of the study isolates. Majority of them co-produced ESBL. The most common Amp C was the CIT family. Screen tests for cefoxitin resistance may be falsely positive due to production of carbapenamases.

  9. Expression of herpes simplex virus. beta. and. gamma. genes integrated in mammalian cells and their induction by an. cap alpha. gene product

    Energy Technology Data Exchange (ETDEWEB)

    Sandri-Goldin, R.M.; Goldin, A.L.; Holland, L.E.

    1983-11-01

    The proteins of herpes simplex virus type 1 (HSV-1) form three kinetic groups termed ..cap alpha..,..beta..,and ..gamma.., whose synthesis is regulated in a cascade fashion, ..cap alpha.. products are synthesized first during infection, and they are required for synthesis of ..beta.. and ..gamma.. proteins. To examine the expression of several HSV-1 ..beta.. and ..gamma.. genes in the absence of ..cap alpha.. functions, we transferred into mammalian cells a plasmid containing a region of the HSV-1 genome that codes for only ..beta.. and ..gamma.. genes (0.315 to 0.421 map units). The authors found stable integration of at least one copy of the intact plasmid in each cell line. Four HSV-1 transcripts of the ..beta.. and ..gamma.. classes were transcribed constitutively in the cells, including the genes for glycoprotein B and DNA-binding protein. No constitutive synthesis of these two proteins could be demonstrated, however. The integrated HSV-1 genes responded to viral regulatory signals in that they could be induced by infection with HSV-1 mutants resulting in a high level of synthesis of both glycoprotein B and DNA-binding protein. The HSV-1 ..cap alpha.. gene product ICP4 was necessary for this induction, and it was found to be most efficient at a low multiplicity of infection. Functional expression of four genes was demonstrated in that the cell lines complemented infecting HSV-1 temperature-sensitive mutants. The same genes were not available for homologous recombination with infecting virus, however, since no recombinant wild-type virus could be detected. These data demonstrate that HSV-1 ..beta.. and ..gamma.. genes can be transcribed in the absence of ..cap alpha.. functions in mammalian cells, but that they still respond to HSV-1 regulatory signals such as the ..cap alpha.. gene product ICP4.

  10. 磷酸烯醇式丙酮酸羧化酶基因的敲除对于谷氨酸棒杆菌V1生理代谢的影响%Effect of phosphoenolpyruvate carboxylase gene knock-out on physiological metabolism in Corynebacterium glutamicum V1

    Institute of Scientific and Technical Information of China (English)

    仇爱梅; 窦文芳; 李会; 许正宏

    2012-01-01

    [Objective] In order to optimize precursor supply for L-valine biosynthesis, a Corynebacterium glutamicum V1 mutant with phosphoenolpyruvate carboxylase gene (pepc) in-frame deletion was constructed through crossover PCR and homologous recombination. The effect of pepc knock-out on physiological characteristics of the mutant was investigated. [Methods] The upstream and downstream fragments of pepc were cloned from C. glutamicum V1 chromosome and ligated to integration vector. The mutant C. glutamicum V1-Δpepc was screened by homologous recombination. The physiological characteristics of the mutant were investigated by fermentation experiments and enzymes activity measurement of pyruvate carboxylase (PC), pyruvate dehydrogenase (PDH) and pyruvate kinase (PK). The mutant with pepc gene in-frame deletion was screened and confirmed by PCR and phosphoenolpyruvate carboxylase activity determination. [Results] The pepc knock-out resulted in L-argine accumulation to 7.48 g/L and no accumulation of L-valine, which accompanied by increase of PDH activity and PC activity in C. glutamicum V1-Δpepc. The knock-out of pepc gene affected the metabolism of the strain to some extent. [Conclusion] Blocking the anaplerotic pathway PEPC participated increased TCA cycle, leading to the increase of L-argine and decrease of amino acids with pyruvic acid as precursor , such as L-valine and alanine.%[目的]L-缬氨酸生物合成的前体物质是丙酮酸.为了增加磷酸烯醇式丙酮酸向丙酮酸的代谢流向,优化L-缬氨酸前体物质的供应,以一株积累L-缬氨酸的谷氨酸棒杆菌V1 (Corynebacterium glutamicum V1)为对象,构建磷酸烯醇式丙酮酸羧化酶(PEPC)基因敲除的重组菌株C.glutamicum V1-△pepc,并研究pepc敲除后菌株生理特性的改变.[方法]运用交叉PCR方法得到pepc基因内部缺失的同源片段△pepc,并构建敲除质粒pK18mobsacB-△pepc.利用同源重组技术获得pepc基因缺陷突变株C.glutamicum V1-△pepc

  11. Pancreatic beta-cell overexpression of the glucagon receptor gene results in enhanced beta-cell function and mass

    DEFF Research Database (Denmark)

    Gelling, Richard W; Vuguin, Patricia M; Du, Xiu Quan

    2009-01-01

    in response to glucagon and glucose, the glucose excursion resulting from both a glucagon challenge and intraperitoneal glucose tolerance test (IPGTT) was significantly reduced in RIP-Gcgr mice compared with controls. However, RIP-Gcgr mice display similar glucose responses to an insulin challenge. beta...... in vivo, we generated mice overexpressing the Gcgr specifically on pancreatic beta-cells (RIP-Gcgr). In vivo and in vitro insulin secretion in response to glucagon and glucose was increased 1.7- to 3.9-fold in RIP-Gcgr mice compared with controls. Consistent with the observed increase in insulin release...... and impaired glucose tolerance (IGT) were reduced in RIP-Gcgr mice compared with controls. Furthermore, the insulin response of RIP-Gcgr mice to an IPGTT was twice that of controls when fed the HFD. These data indicate that increased pancreatic beta-cell expression of the Gcgr increased insulin secretion...

  12. A novel murine model of Cooley anemia and its rescue by lentiviral-mediated human beta-globin gene transfer.

    Science.gov (United States)

    Rivella, Stefano; May, Chad; Chadburn, Amy; Rivière, Isabelle; Sadelain, Michel

    2003-04-15

    Patients affected by beta-thalassemia major require lifelong transfusions because of insufficient or absent production of the beta chain of hemoglobin (Hb). A minority of patients are cured by allogeneic bone marrow transplantation. In the most severe of the hitherto available mouse models of beta-thalassemia, a model for human beta-thalassemia intermedia, we previously demonstrated that globin gene transfer in bone marrow cells is curative, stably raising Hb levels from 8.0-8.5 to 11.0-12.0 g/dL in long-term chimeras. To fully assess the therapeutic potential of gene therapy in the context of a lethal anemia, we now have created an adult model of beta(0)-thalassemia major. In this novel model, mice engrafted with beta-globin-null (Hbb(th3/th3)) fetal liver cells succumb to ineffective erythropoiesis within 60 days. These mice rapidly develop severe anemia (2-4 g/dL), massive splenomegaly, extramedullary hematopoiesis (EMH), and hepatic iron overload. Remarkably, most mice (11 of 13) treated by lentivirus-mediated globin gene transfer were rescued. Long-term chimeras with an average 1.0-2.4 copies of the TNS9 vector in their hematopoietic and blood cells stably produced up to 12 g/dL chimeric Hb consisting of mu alpha(2):hu beta(2) tetramers. Pathologic analyses indicated that erythroid maturation was restored, while EMH and iron overload dramatically decreased. Thus, we have established an adult animal model for the most severe of the hemoglobinopathies, Cooley anemia, which should prove useful to investigate both genetic and pharmacologic treatments. Our findings demonstrate the remarkable efficacy of lentivirus-mediated globin gene transfer in treating a fulminant blood disorder and strongly support the efficacy of gene therapy in the severe hemoglobinopathies.

  13. Cochlear implantation effect on deaf children with gap junction protein beta 2 gene mutation

    Institute of Scientific and Technical Information of China (English)

    KONG Ying; LIU Sha; WANG Su-ju; Li Shu-jing; LIANG Shuang

    2013-01-01

    Background The popularization and promotion of gene diagnosis technology makes it possible to detect deafness genes for children with congenital hearing impairment,and the proportion of gap junction protein beta 2 (GJB2) gene mutations in cochlear implant patients is 26.5% We did follow-up evaluation on auditory rehabilitation effect for all 31 deaf children with GJB2 gene mutation after cochlear implantation to provide a reference for such patients.Methods Application of “the genetic deafness gene chip detection kit” and “gene complete sequence analysis” were applied to conduct detection on common genetic deafness gene mutation hotspots of the hearing impaired children with cochlear implantation.To conduct auditory rehabilitation effect evaluation on all 31 cases of patients with GJB2 genetic deafness after 3,6 and 12 months of the operation respectively.The single factor repeated measure analysis of variance (ANOVA) was applied to analysis whether there were significant difference among the results of initial consonant of a Chinese syllable recognition at 3 different stages after the operation,the results of vowel of a Chinese syllable recognition at 3 different stages after the operation,and the results of two-syllable recognition at 3 different stages after the operation.Results The 235delC is the high-incidence mutational site in 31 cases of patients with GJB2 genetic deafness,and the total detection rate is up to 90.3% (28/31).There were significant differences in the initial consonant and the vowel of a Chinese syllable recognition rate,and the two-syllable recognition rates at 3,6,and 12 months after the operation (P<0.01).Conclusion Cochlear implantation is a safe and effective measure for auditory reconstruction,enabling patients with GJB2 hereditary severe sensorineural deafness to achieve auditory speech recognition effectively.

  14. Characterization of a large deletion in the {beta}-globin gene cluster in a newborn with hemoglobin FE

    Energy Technology Data Exchange (ETDEWEB)

    Louie, E.; Dietz, L.; Shafer, F. [Children`s Hosptial, Oakland, CA (United States)] [and others

    1994-09-01

    A sample on a newborn with hemoglobin FE screen results was obtained to investigate whether E/E or B/{beta}{degrees} thalassemia was present using polymerase chain reaction (PCR) methodology. The newborn appeared homozygous for the hemoglobin E mutation in our initial study, but the parents` genotypes did not support this diagnosis. The father is homozygous for the absence of the hemoglobin E mutation (non E/non E) and the mother is heterozygous (E/non E) for this mutation. The limitation of PCR analysis is an assumption that the amplification of the two {beta}-globin alleles is equivalent. A large deletion on one {beta}-globin gene, which would produce E/{beta}{degrees} thalassemia, would be missed if it included part or the entire region subjected to amplification. The family results were consistent with either non-paternity, sample mix-up or such a deletion of the {beta}-globin gene in the father and child. To rule out the possibility of non-paternity, two polymorphic loci (HLA on chromosome 6 and a VNTR system of chromosome 17) that are outside of the {beta}-globin gene were analyzed and show that inheritance is consistent and the likelihood of a sample mix-up is then reduced. We therefore believe there is a gene deletion in this family. At the present time, analyses of the RFLPs that are 5{prime} of the {beta}-globin gene cluster show that the polymorphisms most distal from the 5{prime} {beta}-globin gene are not being inherited as expected. These results support our interpretation that a deletion exists in the father and was inherited by the child. The father`s clinical picture of possible HPFH (the father has 12% hemoglobin F) also supports the interpretation of a deletion in this family. Deletions of the {beta}-globin gene within this ethnic group are rare. Currently, Southern blots on the family are being probed to determine the extent of the putative deletion.

  15. Massive parallel gene expression profiling of RINm5F pancreatic islet beta-cells stimulated with interleukin-1beta

    DEFF Research Database (Denmark)

    Rieneck, K; Bovin, L F; Josefsen, K

    2000-01-01

    Interleukin 1 (IL-1) is a pleiotropic cytokine with the potential to kill pancreatic beta-cells, and this unique property is thought to be involved in the pathogenesis of type I diabetes mellitus. We therefore determined the quantitative expression of 24,000 mRNAs of RINm5F, an insulinoma cell line...

  16. Acetyl-coenzyme A Carboxylase: A Key Metabolic Enzyme of Fatty Acid and Progress of Its Gene Clone%乙酰辅酶A羧化酶:脂肪酸代谢的关键酶及其基因克隆研究进展

    Institute of Scientific and Technical Information of China (English)

    李洁琼; 郑世学; 喻子牛; 张吉斌

    2011-01-01

    Acetyl-coenzyme A carboxylases (ACCs) have crucial roles in fatty acid metabolism in most living organisms. In this article, structure, types, functions and inhibitors of ACC, as well as research status of ACC gene clone are systematically discussed. ACC is a multi-subunit enzyme in most prokaryotes, whereas it is a large, multi-domain enzyme in most eukaryotes. In addition, there are two special types found from Streptomyces coelicolor and Metallosphaera sedula. All of these types contain three key domains: Biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP) and carboxyltransferase (CT). CT domain, as a candidate target, has been widely used for screening of plant herbicides and drug development against obesity, diabetes and other symptoms of the metabolic syndrome. The gene encoded ACC is also becoming an important target gene applied in the fields of transgenic oil plants and biodiesel. Previous studies showed thatβ-CT in plant plasmid was the limit factor of heteromeric ACC, and BCCP was a negative regulator of fatty acid synthesis. Lipid synthesis metabolism is a very complex network, especially feedback inhibition mechanism exists in it. As a result, cloning and expression of ACC gene may increase the activity of ACC in the host, but not necessarily could obviously promote the accumulation of fatty acid. Fig 2, Ref 52%乙酰辅酶A羧化酶( Acetyl-CoA carboxy lase,ACC)在脂肪酸合成和分解代谢中发挥着重要作用.系统介绍了该酶的结构与分类、生物学作用与应用、抑制剂的类型与作用机理以及基因克隆4个方面的进展.ACC在大多数原核生物中为多亚基型酶,而在大多数真核生物中为多功能型单亚基酶,在天蓝色链霉菌和古菌勤奋金属球菌中为另外两种特殊类型;但都具备3个关键的功能域,即生物素羧化酶(BC)、生物素羧基载体蛋白(BCCP)和羧基转移酶(CT).CT功能域作为潜在的靶标广泛应用于植物除草剂的筛选和哺乳

  17. Candidate Gene Study of TRAIL and TRAIL Receptors: Association with Response to Interferon Beta Therapy in Multiple Sclerosis Patients

    Science.gov (United States)

    Órpez-Zafra, Teresa; Pinto-Medel, María Jesús; Oliver-Martos, Begoña; Ortega-Pinazo, Jesús; Arnáiz, Carlos; Guijarro-Castro, Cristina; Varadé, Jezabel; Álvarez-Lafuente, Roberto; Urcelay, Elena; Sánchez-Jiménez, Francisca

    2013-01-01

    TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO) and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10−4, pc = 0.048, OR = 0.30). This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A), a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells) were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS. PMID:23658636

  18. Candidate gene study of TRAIL and TRAIL receptors: association with response to interferon beta therapy in multiple sclerosis patients.

    Directory of Open Access Journals (Sweden)

    Carlos López-Gómez

    Full Text Available TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10(-4, pc = 0.048, OR = 0.30. This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A, a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS.

  19. Resistance to acetyl-CoA carboxylase-inhibiting herbicides.

    Science.gov (United States)

    Kaundun, Shiv S

    2014-09-01

    Resistance to acetyl-CoA carboxylase herbicides is documented in at least 43 grass weeds and is particularly problematic in Lolium, Alopecurus and Avena species. Genetic studies have shown that resistance generally evolves independently and can be conferred by target-site mutations at ACCase codon positions 1781, 1999, 2027, 2041, 2078, 2088 and 2096. The level of resistance depends on the herbicides, recommended field rates, weed species, plant growth stages, specific amino acid changes and the number of gene copies and mutant ACCase alleles. Non-target-site resistance, or in essence metabolic resistance, is prevalent, multigenic and favoured under low-dose selection. Metabolic resistance can be specific but also broad, affecting other modes of action. Some target-site and metabolic-resistant biotypes are characterised by a fitness penalty. However, the significance for resistance regression in the absence of ACCase herbicides is yet to be determined over a practical timeframe. More recently, a fitness benefit has been reported in some populations containing the I1781L mutation in terms of vegetative and reproductive outputs and delayed germination. Several DNA-based methods have been developed to detect known ACCase resistance mutations, unlike metabolic resistance, as the genes remain elusive to date. Therefore, confirmation of resistance is still carried out via whole-plant herbicide bioassays. A growing number of monocotyledonous crops have been engineered to resist ACCase herbicides, thus increasing the options for grass weed control. While the science of ACCase herbicide resistance has progressed significantly over the past 10 years, several avenues provided in the present review remain to be explored for a better understanding of resistance to this important mode of action.

  20. beta-catenin siRNA regulation of apoptosis- and angiogenesis-related gene expression in hepatocellular carcinoma cells: potential uses for gene therapy.

    Science.gov (United States)

    Wang, Xin-Hong; Sun, Xun; Meng, Xiang-Wei; Lv, Zhi-Wu; Du, Ya-Ju; Zhu, Yan; Chen, Jing; Kong, De-Xia; Jin, Shi-Zhu

    2010-10-01

    The molecular mechanism responsible for hepatocellular carcinoma (HCC) development remains to be defined although a number of gene pathways have been shown to play an active role, such as Wnt/beta-catenin signaling. In this study, beta-catenin small interfering RNA (siRNA) was designed, synthesized, and transfected into HCC HepG2 cells. RT-PCR and western blot assays were performed to detect expression of altered genes and proteins, and the MTT assay was used to detect cell viability. Our data showed that beta-catenin mRNA and protein expression levels were effectively knocked down by beta-catenin siRNA and subsequently, tumor cell proliferation was significantly suppressed. Flow cytometry assay showed that tumor cells were arrested at the G0/G1 phase of the cell cycles. Molecularly, expression of Smad3, p-caspase-3, and Grp78 protein were upregulated after 72 h of beta-catenin siRNA transfection, whereas expression of TERT, caspase-3, XIAP, MMP-2, MMP-9, VEGF-A, VEGF-c, and bFGF protein were reduced. However, there was no change between the expression of STAT3 and the HSP27 protein following transfection. The results from the current study demonstrated the importance of the Wnt/beta-catenin signaling pathway in regulation of gene expression in HCC. Further studies are required to investigate the role of this pathway in HCC development and targeting of this pathway to control HCC.

  1. DNA methylation of retrotransposons, DNA transposons and genes in sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Zakrzewski, Falk; Schmidt, Martin; Van Lijsebettens, Mieke; Schmidt, Thomas

    2017-03-03

    The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses activity of transposable elements (TEs), affects gene expression, and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of the worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%) in particular, satellite DNA, retrotransposons, and DNA transposons. Genome-wide cytosine methylation in the sugar beet genome was studied in leaves and leaf-derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H=A, C, and T), and CHH sites, whereas the TE pattern differed, depending on the classes 1 (retrotransposons) and 2 (DNA transposons), respectively. Along genes and TEs, the CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing toward a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome-wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared to leaves. This article is protected by copyright. All rights reserved.

  2. A review on the origin and spread of deleterious mutants of the beta-globin gene in Indian populations.

    Science.gov (United States)

    Das, S K; Talukder, G

    2001-01-01

    Deleterious mutations of the human beta-globin gene are responsible for beta-thalassaemia and other haemoglobinopathies, which are the most common genetic diseases in Indian populations. A highly heterogeneous distribution of those mutations is observed in India and certain mutations are restricted to some extent to particular groups only. The reasons behind the geographical clustering and origin of the mutations in India is a highly debated issue and the evidence is conflicting. Our present article aims at tracing the origin of the deleterious beta-globin mutation and evaluates the role of different evolutionary forces responsible for the spread and present distribution of those mutations in Indian populations, using data from molecular biology and statistical methods. Mutations are generated essentially randomly, but "hot-spot" sites for mutation are reported for the beta-globin gene cluster, indicating sequence dependency of mutation. A single origin of a deleterious beta-globin mutation, followed by recombination (in a hot spot region) and/or interallelic gene conversion (within beta-globin gene) through time is the most plausible hypothesis to explain the association of those mutations with multiple haplotype backgrounds and frameworks. It is suggested that India is the place of origin of HbE and HbD mutations and that they dispersed to other parts of the would by migration. HbS mutants present in Indian populations are not of Middle East origin but rather a fresh mutation is the probable explanation for the prevalence among tribal groups. beta-thalassaemia represents a heterogeneous group of mutant alleles in India. Five common and twelve rare mutations have been reported in variable frequencies among different Indian populations. Gene flow of those mutant alleles from different populations of the world by political, military and commercial interactions possibly accounts for the heterogenous nature of beta-thalassaemia among Indians. A multiple allelic

  3. Molecular analysis of T-cell receptor beta genes in cutaneous T-cell lymphoma reveals Jbeta1 bias.

    Science.gov (United States)

    Morgan, Suzanne M; Hodges, Elizabeth; Mitchell, Tracey J; Harris, Susan; Whittaker, Sean J; Smith, John L

    2006-08-01

    Molecular characterization of T-cell receptor junctional region sequences in cutaneous T-cell lymphoma had not been previously reported. We have examined in detail the features of the T-cell receptor beta (TCRB) gene rearrangements in 20 individuals with well-defined stages of cutaneous T-cell lymphoma (CTCL) comprising 10 cases with early-stage mycosis fungoides (MF) and 10 cases with late-stage MF or Sezary syndrome. Using BIOMED-2 PCR primers, we detected a high frequency of clonally rearranged TCR gamma and TCRB genes (17/20 and 15/20 cases, respectively). We carried out sequencing analysis of each complete clonal variable (V)beta-diversity (D)beta-joining(J)beta fingerprint generated by PCR amplification, and determined the primary structure of the Vbeta-Dbeta-Jbeta junctional regions. We observed considerable diversity in the T-cell receptor Vbeta gene usage and complementarity-determining region 3 loops. Although we found that TCRB gene usage in CTCL and normal individuals share common features, our analysis also revealed preferential usage of Jbeta1 genes in all cases with advanced stages of disease.

  4. Beta-globin gene cluster haplotypes in Venezuelan sickle cell patients from the State of Aragua

    Directory of Open Access Journals (Sweden)

    Nancy Moreno

    2002-01-01

    Full Text Available Seven polymorphic sites in the beta-globin gene cluster were analyzed on a sample of 96 chromosomes of Venezuelan sickle cell patients from the State of Aragua. The Benin haplotype was predominant with a frequency of 0.479, followed by the Bantu haplotype (0.406; a minority of cases with other haplotypes was also identified: atypical Bantu A2 (0.042, Senegal (0.031, atypical Bantu A7 (0.021 and Saudi Arabia/Indian (0.021 haplotypes; however, the Cameroon haplotype was not identified in this study. Our results are in agreement with the historical records that establish Sudanese and Bantu origins for the African slaves brought into Venezuela.

  5. TGF-beta 1 Gene-Activated Matrices Regulated the Osteogenic Differentiation of BMSCs

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Poly (lactic acid/glycolic acid/asparagic acid-co-polyethylene glycol)(PLGA-[ASP-PEG]) scaffold materials were linked with a novel nonviral vector (K)16GRGDSPC through cross linker Sulfo-LC-SPDP to construct a new type of nonviral gene transfer system. Eukaryotic expressing vector containing transforming growth factor beta 1 (pcDNA3-TGFβ1) was encapsulated by the system. Bone marrow stromal cells (BMSCs) obtained from rabbit were cultured on PLGA-[ASP-PEG] modified by (K)16GRGDSPC and TGF-β1 gene and PLGA-[ASP-PEG] modified by (K)16GRGDSPC and empty vector pcDNA3 as control.The expressions of osteogenic makers of the BMSCs cultured on the TGF-β1 gene-activated scaffold materials were found significantly higher than those of the control group (P<0.05). A brand-new way was provided for regulating seed cells to directionally differentiate into osteoblasts for bone defect restoration in bone tissue engineering.

  6. Relationship Between Polymorphism of Cystathionine beta Synthase Gene and Congenital Heart Disease in Chinese Nuclear Families

    Institute of Scientific and Technical Information of China (English)

    XIAO-MING SONG; XIAO-YING ZHENG; WEN-LI ZHU; LEI HUANG; YONG LI

    2006-01-01

    Objective To study the relationship between polymorphism of cystathionine beta synthase (CBS) gene and development of congenital heart disease (CHD). Methods One hundred and twenty-seven CHD case-parent triads were recruited from Liaoning Province as patient group, and 129 healthy subjects without family history of birth defect were simultaneously recruited as control group together with their biological parents. For all subjects the polymorphism of CBS gene G919A locus was examined by PCR-ARMS method. Results The frequencies of three genotypes (w/w, w/m, and m/m) in control group were 27.2%, 58.4%, and 14.4%, respectively, with no significant difference in gender. A significant difference in the allele frequency was found between CHD patients and controls, the wild allele frequency was 67.9% in patients and 55.7% in controls.CHD parents' genotype distribution was significantly different from that in controls. Further comparison of each type of CHD showed that genotype frequencies in several CHD subtypes were significantly different from those in their corresponding controls. The results of TDT analysis showed that no allele transmission disequilibrium existed in CHD nuclear families.Conclusions CBS gene G919A mutation is associated with the development of CHD, and the mutated allele may decrease the risk of CHD.

  7. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids

    DEFF Research Database (Denmark)

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S

    2008-01-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian...... DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent...

  8. Live Borrelia burgdorferi preferentially activate interleukin-1 beta gene expression and protein synthesis over the interleukin-1 receptor antagonist.

    Science.gov (United States)

    Miller, L C; Isa, S; Vannier, E; Georgilis, K; Steere, A C; Dinarello, C A

    1992-01-01

    Lyme arthritis is one of the few forms of chronic arthritis in which the cause is known with certainty. Because cytokines are thought to contribute to the pathogenesis of chronic arthritis, we investigated the effect of the Lyme disease spirochete, Borrelia burgdorferi, on the gene expression and synthesis of IL-1 beta and the IL-1 receptor antagonist (IL-1ra) in human peripheral blood mononuclear cells. Live B. burgdorferi induced fivefold more IL-1 beta than IL-1 alpha and sevenfold more IL-1 beta than IL-1ra; LPS or sonicated B. burgdorferi induced similar amounts of all three cytokines. This preferential induction of IL-1 beta was most dramatic in response to a low passage, virulent preparation of B. burgdorferi vs. three high passage avirulent strains. No difference in induction of IL-1ra was seen between these strains. The marked induction of IL-1 beta was partially diminished by heat-treatment and abrogated by sonication; IL-1ra was not affected. This suggested that a membrane component(s) accounted for the preferential induction of IL-1 beta. However, recombinant outer surface protein beta induced little IL-1 beta. By 4 h after stimulation, B. burgdorferi induced sixfold more IL-1 beta protein than LPS. In contrast to LPS-induced IL-1 beta mRNA which reached maximal accumulation after 3 h, B. burgdorferi-induced IL-1 beta mRNA showed biphasic elevations at 3 and 18 h. B. burgdorferi-induced IL-1ra mRNA peaked at 12 h, whereas LPS-induced IL-1ra mRNA peaked at 9 h. IL-1 beta synthesis increased in response to increasing numbers of spirochetes, whereas IL-1ra synthesis did not. The preferential induction by B. burgdorferi of IL-1 beta over IL-1ra is an example of excess agonist over antagonist synthesis induced by a microbial pathogen, and may contribute to the destructive lesion of Lyme arthritis. Images PMID:1387885

  9. Early peroxisome proliferator-activated receptor gamma regulated genes involved in expansion of pancreatic beta cell mass

    Directory of Open Access Journals (Sweden)

    Vivas Yurena

    2011-12-01

    Full Text Available Abstract Background The progression towards type 2 diabetes depends on the allostatic response of pancreatic beta cells to synthesise and secrete enough insulin to compensate for insulin resistance. The endocrine pancreas is a plastic tissue able to expand or regress in response to the requirements imposed by physiological and pathophysiological states associated to insulin resistance such as pregnancy, obesity or ageing, but the mechanisms mediating beta cell mass expansion in these scenarios are not well defined. We have recently shown that ob/ob mice with genetic ablation of PPARγ2, a mouse model known as the POKO mouse failed to expand its beta cell mass. This phenotype contrasted with the appropriate expansion of the beta cell mass observed in their obese littermate ob/ob mice. Thus, comparison of these models islets particularly at early ages could provide some new insights on early PPARγ dependent transcriptional responses involved in the process of beta cell mass expansion Results Here we have investigated PPARγ dependent transcriptional responses occurring during the early stages of beta cell adaptation to insulin resistance in wild type, ob/ob, PPARγ2 KO and POKO mice. We have identified genes known to regulate both the rate of proliferation and the survival signals of beta cells. Moreover we have also identified new pathways induced in ob/ob islets that remained unchanged in POKO islets, suggesting an important role for PPARγ in maintenance/activation of mechanisms essential for the continued function of the beta cell. Conclusions Our data suggest that the expansion of beta cell mass observed in ob/ob islets is associated with the activation of an immune response that fails to occur in POKO islets. We have also indentified other PPARγ dependent differentially regulated pathways including cholesterol biosynthesis, apoptosis through TGF-β signaling and decreased oxidative phosphorylation.

  10. Phylogeny of 16S rRNA, Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase, and Adenosine 5′-Phosphosulfate Reductase Genes from Gamma- and Alphaproteobacterial Symbionts in Gutless Marine Worms (Oligochaeta) from Bermuda and the Bahamas

    OpenAIRE

    Blazejak, Anna; Kuever, Jan; Erséus, Christer; Amann, Rudolf; Dubilier, Nicole

    2006-01-01

    Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluore...

  11. Massive parallel gene expression profiling of RINm5F pancreatic islet beta-cells stimulated with interleukin-1beta

    DEFF Research Database (Denmark)

    Rieneck, K; Bovin, L F; Josefsen, K;

    2000-01-01

    the genes into groups according to functional relations on the basis of knowledge of the structure or function ascribed to the individual genes. Many of the differentially regulated genes are known to play a role in immune- and stress-related pathways as well as in insulin secretion and vesicle trafficking......, e.g. alpha-endosulfine and K+ channel Kir6.2 are differentially regulated. A number of transcripts in the biosynthesis pathway for cholesterol are also differentially regulated....

  12. Association of polymorphisms in the beta-2 adrenergic receptor gene with fracture risk and bone mineral density

    NARCIS (Netherlands)

    Veldhuis-Vlug, A G; Oei, L; Souverein, P C; Tanck, M W T; Rivadeneira, F; Zillikens, M C; Kamphuisen, P W; Maitland-van der Zee, A H; de Groot, M C H; Hofman, A; Uitterlinden, A G; Fliers, E; de Boer, A; Bisschop, P H

    2015-01-01

    Signaling through the beta-2 adrenergic receptor (B2AR) on the osteoblast influences bone remodeling in rodents. In the B2AR gene, three polymorphisms influence receptor function. We show that these polymorphisms are not associated with fracture risk or bone mineral density in the UCP, Rotterdam Stu

  13. Association of polymorphisms in the beta-2 adrenergic receptor gene with fracture risk and bone mineral density

    NARCIS (Netherlands)

    A.G. Veldhuis-Vlug; L. Oei (Ling); P. Souverein (Patrick); M.W.T. Tanck (Michael); F. Rivadeneira Ramirez (Fernando); M.C. Zillikens (Carola); P.W. Kamphuisen; A-H. Maitland-van der Zee (Anke-Hilse); M.C.H. de Groot; A. Hofman (Albert); A.G. Uitterlinden (André); E. Fliers (Eric); A.C. de Boer (Anthonius); P.H. Bisschop

    2015-01-01

    textabstractSummary: Signaling through the beta-2 adrenergic receptor (B2AR) on the osteoblast influences bone remodeling in rodents. In the B2AR gene, three polymorphisms influence receptor function. We show that these polymorphisms are not associated with fracture risk or bone mineral density in t

  14. Association of polymorphisms in the beta-2 adrenergic receptor gene with fracture risk and bone mineral density

    NARCIS (Netherlands)

    Veldhuis-Vlug, A. G.; Oei, L.; Souverein, P. C.; Tanck, M. W T; Rivadeneira, F.; Zillikens, M. C.; Kamphuisen, P. W.; Maitland - van der Zee, A. H.; de Groot, M. C H; Hofman, A.; Uitterlinden, A. G.; Fliers, E.; de Boer, A.; Bisschop, P. H.

    2015-01-01

    Summary: Signaling through the beta-2 adrenergic receptor (B2AR) on the osteoblast influences bone remodeling in rodents. In the B2AR gene, three polymorphisms influence receptor function. We show that these polymorphisms are not associated with fracture risk or bone mineral density in the UCP, Rott

  15. The diversity and abundance of phytase genes (beta-propeller phytases) in bacterial communities of the maize rhizosphere

    NARCIS (Netherlands)

    Cotta, S.R.; Cavalcante Franco Dias, A.; Seldin, L.; Andreote, F. D.; van Elsas, J. D.

    2016-01-01

    The ecology of microbial communities associated with organic phosphorus (P) mineralization in soils is still understudied. Here, we assessed the abundance and diversity of bacteria harbouring genes encoding beta-propeller phytases (BPP) in the rhizosphere of traditional and transgenic maize cultivat

  16. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli.

    Science.gov (United States)

    Burgers, P M; Kornberg, A; Sakakibara, Y

    1981-09-01

    An Escherichia coli mutant, dnaN59, stops DNA synthesis promptly upon a shift to a high temperature; the wild-type dnaN gene carried in a transducing phage encodes a polypeptide of about 41,000 daltons [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553; Yuasa, S. & Sakakibara, Y. (1980) Mol. Gen. Genet. 180, 267-273]. We now find that the product of dnaN gene is the beta subunit of DNA polymerase III holoenzyme, the principal DNA synthetic multipolypeptide complex in E. coli. The conclusion is based on the following observations: (i) Extracts from dnaN59 cells were defective in phage phi X174 and G4 DNA synthesis after the mutant cells had been exposed to the increased temperature. (ii) The enzymatic defect was overcome by addition of purified beta subunit but not by other subunits of DNA polymerase III holoenzyme or by other replication proteins required for phi X174 DNA synthesis. (iii) Partially purified beta subunit from the dnaN mutant, unlike that from the wild type, was inactive in reconstituting the holoenzyme when mixed with the other purified subunits. (iv) Increased dosage of the dnaN gene provided by a plasmid carrying the gene raised cellular levels of the beta subunit 5- to 6-fold.

  17. Inter-ethnic polymorphism of the beta-globin gene locus control region (LCR) in sickle-cell anemia patients.

    Science.gov (United States)

    Périchon, B; Ragusa, A; Lapouméroulie, C; Romand, A; Moi, P; Ikuta, T; Labie, D; Elion, J; Krishnamoorthy, R

    1993-06-01

    Sequence polymorphisms within the 5'HS2 segment of human locus control region is described among sickle cell anemia patients. Distinct polymorphic patterns of a simple sequence repeat are observed in strong linkage disequilibrium with each of the five major beta s haplotypes. Potential functional relevance of this polymorphic region in globin gene expression is discussed.

  18. Identification of a Novel Single Nucleotide Polymorphism in Porcine Beta-Defensin-1 Gene.

    Science.gov (United States)

    Pruthviraj, D R; Usha, A P; Venkatachalapathy, R T

    2016-03-01

    Porcine beta-defensin-1 (PBD-1) gene plays an important role in the innate immunity of pigs. The peptide encoded by this gene is an antimicrobial peptide that has direct activity against a wide range of microbes. This peptide is involved in the co-creation of an antimicrobial barrier in the oral cavity of pigs. The objective of the present study was to detect polymorphisms, if any, in exon-1 and exon-2 regions of PBD-1 gene in Large White Yorkshire (LWY) and native Ankamali pigs of Kerala, India. Blood samples were collected from 100 pigs and genomic DNA was isolated using phenol chloroform method. The quantity of DNA was assessed in a spectrophotometer and quality by gel electrophoresis. Exon-1 and exon-2 regions of PBD-1 gene were amplified by polymerase chain reaction (PCR) and the products were subjected to single strand conformation polymorphism (SSCP) analysis. Subsequent silver staining of the polyacrylamide gels revealed three unique SSCP banding patterns in each of the two exons. The presence of single nucleotide polymorphisms (SNPs) was confirmed by nucleotide sequencing of the PCR products. A novel SNP was found in the 5'-UTR region of exon-1 and a SNP was detected in the mature peptide coding region of exon-2. In exon-1, the pooled population frequencies of GG, GT, and TT genotypes were 0.67, 0.30, and 0.03, respectively. GG genotype was predominant in both the breeds whereas TT genotype was not detected in LWY breed. Similarly, in exon-2, the pooled population frequencies of AA, AG, and GG genotypes were 0.50, 0.27, and 0.23, respectively. AA genotype was predominant in LWY pigs whereas GG genotype was predominant in native pigs. These results suggest that there exists a considerable genetic variation at PBD-1 locus and further association studies may help in development of a PCR based genotyping test to select pigs with better immunity.

  19. An ancient repeat sequence in the ATP synthase beta-subunit gene of forcipulate sea stars.

    Science.gov (United States)

    Foltz, David W

    2007-11-01

    A novel repeat sequence with a conserved secondary structure is described from two nonadjacent introns of the ATP synthase beta-subunit gene in sea stars of the order Forcipulatida (Echinodermata: Asteroidea). The repeat is present in both introns of all forcipulate sea stars examined, which suggests that it is an ancient feature of this gene (with an approximate age of 200 Mya). Both stem and loop regions show high levels of sequence constraint when compared to flanking nonrepetitive intronic regions. The repeat was also detected in (1) the family Pterasteridae, order Velatida and (2) the family Korethrasteridae, order Velatida. The repeat was not detected in (1) the family Echinasteridae, order Spinulosida, (2) the family Astropectinidae, order Paxillosida, (3) the family Solasteridae, order Velatida, or (4) the family Goniasteridae, order Valvatida. The repeat lacks similarity to published sequences in unrestricted GenBank searches, and there are no significant open reading frames in the repeat or in the flanking intron sequences. Comparison via parametric bootstrapping to a published phylogeny based on 4.2 kb of nuclear and mitochondrial sequence for a subset of these species allowed the null hypothesis of a congruent phylogeny to be rejected for each repeat, when compared separately to the published phylogeny. In contrast, the flanking nonrepetitive sequences in each intron yielded separate phylogenies that were each congruent with the published phylogeny. In four species, the repeat in one or both introns has apparently experienced gene conversion. The two introns also show a correlated pattern of nucleotide substitutions, even after excluding the putative cases of gene conversion.

  20. The expression of pregnancy-specific {beta}1-glycoprotein genes in Meckel-Gruber syndrome fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Shao-Ming; Cham, Wai-Yee [Georgetown Univ. Medical Center, Washington, DC (United States)

    1994-09-01

    Meckel-Gruber syndrome (MS) is an autosomal recessive disorder with multiple congenital malformations. The only available prenatal diagnostic marker for this disorder is the amniotic fluid level of pregnancy-specific {beta}1-glycoprotein (PSG). PSG is a family of proteins which are expressed at high levels during pregnancy. Increasing maternal serum PSG levels correlate with the progression of pregnancy and can be used as indicators for pregnancy outcome and fetal well-being. The amniotic fluid PSG level is about one-tenth of that of the maternal serum level in normal pregnancy, but are elevated in all cases of MS examined so far. On the other hand, the maternal serum PSG level and third trimester placental PSG content are normal in most cases of MS. This study aims at comparing the expression of PSG in fibroblasts derived from a fetus afflicted with MS. Total cellular RNA was extracted from two MS cultured fibroblast lines (M3206 and GM7817) and four age- and sex-matched control fibroblast lines obtained from the Human Genetic Mutant Cell Repository, Camden, NJ. The expression of eight PSG genes namely, PSG1, PSG2, PSG3, PSG4, PSG5, PSG6, PSG9 and PSG11, were examined with reverse transcription-polymerase chain reaction (RT-PCR). All PSG transcripts present in the cell were first amplified using universal primers in a 28-cycle PCR. Specific PSG gene products were then amplified with PSG gene-specific primers. Results showed that there is no significant difference in PSG expression between control and disease fibroblasts. In both cases, the most abundant transcript was the type II transcript of PSG5 followed by the type I transcripts of PSG1 and PG4. PSG9, PSG11 and PSG 3 were expressed at very low levels or not expressed at all in MS as well as in normal control fibroblasts. These results showed that PSG gene expression was not altered in MS fibroblasts.

  1. Transcriptional profiling of type 1 diabetes genes on chromosome 21 in a rat beta-cell line and human pancreatic islets

    DEFF Research Database (Denmark)

    Bergholdt, R.; Karlsen, A.E.; Hagedorn, Peter;

    2007-01-01

    We recently finemapped a type 1 diabetes (T1D)-linked region on chromosome 21, indicating that one or more T1D-linked genes exist in this region with 33 annotated genes. In the current study, we have taken a novel approach using transcriptional profiling in predicting and prioritizing the most...... likely candidate genes influencing beta-cell function in this region. Two array-based approaches were used, a rat insulinoma cell line (INS-1alphabeta) overexpressing pancreatic duodenum homeobox 1 (pdx-1) and treated with interleukin 1beta (IL-1beta) as well as human pancreatic islets stimulated...... with a mixture of cytokines. Several candidate genes with likely functional significance in T1D were identified. Genes showing differential expression in the two approaches were highly similar, supporting the role of these specific gene products in cytokine-induced beta-cell damage. These were genes involved...

  2. Structural alterations of transforming growth factor-beta receptor genes in human cervical carcinoma

    NARCIS (Netherlands)

    Chen, TP; De Vries, EGE; Hollema, H; Yegen, HA; Vellucci, VF; Strickler, HD; Hildesheim, A; Reiss, M

    1999-01-01

    The development and progression of invasive uterine cervical carcinomas appear to be associated with the progressive loss of sensitivity to transforming growth factor-beta (TGF beta)-mediated cell cycle arrest. In order to identify possible molecular mechanisms responsible for TGF beta resistance, w

  3. Gene expression profiles of Beta-cell enriched tissue obtained by laser capture microdissection from subjects with type 2 diabetes.

    Directory of Open Access Journals (Sweden)

    Lorella Marselli

    Full Text Available BACKGROUND: Changes in gene expression in pancreatic beta-cells from type 2 diabetes (T2D should provide insights into their abnormal insulin secretion and turnover. METHODOLOGY/PRINCIPAL FINDINGS: Frozen sections were obtained from cadaver pancreases of 10 control and 10 T2D human subjects. Beta-cell enriched samples were obtained by laser capture microdissection (LCM. RNA was extracted, amplified and subjected to microarray analysis. Further analysis was performed with DNA-Chip Analyzer (dChip and Gene Set Enrichment Analysis (GSEA software. There were changes in expression of genes linked to glucotoxicity. Evidence of oxidative stress was provided by upregulation of several metallothionein genes. There were few changes in the major genes associated with cell cycle, apoptosis or endoplasmic reticulum stress. There was differential expression of genes associated with pancreatic regeneration, most notably upregulation of members of the regenerating islet gene (REG family and metalloproteinase 7 (MMP7. Some of the genes found in GWAS studies to be related to T2D were also found to be differentially expressed. IGF2BP2, TSPAN8, and HNF1B (TCF2 were upregulated while JAZF1 and SLC30A8 were downregulated. CONCLUSIONS/SIGNIFICANCE: This study made possible by LCM has identified many novel changes in gene expression that enhance understanding of the pathogenesis of T2D.

  4. Assessment of AmpC Beta-Lactamase Genes among Clinical Escherichia coli Isolates

    Directory of Open Access Journals (Sweden)

    HedrooshaMolla Agha-Mirzaeie

    2015-11-01

    Full Text Available Background: AmpC bta lactamases play a significant role in creating resistance to third generation cephalosporins worldwide. They mostly express on chromosome of Enterobacteriaceae especially Escherichia coli and cause consequential problem inclinical treatment and lead to failure in diagnosis and phenotypic test recommended byClinical and Laboratory Standards Institute.Methods:Totally 200 E. coli isolates from different hospitals of Tehran were collected. The isolates were screened by disk diffusion method according to the CLSI guidelines. The profiles and prevalence surveys of AmpC (Dha, CITM, Mox and FOX-type β-lactamase genes in clinical isolates of E. coli by phenotypic and molecular methods.  Results:Out of 200 Ecoli isolated, 115 (89.8% and 13 (10.2% isolates were identified as ESBL- and AmpC- beta-lactamase producers, respectively. Among mpC producers, 13 (100% and 5 (38.5% isolates was reported by PCR assay as bla-CITM and Dha respectively. Mox and FOX genes were not detected in any sample.Conclusions:Our results highlight the importance of using molecular detection methods to identify β-lactamase-producer that have resistance to antibiotics. 

  5. Pyruvate carboxylase is expressed in human skeletal muscle

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2010-01-01

    Pyruvate carboxylase (PC) is a mitochondrial enzyme that catalyses the carboxylation of pyruvate to oxaloacetate thereby allowing supplementation of citric acid cycle intermediates. The presence of PC in skeletal muscle is controversial. We report here, that PC protein is easily detectable...

  6. Beta-globin LCR and intron elements cooperate and direct spatial reorganization for gene therapy.

    Directory of Open Access Journals (Sweden)

    Alla Buzina

    2008-04-01

    Full Text Available The Locus Control Region (LCR requires intronic elements within beta-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional beta-globin intron 2 elements that rescue LCR activity directed by 5'HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igmu 3'MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igmu 3'MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5'HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5'HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate

  7. Analysis of beta-casein gene (CSN2 polymorphism in different breeds of cattle

    Directory of Open Access Journals (Sweden)

    Martina Miluchová

    2014-11-01

    Full Text Available Normal 0 21 false false false SK X-NONE X-NONE MicrosoftInternetExplorer4 The goal of work was identification of b - casein gene polymorphism in different breeds of cow. The beta - casein constitutes up to 45 % of the casein of bovine milk. The most common forms of beta-casein in dairy cattle breeds are A1 and A2, while B is less common. The b-casein A1 variant was associated with the incidence of diabetes mellitus 1st type, coronary heart disease and autism. The A2 variant reduces serum cholesterol. The material involved 287 cows (Simmental breed – 111 cows, Pinzgau breed – 89 cows, Holstein breed – 87 cows. Bovine genomic DNA was extracted from whole blood by using commercial kit and used in order to estimate b - casein genotypes by means of PCR-RFLP method. In the populations included in the study were detected all three genotypes – homozygote genotype A1A1, heterozygote genotype A1A2 and homozygote genotype A2A2 with frequencies 0.1261, 0.3333 and 0.5405 in Simmental breed; 0.1379, 0.4598 and 0.4023 in Holstein breed, 0.3034, 0.5168 and 0.1798 in Pinzgau breed. In population of Simmental breed and Holstein breed was higher frequency of allele A2 (0.7072 and 0.6322. In opposite, in population of Pinzgau breed was present higher frequency of the allele A1 (0.5618.

  8. Quantitative response relationships between degradation rates and functional genes during the degradation of beta-cypermethrin in soil.

    Science.gov (United States)

    Yang, Zhong-Hua; Ji, Guo-Dong

    2015-12-15

    In the present study, the degradation mechanisms of beta-cypermethrin and its metabolites in soil were explored through the quantitative response relationships between the degradation rates and related functional genes. We found that the degradation rate of beta-cypermethrin was rapid in unsterilized soil but not in sterilized soil, which indicated that the degradation process is microbially based. Moreover, three metabolites (3-phenoxybenzoic acid, phenol and protocatechuic acid) were detected during the degradation process and used to identify the degradation pathway and functional genes related to the degradation process. The key rate-limiting functional genes were pytH and pobA, and the relative contributions of these genes to the degradation process were examined with a path analysis. The path analysis revealed that the genes pobA and pytH had the greatest direct effects on the degradation of beta-cypermethrin (pobA), alpha-cypermethrin (pobA), theta-cypermethrin (pytH) and 3-phenoxybenzoic acid (pytH).

  9. Baseline Gene Expression Signatures in Monocytes from Multiple Sclerosis Patients Treated with Interferon-beta

    Science.gov (United States)

    Bustamante, Marta F.; Nurtdinov, Ramil N.; Río, Jordi; Montalban, Xavier; Comabella, Manuel

    2013-01-01

    Background A relatively large proportion of relapsing-remitting multiple sclerosis (RRMS) patients do not respond to interferon-beta (IFNb) treatment. In previous studies with peripheral blood mononuclear cells (PBMC), we identified a subgroup of IFNb non-responders that was characterized by a baseline over-expression of type I IFN inducible genes. Additional mechanistic experiments carried out in IFNb non-responders suggested a selective alteration of the type I IFN signaling pathway in the population of blood monocytes. Here, we aimed (i) to investigate whether the type I IFN signaling pathway is up-regulated in isolated monocytes from IFNb non-responders at baseline; and (ii) to search for additional biological pathways in this cell population that may be implicated in the response to IFNb treatment. Methods Twenty RRMS patients classified according to their clinical response to IFNb treatment and 10 healthy controls were included in the study. Monocytes were purified from PBMC obtained before treatment by cell sorting and the gene expression profiling was determined with oligonucleotide microarrays. Results and discussion Purified monocytes from IFNb non-responders were characterized by an over-expression of type I IFN responsive genes, which confirms the type I IFN signature in monocytes suggested from previous studies. Other relevant signaling pathways that were up-regulated in IFNb non-responders were related with the mitochondrial function and processes such as protein synthesis and antigen presentation, and together with the type I IFN signaling pathway, may also be playing roles in the response to IFNb. PMID:23637780

  10. Pest protection conferred by a Beta vulgaris serine proteinase inhibitor gene.

    Directory of Open Access Journals (Sweden)

    Ann C Smigocki

    Full Text Available Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases.

  11. Pest protection conferred by a Beta vulgaris serine proteinase inhibitor gene.

    Science.gov (United States)

    Smigocki, Ann C; Ivic-Haymes, Snezana; Li, Haiyan; Savić, Jelena

    2013-01-01

    Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI) was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases.

  12. Studies of the variability of the hepatocyte nuclear factor-1beta (HNF-1beta / TCF2) and the dimerization cofactor of HNF-1 (DcoH / PCBD) genes in relation to type 2 diabetes mellitus and beta-cell function

    DEFF Research Database (Denmark)

    Ek, J; Grarup, N; Urhammer, S A

    2001-01-01

    , and 46 type 2 diabetic patients with an impaired beta-cell function by combined single-strand conformation polymorphism (SSCP) and heteroduplex analysis. Analysis of the promoter and nine exons including intron-exon boundaries of the HNF-1beta gene revealed one novel silent polymorphism and three...... previously reported intronic variants. The silent polymorphism (I91I) was found in one patient with late-onset type 2 diabetes. One of the intronic variant (IVS6+26T-->C) was examined further. Among 584 type 2 diabetic patients the allelic frequency was 13.1% (11.2-15.0%) compared to 11.6% (8.6-14.5%) in 229...... comprising the DCoH gene revealed a previously described A-->G polymorphism located in the 3' untranslated region, which was not investigated further. In conclusion, mutations in HNF-1beta and DCoH are not a major cause of MODY or late onset type 2 diabetes in Danish Caucasian subjects....

  13. Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli beta-glucuronidase gene and analysis of its expression in Aspergillus oryzae.

    Science.gov (United States)

    Tada, S; Gomi, K; Kitamoto, K; Takahashi, K; Tamura, G; Hara, S

    1991-10-01

    Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae RIB40 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding beta-glucuronidase (GUS) down-stream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.

  14. ACACβ gene (rs2268388) and AGTR1 gene (rs5186) polymorphism and the risk of nephropathy in Asian Indian patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Shah, Viral N; Cheema, Balneek Singh; Sharma, Rajni;

    2013-01-01

    Patients with type 2 diabetes (T2DM) are usually obese and concurrent obesity results into activation of the renin-angiotensin-system (RAS) which is a risk factor for diabetic nephropathy (DN). Gene-gene interaction between acetyl-coenzymeA carboxylase beta (ACACβ) gene, which is involved in fatty....... We also found significant epistatic interactions between these two genes. TT genotypes of ACACβ gene and CC genotype of AGTR1 gene confers the risk of DN and both genes had significant epistatic interaction in Asian Indian patients with T2DM....... acid metabolism and angiotensin II receptors (AGTR1) gene, which mediates RAS proteins actions on renal tissue, polymorphism with DN have not been studied earlier. The present study was designed with the aim to examine the association of an ACACβ (rs2268388) and AGTR1 (rs5186) gene polymorphism...

  15. Localization of the human {beta}-catenin gene (CTNNB1) to 3p21: A region implicated in tumor development

    Energy Technology Data Exchange (ETDEWEB)

    Kraus, C.; Liehr, T.; Ballhausen, G. [Institut fuer Humangenetik der Universitaet, Erlangen (Germany)] [and others

    1994-09-01

    The human {beta}-catenin locus (CTNNB1) was mapped by in situ fluorescence analysis to band p21 on the short arm of chromosome 3, a region frequently affected by somatic alterations in a variety of tumors. PCR primers for the genomic amplification of {beta}-catenin sequences were selected on the basis of homology to exon 4 of the Drosophila armadillo gene. Analysis of a panel of somatic cell hybrids confirmed the localization of {beta}-catenin on human chromosome 3. Furthermore, exclusion mapping of three hybrids carrying defined fragments of the short arm of human chromosome 3 allowed us to determine the position of the CTNNB1 locus close to the marker D3S2 in 3p21. 22 refs., 3 figs.

  16. Role of transcription factor KLF11 and its diabetes-associated gene variants in pancreatic beta cell function

    DEFF Research Database (Denmark)

    Neve, Bernadette; Fernandez-Zapico, Martin E; Ashkenazi-Katalan, Vered

    2005-01-01

    a role in free radical clearance that may render beta cells more sensitive to oxidative stress. Thus, both functional and genetic analyses reveal that KLF11 plays a role in the regulation of pancreatic beta cell physiology, and its variants may contribute to the development of diabetes.......KLF11 (TIEG2) is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo. However, its functional role in the endocrine pancreas remains to be established. Here, we report, for the first...... in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1...

