Science.gov

Sample records for 3-phosphate dehydrogenase gene

  1. Isolation of a GPD gene from Debaryomyces hansenii encoding a glycerol 3-phosphate dehydrogenase (NAD+).

    PubMed

    Thomé, Patricia E

    2004-01-30

    A gene homologous to GPD1, coding for glycerol-3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD(+) oxidoreductase, EC 1.1.1.8), has been isolated from the halophilic yeast Debaryomyces hansenii by complementation of a Saccharomyces cerevisiae gpd1 Delta mutant. DNA sequencing of the complementing genomic clone indicated the existence of an open reading frame encoding a protein with 369 amino acids. Comparative analysis of the deduced amino acid sequence showed high similarity to homologous genes described for other eukaryotic GPD enzymes. The sequence has been submitted to the GenBank database under Accession No. AY333427.

  2. Cloning and characterization of glyceraldehyde-3-phosphate dehydrogenase encoding gene in Gracilaria/Gracilariopsis lemaneiformis

    NASA Astrophysics Data System (ADS)

    Ren, Xueying; Sui, Zhenghong; Zhang, Xuecheng

    2006-04-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays important roles in various cellular processes. A cytosolic GAPDH encoding gene ( gpd) of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme of G. lemaneiformis had high homology with those of seven red algae. The 5'-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site of Cyanidium caldarium was different from those of the other six algae at the last two residues (CL to LF), thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH of G. lemaneiformis might have undergone an evolution similar to those of Porphyra yezoensis, Chondrus crispus, and Gracilaria verrucosa. C. caldarium had a closer evolutionary relationship with Cyanidioschyzon merolae than with Cyanidium sp. Virtual Northern blot analysis revealed that gpd of G. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis of G. lemaneiformis.

  3. Identification of some ectomycorrhizal basidiomycetes by PCR amplification of their gpd (glyceraldehyde-3-phosphate dehydrogenase) genes.

    PubMed

    Kreuzinger, N; Podeu, R; Gruber, F; Göbl, F; Kubicek, C P

    1996-09-01

    Degenerated oligonucleotide primers designed to flank an approximately 1.2-kb fragment of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) from ascomycetes and basidiomycetes were used to amplify the corresponding gpd fragments from several species of the ectomycorrhizal fungal taxa Boletus, Amanita, and Lactarius. Those from B. edulis, A. muscaria, and L. deterrimus were cloned and sequenced. The respective nucleotide sequences of these gene fragments showed a moderate degree of similarity (72 to 76%) in the protein-encoding regions and only a low degree of similarity in the introns (56 to 66%). Introns, where present, occurred at conserved positions, but the respective positions and numbers of introns in a given taxon varied. The amplified fragment from a given taxon could be distinguished from that of others by both restriction nuclease cleavage analysis and Southern hybridization. A procedure for labeling DNA probes with fluorescein-12-dUTP by PCR was developed. These probes were used in a nonradioactive hybridization assay, with which the gene could be detected in 2 ng of chromosomal DNA of L. deterrimus on slot blots. Taxon-specific amplification was achieved by the design of specific oligonucleotide primers. The application of the gpd gene for the identification of mycorrhizal fungi under field conditions was demonstrated, with Picea abies (spruce) mycorrhizal roots harvested from a northern alpine forest area as well as from a plant-breeding nursery. The interference by inhibitory substances, which sometimes occurred in the DNA extracted from the root-fungus mixture, could be overcome by using very diluted concentrations of template DNA for a first round of PCR amplification followed by a second round with nested oligonucleotide primers. We conclude that gpd can be used to detect ectomycorrhizal fungi during symbiotic interaction. PMID:8795234

  4. Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa.

    PubMed Central

    Schweizer, H P; Po, C

    1994-01-01

    Nitrosoguanidine-induced Pseudomonas aeruginosa mutants which were unable to utilize glycerol as a carbon source were isolated. By utilizing PAO104, a mutant defective in glycerol transport and sn-glycerol-3-phosphate dehydrogenase (glpD), the glpD gene was cloned by a phage mini-D3112-based in vivo cloning method. The cloned gene was able to complement an Escherichia coli glpD mutant. Restriction analysis and recloning of DNA fragments located the glpD gene to a 1.6-kb EcoRI-SphI DNA fragment. In E. coli, a single 56,000-Da protein was expressed from the cloned DNA fragments. An in-frame glpD'-'lacZ translational fusion was isolated and used to determine the reading frame of glpD by sequencing across the fusion junction. The nucleotide sequence of a 1,792-bp fragment containing the glpD region was determined. The glpD gene encodes a protein containing 510 amino acids and with a predicted molecular weight of 56,150. Compared with the aerobic sn-glycerol-3-phosphate dehydrogenase from E. coli, P. aeruginosa GlpD is 56% identical and 69% similar. A similar comparison with GlpD from Bacillus subtilis reveals 21% identity and 40% similarity. A flavin-binding domain near the amino terminus which shared the consensus sequence reported for other bacterial flavoproteins was identified. Images PMID:8157588

  5. Characterization of the highly active fragment of glyceraldehyde-3-phosphate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus.