  17. The Beta-Binomial Distribution for Estimating the Number of False Rejections in Microarray Gene Expression Studies.

    Science.gov (United States)

    Hunt, Daniel L; Cheng, Cheng; Pounds, Stanley

    2009-03-15

    In differential expression analysis of microarray data, it is common to assume independence among null hypotheses (and thus gene expression levels). The independence assumption implies that the number of false rejections V follows a binomial distribution and leads to an estimator of the empirical false discovery rate (eFDR). The number of false rejections V is modeled with the beta-binomial distribution. An estimator of the beta-binomial false discovery rate (bbFDR) is then derived. This approach accounts for how the correlation among non-differentially expressed genes influences the distribution of V. Permutations are used to generate the observed values for V under the null hypotheses and a beta-binomial distribution is fit to the values of V. The bbFDR estimator is compared to the eFDR estimator in simulation studies of correlated non-differentially expressed genes and is found to outperform the eFDR for certain scenarios. As an example, this method is also used to perform an analysis that compares the gene expression of soft tissue sarcoma samples to normal tissue samples.

  18. High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous.

    Science.gov (United States)

    Verwaal, René; Wang, Jing; Meijnen, Jean-Paul; Visser, Hans; Sandmann, Gerhard; van den Berg, Johan A; van Ooyen, Albert J J

    2007-07-01

    To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially beta-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product beta-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of beta-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of beta-carotene by S. cerevisiae.

  19. Association of beta 2 -adrenergic receptor gene polymorphisms and nocturnal asthma in Saudi patients

    Directory of Open Access Journals (Sweden)

    Al-Rubaish Abdullah

    2011-01-01

    Full Text Available Background and Objectives : Two polymorphisms of beta 2 -adrenergic receptor (β2 -AR gene, namely the substitution from arginine (Arg to glycine (Gly at codon 16 and from glutamine (Gln to glutamic (Glu at codon 27, are linked with functional changes in the β2 -AR in the respiratory system even though they are not deemed to be susceptibility genes for asthma per se. The objective of this study was to investigate this association in a subset of asthmatic patients, namely those with nocturnal asthma. Methods : The β2 -AR gene polymorphisms at codon 16 and 27 were assessed in 40 patients clinically diagnosed with nocturnal asthma and 96 normal controls. Genomic DNA was obtained from whole blood and genotyping was carried out by a PCR based restriction fragment length polymorphism technique. Results : There was a statistically significant difference in genotype frequencies at codon 16 (Arg/Gly between nocturnal asthmatic patients and normal control subjects (P < 0.05. However, there was no statistically significant difference in allele frequencies between the two groups. In addition, there was a significant association between Arg16-Gly genotype with nocturnal asthma compared to homozygous Gly16 (codominant model P = 0.0033, OR = 3.69: 95% CI: 1.49-9.12. However, there were no statistically significant differences in genotype and allele frequencies at codon 27 (Gln/Glu between the normal control and nocturnal asthmatic groups (χ2 = 1.81, P = 0.41. The results also indicate that linkage disequilibrium existed between the β2 -AR codon 16 and β2 -AR codon 27 polymorphism (/ D΄/ = 0.577. The data for all haplotypes did not show a statistically significant association. Conclusion : We present the genotype and allele frequencies of β2 -AR gene polymorphisms in normal Saudi subjects and nocturnal asthmatic patients. There was a significant difference in genotype frequencies at codon 16 (Arg/Gly. However, our study indicates a poor association of

  20. Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency

    Science.gov (United States)

    Kim, Chun-Hyung; Zabetian, Cyrus P.; Cubells, Joseph F.; Cho, Sonhae; Biaggioni, Italo; Cohen, Bruce M.; Robertson, David; Kim, Kwang-Soo

    2002-01-01

    Norepinephrine (NE), a key neurotransmitter of the central and peripheral nervous systems, is synthesized by dopamine beta-hydroxylase (DBH) that catalyzes oxidation of dopamine (DA) to NE. NE deficiency is a congenital disorder of unknown etiology, in which affected patients suffer profound autonomic failure. Biochemical features of the syndrome include undetectable tissue and circulating levels of NE and epinephrine, elevated levels of DA, and undetectable levels of DBH. Here, we report identification of seven novel variants including four potentially pathogenic mutations in the human DBH gene (OMIM 223360) from analysis of two unrelated patients and their families. Both patients are compound heterozygotes for variants affecting expression of DBH protein. Each carries one copy of a T-->C transversion in the splice donor site of DBH intron 1, creating a premature stop codon. In patient 1, there is a missense mutation in DBH exon 2. Patient 2 carries missense mutations in exons 1 and 6 residing in cis. We propose that NE deficiency is an autosomal recessive disorder resulting from heterogeneous molecular lesions at DBH. Copyright 2002 Wiley-Liss, Inc.

  1. Beta-globin gene cluster haplotypes in sickle cell patients from southwest Iran.

    Science.gov (United States)

    Rahimi, Z; Karimi, M; Haghshenass, M; Merat, A

    2003-11-01

    Sickle cell anemia in Iran is accompanied by a high level of HbF and mild clinical presentation. Here we report haplotypes of the beta gene cluster found in 81 randomly selected sickle cell patients, including 47 sickle cell anemia (SS), 17 sickle cell trait (AS), and 17 sickle/thalassemia (S/thal) from southwest Iran. We found all five common typical haplotypes as well as five atypical haplotypes in our patients. Except for four patients with homozygous Benin haplotype, none of the other African typical haplotypes were found in a homozygous state. Arab-Indian was found to be the most prevalent haplotype in the study population. This haplotype accounted for 51.1% as the homozygous form in SS patients, where 69.1% of chromosomes in these patients had the Arab-Indian haplotype. Bantu A2 was the second most prevalent haplotype among all patients. The mean %HbF in SS patients was 27.83 and in the homozygous Arab-Indian haplotype it was still higher (30.40%), while in AS patients the %HbF was only 1.20. The high %Ggamma chain (71.81) in the Arab-Indian homozygous haplotype was concomitant with the presence of an Xmn I site in both chromosomes. The presence of the Arab-Indian haplotype as the predominant haplotype might be suggestive of a gene flow to/from Saudi Arabia or India. More haplotype investigations of a normal population can clarify the high incidence of Bantu A2 haplotype in our population.

  2. Effects of intra-gastric beta-casomorphin-7 on somatostatin and gastrin gene expression in rat gastric mucosa

    Institute of Scientific and Technical Information of China (English)

    Ya-Feng Zong; Wei-Hua Chen; Yuan-Shu Zhang; Si-Xiang Zou

    2007-01-01

    AIM: To investigate the in vivo effect of beta-casomorphin-7on the regulation of gastric somatostatin and gastrin messenger RNA in rat gastric mucosa.METHODS: Somatostatin and gastrin mRNA were quantified by RT-PCR and in situ hybridization (ISH)in 24 rats. The rats were divided into three treatment groups: basal diet + physiological saline (n = 8), basal diet + beta-casomorphin-7 (7.5 × 10-7 mol) (n = 8),and basal diet + poly-Gly-7 (containing equal mol of N with 7.5 x 10-7 mol beta-casomorphin-7) (n = 8).After oral administration for 30 days, rats were killed by exsanguinations.RESULTS: After intra-gastric administration of betacasomorphin-7 for 30 d, gastrin mRNA increased by 52.8% (P < 0.05, n = 8), and somatostatin mRNA levels decreased by 30.7% compared with the controls (P <0.01, n = 8). No significant differences in the expression of the two genes were observed in the poly-Gly-treated group, although gastrin mRNA expression was elevated by 35.6% as against the control group (P = 0.15, n =8). The long-term oral administration of a casomorphin solution significantly decreased the even gray of D-cells,but did not lower the number of D-cells both in the antrum and fundus. Interestingly, the number of G-cells increased in the antrum and fundus, but its average density was augmented only in the antrum.CONCLUSION: Beta-casomorphin-7 is capable of modulating gene expression of the regulatory peptides from G and D cells. Data from in situ hybridization studies indicate that beta-casomorphin-7 affects gastrin gene expression indirectly by means of the paracrine action of somatostatin, and depends on its intrinsic molecular function.

  3. Human-specific SNP in obesity genes, adrenergic receptor beta2 (ADRB2, Beta3 (ADRB3, and PPAR γ2 (PPARG, during primate evolution.

    Directory of Open Access Journals (Sweden)

    Akiko Takenaka

    Full Text Available UNLABELLED: Adrenergic-receptor beta2 (ADRB2 and beta3 (ADRB3 are obesity genes that play a key role in the regulation of energy balance by increasing lipolysis and thermogenesis. The Glu27 allele in ADRB2 and the Arg64 allele in ADRB3 are associated with abdominal obesity and early onset of non-insulin-dependent diabetes mellitus (NIDDM in many ethnic groups. Peroxisome proliferator-activated receptor γ (PPARG is required for adipocyte differentiation. Pro12Ala mutation decreases PPARG activity and resistance to NIDDM. In humans, energy-expense alleles, Gln27 in ADRB2 and Trp64 in ADRB3, are at higher frequencies than Glu27 and Arg64, respectively, but Ala12 in PPARG is at lower frequency than Pro12. Adaptation of humans for lipolysis, thermogenesis, and reduction of fat accumulation could be considered by examining which alleles in these genes are dominant in non-human primates (NHP. All NHP (P. troglodytes, G. gorilla, P. pygmaeus, H. agilis and macaques had energy-thrifty alleles, Gly16 and Glu27 in ADRB2, and Arg64 in ADRB3, but did not have energy-expense alleles, Arg16, Gln27 and Trp64 alleles. In PPARG gene, all NHP had large adipocyte accumulating type, the Pro12 allele. CONCLUSIONS: These results indicate that a tendency to produce much more heat through the energy-expense alleles developed only in humans, who left tropical rainforests for savanna and developed new features in their heat-regulation systems, such as reduction of body hair and increased evaporation of water, and might have helped the protection of entrails from cold at night, especially in glacial periods.

  4. Human-Specific SNP in Obesity Genes, Adrenergic Receptor Beta2 (ADRB2), Beta3 (ADRB3), and PPAR γ2 (PPARG), during Primate Evolution

    Science.gov (United States)

    Takenaka, Akiko; Nakamura, Shin; Mitsunaga, Fusako; Inoue-Murayama, Miho; Udono, Toshifumi; Suryobroto, Bambang

    2012-01-01

    Adrenergic-receptor beta2 (ADRB2) and beta3 (ADRB3) are obesity genes that play a key role in the regulation of energy balance by increasing lipolysis and thermogenesis. The Glu27 allele in ADRB2 and the Arg64 allele in ADRB3 are associated with abdominal obesity and early onset of non-insulin-dependent diabetes mellitus (NIDDM) in many ethnic groups. Peroxisome proliferator-activated receptor γ (PPARG) is required for adipocyte differentiation. Pro12Ala mutation decreases PPARG activity and resistance to NIDDM. In humans, energy-expense alleles, Gln27 in ADRB2 and Trp64 in ADRB3, are at higher frequencies than Glu27 and Arg64, respectively, but Ala12 in PPARG is at lower frequency than Pro12. Adaptation of humans for lipolysis, thermogenesis, and reduction of fat accumulation could be considered by examining which alleles in these genes are dominant in non-human primates (NHP). All NHP (P. troglodytes, G. gorilla, P. pygmaeus, H. agilis and macaques) had energy-thrifty alleles, Gly16 and Glu27 in ADRB2, and Arg64 in ADRB3, but did not have energy-expense alleles, Arg16, Gln27 and Trp64 alleles. In PPARG gene, all NHP had large adipocyte accumulating type, the Pro12 allele. Conclusions These results indicate that a tendency to produce much more heat through the energy-expense alleles developed only in humans, who left tropical rainforests for savanna and developed new features in their heat-regulation systems, such as reduction of body hair and increased evaporation of water, and might have helped the protection of entrails from cold at night, especially in glacial periods. PMID:22937051

  5. Interleukin (IL)1beta, IL-1alpha, and IL-1 receptor antagonist gene polymorphisms in patients with temporal lobe epilepsy.

    Science.gov (United States)

    Kanemoto, K; Kawasaki, J; Miyamoto, T; Obayashi, H; Nishimura, M

    2000-05-01

    Proinflammatory cytokines, including interleukin (IL)-1beta, are known to modulate effects of neurotoxic neurotransmitters discharged during excitation or inflammation in the central nervous system (CNS). They also regulate development of glial scars at sites of CNS injury. To elucidate a genetic predisposition of temporal lobe epilepsy with hippocampal sclerosis (TLE-HS+), we studied polymorphisms in the IL-1beta, IL-1alpha, and IL-1 receptor antagonist (IL-1RA) genes in 50 patients with TLE-HS+ and in 112 controls. Fifty-three patients who had TLE without HS were also examined (TLE-HS-) as disease controls. The distribution of the biallelic polymorphism in the promoter region at position -511 of the IL-1beta gene (IL-1B-511) was significantly different both between TLE-HS+ patients and controls and between TLE-HS+ and TLE-HS- patients. The differences were due to overrepresentation of the homozygotes for IL-1B-511*2, which is suggested to be a high producer of IL-1beta, in TLE-HS+ patients compared with both controls and TLE-HS- patients. In contrast, there was no difference between TLE-HS- patients and controls. Our data suggest that, in the homozygotes for IL-IB-511*2, minor events in development such as febrile convulsions could set up a cascade leading to HS.

  6. Occurrence of bacteria producing broad-spectrum beta-lactamases and qnr genes in hospital and urban wastewater samples.

    Science.gov (United States)

    Röderová, Magdaléna; Sedláková, Miroslava Htoutou; Pudová, Vendula; Hricová, Kristýna; Silová, Romana; Imwensi, Peter Eghonghon Odion; Bardoň, Jan; Kolář, Milan

    2016-04-01

    The aims were to investigate the level of antibiotic-resistant bacteria in hospital and urban wastewater and to determine the similarity of isolates obtained from wastewater and hospitalized patients. Wastewater samples were collected in September 2013 and 2014. After identification using MALDI-TOF MS, beta-lactamase production was determined by relevant phenotypic tests. Genes responsible for the production of single beta-lactamase groups and Qnr proteins were established. The epidemiological relationship of the isolates from wastewater and hospitalized patients was determined by PFGE. A total of 51 isolates of enterobacteria were obtained. Overall, 45.1% of them produced broad-spectrum beta-lactamases. Genes encoding TEM, SHV, CTX-M, CIT, DHA and EBC types of enzymes and Qnr proteins were detected. No broad-spectrum beta-lactamase production was confirmed in the urban wastewater treatment plant. The most important finding was the detection of two identical isolates of K. pneumoniae in 2013, one from a patient's urinary catheter and the other from a wastewater sample.

  7. Sequential mutations in the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor beta-subunit genes are necessary for the complete conversion to growth autonomy mediated by a truncated beta C subunit.

    Science.gov (United States)

    Hannemann, J; Hara, T; Kawai, M; Miyajima, A; Ostertag, W; Stocking, C

    1995-05-01

    An amino-terminally truncated beta C receptor (beta C-R) subunit of the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor complex mediates factor-independent and tumorigenic growth in two spontaneous mutants of a promyelocytic cell line. The constitutive activation of the JAK2 protein kinase in these mutants confirms that signaling occurs through the truncated receptor protein. Noteworthily, in addition to a 10-kb deletion in the beta C-R subunit gene encoding the truncated receptor, several secondary and independent mutations that result in the deletion or functional inactivation of the allelic beta C-R subunit and the closely related beta IL3-R subunit genes were observed in both mutants, suggesting that such mutations are necessary for the full oncogenic penetrance of the truncated beta C-R subunit. Reversion of these mutations by the expression of the wild-type beta C-R in the two mutants resulted in a fivefold decrease in cloning efficiency of the mutants in the absence of IL3, confirming a functional interaction between the wild-type and truncated proteins. Furthermore, expression of the truncated beta C-R subunit in factor-dependent myeloid cells did not immediately render the cells autonomous but increased the spontaneous frequency to factor-independent growth by 4 orders of magnitude. Implications for both leukemogenic progression and receptor-subunit interaction and signaling are discussed.

  8. Missense mutations in the MEFV gene are associated with fibromyalgia syndrome and correlate with elevated IL-1beta plasma levels.

    Directory of Open Access Journals (Sweden)

    Jinong Feng

    Full Text Available BACKGROUND: Fibromyalgia syndrome (FMS, a common, chronic, widespread musculoskeletal pain disorder found in 2% of the general population and with a preponderance of 85% in females, has both genetic and environmental contributions. Patients and their parents have high plasma levels of the chemokines MCP-1 and eotaxin, providing evidence for both a genetic and an immunological/inflammatory origin for the syndrome (Zhang et al., 2008, Exp. Biol. Med. 233: 1171-1180. METHODS AND FINDINGS: In a search for a candidate gene affecting inflammatory pathways, among five screened in our patient samples (100 probands with FMS and their parents, we found 10 rare and one common alleles for MEFV, a gene in which various compound heterozygous mutations lead to Familial Mediterranean Fever (FMF. A total of 2.63 megabases of genomic sequence of the MEFV gene were scanned by direct sequencing. The collection of rare missense mutations (all heterozygotes and tested in the aggregate had a significant elevated frequency of transmission to affecteds (p = 0.0085, one-sided, exact binomial test. Our data provide evidence that rare missense variants of the MEFV gene are, collectively, associated with risk of FMS and are present in a subset of 15% of FMS patients. This subset had, on average, high levels of plasma IL-1beta (p = 0.019 compared to FMS patients without rare variants, unaffected family members with or without rare variants, and unrelated controls of unknown genotype. IL-1beta is a cytokine associated with the function of the MEFV gene and thought to be responsible for its symptoms of fever and muscle aches. CONCLUSIONS: Since misregulation of IL-1beta expression has been predicted for patients with mutations in the MEFV gene, we conclude that patients heterozygous for rare missense variants of this gene may be predisposed to FMS, possibly triggered by environmental factors.

  9. Cloning and expression in Escherichia coli of a new gene of Schistosoma japonicum encoding casein kinase Ⅱ beta subunit

    Institute of Scientific and Technical Information of China (English)

    彭寨玉; 余新炳; 吴忠道; 徐劲; 吴德; 李孜

    2004-01-01

    Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E.coli).Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3'RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E.coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24.921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E.coli JM109.The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunit were firstly sequenced, cloned, and expressed in E.coli.

  10. Sequential changes in chromatin structure during transcriptional activation in the beta globin LCR and its target gene.

    Science.gov (United States)

    Kim, Kihoon; Kim, AeRi

    2010-09-01

    Chromatin structure is modulated during transcriptional activation. The changes include the association of transcriptional activators, formation of hypersensitive sites and covalent modifications of histones. To understand the order of the various changes accompanying transcriptional activation, we analyzed the mouse beta globin gene, which is transcriptionally inducible in erythroid MEL cells over a time course of HMBA treatment. Transcription of the globin genes requires the locus control region (LCR) consisting of several hypersensitive sites (HSs). Erythroid specific transcriptional activators such as NF-E2, GATA-1, TAL1 and EKLF were associated with the LCR in the uninduced state before transcriptional activation. The HSs of the LCR were formed in this state as revealed by high sensitivity to DNase I and MNase attack. However the binding of transcriptional activators and the depletion of histones were observed in the promoter of the beta globin gene only after transcriptional activation. In addition, various covalent histone modifications were sequentially detected in lysine residues of histone H3 during the activation. Acetylation of K9, K36 and K27 was notable in both LCR HSs and gene after induction but before transcriptional initiation. Inactive histone marks such as K9me2, K36me2 and K27me2 were removed coincident with transcriptional initiation in the gene region. Taken together, these results indicate that LCR has a substantially active structure in the uninduced state while transcriptional activation serially adds active marks, including histone modifications, and removes inactive marks in the target gene of the LCR.

  11. Concordance of a point mutation 5' to the A gamma-globin gene with A gamma beta + hereditary persistence of fetal hemoglobin in Greeks.

    Science.gov (United States)

    Waber, P G; Bender, M A; Gelinas, R E; Kattamis, C; Karaklis, A; Sofroniadou, K; Stamatoyannopoulos, G; Collins, F S; Forget, B G; Kazazian, H H

    1986-02-01

    In the Greek A gamma beta + type of hereditary persistence of fetal hemoglobin (HPFH), adult heterozygotes produce about 20% fetal hemoglobin (HbF), which is predominantly of the A gamma chain variety. The affected beta-globin gene cluster produces near normal amounts of beta-like globin, but in a A gamma to beta ratio of 20:80 instead of 0.5:99.5. Gelinas et al and Collins et al have shown a G to A change 117 nucleotides 5' to the A gamma gene in two Greeks with A gamma beta + HPFH. To demonstrate that this change is not a neutral polymorphism, we carried out hybridization with oligonucleotide probes (19mers) specific for the normal and the mutant sequences. While normal probe identified the A gamma fragment in genomic DNA of all subjects studied, mutant probe was positive only in Greeks with A gamma beta + HPFH. In sum, 108 beta-globin gene clusters of individuals without HPFH were negative when tested with mutant probe, but all 11 affected individuals of six families with Greek A gamma beta + HPFH (two previously sequenced and four new families) were positive with mutant probe. These data support the conclusion that the -117 mutation is causative of A gamma beta + HPFH in Greeks.

  12. Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase and method of inactivating ribulose-1,5-bisphosphatase carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase activity

    Science.gov (United States)

    Houtz, Robert L.

    1999-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

  13. A second glutamine synthetase gene with expression in the gills of the gulf toadfish (opsanus beta)

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Patrick J.; Mayer, Gregory D.; Medina, Monica; Bernstein, Matthew L.; Barimo, John F.; Mommsen, Thomas P.

    2003-05-08

    Enzyme and molecular biology approaches were used to more completely characterize the expression of the nitrogen metabolism enzyme glutamine synthetase [GSase; L-glutamate: ammonia ligase (ADP-forming), E.C. 6.3.1.2] in a variety of tissues of the gulf toadfish (Opsanus beta) subjected to unconfined (ammonotelic) and confined (ureotelic) conditions. Enzymological results demonstrate that while weight-specific GSase activities rank in the order of brain > liver > stomach {approx} kidney > intestine > gill> heart/spleen > muscle, when tissue mass is used to calculate a glutamine synthetic potential, the liver has the greatest, followed by muscle > stomach and intestine with minor contributions from the remaining tissues. Additionally, during confinement stress, GSase activity only increases significantly in liver (5-fold) and muscle (2-fold), tissues which previously showed significant expression of the other enzymes of urea synthesis. RT PCR and RACE PCR revealed the presence of a second GSas e cDNA from gill tissue that appears to share relatively low nucleotide and amino acid sequence similarity ({approx}73 percent) with the original GSase cloned from liver, and furthermore lacks a mitochondrial leader targeting sequence. RT PCR and restriction digestion experiments demonstrated that mRNA from the original ''liver'' GSase is expressed in all tissues examined (liver, gill, stomach, intestine, kidney, brain and muscle), whereas the new ''gill'' form shows expression primarily in the gill. Enzyme activities of gill GSase also exhibit a different subcellular compartmentation with apparent exclusive expression in the soluble compartment, whereas other tissues expressing the ''liver'' form show both cytoplasmic and mitochondrial activities. Finally, phylogenetic analysis of a number of GSases demonstrates that the toadfish gill GSase has a greater affinity for a clade that includes the Xenopus GSase genes and

  14. Genes involved in the transforming growth factor beta signalling pathway and the risk of intracranial aneurysms

    NARCIS (Netherlands)

    Ruigrok, Y. M.; Tan, S.; Medic, J.; Rinkel, G. J. E.; Wijmenga, C.

    2008-01-01

    Background and purpose: The 19q13.3 locus for intracranial aneurysms (IA) partly overlaps with the 19q13 locus for abdominal aortic aneurysms (AAA). A common genetic risk factor located in this locus for the two aneurysm types seems plausible. The transforming growth factor beta (TGF-beta) signallin

  15. 164Ile allele in the beta2-Adrenergic receptor gene is associated with risk of elevated blood pressure in women. The Copenhagen City Heart Study

    DEFF Research Database (Denmark)

    Sethi, Amar A; Tybjaerg-Hansen, Anne; Jensen, Gorm B;

    2005-01-01

    Since beta2-adrenergic receptors are important regulators of blood pressure, genetic variation in this receptor could explain risk of elevated blood pressure in selected individuals. We tested the hypothesis that Gly16Arg, Gln27Glu, and Thr164Ile in the beta2-adrenergic receptor gene associated w...

  16. Chemical Genetics of Acetyl-CoA Carboxylases

    Directory of Open Access Journals (Sweden)

    Xuyu Zu

    2013-01-01

    Full Text Available Chemical genetic studies on acetyl-CoA carboxylases (ACCs, rate-limiting enzymes in long chain fatty acid biosynthesis, have greatly advanced the understanding of their biochemistry and molecular biology and promoted the use of ACCs as targets for herbicides in agriculture and for development of drugs for diabetes, obesity and cancers. In mammals, ACCs have both biotin carboxylase (BC and carboxyltransferase (CT activity, catalyzing carboxylation of acetyl-CoA to malonyl-CoA. Several classes of small chemicals modulate ACC activity, including cellular metabolites, natural compounds, and chemically synthesized products. This article reviews chemical genetic studies of ACCs and the use of ACCs for targeted therapy of cancers.

  17. Analysis of beta, gamma, and delta T-cell receptor genes in mycosis fungoides and Sezary syndrome.

    Science.gov (United States)

    Whittaker, S J; Smith, N P; Jones, R R; Luzzatto, L

    1991-10-01

    The authors have analyzed the configuration of immunoglobulin (Ig) and beta, gamma and delta T-cell receptor (TCR) genes in DNA extracted from skin, lymph nodes, and peripheral blood mononuclear cells obtained from 41 patients with mycosis fungoides (MF), 14 patients with Sezary syndrome, and 13 patients with benign inflammatory dermatoses. No discrete rearranged bands (DRB) were detected in patients with inflammatory dermatoses. In tissue DNA from 19 patients with MF DRB were detected with beta and gamma, but not delta TCR probes. Only one patient with MF had a rearrangement of gamma and delta with germ line beta TCR genes. In 13 patients multiple biopsies were analyzed and DRB, when present, were identical in different lesions from individual patients. In three patients analysis of DNA from dermatopathic lymph nodes did not reveal DRB. Analysis of peripheral blood DNA from 24 patients revealed a discrete rearrangement of the gamma TCR gene in four patients and both beta and gamma genes in four additional patients. In MF DRB were detected more frequently with advancing stage of disease in tissues (P less than 0.01) but not in peripheral blood (P equals 0.36). Of 14 patients with Sezary syndrome, eight had DRB in peripheral blood DNA with both beta and gamma probes and in three of these patients identical DRB were also detected in DNA from skin biopsy samples. In contrast, DRB were not detected in the peripheral blood of the other six patients. In both MF and Sezary syndrome there was no restricted usage of particular V gamma genes. These results indicate that in MF (1) T-cell clones can be detected in skin biopsy specimens from the majority of patients with early stage disease, (2) gamma delta T-cell clones are only rarely found, and (3) TCR gene analysis can detect T-cell clones in the peripheral blood with a greater degree of specificity than conventional light microscopic study. In Sezary syndrome these studies also suggest that a subset of patients have a

  18. PARP-1 and YY1 are important novel regulators of CXCL12 gene transcription in rat pancreatic beta cells.

    Directory of Open Access Journals (Sweden)

    Jelena Marković

    Full Text Available Despite significant progress, the molecular mechanisms responsible for pancreatic beta cell depletion and development of diabetes remain poorly defined. At present, there is no preventive measure against diabetes. The positive impact of CXCL12 expression on the pancreatic beta cell prosurvival phenotype initiated this study. Our aim was to provide novel insight into the regulation of rat CXCL12 gene (Cxcl12 transcription. The roles of poly(ADP-ribose polymerase-1 (PARP-1 and transcription factor Yin Yang 1 (YY1 in Cxcl12 transcription were studied by examining their in vitro and in vivo binding affinities for the Cxcl12 promoter in a pancreatic beta cell line by the electrophoretic mobility shift assay and chromatin immunoprecipitation. The regulatory activities of PARP-1 and YY1 were assessed in transfection experiments using a reporter vector with a Cxcl12 promoter sequence driving luciferase gene expression. Experimental evidence for PARP-1 and YY1 revealed their trans-acting potential, wherein PARP-1 displayed an inhibitory, and YY1 a strong activating effect on Cxcl12 transcription. Streptozotocin (STZ-induced general toxicity in pancreatic beta cells was followed by changes in Cxcl12 promoter regulation. PARP-1 binding to the Cxcl12 promoter during basal and in STZ-compromised conditions led us to conclude that PARP-1 regulates constitutive Cxcl12 expression. During the early stage of oxidative stress, YY1 exhibited less affinity toward the Cxcl12 promoter while PARP-1 displayed strong binding. These interactions were accompanied by Cxcl12 downregulation. In the later stages of oxidative stress and intensive pancreatic beta cell injury, YY1 was highly expressed and firmly bound to Cxcl12 promoter in contrast to PARP-1. These interactions resulted in higher Cxcl12 expression. The observed ability of PARP-1 to downregulate, and of YY1 to upregulate Cxcl12 promoter activity anticipates corresponding effects in the natural context where the

  19. Ribulose diphosphate carboxylase of the cyanobacterium Spirulina platensis

    Energy Technology Data Exchange (ETDEWEB)

    Terekhova, I.V.; Chernyad' ev, I.I.; Doman, N.G.

    1986-11-20

    The ribulose diphosphate (RDP) carboxylase activity of the cyanobacterium Spirulina platensis is represented by two peaks when a cell homogenate is centrifuged in a sucrose density gradient. In the case of differential centrifugation (40,000 g, 1 h), the activity of the enzyme was distributed between the supernatant liquid (soluble form) and the precipitate (carboxysomal form). From the soluble fraction, in which 80-95% of the total activity of the enzyme is concentrated, electrophoretically homogeneous RDP carboxylase was isolated by precipitation with ammonium sulfate and centrifugation in a sucrose density gradient. The purified enzyme possessed greater electrophoretic mobility in comparison with the RDP carboxylase of beans Vicia faba. The molecular weight of the enzyme, determined by gel filtration, was 450,000. The enzyme consists of monotypic subunits with a molecular weight of 53,000. The small subunits were not detected in electrophoresis in polyacrylamide gel in the presence of SDS after fixation and staining of the gels by various methods.

  20. The chicken beta 2-microglobulin gene is located on a non-major histocompatibility complex microchromosome: a small, G+C-rich gene with X and Y boxes in the promoter

    DEFF Research Database (Denmark)

    Riegert, P; Andersen, R; Bumstead, N

    1996-01-01

    beta 2-Microglobulin is an essential subunit of major histocompatibility complex (Mhc) class I molecules, which present antigenic peptides to T lymphocytes. We sequenced a number of cDNAs and two genomic clones corresponding to chicken beta 2-microglobulin. The chicken beta 2-microglobulin gene has...... a similar genomic organization but smaller introns and higher G+C content than mammalian beta 2-microglobulin genes. The promoter region is particularly G+C-rich and contains, in addition to interferon regulatory elements, potential S/W, X, and Y boxes that were originally described for mammalian class II...

  1. Mutations in the lysosomal [beta]-galactosidase gene that cause the adult form of GMI gangliosidosis

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, S.; Rafi, M.A.; Wenger, D.A. (Thomas Jefferson Univ., Philadelphia, PA (United States))

    1994-06-01

    Three adult patients with acid-galactosidase deficiency/GM1 gangliosidosis who were from two unrelated families of Scandinavian descent were found to share a common point mutation in the coding region of the corresponding gene. The patients share common clinical features, including early dysarthria, mild ataxia, and bone abnormalities. When cDNA from the two patients in family 1 was PCR amplified and sequenced, most (39/41) of the clones showed a C-to-T transition (C[yields]T) at nucleotide 245 (counting from the initiation codon). This mutation changes the codon for the Thr(ACG) to Met(ATG). Mutant and normal sequences were also found in that position in genomic DNA, indicating the presence of another mutant allele. Genomic DNA from the patient in family 2 revealed the same point mutation in one allele. It was determined that in each family only the father carried the C[yields]T mutation. Expression studies showed that this mutation produced 3%-4% of [beta]-galactosidase activity, confirming its deleterious effects. The cDNA clones from the patients in family 1 that did not contain the C[yields]T revealed a 20-bp insertion of intronic sequence between nucleotides 75 and 76, the location of the first intron. Further analysis showed the insertion of a T near the 5[prime] splice donor site which led to the use of a cryptic splice site. It appears that the C[yields]T mutation results in enough functional enzyme to produce a mild adult form of the disease, even in the presence of a second mutation that likely produces nonfunctional enzyme. 31 refs., 7 figs., 1 tab.

  2. Exclusion of the phosphatidylinositol-specific phospholipase C beta 3 (PLC beta 3) gene as candidate for the multiple endocrine neoplasia type 1 (MEN 1) gene

    NARCIS (Netherlands)

    de Wit, M J; Landsvater, R M; Sinke, R J; Geurts van Kessel, A; Lips, C J; Höppener, J W

    1997-01-01

    Multiple endocrine neoplasia type 1 (MEN 1) is inherited as an autosomal dominant disorder, characterized by hyperplasia and neoplasia in several endocrine organs. The MEN 1 gene, which is most probably a tumor suppressor gene, has been localized to a 900-kb region on chromosome 11q13. The human pho

  3. Cloning and characterization of genes encoding alpha and beta subunits of glutamate-gated chloride channel protein in Cylicocyclus nassatus.

    Science.gov (United States)

    Tandon, Ritesh; LePage, Keith T; Kaplan, Ray M

    2006-11-01

    The invertebrate glutamate-gated chloride channels (GluCls) are receptor molecules and targets for the avermectin-milbemycin (AM) group of anthelmintics. Mutations in GluCls are associated with ivermectin resistance in the soil dwelling nematode Caenorhabditis elegans and the parasitic nematode Cooperia oncophora. In this study, full-length cDNAs encoding alpha and beta subunits of GluCl were cloned and sequenced in Cylicocyclus nassatus, a common and important cyathostomin nematode parasite of horses. Both genes possess the sequence characteristics typical of GluCls, and phylogenetic analysis confirms that these genes are evolutionarily closely related to GluCls of other nematodes and flies. Complete coding sequences of C. nassatus GluCl-alpha and GluCl-beta were subcloned into pTL1 mammalian expression vector, and proteins were expressed in COS-7 cells. Ivermectin-binding characteristics were determined by incubating COS-7 cell membranes expressing C. nassatus GluCl-alpha and GluCl-beta proteins with [(3)H]ivermectin. In competitive binding experiments, fitting the data to a one site competition model, C. nassatus GluCl-alpha was found to bind [(3)H]ivermectin with a high amount of displaceable binding (IC(50)=208 pM). Compared to the mock-transfected COS-7 cells, the means of [(3)H]ivermectin binding were significantly different for C. nassatus GluCl-alpha and the Haemonchus contortus GluCl (HcGluCla) (p=0.018 and 0.023, respectively) but not for C. nassatus GluCl-beta (p=0.370). This is the first report of orthologs of GluCl genes and in vitro expression of an ivermectin-binding protein in a cyathostomin species. These data suggest the likelihood of a similar mechanism of action of AM drugs in these parasites, and suggest that mechanisms of resistance may also be similar.

  4. Prevalence PER and VEB beta-lactamase Genes among Acinetobacter baumannii Isolated from Patients in Tehran by PCR

    Directory of Open Access Journals (Sweden)

    Abbas Nazari Monazam

    2014-12-01

    Full Text Available Background and Aim: According to numerous reports of infections caused by spectrum beta lactamases (ESBLs producing Acinetobacter baumannii in our country in recent years, this study was performed to define the antibiotic susceptibility patterns and detect the prevalence PER  and VEB beta-lactamase genes among  A.baumannii isolated from patients in Tehran by PCR. Materials and Methods: 100 A.baumannii clinical isolates collected from various hospitals in Tehran during a year, using special culture media and biochemical tests were identified. The antibiograms of the isolates against 11 antibiotics by disk diffusion method (Disk diffusion according to Clinical and Laboratory Standards Institute (CLSI guidelines was performed. Then minimum inhibitory concentrations (MIC was determined for cefepime and ceftazidime and for the identification of ESBL-producing strains was used in combination disk method, and finally to assess the prevalence of PER and VEB beta-lactamase genes using specific primers PCR was performed. Results: Antibiograms results showed that the greatest resistance to the antibiotics amikacin, cefepime, and less resistance to polymyxin B were obtained. Rates of multi-drug resistant strains of about 70% was achieved. Of the isolates studied, the MIC of ceftazidime in 84% and for cefepime in 91% were ≥ 128 μg/ml. The results of the combined-disk test demonstrated that 20% of samples were ESBL positive. The PCR results showed that 47% and 32% of our isolates had PER and VEB genes respectively. Conclusions: Regarding to existence of PER and VEB genes in this bacterium and possibility of transformation of these genes to the other bacteria, reconsideration in antibiotics consumption patterns and more attention to nosocomial infections control criteria are inevitable.

  5. Mechanism of fiber carcinogenesis: from reactive radical species to silencing of the beta igH3 gene.

    Science.gov (United States)

    Hei, Tom K; Xu, An; Huang, Sarah X; Zhao, Yongliang

    2006-11-01

    Although the U.S. Environmental Protection Agency has restricted the industrial use of regulated forms of asbestos in the United States since the early 1970s, environmental exposure to asbestos remains a health concern in the United States and is a significant health issue among developing countries. Exposure to asbestos is associated with chronic pulmonary diseases and cancer of the lung, pleura, and peritoneum. The mechanism of fiber carcinogenesis is far from clear and is likely to be complex, depending on fiber dimensions, surface properties, and physical durability. The induction of reactive oxygen and nitrogen species upon phagocytosis of fibers plays an important role in fiber genotoxicity. The beta igH3, a secreted protein induced by the transforming growth factor-beta and essential for cell adhesion, is downregulated in asbestos-induced tumorigenic human bronchial epithelial cells. Ectopic expression of the beta igH3 gene abrogates the tumorigenic phenotype and suggests that the gene plays a causal role in fiber carcinogenesis. A better understanding of the carcinogenic mechanism of asbestos and other mineral fibers will provide useful information on interventional and preventive measures for asbestos-mediated diseases such as human pleural and peritoneal mesotheliomas.

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

    Energy Technology Data Exchange (ETDEWEB)

    Albertella, M.R.; Jones, H.; Thomson, W. [Oxford Univ. (United Kingdom)] [and others

    1996-09-01

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

  7. Structural organization of the human and mouse laminin beta2 chain genes, and alternative splicing at the 5' end of the human transcript

    DEFF Research Database (Denmark)

    Durkin, M E; Gautam, M; Loechel, F;

    1996-01-01

    We have determined the structural organization of the human and mouse genes that encode the laminin beta2 chain (s-laminin), an essential component of the basement membranes of the neuromuscular synapse and the kidney glomerulus. The human and mouse genes have a nearly identical exon......-intron organization and are the smallest laminin chain genes characterized to date, due to the unusually small size of their introns. The laminin beta2 chain genes of both species consist of 33 exons that span...

  8. Associations of a polymorphic AP-2 binding site in the 5'-flanking region of the bovine beta-lactoglobulin gene with milk proteins.

    Science.gov (United States)

    Kuss, A W; Gogol, J; Geidermann, H

    2003-06-01

    Studies on a polymorphic position (R10) in an Activator-Protein-2 (AP-2) binding site of the bovine beta-Lactoglobulin (beta-Lg) gene promoter region and quantitative traits of individual milk proteins were based on material from 79 German Holstein Friesian (HF) and 61 Simmental (Sm) cows. At least four milk samples per cow were analyzed with alkaline Urea-PAGE in combination with densitometry for quantification of individual milk proteins. The two alleles of the R10 single nucleotide polymorphism (SNP) carry either G or C in position -435 bp of the beta-Lg promoter region. G- and C-alleles were found in Sm with nearly equal frequencies, while in HF the C-allele frequency was higher (0.73) than that of the G-allele. In both breeds, the R10 G-homozygotes had higher (P beta-Lg secreted per day and proportion of beta-Lg in milk protein compared with the C-homozygotes. A similar association was found for alpha-lactalbumin, whereas the relative proportions and daily secreted amounts of caseins (alphaS1, beta, kappa) showed lower values in beta-Lg R10 G-homozygotes. A positive association (P beta-Lg locus to a candidate gene for this trait. The association between the SNP in the AP-2 binding site of the beta-Lg gene and its gene product can be explained as the result of differences in protein binding activity, and, therefore, allele specific differences in gene expression. Thus, our study clearly links a DNA polymorphism of molecular function very closely with in vivo expression parameters of the same locus.

  9. Candidate gene association study in type 2 diabetes indicates a role for genes involved in beta-cell function as well as insulin action.

    Directory of Open Access Journals (Sweden)

    Inês Barroso

    2003-10-01

    Full Text Available Type 2 diabetes is an increasingly common, serious metabolic disorder with a substantial inherited component. It is characterised by defects in both insulin secretion and action. Progress in identification of specific genetic variants predisposing to the disease has been limited. To complement ongoing positional cloning efforts, we have undertaken a large-scale candidate gene association study. We examined 152 SNPs in 71 candidate genes for association with diabetes status and related phenotypes in 2,134 Caucasians in a case-control study and an independent quantitative trait (QT cohort in the United Kingdom. Polymorphisms in five of 15 genes (33% encoding molecules known to primarily influence pancreatic beta-cell function-ABCC8 (sulphonylurea receptor, KCNJ11 (KIR6.2, SLC2A2 (GLUT2, HNF4A (HNF4alpha, and INS (insulin-significantly altered disease risk, and in three genes, the risk allele, haplotype, or both had a biologically consistent effect on a relevant physiological trait in the QT study. We examined 35 genes predicted to have their major influence on insulin action, and three (9%-INSR, PIK3R1, and SOS1-showed significant associations with diabetes. These results confirm the genetic complexity of Type 2 diabetes and provide evidence that common variants in genes influencing pancreatic beta-cell function may make a significant contribution to the inherited component of this disease. This study additionally demonstrates that the systematic examination of panels of biological candidate genes in large, well-characterised populations can be an effective complement to positional cloning approaches. The absence of large single-gene effects and the detection of multiple small effects accentuate the need for the study of larger populations in order to reliably identify the size of effect we now expect for complex diseases.

  10. Mutations in the latent TGF-beta binding protein 3 (LTBP3) gene cause brachyolmia with amelogenesis imperfecta.

    Science.gov (United States)

    Huckert, Mathilde; Stoetzel, Corinne; Morkmued, Supawich; Laugel-Haushalter, Virginie; Geoffroy, Véronique; Muller, Jean; Clauss, François; Prasad, Megana K; Obry, Frédéric; Raymond, Jean Louis; Switala, Marzena; Alembik, Yves; Soskin, Sylvie; Mathieu, Eric; Hemmerlé, Joseph; Weickert, Jean-Luc; Dabovic, Branka Brukner; Rifkin, Daniel B; Dheedene, Annelies; Boudin, Eveline; Caluseriu, Oana; Cholette, Marie-Claude; Mcleod, Ross; Antequera, Reynaldo; Gellé, Marie-Paule; Coeuriot, Jean-Louis; Jacquelin, Louis-Frédéric; Bailleul-Forestier, Isabelle; Manière, Marie-Cécile; Van Hul, Wim; Bertola, Debora; Dollé, Pascal; Verloes, Alain; Mortier, Geert; Dollfus, Hélène; Bloch-Zupan, Agnès

    2015-06-01

    Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on four families, three of them consanguineous, with an identical phenotype, characterized by significant short stature with brachyolmia and hypoplastic amelogenesis imperfecta (AI) with almost absent enamel. This phenotype was first described in 1996 by Verloes et al. as an autosomal recessive form of brachyolmia associated with AI. Whole-exome sequencing resulted in the identification of recessive hypomorphic mutations including deletion, nonsense and splice mutations, in the LTBP3 gene, which is involved in the TGF-beta signaling pathway. We further investigated gene expression during mouse development and tooth formation. Differentiated ameloblasts synthesizing enamel matrix proteins and odontoblasts expressed the gene. Study of an available knockout mouse model showed that the mutant mice displayed very thin to absent enamel in both incisors and molars, hereby recapitulating the AI phenotype in the human disorder.

  11. [The cloning and expression of the gene for beta-galactosidase from Candida pseudotropicalis yeasts in Saccharomyces cerevisiae cells].

    Science.gov (United States)

    Tretiak, K A; Zakal'skiĭ, A E; Gudz', S P

    1998-01-01

    The gene of beta-galactosidase of lactose-assimilating yeast Candida pseudotropicalis was cloned in pG2 and pBG2-3 hybrid shuttle vectors and expressed in Saccharomyces cerevisiae laboratory strains under the control of own promoter. The plasmids were able to replicate autonomously with relative stability in transformants of baker's yeasts. The availability of glucose or lactose in the medium influenced the recombinant plasmid stability and the expression of the cloned gene. A number of experiments have shown that the LAC+ phenotype in pG2-transformed Saccharomyces cerevisiae was due to the expression of the Candida pseudotropicalis lactose permease gene that is probably located in SaIG1/XhoI DNA fragment about 4.3 kb long. Southern hybridization experiments showed that LAC(+)-transformants of Saccharomyces cerevisiae contained both autonomously-replicative, and integrative pG2 plasmid.

  12. Induction of the expression of genes encoding TGF-beta isoforms and their receptors by inositol hexaphosphate in human colon cancer cells.

    Science.gov (United States)

    Kapral, Małgorzata; Wawszczyk, Joanna; Hollek, Andrzej; Weglarz, Ludmiła

    2013-01-01

    Transforming growth factors-beta (TGF-beta) are multifunctional cytokines involved in the regulation of cell development, differentiation, survival and apoptosis. They are also potent anticancer agents that inhibit uncontrolled proliferation of cells. Incorrect TGF-beta regulation has been implicated in the pathogenesis of many diseases including inflammation and cancer. In humans, the TGF-beta family consists of three members (TGF-beta1, 2, 3) that show high similarity and homology. TGF-betas exert biological activities on various cell types including neoplastic cells via their specific receptors. Inositol hexaphosphate (phytic acid, IP6), a phytochemical has been reported to possess various health benefits. The aim of this study was to examine the effect of IP6 on the expression of genes encoding TGF-beta1, TGF-beta2, TGF-beta3 isoforms and their receptors TbetaRI, TbetaRII, TbetaRIII in human colorectal cancer cell line Caco-2. The cells were treated with 0.5, 1 and 2.5 mM IP6 for 3, 6 and 12 h. The untreated Caco-2 cells were used as the control. Quantification of genes expression was performed by real time QRT-PCR technique with a SYBR Green I chemistry. The experimental data revealed that the TGF-beta1 mRNA was the predominant isoform in Caco-2 cells and that IP6 enhanced transcriptional activity of genes of all three TGF-beta isoforms and their receptors TbetaRI, TbetaRII TbetaRIII in these cells. At concentrations up to 1 mM, IP6 over-expressed the genes in 6 h lasting cultures, and its higher dose (2.5 mM) caused successively increasing transcript level of TGF-beta isoforms and receptors with the duration of experiment up to 12 h. The findings of this study indicate that one of anti-cancer abilities of IP6 can be realized by enhancing the gene expression of TGF-beta isoforms and their receptors at the transcriptional level.

  13. Serotonin Transporter (5-HTT) and gamma-Aminobutyric Acid Receptor Subunit beta3 (GABRB3) Gene Polymorphisms are not Associated with Autism in the IMGSA Families

    DEFF Research Database (Denmark)

    Maestrini, E.; Lai, C.; Marlow, A.;

    1999-01-01

    Previous studies have suggested that the serotonin transporter (5-HTT) gene and the gamma-aminobutyric acid receptor subunit beta3 (GABRB3) gene, or other genes in the 15q11-q13 region, are possibly involved in susceptibility to autism. To test this hypothesis we performed an association study...... and the GABRB3 genes are unlikely to play a major role in the aetiology of autism in our family data set....

  14. Gamma rays and bleomycin nick DNA and reverse the DNase I sensitivity of beta-globin gene chromatin in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Villeponteau, B.; Martinson, H.G.

    1987-05-01

    The active beta-globin genes in chicken erythrocytes, like all active genes, reside in large chromatin domains which are preferentially sensitive to digestion by DNase I. We have recently proposed that the special structure of chromatin in active domains is maintained by torsional stress in the DNA. This hypothesis predicts that nicking of the DNA within any such chromosomal domain in vivo will relax the DNA and lead to loss of the special DNase I-sensitive state. Here we have tested this prediction by using gamma irradiation and bleomycin treatment to cleave DNA within intact chicken embryo erythrocytes. Both treatments cause reversal of DNase I sensitivity. Moreover, reversal occurs at approximately one nick per 150 kilobase pairs for both agents despite their entirely unrelated modes of cell penetration and DNA attack. These results suggest that the domain of DNase I sensitivity surrounding the beta-globin genes comprises 150 kilobase pairs of chromatin under torsional stress and that a single DNA nick in this region is sufficient to reverse the DNase I sensitivity throughout the entire domain.

  15. Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

    Science.gov (United States)

    Houtz, Robert L.

    1999-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

  16. Familial Congenital Hypothyroidism Caused by Abnormal and Bioinactive TSH due to Mutations in the beta-Subunit Gene.

    Science.gov (United States)

    Medeiros-Neto, G; de Lacerda, L; Wondisford, F E

    1997-01-01

    Hereditary TSH deficiency is a rare autosomal recessive disease described in inbred Japanese families and in Greek and Brazilian kindreds. The TSH-beta-subunit gene has been shown to be the site of mutations that will give rise to truncated proteins that cannot dimerize with the alpha subunit or, alternatively, will produce a mutated TSH that is present in the circulation of the affected patients, but it is biologically inactive. Characteristically, the patients with TSH-beta-subunit-defects are born with congenital hypothyroidism, with very low levels of serum thyroid hormones and serum thyroglobulin and, paradoxically, with serum TSH levels that are consistently undetectable or at very low levels. Goiter is not present at birth, but the low radioactive thyroid uptake will increase after bovine TSH stimulation. Other pituitary hormones responses to provocative tests are normal. The subunit levels are at high concentration and are significantly increased following TRH stimulation. In two kindreds, molecular biological studies have indicated mutations in two different sites of exon 2, generating a peptide that would not dimerize with subunits to synthesize TSH molecules. In one kindred, a truncated TSH-beta protein was translated that generated a biologically inactive but detectable serum TSH molecule. (c) 1997, Elsevier Science Inc. (Trends Endocrinol Metab 1997;8:15-20).

  17. Core promoter acetylation is not required for high transcription from the phosphoenolpyruvate carboxylase promoter in maize

    Directory of Open Access Journals (Sweden)

    Horst Ina

    2009-12-01

    Full Text Available Abstract Background Acetylation of promoter nucleosomes is tightly correlated and mechanistically linked to gene activity. However, transcription is not necessary for promoter acetylation. It seems, therefore, that external and endogenous stimuli control histone acetylation and by this contribute to gene regulation. Photosynthetic genes in plants are excellent models with which to study the connection between stimuli and chromatin modifications because these genes are strongly expressed and regulated by multiple stimuli that are easily manipulated. We have previously shown that acetylation of specific histone lysine residues on the photosynthetic phosphoenolpyruvate carboxylase (Pepc promoter in maize is controlled by light and is independent of other stimuli or gene activity. Acetylation of upstream promoter regions responds to a set of other stimuli which include the nutrient availability of the plant. Here, we have extended these studies by analysing histone acetylation during the diurnal and circadian rhythm of the plant. Results We show that histone acetylation of individual lysine residues is removed from the core promoter before the end of the illumination period which is an indication that light is not the only factor influencing core promoter acetylation. Deacetylation is accompanied by a decrease in gene activity. Pharmacological inhibition of histone deacetylation is not sufficient to prevent transcriptional repression, indicating that deacetylation is not controlling diurnal gene regulation. Variation of the Pepc promoter activity during the day is controlled by the circadian oscillator as it is maintained under constant illumination for at least 3 days. During this period, light-induced changes in histone acetylation are completely removed from the core promoter, although the light stimulus is continuously applied. However, acetylation of most sites on upstream promoter elements follows the circadian rhythm. Conclusion Our results

  18. Differential gene expression in human brain pericytes induced by amyloid-beta protein.

    NARCIS (Netherlands)

    Rensink, A.A.M.; Otte-Holler, I.; Donkelaar, H.J. ten; Waal, R.M.W. de; Kremer, H.P.H.; Verbeek, M.M.

    2004-01-01

    Cerebral amyloid angiopathy is one of the characteristics of Alzheimer's disease (AD) and this accumulation of fibrillar amyloid-beta (Alphabeta) in the vascular wall is accompanied by marked vascular damage. In vitro, Abeta1-40 carrying the 'Dutch' mutation (DAbeta1-40) induces degeneration of cult

  19. CLONING, SEQUENCING AND EXPRESSION STUDIES OF THE GENES ENCODING AMICYANIN AND THE BETA-SUBUNIT OF METHYLAMINE DEHYDROGENASE FROM THIOBACILLUS-VERSUTUS

    NARCIS (Netherlands)

    UBBINK, M; VANKLEEF, MAG; KLEINJAN, DJ; HOITINK, CWG; HUITEMA, F; BEINTEMA, JJ; DUINE, JA; CANTERS, GW

    1991-01-01

    The genes encoding amicyanin and the beta-subunit of methylamine dehydrogenase (MADH) from Thiobacillus versutus have been cloned and sequenced. The organization of these genes makes it likely that they are coordinately expressed and it supports earlier findings that the blue copper protein amicyani

  20. A forkhead gene related to HNF-3beta is required for gastrulation and axis formation in the ascidian embryo.

    Science.gov (United States)

    Olsen, C L; Jeffery, W R

    1997-09-01

    We have isolated a member of the HNF-3/forkhead gene family in ascidians as a means to determine the role of winged-helix genes in chordate development. The MocuFH1 gene, isolated from a Molgula oculata cDNA library, exhibits a forkhead DNA-binding domain most similar to zebrafish axial and rodent HNF-3beta. MocuFH1 is a single copy gene but there is at least one other related forkhead gene in the M. oculata genome. The MocuFH1 gene is expressed in the presumptive endoderm, mesenchyme and notochord cells beginning during the late cleavage stages. During gastrulation, MocuFH1 expression occurs in the prospective endoderm cells, which invaginate at the vegetal pole, and in the presumptive notochord and mesenchyme cells, which involute over the anterior and lateral lips of the blastopore, respectively. However, this gene is not expressed in the presumptive muscle cells, which involute over the posterior lip of the blastopore. MocuFH1 expression continues in the same cell lineages during neurulation and axis formation, however, during the tailbud stage, MocuFH1 is also expressed in ventral cells of the brain and spinal cord. The functional role of the MocuFH1 gene was studied using antisense oligodeoxynucleotides (ODNs), which transiently reduce MocuFH1 transcript levels during gastrulation. Embryos treated with antisense ODNs cleave normally and initiate gastrulation. However, gastrulation is incomplete, some of the endoderm and notochord cells do not enter the embryo and undergo subsequent movements, and axis formation is abnormal. In contrast, the prospective muscle cells, which do not express MocuFH1, undergo involution and later express muscle actin and acetylcholinesterase, markers of muscle cell differentiation. The results suggest that MocuFH1 is required for morphogenetic movements of the endoderm and notochord precursor cells during gastrulation and axis formation. The effects of inhibiting MocuFH1 expression on embryonic axis formation in ascidians are

  1. Increased renal gene transcription of protein kinase C-beta in human diabetic nephropathy: relationship to long-term glycaemic control

    DEFF Research Database (Denmark)

    Langham, R.G.; Kelly, D.J.; Gow, R.M.;

    2008-01-01

    was examined in renal biopsies (n=25) with classical histological features of diabetic nephropathy and compared with that in normal control tissue (n=6). Peptide localisation of PKC-alpha, PKC-beta and the activated forms phosphorylated PKC-alpha and -beta was also performed on matched paraffin......-embedded sections of renal biopsies using immunohistochemistry. The effects of high glucose on PRKC-beta expression and peptide production in cultured human proximal tubular epithelial cells were assessed. RESULTS: Quantitative real-time PCR demonstrated a 9.9-fold increase in PRKC-beta mRNA in kidney biopsies...... quantitative analysis of mRNA from archival, formalin-fixed, paraffin-embedded tissue sections. RNA was extracted from scraped 6 microm sections of biopsy tissue, and PRKC-alpha and PRKC-beta (also known as PRKCA and PRKCB) mRNA measured using real-time PCR. Expression of genes encoding PKC isoforms...

  2. Effect of transfection with human interferon-beta gene entrapped in cationic multilamellar liposomes in combination with 5-fluorouracil on the growth of human esophageal cancer cells in vitro.

    Science.gov (United States)

    Tsunoo, Hideo; Komura, Sadaaki; Ohishi, Nobuko; Yajima, Haruyoshi; Akiyama, Seiji; Kasai, Yasushi; Ito, Katsuki; Nakao, Akimasa; Yagi, Kunio

    2002-01-01

    When human esophageal cancer cells were transfected with the human interferon-beta (hIFN-beta) gene entrapped in cationic multilamellar liposomes, the growth of all cancer cells tested was suppressed in a dose-dependent manner. The 50% inhibitory concentration (IC50) of the hIFN-beta gene entrapped in the liposomes ranged from 16 to 176 ng plasmid DNA/ml culture medium. Among the 10 cell lines examined, NUEC3, NUEC4, TE-3 and WSSC cell lines were highly susceptible to transfection with this gene entrapped in the liposomes. The IC50 values of the hIFN-beta gene entrapped in the liposomes with respect to cell growth were positively-correlated with those of exogenous cytokine hIFN-beta, suggesting that the antiproliferative effect of hIFN-beta gene entrapped in the liposomes can be mainly ascribed to the function of hIFN-beta produced by cells transfected with the gene. Two days after transfection with the liposome-entrapped gene, the concentration of hIFN-beta secreted into the medium was determined. Even though the level of hIFN-beta observed in the medium was lower than that of the IC50 of exogenously added hIFN-beta, the inhibitory potency of the hIFN-beta gene entrapped in the liposomes on the cell growth was remarkable. When the esophageal cancer cells were treated with 5-fluorouracil (5-FU) in the presence of a low concentration of liposome-entrapped-gene, the rate of growth inhibition of these cells increased over that caused by either 5-FU or hIFN-beta gene entrapped in the liposomes alone. All these data suggest that combination therapy with the hIFN-beta gene entrapped in cationic multilamellar liposomes and the anticancer drug 5-FU would be beneficial for preoperative treatment of carcinoma of the esophagus.

  3. Experiments on the formation of carboxylase and thiamine pyrophosphate in living bakers' yeast

    NARCIS (Netherlands)

    Leijnse, B.; Terpstra, W.

    1951-01-01

    The formation of carboxylase by living bakers' yeast was demonstrated upon incubation of the yeast with either thiamine or 2-methyl-4-amino-5-ethoxymethylpyrimidine, in the presence and in the absence of glucose. Carboxylase is also formed upon incubation of the yeast with NH4 sulfate and glucose. I

  4. The dynamic organization of fungal acetyl-CoA carboxylase

    Science.gov (United States)

    Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

    2016-04-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.

  5. Effect of beta2-adrenoceptor agonists and other cAMP-elevating agents on inflammatory gene expression in human ASM cells: a role for protein kinase A.

    Science.gov (United States)

    Kaur, Manminder; Holden, Neil S; Wilson, Sylvia M; Sukkar, Maria B; Chung, Kian Fan; Barnes, Peter J; Newton, Robert; Giembycz, Mark A

    2008-09-01

    In diseases such as asthma, airway smooth muscle (ASM) cells play a synthetic role by secreting inflammatory mediators such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, or IL-8 and by expressing surface adhesion molecules, including ICAM-1. In the present study, PGE(2), forskolin, and short-acting (salbutamol) and long-acting (salmeterol and formoterol) beta(2)-adrenoceptor agonists reduced the expression of ICAM-1 and the release of GM-CSF evoked by IL-1beta in ASM cells. IL-1beta-induced IL-8 release was also repressed by PGE(2) and forskolin, whereas the beta(2)-adrenoceptor agonists were ineffective. In each case, repression of these inflammatory indexes was prevented by adenoviral overexpression of PKIalpha, a highly selective PKA inhibitor. These data indicate a PKA-dependent mechanism of repression and suggest that agents that elevate intracellular cAMP, and thereby activate PKA, may have a widespread anti-inflammatory effect in ASM cells. Since ICAM-1 and GM-CSF are highly NF-kappaB-dependent genes, we used an adenoviral-delivered NF-kappaB-dependent luciferase reporter to examine the effects of forskolin and the beta(2)-adrenoceptor agonists on NF-kappaB activation. There was no effect on luciferase activity measured in the presence of forskolin or beta(2)-adrenoceptor agonists. This finding is consistent with the observation that IL-1beta-induced expression of IL-6, a known NF-kappaB-dependent gene in ASM, was also unaffected by beta(2)-adrenoceptor agonists, forskolin, PGE(2), 8-bromo-cAMP, or rolipram. Collectively, these results indicate that repression of IL-1beta-induced ICAM-1 expression and GM-CSF release by cAMP-elevating agents, including beta(2)-adrenoceptor agonists, may not occur through a generic effect on NF-kappaB.

  6. The Effect of Estradiol-17(beta), Goitrogen (T3), and Flutamide on Gene Expression in Medaka, Oryzias latipes

    Energy Technology Data Exchange (ETDEWEB)

    E.Haut, J

    2005-09-06

    Concern has been generated over the discovery of endocrine disrupting chemicals in rivers near sewage outflows. The presence of endocrine disrupting chemicals such as estradiol-17{beta} has been associated with a reduction of reproductive success in fish and an increase in the female phenotype and gonadal intersex in fish downstream of sewage treatment facilities. Such effects are believed to result from a disruption in the normal estrogenic pathways since estrogen plays a vital role in reproduction, sexual differentiation, the developments of secondary sex characteristics, and ovulation. Most studies have focused on the effect of a single endocrine disruptor on a single gene which does not provide for the interaction between genes. Microarray technology has made it possible to put an entire genome on a single chip so that researchers can get a clearer picture of the interaction of genes expressed in a cell and changes of said interactions when those cells are exposed to various conditions. Medaka males were exposed to known endocrine disruptors, estradial-17{beta} and goitrogen, and medaka females were exposed to flutamide. All treatments were then compared to controls. Total RNA was extracted from the livers of both treated and untreated males and hybridized to a microarray chip designed to have EST sequences specific to medaka. ESTs were identified through two-channel microarray analysis and compared to GenBank using blastn searches to identify up regulated genes. Choriogenins H and L, zona radiata, and vitellogenin, previously shown to be estrogen-induced in male fish were identified. Heat shock proteins (hsp70, hsp90, and hsp8) were also induced by estradiol-17{beta}, as was choriogenin Hminor. Exposure to goitrogen (T3) resulted in the induced expression of glutathione S-transferase and a GABA receptor protein in male medaka. Treatment with flutamide, an antiandrogen, caused the up regulation of choriogenin L, choriogenin Hminor, and zona radiata-2 in female

  7. Abundances of tetracycline, sulphonamide and beta-lactam antibiotic resistance genes in conventional wastewater treatment plants (WWTPs) with different waste load

    DEFF Research Database (Denmark)

    Laht, Mailis; Karkman, Antti; Voolaid, Veiko

    2014-01-01

    Antibiotics and antibiotic resistant bacteria enter wastewater treatment plants (WWTPs), an environment where resistance genes can potentially spread and exchange between microbes. Several antibiotic resistance genes (ARGs) were quantified using qPCR in three WWTPs of decreasing capacity located...... conclude that there is neither considerable enrichment nor purification of antibiotic resistance genes in studied conventional WWTPs....... in Helsinki, Tallinn, and Tartu, respectively: sulphonamide resistance genes (sul1 and sul2), tetracycline resistance genes (tetM and tetC), and resistance genes for extended spectrum beta-lactams (blaoxa-58, blashv-34, and blactx-m-32). To avoid inconsistencies among qPCR assays we normalised the ARG...

  8. Cloning, Expression and Purification of Wheat Acetyl-CoA Carboxylases CT Domain in E.coil

    Institute of Scientific and Technical Information of China (English)

    WANG Rui-jian; YANG Xue-ying; ZHENG Liang-yu; YANG Ye; GAO Gui; CAO Shu-gui

    2008-01-01

    The entire gene of carboxyltransferase(CT) domain of acetyl-CoA carboxylase(ACCase) from Chinese Spring wheat(CSW) plastid was cloned firstly,and the 2.3 kb gene was inserted into PET28a+ vector and expressed in E.coil in a soluble state.The (His)6 fusion protein was identified by SDS-PAGE and Western blot.The recombinant protein was purified by affinity chromatography,and the calculated molecular mass(Mr) was 88000.The results of the sequence analysis indicate that the cloned gene(GeneBank accession No.EU124675) was a supplement and revision of the reported ACCase CT partial cDNA from Chinese Spring wheat plastid.The recombinant protein will be significant for us to investigate the recognizing mechanism between ACCase and herbicides,and further to screen new herbicides.

  9. Pdx1 and BETA2/NeuroD1 participate in a transcriptional complex that mediates short-range DNA looping at the insulin gene.

    Science.gov (United States)

    Babu, Daniella A; Chakrabarti, Swarup K; Garmey, James C; Mirmira, Raghavendra G

    2008-03-28

    The activity of the insulin gene, Ins, in islet beta cells is thought to arise in part from the synergistic action of the transcription factors Pdx1 and BETA2/NeuroD1. We asked how the binding of these factors to A and E elements many tens or hundreds of base pairs upstream of the start site could influence activity of transcriptional machinery. We therefore tested the hypothesis that the complex of Pdx1 and BETA2/NeuroD1 maintains a DNA conformation such that distal regions of the gene are brought into proximity of the promoter and coding region. We show by coimmunoprecipitation that Pdx1 and BETA2/NeuroD1 exist within a complex and that the two physically interact with one another in this complex as assessed by fluorescence resonance energy transfer. Consistent with this interaction, we found that the two factors simultaneously occupy the same fragment of the Ins gene in beta cell lines using the chromatin immunoprecipitation/re-chromatin immunoprecipitation assay. Using a modification of the chromosome conformation capture assay in vitro and in beta cells, we observed that Pdx1 and BETA2/NeuroD1 mediate looping of a segment of Ins that brings EcoRI sites located at -623 and +761 bp (relative to the transcriptional start site) in proximity to one another. This looping appears to be dependent in vitro upon an intact A3 binding element, but not upon the E2 element. Based on our findings, we propose a model whereby Pdx1 and BETA2/NeuroD1 physically interact to form a nucleoprotein complex on the Ins gene that mediates formation of a short DNA loop. Our results suggest that such short loop conformations may be a general mechanism to permit interactions between transcription factors and basal transcriptional machinery.

  10. A Polymorphism Within the Promoter of the TGF{beta}1 Gene Is Associated With Radiation Sensitivity Using an Objective Radiologic Endpoint

    Energy Technology Data Exchange (ETDEWEB)

    Kelsey, Chris R., E-mail: kelse003@mc.duke.edu [Department of Radiation Oncology, Duke University Medical Center, Durham, NC (United States); Jackson, Lauren [Department of Pathology, Duke University Medical Center, Durham, NC (United States); Langdon, Scott [Department of Immunology, Duke University Medical Center, Durham, NC (United States); Owzar, Kouros [Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, NC (United States); Hubbs, Jessica [Department of Radiation Oncology, University of North Carolina, Chapel Hill, NC (United States); Vujaskovic, Zeljko; Das, Shiva [Department of Radiation Oncology, Duke University Medical Center, Durham, NC (United States); Marks, Lawrence B. [Department of Radiation Oncology, University of North Carolina, Chapel Hill, NC (United States)

    2012-02-01

    Purpose: To evaluate whether single nucleotide polymorphisms (SNPs) in the transforming growth factor-{beta}1 (TGF{beta}1) gene are associated with radiation sensitivity using an objective radiologic endpoint. Methods and Materials: Preradiation therapy and serial postradiation therapy single photon emission computed tomography (SPECT) lung perfusion scans were obtained in patients undergoing treatment for lung cancer. Serial blood samples were obtained to measure circulating levels of TGF{beta}1. Changes in regional perfusion were related to regional radiation dose yielding a patient-specific dose-response curve, reflecting the patient's inherent sensitivity to radiation therapy. Six TGF{beta}1 SNPs (-988, -800, -509, 869, 941, and 1655) were assessed using high-resolution melting assays and DNA sequencing. The association between genotype and slope of the dose-response curve, and genotype and TGF{beta}1 ratio (4-week/preradiation therapy), was analyzed using the Kruskal-Wallis test. Results: 39 white patients with preradiation therapy and {>=}6-month postradiation therapy SPECT scans and blood samples were identified. Increasing slope of the dose-response curve was associated with the C(-509)T SNP (p = 0.035), but not the other analyzed SNPs. This SNP was also associated with higher TGF{beta}1 ratios. Conclusions: This study suggests that a polymorphism within the promoter of the TGF{beta}1 gene is associated with increased radiation sensitivity (defined objectively by dose-dependent changes in SPECT lung perfusion).

  11. Development a diagnostic pan-dermatophyte TaqMan probe real-time PCR assay based on beta tubulin gene.

    Science.gov (United States)

    Mirhendi, Hossein; Motamedi, Marjan; Makimura, Koichi; Satoh, Kazuo

    2016-08-01

    Early differentiation of dermatophytosis from other cutaneous mycoses is essential to avoid inaccurate therapy. DNA-based techniques including real-time PCR have increasingly been considered for detection of fungal elements in clinical specimens. In this study, after partial sequence analysis of beta tubulin (BT2) gene in 13 common and rare pathogenic dermatophyte species, a pan-dermatophyte primer and probe set was designed in a TaqMan probe-based PCR format. The sensitivity and specificity of the system was tested with 22 reference strains of dermatophytes, 234 positive clinical specimens, 32 DNA samples extracted from normal nails, several fungi other than dermatophytes and human DNAs. Analytical detection limit of the designed PCR on serially diluted DNAs of prepared recombinant plasmid indicated that only five molecules per sample are the minimum number for reliable detection by the assay. A total of 226 out of 234 (96.5%) DNAs extracted from clinical samples, but none of the 32 nail samples, from healthy volunteers were positive in PCR. The real-time PCR targeted beta tubulin gene established in this study could be a sensitive diagnostic tool which is significantly faster than the conventional culture method and should be useful in the clinical settings, in large-scale epidemiological studies and in clinical trials of antifungal therapy.

  12. (AC)n dinucleotide repeat polymorphism in 5' beta-globin gene in native and Mestizo Mexican populations.

    Science.gov (United States)

    Peñaloza, R; Delgado, P; Arenas, D; Barrientos, C; Buentello, L; Loeza, F; Salamanca, F

    2001-12-01

    Repeated sequences are dispersed along the human genome. These sequences are useful as markers in diagnosis of inherited diseases, in forensic medicine, and in tracking the origin and evolution of human populations. The (AC)n repeated element is the most frequent in the human genome. In this paper, the (AC)n repeated element located in the 5' flanking region of the beta-globin gene was studied by single-strand conformation polymorphism (SSCP). Four ethnic Mexican groups (Mixteca, Nahua, Otomí, Purépecha) and a Mestizo population were analyzed. We observed three alleles, A [(AC)16, B [(AC)14], and C [(AC)18], with a frequency of between 68.2% and 86.9%, 13.1% and 18.2%, and 6.7% and 13.7%, respectively. Allele C was present only in Purépecha and Mestizo groups. The absence of this allele in the other ethnic groups studied suggests that there is low genetic admixture of Purépecha and that this is a relatively isolated population. However, it could be that the C allele occurs in low frequencies in the other groups as a result of small sample sizes. The (AC)n repeat polymorphism in the beta-globin gene has not been previously studied in Amerindian populations.

  13. An X11alpha/FSBP complex represses transcription of the GSK3beta gene promoter.

    LENUS (Irish Health Repository)

    Lau, Kwok-Fai

    2010-08-04

    X11alpha is a neuronal adaptor protein that interacts with the amyloid precursor protein (APP) through a centrally located phosphotyrosine binding domain to inhibit the production of Abeta peptide that is deposited in Alzheimer\\'s disease brains. X11alpha also contains two C-terminal postsynaptic density-95, large discs, zona occludens 1 (PDZ) domains, and we show here that through its PDZ domains, X11alpha interacts with a novel transcription factor, fibrinogen silencer binding protein. Moreover, we show that an X11alpha\\/fibrinogen silencer binding protein complex signals to the nucleus to repress glycogen synthase kinase-3beta promoter activity. Glycogen synthase kinase-3beta is a favoured candidate kinase for phosphorylating tau in Alzheimer\\'s disease. Our findings show a new function for X11alpha that may impact on Alzheimer\\'s disease pathogenesis.

  14. Beta-D-glycan synthases and the CesA gene family: lessons to be learned from the mixed-linkage (1-->3),(1-->4)beta-D-glucan synthase.

    Science.gov (United States)

    Vergara, C E; Carpita, N C

    2001-09-01

    Cellulose synthase genes (CesAs) encode a broad range of processive glycosyltransferases that synthesize (1-->4)beta-D-glycosyl units. The proteins predicted to be encoded by these genes contain up to eight membrane-spanning domains and four 'U-motifs' with conserved aspartate residues and a QxxRW motif that are essential for substrate binding and catalysis. In higher plants, the domain structure includes two plant-specific regions, one that is relatively conserved and a second, so-called 'hypervariable region' (HVR). Analysis of the phylogenetic relationships among members of the CesA multi-gene families from two grass species, Oryza sativa and Zea mays, with Arabidopsis thaliana and other dicotyledonous species reveals that the CesA genes cluster into several distinct sub-classes. Whereas some sub-classes are populated by CesAs from all species, two sub-classes are populated solely by CesAs from grass species. The sub-class identity is primarily defined by the HVR, and the sequence in this region does not vary substantially among members of the same sub-class. Hence, we suggest that the region is more aptly termed a 'class-specific region' (CSR). Several motifs containing cysteine, basic, acidic and aromatic residues indicate that the CSR may function in substrate binding specificity and catalysis. Similar motifs are conserved in bacterial cellulose synthases, the Dictyostelium discoideum cellulose synthase, and other processive glycosyltransferases involved in the synthesis of non-cellulosic polymers with (1-->4)beta-linked backbones, including chitin, heparan, and hyaluronan. These analyses re-open the question whether all the CesA genes encode cellulose synthases or whether some of the sub-class members may encode other non-cellulosic (1-->4)beta-glycan synthases in plants. For example, the mixed-linkage (1-->3)(1-->4)beta-D-glucan synthase is found specifically in grasses and possesses many features more similar to those of cellulose synthase than to those of

  15. Identification of Klebsiella pneumoniae strains harboring inactive extended-spectrum beta-lactamase antibiotic-resistance genes

    Institute of Scientific and Technical Information of China (English)

    Xu Li; Zhai Yao; Lyu Yuan; Wang Qi; An Shuchang; Chen Jichao; Chen Yusheng

    2014-01-01

    Background The extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae has increasingly become a major contributor to nosocomial infections and can exhibit multiple antibiotic resistance.Previous studies have focused on the resistance genes in ESBL-producing strains,and the resistance-associated genetic environment of non-ESBL-producing strains has been ignored until now.Here,we investigated the occurrence and characteristics of non-ESBL-producing K.pneumoniae,which potentially carries unexpressed resistance genes.Methods K.pneumoniae strains were collected from five medical institutions in China from February 2010 to August 2013.The VITEK-2 ESBL detection system was used as a primary screen to identify the ESBL-producing phenotype,and the three primary types of ESBL-associated genes (CTX,SHV,and TEM) were detected by polymerase chain reaction (PCR) to confirm the strains presenting with a non-ESBL-producing phenotype.mRNA expression in the non-ESBL-producing strains was further screened by reverse-transcription PCR (RT-PCR) to validate their transcriptional efficiency.Results Out of 224 clinically isolated antibiotic-sensitive K.pneumoniae strains with a non-ESBL-producing phenotype,5 (2.2%) were identified to carry inactivated ESBL blaSHV genes with intact upstream promoter regions and resistance gene sequences.Interestingly,three of the five antibiotic-sensitive K.pneumoniae strains containing ESBL blaSHV genes still exhibited mRNA transcription of blasHv,while the other two exhibited no mRNA transcription.Conclusion These findings suggest that inactivated ESBL genes exist in non-ESBL-producing antibiotic-sensitive K.pneumoniae strains,which have the potential to transform the strain into an ESBL phenotype if an inappropriate application or overdose of antibiotics is implemented during clinical management.

  16. [The effect of CSN1 S2, CSN3 and beta-lg genes on milk performance in Xinong Saanen dairy goat].

    Science.gov (United States)

    Chen, Hong; Lan, Xian-Yong; Li, Rui-Biao; Lei, Chu-Zhao; Sun, Wei-Bin; Zhang, Run-Feng; Zheng, Yuan-Lin; Zhu, Bi-Cai

    2005-08-01

    PCR-RFLP technique was applied to analyze correlation between the polymorphisms of CSN1 S2 (alpha(s2) casein), CSN3 (kappa casein) and beta-lg (beta-lactoglobulin) genes and milk performance in 69 individuals of Xinong Saanen dairy goat. The results showed that there was significant correlation between different genotypes of CSN1 S2 locus and milk yield:average milk yield of individuals with genotype FF was less than that of genotype NN (P gene digested with endonuclease Hind III cleavage showed that no significant difference of milk yield between genotype DE and genotype EE was detected in first, second, third and fourth lactation milk yield and average milk yield (P > 0.05). The results of CSN3 gene with endonuclease Taq I cleavage showed that no significant difference of milk yield among individuals with genotype TT, TC and CC was detected (P > 0.05). No polymorphism was detected in PCR products of CSN3 gene digested with endonuclease Hae III. The analysis of beta-lg gene's 5' flanking region (710 bp) by PCR-RFLP in Xinong Saanen dairy goat showed that milk yield of individuals with genotype AA was higher than that with genotype AB in second, third lactation milk yield and average milk yield (P beta-lg gene's 5' flanking region is probably related to high milk yield.

  17. Expression of Beta-Human Chorionic Gonadotropin Genes in Renal Cell Cancer and Benign Renal Disease Tissues

    Institute of Scientific and Technical Information of China (English)

    姜永光; 曾甫清; 肖传国; 刘俊敏

    2003-01-01

    To study the expression of beta-human chorionic gonadotropin (βhCG) genes in renal cellcarcinomas (RCC) and benign renal disease tissues, nested reverse transcription-polymerase chainreaction (RT-PCR) and restriction endonuclease analysis were employed to detect the expression ofβhCG genes in 44 cases of RCC tissues and 24 cases of benign renal disease tissues. It was foundthat 52% RCC samples revealed positive for βhCG mRNA expression. Positive rate in advancedstage and poorly differentiated RCC was higher, but there was no significant difference. The posi-tive rate of βhCG mRNA expression was 54% in 24 cases of benign renal tissues, including 3 casesout of 6 polycystic kidneys, 7 cases out of 13 renal atrophies, 2 cases out of 2 oncocytomas and 1case out of 2 pyonephrotic kidneys. β7 was most frequently transcribed subtype gene independent onthe histology. These findings suggested βhCG gene transcription is not only involved in RCC but al-so in benign renal diseases.

  18. Computational identification and characterization of conserved miRNAs and their target genes in beet (Beta vulgaris).

    Science.gov (United States)

    Li, J L; Cui, J; Cheng, D Y

    2015-08-07

    Highly conserved endogenous non-coding microRNAs (miRNAs) play important roles in plants and animals by silencing genes via destruction or blocking of translation of homologous mRNA. Sugar beet, Beta vulgaris, is one of the most important sugar crops in China, with properties that include wide adaptability and strong tolerance to salinity and impoverished soils. Seedlings of B. vulgaris can grow in soils containing up to 0.6% salt; it is important to understand the molecular mechanisms of salt tolerance to enrich genetic resources for breeding salt-tolerant sugar beets. Here, we report 13 mature miRNAs from 12 families, predicted using an in silico approach from 29,857 expressed sequence tags and 279,223 genome survey sequences. The psRNATarget server predicted 25 target genes for the 13 miRNAs. Most of the target genes appeared to encode transcription factors or were involved in metabolism, signal transduction, stress response, growth, and development. These results improve our understanding of the molecular mechanisms of miRNA in beet and may aid in the development of novel and precise techniques for understanding post-transcriptional gene-silencing mechanisms in response to stress tolerance.

  19. Low insulin resistance and preserved beta-cell function contribute to human longevity but are not associated with TH-INS genes.

    Science.gov (United States)

    Paolisso, G; Barbieri, M; Rizzo, M R; Carella, C; Rotondi, M; Bonafè, M; Franceschi, C; Rose, G; De Benedictis, G

    2001-12-01

    Tyrosine Hydroxylase (TH) and Insulin (INS) genes lie extremely close in the 11p15.5 chromosomal region. An STR marker of the TH gene had revealed this locus associated with longevity. Thus, it seemed of interest to investigate the association between the TH-STR and INS gene variability (FokI-RFLP) with a phenotypic trait, such as the degree of insulin resistance (IR) and beta-cell function in centenarians (C). We analyzed age-related trajectories of IR and beta-cell function in a large sample (n=466) of individuals whose age ranged from 28 to more than 100 years; furthermore, allele average effects on IR and beta-cell function relevant to TH-STR and INS-FokI polymorphisms were estimated in C. Both IR and beta-cell function increased with advancing age and declined in subjects older than 90 years (p for trend <0.001). C had lower IR (1.5+/-0.7 vs. 3.9+/-1.7, p<0.001) and beta-cell function (26.1+/-8.5 vs. 55.4+/-16, p<0.001) than nC. In nC, but not in C, IR and beta-cell function correlated with the main anthropometric and metabolic confounders. Nevertheless, significant allele average effects by TH-STR and INS-FokI polymorphisms on IR and beta-cell function were not observed in C. In conclusion, C has a lower degree of IR and a preserved beta-cell function in comparison to nC, but the cause of such metabolic differences, which are likely does not lie in this genomic region.

  20. The urea carboxylase and allophanate hydrolase activities of urea amidolyase are functionally independent.

    Science.gov (United States)

    Lin, Yi; Boese, Cody J; St Maurice, Martin

    2016-10-01

    Urea amidolyase (UAL) is a multifunctional biotin-dependent enzyme that contributes to both bacterial and fungal pathogenicity by catalyzing the ATP-dependent cleavage of urea into ammonia and CO2 . UAL is comprised of two enzymatic components: urea carboxylase (UC) and allophanate hydrolase (AH). These enzyme activities are encoded on separate but proximally related genes in prokaryotes while, in most fungi, they are encoded by a single gene that produces a fusion enzyme on a single polypeptide chain. It is unclear whether the UC and AH activities are connected through substrate channeling or other forms of direct communication. Here, we use multiple biochemical approaches to demonstrate that there is no substrate channeling or interdomain/intersubunit communication between UC and AH. Neither stable nor transient interactions can be detected between prokaryotic UC and AH and the catalytic efficiencies of UC and AH are independent of one another. Furthermore, an artificial fusion of UC and AH does not significantly alter the AH enzyme activity or catalytic efficiency. These results support the surprising functional independence of AH from UC in both the prokaryotic and fungal UAL enzymes and serve as an important reminder that the evolution of multifunctional enzymes through gene fusion events does not always correlate with enhanced catalytic function.

  1. The Polymorphisms of Ser49Gly and Gly389Arg in Beta-1-Adrenergic Receptor Gene in Major Depression

    Science.gov (United States)

    KOKUT, Süleyman; ATAY, İnci Meltem; UZ, Efkan; AKPINAR, Abdullah; DEMİRDAŞ, Arif

    2015-01-01

    Introduction It was reported that the genetic susceptibility of major depressive disorder (MDD) is related with genetic polymorphisms. The aim of this study was to investigate the possible association of the genotype and allele frequencies of Ser49Gly and Arg389Gly polymorphisms in MDD by comparing them with healthy subjects. Methods A total of 144 patients with MDD diagnosed according to Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV) criteria and 105 healthy controls were included in the study. Polymerase chain reaction (PCR) with restriction fragment length polymorphism (RFLP) was used for genotyping. Results Of the 144 participants in the MDD group, 77 (53.5%) had homozygous wild type (AA), 57 (39.6%) had heterozygous type (AG), and 10 (6.9%) had mutant (GG) genotype for Ser49Gly, whereas 75 (52.1%) had homozygous wild type (GG), 59 (41.0%) had heterozygous (GC) type, and 10 (6.9%) had mutant homozygous (CC) genotype for Gly386Arg. There were no significant difference in the allele and genotype frequencies of the beta-1-adrenergic receptor (ADRB1) gene for Ser49Gly and Arg389Gly polymorphisms after comparing with healthy controls (p=0.626; p=0.863 and p=0.625; p=0.914). Conclusion The results of our study did not reveal a major effect of the polymorphism of Ser49Gly and Gly389Arg in the ADRB1 gene in MDD. Further studies with larger sample size are required to elucidate the role of other beta-1 adrenergic gene polymorphisms in MDD.

  2. Highly efficient modification of beta-lactoglobulin (BLG) gene via zinc-finger nucleases in cattle

    Institute of Scientific and Technical Information of China (English)

    Shengli Yu; Junjie Luo; Zhiyuan Song; Fangrong Ding; Yunping Dai; Ning Li

    2011-01-01

    Dear Editor,Gene targeting is in widespread use as a gold standard for determining the function of genes in mice and human embryonic stem cells [1].However,the poor efficiency of this technology has hindered its application to domestic animals,for which embryonic stem cells are not available.Although gene-targeted large domestic animals have been produced successfully by combination of homologous recombination-based targeting strategy and cloning [2-4],the efficiency is very low and,more importantly,the disruption of the targeted gene is usually mono-allelic.It thus takes a long time to obtain a null mutant.

  3. Chromosomal integration of a cephalosporinase gene from Acinetobacter baumannii into Oligella urethralis as a source of acquired resistance to beta-lactams.

    Science.gov (United States)

    Mammeri, Hedi; Poirel, Laurent; Mangeney, Nicole; Nordmann, Patrice

    2003-05-01

    Clinical Oligella urethralis isolate COH-1, which was uncommonly resistant to penicillins and narrow-spectrum cephalosporins, was recovered from a 55-year-old patient with a urinary tract infection. Shotgun cloning into Escherichia coli and expression experiments gave recombinant clones expressing either an AmpC beta-lactamase-type phenotype of resistance or a carbenicillin-hydrolyzing beta-lactamase-type phenotype of resistance. The AmpC beta-lactamase identified (ABA-1), which had a pI value of 8.2, had 98% amino acid identity with a chromosomally encoded cephalosporinase of Acinetobacter baumannii. A 820-bp insertion sequence element, ISOur1, belonging to the IS6 family of insertion sequence elements, was identified immediately upstream of bla(ABA-1), providing a -35 promoter sequence and likely giving rise to a hybrid promoter region. The carbenicillin-hydrolyzing beta-lactamase identified (CARB-8), which had a pI value of 6.4, differed from CARB-5 by two amino acid substitutions. Hybridization of CeuI fragment I-restricted DNA fragments of O. urethralis COH-1 with bla(ABA-1)-, bla(CARB-8)-, and 16S rRNA-specific probes indicated the chromosomal integration of the beta-lactamase genes. PCR and hybridization experiments failed to detect bla(CARB-8)- and bla(ABA-1)-like genes in three O. urethralis reference strains, indicating that the beta-lactamase genes identified were the source of acquired resistance in O. urethralis COH-1. This is one of the few examples of the interspecies transfer and the chromosomal integration of a gene encoding a naturally occurring beta-lactamase.

  4. Molecular Cloning and Sequencing of Hemoglobin-Beta Gene of Channel Catfish, Ictalurus Punctatus Rafinesque

    Science.gov (United States)

    : Hemoglobin-y gene of channel catfish , lctalurus punctatus, was cloned and sequenced . Total RNA from head kidneys was isolated, reverse transcribed and amplified . The sequence of the channel catfish hemoglobin-y gene consists of 600 nucleotides . Analysis of the nucleotide sequence reveals one o...

  5. Análise dos haplótipos do gene da betaS-globina no Ceará Analysis of betaS-globin gene haplotypes in Ceará, Brazil

    Directory of Open Access Journals (Sweden)

    Gentil Claudino de Galiza Neto

    2005-10-01

    Full Text Available No presente trabalho abordam-se vários aspectos relacionados à natureza molecular da anemia falciforme (AF, desordem hematológica de caráter hereditário. A descoberta do polimorfismo do DNA no grupamento do gene betaS, originando diferentes haplótipos da doença, permitiu ampliar o conhecimento em torno da heterogeneidade clínica observada nos pacientes falcêmicos nas mais diversas regiões do mundo. Analisaram-se os diferentes haplótipos e seus parâmetros hematológicos, presentes em um grupo de 22 pacientes naturais e procedentes do estado do Ceará. A distribuição das freqüências dos haplótipos encontrados foi de 55,9% para Benin; 41,2% para República Centro-Africana (CAR; e de 2,9% para o haplótipo Senegal. Esses dados, em comparação com os demais estudos realizados no Brasil, mostram associação entre os seus valores para um alfa de 5% (p The present work deals with numerous aspects related to the molecular nature of sickle cell anemia. The discovery of the DNA polymorphism in the betas-globin gene cluster, gave origin to different haplotypes of the disease, making possible to enlarge the knowledge about the clinical heterogenity observed on the patients with sickle cell disease, in the various regions of the world. The different haplotypes and its hematological parameters were analysed in a group of 22 patients born in the State of Ceará, northeast of Brazil. The distribution found in the haplotypes frequency was of 55.9% for the Benin, of 41.2% for the CAR, and of 2.9% for Senegal haplotype. The data, compared to the others works done in Brazil, show relations among their values to alpha of 5% (p < 0,05. The results make possible a full understanding of the pathophisiology of the illness and of its clinical complexity in our State, as well as it allows a better knowledge of the sickle cell disease in our country.

  6. The beta-globin gene cluster haplotypes in sickle cell anemia patients from Northeast Brazil: a clinical and molecular view.

    Science.gov (United States)

    Adorno, Elisângela Vitória; Zanette, Angela; Lyra, Isa; Souza, Cyntia Cajado; Santos, Leandro Ferraz; Menezes, Joelma Figueiredo; Dupuit, Marie France; Almeida, Mari Ney Tavares; Reis, Mitermayer Galvão; Gonçalves, Marilda Souza

    2004-08-01

    The beta(S)-globin haplotypes were studied in 78 sickle cell Brazilian patients from Bahia, Northeast Brazil, that has a large population of African origin. Hemoglobin (Hb) profiles were developed by high-performance liquid chromatography (HPLC), and beta(S)-globin gene haplotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. We identified 44 (55.0%) patients with the CAR/Ben (Central African Republic/Benin) genotype, 16 (20.0%) Ben/Ben, 13 (16.2%) CAR/CAR and seven (8.8%) with other genotypes. Analyses of the phenotypes showed clinical differences related only to Hb F levels and blood transfusion therapy; the presence of -alpha(-3.7)-thalassemia (thal) demonstrated statistical significance when associated with hematocrit (p=0.044), MCV (p=0.0007), MCH (p=0.012) and spleen sequestration events. The haplotype diversity found in the present study can be justified by information about the origin of the slave traffic period in Bahia during the 19th century. The specific characteristics described among the Bahian sickle cell patients could be confirmed by increasing the number of patients with specific genotypes and further studies of genetic markers.

  7. Rearrangement and expression of beta-T-cell receptor and immunoglobulin genes in established Ph1 chronic myelogenous leukemia cell lines.

    Science.gov (United States)

    Berenson, J; Koeffler, H P

    1989-01-01

    We have determined the arrangement and expression of immunoglobulin (Ig) and beta-T-cell receptor (TCR) genes in six established Philadelphia chromosome-positive (Ph1) chronic myelogenous leukemia (CML) cell lines, and correlated these results with their phenotypic characteristics. Three cell lines with nonlymphoid characteristics, EM2, EM3, and K562, did not demonstrate rearrangement or expression of Ig or beta-TCR genes. A new cell line, MB, with a mature B-cell phenotype recently established in our laboratory, contained light and heavy chain immunoglobulin gene rearrangements and expressed mature Ig RNA. In a cell line with an early lymphoid phenotype, BV173, this analysis showed rearrangement of Ig heavy chain and beta-TCR genes, unrearranged Ig light chain DNA, and expression of only an immature beta-TCR transcript. This line provides evidence for T-cell lineage involvement in Ph1 CML. One cell line without markers of any cell type, KCL-22, demonstrated rearranged, unexpressed Ig heavy chain genes, suggesting these cells are at the very earliest stages of lymphoid differentiation. These lines should provide valuable tools to dissect the molecular biology of differentiation in CML and in early lymphocytes.

  8. Haplotypes in tribal Indians bearing the sickle gene: evidence for the unicentric origin of the beta S mutation and the unicentric origin of the tribal populations of India.

    Science.gov (United States)

    Labie, D; Srinivas, R; Dunda, O; Dode, C; Lapoumeroulie, C; Devi, V; Devi, S; Ramasami, K; Elion, J; Ducrocq, R

    1989-08-01

    To determine the origin of sickle cell anemia (SS) in India, we analyzed haplotypes of the beta gene cluster in beta S-carrying individuals belonging to tribal populations living in the Nilgiris region of southern India and complemented the available data on tribes of east-central India. We found that in the Nilgiris tribes chromosomes bearing the beta S gene are linked in 91% of the cases to the "Asian" (Arab-Indian) haplotype (although 25% of the haplotypes had the epsilon polymorphic site negative, making the 5' portion of the haplotype identical with the African Senegal haplotype). These XmnI (+) chromosomes were associated with high G gamma expression (67.2 +/- 5.9%) and a high percentage of Hb F (15.5 +/- 7.9%; range, 6-25.3%). We have similar findings for tribal groups from west-central India (Gujarat). In east-central India we have confirmed the data of others, finding the same haplotype linked to beta S in tribes living in the east (Orissa, Andhra Pradesh). We conclude that the beta S gene in presently isolated and disperse tribal populations in India is associated with one predominant typical haplotype, suggesting a unicentric origin of the mutation in India. In addition, this finding implies a unicentric origin of the tribal populations themselves: The gene must have arisen and spread before tribal dispersion. Furthermore, we find extremely high frequencies of the (-alpha) haplotype in the Nilgiris (0.89) and in Gujarat (0.95). The beta S gene linkage to a high Hb F-expressing haplotype and the high incidence of alpha-thalassemia predict a mild phenotypical expression of sickle cell anemia in India.

  9. Poly purine.pyrimidine sequences upstream of the beta-galactosidase gene affect gene expression in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brahmachari Samir K

    2001-10-01

    Full Text Available Abstract Background Poly purine.pyrimidine sequences have the potential to adopt intramolecular triplex structures and are overrepresented upstream of genes in eukaryotes. These sequences may regulate gene expression by modulating the interaction of transcription factors with DNA sequences upstream of genes. Results A poly purine.pyrimidine sequence with the potential to adopt an intramolecular triplex DNA structure was designed. The sequence was inserted within a nucleosome positioned upstream of the β-galactosidase gene in yeast, Saccharomyces cerevisiae, between the cycl promoter and gal 10Upstream Activating Sequences (UASg. Upon derepression with galactose, β-galactosidase gene expression is reduced 12-fold in cells carrying single copy poly purine.pyrimidine sequences. This reduction in expression is correlated with reduced transcription. Furthermore, we show that plasmids carrying a poly purine.pyrimidine sequence are not specifically lost from yeast cells. Conclusion We propose that a poly purine.pyrimidine sequence upstream of a gene affects transcription. Plasmids carrying this sequence are not specifically lost from cells and thus no additional effort is needed for the replication of these sequences in eukaryotic cells.

  10. Transforming growth factor-beta1 regulation of ATF-3 and identification of ATF-3 target genes in breast cancer cells.

    Science.gov (United States)

    Kwok, Sukyee; Rittling, Susan R; Partridge, Nicola C; Benson, Chellakkan S; Thiyagaraj, Mayuranathan; Srinivasan, Narasimhan; Selvamurugan, Nagarajan

    2009-10-01

    Transforming growth factor-beta1 (TGF-beta1) is a crucial molecule for stimulation of breast cancer invasion and formation of bone metastases. The molecular mechanisms of how TGF-beta1 mediates these effects have yet to be completely determined. We have found that activating transcription factor-3 (ATF-3) is strongly stimulated and its level is sustained by TGF-beta1 in highly invasive and metastatic human breast cancer (MDA-MB231) and in mouse mammary pad tumor cells (r3T). ATF-3 is also overexpressed in human primary breast cancer tissue. Overexpression of ATF-3 increased normal human mammary epithelial cell number and DNA synthesis suggesting a role for ATF-3 in cell proliferation. The functional role of ATF-3 in breast cancer progression was determined by the RNA interference technique. Knockdown of ATF-3 by ATF-3 shRNA in MDA-MB231 cells decreased expression of cell cycle gene, cyclin A1 in MDA-MB231 cells. ATF-3 shRNA also decreased expression of an invasive and metastatic gene, matrix metalloproteinase-13 (MMP-13; collagenase-3) in these cells. Chromatin immunoprecipitation experiments identified the direct physical interaction of ATF-3 protein on the human MMP-13 promoter. Thus, the dysregulation of ATF-3 by TGF-beta1 is likely to activate cyclin A1 and MMP-13 genes in breast cancer cells and that would be key to the subsequent cancer cell invasion and metastasis.

  11. Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression.

    Science.gov (United States)

    Collis, P; Antoniou, M; Grosveld, F

    1990-01-01

    The human beta-globin dominant control region (DCR) was previously identified as a region from the 5' end of the human beta-globin locus which directs high level, site of integration-independent, copy number-dependent expression on a linked human beta-globin gene in transgenic mice and stably transfected mouse erythroleukaemia (MEL) cells. We have now analysed the elements comprising the DCR by systematic deletion mutagenesis in stable MEL transfectants. We have identified two independent elements within the DNase I hypersensitive sites 2 and 3, containing fragments which direct strong transcriptional inducibility of a beta-globin gene. Whilst the remaining two hypersensitive sites do not direct significant transcriptional induction, our data suggest that all four sites may be necessary for the fully regulated expression conferred by the DCR. We have also tested a number of beta-globin minigene constructs under the control of the DCR to assess if any of the local sequences from the gene may be removed without loss of expression. We find that the 3' enhancer may be removed without affecting expression, but there is an absolute requirement for the presence of the second intron, not related to the enhancer present in that intron. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2295312

  12. Genomic organization of the mouse peroxisome proliferator-activated receptor beta/delta gene

    DEFF Research Database (Denmark)

    Larsen, Leif K; Amri, Ez-Zoubir; Mandrup, Susanne

    2002-01-01

    containing eight upstream AUG codons. We show that the presence of the 548 nt leader resulted in a low translational efficiency of the corresponding PPARbeta/delta mRNA and propose, based on structural features of the 5'-untranslated region, that translational initiation may be mediated via an internal...... gene encoding PPARbeta/delta. The gene spans approx. 41 kb and comprises 11 exons of which the six exons located in the 3'-end of the gene are included in all transcripts. Primer-extension and 5'-rapid amplification of cDNA ends experiments revealed the presence of multiple transcription start points...

  13. Expression of gibberellin 3 beta-hydroxylase gene in a gravi-response mutant, weeping Japanese flowering cherry

    Science.gov (United States)

    Sugano, Mami; Nakagawa, Yuriko; Nyunoya, Hiroshi; Nakamura, Teruko

    2004-01-01

    Expressions of the gibberellin biosynthesis gene were investigated in a normal upright type and a gravi-response mutant, a weeping type of Japanese flowering cherry (Prunus spachiana), that is unable to support its own weight and elongates downward. A segment of the gibberellin 3 beta-hydroxylase cDNA of Prunus spachiana (Ps3ox), which is responsible for active gibberellin synthesis, was amplified by using real-time RT-PCR. The content of Ps3ox mRNA in the weeping type was much greater than that in the upright type, while the endogenous gibberellin level was much higher in the elongating zone of the weeping type. These results suggest that the amount and distribution of synthesized gibberellin regulate secondary xylem formation, and the unbalanced distribution of gibberellin affects the gravi-response of the Prunus tree.

  14. Morbidity, beta S haplotype and alpha-globin gene patterns among sickle cell anemia patients in Kuwait.

    Science.gov (United States)

    Adekile, A D; Haider, M Z

    1996-01-01

    Admission records of children with sickle cell anemia (SS), in the two main teaching hospitals in Kuwait, were reviewed for a 1-year period. The haplotypes of 92 beta s chromosomes (from 39 SS, 11 AS, 2 S beta-thalassemia [S beta-thal] and 1 SD individuals) were determined using an allele-specific oligonucleotide (ASO) hybridization technique, while the alpha-globin gene status of 27 SS and 33 AS individuals, i.e. 120 chromosomes, was determined with a combination of polymerase chain reaction and AS techniques. A vasooclusive crisis was the most common (60.0%) cause of hospitalization, followed by infections (20%). Hospital admissions were most common during the hottest month of the year (July). Few complications of the disease were seen among patients on follow-up; however, splenomegaly was present in 24.0%, hepatomegaly in 15.2%, gallstones in 15.2% and aseptic necrosis of the femoral head in 6.1%. Haplotype 31 (Saudi Arabia/India) is the most frequent in this community, being present in 80.4% of the chromosomes tested; Benin haplotype 19 was found in 12.0% and Bantu haplotype 20 in 6.5%. Hb F in the haplotype 31 homozygotes and heterozygotes ranged from 11.4 to 35.1% (mean 22.5 +/- 5.2%). The frequency of alpha-thal determinants in the study was 40.0%, the commonest being the -alpha 3.7-kb deletion (27.5%), the alpha 2 polyadenylation signal (AATAAA-> AATAAG) mutation (10.2%) and the IVS-I 5' end GAGGT-GAGG->GAGG pentanucleotide (5 nt) deletion (3.3%). SS patients with coexistent alpha-thal trait did not have severe recurrent infections and none had gallstones. The high frequencies of the Saudi Arabia/India beta s haplotype and alpha-thalassemia trait contribute to the mild nature of SS disease among Kuwaiti Arabs comparable to that in eastern Saudi Arabia.

  15. Ventilator-associated pneumonia caused by carbapenem-resistant Enterobacteriaceae carrying multiple metallo-beta-lactamase genes

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    Dwivedi Mayank

    2009-07-01

    Full Text Available Context: Ventilator-associated pneumonia (VAP is a leading nosocomial infection in the intensive care unit (ICU. Members of Enterobacteriaceae are the most common causative agents and carbapenems are the most commonly used antibiotics. Metallo-beta-lactamase (MBL production leading to treatment failure may go unnoticed by routine disc diffusion susceptibility testing. Moreover, there is not much information on association of MBL-producing Enterobacteriaceae with ICU-acquired VAP. Therefore, a study was undertaken to find out the association of MBL-producing Enterobacteriaceae with VAP. Settings: This study was conducted in a large tertiary care hospital of North India with an eight-bed critical care unit. Materials and Methods: The respiratory samples (bronchoalveolar lavage, protected brush catheter specimens and endotracheal or transtracheal aspirates obtained from VAP patients (during January 2005-December 2006 were processed, isolated bacteria identified and their antibiotic susceptibilities tested as per standard protocols. The isolates of Enterobacteriaceae resistant to carbapenem were subjected to phenotypic and genotypic tests for the detection of MBLs. Results: Twelve of 64 isolates of Enterobacteriaceae were detected as MBL producers, bla IMP being the most prevalent gene. Additionally, in three strains, simultaneous coexistence of multiple MBL genes was detected. Conclusion: The coexistence of multiple MBL genes in Enterobacteriaceae is an alarming situation. As MBL genes are associated with integrons that can be embedded in transposons, which in turn can be accommodated on plasmids thereby resulting in a highly mobile genetic apparatus, the further spread of these genes in different pathogens is likely to occur.

  16. Thymosin beta 4 protects cardiomyocytes from oxidative stress by targeting anti-oxidative enzymes and anti-apoptotic genes.

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    Chuanyu Wei

    Full Text Available BACKGROUND: Thymosin beta-4 (Tβ4 is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. The mechanism by which Tβ4 modulates cardiac protection under oxidative stress is not known. The purpose of this study is to dissect the cardioprotective mechanism of Tβ4 on H(2O(2 induced cardiac damage. METHODS: Rat neonatal cardiomyocytes with or without Tβ4 pretreatment were exposed to H(2O(2 and expression of antioxidant, apoptotic, and anti-inflammatory genes was evaluated by quantitative real-time PCR and western blotting. ROS levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant, anti-inflammatory and antiapoptotic genes were silenced by siRNA transfections in neonatal cardiomyocytes and effect of Tβ4 on H(2O(2-induced cardiac damage was evaluated. RESULTS: Pre-treatment of Tβ4 resulted in reduction of the intracellular ROS levels induced by H(2O(2 in cardiomyocytes. Tβ4 pretreatment also resulted in an increase in the expression of antiapoptotic proteins and reduction of Bax/BCl(2 ratio in the cardiomyocytes. Pretreatment with Tβ4 resulted in stimulating the expression of antioxidant enzymes copper/zinc SOD and catalase in cardiomyocytes at both transcription and translation levels. Tβ4 treatment resulted in the increased expression of anti-apoptotic and anti-inflammatory genes. Silencing of Cu/Zn SOD and catalase gene resulted in apoptotic cell death in the cardiomyocytes which was prevented by treatment with Tβ4. CONCLUSION: This is the first report that demonstrates the effect of Tβ4 on cardiomyocytes and its capability to selectively upregulate anti-oxidative enzymes, anti-inflammatory genes, and antiapoptotic enzymes in the neonatal cardiomyocytes thus preventing cell death thereby protecting the myocardium. Tβ4 treatment resulted in decreased oxidative stress and inflammation in the

  17. Correlation of phenotypic tests with the presence of the blaZ gene for detection of beta-lactamase

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    Adriano Martison Ferreira

    Full Text Available Abstract Staphylococcus aureus and Staphylococcus saprophyticus are the most common and most important staphylococcal species associated with urinary tract infections. The objective of the present study was to compare and to evaluate the accuracy of four phenotypic methods for the detection of beta-lactamase production in Staphylococcus spp. Seventy-three strains produced a halo with a diameter ≤28 mm (penicillin resistant and all of them were positive for the blaZ gene. Among the 28 susceptible strain (halo ≥29 mm, 23 carried the blaZ gene and five did not. The zone edge test was the most sensitive (90.3%, followed by MIC determination (85.5%, but the specificity of the former was low (40.0%. The nitrocefin test was the least sensitive (28.9%. However, the nitrocefin test together with the disk diffusion method showed the highest specificity (100%. The present results demonstrated that the zone edge test was the most sensitive phenotypic test for detection of beta-lactamase, although it is still not an ideal test to detect this type of resistance since its specificity was low. However, the inhibition halo diameter of the penicillin disk can be used together with the zone edge test since the same disk is employed in the two tests. Combined analysis of the two tests shows a sensitivity of 90.3% and specificity of 100%, proving better sensitivity, especially for S. saprophyticus. This is a low-cost test of easy application and interpretation that can be used in small and medium-sized laboratories where susceptibility testing is usually performed by the disk diffusion method.

  18. Correlation of phenotypic tests with the presence of the blaZ gene for detection of beta-lactamase.

    Science.gov (United States)

    Ferreira, Adriano Martison; Martins, Katheryne Benini; Silva, Vanessa Rocha da; Mondelli, Alessandro Lia; Cunha, Maria de Lourdes Ribeiro de Souza da

    Staphylococcus aureus and Staphylococcus saprophyticus are the most common and most important staphylococcal species associated with urinary tract infections. The objective of the present study was to compare and to evaluate the accuracy of four phenotypic methods for the detection of beta-lactamase production in Staphylococcus spp. Seventy-three strains produced a halo with a diameter ≤28mm (penicillin resistant) and all of them were positive for the blaZ gene. Among the 28 susceptible strain (halo ≥29mm), 23 carried the blaZ gene and five did not. The zone edge test was the most sensitive (90.3%), followed by MIC determination (85.5%), but the specificity of the former was low (40.0%). The nitrocefin test was the least sensitive (28.9%). However, the nitrocefin test together with the disk diffusion method showed the highest specificity (100%). The present results demonstrated that the zone edge test was the most sensitive phenotypic test for detection of beta-lactamase, although it is still not an ideal test to detect this type of resistance since its specificity was low. However, the inhibition halo diameter of the penicillin disk can be used together with the zone edge test since the same disk is employed in the two tests. Combined analysis of the two tests shows a sensitivity of 90.3% and specificity of 100%, proving better sensitivity, especially for S. saprophyticus. This is a low-cost test of easy application and interpretation that can be used in small and medium-sized laboratories where susceptibility testing is usually performed by the disk diffusion method.