    PubMed

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin

    2015-03-01

    Developing efficient native promoters is important for improving recombinant protein expression by fungal genetic engineering. The promoter region of glyceraldehyde-3-phosphate dehydrogenase gene in Pleurotus ostreatus (Pogpd) was isolated and optimized by upstream truncation. The activities of these promoters with different lengths were further confirmed by fluorescence, quantitative real-time PCR and Western blot analysis. A truncated Pogpd-P2 fragment (795 bp) drove enhanced green fluorescence protein (egfp) gene expression in P. ostreatus much more efficiently than full-length Pogpd-P1. Further truncating Pogpd-P2 to 603, 403 and 231 bp reduced the eGFP expression significantly. However, the 403-bp fragment between -356 bp and the start codon was the minimal but sufficient promoter element for eGFP expression. Compact native promoters for genetic engineering of P. ostreatus were successfully developed and validated in this study. This will broaden the preexisting repertoire of fungal promoters for biotechnology application. PMID:25743073

  6. The glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthoraperniciosa, the causal agent of witches' broom disease of Theobroma cacao.

    PubMed

    Lima, Juliana O; Pereira, Jorge F; Rincones, Johana; Barau, Joan G; Araújo, Elza F; Pereira, Gonçalo A G; Queiroz, Marisa V

    2009-04-01

    This report describes the cloning, sequence and expression analysis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Moniliophthora perniciosa, the most important pathogen of cocoa in Brazil. Southern blot analysis revealed the presence of a single copy of the GAPDH gene in the M. perniciosa genome (MpGAPDH). The complete MpGAPDH coding sequence contained 1,461 bp with eight introns that were conserved in the GAPDH genes of other basidiomycete species. The cis-elements in the promoter region of the MpGAPDH gene were similar to those of other basidiomycetes. Likewise, the MpGAPDH gene encoded a putative 339 amino acid protein that shared significant sequence similarity with other GAPDH proteins in fungi, plants, and metazoans. Phylogenetic analyses clustered the MPGAPDH protein with other homobasidiomycete fungi of the family Tricholomataceae. Expression analysis of the MpGAPDH gene by real-time PCR showed that this gene was more expressed (~1.3X) in the saprotrophic stage of this hemibiotrophic plant pathogen than in the biotrophic stage when grown in cacao extracts.

  7. Cloning and characterization of a NAD+-dependent glycerol-3-phosphate dehydrogenase gene from Candida glycerinogenes, an industrial glycerol producer.

    PubMed

    Chen, Xianzhong; Fang, Huiying; Rao, Zhiming; Shen, Wei; Zhuge, Bin; Wang, Zhengxiang; Zhuge, Jian

    2008-08-01

    The osmotolerant yeast Candida glycerinogenes produces glycerol as a major metabolite on an industrial scale, but the underlying molecular mechanisms are poorly understood. We cloned and characterized a 4900-bp genomic fragment containing the CgGPD gene encoding a glycerol-3-phosphate dehydrogenase homologous to GPD genes in other yeasts using degenerate primers in conjunction with inverse PCR. Sequence analysis revealed a 1167-bp open reading frame encoding a putative peptide of 388 deduced amino acids with a molecular mass of 42 695 Da. The CgGPD gene consisted of an N-terminal NAD(+)-binding domain and a central catalytic domain, whereas seven stress response elements were found in the upstream region. Functional analysis revealed that Saccharomyces cerevisiae gpd1Delta and gpd1Delta/gpd2Delta osmosensitive mutants transformed with CgGPD were restored to the wild-type phenotype when cultured in high osmolarity media, suggesting that it is a functional GPD protein. Transformants also accumulated glycerol intracellularly and GPD-specific activity increased significantly when stressed with NaCl, whereas the S. cerevisiae mutants transformed with the empty plasmid showed only slight increases. The full-length CgGPD gene sequence including upstream and downstream regions has been deposited in GenBank under accession no. EU186536.

  8. Effects of salinities on the gene expression of a (NAD+)-dependent glycerol-3-phosphate dehydrogenase in Dunaliella salina.