  19. Hydrocephalus caused by conditional ablation of the Pten or beta-catenin gene

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    Ohtoshi Akihira

    2008-10-01

    Full Text Available Abstract To investigate the roles of Pten and β-Catenin in the midbrain, either the Pten gene or the β-catenin gene was conditionally ablated, using Dmbx1 (diencephalon/mesencephalon-expressed brain homeobox gene 1-Cre mice. Homozygous disruption of the Pten or β-catenin gene in Dmbx1-expressing cells caused severe hydrocephalus and mortality during the postnatal period. Conditional deletion of Pten resulted in enlargement of midbrain structures. β-catenin conditional mutant mice showed malformation of the superior and inferior colliculi and stenosis of the midbrain aqueduct. These results demonstrate that both Pten and β-Catenin are essential for proper midbrain development, and provide the direct evidence that mutations of both Pten and β-catenin lead to hydrocephalus.

  20. Type 1 Diabetes Candidate Genes Linked to Pancreatic Islet Cell Inflammation and Beta-Cell Apoptosis

    Science.gov (United States)

    Størling, Joachim; Pociot, Flemming

    2017-01-01

    Type 1 diabetes (T1D) is a chronic immune-mediated disease resulting from the selective destruction of the insulin-producing pancreatic islet β-cells. Susceptibility to the disease is the result of complex interactions between environmental and genetic risk factors. Genome-wide association studies (GWAS) have identified more than 50 genetic regions that affect the risk of developing T1D. Most of these susceptibility loci, however, harbor several genes, and the causal variant(s) and gene(s) for most of the loci remain to be established. A significant part of the genes located in the T1D susceptibility loci are expressed in human islets and β cells and mounting evidence suggests that some of these genes modulate the β-cell response to the immune system and viral infection and regulate apoptotic β-cell death. Here, we discuss the current status of T1D susceptibility loci and candidate genes with focus on pancreatic islet cell inflammation and β-cell apoptosis. PMID:28212332

  1. Type 1 Diabetes Candidate Genes Linked to Pancreatic Islet Cell Inflammation and Beta-Cell Apoptosis.

    Science.gov (United States)

    Størling, Joachim; Pociot, Flemming

    2017-02-16

    Type 1 diabetes (T1D) is a chronic immune-mediated disease resulting from the selective destruction of the insulin-producing pancreatic islet β-cells. Susceptibility to the disease is the result of complex interactions between environmental and genetic risk factors. Genome-wide association studies (GWAS) have identified more than 50 genetic regions that affect the risk of developing T1D. Most of these susceptibility loci, however, harbor several genes, and the causal variant(s) and gene(s) for most of the loci remain to be established. A significant part of the genes located in the T1D susceptibility loci are expressed in human islets and β cells and mounting evidence suggests that some of these genes modulate the β-cell response to the immune system and viral infection and regulate apoptotic β-cell death. Here, we discuss the current status of T1D susceptibility loci and candidate genes with focus on pancreatic islet cell inflammation and β-cell apoptosis.

  2. Genes, Gender, Environment, and Novel Functions of Estrogen Receptor Beta in the Susceptibility to Neurodevelopmental Disorders

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    Mukesh Varshney

    2017-02-01

    Full Text Available Many neurological disorders affect men and women differently regarding prevalence, progression, and severity. It is clear that many of these disorders may originate from defective signaling during fetal or perinatal brain development, which may affect males and females differently. Such sex-specific differences may originate from chromosomal or sex-hormone specific effects. This short review will focus on the estrogen receptor beta (ERβ signaling during perinatal brain development and put it in the context of sex-specific differences in neurodevelopmental disorders. We will discuss ERβ’s recent discovery in directing DNA de-methylation to specific sites, of which one such site may bear consequences for the susceptibility to the neurological reading disorder dyslexia. We will also discuss how dysregulations in sex-hormone signaling, like those evoked by endocrine disruptive chemicals, may affect this and other neurodevelopmental disorders in a sex-specific manner through ERβ.

  3. Genes, Gender, Environment, and Novel Functions of Estrogen Receptor Beta in the Susceptibility to Neurodevelopmental Disorders

    Science.gov (United States)

    Varshney, Mukesh; Nalvarte, Ivan

    2017-01-01

    Many neurological disorders affect men and women differently regarding prevalence, progression, and severity. It is clear that many of these disorders may originate from defective signaling during fetal or perinatal brain development, which may affect males and females differently. Such sex-specific differences may originate from chromosomal or sex-hormone specific effects. This short review will focus on the estrogen receptor beta (ERβ) signaling during perinatal brain development and put it in the context of sex-specific differences in neurodevelopmental disorders. We will discuss ERβ’s recent discovery in directing DNA de-methylation to specific sites, of which one such site may bear consequences for the susceptibility to the neurological reading disorder dyslexia. We will also discuss how dysregulations in sex-hormone signaling, like those evoked by endocrine disruptive chemicals, may affect this and other neurodevelopmental disorders in a sex-specific manner through ERβ. PMID:28241485

  4. Alternative-splicing in the exon-10 region of GABA(A receptor beta(2 subunit gene: relationships between novel isoforms and psychotic disorders.

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    Cunyou Zhao

    Full Text Available BACKGROUND: Non-coding single nucleotide polymorphisms (SNPs in GABRB2, the gene for beta(2-subunit of gamma-aminobutyric acid type A (GABA(A receptor, have been associated with schizophrenia (SCZ and quantitatively correlated to mRNA expression and alternative splicing. METHODS AND FINDINGS: Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1 and beta(2S2, bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1 expression and decreased beta(2S2 expression in both SCZ and bipolar disorder (BPD compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1 and beta(2S2 expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2 expression. Moreover, site-directed mutagenesis indicated that Thr(365, a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down. CONCLUSION: This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2-subunit splicing diversity and the etiologies of SCZ and BPD.

  5. Energy expenditure, body composition and insulin response to glucose in male twins discordant for the Trp64Arg polymorphism of the beta3-adrenergic receptor gene

    DEFF Research Database (Denmark)

    Højlund, K; Christiansen, C; Bjørnsbo, K S;

    2006-01-01

    and environmental background, the Trp64Arg polymorphism of the beta3AR gene is associated with lower fat mass, fasting insulin levels and an appropriate insulin response to glucose. Thus, heterozygosity for the Trp64Arg variant is unlikely to increase the risk of obesity, insulin resistance or type 2 diabetes.......AIM: The tryptophan to arginine change in position 64 (Trp64Arg) polymorphism of the beta3-adrenergic receptor (beta3AR) gene has been associated with an increased prevalence of obesity, insulin resistance and type 2 diabetes. In this, decreased rates of energy expenditure and impaired insulin......-ray absorptiometry scanning and energy expenditure by indirect and direct calorimetry. RESULTS: Twins heterozygous for the Trp64Arg polymorphism showed significantly lower fat mass independent of the method used, and significantly lower fasting insulin and glucose concentrations compared with their homozygous wild...

  6. VIGS技术在甜菜上的应用%Application of Virus-Induced Gene Silencing in Beta Vulgaris

    Institute of Scientific and Technical Information of China (English)

    龚攀; 崔杰; 李俊良; 罗成飞; 杨虎臣

    2015-01-01

    Virus⁃induced gene silencing is an effective method of reverse genetics research for gene function analysis in plants. Compared with gene knockout, transgene and some other methods, it has many advantages such as faster, low⁃cost and high⁃throughput. With the accomplishment of Beta vulgaris′ complete genome sequencing,the functional characterization of a large number of sequences need to be identified . It is important to develop a rapid method of VIGS functional analysis system of beet genes. In this paper, we mediated the TRV ( Tobacco rattle virus) infection of sugar beet seedlings by Agrobacterium while Tobacco rattle virus is currently the most widely used viral vectors. Total RNA was extracted from the beet leaf and reverse transcribed into cDNA, we designed specific primers used in RT⁃PCR to detect the virus on the infected beet. The results demonstrated that the virus could infect beet, which laid the foundation for the next VIGS system established in Beta vulgaris.%病毒诱导基因沉默( VIGS)是一种应用于研究植物基因功能的反向遗传学手段。相对于基因敲除及转基因等方法,它具有时间短、成本低、高通量等优点。随着甜菜全基因组的测序完成,为尽早对大量序列信息进行注释和功能鉴定,急需建立甜菜VIGS体系。本文采用目前应用最广泛的烟草脆裂病毒载体在农杆菌的介导下侵染甜菜幼苗叶片,同时设立对照。提取甜菜幼苗叶片总RNA,并反转录成cDNA,设计特异引物进行RT⁃PCR检测。结果表明:在侵染植株上检测到了烟草脆裂病毒特异条带,证明该病毒可以对甜菜进行侵染,这一结果为下一步建立甜菜VIGS体系奠定了基础。

  7. Comparative evolutionary analyses of beta globin gene in eutherian, dinosaurian and neopterygii taxa

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    Gauri Awasthi, Garima Srivastava & Aparup Das

    2011-03-01

    Full Text Available Background & objectives: Comparative genomics and evolutionary analyses of conserved genes have enabled usto understand the complexity of genomes of closely related species. For example: -globin gene present inhuman hemoglobin is one such gene that has experienced many genetic changes in many related taxa andproduced more than 600 variants. One of the variant, HBS causes sickle-cell anemia in humans but offersprotection against severe malaria due to Plasmodium falciparum. In the present study, we characterized andperformed evolutionary comparative analyses of the -globin gene in different related and unrelated taxa tohave a comprehensive view of its evolution.Methods: DNA and protein sequences of -globin gene were downloaded from NCBI and characterized in detailin nine eutherian (Homo sapiens, Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus, Bostaurus, Canis familiaris, Equus caballus, Oryctolagus cuniculus, a dinosaurian (Gallus gallus and a neopterygii(Danio rerio taxa. Three more eutherian (Papio anubis, Ovis aries and Sus scrofa taxa were included for ananalysis at the protein level but not included at the gene level owing to lack of genomic information. Computationaland phylogenetic analyses were performed using evolutionary comparative approach.Results: Results of comparative and phylogenetic analyses revealed less conservation of genetic architecture of-globin compared to its protein architecture in all eutherian taxa. Both dinosaurian and neopterygii taxa servedas outgroups and varied at gene and protein levels.Interpretation & conclusion: Most remarkably, all primates from eutherian taxa including P. anubis showedonly nine codon position differences and an absolute similarity between H. sapiens and P. troglodytes. Absoluteconservation of coding region in Equus caballus (horse was observed. The results were discussed with aninference on the role of evolutionary forces in maintaining such close similarities

  8. Use of a non-radioactive hybridisation assay for direct detection of gram-negative bacteria carrying TEM beta-lactamase genes in infected urine.

    Science.gov (United States)

    Carter, G I; Towner, K J; Pearson, N J; Slack, R C

    1989-02-01

    DNA in infected urines from 81 patients with urinary tract infection was hybridised directly with a non-radioactive DNA probe specific for bacterial genes coding for TEM-type beta-lactamase. The results were assessed by means of a computerised image analysis system and compared with those obtained following isolation of the infecting organism, conventional sensitivity testing and isoelectric focusing (IEF) procedures for the detection of TEM-type beta-lactamase. Of the 27 ampicillin-resistant gram-negative organisms isolated in pure culture from the urines, 14 were shown by both hybridisation and IEF to carry a gene for TEM beta-lactamase production. Only four discordant results were obtained: three "false positive" direct hybridisation results, one due to urine pigmentation, and one, possibly, to a TEM beta-lactamase gene which was not being expressed, and one "false negative" result due to insufficient cell numbers in the urine. The system is capable of screening large numbers of samples and is applicable to any gene for which a suitable DNA probe is available.

  9. Thiabendazole resistance and mutations in the beta-tubulin gene of Penicillium expansum strains isolated from apples and pears with blue mold decay.

    Science.gov (United States)

    Cabañas, Romualdo; Castellá, Gemma; Abarca, M Lourdes; Bragulat, M Rosa; Cabañes, F Javier

    2009-08-01

    Penicillium expansum is the causal agent of blue mold rot, a postharvest decay of stored fruits. This fungus also produces the mycotoxins patulin and citrinin. Control of P. expansum still relies mainly on the use of fungicides such as thiabendazole. Since its introduction, resistant strains have been reported. The aim of this work was to investigate the thiabendazole resistance and mutations in the beta-tubulin gene of P. expansum strains isolated from apples and pears with blue mold decay from Spain. A total of 71 strains of P. expansum were scored for resistance to thiabendazole and the beta-tubulin gene was sequenced. Out of 71 strains, 37 were sensitive and 34 were resistant to thiabendazole. Regarding the beta-tubulin gene sequence, 10 different genetic types were determined, with a 99.7-100% similarity. When the amino acid sequence was deduced, five different amino acid sequences were found. All except one of the sensitive strains lacked mutations in the region sequenced. Of the 34 resistant strains, only eight had mutations that involved the residues 198 and 240. All the strains with mutations at position 198 always corresponded to resistant isolates. However, a high percentage of resistant strains had no mutations in the region of the beta-tubulin gene sequenced, and so other mechanisms may be involved in thiabendazole resistance.

  10. Effect of beta-globin gene cluster haplotype on the hematological and clinical features of sickle cell anemia.

    Science.gov (United States)

    Rieder, R F; Safaya, S; Gillette, P; Fryd, S; Hsu, H; Adams, J G; Steinberg, M H

    1991-03-01

    In 113 black American adults with sickle cell anemia (HbSS), we examined nine polymorphic restriction sites, including the Xmnl site 5' to the G gamma gene, to see whether haplotype is related to the level of HbF and the proportion of G gamma chains or if it influences the hematological and clinical features of the disease. Seventy-five percent of the patients were homozygous or heterozygous for the Benin (no. 19) or Central African Republic (Bantu, no. 20) haplotypes; 13.3% were homozygous or heterozygous for the Senegal (no. 3) haplotype, while 11.5% had other genotypes. Of the subjects, 14.2% were either homozygous or heterozygous for the Xmnl restriction site 5' to the G gamma gene. We found no effect of haplotype on HbF levels. The level of G gamma chains was 60.5% +/- 17.0% in individuals heterozygous or homozygous for haplotype no. 3 and was 46.9% +/- 11.6% in individuals with other haplotypes. Subjects with the Xmnl site 5' to the G gamma gene had G gamma globin levels of 59.5% +/- 16.7% while those lacking that site had an average of 47.2% +/- 12.1%. There were no significant differences among these groups in hemoglobin concentration, packed cell volume, mean cell volume, or clinical indicators of vaso-occlusive severity, including crises, hospitalizations per year, aseptic bone necrosis, acute chest syndrome, or leg ulcers. While the presence of haplotype 3 and the 5' G gamma Xmnl site were associated with increased G gamma chains, there was no effect on HbF level or other hematological and clinical features that might reflect disease severity. It is likely that determinants unrelated to haplotype, linked or unlinked to the beta-globin gene cluster, are the major effectors of differences in the levels of HbF in American patients with sickle cell anemia.

  11. Decreased renal vitamin K-dependent γ-glutamyl carboxylase activity in calcium oxalate calculi patients

    Institute of Scientific and Technical Information of China (English)

    陈俊汇; 刘继红; 章咏裳; 叶章群; 王少刚

    2003-01-01

    Objective To study the activity of vitamin K-dependent γ-glutamyl carboxylase in patients with calcium oxalate (CaOx) urolithiasis compared with healthy individuals and to assess its relationship to the renal calcium oxalate urolithiasis. Methods Renal parenchymas were harvested from urolithic patients and renal tumor patients undergoing nephrectomy. The renal carboxylase activity was evaluated as the radioactivity of [14C] labeled sodium bicarbonate in carboxylic reactions in vitro using β-liquid scintillation counting. Results Significantly reduced activity of renal vitamin K-dependent γ-glutamyl carboxylase was observed in the urolithic group as compared with normal controls (P<0.01). Conclusion It suggests that the reduced carboxylase activity observed in the urolithic patients may play an important role in the course of renal calcium oxalate urolithiasis.

  12. Characterization of a Beta vulgaris PGIP defense gene promoter in transgenic plants

    Science.gov (United States)

    Polygalacturonase-inhibiting protein (BvPGIP) genes were cloned from a sugar beet breeding line F1016 with increased tolerance to the sugar beet root maggot. Polygalacturonase-inhibiting proteins are cell wall leucine-rich repeat (LRR) proteins with crucial roles in development, pathogen defense an...

  13. Beta vulgaris L. serine proteinase inhibitor gene expression correlates to insect pest resistance in sugar beet

    Science.gov (United States)

    Analyzing genes that can be used for improving sugar beet resistance to the sugar beet root maggot (SBRM, Tetanops myopaeformis Roder), one of the most destructive insect pests of sugar beet in North America, was a major goal in our investigation. We report on the expression patterns of a sugar beet...

  14. A PUTATIVE BETA-GLUCANASE PSEUDOGENE BEHIND THE POTATO GBSS GENE

    NARCIS (Netherlands)

    VANDERLEIJ, FR; ABELN, ECA; HESSELINGMEINDERS, A; FEENSTRA, WJ

    1993-01-01

    We identified an open reading frame (ORF) which is located closely behind the gene encoding granule-bound starch synthase (GBSS) of potato (Solanum tuberosum L.). The ORF ends with a perfect 43 bp direct repeat, which carries the stop triplet precisely at the beginning of the second repeat. The dedu

  15. Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a beta-carotene 3-hydroxylase/4-ketolase.

    Science.gov (United States)

    Ojima, Kazuyuki; Breitenbach, Jürgen; Visser, Hans; Setoguchi, Yutaka; Tabata, Kazuyuki; Hoshino, Tatsuo; van den Berg, Johan; Sandmann, Gerhard

    2006-02-01

    A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a beta-carotene accumulating mutant. It consists of 3,166 bp and contains 17 introns. For the beta-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found. The resulting improper splicing of the mRNA results in an inactive protein. The cDNA of this beta-carotene oxygenase encodes a cytochrome P450 monooxygenase belonging to the 3A subfamily. P450-specific domains were identified including a cytochrome P450 and an oxygen binding motif. Electrons are provided by a cytochrome P450 reductase. Functional characterization of the enzyme by genetic modification of X. dendrorhous demonstrated that this P450 monooxygenase is multifunctional catalyzing all steps from beta-carotene to astaxanthin formation by oxygenation of carbon 3 and 4. The reaction sequence is first 4-ketolation of beta-carotene followed by 3-hydroxylation. A hydroxylation mechanism at allylic carbon atoms has been proposed for the generation of 4-keto and 3-hydroxy groups at both beta-ionone ends.

  16. Molecular variations linked to the grouping of beta- and alpha-globin genes in neonatal patients with sickle cell disease in the State of Pernambuco, Brazil.

    Science.gov (United States)

    Bezerra, Marcos André C; Santos, Magnun N N; Araújo, Aderson S; Gomes, Yara M; Abath, Frederico G C; Bandeira, Flavia M G C

    2007-01-01

    Various factors have been described as phenotypic modulators of sickle cell disease, such as levels of fetal hemoglobin (Hb F), presence of alpha-thalassemia (thal), and haplotypes of the beta-globin genes. In order to characterize and determine the frequency of the betaS and betaC mutations and the prevalence of -alpha3.7-thal, 74 patients with sickle cell disease detected during neonatal screening in the State of Pernambuco, Brazil, were studied. The haplotypes of the beta gene and -alpha3.7-thal were determined using polymerase chain reaction (PCR), and specific restriction endonucleases were used to establish the polymorphic sites of the haplotypes. The results showed the high frequency of the Central African Republic (CAR) or Bantu haplotype in the State of Pernambuco, Brazil. The low frequency of the Benin haplotype recorded in this study, in comparison with other states in northeast Brazil, suggests the diversity of origins of Afro-Brazilians in this region.

  17. Transforming growth factor beta stimulation of biglycan gene expression is potentially mediated by sp1 binding factors

    DEFF Research Database (Denmark)

    Heegaard, Anne-Marie; Xie, Zhongjian; Young, Marian Frances;

    2004-01-01

    Biglycan is a small leucine-rich proteoglycan which is localized in the extracellular matrix of bone and other specialized connective tissues. Both biglycan mRNA and protein are up-regulated by transforming growth factor-beta(1) (TGF-beta(1)) and biglycan appears to influence TGF-beta(1) activity...

  18. Crystallization and structure of a recombinant ribulose-1,5-bisphosphate carboxylase

    Science.gov (United States)

    Schneider, Gunter; Lindqvist, Ylva; Brändén, Carl-Ivar; Lorimer, George

    1988-07-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase is the key enzyme in photosynthetic carbon dioxide fixation and photorespiration. The dimeric carboxylase from the photosynthetic bacterium Rhodospirillum rubrum has been cloned and expressed in E. coli. The recombinant enzyme has been crystallized in a number of different crystal forms. The three-dimensional structure of the enzyme has been determined by X-ray crystallographic methods to 2.9Åresolution.

  19. Chemical and Physical Characterization of the Activation of Ribulosebiphosphate Carboxylase/Oxygenase

    Science.gov (United States)

    Donnelly, M. I.; Ramakrishnan, V.; Hartman, F. C.

    1983-08-01

    Molecular structure of ribulosebiphosphate carboxylase/oxygenase isolated from Rhodospirillium was compared with the enzyme isolated from Alcaligens eutrophus. Peptides derived from the active center of the bacterial enzyme were highly homologous with those isolated from spinach. Molecular shapes of the carboxylases were estimated using neutron scattering data. These studies suggested that the enzyme as isolated from R. rubrum is a solid prolate ellipsoid or cylinder, while the spinach enzyme resembles a hollow sphere.

  20. Cloning and Expression Analysis of a Gene Encoding for Ascorbate Peroxidase and Responsive to Salt Stress in Beet (Beta vulgaris).

    Science.gov (United States)

    Dunajska-Ordak, Kamila; Skorupa-Kłaput, Monika; Kurnik, Katarzyna; Tretyn, Andrzej; Tyburski, Jarosław

    2014-01-01

    BvpAPX is a full-length cDNA-encoding peroxisomal ascorbate peroxidase isolated from leaves of salt-stressed beet (Beta vulgaris) plants. A high level of identity has been reported between the deduced amino acid sequence of BvpAPX and other known ascorbate peroxidases. The genomic sequence of BvpAPX revealed a gene composed of 5 exons and 4 introns. Several sequence motifs revealed in the 5'UTR region of the gene confer to BvpAPX a putative responsiveness to various abiotic stresses. We determined the effect of salt stress on BvpAPX expression in leaves of the cultivated beet varieties, Huzar and Janosik, and their wild salt-tolerant relative B. vulgaris ssp. maritima. Plants were subjected to salt stress during a 32-day culture period (long-term salt treatment). An alternative salinization protocol consisted of an 18-h incubation of detached beet leaves in media supplemented with toxic salt concentrations (short-term salt treatment). RT-Q-PCR analysis revealed that BvpAPX expression markedly increased in leaves of plants subjected to conditions of long-term treatment with salinity, whereas BvpAPX transcript levels remained unaffected in detached leaves during short-term salt treatment. In addition, several leaf redox system parameters, such as ascorbate peroxidase activity or ascorbic acid, hydrogen peroxide, and lipid hydroperoxide concentration, were determined in the leaves of beet plants subjected to salt stress conditions.

  1. Differential effects of histone deacetylase inhibitors on phorbol ester- and TGF-beta1 induced murine tissue inhibitor of metalloproteinases-1 gene expression.

    Science.gov (United States)

    Young, David A; Billingham, Olivia; Sampieri, Clara L; Edwards, Dylan R; Clark, Ian M

    2005-04-01

    Expression of the tissue inhibitor of metalloproteinases-1 (Timp-1) gene can be induced by either phorbol myristate acetate (PMA) or transforming growth factor beta1 (TGF-beta1), although the signalling pathways involved are not clearly defined. Canonically, histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) or sodium butyrate (NaB) increase total cellular histone acetylation and activate expression of susceptible genes. Remarkably, PMA and TGF-beta1 stimulation of Timp-1 show a differential response to TSA or NaB. TSA or NaB potentiate PMA-induced Timp-1 expression but repress TGF-beta1-induced Timp-1 expression. The repression of TGF-beta1-induced Timp-1 by TSA was maximal at 5 ng.mL(-1), while for the superinduction of PMA-induced Timp-1 expression, the maximal dose is > 500 ng x mL(-1) TSA. A further HDACi, valproic acid, did not block TGF-beta1-induced Timp-1 expression, demonstrating that different HDACs impact on the induction of Timp-1. For either PMA or TGF-beta1 to induce Timp-1 expression, new protein synthesis is required, and the induction of AP-1 factors closely precedes that of Timp-1. The effects of the HDACi can be reiterated in transient transfection using Timp-1 promoter constructs. Mutation or deletion of the AP-1 motif (-59/-53) in the Timp-1 promoter diminishes PMA-induction of reporter constructs, however, the further addition of TSA still superinduces the reporter. In c-Jun-/- cells, PMA still stimulates Timp-1 expression, but TSA superinduction is lost. Transfection of a series of Timp-1 promoter constructs identified three regions through which TSA superinduces PMA-induced Timp-1 and we have demonstrated specific protein binding to two of these regions which contain either an avian erythroblastosis virus E26 (v-ets) oncogene homologue (Ets) or Sp1 binding motif.

  2. Structural Analysis of Substrate, Reaction Intermediate, and Product Binding in Haemophilus influenzae Biotin Carboxylase.

    Science.gov (United States)

    Broussard, Tyler C; Pakhomova, Svetlana; Neau, David B; Bonnot, Ross; Waldrop, Grover L

    2015-06-23

    Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1'-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1'-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO₂ from the carboxyphosphate intermediate to biotin.

  3. Searching for Beta-Haemolysin hlb Gene in Staphylococcus pseudintermedius with Species-Specific Primers.

    Science.gov (United States)

    Kmieciak, Wioletta; Szewczyk, Eligia M; Ciszewski, Marcin

    2016-07-01

    The paper presents an analysis of 51 Staphylococcus pseudintermedius clinically isolated strains from humans and from animals. Staphylococcus pseudintermedius strains' ability to produce β-haemolysin was evaluated with phenotypic methods (hot-cold effect, reverse CAMP test). In order to determine the hlb gene presence (coding for β-haemolysin) in a genomic DNA, PCR reactions were conducted with two different pairs of primers: one described in the literature for Staphylococcus aureus and recommended for analysing SIG group staphylococci and newly designed one in CLC Main Workbench software. Only reactions with newly designed primers resulted in product amplification, the presence of which was fully compatible with the results of phenotypic β-haemolysin test. Negative results for S. aureus and S. intermedius reference ATCC strains suggest that after further analysis the fragment of hlb gene amplified with primers described in this study might be included in the process of S. pseudintermedius strains identification.

  4. Sequence analysis of a 9873 bp fragment of the left arm of yeast chromosome XV that contains the ARG8 and CDC33 genes, a putative riboflavin synthase beta chain gene, and four new open reading frames.

    Science.gov (United States)

    Casas, C; Aldea, M; Casamayor, A; Lafuente, M J; Gamo, F J; Gancedo, C; Ariño, J; Herrero, E

    1995-09-15

    The DNA sequence of a 9873 bp fragment located near the left telomere of chromosome XV has been determined. Sequence analysis reveals seven open reading frames. One is the ARG8 gene coding for N-acetylornithine aminotransferase. Another corresponds to CDC33, which codes for the initiation factor 4E or cap binding protein. The open reading frame AOE169 can be considered as the putative gene for the Saccharomyces cerevisiae riboflavin synthase beta chain, since its translation product shows strong homology with four prokaryotic riboflavin synthase beta chains.

  5. Characterization of the human laminin beta2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS)

    DEFF Research Database (Denmark)

    Durkin, M E; Jäger, A C; Khurana, T S;

    1999-01-01

    7q22-->q31 region that contains the laminin beta1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3' end of LAMB2 lies 16 kb from the 5' end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we...... found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3' end of LAMB2L was similar to exons 8-12 of the laminin beta2 chain gene. The LAMB2L-LAMB2-QARS cluster lies telomeric to the gene encoding the laminin-binding protein...

  6. Upregulation of B7 molecules (CD80 and CD86) and exacerbated eosinophilic pulmonary inflammatory response in mice lacking the IFN-beta gene

    DEFF Research Database (Denmark)

    Matheu, Victor; Treschow, Alexandra; Navikas, Vaidrius

    2003-01-01

    )-sensitized and OVA-challenged mice. RESULTS: OVA-sensitized and OVA-challenged mice with lack of the IFNB gene had more severe pulmonary inflammation with increased lung local response, including IL-4, IL-5, IL-13, IgE, eosinophilia, and goblet cells, than their litter mates (IFN-beta+/-), whereas no differences...... were observed in regard to local levels of IFN-gamma. Moreover, systemic response with IgE production is also enhanced. Lack of IFN-beta also results in significantly higher antigen-specific T cells, with higher levels of IL-4, IL-5, and IL-13, whereas no significant differences in IFN-gamma response...

  7. Non-random association of the Rsa I polymorphic site 5' to the beta-globin gene with major sickle cell haplotypes.

    Science.gov (United States)

    Sharon, B; Poncz, M; Surrey, S; Schwartz, E

    1988-01-01

    There are three main African haplotypes associated with the sickle mutation on chromosome 11. We have examined an Rsa I polymorphism 550 bp 5' to the beta-globin gene to study the degree of linkage disequilibrium between this Rsa I site and the three haplotypes. This Rsa I site is contained within the 10.3 kb or less area of randomization separating the 5'- and 3'-haplotype clusters. The beta S-containing chromosomes of the Benin and Senegal haplotypes are not cut, while those of the Central African Republic are cleaved by Rsa I at this site. Possible explanations of these findings are discussed.

  8. Imputing Variants in HLA-DR Beta Genes Reveals That HLA-DRB1 Is Solely Associated with Rheumatoid Arthritis and Systemic Lupus Erythematosus.

    Directory of Open Access Journals (Sweden)

    Kwangwoo Kim

    Full Text Available The genetic association of HLA-DRB1 with rheumatoid arthritis (RA and systemic lupus erythematosus (SLE is well documented, but association with other HLA-DR beta genes (HLA-DRB3, HLA-DRB4 and HLA-DRB5 has not been thoroughly studied, despite their similar functions and chromosomal positions. We examined variants in all functional HLA-DR beta genes in RA and SLE patients and controls, down to the amino-acid level, to better understand disease association with the HLA-DR locus. To this end, we improved an existing HLA reference panel to impute variants in all protein-coding HLA-DR beta genes. Using the reference panel, HLA variants were inferred from high-density SNP data of 9,271 RA-control subjects and 5,342 SLE-control subjects. Disease association tests were performed by logistic regression and log-likelihood ratio tests. After imputation using the newly constructed HLA reference panel and statistical analysis, we observed that HLA-DRB1 variants better accounted for the association between MHC and susceptibility to RA and SLE than did the other three HLA-DRB variants. Moreover, there were no secondary effects in HLA-DRB3, HLA-DRB4, or HLA-DRB5 in RA or SLE. Of all the HLA-DR beta chain paralogs, those encoded by HLA-DRB1 solely or dominantly influence susceptibility to RA and SLE.

  9. Imputing Variants in HLA-DR Beta Genes Reveals That HLA-DRB1 Is Solely Associated with Rheumatoid Arthritis and Systemic Lupus Erythematosus

    Science.gov (United States)

    Kim, Kwangwoo; Bang, So-Young; Yoo, Dae Hyun; Cho, Soo-Kyung; Choi, Chan-Bum; Sung, Yoon-Kyoung; Kim, Tae-Hwan; Jun, Jae-Bum; Kang, Young Mo; Suh, Chang-Hee; Shim, Seung-Cheol; Lee, Shin-Seok; Lee, Jisoo; Chung, Won Tae; Kim, Seong-Kyu; Choe, Jung-Yoon; Nath, Swapan K.; Lee, Hye-Soon; Bae, Sang-Cheol

    2016-01-01

    The genetic association of HLA-DRB1 with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) is well documented, but association with other HLA-DR beta genes (HLA-DRB3, HLA-DRB4 and HLA-DRB5) has not been thoroughly studied, despite their similar functions and chromosomal positions. We examined variants in all functional HLA-DR beta genes in RA and SLE patients and controls, down to the amino-acid level, to better understand disease association with the HLA-DR locus. To this end, we improved an existing HLA reference panel to impute variants in all protein-coding HLA-DR beta genes. Using the reference panel, HLA variants were inferred from high-density SNP data of 9,271 RA-control subjects and 5,342 SLE-control subjects. Disease association tests were performed by logistic regression and log-likelihood ratio tests. After imputation using the newly constructed HLA reference panel and statistical analysis, we observed that HLA-DRB1 variants better accounted for the association between MHC and susceptibility to RA and SLE than did the other three HLA-DRB variants. Moreover, there were no secondary effects in HLA-DRB3, HLA-DRB4, or HLA-DRB5 in RA or SLE. Of all the HLA-DR beta chain paralogs, those encoded by HLA-DRB1 solely or dominantly influence susceptibility to RA and SLE. PMID:26919467

  10. Pyruvate carboxylase deficiency: An underestimated cause of lactic acidosis

    Directory of Open Access Journals (Sweden)

    F. Habarou

    2015-03-01

    Full Text Available Pyruvate carboxylase (PC is a biotin-containing mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate, thereby being involved in gluconeogenesis and in energy production through replenishment of the tricarboxylic acid (TCA cycle with oxaloacetate. PC deficiency is a very rare metabolic disorder. We report on a new patient affected by the moderate form (the American type A. Diagnosis was nearly fortuitous, resulting from the revision of an initial diagnosis of mitochondrial complex IV (C IV defect. The patient presented with severe lactic acidosis and pronounced ketonuria, associated with lethargy at age 23 months. Intellectual disability was noted at this time. Amino acids in plasma and organic acids in urine did not show patterns of interest for the diagnostic work-up. In skin fibroblasts PC showed no detectable activity whereas biotinidase activity was normal. We had previously reported another patient with the severe form of PC deficiency and we show that she also had secondary C IV deficiency in fibroblasts. Different anaplerotic treatments in vivo and in vitro were tested using fibroblasts of both patients with 2 different types of PC deficiency, type A (patient 1 and type B (patient 2. Neither clinical nor biological effects in vivo and in vitro were observed using citrate, aspartate, oxoglutarate and bezafibrate. In conclusion, this case report suggests that the moderate form of PC deficiency may be underdiagnosed and illustrates the challenges raised by energetic disorders in terms of diagnostic work-up and therapeutical strategy even in a moderate form.

  11. CO2 Exchange and Chlorophyll Fluorescence of Phosphoenolpyruvate Carboxylase Transgenic Rice Pollen Lines

    Institute of Scientific and Technical Information of China (English)

    Li-Li Ling; Hong-Hui Lin; Ben-Hua Ji; De-Mao Jiao

    2006-01-01

    To elucidate the photosynthetic physiological characteristics and the physiological inherited traits of rice(Oryza sativa L.) hybrids and their parents, physiological indices of photosynthetic CO2 exchange and chlorophyll fluorescence parameters were measured in leaves of the maize phosphoenolpyruvate carboxylase (PEPC) transgenic rice as the male parent, sp. japonica rice cv. 9516 as the female parent, and the stable JAAS45 pollen line. The results revealed that the PEPC gene could be stably inherited and transferred from the male parent to the JAAS45 pollen line. Moreover, the JAAS45 pollen line exhibited high levels of PEPC activity, manifesting higher saturated photosynthetic rates, photosynthetic apparent quantum yield (AQY), photochemical efficiency of photosystem Ⅱ and photochemical and non-photochemical quenching, which indicated that the JAAS45 pollen line has a high tolerance to photo-inhibition/photooxidation under strong light and high temperature. Furthermore, JAAS45 was confirmed to still be a C3 plant by δ13C carbon isotope determination and was demonstrated to have a limited photosynthetic C4 microcycle by feeding with exogenous C4 primary products, such as oxaloacetate or maiate, or phosphoenolpyruvate.The present study explains the physiological inherited properties of PEPC transgenic rice and provides an expectation for the integration of traditional breeding and biological technology.

  12. Regulatory enzymes of mitochondrial beta-oxidation as targets for treatment of the metabolic syndrome

    NARCIS (Netherlands)

    Schreurs, M.; Kuipers, F.; van der Leij, F. R.

    2010-01-01

    P>Insulin sensitizers like metformin generally act through pathways triggered by adenosine monophosphate-activated protein kinase. Carnitine palmitoyltransferase 1 (CPT1) controls mitochondrial beta-oxidation and is inhibited by malonyl-CoA, the product of acetyl-CoA carboxylase (ACC). The adenosine

  13. Retrovirus-mediated gene transfer of the cytokine genes interleukin-1beta and tumor necrosis factor-alpha into human neuroblastoma cells: consequences for cell line behavior and immunomodulatory properties.

    Science.gov (United States)

    Coze, C; Leimig, T; Jimeno, M T; Mannoni, P

    2001-03-01

    We have investigated the value of a gene therapy approach for neuroblastoma (NB), based on retroviral transduction of the IL-1beta or TNF-alpha cytokine genes into human NB lines. Secretion of the corresponding cytokine, was demonstrated in all lines, although with considerable quantitative variations. Cytokine gene expression significantly reduced the proliferation index (p = 0.0001); this effect was associated with either terminal neuronal (one TNF-alpha line) or fibroblast-like differentiation (two IL-1beta lines), leading to growth arrest after a few weeks. Cell surface levels of CD54 and HLA class II remained unaffected, but HLA class I (p < 0.001) and CD58 expression (p = 0.01) increased on SKNSH after TNF-alpha gene transfer. Mononuclear cells from normal allogeneic donors cocultured with both IL-1beta (p < 0.001) and TNF-alpha lines (p < 0.01), showed a significant increase in the proportion of activated T cells (CD3+DR+); however, their cytotoxicity and proliferation rate remained unchanged. Immunotherapy of neuroblastoma will require identification of transduced lines in which cytokine secretion induces phenotypic changes in such a way as to augment their likely immunomodulatory properties without impeding cell growth. Because of the limited efficacy of IL-1beta or TNF-alpha gene transfer alone, further studies should focus on combination with other immunomodulatory agents, to improve their potential efficacy in neuroblastoma.

  14. Commensal Enterobacteriaceae as reservoirs of extended-spectrum beta-lactamases, integrons and sul genes in Portugal

    Directory of Open Access Journals (Sweden)

    Elisabete eMachado

    2013-04-01

    Full Text Available Bacteria colonizing the human intestine have a relevant role in the spread of antimicrobial resistance. We investigated the faecal carriage of extended-spectrum beta-lactamase (ESBL-producing Enterobacteriaceae in healthy humans from Portugal and analysed the distribution of sul genes and class 1 and 2 integrons. Faecal samples (n=113 were recovered from healthy persons (North/Centre of Portugal, 2001-04 and plated on MacConkey agar with and without ceftazidime (1mg/L or cefotaxime (1mg/L. Isolates representing different morphotypes/plate and antibiotic susceptibility patterns (n=201 were selected. Isolates resistant to sulfonamides and/or streptomycin, gentamicin and trimethoprim were screened (PCR, sequencing for sul genes (sul1, sul2, sul3 and class 1 and 2 integrons. Presence of ESBLs was inferred using the DDST and further confirmed by PCR and sequencing. ESBL producers were selected for clonal analysis, plasmid characterization and conjugation assays by standard methods. ESBL-producing isolates were found in 1.8% (2/113 of samples, corresponding to Escherichia coli of phylogroups A (n=1 and B1 (n=1 carrying transferable blaCTX-M-14 and the new blaTEM-153, respectively. A 80kb IncK-blaCTX-M-14 was found, being highly related to that widely spread among CTX-M-14 producers of humans and animals from Portugal and other European countries. sul genes were found in 88% (22/25;sul2-60%, sul1-48%, sul3-4% of the sulfonamide resistant isolates. Class 1 integrons were more frequently found than class 2 (7% vs 3%. Interestingly, gene cassette arrangements within these platforms were identical to those commonly observed among Enterobacteriaceae from Portuguese food-producing animals, although aadA13 is here firstly described in Morganella morganii. These results reinforce the relevance of human commensal flora as reservoir of clinically relevant antibiotic resistance genes including blaESBLs, and highly transferable genetic platforms as IncK epidemic

  15. Thymosin beta 4 prevents oxidative stress by targeting antioxidant and anti-apoptotic genes in cardiac fibroblasts.

    Directory of Open Access Journals (Sweden)

    Sandeep Kumar

    Full Text Available RATIONALE: Thymosin beta-4 (Tβ4 is a ubiquitous protein with diverse functions relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory responses. The effecter molecules targeted by Tβ4 for cardiac protection remains unknown. The purpose of this study is to determine the molecules targeted by Tβ4 that mediate cardio-protection under oxidative stress. METHODS: Rat neonatal fibroblasts cells were exposed to hydrogen peroxide (H(2O(2 in presence and absence of Tβ4 and expression of antioxidant, apoptotic and pro-fibrotic genes was evaluated by quantitative real-time PCR and western blotting. Reactive oxygen species (ROS levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant and antiapoptotic genes were silenced by siRNA transfections in cardiac fibroblasts and the effect of Tβ4 on H(2O(2-induced profibrotic events was evaluated. RESULTS: Pre-treatment with Tβ4 resulted in reduction of the intracellular ROS levels induced by H(2O(2 in the cardiac fibroblasts. This was associated with an increased expression of antioxidant enzymes Cu/Zn superoxide dismutase (SOD and catalase and reduction of Bax/Bcl(2 ratio. Tβ4 treatment reduced the expression of pro-fibrotic genes [connective tissue growth factor (CTGF, collagen type-1 (Col-I and collagen type-3 (Col-III] in the cardiac fibroblasts. Silencing of Cu/Zn-SOD and catalase gene triggered apoptotic cell death in the cardiac fibroblasts, which was prevented by treatment with Tβ4. CONCLUSION: This is the first report that exhibits the targeted molecules modulated by Tβ4 under oxidative stress utilizing the cardiac fibroblasts. Tβ4 treatment prevented the profibrotic gene expression in the in vitro settings. Our findings indicate that Tβ4 selectively targets and upregulates catalase, Cu/Zn-SOD and Bcl(2, thereby, preventing H(2O(2-induced profibrotic changes in the myocardium. Further studies are

  16. Gene Network Analysis of Metallo Beta Lactamase Family Proteins Indicates the Role of Gene Partners in Antibiotic Resistance and Reveals Important Drug Targets.

    Science.gov (United States)

    Parimelzaghan, Anitha; Anbarasu, Anand; Ramaiah, Sudha

    2016-06-01

    Metallo Beta (β) Lactamases (MBL) are metal dependent bacterial enzymes that hydrolyze the β-lactam antibiotics. In recent years, MBL have received considerable attention because it inactivates most of the β-lactam antibiotics. Increase in dissemination of MBL encoding antibiotic resistance genes in pathogenic bacteria often results in unsuccessful treatments. Gene interaction network of MBL provides a complete understanding on the molecular basis of MBL mediated antibiotic resistance. In our present study, we have constructed the MBL network of 37 proteins with 751 functional partners from pathogenic bacterial spp. We found 12 highly interconnecting clusters. Among the 37 MBL proteins considered in the present study, 22 MBL proteins are from B3 subclass, 14 are from B1 subclass and only one is from B2 subclass. Global topological parameters are used to calculate and compare the probability of interactions in MBL proteins. Our results indicate that the proteins associated within the network have a strong influence in antibiotic resistance mechanism. Interestingly, several drug targets are identified from the constructed network. We believe that our results would be helpful for researchers exploring MBL-mediated antibiotic resistant mechanisms.

  17. Transforming growth factor-beta 1 downregulates dexamethasone-induced tetranectin gene expression during the in vitro mineralization of the human osteoblastic cell line SV-HFO

    DEFF Research Database (Denmark)

    Iba, K; Sawada, N; Chiba, H;

    1995-01-01

    treatment as evidenced by Northern blotting. When transforming growth factor-beta 1 (TGF-beta 1) was added together with dexamethasone to the SV-HFO cell cultures, the mineralization process was markedly suppressed and the expression of tetra nectin and alkaline phosphatase was downregulated in a dose......-dependent manner. These results demonstrate that the expression of tetranectin in these osteoblastic cells is regulated by dexamethasone and TGF-beta 1 and that tetranectin expression is tightly linked to the process of mineralization.......In the present study, we examined the regulation of tetranectin gene expression using a human osteoblastic cell line, SV-HFO, that undergoes mineralization upon treatment with dexamethasone. We found that the expression of tetranectin and alkaline phosphatase mRNA was induced by dexamethasone...

  18. T-cell receptor v-alpha and v-Beta gene usage in interleukin-2-cultured tumor-infiltrating lymphocytes from patients with breast-cancer

    DEFF Research Database (Denmark)

    Andersen, E; Scholler, J; Straten, P;

    1994-01-01

    surface through the T cell receptor (TCR) complex. We have studied the phenotype, cytotoxicity, and expression of TCR variable (V) alpha and beta chain on in vitro IL-2-cultured TIL isolated from primary malignant breast tumors from 11 patients. 10/11 cultures were dominated by CD4(+) (T-helper) cells....... The different TIL cultures exhibited varying levels of cytotoxicity against the natural killer (NK)-sensitive cell line K562 and breast cancer cell line T47D. The level of clonality, as measured by PCR-based analyses of usage of the different V segments was low, as only a few tumors showed patterns...... of restricted V gene expression. The mean number of V alpha segments per TIL culture was higher than the number of V beta segments per culture. A significant negative correlation was observed between the number of CD4+ cells and the number of V beta segments per culture, and no other correlations between...

  19. In vitro and in vivo gene therapy with CMV vector-mediated presumed dog beta-nerve growth factor in pyridoxine-induced neuropathy dogs.

    Science.gov (United States)

    Chung, Jin Young; Choi, Jung Hoon; Shin, Il Seob; Choi, Eun Wha; Hwang, Cheol Yong; Lee, Sang Koo; Youn, Hwa Young

    2008-12-01

    Due to the therapeutic potential of gene therapy for neuronal injury, many studies of neurotrophic factors, vectors, and animal models have been performed. The presumed dog beta-nerve growth factor (pdbeta-NGF) was generated and cloned and its expression was confirmed in CHO cells. The recombinant pdbeta-NGF protein reacted with a human beta-NGF antibody and showed bioactivity in PC12 cells. The pdbeta-NGF was shown to have similar bioactivity to the dog beta-NGF. The recombinant pdbeta-NGF plasmid was administrated into the intrathecal space in the gene therapy group. Twenty-four hours after the vector inoculation, the gene therapy group and the positive control group were intoxicated with excess pyridoxine for seven days. Each morning throughout the test period, the dogs' body weight was taken and postural reaction assessments were made. Electrophysiological recordings were performed twice, once before the experiment and once after the test period. After the experimental period, histological analysis was performed. Dogs in the gene therapy group had no weight change and were normal in postural reaction assessments. Electrophysiological recordings were also normal for the gene therapy group. Histological analysis showed that neither the axons nor the myelin of the dorsal funiculus of L4 were severely damaged in the gene therapy group. In addition, the dorsal root ganglia of L4 and the peripheral nerves (sciatic nerve) did not experience severe degenerative changes in the gene therapy group. This study is the first to show the protective effect of NGF gene therapy in a dog model.

  20. cDNA sequence and gene locus of the human retinal phosphoinositide-specific phospholipase-C{beta}4 (PLCB4)

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, R.A.; Ghalayini, A.J.; Anderson, R.E. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1995-09-01

    Defects in the Drosophila norpA (no receptor potential A) gene encoding a phosphoinositide-specific phospholipase C (PLC) block invertebrate phototransduction and lead to retinal degeneration. The mammalian homolog, PLCB4, is expressed in rat brain, bovine cerebellum, and the bovine retina in several splice variants. To determine a possible role of PLCB4 gene defects in human disease, we isolated several overlapping cDNA clones from a human retina library. The composite cDNA sequence predicts a human PLC{beta}4 polypeptide of 1022 amino acid residues (MW 117,000). This PLC{beta}4 variant lacks a 165-amino-acid N-terminal domain characteristic for the rat brain isoforms, but has a distinct putative exon 1 unique for human and bovine retina isoforms. A PLC{beta}4 monospecific antibody detected a major (130 kDa) and a minor (160 kDa) isoform in retina homogenates. Somatic cell hybrids and deletion panels were used to localize the PCLB4 gene to the short arm of chromosome 20. The gene was further sublocalized to 20p12 by florescence in situ hybridization. 4 refs., 5 figs.

  1. Analysis of the beta-tubulin gene and morphological changes of the microsporidium Anncaliia algerae both suggest albendazole sensitivity.

    Science.gov (United States)

    Santiana, Marianita; Pau, Cyrilla; Takvorian, Peter M; Cali, Ann

    2015-01-01

    The Microsporidium, Anncaliia algerae, an obligate intracellular parasite, has been identified as an opportunistic human pathogen, but treatment has not been evaluated for infections with this organism. Albendazole, an antitubulin polymerization drug used against parasitic worm infections, has been the medication of choice used to treat some microsporidial infections affecting humans, with varying results ranging from clearing infection (Encephalitozoon) to resistance (Enterocytozoon). This study illustrates the effect of albendazole treatment on A. algerae infection in Rabbit Kidney (RK13) cells and Human Fetal Lung (HFL-1) fibroblasts. Albendazole appears to have an attenuating effect on A. algerae infection and albendazole's IC50 in RK13 cells is 0.1 μg/ml. Long-term treatment inhibits up to 98% of spore production, but interrupting treatment reestablishes the infection without new exposure to the parasite as supported by microscopic observations. The parasite's beta-tubulin gene was purified, cloned, and sequenced. Five of the six specific amino acids, associated with benzimidazole sensitivity, are conserved in A. algerae. These findings suggest that A. algerae is sensitive to albendazole; however, the organism is not completely cleared from cultures.

  2. Beta-thymosin gene polymorphism associated with freshwater invasiveness of alewife (Alosa pseudoharengus)

    Science.gov (United States)

    Michalak, Katarzyna; Czesny, Sergiusz J.; Epifanio, John; Snyder, Randal J.; Schultz, Eric T.; Velotta, Jonathan P.; McCormick, Stephen D.; Brown, Bonnie L.; Santopietro, Graciela; Michalak, Pawel

    2014-01-01

    Predicting the success of a species’ colonization into a novel environment is routinely considered to be predicated on niche-space similarity and vacancy, as well as propagule pressure. The role genomic variation plays in colonization success (and the interaction with environment) may be suggested, but has not rigorously been documented. To test an hypothesis that previously observed ecotype-specific polymorphisms between anadromous and landlocked alewife (Alosa pseudoharengus) populations are an adaptive response to osmoregulatory challenges rather than a result of allele sampling at founding, we examined multiple anadromous and landlocked (colonized) populations for their allelic profiles at a conserved region (3’-UTR end) of a β-thymosin gene whose protein product plays a central role in the organization of cytoskeleton. The putatively ancestral β-thymosin allele was prevalent in anadromous populations, whereas a newly derived allele was overrepresented in landlocked populations; a third allele was exclusive to the anadromous populations. We also conducted a complementary set of salinity exposure experiments to test osmoregulatory performance of the alewife ecotypes in contrasting saline environments. The pattern of variation and results from these challenges indicate a strong association of β-thymosin with colonization success and a transition for species with an anadromous life-history to one with only a freshwater component.

  3. Emergence of Acinetobacter pittii harboring New Delhi metallo-beta-lactamase genes in Daejeon, Korea.

    Science.gov (United States)

    Sung, Ji Youn; Koo, Sun Hoe; Kim, Semi; Kwon, Gye Cheol

    2015-09-01

    Carbapenemase production has been reported worldwide in gram-negative bacteria, including Acinetobacter species. We detected carbapenemase-producing Acinetobacter pittii in clinical isolates in Daejeon, Korea. Twenty-one ertapenem-resistant A. pittii isolates screened with a disk diffusion method were characterized by using the Epsilon test, four multiplex PCR assays, and a multilocus sequence typing (MLST) scheme. A total of 21 A. pittii isolates harbored the metallo-β-lactamase (MBL) gene bla(IMP-1) or bla(NDM-1). Nineteen isolates containing bla(IMP-1) were resistant to imipenem and meropenem, but two isolates harboring bla(NDM-1) were susceptible to them. The sequence types (STs) of the two New Delhi MBL (NDM-1)-producing A. pittii isolates were ST70 and ST207, which differed from the STs (ST63, ST119, ST396, and a novel ST) of the IMP-1-producing A. pittii. This is the first report on NDM-1-producing A. pittii isolates in Korea. Our results emphasize that the study of NDM-1-producing gram-negative bacteria should involve carbapenem-susceptible as well as carbapenem-resistant isolates.

  4. Polymorphisms within beta-catenin encoding gene affect multiple myeloma development and treatment.

    Science.gov (United States)

    Butrym, Aleksandra; Rybka, Justyna; Łacina, Piotr; Gębura, Katarzyna; Frontkiewicz, Diana; Bogunia-Kubik, Katarzyna; Mazur, Grzegorz

    2015-12-01

    Recent studies have suggested that cereblon (CRBN) is essential for the anti-myeloma (MM) activity of immunomodulatory drugs (IMiDs), such as thalidomide and lenalidomide, and that dysregulation of Wnt/β-catenin pathway may be one of possible reasons of lenalidomide resistance. This prompted us to analyze the effect of polymorphisms within the genes coding for cereblon (CRBN (rs121918368 C>T)) and β-catenin (CTNNB1 (rs4135385 A>G; rs4533622 A>C)). MM patients (n=142) and healthy individuals (n=123) were genotyped using the Light SNiP assays. The presence of the CTNNB1 (rs4533622) A allele was more frequently detected in patients presented with stage II-III disease according to International Staging System (63/82 vs. 26/44, p=0.043) and Durie-Salmon criteria (75/99 vs. 14/26, p=0.049). The CTNNB1 (rs4135385) AA homozygosity was more frequent among patients with better response to CTD, i.e., cyclophosphamide-thalidomide-dexamethasone (18/23 vs. 32/60, p=0.047). Patients carrying the CTNNB1 (rs4533622) AA genotype were better responders to the first line therapy with thalidomide containing regimens (pCRBN polymorphism. These results suggest that the CTNNB1 polymorphisms may affect the clinical course and response to chemotherapy in patients with multiple myeloma.

  5. [Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. II. Transgenic tobacco plants expressing the bacterial beta-glucanase gene from Clostridium thermocellum,--a model for studying the differential expression of stress response-related genes].

    Science.gov (United States)

    Darbinian, N S; Popov, Iu G; Mochul'skiĭ, A V; Oming, D; Piruzian, E S; Vasilevko, V T

    1996-02-01

    The modified hybrid beta-1,3-glucanase gene (glc) of Clostridium thermocellum was expressed in tobacco Nicotiana tabacum. The glc gene was cloned into two plasmids, pC27-glc and pC29-glc, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter of Arabidopsis, respectively. These constructions were used for transformation of agrobacteria followed by transfer into plants. In transformed plants, each plasmid caused a high level of activity of thermostable bacterial glucanase not observed in reference plants. The plants obtained were used to study activation of some defense-related genes induced by their interaction with either tobacco mosaic virus (TMV) or a pathogenic fungus.

  6. Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032.

    Science.gov (United States)

    Wada, Masaru; Sawada, Kazunori; Ogura, Kotaro; Shimono, Yuta; Hagiwara, Takuya; Sugimoto, Masakazu; Onuki, Akiko; Yokota, Atsushi

    2016-02-01

    Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum.

  7. Mutant mice lacking acetyl-CoA carboxylase 1 are embryonically lethal

    Science.gov (United States)

    Abu-Elheiga, Lutfi; Matzuk, Martin M.; Kordari, Parichher; Oh, WonKeun; Shaikenov, Tattym; Gu, Ziwei; Wakil, Salih J.

    2005-01-01

    Acetyl-CoA carboxylases (ACC1 and ACC2) catalyze the carboxylation of acetyl-CoA to form malonyl-CoA, an intermediate metabolite that plays a pivotal role in the regulation of fatty acid metabolism. We previously reported that ACC2 null mice are viable, and that ACC2 plays an important role in the regulation of fatty acid oxidation through the inhibition of carnitine palmitoyltransferase I, a mitochondrial component of the fatty-acyl shuttle system. Herein, we used gene targeting to knock out the ACC1 gene. The heterozygous mutant mice (Acc1+/–) had normal fertility and lifespans and maintained a similar body weight to that of their wild-type cohorts. The mRNA level of ACC1 in the tissues of Acc1+/– mice was half that of the wild type; however, the protein level of ACC1 and the total malonyl-CoA level were similar. In addition, there was no difference in the acetate incorporation into fatty acids nor in the fatty acid oxidation between the hepatocytes of Acc1+/– mice and those of the wild type. In contrast to Acc2–/– mice, Acc1–/– mice were not detected after mating. Timed pregnancies of heterozygotes revealed that Acc–/– embryos are already undeveloped at embryonic day (E)7.5, they die by E8.5, and are completely resorbed at E11.5. Our previous results of the ACC2 knockout mice and current studies of ACC1 knockout mice further confirm our hypotheses that malonyl-CoA exists in two independent pools, and that ACC1 and ACC2 have distinct roles in fatty acid metabolism. PMID:16103361

  8. Efficient delivery of C/EBP beta gene into human mesenchymal stem cells via polyethylenimine-coated gold nanoparticles enhances adipogenic differentiation

    Science.gov (United States)

    Joydeep, Das; Choi, Yun-Jung; Yasuda, Hideyo; Han, Jae Woong; Park, Chankyu; Song, Hyuk; Bae, Hojae; Kim, Jin-Hoi

    2016-01-01

    The controlled differentiation of stem cells via the delivery of specific genes encoding appropriate differentiation factors may provide useful models for regenerative medicine and aid in developing therapies for human patients. However, the majority of non-viral vectors are not efficient enough to manipulate difficult-to-transfect adult human stem cells in vitro. Herein, we report the first use of 25 kDa branched polyethylenimine-entrapped gold nanoparticles (AuPEINPs) and covalently bound polyethylenimine-gold nanoparticles (AuMUAPEINPs) as carriers for efficient gene delivery into human mesenchymal stem cells (hMSCs). We determined a functional application of these nanoparticles by transfecting hMSCs with the C/EBP beta gene, fused to EGFP, to induce adipogenic differentiation. Transfection efficacy with AuPEINPs and AuMUAPEINPs was 52.3% and 40.7%, respectively, which was 2.48 and 1.93 times higher than that by using Lipofectamine 2000. Luciferase assay results also demonstrated improved gene transfection efficiency of AuPEINPs/AuMUAPEINPs over Lipofectamine 2000 and polyethylenimine. Overexpression of exogenous C/EBP beta significantly enhanced adipogenesis in hMSCs as indicated by both of Oil Red O staining and mRNA expression analyses. Nanoparticle/DNA complexes exhibited favorable cytocompatibility in hMSCs. Taken together, AuPEINPs and AuMUAPEINPs potentially represent safe and highly efficient vehicles for gene delivery to control hMSC differentiation and for therapeutic gene delivery applications. PMID:27677463

  9. Genetic variation in codons 167, 198 and 200 of the beta-tubulin gene in whipworms (Trichuris spp.) from a range of domestic animals and wildlife.

    Science.gov (United States)

    Hansen, Tina V A; Nejsum, Peter; Olsen, Annette; Thamsborg, Stig Milan

    2013-03-31

    A recurrent problem in the control of whipworm (Trichuris spp.) infections in many animal species and man is the relatively low efficacy of treatment with a single application of benzimidazoles (BZs). The presence of single nucleotide polymorphisms (SNPs) in codons 167, 198 and 200 in the beta-tubulin gene has been associated with BZ anthelmintic resistance in intestinal nematodes of veterinary importance. We hypothesized that the low susceptibility to BZ could be related to a natural tolerance or induced resistance caused by BZ-resistant associated SNPs. The aim of the present study was therefore to investigate the presence of these SNPs in the beta-tubulin gene of Trichuris spp. obtained from a range of animals. DNA was extracted from a total of 121 Trichuris spp. adult whipworm specimens obtained from 6 different host species. The number of worms from each host was pig: 31, deer: 21, sheep: 18, mouse: 17, dog: 19 and Arabian camels: 14. A pooled sample of Trichuris eggs from 3 moose was also used. In order to amplify the beta-tubulin fragments which covered codons 167, 198 and 200 of the gene, degenerate primers were designed. The sequences obtained were used to design species specific primers and used to amplify a ~476 bp fragment of the beta-tubulin gene. The PCR products were sequenced, analysed and evaluated. We did not identify SNPs in codons 167, 198 or 200 that led to amino acid substitutions in any of the studied Trichuris spp., but genetic variation expected to be related to species differences was observed. The cluster analysis showed close evolutionary relationship between Trichuris spp. from ruminants and between mouse and dog whereas the pig-derived worms, T. suis, clustered with T. trichiura obtained from Genbank.

  10. Major contribution of the type II beta carbonic anhydrase CanB (Cj0237) to the capnophilic growth phenotype of Campylobacter jejuni.

    Science.gov (United States)

    Al-Haideri, Halah; White, Michael A; Kelly, David J

    2016-02-01

    Campylobacter jejuni, the leading cause of human bacterial gastroenteritis, requires low environmental oxygen and high carbon dioxide for optimum growth, but the molecular basis for the carbon dioxide requirement is unclear. One factor may be inefficient conversion of gaseous CO2 to bicarbonate, the required substrate of various carboxylases. Two putative carbonic anhydrases (CAs) are encoded in the genome of C. jejuni strain NCTC 11168 (Cj0229 and Cj0237). Here, we show that the deletion of the cj0237 (canB) gene alone prevents growth in complex media at low (1% v/v) CO2 and significantly reduces the growth rate at high (5% v/v) CO2. In minimal media incubated under high CO2, the canB mutant grew on L-aspartate but not on the key C3 compounds L-serine, pyruvate and L-lactate, showing that CanB is crucial in bicarbonate provision for pyruvate carboxylase-mediated oxaloacetate synthesis. Nevertheless, purified CanB (a dimeric, anion and acetazolamide sensitive, zinc-containing type II beta-class enzyme) hydrates CO2 actively only above pH 8 and with a high Km (∼ 34 mM). At typical cytoplasmic pH values and low CO2, these kinetic properties might limit intracellular bicarbonate availability. Taken together, our data suggest CanB is a major contributor to the capnophilic growth phenotype of C. jejuni.

  11. Generation of a genomic reporter assay system for analysis of gamma- and beta-globin gene regulation

    NARCIS (Netherlands)

    Chan, Kasey S. K.; Xu, Jian; Wardan, Hady; McColl, Bradley; Orkin, Stuart; Vadolas, Jim

    2012-01-01

    A greater understanding of the regulatory mechanisms that govern gamma-globin expression in humans, especially the switching from gamma- to beta-globin, which occurs after birth, would help to identify new therapeutic targets for patients with beta-hemoglobinopathy. To further elucidate the mechanis

  12. Restriction fragment length polymorphism of ovine casein genes: close linkage between the alpha s1-, alpha s2-, beta- and kappa-casein loci.

    Science.gov (United States)

    Leveziel, H; Metenier, L; Guerin, G; Cullen, P; Provot, C; Bertaud, M; Mercier, J C

    1991-01-01

    Restriction fragment length polymorphism (RFLP) of ovine casein genes was investigated. Genomic DNA from 56 rams was digested with 10 restriction endonucleases and Southern blots probed with the four ovine casein cDNAs (alpha s1-, beta-, alpha s2- and kappa-Cn). Five enzymes, namely, BglI, PvuII, RsaI, TaqI and HindIII revealed nine different RFLPs. The inheritance of six of these polymorphisms was studied by segregation analysis of gametes in nine rams' families, and each of them could be related to the existence of alleles at the relevant casein locus. A close linkage between the four ovine casein genes was demonstrated since no recombination within the four pairs of loci examined, alpha s1-beta-Cn, alpha s1-kappa-Cn, beta-kappa-Cn and alpha s2-kappa-Cn, was observed in the progeny of double heterozygous rams. The casein genes are thus clustered in the ovine species as in the case of other mammals.

  13. Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet "Beta vulgaris L.": GMO application.

    Science.gov (United States)

    Chaouachi, Maher; Alaya, Akram; Ali, Imen Ben Haj; Hafsa, Ahmed Ben; Nabi, Nesrine; Bérard, Aurélie; Romaniuk, Marcel; Skhiri, Fethia; Saïd, Khaled

    2013-01-01

    KEY MESSAGE : Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis. The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R (2) > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.

  14. Geographical variation in the presence of genes encoding superantigenic exotoxins and beta-hemolysin among Staphylococcus aureus isolated from bovine mastitis in Europe and USA

    DEFF Research Database (Denmark)

    Larsen, H. D.; Aarestrup, Frank Møller; Jensen, N. E.

    2002-01-01

    for the presence of genes encoding staphylococcal enterotoxins A-E, and H, toxic shock toxin-1 (TSST-1), and beta-hemolysin, and 128 of these were examined for exfoliative toxins A and B. The detection was done by PCR. Phenotypic methods were used to confirm the PCR-results. None of the 128 isolates carried...... for the individual exotoxins. The genes encoding enterotoxin C, TSST-1, and enterotoxin D were the most common superantigens. The present and earlier studies demonstrate that the superantigenic exotoxins that were investigated in this study, do not play a role in the pathogenesis of bovine S. aureus mastitis...

  15. Regulation of gene expression by glucose in pancreatic beta -cells (MIN6) via insulin secretion and activation of phosphatidylinositol 3'-kinase.

    Science.gov (United States)

    da Silva Xavier, G; Varadi, A; Ainscow, E K; Rutter, G A

    2000-11-17

    Increases in glucose concentration control the transcription of the preproinsulin (PPI) gene and several other genes in the pancreatic islet beta-cell. Although recent data have demonstrated that secreted insulin may regulate the PPI gene (Leibiger, I. B., Leibiger, B., Moede, T., and Berggren, P. O. (1998) Mol. Cell 1, 933-938), the role of insulin in the control of other beta-cell genes is unexplored. To study the importance of insulin secretion in the regulation of the PPI and liver-type pyruvate kinase (L-PK) genes by glucose, we have used intranuclear microinjection of promoter-luciferase constructs into MIN6 beta-cells and photon-counting imaging. The activity of each promoter was increased either by 30 (versus 3) mm glucose or by 1-20 nm insulin. These effects of insulin were not due to enhanced glucose metabolism since culture with the hormone had no impact on the stimulation of increases in intracellular ATP concentration caused by 30 mm glucose. Furthermore, the islet-specific glucokinase promoter and cellular glucokinase immunoreactivity were unaffected by 30 mm glucose or 20 nm insulin. Inhibition of insulin secretion with the Ca(2+) channel blocker verapamil, the ATP-sensitive K(+) channel opener diazoxide, or the alpha(2)-adrenergic agonist clonidine blocked the effects of glucose on L-PK gene transcription. Similarly, 30 mm glucose failed to induce the promoter after inhibition of phosphatidylinositol 3'-kinase activity with LY294002 and the expression of dominant negative-acting phosphatidylinositol 3'-kinase (Deltap85) or the phosphoinositide 3'-phosphatase PTEN (phosphatase and tensin homologue). LY294002 also diminished the activation of the L-PK gene caused by inhibition of 5'-AMP-activated protein kinase with anti-5'-AMP-activated protein kinase alpha2 antibodies. Conversely, stimulation of insulin secretion with 13 mm KCl or 10 microm tolbutamide strongly activated the PPI and L-PK promoters. These data indicate that, in MIN6 beta

  16. [Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. I. Construction of vector plasmids for transfer into plants and expression of a modified gene for beta-1,3-glucanase from Clostridium thermocellum in tobacco protoplasts].

    Science.gov (United States)

    Darbinian, N S; Popov, Iu G; Mochul'skiĭ, A V; Volkova, L V; Piruzian, E S; Vasilevko, V T

    1996-02-01

    We constructed two vectors, pC27-glc and pC29-glc, that allow expression of the beta-1,3-glucanase gene (glc) in plant cells. The glc gene was previously cloned from anaerobic thermophilous bacterium Clostridium thermocellum. To increase the efficiency of expression, the N-terminal fragment of the glc gene encoding bacterial transient peptide was deleted, and hybrid variants of lacZ-glc were obtained. Analysis of expression of the hybrid genes in Escherichia coli showed that deletion of the fragment corresponding to 31 amino acids (a.a.) of beta-glucanase affected neither activity nor thermostability of the enzyme. The modified gene was subcloned into two vectors, pC27 and pC29, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter from Arabidopsis, respectively. Each of the resulting plasmids, pC27-glc and pC29-glc, was transfected into protoplasts of Nicotiana plumbaginifolia. Both the plasmids were shown to allow a high level of activity of the thermostable beta-1,3-glucanase. We plan to use the vectors obtained for transformation of agrobacteria and construction of transgenic plants.

  17. Changing ribulose diphosphate carboxylase/oxygenase activity in ripening tomato fruit.

    Science.gov (United States)

    Bravdo, B A; Palgi, A; Lurie, S

    1977-08-01

    Tomato fruit (Lycopersicum esculentum Mill) from green, pink, and red stages were assayed for changes in the activity of ribulose diphosphate carboxylase and oxygenase, phosphoenolpyruvate carboxylase, changes in the levels of glycolate and respiratory gas exchange. The ribulose diphosphate carboxylase activity decreased as the fruit ripened. By comparison, the ribulose diphosphate oxygenase activity increased during the transition from the green to the pink stage, and declined afterward. The changes in the endogenous glycolate levels and the respiratory gas exchange, as observed at different stages of ripening, resembled the changes in the ribulose diphosphate oxygenase activity. The utilization of glycolate in further metabolic activity may result in the formation of peroxidases required for the onset of ripening.

  18. Detection of Extended Spectrum Beta-Lactamases Resistance Genes among Bacteria Isolated from Selected Drinking Water Distribution Channels in Southwestern Nigeria

    Science.gov (United States)

    Ogunjobi, Adeniyi A.

    2016-01-01

    Extended Spectrum Beta-Lactamases (ESBL) provide high level resistance to beta-lactam antibiotics among bacteria. In this study, previously described multidrug resistant bacteria from raw, treated, and municipal taps of DWDS from selected dams in southwestern Nigeria were assessed for the presence of ESBL resistance genes which include blaTEM, blaSHV, and blaCTX by PCR amplification. A total of 164 bacteria spread across treated (33), raw (66), and municipal taps (68), belonging to α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Flavobacteriia, Bacilli, and Actinobacteria group, were selected for this study. Among these bacteria, the most commonly observed resistance was for ampicillin and amoxicillin/clavulanic acid (61 isolates). Sixty-one isolates carried at least one of the targeted ESBL genes with blaTEM being the most abundant (50/61) and blaCTX being detected least (3/61). Klebsiella was the most frequently identified genus (18.03%) to harbour ESBL gene followed by Proteus (14.75%). Moreover, combinations of two ESBL genes, blaSHV + blaTEM or blaCTX + blaTEM, were observed in 11 and 1 isolate, respectively. In conclusion, classic blaTEM ESBL gene was present in multiple bacterial strains that were isolated from DWDS sources in Nigeria. These environments may serve as foci exchange of genetic traits in a diversity of Gram-negative bacteria. PMID:27563674

  19. Biological response to ionizing radiation in mouse embryo fibroblasts with a targeted disruption of the DNA polymerase beta gene.

    Science.gov (United States)

    Miura, M; Watanabe, H; Okochi, K; Sasaki, T; Shibuya, H

    2000-06-01

    Base excision repair (BER) is carried out by two distinct pathways in mammalian cells, one dependent on DNA polymerase beta (Polb) and the other on proliferating cell nuclear antigen (Pcna). We studied whether the Polb-dependent pathway plays an important role in BER in vivo after exposure to ionizing radiation. For this purpose, we used mouse embryo fibroblasts derived from wild-type and Polb gene knockout littermates. Both cell lines had essentially the same clonogenic cell survival and low levels of apoptosis as determined by a colony formation assay and by a change in mitochondrial membrane potential, respectively. No significant cleavage of protein kinase C delta (Pkcd) in vivo, which is a substrate for caspase 3, was detected, and intact Pkcd was retained in both cell lines for at least 72 h after irradiation. Similar significant increases in caspase 3-like activities as measured by Asp-Glu-Val-Asp (DEVD) cleaving activity in vitro were observed in both cell lines after irradiation. Radiation induced cell cycle arrest in the form of a G(2)-phase block, and G(2)/M-phase fractions reached a peak approximately 10 h after irradiation and decreased thereafter with a similar time course in both cell lines. Similar levels of chromatin-bound Pcna were observed immediately after irradiation in non-S-phase cells of both cell lines and disappeared by 4 h after irradiation. We conclude that the deficiency in Polb does not have a significant influence on the radiation responses of these cells. Together with evidence accumulated in vitro, these results strongly support the idea that the Pcna-dependent pathway predominantly acts in BER of radiation-induced DNA damage in vivo.

  20. Immature transformed rat islet beta-cells differentially express C-peptides derived from the genes coding for insulin I and II as well as a transfected human insulin gene

    DEFF Research Database (Denmark)

    Blume, N; Petersen, J S; Andersen, L C;

    1992-01-01

    Synthetic peptides representing unique sequences in rat proinsulin C-peptide I and II were used to generate highly specific antisera, which, when applied on sections of normal rat pancreas, confirm a homogeneous coexpression of the two C-peptides in all islet beta-cells. Insulin gene expression...... is induced in the transformed heterogeneous rat islet cell clone, NHI-6F, by transient in vivo passage. During this process a transfected human insulin gene is coactivated with the endogenous nonallelic rat insulin I and II genes. Newly established cultures from NHI-6F insulinomas having a high frequency...

  1. Beta-globin gene cluster haplotypes and HbF levels are not the only modulators of sickle cell disease in Lebanon.

    Science.gov (United States)

    Inati, A; Taher, A; Bou Alawi, W; Koussa, S; Kaspar, H; Shbaklo, H; Zalloua, P A

    2003-02-01

    Sickle cell disease (SCD) is an inherited autosomal recessive disorder of the beta-globin chain. Despite the fact that all subjects with SCD have the same single base pair mutation, the severity of the clinical and hematological manifestations is extremely variable. This study examined for the first time in Lebanon the correlation between the clinical manifestation of SCD and the beta-globin gene haplotypes. The haplotypes of 50 patients diagnosed with SCD were determined using polymerase chain reaction amplification of fragments containing nine polymorphic restriction sites around and within the epsilon-Ggamma-Agamma-psibeta-delta-beta-globin gene complex. Most reported haplotypes were found in our population with the Benin haplotype as the most prevalent one. When the patients were divided according to their HbF levels into three groups (Group A: HbF 15%), surprisingly, the highest levels of HbF were associated with the most severe clinical cases. Our findings suggest that fetal hemoglobin levels are important but not the only parameters that affect the severity of the disease. In addition, the high levels of HbF in patients with CAR haplotypes did not seem to ameliorate the severity of symptoms, suggesting that genetic factors other than haplotypes are the major determinants of increased HbF levels in Lebanon.

  2. Prevalence of 16S rRNA methylase, modifying enzyme, and extended-spectrum beta-lactamase genes among Acinetobacter baumannii isolates.

    Science.gov (United States)

    Liu, Zhenru; Ling, Baodong; Zhou, Liming

    2015-08-01

    Multidrug-resistant Acinetobacter baumannii has become a worldwide problem, and methylation of 16S rRNA has recently emerged as a new mechanism of resistance to aminoglycosides, which is mediated by a newly recognized group of 16S rRNA methylases. 16S rRNA methylase confers a high-level resistance to all 4,6-substituted deoxystreptamine aminoglycosides that are currently used in clinical practice. Some of the A. baumannii isolates have been found to coproduce extended-spectrum beta-lactamases (ESBLs), contributing to their multidrug resistance. The aim of this study was to detect the determinants of the 16S rRNA methylase genes armA, rmtA, rmtB, rmtC, rmtD, rmtE, and npmA, the modifying enzyme genes aac(6')-Ib, ant(3″)-Ia, aph(3')-I, and the extended-spectrum beta-lactamase genes bla(TEM), bla(SHV), and bla(CTX-M-3) among A. baumannii isolates in northeastern Sichuan, China. Minimum inhibitory concentrations (MICs) of 21 different antimicrobial agents against the A. baumannii isolates were determined. The clinical isolates showed a high level of resistance (MIC≧256 μg/ml) to aminoglycosides, which ranged from 50·1 to 83·8%. The resistances to meropenem and imipenem, two of the beta-lactam antibiotics and the most active antibiotics against A. baumannii, were 9·1 and 8·2%, respectively. Among 60 amikacin-resistant isolates, only the 16S rRNA methylase gene armA was found to be prevalent (66·7%), but the other 16S rRNA methylase genes rmtA, rmtB, rmtC, rmtD, rmtE, and npmA were not detected. The prevalences of the modifying enzyme genes aac (6')-Ib, ant (3″)-Ia, and aph (3')-I were 51·7, 81·7, and 58·3%, respectively, which are different from a previous study in which the occurrences of these genes were 3, 64, and 72%, respectively. Among the 40 isolates that were armA-positive, the prevalences of bla(TEM), bla(SHV), and bla(CTX-M-3) genes were detected for the first time in China, and their occurrences were 45, 65, and 52·5%, respectively. In all, A

  3. Carbon fixation in Pinus halepensis submitted to ozone. Opposite response of ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Fontaine, V.; Pelloux, J.; Afif, D.; Gerant, D.; Dizengremel, P. [Univ. Henri Poincare-Nancy 1, Lab. de Biologie Forestiere, Vandauvre les Nancy cedex (France); Podor, M.; Grieu, P. [ENSAIA-INRA, Lab. Agronomie Environnement, Vandauvre les Nancy cedex (France)

    1999-06-01

    The effects of ozone exposure on carbon-fixation-related processes in Pinus halepensis Mill. needles were assessed over 3 months under controlled conditions. Ozone fumigation (200 ppb) did not induce a modification of either net CO{sub 2} assimilation or stomatal conductance in 1-year-old needles, whereas ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) activity was shown to be reduced by a half. Moreover, this ozone-induced reduction in Rubisco activity was associated with a decrease in the quantity of Rubisco, as determined by the decrease in the large subunit (LSU). On the other hand, 200-ppb ozone fumigation induced a strong increase in both activity and quantity of another carboxylating enzyme, phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), generally considered in C{sub 3} plants to participate in carbon catabolism processes. Ozone induced a significant decrease in the Rubisco/PEPC activity ratio which promotes the role of PEPC in trees under ozone stress. The role of this carboxylase will be discussed. (au) 42 refs.

  4. Angiotensin II modulates interleukin-1{beta}-induced inflammatory gene expression in vascular smooth muscle cells via interfering with ERK-NF-{kappa}B crosstalk

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Shanqin [Vascular Biology Unit, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA (United States); Zhi, Hui [Cardiovascular Division, Department of Medicine, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA (United States); Hou, Xiuyun [Vascular Biology Unit, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA (United States); Jiang, Bingbing, E-mail: bjiang1@rics.bwh.harvard.edu [Vascular Biology Unit, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA (United States); Cardiovascular Division, Department of Medicine, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA (United States)

    2011-07-08

    Highlights: {yields} We examine how angiotensin II modulates ERK-NF-{kappa}B crosstalk and gene expression. {yields} Angiotensin II suppresses IL-1{beta}-induced prolonged ERK and NF-{kappa}B activation. {yields} ERK-RSK1 signaling is required for IL-1{beta}-induced prolonged NF-{kappa}B activation. {yields} Angiotensin II modulates NF-{kappa}B responsive genes via regulating ERK-NF-{kappa}B crosstalk. {yields} ERK-NF-{kappa}B crosstalk is a novel mechanism regulating inflammatory gene expression. -- Abstract: Angiotensin II is implicated in cardiovascular diseases, which is associated with a role in increasing vascular inflammation. The present study investigated how angiotensin II modulates vascular inflammatory signaling and expression of inducible nitric oxide synthase (iNOS) and vascular cell adhesion molecule (VCAM)-1. In cultured rat aortic vascular smooth muscle cells (VSMCs), angiotensin II suppressed interleukin-1{beta}-induced prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK)-1, and nuclear translocation of nuclear factor (NF)-{kappa}B, leading to decreased iNOS but enhanced VCAM-1 expression, associated with an up-regulation of mitogen-activated protein kinase phosphatase-1 expression. Knock-down of RSK1 selectively down regulated interleukin-1{beta}-induced iNOS expression without influencing VCAM-1 expression. In vivo experiments showed that interleukin-1{beta}, iNOS, and VCAM-1 expression were detectable in the aortic arches of both wild-type and apolipoprotein E-deficient (ApoE{sup -/-}) mice. VCAM-1 and iNOS expression were higher in ApoE{sup -/-} than in wild type mouse aortic arches. Angiotensin II infusion (3.2 mg/kg/day, for 6 days, via subcutaneous osmotic pump) in ApoE{sup -/-} mice enhanced endothelial and adventitial VCAM-1 and iNOS expression, but reduced medial smooth muscle iNOS expression associated with reduced phosphorylation of ERK and RSK-1. These results indicate that angiotensin

  5. The molecular phenomena of the blaZ genes forming betalactamase enzymes structure in Staphylococcus aureus resistant to beta-lactam antibiotics (ampicillin

    Directory of Open Access Journals (Sweden)

    Mieke Satari

    2010-09-01

    Full Text Available Background: Nowadays, infectious disease still an important problem. One of the bacteria causing infectious diseases is Staphylococcus aureus (S. aureus. In the effort to deal with infections caused by S. aureus, beta-lactam antibiotics, such as ampicillin, are used. In fact, it is unfortunately known that many of S. aureus bacteria are resistant to this group of antibiotics. Because of nucleotide base changes in the structure of the genes blaZ which encode beta-lactamase enzymes in S. aureus. Purpose: The objective of this study was to analyze the nucleotide base changes in the structure of the genes blaZ forming beta-lactamase enzymes in S. aureus resistant to ampicillin based on molecular point of view. Methods: Molecular examinations was conducted by isolating the genes, forming beta-lactamase enzyme, which length was 845bp, from 7 isolates of S. aureus resistant to ampicillin by using PCR technique. The results of blaZ amplification were then subjected to homology by using Tn 552 of S. aureus obtained from bank of genes. Results: Based on the result of the homology, it was found that there was a change in purine base TG, which was a pyrimidine base at the -37 position of the initial codon of blaZ. This change, however, did not affect the strength of the promoter since the number of A and T is still more than the number of G and C. In the structure of the blaZ gene there was even no mutation or deletion or nucleotide base substitution found, so it would not affect the effectiveness of beta-lactamase enzyme. Conclusion: It can be concluded that the resistance of S. aureus towards ampicillin was not caused by nucleotide base deletion/substation. It is suspected that there were other causes leading to the resistance, including the overproduction of beta-lactamase enzyme of the blaZ gene, causing the degradation of beta-lactam antibiotics.Latar belakang: Penyakit infeksi sampai saat ini masih merupakan masalah. Salah satu bakteri penyebab

  6. Suggestive association between the C825T polymorphism of the G-protein beta3 subunit gene (GNB3) and clinical improvement with antipsychotics in schizophrenia.

    Science.gov (United States)

    Müller, Daniel J; De Luca, Vincenzo; Sicard, Tricia; King, Nicole; Hwang, Rudi; Volavka, Jan; Czobor, Pal; Sheitman, Brian B; Lindenmayer, Jean-Pierre; Citrome, Leslie; McEvoy, Joseph P; Lieberman, Jeffrey A; Meltzer, Herbert Y; Kennedy, James L

    2005-10-01

    G-proteins are composed of alpha, beta and gamma subunits. Once activated, these subunits play a major role in the conversion of external receptor activation into intracellular signals. The functional C825T polymorphism of the beta3 subunit gene (GNB3) has recently been shown to modulate antidepressant response, with the T-allele conferring an increased signaling and being associated with favorable antidepressant response. We hypothesized that this polymorphism may be associated with response to antipsychotics in a population of 145 chronic schizophrenic patients deriving from two study-samples and being mainly treated with clozapine for up to 6 months. Overall, the C/C genotype was significantly associated with relative clinical improvement as measured by Brief Psychiatric Rating Scale (BPRS) change scores after 6 and 12 weeks (ppoint to the role of intracellular mechanisms in antipsychotic response.

  7. Familial Polycythemia Caused by a Novel Mutation in the Beta Globin Gene: Essential Role of P50 in Evaluation of Familial Polycythemia

    Directory of Open Access Journals (Sweden)

    Neeraj Agarwal, Mariluz P. Mojica-Henshaw, Elizabeth. D. Simmons, Dottie Hussey, Ching N. Ou, Josef T. Prchal

    2007-01-01

    Full Text Available Two polycythemic subjects from a family with multiple polycythemic subjects were evaluated. Estimation of oxygen affinity of Hb from venous blood gas parameters (P50 revealed low P50 suggesting a high affinity Hb variant. Further work up, which included beta globin gene sequencing, revealed a novel mutation changing a codon to the previously reported high affinity Hb - Hb Johnstown (beta109 Val->Leu. Polycythemic subjects with high affinity Hb variant are asymptomatic with normal life expectancy. Their differentiation from polycythemia vera (PV is crucial to avoid therapy which is otherwise reserved for PV patients. We provide an electronic version (in Microsoft excel program of a previously reported mathematical formula for rapid calculation of P50 from venous blood gases. Estimation of P50 is an essential initial step in the evaluation of a subject with personal and family history of polycythemia.

  8. Growth arrest- and DNA-damage-inducible 45beta gene inhibits c-Jun N-terminal kinase and extracellular signal-regulated kinase and decreases IL-1beta-induced apoptosis in insulin-producing INS-1E cells

    DEFF Research Database (Denmark)

    Larsen, Claus Morten; Døssing, M G; Papa, S;

    2006-01-01

    IL-1beta is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1beta activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen...

  9. Low autocrine interferon beta production as a gene therapy approach for AIDS: Infusion of interferon beta-engineered lymphocytes in macaques chronically infected with SIVmac251

    Directory of Open Access Journals (Sweden)

    Dormont Dominique

    2004-09-01

    Full Text Available Abstract Background The aim of this study was to evaluate gene therapy for AIDS based on the transduction of circulating lymphocytes with a retroviral vector giving low levels of constitutive macaque interferon β production in macaques chronically infected with a pathogenic isolate of SIVmac251. Results Two groups of three animals infected for more than one year with a pathogenic primary isolate of SIVmac251 were included in this study. The macaques received three infusions of their own lymphocytes transduced ex vivo with the construct encoding macaque IFN-β (MaIFN-β or with a vector carrying a version of the MaIFN-β gene with a deletion preventing translation of the mRNA. Cellular or plasma viremia increased transiently following injection in most cases, regardless of the retroviral construct used. Transduced cells were detected only transiently after each infusion, among the peripheral blood mononuclear cells of all the animals, with copy numbers of 10 to 1000 per 106 peripheral mononuclear cells. Conclusion Long-term follow-up indicated that the transitory presence of such a small number of cells producing such small amounts of MaIFN-β did not prevent animals from the progressive decrease in CD4+ cell count typical of infection with simian immunodeficiency virus. These results reveal potential pitfalls for future developments of gene therapy strategies of HIV infection.

  10. Transforming growth factor beta-1 and interleukin-17 gene transcription in peripheral blood mononuclear cells and the human response to infection.

    LENUS (Irish Health Repository)

    White, Mary

    2012-02-01

    INTRODUCTION: The occurrence of severe sepsis may be associated with deficient pro-inflammatory cytokine production. Transforming growth factor beta-1 (TGFbeta-1) predominantly inhibits inflammation and may simultaneously promote IL-17 production. Interleukin-17 (IL-17) is a recently described pro-inflammatory cytokine, which may be important in auto-immunity and infection. We investigated the hypothesis that the onset of sepsis is related to differential TGFbeta-1 and IL-17 gene expression. METHODS: A prospective observational study in a mixed intensive care unit (ICU) and hospital wards in a university hospital. Patients (59) with severe sepsis; 15 patients with gram-negative bacteraemia but without critical illness and 10 healthy controls were assayed for TGFbeta-1, IL-17a, IL-17f, IL-6 and IL-1beta mRNA in peripheral blood mononuclear cells (PBMC) by quantitative real-time PCR and serum protein levels by ELISA. RESULTS: TGFbeta-1 mRNA levels are reduced in patients with bacteraemia and sepsis compared with controls (p=0.02). IL-6 mRNA levels were reduced in bacteraemic patients compared with septic patients and controls (p=0.008). IL-1beta mRNA levels were similar in all groups, IL-17a and IL-17f mRNA levels are not detectable in peripheral blood mononuclear cells. IL-6 protein levels were greater in patients with sepsis than bacteraemic and control patients (p<0.0001). Activated TGFbeta-1 and IL-17 protein levels were similar in all groups. IL-1beta protein was not detectable in the majority of patients. CONCLUSIONS: Down regulation of TGFbeta-1 gene transcription was related to the occurrence of infection but not the onset of sepsis. Interleukin-17 production in PBMC may not be significant in the human host response to infection.

  11. A Patient With Pyruvate Carboxylase Deficiency and Nemaline Rods on Muscle Biopsy

    DEFF Research Database (Denmark)

    Unal, Ozlem; Orhan, Diclehan; Ostergaard, Elsebet;

    2013-01-01

    and nemaline rods detected on muscle biopsy. The nemaline rods may be due to cellular energy shortage and altered energy metabolism in pyruvate carboxylase deficiency, similar to that in the previously reported patients. The mechanism of nemaline rod formation may be associated with the role of pyruvate...

  12. Ribulose 1,5-bisphosphate carboxylase and polyhedral bodies of Chlorogloeopsis fritschii.

    Science.gov (United States)

    Lanaras, T; Codd, G A

    1981-11-01

    Ribulose 1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) activity was approximately equally distributed between supernatant and pellet fractions produced by differential centrifugation of disrupted cells of Chlorogloeopsis fritschii. Low ionic strength buffer favoured the recovery of particulate RuBP carboxylase. Density gradient centrifugation of resuspended cell-free particulate material produced a single band of RuBP carboxylase activity, which was associated with the polyhedral body fraction, rather than with the thylakoids or other observable particles. Isolated polyhedral body stability was improved by density gradient centrifugation through gradients of Percoll plus sucrose in buffer, which yielded apparently intact polyhedral bodies. These were 100 to 150 nm in diameter and contained ring-shaped, 12 nm diameter particles. It is inferred that the C. fritschii polyhedral bodies are carboxysomes. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of SDS-dissociated polyhedral bodies revealed 8 major polypeptides. The most abundant, with molecular weights of 52,000 and 13,000, correspond with the large and small subunits, respectively, of RuBP carboxylase.

  13. Isolation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Leaves

    Science.gov (United States)

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a multi-functional enzyme that catalyzes the fixation of CO2 and O2 in photosynthesis and photorespiration, respectively. As the rate-limiting step in photosynthesis, improving the catalytic properties of Rubisco has long been viewed as a...

  14. Pyruvate Carboxylase Activates the RIG-I-like Receptor-Mediated Antiviral Immune Response by Targeting the MAVS signalosome

    Science.gov (United States)

    Cao, Zhongying; Zhou, Yaqin; Zhu, Shengli; Feng, Jian; Chen, Xueyuan; Liu, Shi; Peng, Nanfang; Yang, Xiaodan; Xu, Gang; Zhu, Ying

    2016-01-01

    When retinoic acid-inducible gene 1 protein (RIG-I)-like receptors sense viral dsRNA in the cytosol, RIG-I and melanoma differentiation-associated gene 5 (MDA5) are recruited to the mitochondria to interact with mitochondrial antiviral signaling protein (MAVS) and initiate antiviral immune responses. In this study, we demonstrate that the biotin-containing enzyme pyruvate carboxylase (PC) plays an essential role in the virus-triggered activation of nuclear factor kappa B (NF-κB) signaling mediated by MAVS. PC contributes to the enhanced production of type I interferons (IFNs) and pro-inflammatory cytokines, and PC knockdown inhibits the virus-triggered innate immune response. In addition, PC shows extensive antiviral activity against RNA viruses, including influenza A virus (IAV), human enterovirus 71 (EV71), and vesicular stomatitis virus (VSV). Furthermore, PC mediates antiviral action by targeting the MAVS signalosome and induces IFNs and pro-inflammatory cytokines by promoting phosphorylation of NF-κB inhibitor-α (IκBα) and the IκB kinase (IKK) complex, as well as NF-κB nuclear translocation, which leads to activation of interferon-stimulated genes (ISGs), including double-stranded RNA-dependent protein kinase (PKR) and myxovirus resistance protein 1 (Mx1). Our findings suggest that PC is an important player in host antiviral signaling. PMID:26906558

  15. Promotive Effect of Low Concentrations of NaHSO3 on Photophosphorylation and Photosynthesis in Phosphoenolpyruvate Carboxylase Transgenic Rice Leaves

    Institute of Scientific and Technical Information of China (English)

    Ben-Hua JI; Hong-He TAN; Rong ZHOU; De-Mao JIAO; Yun-Gang SHEN

    2005-01-01

    Spraying a 1-2 mmol/L solution of NaHSO3 on the leaves of wild-type rice (Oryza sativa L.)Kitaake (WT), phosphoenolpyruvate carboxylase (PEPC) transgenic (PC) rice and PEPC+phosphate dikinase (PPDK) transgenic rice (PC+PK), in which the germplasm was transformed with wild-type Kitaake as the gene receptor, resulted in an enhancement of the net photosynthetic rate by 23.0%, 28.8%, and 34.4%,respectively, for more than 3 d. It was also observed that NaHSO3 application caused an increase in the ATP content in leaves. Spraying PMS (a cofactor catalysing the photophosphorylation cycle) and NaHSO3 separately or together on leaves resulted in an increase in photosynthesis with all treatments. There was no additional effect on photosynthetic rate when the mixture was applied, suggesting that the mechanism by which NaHSO3 promotes photosynthesis is similar to the mechanism by which PMS acts and that both of compounds enhanced the supply of ATP. After spraying a solution of NaHSO3 on leaves, compared with the WT Kitaake rice, a greater enhancement of net photosynthetic rate was observed in PEPC transgenic (PC) and PEPC+PPDK transgenic (PC+PK) rice, with the greatest increase being observed in the latter group. Therefore ATP supply may become the limiting factor that concentrates CO2 in rice leaves transformed with an exogenous PEPC gene and exogenous PEPC+PPDK genes.

  16. Prevalence of SHV/CTX-M/TEM (ESBL Beta-lactamase Resistance Genes in Escherichia Coli Isolated from Urinary Tract Infections in Tehran, Iran

    Directory of Open Access Journals (Sweden)

    Yazdi M

    2010-01-01

    Full Text Available Background and objectives: Beta-lactamase enzymes are the most causes ofresistance to antibiotics among gram-negative bacteria. Nowadays, Infectionsdue to ESBLs are being increased throughout the world and is considered as anew burden to the health systems. This study aimed at determining thesensitivity pattern of E.coli isolates to beta-lactam antibiotics, andinvestigating the presence of blaCTX-M, blaTEM, and blaSHV genes in the urinesamples..Material and Methods: In this study, 244 E.coli isolates were screened in2009-2010. The antibiotic susceptibility of E. coli isolates were determined bydisc-diffusion method. Antimicrobial agents tested were cefoxatime,ceftazidime, imipenem, nalidixic acid, and ciprofloxacin. The combined disctest was used to confirm the results. The results were compared to the Clinicaland Laboratory Standards Institute (CLSI and ESBL positive isolates werefurther investigated for the presence of blaCTX-M, blaTEM, and blaSHV genes byPCR.Results: Of 244 E. coli isolates, 116 (47.1% are resistant to Ceftazidime, and96 (39.2% to cefoxatime. Also, 109 (44.3% isolates are ESBL positive.blaCTX-M, blaTEM, and blaSHV genes are found among 95 (87.1%, 75 (68.8%,and 77 (70.6% ESBL positive isolates, respectively. Forty (36.6% isolateshave all three genes, while 68 (62.3% include blaTEM and blaSHV genes.Moreover, 61 (55.9% isolates carry blaCTX-M and blaTEM genes, and 54(49.5%have blactx-M and blashv.Conclusion: Regarding the high frequency of resistance to the thirdgeneration cephalosporin antibiotics, precise antibiogram testing is highlyrecommended before any antibiotic prescription in cases of infections withESBL producing microorganisms.Key words: ESBL; Escherichia coli; blaCTX-M; blaTEM; blaSHV

  17. Phenotypic and gene expression changes between low (glucose-responsive) and High (glucose non-responsive) MIN-6 beta cells

    DEFF Research Database (Denmark)

    O´Driscoll, L.; Gammell, p.; McKierman, E.

    2006-01-01

    The long-term potential to routinely use replacement beta cells/islets as cell therapy for type 1 diabetes relies on our ability to culture such cells/islets, in vitro, while maintaining their functional status. Previous beta cell studies, by ourselves and other researchers, have indicated......, high passage) were determined by ELISA (assessing GSIS and cellular (pro)insulin content), proliferation assays, phase contrast light microscopy and analysis of alkaline phosphatase expression. Differential mRNA expression was investigated using microarray, bioinformatics and real-time PCR technologies......, to be significantly affected by passaging/ long-term culture. Loss/reduced levels, in high passage cells, of certain transcripts associated with the mature beta cell, together with increased levels of neuron/glia-associated mRNAs, suggest that, with time in culture, MIN-6 cells may revert to an early (possibly multi...

  18. Effect of beta-adrenoceptor blockers on human ether-a-go-go-related gene (HERG) potassium channels

    DEFF Research Database (Denmark)

    Dupuis, Delphine S; Klaerke, Dan A; Olesen, Søren-Peter

    2005-01-01

    Patients with congenital long QT syndrome may develop arrhythmias under conditions of increased sympathetic tone. We have addressed whether some of the beta-adrenoceptor blockers commonly used to prevent the development of these arrhythmias could per se block the cardiac HERG (Human Ether....... These data showed that HERG blockade by beta-adrenoceptor blockers occurred only at high micromolar concentrations, which are significantly above the recently established safe margin of 100 (Redfern et al., 2003).......-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride) blocked the HERG channel with similar affinity, whereas the beta1-receptor antagonists metoprolol and atenolol showed weak effects. Further, the four compounds blocked HERG channels expressed in a mammalian HEK293 cell line...

  19. [blaVIM-2 gene detection in metallo-beta-lactamase-producing Pseudomonas aeruginosa strains isolated in an intensive care unit in Ciudad Bolívar, Venezuela].

    Science.gov (United States)

    Guevara, Armando; de Waard, Jacobus; Araque, María

    2009-08-01

    Ten Pseudomonas aeruginosa strains with resistance to broad-spectrum cephalosporin and carbapenems were studied to determine the presence of genes that mediate the production of metallo-beta-lactamases. These strains were isolated from patients with nosocomial infection at the Intensive Care Unit of the Complejo Hospitalario "Ruiz y Paéz" of Ciudad Bolívar, Bolívar State, Venezuela, from 2003 to 2006. In all isolates a metallo-enzyme activity was detected by using the double disk synergism test. PCR amplification of genes encoding the families IMP, VIM and SPM metallo-beta-lactamases showed the presence of a blaVIM gene in all strains studied. DNA sequencing revealed that all isolates showed the presence of blaVIM-2. These results suggest that it is necessary to keep these strains under epidemiologic surveillance, establish laboratory strategies for opportune detection and the implementation of new policies to ensure the appropriate use of antibiotics in this institution.

  20. Abundances of tetracycline, sulphonamide and beta-lactam antibiotic resistance genes in conventional wastewater treatment plants (WWTPs) with different waste load.

    Science.gov (United States)

    Laht, Mailis; Karkman, Antti; Voolaid, Veiko; Ritz, Christian; Tenson, Tanel; Virta, Marko; Kisand, Veljo

    2014-01-01

    Antibiotics and antibiotic resistant bacteria enter wastewater treatment plants (WWTPs), an environment where resistance genes can potentially spread and exchange between microbes. Several antibiotic resistance genes (ARGs) were quantified using qPCR in three WWTPs of decreasing capacity located in Helsinki, Tallinn, and Tartu, respectively: sulphonamide resistance genes (sul1 and sul2), tetracycline resistance genes (tetM and tetC), and resistance genes for extended spectrum beta-lactams (blaoxa-58, blashv-34, and blactx-m-32). To avoid inconsistencies among qPCR assays we normalised the ARG abundances with 16S rRNA gene abundances while assessing if the respective genes increased or decreased during treatment. ARGs were detected in most samples; sul1, sul2, and tetM were detected in all samples. Statistically significant differences (adjusted p<0.01) between the inflow and effluent were detected in only four cases. Effluent values for blaoxa-58 and tetC decreased in the two larger plants while tetM decreased in the medium-sized plant. Only blashv-34 increased in the effluent from the medium-sized plant. In all other cases the purification process caused no significant change in the relative abundance of resistance genes, while the raw abundances fell by several orders of magnitude. Standard water quality variables (biological oxygen demand, total phosphorus and nitrogen, etc.) were weakly related or unrelated to the relative abundance of resistance genes. Based on our results we conclude that there is neither considerable enrichment nor purification of antibiotic resistance genes in studied conventional WWTPs.

  1. Abundances of tetracycline, sulphonamide and beta-lactam antibiotic resistance genes in conventional wastewater treatment plants (WWTPs with different waste load.

    Directory of Open Access Journals (Sweden)

    Mailis Laht

    Full Text Available Antibiotics and antibiotic resistant bacteria enter wastewater treatment plants (WWTPs, an environment where resistance genes can potentially spread and exchange between microbes. Several antibiotic resistance genes (ARGs were quantified using qPCR in three WWTPs of decreasing capacity located in Helsinki, Tallinn, and Tartu, respectively: sulphonamide resistance genes (sul1 and sul2, tetracycline resistance genes (tetM and tetC, and resistance genes for extended spectrum beta-lactams (blaoxa-58, blashv-34, and blactx-m-32. To avoid inconsistencies among qPCR assays we normalised the ARG abundances with 16S rRNA gene abundances while assessing if the respective genes increased or decreased during treatment. ARGs were detected in most samples; sul1, sul2, and tetM were detected in all samples. Statistically significant differences (adjusted p<0.01 between the inflow and effluent were detected in only four cases. Effluent values for blaoxa-58 and tetC decreased in the two larger plants while tetM decreased in the medium-sized plant. Only blashv-34 increased in the effluent from the medium-sized plant. In all other cases the purification process caused no significant change in the relative abundance of resistance genes, while the raw abundances fell by several orders of magnitude. Standard water quality variables (biological oxygen demand, total phosphorus and nitrogen, etc. were weakly related or unrelated to the relative abundance of resistance genes. Based on our results we conclude that there is neither considerable enrichment nor purification of antibiotic resistance genes in studied conventional WWTPs.

  2. Copy number variation of the beta defensin gene cluster on chromosome 8p influences the bacterial microbiota within the nasopharynx of otitis-prone children.

    Science.gov (United States)

    Jones, Eric A; Kananurak, Anchasa; Bevins, Charles L; Hollox, Edward J; Bakaletz, Lauren O

    2014-01-01

    As there is increasing evidence that aberrant defensin expression is related to susceptibility for infectious disease and inflammatory disorders, we sought to determine if copy number of the beta-defensin gene cluster located on chromosome 8p23.1 (DEFB107, 106, 105, 104, 103, DEFB4 and SPAG11), that shows copy number variation as a block, was associated with susceptibility to otitis media (OM). The gene DEFB103 within this complex encodes human beta defensin-3 (hBD-3), an antimicrobial peptide (AP) expressed by epithelial cells that line the mammalian airway, important for defense of mucosal surfaces and previously shown to have bactericidal activity in vitro against multiple human pathogens, including the three that predominate in OM. To this end, we conducted a retrospective case-control study of 113 OM prone children and 267 controls aged five to sixty months. We identified the copy number of the above defined beta-defensin gene cluster (DEFB-CN) in each study subject by paralogue ratio assays. The mean DEFB-CN was indistinguishable between subjects classified as OM prone based on a recent history of multiple episodes of OM and control subjects who had no history of OM (4.4 ± 0.96 versus 4.4 ± 1.08, respectively: Odds Ratio [OR]: 1.16 (95% CI: 0.61, 2.20). Despite a lack of direct association, we observed a statistically significant correlation between DEFB-CN and nasopharyngeal bacterial colonization patterns. Collectively, our findings suggested that susceptibility to OM might be mediated by genetic variation among individuals, wherein a DEFB-CN less than 4 exerts a marked influence on the microbiota of the nasopharynx, specifically with regard to colonization by the three predominant bacterial pathogens of OM.

  3. Copy number variation of the beta defensin gene cluster on chromosome 8p influences the bacterial microbiota within the nasopharynx of otitis-prone children.

    Directory of Open Access Journals (Sweden)

    Eric A Jones

    Full Text Available As there is increasing evidence that aberrant defensin expression is related to susceptibility for infectious disease and inflammatory disorders, we sought to determine if copy number of the beta-defensin gene cluster located on chromosome 8p23.1 (DEFB107, 106, 105, 104, 103, DEFB4 and SPAG11, that shows copy number variation as a block, was associated with susceptibility to otitis media (OM. The gene DEFB103 within this complex encodes human beta defensin-3 (hBD-3, an antimicrobial peptide (AP expressed by epithelial cells that line the mammalian airway, important for defense of mucosal surfaces and previously shown to have bactericidal activity in vitro against multiple human pathogens, including the three that predominate in OM. To this end, we conducted a retrospective case-control study of 113 OM prone children and 267 controls aged five to sixty months. We identified the copy number of the above defined beta-defensin gene cluster (DEFB-CN in each study subject by paralogue ratio assays. The mean DEFB-CN was indistinguishable between subjects classified as OM prone based on a recent history of multiple episodes of OM and control subjects who had no history of OM (4.4 ± 0.96 versus 4.4 ± 1.08, respectively: Odds Ratio [OR]: 1.16 (95% CI: 0.61, 2.20. Despite a lack of direct association, we observed a statistically significant correlation between DEFB-CN and nasopharyngeal bacterial colonization patterns. Collectively, our findings suggested that susceptibility to OM might be mediated by genetic variation among individuals, wherein a DEFB-CN less than 4 exerts a marked influence on the microbiota of the nasopharynx, specifically with regard to colonization by the three predominant bacterial pathogens of OM.

  4. Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta

    DEFF Research Database (Denmark)

    Hermanrud, Christina; Ryner, Malin; Luft, Thomas

    2016-01-01

    Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach...... was validated at Innsbruck Medical University (LUCIMU) and at Rigshospitalet (LUCRH) Copenhagen, and the iLite assay at Karolinska Institutet, Stockholm. For both assays, the optimal serum sample concentration in relation to sensitivity and recovery was 2.5% (v/v) in assay media. A Shapiro-Wilk test indicated...

  5. Retracted: Impact of polymorphisms in the oestrogen receptors alpha and beta (ESR1, ESR2) genes on risk of vasculogenic erectile dysfunction.