    PubMed

    Chen, Hui; Lao, Yong-Min; Jiang, Jian-Guo

    2011-03-01

    Glycerol-3-phosphate dehydrogenase (G3pdh) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. In this study, the effects of salinity changes on variation of cell shape and single cell glycerol content of Dunaliella salina were observed, and the effects of salinity changes on the gene expressions of a (NAD+)-dependent G3pdh (EC1.1.1.8) among G3pdh isozymes in D. salina were detected by real-time quantitative PCR. Results showed that the changes of shape and volume of D. salina cell cultured chronically at various salinities were minor, but when the salinity was changed rapidly, the variations of cell shape and cell volume of D. salina were significant, which were recovered basically after 2h except treating by high salinity. Also, it was found some lipid globules in the surface of D. salina cells when the salinity increased from 2.0 to 4.0-5.0 M NaCl rapidly. When D. salina was cultured chronically at various salinities, the accumulation of single cell glycerol increased with increased salinity, and D. salina also could rapidly decrease or increase single cell glycerol contents to adapt to hypoosmotic or hyperosmotic shock. The expression level of G3pdh in D. salina grown at various salinities was significantly inversely correlated to the salinity, but there was no significant correlation between the expression level of G3pdh and salinity after 2 h of treatment by hyperosmotic or hypoosmotic shock.

  9. Sequence analysis and structural characterization of a glyceraldehyde-3-phosphate dehydrogenase gene from the phytopathogenic fungus Eremothecium ashbyi.

    PubMed

    Sengupta, Sudeshna; Chandra, T S

    2011-02-01

    Eremothecium ashbyi is a phytopathogenic fungus infesting cotton, soybeans and several other plants. This highly flavinogenic fungus has been phylogenetically characterized, but the genetic aspects of its central metabolic and riboflavin biosynthetic pathways are unknown. An ORF of 996 bp was obtained from E. ashbyi by using degenerate primers for glyceraldehyde-3-phosphate dehydrogenase (GPD) through reverse transcriptase polymerase chain reaction (RT-PCR) and 5'-3' rapid amplification of cDNA ends (RACE-PCR). This nucleotide sequence had a high similarity of 88% with GPD sequence of Ashbya gossypii. The putative GPD peptide of 331-aa had a high similarity of 85% with the GPD sequence from other ascomycetes. The ORF had an unusually strong codon bias with 5 amino acids showing strict preference of a single codon. The theoretical molecular weight for the putative peptide was 35.58 kDa with an estimated pI of 5.7. A neighbor-joining tree showed that the putative peptide from E. ashbyi displayed the highest similarity to GPD of A. gossypii. The gene sequence is available at the GenBank, accession number EU717696. Homology modeling done with Kluyveromyces marxianus GPD (PDB: 2I5P) as template indicated high structural similarity. PMID:20820924

  10. Cell cycle regulation of the glyceraldehyde-3-phosphate dehydrogenase/uracil DNA glycosylase gene in normal human cells.

    PubMed Central

    Mansur, N R; Meyer-Siegler, K; Wurzer, J C; Sirover, M A

    1993-01-01

    The cell cycle regulation of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/uracil DNA glycosylase (UDG) gene was examined in normal human cells. Steady state RNA levels were monitored by Northern blot analysis using a plasmid (pChug 20.1) which contained the 1.3 kb GAPDH/UDG cDNA. The biosynthesis of the 37 kDa GAPDH/UDG protein was determined using an anti-human placental GAPDH/UDG monoclonal antibody to immunoprecipitate the radiolabeled protein. Increases in steady state GAPDH/UDG mRNA levels were cell cycle specific. A biphasic pattern was observed resulting in a 19-fold increase in the amount of GAPDH/UDG mRNA. The biosynthesis of the 37 kDa GAPDH/UDG protein displayed a similar biphasic regulation with a 7-fold increase. Pulse-chase experiments revealed a remarkably short half life of less than 1 hr. for the newly synthesized 37 kDa protein, comparable to that previously documented for a number of oncogenes. GAPDH/UDG mRNA levels were markedly reduced at 24 hr. when DNA synthesis was maximal. These results define the GAPDH/UDG gene as cell cycle regulated with a characteristic temporal sequence of expression in relation to DNA synthesis. The cell cycle synthesis of a labile 37 kDa monomer suggests a possible regulatory function for this multidimensional protein. Further, modulation of the GAPDH/UDG gene in the cell cycle may preclude its use as a reporter gene when the proliferative state of the cell is not kept constant. Images PMID:8451199

  11. HUMAN GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-2 (GAPD2) GENE IS EXPRESSED SPECIFICALLY IN SPERMATOGENIC CELLS

    EPA Science Inventory

    Although the process of glycolysis is highly conserved in eukaryotes, several glycolytic enzymes have unique structural or functional features in spermatogenic cells. We previously identified and characterized the mouse complementary DNA (cDNA) and a gene for 1 of these enzymes, ...

  12. Heterologous expression of glycerol 3-phosphate dehydrogenase gene [DhGPD1] from the osmotolerant yeast Debaryomyces hansenii in Saccharomyces cerevisiae.