    Science.gov (United States)

    2014-01-01

    The above article from Andrology, 'Impact of polymorphisms in the oestrogen receptors alpha and beta (ESR1, ESR2) genes on risk of vasculogenic erectile dysfunction' by M. R. Safarinejad, A. Taghva, N. Shafiei and S. Safarinejad published online on 20 May 2013 in Wiley Online Library has been retracted by agreement between the journal Editors-in-Chief, Douglas Carrell and Ewa Rajpert-De Meyts and John Wiley and Sons Ltd. The retraction has been decided due to failure by the lead author to verify the data contained in the study, and to provide evidence of the role of co-authors and their institutional affiliations.

  6. Inhibition of beta-site amyloid precursor protein-cleaving enzyme and beta-amyloid precursor protein genes in SK-N-SH cells

    Institute of Scientific and Technical Information of China (English)

    Suqin Gao; Lin Sun; Enji Han; Hongshun Qi; Jinbo Feng; Shunliang Xu; Wen Xia

    2009-01-01

    BACKGROUND:Previous studies have demonstrated that Piper futokadsura stem selectively inhibits expression of amyloid precursor protein (APP) at the mRNA level.In addition,the piperlonguminine (A) and dihydropiperlonguminine (B) components (1:0.8),which can be separated from Futokadsura stem,selectively inhibit expression of the APP at mRNA and protein levels.OBJECTIVE:Based on previous findings,the present study investigated the effects of β-site amyloid precursor protein cleaving enzyme (BACE1) and APP genes on the production of β-amyloid peptide 42 (Aβ42) in human neuroblastoma cells (SK-N-SH cells) using small interfering RNAs (siRNAs) and A/B components separated from Futokadsura stem,respectively.DESIGN,TIME AND SETTING:A gene interference-based randomized,controlled,in vitro experiment was performed at the Key Laboratory of Cardiovascular Remodeling and Function Research,Ministries of Education and Public Health,and Institute of Pharmacologic Research,School of Pharmaceutical Science & Department of Biochemistry,School of Medicine,Shandong University between July 2006 and December 2007.MATERIALS:SK-N-SH cells were provided by Shanghai Institutes of Biological Sciences,Chinese Academy of Sciences,Shanghai,China;mouse anti-human BACE1 monoclonal antibody was purchased from R&D Systems,USA;mouse anti-human APP monoclonal antibody was purchased from Cell Signaling Technology,USA;and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was provided by Sigma,USA.METHODS:The human BACE1 cDNA sequence was obtained from NCBI website (www.ncbi.nlm.nih.gov/sites/entrez).Three pairs of siRNAs,specific to human BACE1 gene,were synthesized through the use of Silencer? pre-designed siRNA specification,and were transfected into SK-N-SH cells with siPORT NeoFX transfection agent to compare the effects of different concentrations of siRNAs (10-50 nmol/L) on SK-N-SH cells.Futokadsura stem was separated and purified with chemical methods,and the crystal was composed of

  7. Phylogenetic studies of pantherine cats (Felidae) based on multiple genes, with novel application of nuclear beta-fibrinogen intron 7 to carnivores.

    Science.gov (United States)

    Yu, Li; Zhang, Ya-Ping

    2005-05-01

    The pantherine lineage of the cat family Felidae (order: Carnivora) includes five big cats of genus Panthera and a great many midsized cats known worldwide. Presumably because of their recent and rapid radiation, the evolutionary relationship among pantherines remains ambiguous. We provide an independent assessment of the evolutionary history of pantherine lineage using two complete mitochondrial (mt) genes (ND2 and ND4) and the nuclear beta-fibrinogen intron 7 gene, whose utility in carnivoran phylogeny was first explored. The available four mt (ND5, cytb, 12S, and 16SrRNA) and two nuclear (IRBP and TTR) sequence loci were also combined to reconstruct phylogeny of 14 closely related cat species. Our analyses of combined mt data (six genes; approximately 3750 bp) and combined mt and nuclear data (nine genes; approximately 6500 bp) obtained identical tree topologies, which were well-resolved and strongly supported for almost all nodes. Monophyly of Panthera genus in pantherine lineage was confirmed and interspecific affinities within this genus revealed a novel branching pattern, with P. tigris diverging first in Panthera genus, followed by P. onca, P. leo, and last two sister species P. pardus and P. uncia. In addition, close association of Neofelis nebulosa to Panthera, the phylogenetic redefinition of Otocolobus manul within the domestic cat group, and the relatedness of Acinonyx jubatus and Puma concolor were all important findings in the resulting phylogenies. The potential utilities of nine different genes for phylogenetic resolution of closely related pantherine species were also evaluated, with special interest in that of the novel nuclear beta-fibrinogen intron 7.

  8. Search for genetic variants in the retinoid X receptor-gamma-gene by polymerase chain reaction-single-strand conformation polymorphism in patients with resistance to thyroid hormone without mutations in thyroid hormone receptor beta gene.

    Science.gov (United States)

    Romeo, Stefano; Menzaghi, Claudia; Bruno, Rocco; Sentinelli, Federica; Fallarino, Mara; Fioretti, Francesca; Filetti, Sebastiano; Balsamo, Armando; Di Mario, Umberto; Baroni, Marco G

    2004-05-01

    Resistance to thyroid hormone (RTH) is an inherited disease characterized by reduced tissue sensitivity to thyroid hormone. Approximately 90% of subjects with RTH have mutation in the thyroid hormone receptor beta (TRbeta) gene. Approximately 10% of subjects diagnosed as having RTH do not carry mutation in the TRbeta gene. A possible linkage was reported with the retinoid X receptor-gamma (RXR-gamma) gene in two families. The aim of this study is to search for mutation within the RXR-gamma gene in unrelated subjects with diagnosed RTH without mutations in the TRbeta gene. Four subjects with RTH were studied, and sequence variants in the RXR-gamma gene were searched by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Analysis of all the 10 exons of the RXR-gamma gene, including intron-exon boundaries, promoter region and 3' untranslated region (UTR) reveled two variant bands in subjects II and III. Sequencing of these variants showed two single nucleotide polymorphisms (SNPs): 447C > T in exon 3 for patients II and IVS9 + 6A > G for patient III. Both SNPs were also present at high frequency in a group of normal subjects and in nonaffected relatives of subject III. In conclusion, in patients with RTH we have found two SNPs in the RXR-gamma gene; these SNPS are common in the general population, thus excluding a role for the RXR-gamma gene in these patients.

  9. Water deficit modulates gene expression in growing zones of soybean seedlings. Analysis of differentially expressed cDNAs, a new beta-tubulin gene, and expression of genes encoding cell wall proteins.

    Science.gov (United States)

    Creelman, R A; Mullet, J E

    1991-10-01

    Transfer of soybean seedlings to low-water-potential vermiculite (psi w = -0.3 MPa) results in a reversible decrease in hypocotyl growth and modulation of several polysomal mRNAs (Plant Physiol 92: 205-214). We report here the isolation of two cDNA clones (pGE16 and pGE95) which correspond to genes whose mRNA levels are increased, and one cDNA clone (pGE23) which corresponds to a gene whose mRNA level is decreased in the hypocotyl zone of cell elongation by water deficit. In well-watered seedlings mRNAs hybridizing to pGE16 and pGE95 are most abundant in mature regions of the seedling, but in water-deficient seedlings mRNA levels are reduced in mature regions and enhanced in elongating regions. RNA corresponding to soybean proline-rich protein 1 (sbPRP1) shows a similar tissue distribution and response to water deficit. In contrast, in well-watered seedlings, the gene corresponding to pGE23 was highly expressed in the hypocotyl and root growing zones. Transfer of seedlings to low-water-potential vermiculite caused a rapid decrease in mRNA hybridizing to pGE23. Sequence analysis revealed that pGE23 has high homology with beta-tubulin. Water deficit also reduced the level of mRNA hybridizing to JCW1, an auxin-modulated gene, although with different kinetics. Furthermore, mRNA encoding actin, glycine-rich proteins (GRPs), and hydroxyproline-rich glycoproteins (HRGPs) were down-regulated in the hypocotyl zone of elongation of seedlings exposed to water deficit. No effect of water deficit was observed on the expression of chalcone synthase. Decreased expression of beta-tubulin, actin, JCW1, HRGP and GRP and increased expression of sbPRP1, pGE95 and pGE16 in the hypocotyl zone of cell elongation could participate in the reversible growth inhibition observed in water-deficient soybean seedlings.

  10. The association of single nucleotide polymorphisms of the maternal cystathionine-beta-synthase gene with early-onset preeclampsia

    NARCIS (Netherlands)

    Holwerda, Kim M.; Weedon-Fekjaer, M. Susanne; Staff, Anne C.; Nolte, Ilja M.; van Goor, Harry; Lely, A. Titia; Faas, Marijke M.

    2016-01-01

    Objectives: Preeclampsia (PE) is a pregnancy complication, characterized by hypertension and proteinuria. The transsulfuration pathway may be involved in its pathophysiology, since homocysteine, cystathionine and cysteine are increased in PE. Cystathionine-beta-synthase (CBS) is a key-enzyme in the

  11. Associação dos polimorfismos dos genes TGF-beta1, CD14, IL-4, IL-4R e ADAM33 com a gravidade da asma em crianças e adolescentes Association of TGF-beta1, CD14, IL-4, IL-4R and ADAM33 gene polymorphisms with asthma severity in children and adolescents

    Directory of Open Access Journals (Sweden)

    Isabel C. J. de Faria

    2008-06-01

    Full Text Available OBJETIVO: Verificar, em uma amostra de pacientes com asma atópica persistente leve, moderada e grave, a associação entre os polimorfismos dos genes fator de crescimento transformante-beta1 (TGF-beta1 (C-509T e T869C, CD14 (C-159T, IL-4 (C-590T, IL-4R (ILe50Val e ADAM33 (S_2 com a gravidade da asma. MÉTODOS: Realizou-se um estudo clínico laboratorial prospectivo em pacientes com asma atópica persistente, comparados a um grupo controle no Hospital Universitário da Universidade Estadual de Campinas nos anos de 2006 e 2007. A análise do polimorfismo T869C do gene TGF-beta1 foi realizada pela técnica de reação em cadeia da polimerase (PCR + sistema de amplificação refratária de mutação (ARMS. Os outros polimorfismos, C-509T do gene TGF-beta1, C-159T do gene CD14, C-590T da IL-4, ILe50Val da IL-4Ra e S2 do gene ADAM33, foram detectados por PCR e enzima de restrição. RESULTADOS: Foram incluídos 88 pacientes com asma atópica persistente (27 leves, 23 moderados e 38 graves e 202 indivíduos saudáveis, doadores de sangue. Em relação ao polimorfismo T869C (TGF-beta1, observou-se uma associação entre o genótipo CC e os pacientes com asma grave. Nenhuma associação foi encontrada com os polimorfismos C-509T (TGF-beta1, C-590T (IL4 e S_2 (ADAM33. Quando se comparou a distribuição da freqüência genotípica do polimorfismo C-159T (CD14 na asma grave com o grupo controle, foi observado um resultado significativo com o genótipo TT. Houve associação significativa do genótipo Val/Val (IL-4R com a asma leve. CONCLUSÃO: Nossos resultados indicam que os polimorfismos T869C (TGF-beta1, C-159T (CD14 e Val/Val (IL-4R podem estar envolvidos na modulação da gravidade da asma.OBJECTIVE: To verify the association of transforming growth factor-beta1 (TGF-beta1 (C-509T and T869C, CD14 (C-159T, IL-4 (C-590T, IL-4R (ILe50Val and ADAM33 (S_2 gene polymorphisms with asthma severity in a sample of patients with mild, moderate and severe

  12. Methyl jasmonate- or gibberellins A3-induced astaxanthin accumulation is associated with up-regulation of transcription of beta-carotene ketolase genes (bkts) in microalga Haematococcus pluvialis.

    Science.gov (United States)

    Lu, Yandu; Jiang, Peng; Liu, Shaofang; Gan, Qinhua; Cui, Hongli; Qin, Song

    2010-08-01

    The microalga Haematococcus pluvialis accumulates astaxanthin in response to abiotic stresses. Since methyl jasmonate (MJ) and gibberellins A(3) (GA(3)) are involved in the stress responses of plants, the impact of these compounds on astaxanthin metabolism was studied. Alga cells treated separately with MJ and GA(3) accumulated more astaxanthin than the controls. MJ and GA(3) treatment increased the transcription of three beta-carotene ketolase genes (bkts). MJ- and GA(3)-responsive cis-acting elements were identified in the 5'-flanking regions of bkt genes. These results suggest that MJ and GA(3) constitute molecular signals in the network of astaxanthin accumulation. Induction of astaxanthin accumulation by MJ or GA(3) without any other stimuli presents an attractive application potential.

  13. Integron-associated mobile gene cassettes code for folded proteins: the structure of Bal32a, a new member of the adaptable alpha+beta barrel family.

    Science.gov (United States)

    Robinson, Andrew; Wu, Peter S-C; Harrop, Stephen J; Schaeffer, Patrick M; Dosztányi, Zsuzsanna; Gillings, Michael R; Holmes, Andrew J; Nevalainen, K M Helena; Stokes, H W; Otting, Gottfried; Dixon, Nicholas E; Curmi, Paul M G; Mabbutt, Bridget C

    2005-03-11

    The wide-ranging physiology and large genetic variability observed for prokaryotes is largely attributed, not to the prokaryotic genome itself, but rather to mechanisms of lateral gene transfer. Cassette PCR has been used to sample the integron/gene cassette metagenome from different natural environments without laboratory cultivation of the host organism, and without prior knowledge of any target protein sequence. Since over 90% of cassette genes are unrelated to any sequence in the current databases, it is not clear whether these genes code for folded functional proteins. We have selected a sample of eight cassette-encoded genes with no known homologs; five have been isolated as soluble protein products and shown by biophysical techniques to be folded. In solution, at least three of these proteins organise as stable oligomeric assemblies. The tertiary structure of one of these, Bal32a derived from a contaminated soil site, has been solved by X-ray crystallography to 1.8 A resolution. From the three-dimensional structure, Bal32a is found to be a member of the highly adaptable alpha+beta barrel family of transport proteins and enzymes. In Bal32a, the barrel cavity is unusually deep and inaccessible to solvent. Polar side-chains in its interior are reminiscent of catalytic sites of limonene-1,2-epoxide hydrolase and nogalonic acid methyl ester cyclase. These studies demonstrate the viability of direct sampling of mobile DNA as a route for the discovery of novel proteins.

  14. Up-regulation of the ectodermal-neural cortex 1 (ENC1) gene, a downstream target of the beta-catenin/T-cell factor complex, in colorectal carcinomas.

    Science.gov (United States)

    Fujita, M; Furukawa, Y; Tsunoda, T; Tanaka, T; Ogawa, M; Nakamura, Y

    2001-11-01

    To clarify the molecular mechanisms of human carcinogenesis associated with abnormal Wnt/wingless signaling, we searched for genes the expression of which was significantly altered by introduction of wild-type AXIN1 into LoVo colon cancer cells. By means of a cDNA microarray, we compared expression profiles of LoVo cells infected with either adenoviruses expressing wild-type AXIN1 (Ad-Axin) or those expressing a control gene (Ad-LacZ). Among the genes showing altered expression, the ectodermal-neural cortex 1 (ENC1) gene was down-regulated in response to Ad-Axin. The promoter activity of ENC1 was elevated approximately 3-fold by transfection of an activated form of beta-catenin together with wild-type T-cell factor (Tcf)4 in HeLa cells. Semiquantitative reverse transcription-PCR experiments revealed that expression of ENC1 was increased in more than two-thirds of 24 primary colon cancer tissues that we examined compared with corresponding noncancerous mucosae. Introduction of exogenous ENC1 increased the growth rate of HCT116 colon cancer cells in serum-depleted medium. In other experiments, overexpression of ENC1 in HT-29 colon cancer cells suppressed the usual increase of two differentiation markers, in response to treatment with sodium butyrate, a differentiation-inducible agent. These data suggest that ENC1 is regulated by the beta-catenin/Tcf pathway and that its altered expression may contribute to colorectal carcinogenesis by suppressing differentiation of colonic cells.

  15. Palaeohexaploid ancestry for Caryophyllales inferred from extensive gene-based physical and genetic mapping of the sugar beet genome (Beta vulgaris).

    Science.gov (United States)

    Dohm, Juliane C; Lange, Cornelia; Holtgräwe, Daniela; Sörensen, Thomas Rosleff; Borchardt, Dietrich; Schulz, Britta; Lehrach, Hans; Weisshaar, Bernd; Himmelbauer, Heinz

    2012-05-01

    Sugar beet (Beta vulgaris) is an important crop plant that accounts for 30% of the world's sugar production annually. The genus Beta is a distant relative of currently sequenced taxa within the core eudicotyledons; the genomic characterization of sugar beet is essential to make its genome accessible to molecular dissection. Here, we present comprehensive genomic information in genetic and physical maps that cover all nine chromosomes. Based on this information we identified the proposed ancestral linkage groups of rosids and asterids within the sugar beet genome. We generated an extended genetic map that comprises 1127 single nucleotide polymorphism markers prepared from expressed sequence tags and bacterial artificial chromosome (BAC) end sequences. To construct a genome-wide physical map, we hybridized gene-derived oligomer probes against two BAC libraries with 9.5-fold cumulative coverage of the 758 Mbp genome. More than 2500 probes and clones were integrated both in genetic maps and the physical data. The final physical map encompasses 535 chromosomally anchored contigs that contains 8361 probes and 22 815 BAC clones. By using the gene order established with the physical map, we detected regions of synteny between sugar beet (order Caryophyllales) and rosid species that involves 1400-2700 genes in the sequenced genomes of Arabidopsis, poplar, grapevine, and cacao. The data suggest that Caryophyllales share the palaeohexaploid ancestor proposed for rosids and asterids. Taken together, we here provide extensive molecular resources for sugar beet and enable future high-resolution trait mapping, gene identification, and cross-referencing to regions sequenced in other plant species.

  16. A crotonyl-CoA reductase-carboxylase independent pathway for assembly of unusual alkylmalonyl-CoA polyketide synthase extender units

    Science.gov (United States)

    Ray, Lauren; Valentic, Timothy R.; Miyazawa, Takeshi; Withall, David M.; Song, Lijiang; Milligan, Jacob C.; Osada, Hiroyuki; Takahashi, Shunji; Tsai, Shiou-Chuan; Challis, Gregory L.

    2016-12-01

    Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues.

  17. Induction of nuclear factor-kappaB and its downstream genes by TNF-alpha and IL-1beta has a pro-apoptotic role in pancreatic beta cells

    DEFF Research Database (Denmark)

    Ortis, F; Pirot, P; Naamane, N

    2008-01-01

    AIMS/HYPOTHESIS: IL-1beta and TNF-alpha contribute to pancreatic beta cell death in type 1 diabetes. Both cytokines activate the transcription factor nuclear factor-kappaB (NF-kappaB), but recent observations suggest that NF-kappaB blockade prevents IL-1beta + IFN-gamma- but not TNF-alpha + IFN-g...

  18. Molecular mechanisms underlying the glucose-dependent transcription of the insulin and glucokinase genes in the pancreatic beta-cell

    OpenAIRE

    2002-01-01

    Background: Insulin is of vital importance in the maintenance of the glucose homeostasis in mammals. This necessitates a tight regulation of both insulin release and biosynthesis. Although pancreatic beta-cells secrete only a fraction of the stored insulin upon glucose stimulation, insulin biosynthesis starts immediately in order to replenish the insulin store. It is well documented that glucose exerts its immediate effects at the posttranscriptional and translational levels...

  19. Genomic organization and differential signature of positive selection in the alpha and beta globin gene clusters in two cetacean species.

    Science.gov (United States)

    Nery, Mariana F; Arroyo, José Ignacio; Opazo, Juan C

    2013-01-01

    The hemoglobin of jawed vertebrates is a heterotetramer protein that contains two α- and two β-chains, which are encoded by members of α- and β-globin gene families. Given the hemoglobin role in mediating an adaptive response to chronic hypoxia, it is likely that this molecule may have experienced a selective pressure during the evolution of cetaceans, which have to deal with hypoxia tolerance during prolonged diving. This selective pressure could have generated a complex history of gene turnover in these clusters and/or changes in protein structure themselves. Accordingly, we aimed to characterize the genomic organization of α- and β-globin gene clusters in two cetacean species and to detect a possible role of positive selection on them using a phylogenetic framework. Maximum likelihood and Bayesian phylogeny reconstructions revealed that both cetacean species had retained a similar complement of putatively functional genes. For the α-globin gene cluster, the killer whale presents a complement of genes composed of HBZ, HBK, and two functional copies of HBA and HBQ genes, whereas the dolphin possesses HBZ, HBK, HBA and HBQ genes, and one HBA pseudogene. For the β-globin gene cluster, both species retained a complement of four genes, two early expressed genes-HBE and HBH-and two adult expressed genes-HBD and HBB. Our natural selection analysis detected two positively selected sites in the HBB gene (56 and 62) and four in HBA (15, 21, 49, 120). Interestingly, only the genes that are expressed during the adulthood showed the signature of positive selection.

  20. Crystal Structures of Human and Staphylococcus aureus Pyruvate Carboxylase and Molecular Insights into the Carboxyltransfer Reaction

    Energy Technology Data Exchange (ETDEWEB)

    Xiang,S.; Tong, L.

    2008-01-01

    Pyruvate carboxylase (PC) catalyzes the biotin-dependent production of oxaloacetate and has important roles in gluconeogenesis, lipogenesis, insulin secretion and other cellular processes. PC contains the biotin carboxylase (BC), carboxyltransferase (CT) and biotin-carboxyl carrier protein (BCCP) domains. We report here the crystal structures at 2.8-Angstroms resolution of full-length PC from Staphylococcus aureus and the C-terminal region (missing only the BC domain) of human PC. A conserved tetrameric association is observed for both enzymes, and our structural and mutagenesis studies reveal a previously uncharacterized domain, the PC tetramerization (PT) domain, which is important for oligomerization. A BCCP domain is located in the active site of the CT domain, providing the first molecular insights into how biotin participates in the carboxyltransfer reaction. There are dramatic differences in domain positions in the monomer and the organization of the tetramer between these enzymes and the PC from Rhizobium etli.

  1. Generation of Diversity in Streptococcus mutans Genes Demonstrated by MLST

    OpenAIRE

    Thuy Do; Gilbert, Steven C.; Douglas Clark; Farida Ali; Clarissa C Fatturi Parolo; Marisa Maltz; Russell, Roy R.; Peter Holbrook; Wade, William G.; David Beighton

    2010-01-01

    Streptococcus mutans, consisting of serotypes c, e, f and k, is an oral aciduric organism associated with the initiation and progression of dental caries. A total of 135 independent Streptococcus mutans strains from caries-free and caries-active subjects isolated from various geographical locations were examined in two versions of an MLST scheme consisting of either 6 housekeeping genes [accC (acetyl-CoA carboxylase biotin carboxylase subunit), gki (glucokinase), lepA (GTP-binding protein), r...

  2. Breast Cancer-Derived Lung Metastases Show Increased Pyruvate Carboxylase-Dependent Anaplerosis

    OpenAIRE

    Stefan Christen; Doriane Lorendeau; Roberta Schmieder; Dorien Broekaert; Kristine Metzger; Koen Veys; Ilaria Elia; Joerg Martin Buescher; Martin Franz Orth; Shawn Michael Davidson; Thomas Georg Philipp Grünewald; Katrien De Bock; Sarah-Maria Fendt

    2016-01-01

    Cellular proliferation depends on refilling the tricarboxylic acid (TCA) cycle to support biomass production (anaplerosis). The two major anaplerotic pathways in cells are pyruvate conversion to oxaloacetate via pyruvate carboxylase (PC) and glutamine conversion to α-ketoglutarate. Cancers often show an organ-specific reliance on either pathway. However, it remains unknown whether they adapt their mode of anaplerosis when metastasizing to a distant organ. We measured PC-dependent anaplerosis ...

  3. (4-Piperidinyl)-piperazine: a new platform for acetyl-CoA carboxylase inhibitors.

    Science.gov (United States)

    Chonan, Tomomichi; Oi, Takahiro; Yamamoto, Daisuke; Yashiro, Miyoko; Wakasugi, Daisuke; Tanaka, Hiroaki; Ohoka-Sugita, Ayumi; Io, Fusayo; Koretsune, Hiroko; Hiratate, Akira

    2009-12-01

    Acetyl-CoA carboxylases (ACCs), the rate limiting enzymes in de novo lipid synthesis, play important roles in modulating energy metabolism. The inhibition of ACC has demonstrated promising therapeutic potential for treating obesity and type 2 diabetes mellitus in transgenic mice and preclinical animal models. We describe herein the synthesis and structure-activity relationships of a series of disubstituted (4-piperidinyl)-piperazine derivatives as a new platform for ACC1/2 non-selective inhibitors.

  4. beta (+)-Thalassaemia in the Po river delta region (northern Italy): genotype and beta globin synthesis.

    Science.gov (United States)

    Del Senno, L; Pirastu, M; Barbieri, R; Bernardi, F; Buzzoni, D; Marchetti, G; Perrotta, C; Vullo, C; Kan, Y W; Conconi, F

    1985-01-01

    Six beta(+)-thalassaemic patients from the Po river delta region have been studied. Using synthetic oligonucleotides as specific hybridisation probes, the beta(+) IVS I mutation (G----A at position 108) was demonstrated. This lesion and the enzyme polymorphism pattern in the subjects examined are the same as have been described for other Mediterranean beta(+)-thalassaemias. Antenatal diagnosis through DNA analysis of beta(+)-thalassaemia is therefore possible. The production of beta globin in a beta(+), homozygote and in a beta (+), beta(0) 39 (nonsense mutation at codon 39) double heterozygote is approximately 20% and 10% respectively of total non-alpha globin synthesis. Despite some overlapping of the results, similar beta globin synthesis levels have been obtained in 43 beta(+)-thalassaemia patients. This suggests that in the Po river delta region the most common thalassaemic genes are beta(0) 39 and beta(+) IVS I. Images PMID:2580095

  5. Mutagenesis of beta-1,3-Glucanase Genes in Lysobacter enzymogenes Strain C3 Results in Reduced Biological Control Activity Toward Bipolaris Leaf Spot of Tall Fescue and Pythium Damping-Off of Sugar Beet.

    Science.gov (United States)

    Palumbo, Jeffrey D; Yuen, Gary Y; Jochum, C Christine; Tatum, Kristin; Kobayashi, Donald Y

    2005-06-01

    ABSTRACT Lysobacter enzymogenes produces extracellular lytic enzymes capable of degrading the cell walls of fungi and oomycetes. Many of these enzymes, including beta-1,3-glucanases, are thought to contribute to the biological control activity expressed by several strains of the species. L. enzymogenes strain C3 produces multiple extracellular beta-1,3-glucanases encoded by the gluA, gluB, and gluC genes. Analysis of the genes indicates they are homologous to previously characterized genes in the related strain N4-7, each sharing >95% amino acid sequence identity to their respective counterparts. The gluA and gluC gene products encode enzymes belonging to family 16 glycosyl hydrolases, whereas gluB encodes an enzyme belonging to family 64. Mutational analysis indicated that the three genes accounted for the total beta-1,3-glucanase activity detected in culture. Strain G123, mutated in all three glucanase genes, was reduced in its ability to grow in a minimal medium containing laminarin as a sole carbon source. Although strain G123 was not affected in antimicrobial activity toward Bipolaris sorokiniana or Pythium ultimum var. ultimum using in vitro assays, it was significantly reduced in biological control activity against Bipolaris leaf spot of tall fescue and Pythium damping-off of sugar beet. These results provide direct supportive evidence for the role of beta-1,3-glucanases in biocontrol activity of L. enzymogenes strain C3.

  6. Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxgenase large subunit .epsilon. n-methyltransferase and method of inactivating ribulose-1,5-bishosphatase .epsilon. n-methyltransferase activity

    Science.gov (United States)

    Houtz, Robert L.

    2001-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltansferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

  7. The DUB/USP17 deubiquitinating enzymes: A gene family within a tandemly repeated sequence, is also embedded within the copy number variable Beta-defensin cluster

    Directory of Open Access Journals (Sweden)

    Scott Christopher J

    2010-04-01

    Full Text Available Abstract Background The DUB/USP17 subfamily of deubiquitinating enzymes were originally identified as immediate early genes induced in response to cytokine stimulation in mice (DUB-1, DUB-1A, DUB-2, DUB-2A. Subsequently we have identified a number of human family members and shown that one of these (DUB-3 is also cytokine inducible. We originally showed that constitutive expression of DUB-3 can block cell proliferation and more recently we have demonstrated that this is due to its regulation of the ubiquitination and activity of the 'CAAX' box protease RCE1. Results Here we demonstrate that the human DUB/USP17 family members are found on both chromosome 4p16.1, within a block of tandem repeats, and on chromosome 8p23.1, embedded within the copy number variable beta-defensin cluster. In addition, we show that the multiple genes observed in humans and other distantly related mammals have arisen due to the independent expansion of an ancestral sequence within each species. However, it is also apparent when sequences from humans and the more closely related chimpanzee are compared, that duplication events have taken place prior to these species separating. Conclusions The observation that the DUB/USP17 genes, which can influence cell growth and survival, have evolved from an unstable ancestral sequence which has undergone multiple and varied duplications in the species examined marks this as a unique family. In addition, their presence within the beta-defensin repeat raises the question whether they may contribute to the influence of this repeat on immune related conditions.

  8. The DYRK1A gene, encoded in chromosome 21 Down syndrome critical region, bridges between beta-amyloid production and tau phosphorylation in Alzheimer disease.

    Science.gov (United States)

    Kimura, Ryo; Kamino, Kouzin; Yamamoto, Mitsuko; Nuripa, Aidaralieva; Kida, Tomoyuki; Kazui, Hiroaki; Hashimoto, Ryota; Tanaka, Toshihisa; Kudo, Takashi; Yamagata, Hidehisa; Tabara, Yasuharu; Miki, Tetsuro; Akatsu, Hiroyasu; Kosaka, Kenji; Funakoshi, Eishi; Nishitomi, Kouhei; Sakaguchi, Gaku; Kato, Akira; Hattori, Hideyuki; Uema, Takeshi; Takeda, Masatoshi

    2007-01-01

    We scanned throughout chromosome 21 to assess genetic associations with late-onset Alzheimer disease (AD) using 374 Japanese patients and 375 population-based controls, because trisomy 21 is known to be associated with early deposition of beta-amyloid (Abeta) in the brain. Among 417 markers spanning 33 Mb, 22 markers showed associations with either the allele or the genotype frequency (P KCNJ6 genes. In logistic regression, the DYRK1A (dual-specificity tyrosine-regulated kinase 1A) gene, located in the Down syndrome critical region, showed the highest significance [OR = 2.99 (95% CI: 1.72-5.19), P = 0.001], whereas the RUNX1 gene showed a high odds ratio [OR = 23.3 (95% CI: 2.76-196.5), P = 0.038]. DYRK1A mRNA level in the hippocampus was significantly elevated in patients with AD when compared with pathological controls (P < 0.01). DYRK1A mRNA level was upregulated along with an increase in the Abeta-level in the brain of transgenic mice, overproducing Abeta at 9 months of age. In neuroblastoma cells, Abeta induced an increase in the DYRK1A transcript, which also led to tau phosphorylation at Thr212 under the overexpression of tau. Therefore, the upregulation of DYRK1A transcription results from Abeta loading, further leading to tau phosphorylation. Our result indicates that DYRK1A could be a key molecule bridging between beta-amyloid production and tau phosphorylation in AD.

  9. A substrate-induced biotin binding pocket in the carboxyltransferase domain of pyruvate carboxylase.

    Science.gov (United States)

    Lietzan, Adam D; St Maurice, Martin

    2013-07-05

    Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp(590) and Tyr(628) and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes.

  10. The promoter of the nematode resistance gene Hs1pro-1 activates a nematode-responsive and feeding site-specific gene expression in sugar beet (Beta vulgaris L.) and Arabidopsis thaliana.

    Science.gov (United States)

    Thurau, Tim; Kifle, Sirak; Jung, Christian; Cai, Daguang

    2003-06-01

    The Hs1pro-1 gene confers resistance to the beet cyst nematode Heterodera schachtii in sugar beet (Beta vulgaris L.) on the basis of a gene-for-gene relationship. RNA-gel blot analysis revealed that the transcript of Hs1pro-1 was present in uninfected roots of resistant beet at low levels but increased by about fourfold one day after nematode infection. Treatments of plants with external stimuli including salicylic acid, jasmonic acid, gibberellic acid and abscisic acid as well as wounding or salt stress did not result in changes in the gene transcription, indicating de novo transcription of Hs1pro-1 upon nematode infection specifically. To study transcriptional regulation of Hs1pro-1 expression at the cellular level, a 3082 bp genomic fragment representing the Hs1pro-1 promoter, isolated from the YAC-DNA housing the Hs1pro-1 gene, was fused to the beta-glucuronidase reporter gene (1832prm1::GUS) and transformed into susceptible beet roots and Arabidopsis plants, respectively. Fluorometric and histochemical GUS assays on transgenic beet roots and Arabidopsis plants carrying the 1832prm1::GUS construct demonstrated that the Hs1pro-1 promoter is functional in both species and drives a nematode responsive and feeding site-specific GUS-expression. GUS activity was detected as early as at initiation of the nematode feeding sites and GUS staining was restricted to the nematode feeding sites. To delineate the regulatory domains of the Hs1pro-1 promoter, fusion genes with various 5' deletions of the Hs1pro-1 promoter and the GUS gene were constructed and analysed in transgenic beet roots as well. Cis elements responsible for feeding site-specific gene expression reside between -355 and +247 from the transcriptional initiation site of Hs1pro-1 whereas an enhancer region necessary for higher gene expression is located between -1199 and -705 of the promoter. The Hs1pro-1 promoter drives a nematode feeding site-specific GUS expression in both sugar beet and Arabidopsis

  11. Interaction between potyvirus P3 and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) of host plants.

    Science.gov (United States)

    Lin, Lin; Luo, Zhaopeng; Yan, Fei; Lu, Yuwen; Zheng, Hongying; Chen, Jianping

    2011-08-01

    The P3 protein encoded by Shallot yellow stripe virus onion isolate (SYSV-O) interacted in the Yeast Two-hybrid (Y2H) system and in co-immunoprecipitation (Co-IP) assays with the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) protein that is encoded by the rbcL gene of its onion host. Dissection analysis by Y2H showed that the main part of SYSV P3 (amino acids 1-390) and onion RbcL (amino acids 1-137) were responsible for the interaction. The P3 proteins encoded by Onion yellow dwarf virus (OYDV), Soybean mosaic virus Pinellia isolate (SMV-P), and Turnip mosaic virus (TuMV) also interacted with RbcL, suggesting that a P3/RbcL interaction might exist generally for potyviruses. An interaction between P3 of these potyviruses and the small subunit of RubisCO (RbcS) was also demonstrated. Moreover, the P3N-PIPO protein encoded by a newly identified open reading frame embedded within the P3 cistron also interacted with both RbcL and RbcS. It is possible that the potyvirus P3 protein affects the normal functions of RubisCO which thus contributes to symptom development.

  12. Mechanism of metamifop inhibition of the carboxyltransferase domain of acetyl-coenzyme A carboxylase in Echinochloa crus-galli

    Science.gov (United States)

    Xia, Xiangdong; Tang, Wenjie; He, Shun; Kang, Jing; Ma, Hongju; Li, Jianhong

    2016-09-01

    Acetyl-coenzyme A carboxylase (ACCase) plays crucial roles in fatty acid metabolism and is an attractive target for herbicide discovery. Metamifop is a novel ACCase-inhibiting herbicide that can be applied to control sensitive weeds in paddy fields. In this study, the effects of metamifop on the chloroplasts, ACCase activity and carboxyltransferase (CT) domain gene expression in Echinochloa crus-galli were investigated. The results showed that metamifop interacted with the CT domain of ACCase in E. crus-galli. The three-dimensional structure of the CT domain of E. crus-galli ACCase in complex with metamifop was examined by homology modelling, molecular docking and molecular dynamics (MD) simulations. Metamifop has a different mechanism of inhibiting the CT domain compared with other ACCase inhibitors as it interacted with a different region in the active site of the CT domain. The protonation of nitrogen in the oxazole ring of metamifop plays a crucial role in the interaction between metamifop and the CT domain. The binding mode of metamifop provides a foundation for elucidating the molecular mechanism of target resistance and cross-resistance among ACCase herbicides, and for designing and optimizing ACCase inhibitors.

  13. Acute Radiation-Induced Nocturia in Prostate Cancer Patients Is Associated With Pretreatment Symptoms, Radical Prostatectomy, and Genetic Markers in the TGF{beta}1 Gene

    Energy Technology Data Exchange (ETDEWEB)

    De Langhe, Sofie, E-mail: Sofie.DeLanghe@UGent.be [Department of Basic Medical Sciences, Ghent University, Gent (Belgium); De Ruyck, Kim [Department of Basic Medical Sciences, Ghent University, Gent (Belgium); Ost, Piet; Fonteyne, Valerie [Department of Radiation Oncology, Ghent University Hospital, Gent (Belgium); Werbrouck, Joke [Department of Basic Medical Sciences, Ghent University, Gent (Belgium); De Meerleer, Gert; De Neve, Wilfried [Department of Radiation Oncology, Ghent University Hospital, Gent (Belgium); Thierens, Hubert [Department of Basic Medical Sciences, Ghent University, Gent (Belgium)

    2013-02-01

    Purpose: After radiation therapy for prostate cancer, approximately 50% of the patients experience acute genitourinary symptoms, mostly nocturia. This may be highly bothersome with a major impact on the patient's quality of life. In the past, nocturia is seldom reported as a single, physiologically distinct endpoint, and little is known about its etiology. It is assumed that in addition to dose-volume parameters and patient- and therapy-related factors, a genetic component contributes to the development of radiation-induced damage. In this study, we investigated the association among dosimetric, clinical, and TGF{beta}1 polymorphisms and the development of acute radiation-induced nocturia in prostate cancer patients. Methods and Materials: Data were available for 322 prostate cancer patients treated with primary or postoperative intensity modulated radiation therapy (IMRT). Five genetic markers in the TGF{beta}1 gene (-800 G>A, -509 C>T, codon 10 T>C, codon 25 G>C, g.10780 T>G), and a high number of clinical and dosimetric parameters were considered. Toxicity was scored using an symptom scale developed in-house. Results: Radical prostatectomy (P<.001) and the presence of pretreatment nocturia (P<.001) are significantly associated with the occurrence of radiation-induced acute toxicity. The -509 CT/TT (P=.010) and codon 10 TC/CC (P=.005) genotypes are significantly associated with an increased risk for radiation-induced acute nocturia. Conclusions: Radical prostatectomy, the presence of pretreatment nocturia symptoms, and the variant alleles of TGF{beta}1 -509 C>T and codon 10 T>C are identified as factors involved in the development of acute radiation-induced nocturia. These findings may contribute to the research on prediction of late nocturia after IMRT for prostate cancer.

  14. SNPs within the beta myosin heavy chain (MYH7 and the pyruvate kinase muscle (PKM2 genes in horse

    Directory of Open Access Journals (Sweden)

    Vincenzo Russo

    2010-01-01

    Full Text Available Two highly expressed skeletal muscle genes (the MYH7 gene encoding the myosin heavy chain slow/β-cardiac isoform and the PKM2 gene encoding the pyruvate kinase muscle isoforms were investigated with the objective to identify DNA markers in horses. A panel of DNA samples from different horse breeds was analysed using a PCR-single strand conformation polymorphism (SSCP approach. Four and two alleles were identified for the MYH7 and PKM2 loci, respectively. Mendelian inheritance of alleles of the two investigated genes was confirmed analysing horse families. Sequencing of PCR products obtained from the MYH7 and PKM2 genes made it possible to characterise two SSCP alleles for each gene. The polymorphisms found in the MYH7 and PKM2 genes were further studied in 61 and 68 horses of three (Italian Heavy Draught Horse, Italian Saddler and Murgese and five (Franches-Montagnes, Haflinger, Italian Heavy Draught Horse, Murgese and Standardbred breeds, respectively. Allele frequencies of the two loci varied among the considered breeds. The SNPs discovery in MYH7 and PKM2 genes makes it possible to locate new molecular markers to ECA1. The identified markers could be used in association analysis with performance traits in horses.

  15. Identification and characterization of adult alpha-and beta-globin genes and their genomic arrangement in Pseudosciaena crocea

    Institute of Scientific and Technical Information of China (English)

    CHU Wuying; QIAN Ronghua; WANG Lianshen; YU Xiameng; YOU Zhenqiang; YU Lian

    2006-01-01

    The α- and β-globin genes from Pseudosciaena crocea were cloned by rapid amplification of cDNA 3'-end (3'-RACE). The cDNA of the α-globin is 595 bp with the ATG start codon located at Position 37, the TAA stop codon at Position 469 and the AATAAA polyadenylation signal at Position 560, which codifies 145 amino acids. The entire open reading frame of the β-globin gene is 447 bp long, which encodes 148 amino acids. Amino acid identity of the α- globin or β-globin gene compared with those reported in other fish species, ranged from 31.9% to 76.4%. When comparing with human α- and β-globins, three important alterations in the structural regions can be noted: α39 Thr→Gln, α113 His→Tyr and β117 His→Lys. The α-globin has a unique inserted amino acid residue in the 47th position. To understand the process of globin gene duplication and identify the regulatory elements present in the intergenic and intragenic regions of globin genes, the genomic arrangement of α- and β-globin genes was investigated. The results showed that the orientation of the two genes was head-to-head relative to each other. The intergenic region between the translation initiation codons of the linked α- and β-globin genes contains classical promoter elements and the length of it is much shorter than that reported in other fish.

  16. An experimentally validated panel of subfamily-specific oligonucleotide primers (V alpha 1-w29/V beta 1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction.

    Science.gov (United States)

    Genevée, C; Diu, A; Nierat, J; Caignard, A; Dietrich, P Y; Ferradini, L; Roman-Roman, S; Triebel, F; Hercend, T

    1992-05-01

    We report here the characterization of a series of T cell receptor (TcR) V alpha or V beta subfamily-specific oligonucleotide primers. Criteria that have guided the design of each oligonucleotide include appropriate thermodynamic parameters as well as differential base-pairing scores with related and unrelated target sequences. The specificity of the oligonucleotides for each V alpha or V beta subfamily was tested by polymerase chain reaction (PCR) on both a series of TcR encoding plasmid DNA and clonal T cell populations. Unexpected cross-reactivities were observed with plasmid cDNA sequences corresponding to unrelated subfamily gene segments. This led to the synthesis of additional series of oligonucleotides to obtain a relevant panel. A series of V alpha 1-w29/V beta 1-w24 TcR subfamily-specific oligonucleotides was eventually selected which generates little, if any, cross-reactivity. The use of C alpha or C beta primers for the amplification of internal positive control templates (i.e. C beta for the V alpha series and C alpha for the V beta series) has been tested in PCR performed with cDNA derived from peripheral blood lymphocytes; it was shown not to alter the amplification of the V subfamily-specific DNA fragments. This panel of oligonucleotides will be helpful in the study of TcRV gene segment usage and, thus, may lead to a better characterization of T cell responses in physiological and pathological situations.

  17. Differential effect of 1{alpha},25-dihydroxyvitamin D{sub 3} on Hsp28 and PKC{beta} gene expression in the phorbol ester-resistant human myeloid HL-525 leukemic cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yong J.; Galoforo, S.S.; Berns, C.M. [William Beaumont Hospital, Royal Oak, MI (United States). Dept. of Radiation Oncology] [and others

    1997-08-01

    We investigated the effect of 1{alpha},25-dihydroxyvitamin D{sub 3} [1,25-(OH){sub 2}D{sub 3}] on the expression of the 28-kDa heat shock protein gene (hsp28) and the protein kinase C beta gene (PKC{beta}) in the human myeloid HL-60 leukemic cell variant HL-525, which is resistance to phorbol ester-induced macrophage differentiation. Northern and Western blot analysis showed little or no hsp28 gene expression in the HL-60 cell variant, HL-205, which is susceptible to such differentiation, while a relatively high basal level of hps28 gene expression was observed in the HL-525 cells. However, both cell lines demonstrated heat shock-induced expression of this gene. During treatment with 50-300 nM 1,25-(OH){sub 2}D{sub 3}, a marked reduction of hsp28 gene expression was not associated with heat shock transcription factor-heat shock element (HSF-HSE) binding activity. Our results suggest that the differential effect of 1,25-(OH){sub 2}D{sub 3} on hsp28 and PKC{beta} gene expression is due to the different sequence composition of the vitamin D response element in the in the promoter region as well as an accessory factor for each gene or that 1,25-(OH){sub 2}D{sub 3} increases PKC{beta} gene expression, which in turn negatively regulates the expression of the hsp28 gene, or vice versa.

  18. Solving a case of allelic dropout in the GNPTAB gene: implications in the molecular diagnosis of mucolipidosis type III alpha/beta.

    Science.gov (United States)

    Coutinho, Maria Francisca; Encarnação, Marisa; Laranjeira, Francisco; Lacerda, Lúcia; Prata, Maria João; Alves, Sandra

    2016-10-01

    While being well known that the diagnosis of many genetic disorders relies on a combination of clinical suspicion and confirmatory genetic testing, not rarely, however, genetic testing needs much perseverance and cunning strategies to identify the causative mutation(s). Here we present a case of a thorny molecular diagnosis of mucolipidosis type III alpha/beta, which is an autosomal recessive lysosomal storage disorder, caused by a defect in the GNPTAB gene that codes for the α/β-subunits of the GlcNAc-1-phosphotransferase. We used both cDNA and gDNA analyses to characterize a mucolipidosis type III alpha/beta patient whose clinical diagnosis was already confirmed biochemically. In a first stage only one causal mutation was identified in heterozygosity, the already described missense mutation c.1196C>T(p.S399F), both at cDNA and gDNA levels. Only after conducting inhibition of nonsense-mediated mRNA decay (NMD) assays and after the utilization of another pair of primers the second mutation, the c.3503_3504delTC deletion, was identified. Our findings illustrate that allelic dropout due to the presence of polymorphisms and/or of mutations that trigger the NMD pathway can cause difficulties in current molecular diagnosis tests.

  19. [Molecular cloning of the DNA sequence of activin beta A subunit gene mature peptides from panda and related species and its application in the research of phylogeny and taxonomy].

    Science.gov (United States)

    Wang, Xiao-Jing; Wang, Xiao-Xing; Wang, Ya-Jun; Wang, Xi-Zhong; He, Guang-Xin; Chen, Hong-Wei; Fei, Li-Song

    2002-09-01

    Activin, which is included in the transforming growth factor-beta (TGF beta) superfamily of proteins and receptors, is known to have broad-ranging effects in the creatures. The mature peptide of beta A subunit of this gene, one of the most highly conserved sequence, can elevate the basal secretion of follicle-stimulating hormone (FSH) in the pituitary and FSH is pivotal to organism's reproduction. Reproduction block is one of the main reasons which cause giant panda to extinct. The sequence of Activin beta A subunit gene mature peptides has been successfully amplified from giant panda, red panda and malayan sun bear's genomic DNA by using polymerase chain reaction (PCR) with a pair of degenerate primers. The PCR products were cloned into the vector pBlueScript+ of Esherichia coli. Sequence analysis of Activin beta A subunit gene mature peptides shows that the length of this gene segment is the same (359 bp) and there is no intron in all three species. The sequence encodes a peptide of 119 amino acid residues. The homology comparison demonstrates 93.9% DNA homology and 99% homology in amino acid among these three species. Both GenBank blast search result and restriction enzyme map reveal that the sequences of Activin beta A subunit gene mature peptides of different species are highly conserved during the evolution process. Phylogeny analysis is performed with PHYLIP software package. A consistent phylogeny tree has been drawn with three different methods. The software analysis outcome accords with the academic view that giant panda has a closer relationship to the malayan sun bear than the red panda. Giant panda should be grouped into the bear family (Uersidae) with the malayan sun bear. As to the red panda, it would be better that this animal be grouped into the unique family (red panda family) because of great difference between the red panda and the bears (Uersidae).

  20. Unusual and typical features of a novel restorer-of-fertility gene of sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Matsuhira, Hiroaki; Kagami, Hiroyo; Kurata, Masayuki; Kitazaki, Kazuyoshi; Matsunaga, Muneyuki; Hamaguchi, Yuko; Hagihara, Eiki; Ueda, Minoru; Harada, Michiyo; Muramatsu, Aki; Yui-Kurino, Rika; Taguchi, Kazunori; Tamagake, Hideto; Mikami, Tetsuo; Kubo, Tomohiko

    2012-12-01

    Male gametogenesis in plants can be impaired by an incompatibility between nuclear and mitochondrial genomes, termed cytoplasmic male sterility (CMS). A sterilizing factor resides in mitochondria, whereas a nuclear factor, Restorer-of-fertility (Rf), restores male fertility. Although a majority of plant Rf genes are thought to encode a family of RNA-binding proteins called pentatrico-peptide repeat (PPR) proteins, we isolated a novel type of Rf from sugar beet. Two BACs and one cosmid clone that constituted a 383-kbp contig covering the sugar beet Rf1 locus were sequenced. Of 41 genes borne by the contig, quadruplicated genes were found to be associated with specific transcripts in Rf1 flower buds. The quadruplicated genes encoded a protein resembling OMA1, a protein known from yeast and mammals to be involved in mitochondrial protein quality control. Construction of transgenic plants revealed that one of the four genes (bvORF20) was capable of restoring partial pollen fertility to CMS sugar beet; the level of restoration was comparable to that evaluated by a crossing experiment. However, the other genes lacked such a capability. A GFP-fusion experiment showed that bvORF20 encoded a mitochondrial protein. The corresponding gene was cloned from rf1rf1 sugar beet and sequenced, and a solitary gene that was similar but not identical to bvORF20 was found. Genetic features exhibited by sugar beet Rf1, such as gene clustering and copy-number variation between Rf1 and rf, were reminiscent of PPR-type Rf, suggesting that a common evolutionary mechanism(s) operates on plant Rfs irrespective of the translation product.

  1. IL-20 gene expression is induced by IL-1beta through mitogen-activated protein kinase and NF-kappaB-dependent mechanisms

    DEFF Research Database (Denmark)

    Otkjaer, Kristian; Kragballe, Knud; Johansen, Claus;

    2007-01-01

    -20 was rapidly induced by proinflammatory stimuli, in particular IL-1beta, IL-6, and UVB irradiation. Using kinase inhibitors and small-interfering RNA, we discovered that the p38 mitogen-activated protein kinase (MAPK) as well as inhibitory kappaB kinase-NF-kappaB signaling pathways are crucial...... activation of the downstream kinase mitogen- and stress-activated kinase 1 (MSK1), indicating transactivation of NF-kappaB driven IL-20 messenger RNA transcription as an important mechanism of action. IL-20 is assumed to be a key cytokine in the pathogenesis of psoriasis and possibly cancer, and therefore...... for IL-20 expression. By electrophoretic mobility shift assay two kappaB-binding sites were identified upstream from the start codon in the IL-20 gene. Supershift analysis revealed binding of the p50/p65 heterodimer. Furthermore, the p38 MAPK was shown to exert its effects on IL-20 expression through...

  2. Quantitative gene-expression of the tumor angiogenesis markers vascular endothelial growth factor, integrin alphaV and integrin beta3 in human neuroendocrine tumors

    DEFF Research Database (Denmark)

    Oxboel, Jytte; Binderup, Tina; Knigge, Ulrich;

    2009-01-01

    Anti-angiogenesis treatment is a promising new therapy for cancer that recently has also been suggested for patients with neuroendocrine tumors. The aim of the present study was therefore to investigate the level of tumor angiogenesis, and thereby the molecular basis for anti-angiogenesis treatment......, in neuroendocrine tumors. We used quantitative real-time PCR for measuring mRNA gene-expression of vascular endothelial growth factor (VEGF), integrin alphaV, and integrin beta3, and CD34 for a group of patients with neuroendocrine tumors (n=13). Tissue from patients with colorectal cancer liver metastases (n=14......) and normal liver tissues (n=16) was used as control. We found a lower mRNA level of VEGF in neuroendocrine tumors compared to both colorectal liver metastases (ptumors...

  3. Post-harvest regulated gene expression and splicing efficiency in storage roots of sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Rotthues, Alexander; Kappler, Jeannette; Lichtfuss, Anna; Kloos, Dorothee U; Stahl, Dietmar J; Hehl, Reinhard

    2008-05-01

    Sixteen post-harvest upregulated genes from sugar beet comprising five novel sequences were isolated by subtractive cloning. Transcription profiles covering a period of up to 49 days after harvest under controlled storage conditions and in field clamps are reported. Post-harvest induced genes are involved in wound response, pathogen defense, dehydration stress, and detoxification of reactive oxygen species. An early induction of a cationic peroxidase indicates a response to post-harvest damage. Wound response reactions may also involve genes required for cell division such as a regulator of chromatin condensation and a precursor of the growth stimulating peptide phytohormone phytosulfokine-alpha. Surprisingly, also three putative non-protein coding genes were isolated. Two of these genes show intron specific and storage temperature dependent splicing of a precursor mRNA. The temperature dependent splicing of an intron containing sugar beet mRNA is also maintained in transgenic Arabidopsis thaliana. The storage induced genes are integrated into a model that proposes the response to several post-harvest stress conditions. Temperature regulated splicing may be a mechanism to sense seasonal temperature changes.