    PubMed

    Thomé, Patricia E

    2005-08-01

    The role for the gene encoding glycerol 3-phosphate dehydrogenase (DhGPD1) from the osmotolerant yeast Debaryomyces hansenii, in glycerol production and halotolerance, was studied through its heterologous expression in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Delta). The expression of the DhGPD1 gene in the gpd1Delta background restored glycerol production and halotolerance to wild type levels, corroborating its role in the salt-induced production of glycerol. Although the gene was functional in S. cerevisiae, its heterologous expression was not efficient, suggesting that the regulatory mechanism may not be shared by these two yeasts.

  13. Over-expression of PsGPD, a mushroom glyceraldehyde-3-phosphate dehydrogenase gene, enhances salt tolerance in rice plants.

    PubMed

    Cho, Jung-Il; Lim, Hye-Min; Siddiqui, Zamin Shaheed; Park, Sung-Han; Kim, A-Ram; Kwon, Taek-Ryoun; Lee, Seong-Kon; Park, Soo-Chul; Jeong, Mi-Jeong; Lee, Gang-Seob

    2014-08-01

    Transgenic potatoes expressing glyceraldehyde-3-phosphate dehydrogenase (GPD), isolated from the oyster mushroom, Pleurotus sajor-caju, had increased tolerance to salt stress (Jeong et al. Biochem Biophys Res Commun 278:192-196, 2000). To examine the physiological mechanisms enhancing salt tolerance in GPD-transgenic rice plants, the salt tolerance of five GPD transgenic rice lines (T1-T5) derived from Dongjin rice cultivar were evaluated in a fixed 150 mM saline environment in comparison to two known wild-type rice cultivars, Dongjin (salt sensitive) and Pokali (salt tolerant). Transgenic lines, T2, T3, and T5, had a substantial increase in biomass and relative water content compared to Dongjin. Stomatal conductance and osmotic potential were higher in the GPD transgenic lines and were similar to those in Pokali. The results are discussed based on the comparative physiological response of GPD transgenic lines with those of the salt-sensitive and salt-tolerant rice cultivars.

  14. Over-expression of PsGPD, a mushroom glyceraldehyde-3-phosphate dehydrogenase gene, enhances salt tolerance in rice plants.

    PubMed

    Cho, Jung-Il; Lim, Hye-Min; Siddiqui, Zamin Shaheed; Park, Sung-Han; Kim, A-Ram; Kwon, Taek-Ryoun; Lee, Seong-Kon; Park, Soo-Chul; Jeong, Mi-Jeong; Lee, Gang-Seob

    2014-08-01

    Transgenic potatoes expressing glyceraldehyde-3-phosphate dehydrogenase (GPD), isolated from the oyster mushroom, Pleurotus sajor-caju, had increased tolerance to salt stress (Jeong et al. Biochem Biophys Res Commun 278:192-196, 2000). To examine the physiological mechanisms enhancing salt tolerance in GPD-transgenic rice plants, the salt tolerance of five GPD transgenic rice lines (T1-T5) derived from Dongjin rice cultivar were evaluated in a fixed 150 mM saline environment in comparison to two known wild-type rice cultivars, Dongjin (salt sensitive) and Pokali (salt tolerant). Transgenic lines, T2, T3, and T5, had a substantial increase in biomass and relative water content compared to Dongjin. Stomatal conductance and osmotic potential were higher in the GPD transgenic lines and were similar to those in Pokali. The results are discussed based on the comparative physiological response of GPD transgenic lines with those of the salt-sensitive and salt-tolerant rice cultivars. PMID:24737077

  15. The tigA gene is a transcriptional fusion of glycolytic genes encoding triose-phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase in oomycota.

    PubMed Central

    Unkles, S E; Logsdon, J M; Robison, K; Kinghorn, J R; Duncan, J M

    1997-01-01

    Genes encoding triose-phosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are fused and form a single transcriptional unit (tigA) in Phytophthora species, members of the order Pythiales in the phylum Oomycota. This is the first demonstration of glycolytic gene fusion in eukaryotes and the first case of a TPI-GAPDH fusion in any organism. The tigA gene from Phytophthora infestans has a typical Oomycota transcriptional start point consensus sequence and, in common with most Phytophthora genes, has no introns. Furthermore, Southern and PCR analyses suggest that the same organization exists in other closely related genera, such as Pythium, from the same order (Oomycota), as well as more distantly related genera, Saprolegnia and Achlya, in the order Saprolegniales. Evidence is provided that in P. infestans, there is at least one other discrete copy of a GAPDH-encoding gene but not of a TPI-encoding gene. Finally, a phylogenetic analysis of TPI does not place Phytophthora within the assemblage of crown eukaryotes and suggests TPI may not be particularly useful for resolving relationships among major eukaryotic groups. PMID:9352934

  16. Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000.