  4. Association of high myopia with crystallin beta A4 (CRYBA4 gene polymorphisms in the linkage-identified MYP6 locus.

    Directory of Open Access Journals (Sweden)

    Daniel W H Ho

    Full Text Available BACKGROUND: Myopia is the most common ocular disorder worldwide and imposes tremendous burden on the society. It is a complex disease. The MYP6 locus at 22 q12 is of particular interest because many studies have detected linkage signals at this interval. The MYP6 locus is likely to contain susceptibility gene(s for myopia, but none has yet been identified. METHODOLOGY/PRINCIPAL FINDINGS: Two independent subject groups of southern Chinese in Hong Kong participated in the study an initial study using a discovery sample set of 342 cases and 342 controls, and a follow-up study using a replication sample set of 316 cases and 313 controls. Cases with high myopia were defined by spherical equivalent ≤ -8 dioptres and emmetropic controls by spherical equivalent within ±1.00 dioptre for both eyes. Manual candidate gene selection from the MYP6 locus was supported by objective in silico prioritization. DNA samples of discovery sample set were genotyped for 178 tagging single nucleotide polymorphisms (SNPs from 26 genes. For replication, 25 SNPs (tagging or located at predicted transcription factor or microRNA binding sites from 4 genes were subsequently examined using the replication sample set. Fisher P value was calculated for all SNPs and overall association results were summarized by meta-analysis. Based on initial and replication studies, rs2009066 located in the crystallin beta A4 (CRYBA4 gene was identified to be the most significantly associated with high myopia (initial study: P = 0.02; replication study: P = 1.88e-4; meta-analysis: P = 1.54e-5 among all the SNPs tested. The association result survived correction for multiple comparisons. Under the allelic genetic model for the combined sample set, the odds ratio of the minor allele G was 1.41 (95% confidence intervals, 1.21-1.64. CONCLUSIONS/SIGNIFICANCE: A novel susceptibility gene (CRYBA4 was discovered for high myopia. Our study also signified the potential importance of

  5. Beta-lactamases in Enterobacteriaceae in broilers

    NARCIS (Netherlands)

    Dierikx, C.M.

    2013-01-01

    Resistance to cephalosprins due to the production of extended spectrum beta-lactamases (ESBLs) or plasmid mediated AmpC beta-lactamases is increasingly found in infections in humans outside the hospital. The genes encoding for these beta-lactamases are located on mobile DNA (plasmids), which can be

  6. Studies of the Ala/Val98 polymorphism of the hepatocyte nuclear factor-1alpha gene and the relationship to beta-cell function during an OGTT in glucose-tolerant women with and without previous gestational diabetes mellitus

    DEFF Research Database (Denmark)

    Lauenborg, J; Damm, P; Ek, J;

    2004-01-01

    In pregnancies complicated by gestational diabetes mellitus (GDM) an increased demand for insulin is not met due to beta-cell dysfunction. An Ala/Val polymorphism at codon 98 of the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene has been associated with decreased serum insulin and C...

  7. Antimicrobial Resistance Pattern and Their Beta-Lactamase Encoding Genes among Pseudomonas aeruginosa Strains Isolated from Cancer Patients

    Directory of Open Access Journals (Sweden)

    Mai M. Zafer

    2014-01-01

    Full Text Available This study was designed to investigate the prevalence of metallo-β-lactamases (MBL and extended-spectrum β-lactamases (ESBL in P. aeruginosa isolates collected from two different hospitals in Cairo, Egypt. Antibiotic susceptibility testing and phenotypic screening for ESBLs and MBLs were performed on 122 P. aeruginosa isolates collected in the period from January 2011 to March 2012. MICs were determined. ESBLs and MBLs genes were sought by PCR. The resistant rate to imipenem was 39.34%. The resistance rates for P. aeruginosa to cefuroxime, cefoperazone, ceftazidime, aztreonam, and piperacillin/tazobactam were 87.7%, 80.3%, 60.6%, 45.1%, and 25.4%, respectively. Out of 122 P. aeruginosa, 27% and 7.4% were MBL and ESBL, respectively. The prevalence of blaVIM-2, blaOXA-10-, blaVEB-1, blaNDM-, and blaIMP-1-like genes were found in 58.3%, 41.7%, 10.4%, 4.2%, and 2.1%, respectively. GIM-, SPM-, SIM-, and OXA-2-like genes were not detected in this study. OXA-10-like gene was concomitant with VIM-2 and/or VEB. Twelve isolates harbored both OXA-10 and VIM-2; two isolates carried both OXA-10 and VEB. Only one strain contained OXA-10, VIM-2, and VEB. In conclusion, blaVIM-2- and blaOXA-10-like genes were the most prevalent genes in P. aeruginosa in Egypt. To our knowledge, this is the first report of blaVIM-2, blaIMP-1, blaNDM, and blaOXA-10 in P. aeruginosa in Egypt.

  8. Identification of Mutations in Myocilin and Beta-1,4-galactosyltransferase 3 Genes in a Chinese Family with Primary Open-angle Glaucoma

    Institute of Scientific and Technical Information of China (English)

    Rong-Feng Liao; Zi-Lin Zhong; Min-Jie Ye; Li-Yun Han; Dong-Qing Ye; Jian-Jun Chen

    2016-01-01

    Background:Glaucoma is a major cause of irreversible blindness worldwide.There is evidence showing that a subset of the disease is genetically determined.In this study,we screened for mutations in chromosome 1q-linked open-angle glaucoma (GLC1A) in a Chinese family with primary open-angle glaucoma (POAG).Methods:A total of 23 members from five generations of a family were enrolled and underwent thorough ophthalmologic examinations.In addition,200 unrelated healthy Chinese controls were also recruited as normal control.GLC1A gene was amplified by polymerase chain reaction,and DNA sequencing was performed to screen for mutations.Results:Six members were diagnosed as POAG,with severe clinical manifestations,and history of high intraocular pressures.The mean age of disease onset was 26.3 years.However,the others were asymptomatic.In six affected and three asymptomatic members,gene sequencing revealed a mutation c.C1456T in exon 3 of myocilin gene (MYOC).Furthermore,we also identified a novel mutation c.G322A in beta-1,4-galactosyltransferase 3 (B4GALT3) gene in all six affected and three asymptomatic members,which was not reported previously in POAG patients.The two newly identified variants were absent in other family members as well as controls.Conclusion:The mutations c.1456C<T (p.L486F) in MYOC and c.322G<A (p.V108I) in B4GALT3 are likely responsible for the pathogenesis of POAG in this family.

  9. Identification of Mutations in Myocilin and Beta-1,4-galactosyltransferase 3 Genes in a Chinese Family with Primary Open-angle Glaucoma

    Science.gov (United States)

    Liao, Rong-Feng; Zhong, Zi-Lin; Ye, Min-Jie; Han, Li-Yun; Ye, Dong-Qing; Chen, Jian-Jun

    2016-01-01

    Background: Glaucoma is a major cause of irreversible blindness worldwide. There is evidence showing that a subset of the disease is genetically determined. In this study, we screened for mutations in chromosome 1q-linked open-angle glaucoma (GLC1A) in a Chinese family with primary open-angle glaucoma (POAG). Methods: A total of 23 members from five generations of a family were enrolled and underwent thorough ophthalmologic examinations. In addition, 200 unrelated healthy Chinese controls were also recruited as normal control. GLC1A gene was amplified by polymerase chain reaction, and DNA sequencing was performed to screen for mutations. Results: Six members were diagnosed as POAG, with severe clinical manifestations, and history of high intraocular pressures. The mean age of disease onset was 26.3 years. However, the others were asymptomatic. In six affected and three asymptomatic members, gene sequencing revealed a mutation c.C1456T in exon 3 of myocilin gene (MYOC). Furthermore, we also identified a novel mutation c.G322A in beta-1,4-galactosyltransferase 3 (B4GALT3) gene in all six affected and three asymptomatic members, which was not reported previously in POAG patients. The two newly identified variants were absent in other family members as well as controls. Conclusion: The mutations c.1456C

  10. Role of Flightless-I (Drosophila) homolog in the transcription activation of type I collagen gene mediated by transforming growth factor beta

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Mi-Sun; Jeong, Kwang Won, E-mail: kwjeong@gachon.ac.kr

    2014-11-21

    Highlights: • FLII activates TGFβ-mediated expression of COL1A2 gene. • TGFβ induces the association of FLII with SMAD3 and BRG1 in A549 cells. • FLII is required for the recruitment of SWI/SNF complex and chromatin accessibility to COL1A2 promoter. - Abstract: Flightless-I (Drosophila) homolog (FLII) is a nuclear receptor coactivator that is known to interact with other transcriptional regulators such as the SWI/SNF complex, an ATP-dependent chromatin-remodeling complex, at the promoter or enhancer region of estrogen receptor (ER)-α target genes. However, little is known about the role of FLII during transcription initiation in the transforming growth factor beta (TGFβ)/SMAD-dependent signaling pathway. Here, we demonstrate that FLII functions as a coactivator in the expression of type I collagen gene induced by TGFβ in A549 cells. FLII activates the reporter gene driven by COL1A2 promoter in a dose-dependent manner. Co-expression of GRIP1, CARM1, or p300 did not show any synergistic activation of transcription. Furthermore, the level of COL1A2 expression correlated with the endogenous level of FLII mRNA level. Depletion of FLII resulted in a reduction of TGFβ-induced expression of COL1A2 gene. In contrast, over-expression of FLII caused an increase in the endogenous expression of COL1A2. We also showed that FLII is associated with Brahma-related gene 1 (BRG1) as well as SMAD in A549 cells. Notably, the recruitment of BRG1 to the COL1A2 promoter region was decreased in FLII-depleted A549 cells, suggesting that FLII is required for TGFβ-induced chromatin remodeling, which is carried out by the SWI/SNF complex. Furthermore, formaldehyde-assisted isolation of regulatory elements (FAIRE)-quantitative polymerase chain reaction (qPCR) experiments revealed that depletion of FLII caused a reduction in chromatin accessibility at the COL1A2 promoter. These results suggest that FLII plays a critical role in TGFβ/SMAD-mediated transcription of the COL1A2 gene

  11. Common genetic variation in the Estrogen Receptor Beta (ESR2) gene and osteoarthritis: Results of a meta-analysis

    NARCIS (Netherlands)

    J.M. Kerkhof (Hanneke); I. Meulenbelt (Ingrid); A. Gonzalez (Antonio); D.J. Hart (Deborah); A. Hofman (Albert); M. Kloppenburg (Margreet); N.E. Lane; J. Loughlin (John); M.C. Nevitt (Michael); H.A.P. Pols (Huib); F. Rivadeneira Ramirez (Fernando); E. Slagboom (Eline); T.D. Spector (Tim); L. Stolk (Lisette); A. Tsezou (Aspasia); A.G. Uitterlinden (André); A.M. Valdes (Ana Maria); J.B.J. van Meurs (Joyce); A.J. Carr (Andrew Jonathan)

    2010-01-01

    textabstractBackground: The objective of this study was to examine the relationship between common genetic variation of the ESR2 gene and osteoarthritis.Methods: In the discovery study, the Rotterdam Study-I, 7 single nucleotide polymorphisms (SNPs) were genotyped and tested for association with hip

  12. Variants in the interleukin-1 alpha and beta genes, and the risk for periodontal disease in dogs

    Indian Academy of Sciences (India)

    C. Albuquerque; F. Morinha; J. Magalhães; J. Requicha; I. Dias; H. Guedes-Pinto; E. Bastos; C. Viegas

    2015-12-01

    Elevated levels of interleukin-1 (IL-1) have been shown to amplify the inflammatory response against periodontopathogenic bacteria. In humans, polymorphisms in the 1 and 1 genes are the most well-studied genetic polymorphisms associated with periodontal disease (PD). In contrast to human, there is a lack of knowledge on the genetic basis of canine PD. A case–control study was conducted in which a molecular analysis of dog 1 and 1 genes was performed. Of the eight genetic variants identified, seven in 1 gene and one in 1 gene, 1/1_g.388A>C and 1/1_g.521T>A showed statistically significant differences between groups (adjusted OR (95% CI): 0.15 (0.03–0.76), = 0.022; 5.76 (1.03–32.1), P = 0.046, respectively). It suggests that in the studied population the 1/1_g.388C allele is associated with a decreased PD risk, whereas the 1/1_g.521A allele can confer an increased risk. Additionally, the 1/2_g.515G>T variation resulted in a change of amino acid, i.e. glycine to valine. In silico analysis suggests that this change can alter protein structure and function, predicting it to be deleterious or damaging. This work suggests that 1 genetic variants may be important in PD susceptibility in canines.

  13. High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous

    NARCIS (Netherlands)

    Verwaal, R.; Wang, J.; Meijnen, J.P.; Visser, H.; Sandmann, G.; Berg, van den J.A.; Ooyen, van A.J.J.

    2007-01-01

    To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially ß-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these g

  14. Drug and cell type-specific regulation of genes with different classes of estrogen receptor beta-selective agonists.

    Directory of Open Access Journals (Sweden)

    Sreenivasan Paruthiyil

    Full Text Available Estrogens produce biological effects by interacting with two estrogen receptors, ERalpha and ERbeta. Drugs that selectively target ERalpha or ERbeta might be safer for conditions that have been traditionally treated with non-selective estrogens. Several synthetic and natural ERbeta-selective compounds have been identified. One class of ERbeta-selective agonists is represented by ERB-041 (WAY-202041 which binds to ERbeta much greater than ERalpha. A second class of ERbeta-selective agonists derived from plants include MF101, nyasol and liquiritigenin that bind similarly to both ERs, but only activate transcription with ERbeta. Diarylpropionitrile represents a third class of ERbeta-selective compounds because its selectivity is due to a combination of greater binding to ERbeta and transcriptional activity. However, it is unclear if these three classes of ERbeta-selective compounds produce similar biological activities. The goals of these studies were to determine the relative ERbeta selectivity and pattern of gene expression of these three classes of ERbeta-selective compounds compared to estradiol (E(2, which is a non-selective ER agonist. U2OS cells stably transfected with ERalpha or ERbeta were treated with E(2 or the ERbeta-selective compounds for 6 h. Microarray data demonstrated that ERB-041, MF101 and liquiritigenin were the most ERbeta-selective agonists compared to estradiol, followed by nyasol and then diarylpropionitrile. FRET analysis showed that all compounds induced a similar conformation of ERbeta, which is consistent with the finding that most genes regulated by the ERbeta-selective compounds were similar to each other and E(2. However, there were some classes of genes differentially regulated by the ERbeta agonists and E(2. Two ERbeta-selective compounds, MF101 and liquiritigenin had cell type-specific effects as they regulated different genes in HeLa, Caco-2 and Ishikawa cell lines expressing ERbeta. Our gene profiling studies

  15. Co-inheritance of novel ATRX gene mutation and globin (α & β) gene mutations in transfusion dependent beta-thalassemia patients.

    Science.gov (United States)

    Al-Nafie, Awatif N; Borgio, J Francis; AbdulAzeez, Sayed; Al-Suliman, Ahmed M; Qaw, Fuad S; Naserullah, Zaki A; Al-Jarrash, Sana; Al-Madan, Mohammed S; Al-Ali, Rudaynah A; AlKhalifah, Mohammed A; Al-Muhanna, Fahad; Steinberg, Martin H; Al-Ali, Amein K

    2015-06-01

    α-Thalassemia X-linked mental retardation syndrome is a rare inherited intellectual disability disorder due to mutations in the ATRX gene. In our previous study of the prevalence of β-thalassemia mutations in the Eastern Province of Saudi Arabia, we confirmed the widespread coinheritance of α-thalassemia mutation. Some of these subjects have a family history of mental retardation, the cause of which is unknown. Therefore, we investigated the presence or absence of mutations in the ATRX gene in these patients. Three exons of the ATRX gene and their flanking regions were directly sequenced. Only four female transfusion dependent β-thalassemia patients were found to be carriers of a novel mutation in the ATRX gene. Two of the ATRX gene mutations, c.623delA and c.848T>C were present in patients homozygous for IVS I-5(G→C) and homozygous for Cd39(C → T) β-thalassemia mutation, respectively. While the other two that were located in the intronic region (flanking regions), were present in patients homozygous for Cd39(C → T) β-thalassemia mutation. The two subjects with the mutations in the coding region had family members with mental retardation, which suggests that the novel frame shift mutation and the missense mutation at coding region of ATRX gene are involved in ATRX syndrome.

  16. Positive association between--1021TT genotype of dopamine beta hydroxylase gene and progressive behavior of injection heroin users.

    Science.gov (United States)

    Xie, Xiaohu; Xu, Limin; Liu, Huifen; Chen, Weisheng; Zhuang, Dingding; Zhang, Jianbing; Duan, Shiwei; Zhou, Wenhua

    2013-04-29

    By balancing the ratios of dopamine and norepinephrine, dopamine beta hydroxylase (DBH) plays an important role in brain reward circuit that is involved with behavioral effects of heroin addiction. DBH -1021C/T (rs1611115) is a functional variant with strong correlation with plasma DBH activity and several nerval and psychic disorders. In the present study, we have collected 333 male cases with heroin addiction and 200 male healthy controls to explore the role of -1021C/T in heroin addiction. There is no evidence of association between -1021C/T and heroin addiction on both genotype and allele levels (P>0.05). In the injection subgroup of cases, -1021TT carriers have longer heroin addiction time (P<0.001) and higher dosage of self-administered heroin (P=0.045) than carriers with -1021CC or -1021CT, suggesting that patients with TT genotype are likely to have more progressive style of heroin users with injection route. In conclusion, our results support -1021TT genotype may be implicated with a more progressive nature of heroin addiction, although DBH -1021C/T is unlikely to be involved in the risk of heroin addiction.

  17. Genetic polymorphism of beta-casein gene and its associations with milk traits in Holstein-Friesian cows

    Directory of Open Access Journals (Sweden)

    Teodor Bugeac

    2015-05-01

    Full Text Available In animal breeding finding and using effective genetic markers for improving important traits it is a continuous challenge. In this respect, several genetic markers were associated in cattle with increased milk production or a better milk quality. This proved to be a useful tool for improving certain traits by selecting individuals carriers of allelic variants that have an effect on a desirable trait. In particular, positive associations between certain alleles found at the milk protein loci with some milk production traits convincingly demonstrated in several cattle breeds. Although, in some cases the results obtained in various studies were not in agreement and varied between breeds or populations. Therefore the objective of this study was to establish associations (if any between alleles found at the beta-casein (CSN2 locus and some milk production traits (milk yield and fat, protein, casein and lactose content in a Holstein-Friesian population reared in Romania. Genetic variants at CSN2 locus were identified by isoelectric focusing (IEF of milk samples. In order to determine milk composition the samples were analysed with MilkoScan FT 6000. For the statistical analysis of data SPSS v.19 for Windows was used. At the CSN2 locus four alleles and seven genotypes were identified in the analyzed cattle population. The cows carriers of CSN2 A2 allele produced the highest milk yield and the highest milk protein content, this result being in agreement with other previous studies.

  18. Induction of dental epithelial cell differentiation marker gene expression in non-odontogenic human keratinocytes by transfection with thymosin beta 4

    Directory of Open Access Journals (Sweden)

    Tamotsu Kiyoshima

    2014-01-01

    Full Text Available Previous studies have shown that the recombination of cells liberated from developing tooth germs develop into teeth. However, it is difficult to use human developing tooth germ as a source of cells because of ethical issues. Previous studies have reported that thymosin beta 4 (Tmsb4x is closely related to the initiation and development of the tooth germ. We herein attempted to establish odontogenic epithelial cells from non-odontogenic HaCaT cells by transfection with TMSB4X. TMSB4X-transfected cells formed nodules that were positive for Alizarin-red S (ALZ and von Kossa staining (calcium phosphate deposits when cultured in calcification-inducing medium. Three selected clones showing larger amounts of calcium deposits than the other clones, expressed PITX2, Cytokeratin 14, and Sonic Hedgehog. The upregulation of odontogenesis-related genes, such as runt-related transcription factor 2 (RUNX2, Amelogenin (AMELX, Ameloblastin (AMBN and Enamelin (ENAM was also detected. These proteins were immunohistochemically observed in nodules positive for the ALZ and von Kossa staining. RUNX2-positive selected TMSB4X-transfected cells implanted into the dorsal subcutaneous tissue of nude mice formed matrix deposits. Immunohistochemically, AMELX, AMBN and ENAM were observed in the matrix deposits. This study demonstrated the possibility of induction of dental epithelial cell differentiation marker gene expression in non-odontogenic HaCaT cells by TMSB4X.

  19. The crtS gene of Xanthophyllomyces dendrorhous encodes a novel cytochrome-P450 hydroxylase involved in the conversion of beta-carotene into astaxanthin and other xanthophylls.

    Science.gov (United States)

    Alvarez, Vanessa; Rodríguez-Sáiz, Marta; de la Fuente, Juan Luis; Gudiña, Eduardo J; Godio, Ramiro P; Martín, Juan F; Barredo, José Luis

    2006-04-01

    The conversion of beta-carotene into xanthophylls is a subject of great scientific and industrial interest. We cloned the crtS gene involved in astaxanthin biosynthesis from two astaxanthin producing strains of Xanthophyllomyces dendrorhous: VKPM Y2410, an astaxanthin overproducing strain, and the wild type ATCC 24203. In both cases, the ORF has a length of 3166 bp, including 17 introns, and codes for a protein of 62.6 kDa with similarity to cytochrome-P450 hydroxylases. crtS gene sequences from strains VKPM Y2410, ATCC 24203, ATCC 96594, and ATCC 96815 show several nucleotide changes, but none of them causes any amino acid substitution, except a G2268 insertion in the 13th exon of ATCC 96815 which causes a change in the reading frame. A G1470 --> A change in the 5' splicing region of intron 8 was also found in ATCC 96815. Both point mutations explain astaxanthin idiotrophy and beta-carotene accumulation in ATCC 96815. Mutants accumulating precursors of the astaxanthin biosynthetic pathway were selected from the parental strain VKPM Y2410 (red) showing different colors depending on the compound accumulated. Two of them were blocked in the biosynthesis of astaxanthin, M6 (orange; 1% astaxanthin, 71 times more beta-carotene) and M7 (orange; 1% astaxanthin, 58 times more beta-carotene, 135% canthaxanthin), whereas the rest produced lower levels of astaxanthin (5-66%) than the parental strain. When the crtS gene was expressed in M7, canthaxanthin accumulation disappeared and astaxanthin production was partially restored. Moreover, astaxanthin biosynthesis was restored when X. dendrorhous ATCC 96815 was transformed with the crtS gene. The crtS gene was heterologously expressed in Mucor circinelloides conferring to this fungus an improved capacity to synthesize beta-cryptoxanthin and zeaxanthin, two hydroxylated compounds from beta-carotene. These results show that the crtS gene is involved in the conversion of beta-carotene into xanthophylls, being potentially useful to

  20. N-Acetyl-L-Cystein downregulates beta-amyloid precursor protein gene transcription in human neuroblastoma cells.

    Science.gov (United States)

    Studer, R; Baysang, G; Brack, C

    2001-01-01

    The causes for the sporadic form of Alzheimer's disease (AD) are still poorly understood, except from the fact that age is an important risk factor. The main component of the characteristic amyloid plaques in brains of AD patients are Abeta peptides, derivatives of the amyloid precursor protein APP. Oxidative stress may contribute to the aetiology of AD by dysregulation of APP metabolism. Overexpression of the APP gene could result in an increased secretion of neurotoxic Abeta peptides, while preventing the overexpression might be protective. We here report that the antioxidant N-Acetyl-L-Cystein (NAC) downregulates APP gene transcription in human neuroblastoma cells. The effect is reversible when cells are returned to NAC free medium. These results open up new possibilities for the development of therapeutic agents that intervene at the transcriptional level.

  1. Beta 2-adrenergic receptor gene association with overweight and asthma in children and adolescents and its relationship with physical fitness

    Directory of Open Access Journals (Sweden)

    Neiva Leite

    2015-12-01

    Full Text Available Objective: To investigate the association of Arg16Gly and Gln27Glu polymorphisms of β2-adrenergic receptor gene (ADRB2 with the occurrence of asthma and overweight and the gene's influence on anthropometric, clinic, biochemical and physical fitness variables in children and adolescents. Methods: Subjects were evaluated for allelic frequencies of the β2-adrenergic receptor gene, height, weight, body mass index (BMI, BMI Z-score, waist circumference (WC, pubertal stage, resting heart rate (HRres, blood pressure (BP, total cholesterol (TC, glucose, insulin, high density lipoprotein (HDL-C, low density lipoprotein (LDL-C, triglyceride (TG, Homeostasis Metabolic Assessment (HOMA2-IR, Quantitative Insulin Sensitivity Check Index (QUICKI and maximal oxygen uptake (VO2max. The participants were divided in four groups: overweight asthmatic (n=39, overweight non-asthmatic (n=115, normal weight asthmatic (n=12, and normal weight non-asthmatic (n=40. Results: Regarding the Gln27Glu polymorphism, higher total cholesterol was observed in usual genotype individuals than in genetic variant carriers (p=0.04. No evidence was found that the evaluated polymorphisms are influencing the physical fitness. The Arg16 allele was found more frequently among the normal weight asthmatic group when compared to the normal weight non-asthmatic group (p=0.02, and the Glu27 allele was more frequently found in the overweight asthmatics group when compared to the normal weight non-asthmatic group (p=0.03. Conclusions: The association of Arg16 allele with the occurrence of asthma and of the Glu27 allele with overweight asthmatic adolescents evidenced the contribution of the β2-adrenergic receptor gene to the development of obesity and asthma.

  2. Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genes

    Directory of Open Access Journals (Sweden)

    Amanda M. Ackermann

    2016-03-01

    Conclusions: We have determined the genetic landscape of human α- and β-cells based on chromatin accessibility and transcript levels, which allowed for detection of novel α- and β-cell signature genes not previously known to be expressed in islets. Using fine-mapping of open chromatin, we have identified thousands of potential cis-regulatory elements that operate in an endocrine cell type-specific fashion.

  3. Variation within the PPARG gene is associated with residual beta-cell function and glycemic control in children and adolescents during the first year of clinical type 1 diabetes

    DEFF Research Database (Denmark)

    Porksen, S.; Nielsen, L.B.; Mortensen, H.B.;

    2008-01-01

    the PPARG gene in relation to residual beta-cell function and glycemic control in newly diagnosed T1D. Design: Prospective, non-interventional, 12-month follow-up study, conducted in 18 centers in 15 countries. Patients: Two hundred and fifty-seven children and adolescents (aged ... diagnosed T1D. Main outcome measures: Beta-cell function was determined as 90-min meal-stimulated C-peptide (Boost test) 1, 6, and 12 months after diagnosis. Hemoglobin A1c (HbA1c) and daily insulin dose (IU/kg/d) were recorded at 1, 3, 6, 9, and 12 months after diagnosis. Haplotypes within PPARG were...

  4. Characterization of mice with targeted deletion of the gene encoding core 2 beta1,6-N-acetylglucosaminyltransferase-2.

    Science.gov (United States)

    Stone, Erica L; Lee, Seung Ho; Ismail, Mohd Nazri; Fukuda, Minoru

    2010-01-01

    The three glycosyltransferases of the Core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT) family, C2GnT1, C2GnT2, and C2GnT3, are able to initiate the Core 2 branch of O-glycans. However, C2GnT2, which is highly expressed in the digestive tract, has a broader acceptor substrate specificity that allows it to also generate Core 4 O-glycans and I branches. We discovered that C2GnT2 KO mice have decreased mucosal barrier function in the digestive tract, reduced levels of circulating IgGs and fecal IgA, and increased susceptibility to experimental colitis. Mass spectrometric analyses also revealed that C2GnT2 KO mice had a reduction in Core 2 O-glycans in the digestive tract with a corresponding increase in elongated Core 1 O-glycans. Unexpectedly, we saw that the loss of C2GnT2 and especially the loss of all three C2GnTs resulted in the expression of elongated O-mannose structures in the stomach, suggesting that the elongation of these structures is controlled by competition for UDP-GlcNAc [Stone, E. L., Ismail, M. N., Lee, S. H., Luu, Y., Ramirez, K., Haslam, S. M., Ho, S. B., Dell, A., Fukuda, M. and Marth, J. D. (2009). Glycosyltransferase function in Core 2-type protein O-glycosylation. Mol. Cell. Biol. 29, 3370-3782].

  5. Glucose-induced repression of PPARalpha gene expression in pancreatic beta-cells involves PP2A activation and AMPK inactivation

    DEFF Research Database (Denmark)

    Ravnskjaer, Kim; Boergesen, Michael; Dalgaard, Louise T;

    2006-01-01

    Tight regulation of fatty acid metabolism in pancreatic beta-cells is important for beta-cell viability and function. Chronic exposure to elevated concentrations of fatty acid is associated with beta-cell lipotoxicity. Glucose is known to repress fatty acid oxidation and hence to augment the toxi......Tight regulation of fatty acid metabolism in pancreatic beta-cells is important for beta-cell viability and function. Chronic exposure to elevated concentrations of fatty acid is associated with beta-cell lipotoxicity. Glucose is known to repress fatty acid oxidation and hence to augment...... but not AMPKalpha1 using RNAi suppressed PPARalpha expression, thereby mimicking the effect of glucose. These results indicate that activation of protein phosphatase 2A and subsequent inactivation of AMPK is necessary for glucose repression of PPARalpha expression in pancreatic beta-cells....

  6. Characterization of the beta amyloid precursor protein-like gene in the central nervous system of the crab Chasmagnathus. Expression during memory consolidation

    Directory of Open Access Journals (Sweden)

    Fustiñana Maria

    2010-09-01

    Full Text Available Abstract Background Human β-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the β-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information. Results Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl, showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels Conclusions cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation.

  7. Effect of transforming growth factor-beta on activity of connective tissue growth factor gene promoter in mouse NIH/3T3 fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Qing ZHAO; Nan CHEN; Wei-ming WANG; Jian LU; Bing-bing DAI

    2004-01-01

    AIM: To investigate the regulatory mechanism of transforming growth factor-beta on activity of connective tissue growth factor promoter in mouse NIH/3T3 fibroblasts. METHODS: The regulation fragment of the 5' flanking region of the human CTGF gene was linked to pGL3-Basic vector, a firefly luciferase reporter construct without promoter. The recombinant plasmid pCTGF-luc was transiently transfected to NIH/3T3 fibroblasts. The activity of CTGF promoter after treatment of TGF-β1 and MAPK pathway inhibitors were assayed with luciferase reporter gene assay system. RESULTS: TGF-β1-induced increase of CTGF promoter activity was concentration-dependent,with a plateau at 5 μg/L by 2.67-fold vs control (P<0.05). The TGF-β1 stimulation of CTGF promoter activity was time-dependent, too. After exposure to TGF-β1 (5 μg/L), the maximal level of luciferase activity was reached at 12 h and maintained to 24 h by 2.76- and 2.20-fold vs control, respectively (P<0.05). Blockade of mitogen-activated protein kinases (MAPK) pathway with PD98059 (10 μmnol/L), the MAP kinase kinase 1 inhibitor, and SB203580 (10μmol/L), the p38 MAP kinase inhibitor, decreased basal and TGF-β1-induced activation of CTGF promoter. However,inhibition of c-Jun-N-terminal kinase/stress-activated protein kinase by SP600125 (20 μmol/L) was without effect.CONCLUSION: TGF-β1 stimulated the transcriptional activity of CTGF gene promoter in NIH/3T3 fibroblasts in a dose- and time-dependent manner. MAPK pathway may play a role in the regulation of TGF-β1-induced CTGF expression.

  8. Review Paper: Association of Transforming Growth Factor Beta-1 -509C/T Gene Polymorphism with Ischemic Stroke: A Meta Analysis

    Directory of Open Access Journals (Sweden)

    Pradeep Kumar

    2016-05-01

    Full Text Available Introduction: Transforming Growth Factor-Beta 1 (TGF-β1 is a pleiotropic cytokine with potent anti-inflammatory property, which has been considered as an essential risk factor in the inflammatory process of Ischemic Stroke (IS, by involving in the pathophysiological progression of hypertension, atherosclerosis, and lipid metabolisms. -509C/T TGF-β1 gene polymorphism has been found to be associated with the risk of IS. The aim of this meta-analysis was to provide a relatively comprehensive account of the relation between -509C/T gene polymorphisms of TGF-β1 and susceptibility to IS. Methods: A review of literature for eligible genetic association Studies published before October 20, 2014 was conducted in the PubMed, EMBASE, Google Scholar and Trip database. The strength of association was calculated by pooled odds ratios (ORs with 95% confidence intervals using RevMan 5.3 software. Heterogeneity was examined using Higgins I-squared, Tau-squared, and Chi-squared tests. Results: A total of 2 studies involving 614 cases and 617 controls were found. The overall estimates did not show any significant relation between TGF-β1-509C/T polymorphism and risk of IS under dominant (CC+CT vs. TT: OR=1.01, 95%CI=0.31 to 3.26; P=0.99, recessive (CC vs. CT+TT: OR=0.94, 95%CI=0.47 to 1.90; P=0.87, and allelic models (T vs. C: OR=1.06, 95%CI=0.55 to 2.04; P=0.86. Conclusion: This meta-analysis showed that TGF-β1-509C/T gene polymorphism has no significant association with the susceptibility of IS. Further well-designed prospective studies with larger sample size are needed to confirm these findings.

  9. New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination.

    Science.gov (United States)

    Ruiz-Ballesta, Isabel; Baena, Guillermo; Gandullo, Jacinto; Wang, Liqun; She, Yi-Min; Plaxton, William Charles; Echevarría, Cristina

    2016-05-01

    Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV-V when coleoptiles initiate the formation of the photosynthetic tissues.

  10. New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination

    Science.gov (United States)

    Ruiz-Ballesta, Isabel; Baena, Guillermo; Gandullo, Jacinto; Wang, Liqun; She, Yi-Min; Plaxton, William Charles; Echevarría, Cristina

    2016-01-01

    Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV–V when coleoptiles initiate the formation of the photosynthetic tissues. PMID:27194739

  11. Attempts to apply affinity labeling techniques to ribulosebisphosphate carboxylase/oxygenase. [Comparison of spinach leaf and Rhodospirillum rubrum

    Energy Technology Data Exchange (ETDEWEB)

    Hartman, F. C.; Norton, I. L.; Stringer, C. D.; Schloss, J. V.

    1978-01-01

    Studies on carboxylases/oxygenases from different species may be necessary to confirm that a residue implicated as essential is indeed an active-site component. To provide an especially stringent test case for the identification of species invariant structural features the enzymes from two phylogenetically distant species, spinach and Rhodospirillum rubrum, were compared. To date, the reactions of Br-butanone-P/sub 2/ and BrAcNHEtOP with the spinach enayme have been rather thoroughly characterized; only preliminary experiments have been completed with the R. rubrum enzyme. Both enzymes were isolated and assayed for carboxylase activity (spectrophotometrically or /sup 14/CO/sub 2/-fixation) and for oxygenase activity.

  12. Dark/Light Modulation of Ribulose Bisphosphate Carboxylase Activity in Plants from Different Photosynthetic Categories 1

    Science.gov (United States)

    Vu, J. Cu V.; Allen, Leon H.; Bowes, George

    1984-01-01

    Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from lightexposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO3− and Mg2+ concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C3); P. maximum (C4 phosphoenolpyruvate carboxykinase); P. milioides (C3/C4); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C3); P. miliaceum (C4 NAD malic enzyme); Zea mays and Sorghum bicolor (C4 NADP malic enzyme); Moricandia arvensis (C3/C4); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C3 species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO2 and Mg2+ activation, but which can be converted to an activatable state upon exposure of the leaf to light. PMID:16663937

  13. Dark/Light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories.

    Science.gov (United States)

    Vu, J C; Allen, L H; Bowes, G

    1984-11-01

    Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from lightexposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO(3) (-) and Mg(2+) concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C(3)); P. maximum (C(4) phosphoenolpyruvate carboxykinase); P. milioides (C(3)/C(4)); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C(3)); P. miliaceum (C(4) NAD malic enzyme); Zea mays and Sorghum bicolor (C(4) NADP malic enzyme); Moricandia arvensis (C(3)/C(4)); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C(3) species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO(2) and Mg(2+) activation, but which can be converted to an activatable state upon exposure of the leaf to light.

  14. The role of biotin and oxamate in the carboxyltransferase reaction of pyruvate carboxylase.

    Science.gov (United States)

    Lietzan, Adam D; Lin, Yi; St Maurice, Martin

    2014-11-15

    Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. During catalysis, carboxybiotin is translocated to the carboxyltransferase domain where the carboxyl group is transferred to the acceptor substrate, pyruvate. Many studies on the carboxyltransferase domain of PC have demonstrated an enhanced oxaloacetate decarboxylation activity in the presence of oxamate and it has been shown that oxamate accepts a carboxyl group from carboxybiotin during oxaloacetate decarboxylation. The X-ray crystal structure of the carboxyltransferase domain from Rhizobium etli PC reveals that oxamate is positioned in the active site in an identical manner to the substrate, pyruvate, and kinetic data are consistent with the oxamate-stimulated decarboxylation of oxaloacetate proceeding through a simple ping-pong bi bi mechanism in the absence of the biotin carboxylase domain. Additionally, analysis of truncated PC enzymes indicates that the BCCP domain devoid of biotin does not contribute directly to the enzymatic reaction and conclusively demonstrates a biotin-independent oxaloacetate decarboxylation activity in PC. These findings advance the description of catalysis in PC and can be extended to the study of related biotin-dependent enzymes.

  15. Biotin deficiency in the cat and the effect on hepatic propionyl CoA carboxylase.

    Science.gov (United States)

    Carey, C J; Morris, J G

    1977-02-01

    Biotin deficiency was produced in growing kittens by feeding a diet containing dried, raw egg white. After receiving either an 18.5% egg white diet for 25 weeks, or a 32% egg white diet for 12 weeks, they exhibited dermal lesions characterized by alopecia, scaly dermatitis and achromotrichia, which increased in severity with the deficiency. Females developed accumulations of dried salivary, nasal and lacrymal secretions in the facial region although a male did not. There was a loss of body weight in all cats as the deficiency progressed. Hepatic propionyl CoA carboxylase activities were measured on biopsy samples of liver during biotin deficiency and after biotin supplementation. In the deficient state, activities were 4% and 24% of that following biotin supplementation. Propionyl carboxylase activity in the liver of the cat was comparable to that reported in the rat and chick in the deficient and normal states. Subcutaneous injection of 0.25 mg biotin every other day while continuing to receive the egg white diet caused remission of clinical signs, a body weight gain and increased food intake.

  16. Hybrid Structure of a Dynamic Single-Chain Carboxylase from Deinococcus radiodurans.

    Science.gov (United States)

    Hagmann, Anna; Hunkeler, Moritz; Stuttfeld, Edward; Maier, Timm

    2016-08-01

    Biotin-dependent acyl-coenzyme A (CoA) carboxylases (aCCs) are involved in key steps of anabolic pathways and comprise three distinct functional units: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT). YCC multienzymes are a poorly characterized family of prokaryotic aCCs of unidentified substrate specificity, which integrate all functional units into a single polypeptide chain. We employed a hybrid approach to study the dynamic structure of Deinococcus radiodurans (Dra) YCC: crystal structures of isolated domains reveal a hexameric CT core with extended substrate binding pocket and a dimeric BC domain. Negative-stain electron microscopy provides an approximation of the variable positioning of the BC dimers relative to the CT core. Small-angle X-ray scattering yields quantitative information on the ensemble of Dra YCC structures in solution. Comparison with other carrier protein-dependent multienzymes highlights a characteristic range of large-scale interdomain flexibility in this important class of biosynthetic enzymes.

  17. Dark/light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories

    Energy Technology Data Exchange (ETDEWEB)

    Vu, J.C.V.; Allen, L.H. Jr.; Bowes, G.

    1984-11-01

    Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from light-exposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO/sub 3//sup -/ and Mg/sup 2 +/ concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C/sub 3/); P. maximum (C/sub 4/ phosphoenolpyruvate carboxykinase); P. milioides (C/sub 3//C/sub 4/); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C/sub 3/); P. miliaceum (C/sub 4/ NAD malic enzyme); Zea mays and Sorghum bicolor (C/sub 4/ NADP malic enzyme); Moricandia arvensis (C/sub 3//C/sub 4/); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C/sub 3/ species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO/sub 2/ and Mg/sup 2 +/ activation, but which can be converted to an activatable state upon exposure of the leaf to light. 16 references, 2 tables.

  18. A Symmetrical Tetramer for S. aureus Pyruvate Carboxylase in Complex with Coenzyme A

    Energy Technology Data Exchange (ETDEWEB)

    Yu, L.; Xiang, S; Lasso, G; Gil, D; Valle, M; Tong, L

    2009-01-01

    Pyruvate carboxylase (PC) is a conserved metabolic enzyme with important cellular functions. We report crystallographic and cryo-electron microscopy (EM) studies of Staphylococcus aureus PC (SaPC) in complex with acetyl-CoA, an allosteric activator, and mutagenesis, biochemical, and structural studies of the biotin binding site of its carboxyltransferase (CT) domain. The disease-causing A610T mutation abolishes catalytic activity by blocking biotin binding to the CT active site, and Thr908 might play a catalytic role in the CT reaction. The crystal structure of SaPC in complex with CoA reveals a symmetrical tetramer, with one CoA molecule bound to each monomer, and cryo-EM studies confirm the symmetrical nature of the tetramer. These observations are in sharp contrast to the highly asymmetrical tetramer of Rhizobium etli PC in complex with ethyl-CoA. Our structural information suggests that acetyl-CoA promotes a conformation for the dimer of the biotin carboxylase domain of PC that might be catalytically more competent.

  19. Characterization of acetyl-CoA and propionyl-CoA carboxylases encoded by Leptospira interrogans serovar Lai: an initial biochemical study for leptospiral gluconeogenesis via anaplerotic CO2 assimilation

    Institute of Scientific and Technical Information of China (English)

    Nanqiu Peng; Yi Zhong; Qing Zhang; Mingyue Zheng; Wei Zhao; Hualiang Jiang; Chen Yang; Xiaokui Guo; Guoping Zhao

    2012-01-01

    Leptospira interrogans is the causative agent of leptospirosis.The in vitro growth of L.interrogans requires CO2 and a partial 3-hydroxypropionate pathway involving two acyl-CoA carboxylases was suggested by genomic analysis to assimilate CO2.Either set of the candidate genes heterologously co-expressed in Escherichia coli was able to demonstrate both acetyl-CoA carboxylase (ACC)and propionyl-CoA carboxylase (PCC) activities.The trisubunit holoenzyme (LA_2736-LA_2735 and LA_3803),although failed to be purified,was designated ACC based on its substrate preference toward acetyl-CoA.The partially purified bi-subunit holoenzyme (LA_2432-LA_2433) has a considerably higher activity against propionyi-CoA as the substrate than that of acetyl-CoA,and thus,designated PCC.Native polyacrylamide gel electrophoresis indicated that this PCC has a molecular mass of around 669 kDa,suggesting an α4β4 quaternary structure and both structural homology modeling and site-directed mutagenesis analysis of its carboxyltransferase subunit (LA_2433) indicated that the A431 residue located at the bottom of the putative substrate binding pocket may play an important role in substrate specificity determination.Both transcriptomic and proteomic data indicated that enzymes involved in the suggested partial 3-hydroxypropionate pathway were expressed in vivo in addition to ACC/PCC and the homologous genes in genomes of other Leptospira species were re-annotated accordingly.However,as the in vitro detected specific activity of ACC in the crude cell extract was too low to account for the growth of the bacterium in Ellinghausen-McCulloughJohnson-Harris minimal medium,further systematic analysis is required to unveil the mechanism of gluconeogenesis via anaplerotic CO2 assimilation in Leptospira species.

  20. Association of Gln27Glu and Arg16Gly polymorphisms in Beta2-adrenergic receptor gene with obesity susceptibility: a meta-analysis.

    Directory of Open Access Journals (Sweden)

    Hongxiu Zhang

    Full Text Available BACKGROUND: The beta2-adrenergic receptor (ADRB2 gene polymorphism has been implicated in susceptibility to obesity, but study results are still controversial. OBJECTIVE: The present meta-analysis is performed to determine whether there are any associations between the Gln27Glu (rs1042714 or the Arg16Gly (rs1042713 polymorphisms in ADRB2 and obesity susceptibility. METHODS: The PubMed (1950-2014, Embase (1974-2014, and China National Knowledge Infrastructure (CNKI, 1994-2014 databases were searched using the search terms ("Beta2-adrenergic receptor", "β2-adrenergic receptor" or "ADRB2", "polymorphism," and "obesity". Fixed- or random-effects pooled measures were determined on the bias of heterogeneity tests across studies. Publication bias was examined by Egger's test and the modified Begg's test. RESULTS: Eighteen published articles were selected for meta-analysis. Overall analyses showed that rs1042714 (Gln27Glu was associated with significantly increased obesity risk in the heterozygote model (Gln/Glu vs. Gln/Gln: OR: 1.16, 95% CI: 1.04-1.30, I2 = 49%, P = 0.009 and the dominant model (Gln/Glu + Glu/Glu vs. Gln/Gln: OR: 1.2, 95% CI: 1.00-1.44, I2 = 55%, P = 0.04, whereas no significant association was found in the other models for rs1042714. Also, no significant association was found between the rs1042713 (Arg16Gly gene polymorphism and the risk of obesity in all genetic models. In addition, neither rs1042713 (Arg16Gly nor rs1042714 (Gln27Glu showed any significant association with obesity susceptibility when the population were stratified based on gender. CONCLUSION: Our meta-analysis revealed that the rs1042714 (Gln27Glu polymorphism is associated with obesity susceptibility. However, our results do not support an association between rs1042713 (Arg16Gly polymorphisms and obesity in the populations investigated. This conclusion warrants confirmation by more case-control and cohort studies.

  1. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

    Directory of Open Access Journals (Sweden)

    Roger Huerlimann

    Full Text Available The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT, and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid and in two forms (homomeric and heteromeric. All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa and Chromista (Stramenopiles, Haptophyta and Cryptophyta have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO, Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta. These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was

  2. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

    KAUST Repository

    Huerlimann, Roger

    2015-07-01

    The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT), and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid) and in two forms (homomeric and heteromeric). All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa) and Chromista (Stramenopiles, Haptophyta and Cryptophyta) have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO), Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta). These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was acquired by the

  3. Dual functions of transcription factors, transforming growth factor-beta-inducible early gene (TIEG)2 and Sp3, are mediated by CACCC element and Sp1 sites of human monoamine oxidase (MAO) B gene.

    Science.gov (United States)

    Ou, Xiao-Ming; Chen, Kevin; Shih, Jean C

    2004-05-14

    Monoamine oxidases (MAO) A and B catalyze the oxidative deamination of many biogenic and dietary amines. Abnormal expression of MAO has been implicated in several psychiatric and neurodegenerative disorders. Human MAO B core promoter (-246 to -99 region) consists of CACCC element flanked by two clusters of overlapping Sp1 sites. Here, we show that cotransfection with transforming growth factor (TGF)-beta-inducible early gene (TIEG)2 increased MAO B gene expression at promoter, mRNA, protein, and catalytic activity levels in both SH-SY5Y and HepG2 cells. Mutation of the CACCC element increased the MAO B promoter activity, and cotransfection with TIEG2 further increased the promoter activity, suggesting that CACCC was a repressor element. This increase was reduced when the proximal Sp1 overlapping sites was mutated. Similar interactions were found with Sp3. These results showed that TIEG2 and Sp3 were repressors at the CACCC element but were activators at proximal Sp1 overlapping sites of MAO B. Gel-shift and chromatin immunoprecipitation assays showed that TIEG2 and Sp3 bound directly to CACCC element and the proximal Sp1 sites in both synthetic oligonucleotides and natural MAO B core promoter. TIEG2 had a higher affinity to Sp1 sites than CACCC element, whereas Sp3 had an equal affinity to both elements. Thus, TIEG2 was an activator, but Sp3 had no effect on MAO B gene expression. This study provides new insights into MAO B gene expression and illustrates the complexity of gene regulation.

  4. Polygenic inheritance of Tourette syndrome, stuttering, attention deficit hyperactivity, conduct, and oppositional defiant disorder: The additive and subtractive effect of the three dopaminergic genes - DRD2, D{beta}H, and DAT1

    Energy Technology Data Exchange (ETDEWEB)

    Comings, D.E.; Wu, S.; Chiu, C.; Ring, R.H.; Gade, R.; Ahn, C.; Dietz, G.; Muhleman, D. [Hope Medical Center, Duarte, CA (United States)] [and others

    1996-05-31

    Polymorphisms of three different dopaminergic genes, dopamine D{sub 2} receptor (DRD2), dopamine {beta}-hydroxylase (D{beta}H), and dopamine transporter (DAT1), were examined in Tourette syndrome (TS) probands, their relatives, and controls. Each gene individually showed a significant correlation with various behavioral variables in these subjects. The additive and subtractive effects of the three genes were examined by genotyping all three genes in the same set of subjects. For 9 of 20 TS associated comorbid behaviors there was a significant linear association between the degree of loading for markers of three genes and the mean behavior scores. The behavior variables showing the significant associations were, in order, attention deficit hyperactivity disorder (ADHD), stuttering, oppositional defiant, tics, conduct, obsessive-compulsive, mania, alcohol abuse, and general anxiety - behaviors that constitute the most overt clinical aspects of TS. For 16 of the 20 behavior scores there was a linear progressive decrease in the mean score with progressively lesser loading for the three gene markers. These results suggest that TS, ADHD, stuttering, oppositional defiant and conduct disorder, and other behaviors associated with TS, are polygenic, due in part to these three dopaminergic genes, and that the genetics of other polygenic psychiatric disorders may be deciphered using this technique. 144 refs., 2 figs., 13 tabs.

  5. Characterization and expression of MHC class II alpha and II beta genes in mangrove red snapper (Lutjanus argentimaculatus).

    Science.gov (United States)

    Wang, Tianyan; Tan, Shangjin; Cai, Zhonghua

    2015-12-01

    The major histocompatibility complex (MHC) class II plays a key role in adaptive immunity by presenting foreign peptides to CD4(+) T cells and by triggering the adaptive immune response. While the structure and function of MHC class II have been well characterized in mammalian, limited research has been done on fishes. In this study, we characterized the gene structure and expression of MHC class II α (Lunar-DAA) and II β (Lunar-DAB) of mangrove red snapper (Lutjanus argentimaculatus). Both genes shared, respectively, a high similarity and typical features with other vertebrate MHC class II α and II β. The phylogenetic analysis of the deduced peptides revealed that both Lunar-DAA and Lunar-DAB were located in the teleost subclass. Western blotting analyses indicated that both MHC class II α and II β were expressed ubiquitously in immune-related cells, tissues and organs, and that MHC class II α and II β chains existed mainly as heterodimers. While it was highly expressed in gills, thymus, head kidney (HK), spleen, head kidney macrophage and spleen leucocytes, MHC class II β chain was expressed with a low abundance in skin, intestine, stomach and heart. The highest expression of MHC class II β in thymus confirmed the conclusion that thymus is one of the primary lymphoid organs in fishes. The detection of MHC class II αβ dimers in HK macrophages and spleen leucocytes indicated that HK macrophages and spleen leucocytes play a critical role in the adaptive immunity in fishes. All these results provide valuable information for understanding the structure of MHC class II α and II β and their function in immune responses.

  6. Common genetic variation in the Estrogen Receptor Beta (ESR2 gene and osteoarthritis: results of a meta-analysis

    Directory of Open Access Journals (Sweden)

    Rivadeneira Fernando

    2010-11-01

    Full Text Available Abstract Background The objective of this study was to examine the relationship between common genetic variation of the ESR2 gene and osteoarthritis. Methods In the discovery study, the Rotterdam Study-I, 7 single nucleotide polymorphisms (SNPs were genotyped and tested for association with hip (284 cases, 2772 controls, knee (665 cases, 2075 controls, and hand OA (874 cases, 2184 controls using an additive model. In the replication stage one SNP (rs1256031 was tested in an additional 2080 hip, 1318 knee and 557 hand OA cases and 4001, 2631 and 1699 controls respectively. Fixed- and random-effects meta-analyses were performed over the complete dataset including 2364 hip, 1983 knee and 1431 hand OA cases and approximately 6000 controls. Results The C allele of rs1256031 was associated with a 36% increased odds of hip OA in women of the Rotterdam Study-I (OR 1.36, 95% CI 1.08-1.70, p = 0.009. Haplotype analysis and analysis of knee- and hand OA did not give additional information. With the replication studies, the meta-analysis did not show a significant effect of this SNP on hip OA in the total population (OR 1.06, 95% CI 0.99-1.15, p = 0.10. Stratification according to gender did not change the results. In this study, we had 80% power to detect an odds ratio of at least 1.14 for hip OA (α = 0.05. Conclusion This study showed that common genetic variation in the ESR2 gene is not likely to influence the risk of osteoarthritis with effects smaller than a 13% increase.

  7. Confirmation of the potential usefulness of two human beta globin pseudogene markers to estimate gene flows to and from sub-Saharan Africans.

    Science.gov (United States)

    Ciminelli, Bianca Maria; Pompei, Fiorenza; Relucenti, Michela; Lum, J Koji; Simporé, Jacques; Spedini, Gabriella; Martínez-Labarga, Cristina; Pardo, Miguel G

    2002-04-01

    Two polymorphic sites, -107 and -100 with respect to the "cap" site of the human beta globin pseudogene, recently discovered in our laboratory, turned out to have an ethnically complementary distribution. The first site is polymorphic in Europeans, North Africans, Indians (Hindu), and Oriental Asians, and monomorphic in sub-Saharan Africans. Conversely, the second site is polymorphic in sub-Saharan African populations and monomorphic in the aforementioned populations. Here we report the gene frequencies of these two polymorphic sites in nine additional populations (Egyptians, Spaniards, Japanese, Chinese, Filipinos, Vietnamese, Africans from Togo and from Benin, and Pygmies), confirming their ethnospecificity and, through the analysis of these two markers in Oromo and Amhara of Ethiopia (two mixed populations), their usefulness in genetic admixture studies. Moreover, we studied another marker polymorphic in sub-Saharan African populations only, a TaqI restriction fragment length polymorphism located in the same region as the present markers, demonstrating the absence of linkage disequilibrium between it and the -100 site, so that we can exclude that the information they provide is redundant.

  8. Hereditary Xerocytosis due to Mutations in PIEZO1 Gene Associated with Heterozygous Pyruvate Kinase Deficiency and Beta-Thalassemia Trait in Two Unrelated Families

    Directory of Open Access Journals (Sweden)

    Elisa Fermo

    2017-01-01

    Full Text Available Hereditary xerocytosis (HX is a rare disorder caused by defects of RBC permeability, associated with haemolytic anaemia of variable degree and iron overload. It is sometimes misdiagnosed as hereditary spherocytosis or other congenital haemolytic anaemia. Splenectomy is contraindicated due to increased risk of thromboembolic complications. We report the clinical, haematological, and molecular characteristics of four patients from two unrelated Italian families affected by HX, associated with beta-thalassemia trait and heterozygous pyruvate kinase deficiency, respectively. Two patients had been splenectomised and displayed thrombotic episodes. All patients had iron overload in the absence of transfusion, two of them requiring iron chelation. The diagnosis of HX was confirmed by LoRRca Osmoscan analysis showing a left-shifted curve. PIEZO1 gene sequencing revealed the presence of mutation p.E2496ELE, showing that this is one of the most frequent mutations in this disease. The concomitant defects did not aggravate the clinical phenotype; however, in one patient, the initial diagnosis of pyruvate kinase deficiency delayed the correct diagnosis of HX for many years and resulted in splenectomy followed by thrombotic complications. The study underlines the importance of a precise diagnosis in HX, particularly in view of splenectomy, and the need of a molecular confirmation of suspected RBC enzymopathy.

  9. The role of horizontal gene transfer in the dissemination of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates in an endemic setting

    Science.gov (United States)

    Doi, Yohei; Adams-Haduch, Jennifer M.; Peleg, Anton Y.; D’Agata, Erika MC

    2012-01-01

    The contribution of horizontal gene transmission (HGT) in the emergence and spread of extended-spectrum beta-lactamase (ESBL)-producing gram-negative bacteria during periods of endemicity is unclear. Over a 12-month period, rectal colonization with SHV-5 and SHV-12 producing-Escherichia coli and Klebsiella pneumoniae was quantified among a cohort of residents in a long-term care facility. Demographic and clinical data were collected on colonized residents. Transferability of SHV-encoding plasmids and pulsed-field gel electrophoresis was performed to quantify the contribution of HGT and cross-transmission, respectively. A total of 25 (12%) of 214 enrolled patients were colonized with 11 SHV-5- and 17 SVH-12-producing E. coli and K. pneumoniae. Clonally-related isolates were detected among multiple residents residing on the same and different wards. Among 12 clonally-distinct isolates, HGT of SHV-5- and SHV-12-encoding plasmids was identified among 6 (50%) isolates. HGT among clonally-distinct strains contributes to the transmission dynamics of these ESBL-producing gram-negative bacteria and should be considered when evaluating the spread of these pathogens. PMID:22722012

  10. Hereditary Xerocytosis due to Mutations in PIEZO1 Gene Associated with Heterozygous Pyruvate Kinase Deficiency and Beta-Thalassemia Trait in Two Unrelated Families

    Science.gov (United States)

    Vercellati, Cristina; Marcello, Anna Paola; Zaninoni, Anna; van Wijk, Richard; Mirra, Nadia; Curcio, Cristina; Cortelezzi, Agostino; Zanella, Alberto; Barcellini, Wilma; Bianchi, Paola

    2017-01-01

    Hereditary xerocytosis (HX) is a rare disorder caused by defects of RBC permeability, associated with haemolytic anaemia of variable degree and iron overload. It is sometimes misdiagnosed as hereditary spherocytosis or other congenital haemolytic anaemia. Splenectomy is contraindicated due to increased risk of thromboembolic complications. We report the clinical, haematological, and molecular characteristics of four patients from two unrelated Italian families affected by HX, associated with beta-thalassemia trait and heterozygous pyruvate kinase deficiency, respectively. Two patients had been splenectomised and displayed thrombotic episodes. All patients had iron overload in the absence of transfusion, two of them requiring iron chelation. The diagnosis of HX was confirmed by LoRRca Osmoscan analysis showing a left-shifted curve. PIEZO1 gene sequencing revealed the presence of mutation p.E2496ELE, showing that this is one of the most frequent mutations in this disease. The concomitant defects did not aggravate the clinical phenotype; however, in one patient, the initial diagnosis of pyruvate kinase deficiency delayed the correct diagnosis of HX for many years and resulted in splenectomy followed by thrombotic complications. The study underlines the importance of a precise diagnosis in HX, particularly in view of splenectomy, and the need of a molecular confirmation of suspected RBC enzymopathy. PMID:28367341

  11. Spontaneous perforation of the small intestine, a novel manifestation of classical homocystinuria in an adult with new cystathionine beta-synthetase gene mutations.

    Science.gov (United States)

    Muacević-Katanec, Diana; Kekez, Tihomir; Fumić, Ksenija; Barić, Ivo; Merkler, Marijan; Jakić-Razumović, Jasminka; Krznarić, Zeljko; Zadro, Renata; Katanec, Davor; Reiner, Zeljko

    2011-03-01

    The clinical picture of classical homocystinuria is diverse. This is the first report of an adult homocystinuric patient with non-traumatic spontaneous small bowel perforation. A 47-year old man presented with abdominal rebound tenderness, hypotension and tachycardia, anemia, and elevated markers of inflammation. Other routine laboratory tests were normal. Abdominal x-ray showed no free air. An emergency laparotomy revealed jejunal perforation in the left upper quadrant. Histologic specimen showed full-thickness nonspecific inflammation of the intestinal wall with granulocytic infiltration, hemorrhage and necrosis. Tuberculosis, actinomycosis and typhus were histologically and clinically excluded. After excluding all known possible causes of perforation, we presumed a causative relationship between homocystinuria and small bowel perforation. It could be hypothesized that connective tissue weakness in homocystinuria is a result of homocysteine interference with recombinant human fibrillin-1 fragments or cross-linking of collagen through permanent degradation of disulfide bridges and lysine amino acid residues in proteins. DNA analysis showed three detectable mutations in the cystathionine beta-synthetase gene, 1278T:c.833T>C, and two new mutations, V372G:c.1133T > G, and D520G:c.1558A > G in the aternatively spliced exon 15.

  12. Geographic distribution of 119 alleles of the alpha and beta globin genes detected in 432 French Caucasian carriers of haemoglobin variant.

    Science.gov (United States)

    Chami, B; Braconnier, F; Riou, J; Bardakdjian-Michau, J; Préhu, C; Blouquit, Y; Rosa, J; Wajcman, H; Galactéros, F

    1995-01-01

    The French population is the result of mixing of different peoples including the Celts, Saxons, Germans, Italians and Hispanics. Between 1981 and 1993 patients were selected during investigations in France for haematological disorders associated or otherwise with the presence of a haemoglobin (Hb) variant. Further carriers of abnormal Hb were identified by HPLC measurement of glycated Hb in diabetics and by neonatal screening. Four-hundred and thirty-two subjects were found to be heterozygous for one of the 119 different alpha and the beta gene alleles encountered. These variants were characterised by a combination of 6 electrophoretic methods and in some cases by protein structure determinations. Some mutants reflected the population movements in and into France. A few mutants are frequently described in the French Caucasian population: Hb Lepore Boston, Hb D Punjab, whereas others appear to be anthropological markers. Hb Winnipeg has only been found in the West of France (Normandy); Hb J Baltimore is mainly found in French subjects of Spanish origin. Several cases of sporadic and previously undescribed mutations of Hb were identified. The last immigration waves from Africa and Asia appear to have contributed to the evolution of the pattern of haemoglobinopathies in the French population (Hb S, Hb O Arab or Hb C).

  13. Prevention of beta cell dysfunction and apoptosis by adenoviral gene transfer of rat insulin-like growth factor 1

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhi-hong; LI Tang; CHEN Zong-bo; LUO Bing; SUN Ruo-peng

    2009-01-01

    Background Islet β-cells are almost completely destroyed when patients with type 1 diabete are diagnosed. To date, insulin substitute therapy is still one of the main treatments. The cure of type 1 diabetes requires β-cell regeneration from islet cell precursors and prevention of recurring autoimmunity, Therefore, β-cell regeneration and proliferation emerge as a new research focus on therapy for type 1 diabetes. Islet β-cell regeneration and development are controlled by many growth factors, especially insulin-like growth factor-1 (IGF-1).Methods Recombinant adenovirus encoding rat IGF-1 (rlGF-1) was constructed and transduced into rat β-cells, RINm5F cells. Western blotting analysis and ELISA were used to detect rlGF-1 protein. Streptozotocin (STZ) was used to induce RINm5F cell destruction. The level of nitric oxide (NO) was detected in cell culture supernatants by the Griess reaction. Islet cell function was evaluated by glucose-stimulated insulin production. Flow cytometry analysis was further used to investigate the apoptosis of RINm5F cells. Thiaoollyl blue viability assay was applied to determine cell viability.Results The recombined adenovirus-rlGF-1 was successfully constructed and the titer was 4.0×108pfu/ml. The rlGF-1 protein was effectively expressed in the RINm5F cells and cell culture supernatants, rlGF-1 expression remarkably inhibited STZ-induced islet cell apoptosis and significantly decreased the level of NO. Furthermore, IGF-1 expression also significantly protected insulin secretion and cell proliferation in a time-dependent manner.Conclusions Our study suggests that locally produced rlGF-1 from RINm5F cells may be beneficial in maintaining β-cell function, protecting β-cells from the destruction of apoptosis factors and promoting β-cell survival and proliferation. IGF-1 might be considered as a candidate gene in gene therapy for type 1 diabetes. In addition, it appears that the apoptosis induced by STZ may be NO-dependent.

  14. Effect of Prolactin Receptor (PRLR) and Beta-Casein (CSN2) Gene Polymorphism on the Chemical Composition of Milk Sows.

    Science.gov (United States)

    Skrzypczak, Ewa; Babicz, Marek; Pastwa, Marcin

    2015-01-01

    The objective of the studies was to evaluate the impact of the prolactin receptor and β-casein genes on the basic chemical composition and pH of the colostrum and milk of sows. Experiments were carried out on 103 Złotnicka White breed sows. These animals are under the Domestic Program of Protection of Genetic Resources. Analysis of the influence of polymorphism in the PRLR and CSN2 loci revealed that sows of the TT homozygote were characterised by the highest dry matter content. Analysis of polymorphism in the PRLR locus for protein showed that the highest values were in milk of sows of the TT genotype, and GG homozygotes in the case of the CSN2 locus. Inference of the impact of polymorphism in the PRLR and CSN2 loci on the fat and lactose content of sow milk demonstrated considerable variability. These differences were statistically significant at the level of α = 0.01 and α = 0.05. Periodical changes in individual pH values were apparent for particular genotypes in both loci (PRLR and CSN2). The perceptible changes that occurred between individual genotypes were statistically significant at the levels of α = 0.01 and α = 0.05. The investigations confirmed that the nutritive values of sow colostrum and milk were determined by genetic factors. This issue warrants comprehensive analysis, especially in terms of evaluation of the breeding value of maternal breeds.

  15. Analysis of Beta-Lactoglobuline Gene (LGB Polymorphism in Different Breeds of Bulls by High Resolution Melting

    Directory of Open Access Journals (Sweden)

    Martina Miluchová

    2012-05-01

    Full Text Available The goal of the paper was to identify  - lactoglobulin gene polymorphism in bulls. The  - lactoglobulin (LGB is expressed in milk and is important in the evaluation of milk production potential and butterfat and protein content. LGB is localized on bovine chromosome 11. The AA genotype of LGB is associated with higher milk yield, the BB genotype with higher fat and casein content and is more desirable for cheese making. The material involved 46 bulls (Slovak spotted breed – 41 bulls, Pinzgau breed – 3 bulls and Holstein breed – 2 bulls. Bovine genomic DNA was isolated from sperm using commercial kit NucleoSpin Tissue and used in order to estimate LGB genotypes by means of PCR RFLP method and high resolution melting analysis (HRMA. In the population of Slovak spotted breed we detected all genotypes AA, AB and BB with frequency 0.3415, 0.4390 and 0.2195, subsequently. In Pinzgau breed was detected homozygote genotypes AA and BB with frequency 0.3333 and 0.6667. In Holstein breed was observed only heterozygote genotype AB with frequency 1.

  16. Expression, purification, and characterization of human acetyl-CoA carboxylase 2.

    Science.gov (United States)

    Kim, Ki Won; Yamane, Harvey; Zondlo, James; Busby, James; Wang, Minghan

    2007-05-01

    The full-length human acetyl-CoA carboxylase 1 (ACC1) was expressed and purified to homogeneity by two separate groups (Y.G. Gu, M. Weitzberg, R.F. Clark, X. Xu, Q. Li, T. Zhang, T.M. Hansen, G. Liu, Z. Xin, X. Wang, T. McNally, H. Camp, B.A. Beutel, H.I. Sham, Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1-methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors, J. Med. Chem. 49 (2006) 3770-3773; D. Cheng, C.H. Chu, L. Chen, J.N. Feder, G.A. Mintier, Y. Wu, J.W. Cook, M.R. Harpel, G.A. Locke, Y. An, J.K. Tamura, Expression, purification, and characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes, Protein Expr. Purif., in press). However, neither group was successful in expressing the full-length ACC2 due to issues of solubility and expression levels. The two versions of recombinant human ACC2 in these reports are either truncated (lacking 1-148 aa) or have the N-terminal 275 aa replaced with the corresponding ACC1 region (1-133 aa). Despite the fact that ACC activity was observed in both cases, these constructs are not ideal because the N-terminal region of ACC2 could be important for the correct folding of the catalytic domains. Here, we report the high level expression and purification of full-length human ACC2 that lacks only the N-terminal membrane attachment sequence (1-20 and 1-26 aa, respectively) in Trichoplusia ni cells. In addition, we developed a sensitive HPLC assay to analyze the kinetic parameters of the recombinant enzyme. The recombinant enzyme is a soluble protein and has a K(m) value of 2 microM for acetyl-CoA, almost 30-fold lower than that reported for the truncated human ACC2. Our recombinant enzyme also has a lower K(m) value for ATP (K(m)=52 microM). Although this difference could be ascribed to different assay conditions, our data suggest that the longer human ACC2 produced in our system may have higher affinities for the substrates and could

  17. Immunochemical localization of ribulose-1,5-bisphosphate carboxylase in the symbiont-containing gills of Solemya velum (Bivalvia : Mollusca)

    NARCIS (Netherlands)

    Cavanaugh, Colleen M.; Abbott, Marilyn S.; Veenhuis, Marten

    1988-01-01

    The distribution of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase (RbuP2Case; EC 4.1.1.39) was examined by using two immunological methods in tissues of Solemya velum, an Atlantic coast bivalve containing putative chemoautotrophic symbionts. Antibodies elicited by the purified large

  18. Multiplex PCR Study of Plasmid-Mediated AmpC Beta-Lactamases Genes in Clinical Isolates of Escherichia coli

    Directory of Open Access Journals (Sweden)

    Maryam Dehghani

    2017-02-01

    Full Text Available Background:   AmpC β-lactamases are important cephalosporinases chromosomally encoded in many of Enterobacteriaceae and a few other organisms where they mediate resistance to cephalothin, cefazolin, cefoxitin and penicillins. The six different families of plasmid-mediated AmpC β-lactamases have been described, but no phenotypic test can discriminate among them. AmpC multiplex PCR has been successfully used to discriminate plasmid-mediated ampC specific families in organisms such as Klebsiella pneumonia and Escherichia coli. The aim of this study was to indicate the prevalence of AmpC β-lactamase genes by specifically designed primers through PCR test.Methods:   243 total clinical urine samples were collected, and 227 isolates were identified as Escherichia coli based on standard biochemical tests. Subsequently, the isolates were screened by disc diffusion and combined disc test for β-lactamase production. Resistant isolates were evaluated by PCR for ampC family determination. Results:  Antibiotic resistance pattern were observed as follows: cefepime (%25, ceftazidime (%31, ceftriaxone (%37, cefotaxime (%38. The ratio of isolates was detected as ESBLs and AmpC producers were 34% and 5.2%, respectively. PCR performed on 12 selected isolates via phenotypic tests and the results revealed that among 12 isolates, 11 contained blaCMY-42. Conclusion:  Unfortunately, antibiotic resistance has become an increasingly critical problem in many countries like Iran and occurrence of isolates co-expressing AmpC-β-lactamases and ESBLs can create serious problems in the future. As antibiotic options in the treatment of AmpC β-lactamases and ESBLs producing organisms are extremely limited, molecular screening by laboratories is suggested to reduce the risk of therapeutic defeat.

  19. Mutant U5A cells are complemented by an interferon-alpha beta receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster.

    OpenAIRE

    Lutfalla, G; Holland, S J; Cinato, E; Monneron, D; Reboul, J.; Rogers, N C; J. M. Smith; Stark, G R; Gardiner, K.; Mogensen, K E

    1995-01-01

    The cellular receptor for the alpha/beta interferons contains at least two components that interact with interferon. The ifnar1 component is well characterized and a putative ifnar2 cDNA has recently been identified. We have cloned the gene for ifnar2 and show that it produces four different transcripts encoding three different polypeptides that are generated by exon skipping, alternative splicing and differential use of polyadenylation sites. One polypeptide is likely to be secreted and two ...

  20. Preferential Expression of a Beta-tubulin Gene in Developing Cotton Fibers%棉花发育纤维中一个优势表达的β-Tubulin基因

    Institute of Scientific and Technical Information of China (English)

    Yuan-li LI; Jie SUN; Chun-hong LI; Yong-qing ZHU; Gui-xian XIA

    2002-01-01

    @@ The developing cotton fiber is considered as a model system for studying the function of microtubules in plant cell elongation and cell wall biosynthesis. It has been shown that microtubules undergo active dynamic changes during fiber development. At the molecular level, Dixon et al demonstrated that two alphatubulin and two beta-tubulin isoforms accumulated preferentially in fibers, and Whittaker and Triplett showed that the accumulation of alpha-tubulin transcripts changed in a gene-specific manner in the developing cotton fibers.

  1. Pollination-, development-, and auxin-specific regulation of gibberellin 3beta-hydroxylase gene expression in pea fruit and seeds.

    Science.gov (United States)

    Ozga, Jocelyn A; Yu, Jody; Reinecke, Dennis M

    2003-03-01

    To understand further how pollination, seeds, auxin (4-chloroindole-3-acetic acid [4-Cl-IAA]), and gibberellins (GAs) regulate GA biosynthesis in pea (Pisum sativum) fruit, we studied expression of the gene PsGA3ox1 that codes for the enzyme that converts GA(20) to biologically active GA(1) using real-time reverse transcription-polymerase chain reaction analysis. PsGA3ox1 mRNA levels were minimally detectable in prepollinated pericarps and ovules (-2 d after anthesis [DAA]), increased dramatically after pollination (0 DAA), then decreased by 1 DAA. Seed PsGA3ox1 mRNA levels increased at 4 DAA and again 8 to 12 DAA, when seed development was rapid. Pericarp PsGA3ox1 mRNA levels peaked coincidentally with rapid pod diameter expansion (6-10 DAA) to accommodate the growing seeds. The effects of seeds and hormones on the expression of pericarp PsGA3ox1 were investigated over a 24-h treatment period. Pericarp PsGA3ox1 mRNA levels gradually increased from 2 to 3 DAA when seeds were present; however, when the seeds were removed, the pericarp transcript levels dramatically declined. When 2-DAA deseeded pericarps were treated with 4-Cl-IAA, PsGA3ox1 mRNA levels peaked 4 h after hormone treatment (270-fold increase), then decreased. PsGA3ox1 mRNA levels in deseeded pericarps treated with indole-3-acetic acid or GA(3) were the same or lower than deseeded controls. These data show that PsGA3ox1 is expressed and developmentally regulated in pea pericarps and seeds. These data also show that pericarp PsGA3ox1 expression is hormonally regulated and suggest that the conversion of GA(20) to GA(1) occurs in the pericarp and is regulated by the presence of seeds and 4-Cl-IAA for fruit growth.

  2. Beta-Lactamase Encoded Genes blaTEM and blaCTX Among Acinetobacter baumannii Species Isolated From Medical Devices of Intensive Care Units in Tehran Hospitals

    Science.gov (United States)

    Khalilzadegan, Sara; Sade, Mojtaba; Godarzi, Hussein; Eslami, Gita; Hallajzade, Masoumeh; Fallah, Fatemeh; Yadegarnia, Davood

    2016-01-01

    Background Excessive consumption of antimicrobial materials in hospitals is considered as the main encoder leading to the emergence, development and acquisition of new bacterial resistance to beta-lactamase. Objectives Owing to the lack of proper information regarding the mechanism of the bacterial resistance to antibiotics and responsible genes in the country, the current study aimed to consider the resistance or sensitivity of the Acinetobacter baumannii multi drug resistant (MDR) isolates facing 2% glutaraldehyde. The study was conducted in the selected intensive care units in Tehran hospitals, Iran, in 2013. Materials and Methods In this study conducted over a period of 10 months, A. baumannii species were isolated by bacterial culture following biochemical tests from intensive care units (ICUs) of some hospitals in Tehran, Iran (Fayazbaksh, Taleghani, Imam Khomeini, Valiasr, Labafinejad). The resistance and sensitivity of the isolates to antibiotics were considered according to the clinical and laboratory standard institute CLSI (2012) guidelines. By multiplex PCR method, blaCTX and blaTEM genes were detected and finally, MDR strains were treated with 2% glutaraldehyde. PCR was used for each strain of MDR using specific primers. Results In the current study, 131 A. baumannii isolates (22.3%) out of 588 were studied. The level of resistance to various antibiotics was in the range of 69.4% to 100%. The frequencies of blaTEM and blaCTX genes were 3.2% and 19.4%, respectively. MIC50% and MIC90% of imipenem and meropenem antibiotics were 32 ± 1 µg/mL and 64 ± 1 µg/mL, respectively (P < 0.9). However no resistance to glutaraldehyde was observed. Different bands of MDR strains were observed in the PCR product by electrophoresis. Conclusions It seems that besides the variety and prevalence of blaTEM and blaCTX, enormous mechanisms such as porin and leaking systems (efflux pumps) are responsible for the information of the A. baumannii resistance to disinfectants

  3. Targeted disruption of the IA-2beta gene causes glucose intolerance and impairs insulin secretion but does not prevent the development of diabetes in NOD mice.

    NARCIS (Netherlands)

    Kubosaki, A.; Gross, S.; Miura, J.; Saeki, K.; Zhu, M.; Nakamura, S.; Hendriks, W.J.A.J.; Notkins, A.L.

    2004-01-01

    Insulinoma-associated protein (IA)-2beta, also known as phogrin, is an enzymatically inactive member of the transmembrane protein tyrosine phosphatase family and is located in dense-core secretory vesicles. In patients with type 1 diabetes, autoantibodies to IA-2beta appear years before the developm

  4. Inflammatory arthritis in caspase 1 gene-deficient mice: contribution of proteinase 3 to caspase 1-independent production of bioactive interleukin-1beta.

    NARCIS (Netherlands)

    Joosten, L.A.B.; Netea, M.G.; Fantuzzi, G.; Koenders, M.I.; Helsen, M.M.A.; Sparrer, H.; Pham, C.T.; Meer, J.W.M. van der; Dinarello, C.A.; Berg, W.B. van den

    2009-01-01

    OBJECTIVE: Caspase 1, a known cysteine protease, is a critical component of the inflammasome. Both caspase 1 and neutrophil serine proteases such as proteinase 3 (PR3) can process pro-interleukin-1beta (proIL-1beta), a crucial cytokine linked to the pathogenesis of rheumatoid arthritis. This study w

  5. Acetyl CoA Carboxylase 2 Is Dispensable for CD8+ T Cell Responses.

    Directory of Open Access Journals (Sweden)

    Jang Eun Lee

    Full Text Available Differentiation of T cells is closely associated with dynamic changes in nutrient and energy metabolism. However, the extent to which specific metabolic pathways and molecular components are determinative of CD8+ T cell fate remains unclear. It has been previously established in various tissues that acetyl CoA carboxylase 2 (ACC2 regulates fatty acid oxidation (FAO by inhibiting carnitine palmitoyltransferase 1 (CPT1, a rate-limiting enzyme of FAO in mitochondria. Here, we explore the cell-intrinsic role of ACC2 in T cell immunity in response to infections. We report here that ACC2 deficiency results in a marginal increase of cellular FAO in CD8+ T cells, but does not appear to influence antigen-specific effector and memory CD8+ T cell responses during infection with listeria or lymphocytic choriomeningitis virus. These results suggest that ACC2 is dispensable for CD8+ T cell responses.

  6. In Vitro Reassembly of Tobacco Ribulose-1,5-bisphosphate Carboxylase/ Oxygenase from Fully Denatured Subunits

    Institute of Scientific and Technical Information of China (English)

    Zhen-Hua YONG; Gen-Yun CHEN; Jiao-Nai SHI; Da-Quan XU

    2006-01-01

    It has been generally proved impossible to reassemble ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from fully denatured subunits in vitro in higher plant, because large subunit of fully denatured Rubisco is liable to precipitate when the denaturant is removed by common methods of direct dilution and one-step dialysis. In our experiment, the problem of precipitation was resolved by an improved gradual dialysis method, which gradually decreased the concentration of denaturant. However, fully denatured Rubisco subunits still could not be reassembled into holoenzyme using gradual dialysis unless chaperonin 60was added. The restored activity of reassembled Rubisco was approximately 8% of natural enzyme. The quantity of reassembled Rubisco increased greatly when heat shock protein 70 was present in the reassembly process. ATP and Mg2+ were unnecessary for in vitro reassembly of Rubisco, and Mg2+ inhibited the reassembly process. The reassembly was weakened when ATP, Mg2+ and K+ existed together in the reassembly process.

  7. Acetyl-CoA carboxylase inhibitors from avocado (Persea americana Mill) fruits.

    Science.gov (United States)

    Hashimura, H; Ueda, C; Kawabata, J; Kasai, T

    2001-07-01

    A methanol extract of avocado fruits showed potent inhibitory activity against acetyl-CoA carboxylase, a key enzyme in fatty acid biosynthesis. The active principles were isolated and identified as (5E,12Z,15Z)-2-hydroxy-4-oxoheneicosa-5,12,15-trienyl (1), (2R,12Z,15Z)-2-hydroxy-4-oxoheneicosa-12,15-dienyl (2), (2R*,4R*)-2,4-dihydroxyheptadec-16-enyl (3) and (2R*,4R*)-2,4-dihydroxyheptadec-16-ynyl (4) acetates by instrumental analyses. The IC50 of the compounds were 4.0 x 10(-6), 4.9 x 10(-6), 9.4 x 10(-6), and 5.1 x 10(-6) M, respectively.

  8. Two new beta0-thalassemic mutations: a deletion (-CC) at codon 142 or overlapping codons 142-143, and an insertion (+T) at codon 45 or overlapping codons 44-45/45-46 of the beta-globin gene.

    Science.gov (United States)

    Lacan, Philippe; Aubry, Martine; Couprie, Nicole; Francina, Alain

    2007-01-01

    We report here two new beta(0)-thalassemic mutations. In the first case, a deletion of two nucleotides (-CC) at codon 142 was found in a French Caucasian woman. In the second case, an insertion of a single nucleotide (+T) at codon 45 was found in a Turkish girl. In both cases, no dominant thalassemia-like phenotype was observed.

  9. Mutant U5A cells are complemented by an interferon-alpha beta receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster.

    Science.gov (United States)

    Lutfalla, G; Holland, S J; Cinato, E; Monneron, D; Reboul, J; Rogers, N C; Smith, J M; Stark, G R; Gardiner, K; Mogensen, K E

    1995-10-16

    The cellular receptor for the alpha/beta interferons contains at least two components that interact with interferon. The ifnar1 component is well characterized and a putative ifnar2 cDNA has recently been identified. We have cloned the gene for ifnar2 and show that it produces four different transcripts encoding three different polypeptides that are generated by exon skipping, alternative splicing and differential use of polyadenylation sites. One polypeptide is likely to be secreted and two are transmembrane proteins with identical extracellular and transmembrane domains but divergent cytoplasmic tails of 67 and 251 amino acids. A mutant cell line U5A, completely defective in IFN-alpha beta binding and response, has been isolated and characterized. Expression in U5A cells of the polypeptide with the long cytoplasmic domain reconstitutes a functional receptor that restores normal interferon binding, activation of the JAK/STAT signal transduction pathway, interferon-inducible gene expression and antiviral response. The IFNAR2 gene maps at 0.5 kb from the CRFB4 gene, establishing that together IFNAR2, CRFB4, IFNAR1 and AF1 form a cluster of class II cytokine receptor genes on human chromosome 21.

  10. Molecular Analysis of Gene Frequencies of TEM, CTX-M and SHV in Beta-Lactam Antibiotic-Resistant Strains of E. Coli Isolated from Urinary Tract Infections in Yasuj Hospitals

    Directory of Open Access Journals (Sweden)

    R Mortezavi

    2014-06-01

    Full Text Available Background & aim: Urinary tract infections is one of the most common infectious diseases which many factors are involved, but bacteria such as E.coli is the most important agent of urinary tract infections. Antibiotic resistance as a major problem in the treatment and control of these infections is considered. The aim of this study was to determine the genes that cause resistance to beta-lactam family of antibiotics on E.coli isolated from urinary tract infections in Yasuj city. Methods: In the present Cross-sectional study which was conducted over a period of seven months in 2013, 123 samples of E.coli were collected from Yasuj hospitals for molecular analysis of TEM, SHV CTX-M genes, causing antibiotic resistance by (PCR method.Data were analyzed using SPSS statistical test. Results: PCR showed that the gene frequency of TEM (50.94%, SHV (47.16%, CTX-M-9 (35.84%, and CTX-M-10, (32.07% and the highest and lowest prevalent of genes were related to TEM and CTX-M10 in E.coli isolated from urinary tract infections respectively. Conclusion: According to the high prevalence of resistance to beta-lactam antibiotics, the current study showed that the noted genes play an important role in facilitating the spread of antimicrobial resistance in this region.

  11. Antibacterial activities of multi drug resistant Myroides odoratimimus bacteria isolated from adult flesh flies (Diptera: Sarcophagidae are independent of metallo beta-lactamase gene Atividades antibacterianas de Myroides odoratimimus isolada de moscas varejeiras adultas (Diptera: Sarcophagidae são independentes do gene metalo beta lactamase

    Directory of Open Access Journals (Sweden)

    M.S. Dharne

    2008-06-01

    avaliação de parâmetros nutricionais pelo sistema BIOLOG GN, ao sequenciamento genético 16S rRNA, à sensibilidade a vários antimicrobianos pelo método de difusão de discos e à detecção dos genes de metalo beta lactamases (TUS/MUS. Os efeitos antagonistas foram testados contra bactérias Gram negativas e Gram positivas isoladas de material clínico humano, amostras ambientais e intestino do inseto. As espécies bacterianas incluíram Aeromonas hydrophila, A. culicicola, Morganella morganii subsp sibonii, Ochrobactrum anthropi, Weissella confusa, Escherichia coli, Ochrobactrum sp, Serratia sp, Kestersia sp, Ignatzschineria sp e Bacillus sp. A cepa Myroides sp foi resistente à penicilina G, eritromicina, estreptomicina, amicacina, canamicina, gentamicina, ampicilina, trimetoprim e tobramicina. Esta cepa apresentou atividade antimicrobiana contra todas as cepas exceto W.confusa, Ignatzschineria sp, A. hydrophila e M. morgani subsp sibonii. A resistência múltipla da cepa foi semelhante à de isolados clínicos, inibindo bactérias das amostras clínicas, ambientais e do intestino do inseto. Os genes de metalo beta lactamases (TUS/MUS estavam ausentes, excluindo-se a resistência mediada por esses genes, o que indica o envolvimento de um mecanismo alternativo de secreção.

  12. Two thyroid hormone regulated genes, the beta-subunits of nerve growth factor (NGFB) and thyroid stimulating hormone (TSHB), are located less than 310 kb apart in both human and mouse genomes.

    Science.gov (United States)

    Dracopoli, N C; Rose, E; Whitfield, G K; Guidon, P T; Bale, S J; Chance, P A; Kourides, I A; Housman, D E

    1988-08-01

    Two thyroid hormone regulated genes, the beta-subunits of nerve growth factor (NGFB) and thyroid stimulating hormone (TSHB), have been assigned to mouse chromosome 3 and human chromosome 1p22. We have used the techniques of linkage analysis and pulsed field gel electrophoresis to determine the proximity of these two antithetically regulated genes in this conserved linkage group. Four novel restriction fragment length polymorphisms were identified at the human TSHB gene. Two-point linkage analysis between TSHB and NGFB in 46 families, including the Centre d'Etude du Polymorphisme Humain (CEPH) reference panel, demonstrated no recombination (theta = 0.00, Z = 42.8). Analysis of this region by pulsed field gel electrophoresis showed that the genes for TSHB and NGFB are located less than 310 kb apart in man and 220 kb in the mouse.

  13. G-protein beta3 subunit gene variant is unlikely to have a significant influence on serum uric acid level in Japanese workers.

    Science.gov (United States)

    Suwazono, Yasushi; Kobayashi, Etsuko; Uetani, Mirei; Miura, Katsuyuki; Morikawa, Yuko; Ishizaki, Masao; Kido, Teruhiko; Nakagawa, Hideaki; Nogawa, Koji

    2006-06-01

    The C825T variant of the G-protein beta3 subunit (GNB3) gene has attracted renewed attention as a candidate gene for obesity, hypertension and hyperuricemia. The main role of G-protein is to translate signals from the cell surface into a cellular response. The 825T allele is associated with a splice variant of GNB3 protein and enhanced G-protein activation. We examined the relationship between this variant and the risk of hyperuricemia in Japanese workers. The study subjects were 1,452 men and 1,169 women selected from 3,834 men and 2,591 women in 1997. On the basis of common clinical criteria, hyperuricemia I was defined as serum uric acid >or= 7.0 mg/dl in men and 6.0 mg/dl in women or taking antihyperuricemic medication. The hyperuricemia I group consisted of 186 men and 20 women and its control of 1,266 men and 1,149 women. Hyperuricemia II was defined as serum uric acid > 5.7 mg/dl (median) in men and 3.9 mg/dl (median) in women or taking antihyperuricemic medication. The hyperuricemic II group consisted of 684 men and 570 women and its control of 768 men and 599 women. To replicate previous significant results in young Caucasian men, we selected these criteria because the authors of the study in young Caucasian men adopted the median in their subjects as a cut-off. The statistical power was estimated as 99% based on the significant results in Caucasians. Genotype and allele distributions in men and women with hyperuricemia I and II were not significantly different from those in the corresponding control groups. Logistic regression analysis on hyperuricemia I and II, and multiple regression on serum uric acid level demonstrated no significant effect of the C825T genotype. Despite the sufficient statistical power, this study could not demonstrate the significant influence of C825T on hyperuricemia or serum uric acid. The targeting of this polymorphism is unlikely to be beneficial in the prevention of hyperuricemia in the general Japanese population.

  14. Transcription of the human beta enolase gene (ENO-3) is regulated by an intronic muscle-specific enhancer that binds myocyte-specific enhancer factor 2 proteins and ubiquitous G-rich-box binding factors.

    Science.gov (United States)

    Feo, S; Antona, V; Barbieri, G; Passantino, R; Calì, L; Giallongo, A

    1995-01-01

    To provide evidence for the cis-regulatory DNA sequences and trans-acting factors involved in the complex pattern of tissue- and stage-specific expression of the beta enolase gene, constructs containing fragments of the gene fused to the chloramphenicol acetyltransferase gene were used in transient-transfection assays of C2C12 myogenic cells. Deletion analysis revealed the presence of four major regions: two negative regions in the 5'-flanking sequence, a basal promoter region which directs expression at low levels in proliferating and differentiated muscle cells, and a positive region within the first intron that confers cell-type-specific and differentiation-induced expression. This positive regulatory element is located in the 3'-proximal portion of the first intron (nucleotides +504 to +637) and acts as an enhancer irrespective of orientation and position from the homologous beta enolase promoter or the heterologous thymidine kinase promoter, conferring in both cases muscle-specific expression to the linked reporter gene. Deletion of a putative myocyte-specific enhancer factor 1 (MEF-1) binding site, containing a canonical E-box motif, had no effects on muscle-specific transcription, indicating that this site is not required for the activity of the enhancer. Gel mobility shift assays, competition analysis, DNase I footprinting, and mutagenesis studies indicated that this element interacts through an A/T-rich box with a MEF-2 protein(s) and through a G-rich box with a novel ubiquitous factor(s). Mutation of either the G-rich box or the A/T-rich box resulted in a significantly reduced activity of the enhancer in transient-transfection assays. These data indicate that MEF-2 and G-rich-box binding factors are each necessary for tissue-specific expression of the beta enolase gene in skeletal muscle cells. PMID:7565752

  15. Resistance to spiromesifen in Trialeurodes vaporariorum is associated with a single amino acid replacement in its target enzyme acetyl-coenzyme A carboxylase.

    Science.gov (United States)

    Karatolos, N; Williamson, M S; Denholm, I; Gorman, K; ffrench-Constant, R; Nauen, R

    2012-06-01

    Spiromesifen is a novel insecticide and is classed as a tetronic acid derivative. It targets the insects' acetyl-coenzyme A carboxylase (ACCase) enzyme, causing a reduction in lipid biosynthesis. At the time of this publication, there are no reports of resistance to this class of insecticides in insects although resistance has been observed in several mite species. The greenhouse whitefly Trialeurodes vaporariorum (Westwood) is a serious pest of protected vegetable and ornamental crops in temperate regions of the world and spiromesifen is widely used in its control. Mortality rates of UK and European populations of T. vaporariorum to spiromesifen were calculated and up to 26-fold resistance was found. We therefore sought to examine the molecular mechanism underlying spiromesifen resistance in this important pest. Pre-treatment with piperonyl butoxide did not synergize spiromesifen, suggesting a target-site resistance mechanism. The full length ACCase gene was sequenced for a range of T. vaporariorum strains and a strong association was found between spiromesifen resistance and a glutamic acid substitution with lysine in position 645 (E645K) of this gene. A TaqMan allelic discrimination assay confirmed these findings. Although this resistance is not considered sufficient to compromise the field performance of spiromesifen, this association of E645K with resistance is the first report of a potential target site mechanism affecting an ACCase inhibitor in an arthropod species.

  16. The glossyhead1 allele of acc1 reveals a principal role for multidomain acetyl-coenzyme a carboxylase in the biosynthesis of cuticular waxes by Arabidopsis

    KAUST Repository

    Lu, Shiyou

    2011-09-23

    A novel mutant of Arabidopsis (Arabidopsis thaliana), having highly glossy inflorescence stems, postgenital fusion in floral organs, and reduced fertility, was isolated from an ethyl methanesulfonate-mutagenized population and designated glossyhead1 (gsd1). The gsd1 locus was mapped to chromosome 1, and the causal gene was identified as a new allele of Acetyl-Coenzyme A Carboxylase1 (ACC1), a gene encoding the main enzyme in cytosolic malonyl-coenzyme A synthesis. This, to our knowledge, is the first mutant allele of ACC1 that does not cause lethality at the seed or early germination stage, allowing for the first time a detailed analysis of ACC1 function in mature tissues. Broad lipid profiling of mature gsd1 organs revealed a primary role for ACC1 in the biosynthesis of the very-long-chain fatty acids (C 20:0 or longer) associated with cuticular waxes and triacylglycerols. Unexpectedly, transcriptome analysis revealed that gsd1 has limited impact on any lipid metabolic networks but instead has a large effect on environmental stress-responsive pathways, especially senescence and ethylene synthesis determinants, indicating a possible role for the cytosolic malonyl-coenzyme A-derived lipids in stress response signaling. © 2011 American Society of Plant Biologists. All Rights Reserved.

  17. Genetic dissection of methylcrotonyl CoA carboxylase indicates a complex role for mitochondrial leucine catabolism during seed development and germination.

    Science.gov (United States)

    Ding, Geng; Che, Ping; Ilarslan, Hilal; Wurtele, Eve S; Nikolau, Basil J

    2012-05-01

    3-methylcrotonyl CoA carboxylase (MCCase) is a nuclear-encoded, mitochondrial-localized biotin-containing enzyme. The reaction catalyzed by this enzyme is required for leucine (Leu) catabolism, and it may also play a role in the catabolism of isoprenoids and the mevalonate shunt. In Arabidopsis, two MCCase subunits (the biotinylated MCCA subunit and the non-biotinylated MCCB subunit) are each encoded by single genes (At1g03090 and At4g34030, respectively). A reverse genetic approach was used to assess the physiological role of MCCase in plants. We recovered and characterized T-DNA and transposon-tagged knockout alleles of the MCCA and MCCB genes. Metabolite profiling studies indicate that mutations in either MCCA or MCCB block mitochondrial Leu catabolism, as inferred from the increased accumulation of Leu. Under light deprivation conditions, the hyper-accumulation of Leu, 3-methylcrotonyl CoA and isovaleryl CoA indicates that mitochondrial and peroxisomal Leu catabolism pathways are independently regulated. This biochemical block in mitochondrial Leu catabolism is associated with an impaired reproductive growth phenotype, which includes aberrant flower and silique development and decreased seed germination. The decreased seed germination phenotype is only observed for homozygous mutant seeds collected from a parent plant that is itself homozygous, but not from a parent plant that is heterozygous. These characterizations may shed light on the role of catabolic processes in growth and development, an area of plant biology that is poorly understood.

  18. Common haplotype dependency of high G gamma-globin gene expression and high Hb F levels in beta-thalassemia and sickle cell anemia patients.

    OpenAIRE

    Labie, D; Pagnier, J.; Lapoumeroulie, C; Rouabhi, F; Dunda-Belkhodja, O; Chardin, P; Beldjord, C; Wajcman, H; Fabry, M E; Nagel, R L

    1985-01-01

    We have studied 42 homozygous beta-thalassemia patients from Algeria and 34 sickle cell anemia patients from Senegal and Benin, determining the relationship between haplotypes, Hb F, and G gamma-globin/A gamma-globin ratios. Populations selected have a high frequency of haplotype homozygotes because of consanguinity (Algeria) and geographic homogeneity (West Africa). We find in beta-thalassemia patients, that haplotype IX in haplotypic homozygotes and heterozygotes, haplotype III in heterozyg...

  19. An analysis of fetal hemoglobin variation in sickle cell disease: the relative contributions of the X-linked factor, beta-globin haplotypes, alpha-globin gene number, gender, and age.

    Science.gov (United States)

    Chang, Y C; Smith, K D; Moore, R D; Serjeant, G R; Dover, G J

    1995-02-15

    Five factors have been shown to influence the 20-fold variation of fetal hemoglobin (Hb F) levels in sickle cell anemia (SS): age, sex, the alpha-globin gene number, beta-globin haplotypes, and an X-linked locus that regulates the production of Hb F-containing erythrocytes (F cells), ie, the F-cell production (FCP) locus. To determine the relative importance of these factors, we studied 257 Jamaican SS subjects from a Cohort group identified by newborn screening and from a Sib Pair study. Linear regression analyses showed that each variable, when analyzed alone, had a significant association with Hb F levels (P haplotypes, alpha-globin genes, and age accounted for less than 10% of the variation. The association between the beta-globin haplotypes and Hb F levels becomes apparent if the influence of the FCP locus is removed by analyzing only individuals with the same FCP phenotype. Thus, the FCP locus is the most important factor identified to date in determining Hb F levels. The variation within each FCP phenotype is modulated by factors associated with the three common beta-globin haplotypes and other as yet unidentified factor(s).

  20. Genetic predisposition to essential hypertension in a Mongolian population Detecting the C825T polymorphism of the G-protein beta 3 subunit gene

    Institute of Scientific and Technical Information of China (English)

    Chunyu Zhang; Shigang Zhao; Guangming Niu; Rile Hu; Zhiguang Wang; Mingfang Jiang; Rile Hu

    2007-01-01

    BACKGROUND: The prevalences of hypertension, cerebrovascular diseases, etc. are higher in Mongolian population because of the influence of various factors including genetics, geography, diet, etc. Therefore, it is helpful for prevention to develop researches on the genetics of various diseases including hypertension in Mongolian population.OBJECTIVE: To analyze the association between C825T polymorphisms of G-protein beta 3 subunit gene (GNB3), the important candidate gene of various disease of cardiovascular system, and Mongolian patients with essential hypertension.DESIGN: A comparative observation.SETTINGS: Department of Neurology, the First Affiliated Hospital of Inner Mongolia Medical College;Wulate Houqi Red Cross Society.PARTICIPANTS: Totally 267 Mongolian residents, whose blood relations of 3 generations were all Mongolians, were selected from Wulate Houqi, Inner Mongolia. The patients were screened based on the diagnostic standard of hypertension set by WHO in 1999, and the enrolled subjects were divided into two groups according to the level of blood pressure: ① Normal blood pressure group (n =124): 64 males and 60 females, systolic blood pressure (SBP) < 140 mm Hg (1 mm Hg=0.133 kPa), diastolic blood pressure (DBP) <90 mm Hg; ② Essential hypertension group (n =143): 71 males and 72 females, including 60 patients with simple high SBP (SBP ranged 145 to 195 mm Hg, whereas DBP < 90 mm Hg).METHODS: Peripheral venous blood (5 mL) was drawn from all the subjects, the genome DNA was extracted, and the polymorphisms of the GNB3 C825T genotype were detected with the Sequenom system.Polymerase chain reaction (PCR) experiment and SNP detection were performed in Beijing Huada gene laboratory. Then the univariate analysis of variance was applied in the sample comparison among groups, and the chi-square test was used to compare the genotypes and allele frequencies. The odd ratio (OR) and 95% confidence interval (CI)were calculated.MAIN OUTCOME MEASURES: The

  1. Transcriptional regulation of acetyl-CoA carboxylase α isoforms in dairy ewes during conjugated linoleic acid induced milk fat depression.

    Science.gov (United States)

    Ticiani, E; Urio, M; Ferreira, R; Harvatine, K J; De Oliveira, D E

    2016-10-01

    Feeding trans-10, cis-12 CLA to lactating ewes reduces milk fat by down-regulating expression of enzymes involved in lipid synthesis in the mammary gland and increases adipose tissue lipogenesis. Acetyl-CoA carboxylase α (ACC-α) is a key regulated enzyme in de novo fatty acid synthesis and is decreased by CLA. In the ovine, the ACC-α gene is expressed from three tissue-specific promoters (PI, PII and PIII). This study evaluated promoter-specific ACC-α expression in mammary and adipose tissue of lactating cross-bred Lacaune/Texel ewes during milk fat depression induced by rumen-unprotected trans-10, cis-12 CLA supplement. In all, 12 ewes arranged in a completely randomized design were fed during early, mid and late lactation one of the following treatments for 14 days: Control (forage+0.9 kg of concentrate on a dry matter basis) and CLA (forage+0.9 kg of concentrate+27 g/day of CLA (29.9% trans-10, cis-12)). Mammary gland and adipose tissue biopsies were taken on day 14 for gene expression analysis by real-time PCR. Milk fat yield and concentration were reduced with CLA supplementation by 27%, 21% and 35% and 28%, 26% and 42% during early, mid and late lactation, respectively. Overall, our results suggest that trans-10, cis-12 CLA down-regulates mammary ACC-α gene expression by decreasing expression from PII and PIII in mammary gland and up-regulates adipose ACC-α gene expression by increasing expression from PI.

  2. Chromosomal localization and molecular marker development of the lipopolysaccharide and beta-1,3-glucan binding protein gene in the Zhikong scallop Chlamys farreri (Jones et Preston (Pectinoida, Pectinidae

    Directory of Open Access Journals (Sweden)

    Pin Huan

    2010-01-01

    Full Text Available Zhikong scallop Chlamys farreri (Jones et Preston is an economically important species in China. Understanding its immune system would be of great help in controlling diseases. In the present study, an important immunity-related gene, the Lipopolysaccharide and Beta-1,3-glucan Binding Protein (LGBP gene, was located on C. farreri chromosomes by mapping several lgbp-containing BAC clones through fluorescence in situ hybridization (FISH. Through the localization of various BAC clones, it was shown that only one locus of this gene existed in the genome of C. farreri, and that this was located on the long arm of a pair of homologous chromosomes. Molecular markers, consisting of eight single nucleotide polymorphism (SNPs markers and one insertion-deletion (indel, were developed from the LGBP gene. Indel marker testing in an F1 family revealed slightly distorted segregation (p = 0.0472. These markers can be used to map the LGBP gene to the linkage map and assign the linkage group to the corresponding chromosome. Segregation distortion of the indel marker indicated genes with deleterious alleles might exist in the surrounding region of the LGBP gene.

  3. Effects of estrogen receptor alpha and beta gene deletion on estrogenic induction of progesterone receptors in the locus coeruleus in female mice.

    Science.gov (United States)

    Helena, Cleyde; Gustafsson, Jan-Ake; Korach, Kenneth; Pfaff, Donald; Anselmo-Franci, Janete A; Ogawa, Sonoko

    2009-08-01

    Locus coeruleus (LC) is involved in the LHRH regulation by gonadal steroids. We investigated the expression of progesterone and estrogen receptors (PR; ER) in LC neurons of ERalpha (alphaERKO) or ERbeta (betaERKO) knockout mice, and their wild-type (alphaWT and betaWT). Immunocytochemical studies showed that LC expresses PR and both ERs, although ERbeta was more abundant. Estradiol benzoate (EB) decreased ERalpha-positive cells in WT and betaERKO mice, and progesterone caused a further reduction, whereas none of the steroids influenced ERbeta expression. ERbeta deletion increased ERalpha while ERalpha deletion did not alter ERbeta expression. In both WT mice, EB increased PR expression, which was diminished by progesterone. These steroid effects were also observed in alphaERKO animals but to a lesser extent, suggesting that ERalpha is partially responsible for the estrogenic induction of PR in LC. Steroid effects on PR in betaERKO mice were similar to those in the alphaERKO but to a lesser extent, probably because PR expression was already high in the oil-treated group. This expression seems to be specific of LC neurons, since it was not observed in other areas studied, the preoptic area and ventromedial nucleus of hypothalamus. These findings show that LC in mice expresses alphaER, betaER, and PR, and that a balance between them may be critical for the physiological control of reproductive function.

  4. Horizontol dissemination of TEM- and SHV-typr beta-lactamase genes-carrying resistance plasmids amongst clonical isolates of Enterobacteriaceae Disseminação horizontal de plasmídios de resistência contendo genes de beta-lactamase dos tipos TEM e SHV entre isolados clínicos de Enterobacteriaceae

    Directory of Open Access Journals (Sweden)

    Osman Birol Ozgumus

    2008-12-01

    Full Text Available The extended-spectrum β-lactamase (ESBL-producing bacteria have been isolated at increasing frequency worldwide. Expression of ESBL is often associated with multidrug resistance and dissemination by resistance plasmids. During a two-month period in 2000, 133 clinical isolates of enterobacterial strains were randomly collected from outpatients and inpatients at a university hospital in Turkey. The ESBL producing strains were determined by double-disk synergy (DDS testing. Twenty ESBL producing strains (15% including Escherichia coli (n = 9, Klebsiella pneumoniae (n = 7, Klebsiella oxytoca (n = 2 and Enterobacter aerogenes (n = 2 were detected and further analyzed for their resistance transfer features, plasmid profile and nature of the resistance genes. Plasmid transfer assays were performed using broth mating techniques. TEM- and SHV- genes were analyzed by polymerase chain reaction (PCR and hybridization using specific probes. EcoRI restriction enzyme analyses of R plasmids were used in the detection of epidemic plasmids. Fourteen plasmid profiles (A, B1, B2, C1, and C2 to L were obtained with EcoRI restriction enzyme analysis. Most of these plasmids were detected to carry both TEM- and SHV-derived genes by PCR, and confirmed by localizing each gene by hybridization assay. Epidemiological evidence indicated that there was an apparent horizontal dissemination of conjugative R plasmids among multidrug-resistant enterobacterial genera and species in this hospitalO isolamento de bactérias produtoras de beta-lactamases de espectro expandido (ESBL está aumentando no mundo todo. Freqüentemente, a expressão de ESBL está associada com resistência a múltiplas drogas e disseminação por plasmídios de resistência. Durante um período de dois meses em 2000, 133 isolados clínicos de cepas de enterobactérias foram obtidos aleatoriamente de pacientes internos e externos de um hospital universitário na Turquia. As cepas produtoras de ESBL foram

  5. Characterization of lysine acetylation of a phosphoenolpyruvate carboxylase involved in glutamate overproduction in Corynebacterium glutamicum.

    Science.gov (United States)

    Nagano-Shoji, Megumi; Hamamoto, Yuma; Mizuno, Yuta; Yamada, Ayuka; Kikuchi, Masaki; Shirouzu, Mikako; Umehara, Takashi; Yoshida, Minoru; Nishiyama, Makoto; Kosono, Saori

    2017-03-03

    Protein Nε-acylation is emerging as a ubiquitous post-translational modification. In Corynebacterium glutamicum, which is utilized for industrial production of L-glutamate, the levels of protein acetylation and succinylation change drastically under the conditions that induce glutamate overproduction. Here, we characterized the acylation of phosphoenolpyruvate carboxylase (PEPC), an anaplerotic enzyme that supplies oxaloacetate for glutamate overproduction. We showed that acetylation of PEPC at lysine 653 decreased enzymatic activity, leading to reduced glutamate production. An acetylation-mimic (KQ) mutant of K653 showed severely reduced glutamate production, while the corresponding KR mutant showed normal production levels. Using an acetyllysine-incorporated PEPC protein, we verified that K653-acetylation negatively regulates PEPC activity. In addition, NCgl0616, a sirtuin-type deacetylase, deacetylated K653-acetylated PEPC in vitro. Interestingly, the specific activity of PEPC was increased during glutamate overproduction, which was blocked by the K653R mutation or deletion of sirtuin-type deacetylase homologues. These findings suggested that deacetylation of K653 by NCgl0616 likely plays a role in the activation of PEPC, which maintains carbon flux under glutamate-producing conditions. PEPC deletion increased protein acetylation levels in cells under glutamate-producing conditions, supporting our hypothesis that PEPC is responsible for a large carbon flux change under glutamate-producing conditions. This article is protected by copyright. All rights reserved.