    PubMed

    Elkhalfi, Bouchra; Araya-Garay, José Miguel; Rodríguez-Castro, Jorge; Rey-Méndez, Manuel; Soukri, Abdelaziz; Serrano Delgado, Aurelio

    2013-06-01

    The gammaproteobacterium Pseudomonas syringae pv. tomato DC3000 is the causal agent of bacterial speck, a common disease of tomato. The mode of infection of this pathogen is not well understood, but according to molecular biological, genomic and proteomic data it produces a number of proteins that may promote infection and draw nutrients from the plant. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a major enzyme of carbon metabolism that was reported to be a surface antigen and virulence factor in other pathogenic microorganisms, but its possible role in the infection process of P. syringae has so far not been studied. Whole-genome sequence analyses revealed the occurrence in this phytopathogenic bacterium of three paralogous gap genes encoding distinct GAPDHs, namely two class I enzymes having different molecular mass subunits and one class III bifunctional D-erythrose-4-phosphate dehydrogenase/GAPDH enzyme. By using genome bioinformatics data, as well as alignments of both DNA and deduced protein sequences, the three gap genes of P. syringae were one-step cloned with a His-Tag in pET21a vector using a PCR-based strategy, and its expression optimized in Escherichia coli BL21 to achieve high yield of the heterologous proteins. In accordance with their distinct molecular phylogenies, these bacterial gap genes encode functional GAPDHs of diverse molecular masses and nicotinamide-coenzyme specificities, suggesting specific metabolic and/or cellular roles. PMID:23507306

  17. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Alzheimer's disease.

    PubMed

    El Kadmiri, N; Slassi, I; El Moutawakil, B; Nadifi, S; Tadevosyan, A; Hachem, A; Soukri, A

    2014-12-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme that catalyzes the sixth step of glycolysis and thus, serves to break down glucose for energy production. Beyond the traditional aerobic metabolism of glucose, recent studies have highlighted additional roles played by GAPDH in non-metabolic processes, such as control of gene expression and redox post-translational modifications. Neuroproteomics have revealed high affinity interactions between GAPDH and Alzheimer's disease-associated proteins, including the β-amyloid, β-amyloid precursor protein and tau. This neuronal protein interaction may lead to impairment of the GAPDH glycolytic function in Alzheimer's disease and may be a forerunner of its participation in apoptosis. The present review examines the crucial implication of GAPDH in neurodegenerative processes and clarifies its role in apoptotic cell death.

  18. GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S, A SPERM-SPECIFIC GLYCOLYTIC ENZYME, IS REQUIRED FOR SPERM MOTILITY AND MALE FERTILITY

    EPA Science Inventory

    While glycolysis is highly conserved, it is remarkable that several novel isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like it...

  19. Cis-acting elements essential for light regulation of the nuclear gene encoding the A subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase in Arabidopsis thaliana.

    PubMed Central

    Park, S C; Kwon, H B; Shih, M C

    1996-01-01

    We report the characterization of cis-acting elements involved in light regulation of the nuclear gene (GapA) that encodes the A subunit of glyceraldehyde 3-phosphate dehydrogenase in Arabidopsis thaliana. Our previous deletion analyses indicate that the -277 to -195 upstream region of GapA is essential for light induction of the beta-glucuronidase reporter gene in transgenic tobacco (Nicotiana tabacum) plants. This region contains three direct repeats with the consensus sequence 5'-CAAATGAA(A/G)A-3' (Gap boxes). Our results show that 2-bp substitutions of the last four nucleotides (AA or GA) of the Gap boxes by CC abolish light induction of the beta-glucuronidase reporter gene in vivo and affect binding of the Gap box binding factor in vitro. We have also identified an additional cis-acting element, AE (Activation Element) box, that is involved in regulation of GapA. A combination of a Gap box trimer and an AE box dimer can confer light responsiveness of the cauliflower mosaic virus 35S promoter containing the -92 to +6 upstream sequence, whereas oligomers of Gap boxes or AE boxes alone cannot confer light responsiveness on the same promoter. These results suggest that Gap boxes and AE boxes function together as the light-responsive element of GapA. PMID:8972600

  20. Targeted gene disruption of glycerol-3-phosphate dehydrogenase in Colletotrichum gloeosporioides reveals evidence that glycerol is a significant transferred nutrient from host plant to fungal pathogen.

    PubMed

    Wei, Yangdou; Shen, Wenyun; Dauk, Melanie; Wang, Feng; Selvaraj, Gopalan; Zou, Jitao

    2004-01-01

    Unidirectional transfer of nutrients from plant host to pathogen represents a most revealing aspect of the parasitic lifestyle of plant pathogens. Whereas much effort has been focused on sugars and amino acids, the identification of other significant metabolites is equally important for comprehensive characterization of metabolic interactions between plants and biotrophic fungal pathogens. Employing a strategy of targeted gene disruption, we generated a mutant strain (gpdhDelta) defective in glycerol-3-phosphate dehydrogenase in a hemibiotrophic plant pathogen, Colletotrichum gloeosporioides f.sp. malvae. The gpdhDelta strain had severe defects in carbon utilization as it could use neither glucose nor amino acids for sustained growth. Although the mutant mycelia were able to grow on potato dextrose agar medium, they displayed arrhythmicity in growth and failure to conidiate. The metabolic defect of gpdhDelta could be entirely ameliorated by glycerol in chemically defined minimal medium. Furthermore, glycerol was the one and only metabolite that could restore rhythmic growth and conidiation of gpdhDelta. Despite the profound defects in carbon source utilization, in planta the gpdhDelta strain exhibited normal pathogenicity, proceeded normally in its life cycle, and produced abundant conidia. Analysis of plant tissues at the peripheral zone of fungal infection sites revealed a time-dependent reduction in glycerol content. This study provides strong evidence for a role of glycerol as a significant transferred metabolite from plant to fungal pathogen.

  1. Proteome analysis of a Lactococcus lactis strain overexpressing gapA suggests that the gene product is an auxiliary glyceraldehyde 3-phosphate dehydrogenase.

    PubMed

    Willemoës, Martin; Kilstrup, Mogens; Roepstorff, Peter; Hammer, Karin

    2002-08-01

    The sequence of the genome from the Lactococcus lactis subspecies lactis strain IL1403 shows the presence of two reading frames, gapA and gapB, putatively encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Previous proteomic analysis of the L. lactis subspecies cremoris strain MG1363 has revealed two neighbouring protein spots, GapBI and GapBII, with amino terminal sequences identical to the product of gapA from the L. lactis subspecies cremoris strain LM0230 and that of the two IL1403 sequences. In order to assign the two protein spots to their respective genes we constructed an L. lactis strain that overexpessed the gapA gene derived from MG1363 upon nisin induction. Compared to the wild-type, the overexpressing strain had a 3.4-fold elevated level of specific GAPDH activity when grown in the presence of nisin. In both MG1363 and the gapA overexpressing strain the GAPDH activity was specific for NAD. No NADP dependent activity was detected. Proteome analysis of the gapA overexpressing strain revealed two new protein spots, GapAI and GapAII, not previously detected in proteome analysis of MG1363. Results from mass spectrometry analysis of GapA and GapB and comparison with the deduced protein sequences for the GAPDH isozymes from the genome sequence of strain IL1403 allowed us to assign GapA and GapB to their apparent IL1403 homologues encoded by gapA and gapB, respectively. Furthermore, we suggest that a homologue of a gapB product, represented by GapB, is the main source of GAPDH activity in L. lactis during normal growth.

  2. Identification of a light-responsive region of the nuclear gene encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana.

    PubMed Central

    Kwon, H B; Park, S C; Peng, H P; Goodman, H M; Dewdney, J; Shih, M C

    1994-01-01

    We report here the identification of a cis-acting region involved in light regulation of the nuclear gene (GapB) encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana. Our results show that a 664-bp GapB promoter fragment is sufficient to confer light induction and organ-specific expression of the Escherichia coli beta-glucuronidase reporter gene (Gus) in transgenic tobacco (Nicotiana tabacum) plants. Deletion analysis indicates that the -261 to -173 upstream region of the GapB gene is essential for light induction. This region contains four direct repeats with the consensus sequence 5'-ATGAA(A/G)A-3' (Gap boxes). Deletion of all four repeats abolishes light induction completely. In addition, we have linked a 109-bp (-263 to -152) GapB upstream fragment containing the four direct repeats in two orientations to the -92 to +6 upstream sequence of the cauliflower mosaic virus 35S basal promoter. The resulting chimeric promoters are able to confer light induction and to enhance leaf-specific expression of the Gus reporter gene in transgenic tobacco plants. Based on these results we conclude that Gap boxes are essential for light regulation and organ-specific expression of the GapB gene in A. thaliana. Using gel mobility shift assays we have also identified a nuclear factor from tobacco that interacts with GapA and GapB DNA fragments containing these Gap boxes. Competition assays indicate that Gap boxes are the binding sites for this factor. Although this binding activity is present in nuclear extracts from leaves and roots of light-grown or dark-treated tobacco plants, the activity is less abundant in nuclear extracts prepared from leaves of dark-treated plants or from roots of greenhouse-grown plants. In addition, our data show that this binding factor is distinct from the GT-1 factor, which binds to Box II and Box III within the light-responsive element of the RbcS-3A gene of pea. PMID:8029358

  3. Molecular clone and expression of a NAD+-dependent glycerol-3-phosphate dehydrogenase isozyme gene from the halotolerant alga Dunaliella salina.

    PubMed

    Cai, Ma; He, Li-Hong; Yu, Tu-Yuan

    2013-01-01

    Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD(+)-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value.

  4. Structure of rabbit-muscle glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Cowan-Jacob, Sandra W; Kaufmann, Markus; Anselmo, Anthony N; Stark, Wilhelm; Grütter, Markus G

    2003-12-01

    The crystal structure of the tetrameric form of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isolated from rabbit muscle was solved at 2.4 A resolution after careful dynamic light-scattering experiments to find a suitable buffer for crystallization trials. The refined model has a crystallographic R factor of 20.3%. Here, the first detailed model of a mammalian GAPDH is presented. The cofactor NAD(+) (nicotinamide adenine dinucleotide) is bound to two subunits of the tetrameric enzyme, which is consistent with the negative cooperativity of NAD(+) binding to this enzyme. The structure of rabbit-muscle GAPDH is of interest because it shares 91% sequence identity with the human enzyme; human GAPDH is a potential target for the development of anti-apoptotic drugs. In addition, differences in the cofactor-binding pocket compared with the homology-model structure of GAPDH from the malaria parasite Plasmodium falciparum could be exploited in order to develop novel selective and potential antimalaria drugs.

  5. Glyceraldehyde 3-phosphate dehydrogenase is bound to the fibrous sheath of mammalian spermatozoa.

    PubMed

    Westhoff, D; Kamp, G

    1997-08-01

    Evidence is provided that the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase is covalently linked to the fibrous sheath. The fibrous sheath is a typical structure of mammalian spermatozoa surrounding the axoneme in the principal piece of the flagellum. More than 90% of boar sperm glyceraldehyde 3-phosphate dehydrogenase activity is sedimented after cell disintegration by centrifugation. Detergents, different salt concentrations or short term incubation with chymotrypsin do not solubilize the enzyme, whereas digestion with trypsin or elastase does. Short term incubation with trypsin (15 minutes) even resulted in an activation of glyceraldehyde 3-phosphate dehydrogenase. Purification on phenyl-Sepharose yielded a homogeneous glyceraldehyde 3-phosphate dehydrogenase as judged from gel electrophoresis SDS-PAGE and native gradient PAGE. The molecular masses are 41.5 and 238 kDa, respectively, suggesting native glyceraldehyde 3-phosphate dehydrogenase to be a hexamer. Rabbit polyclonal antibodies raised to purified glyceraldehyde 3-phosphate dehydrogenase show a high specificity for mammalian spermatozoal glyceraldehyde 3-phosphate dehydrogenase, while other proteins of boar spermatozoa or the muscle glyceraldehyde 3-phosphate dehydrogenase are not labelled. Immunogold staining performed in a post-embedding procedure reveals the localization of glyceraldehyde 3-phosphate dehydrogenase along the fibrous sheath in spermatozoa of boar, bull, rat, stallion and man. Other structures such as the cell membrane, dense fibres, the axoneme or the mitochondria are free of label. During the process of sperm maturation, most of the cytoplasm of the sperm midpiece is removed as droplets during the passage through the epididymis. The labelling of this cytoplasm, in immature boar spermatozoa and in the droplets, indicates that glyceraldehyde 3-phosphate dehydrogenase is completely removed from the midpiece during sperm maturation in the epididymis. The inverse

  6. Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules.

    PubMed

    Andrade, Josefa; Pearce, Sandy Timm; Zhao, Hu; Barroso, Margarida

    2004-12-01

    Previously, we have shown that p22, an EF-hand Ca2+-binding protein, interacts indirectly with microtubules in an N-myristoylation-dependent and Ca2+-independent manner. In the present study, we report that N-myristoylated p22 interacts with several microtubule-associated proteins within the 30-100 kDa range using overlay blots of microtubule pellets containing cytosolic proteins. One of those p22-binding partners, a 35-40 kDa microtubule-binding protein, has been identified by MS as GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Several lines of evidence suggest a functional relationship between GAPDH and p22. First, endogenous p22 interacts with GAPDH by immunoprecipitation. Secondly, p22 and GAPDH align along microtubule tracks in analogous punctate structures in BHK cells. Thirdly, GAPDH facilitates the p22-dependent interactions between microtubules and microsomal membranes, by increasing the ability of p22 to bind microtubules but not membranes. We have also shown a direct interaction between N-myristoylated p22 and GAPDH in vitro with a K(D) of approximately 0.5 microM. The removal of either the N-myristoyl group or the last six C-terminal amino acids abolishes the binding of p22 to GAPDH and reduces the ability of p22 to associate with microtubules. In summary, we report that GAPDH is involved in the ability of p22 to facilitate microtubule-membrane interactions by affecting the p22-microtubule, but not the p22-membrane, association. PMID:15312048

  7. THE HEME BINDING PROPERTIES OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

    PubMed Central

    Hannibal, Luciana; Collins, Daniel; Brassard, Julie; Chakravarti, Ritu; Vempati, Rajesh; Dorlet, Pierre; Santolini, Jérôme; Dawson, John H.; Stuehr, Dennis J.

    2012-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for cellular heme insertion into inducible nitric oxide synthase (Chakravarti et al, PNAS 2010, 107(42):18004-9), we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (1 heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418 and 537 nm, and when reduced to ferrous gave maxima at 424, 527 and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were kon =17,800 M−1s−1 and koff1 = 7.0 × 10−3 s−1; koff2 = 3.3 × 10−4 s−1 respectively, giving approximate affinities of 19–390 nM. Ferrous heme bound more poorly to GAPDH and dissociated with a koff = 4.2 × 10−3 s−1. Magnetic circular dichroism (MCD), resonance Raman (rR) and EPR spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in ferric complex was not displaced by CN− or N3− but in ferrous complex was displaceable by CO at a rate of 1.75 s−1 (for [CO]>0.2 mM). Studies with heme analogs revealed selectivity toward the coordinating metal and porphyrin ring structure. GAPDH-heme was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-amino levulinic acid. Our finding of heme binding to GAPDH expands the protein’s potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH is consistent with it performing heme sensing or heme chaperone-like functions in cells. PMID:22957700

  8. Heme binding properties of glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Hannibal, Luciana; Collins, Daniel; Brassard, Julie; Chakravarti, Ritu; Vempati, Rajesh; Dorlet, Pierre; Santolini, Jérôme; Dawson, John H; Stuehr, Dennis J

    2012-10-30

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for the insertion of cellular heme into inducible nitric oxide synthase [Chakravarti, R., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 18004-18009], we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (one heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418, and 537 nm and when reduced to ferrous gave maxima at 424, 527, and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were as follows: k(on) = 17800 M(-1) s(-1), k(off1) = 7.0 × 10(-3) s(-1), and k(off2) = 3.3 × 10(-4) s(-1) (giving approximate affinities of 19-390 nM). Ferrous heme bound more poorly to GAPDH and dissociated with a k(off) of 4.2 × 10(-3) s(-1). Magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in the ferric complex was not displaced by CN(-) or N(3)(-) but in the ferrous complex could be displaced by CO at a rate of 1.75 s(-1) (for >0.2 mM CO). Studies with heme analogues revealed selectivity toward the coordinating metal and porphyrin ring structure. The GAPDH-heme complex was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-aminolevulinic acid. Our finding of heme binding to GAPDH expands the protein's potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH are consistent with it performing heme sensing or heme chaperone-like functions in cells.

  9. Expanding the molecular diversity and phenotypic spectrum of glycerol 3-phosphate dehydrogenase 1 deficiency.

    PubMed

    Dionisi-Vici, Carlo; Shteyer, Eyal; Niceta, Marcello; Rizzo, Cristiano; Pode-Shakked, Ben; Chillemi, Giovanni; Bruselles, Alessandro; Semeraro, Michela; Barel, Ortal; Eyal, Eran; Kol, Nitzan; Haberman, Yael; Lahad, Avishai; Diomedi-Camassei, Francesca; Marek-Yagel, Dina; Rechavi, Gideon; Tartaglia, Marco; Anikster, Yair

    2016-09-01

    Transient infantile hypertriglyceridemia (HTGT1; OMIM #614480) is a rare autosomal recessive disorder, which manifests in early infancy with transient hypertriglyceridemia, hepatomegaly, elevated liver enzymes, persistent fatty liver and hepatic fibrosis. This rare clinical entity is caused by inactivating mutations in the GPD1 gene, which encodes the cytosolic isoform of glycerol-3-phosphate dehydrogenase. Here we report on four patients from three unrelated families of diverse ethnic origins, who presented with hepatomegaly, liver steatosis, hypertriglyceridemia, with or without fasting ketotic hypoglycemia. Whole exome sequencing revealed the affected individuals to harbor deleterious biallelic mutations in the GPD1 gene, including the previously undescribed c.806G > A (p.Arg269Gln) and c.640T > C (p.Cys214Arg) mutations. The clinical features in three of our patients showed several differences compared to the original reports. One subject presented with recurrent episodes of fasting hypoglycemia along with hepatomegaly, hypetriglyceridemia, and elevated liver enzymes; the second showed a severe liver disease, with intrahepatic cholestasis associated with kidney involvement; finally, the third presented persistent hypertriglyceridemia at the age of 30 years. These findings expand the current knowledge of this rare disorder, both with regard to the phenotype and molecular basis. The enlarged phenotypic spectrum of glycerol-3-phosphate dehydrogenase 1 deficiency can mimic other inborn errors of metabolism with liver involvement and should alert clinicians to recognize this entity by considering GPD1 mutations in appropriate clinical settings. PMID:27368975

  10. [Activity of NADP-dependent glycerol-3-phosphate dehydrogenase in skeletal muscles of animals].

    PubMed

    Epifanova, Iu E; Glushankov, E P; Kolotilova, A I

    1978-01-01

    The NADP-dependent glycerol-3-phosphate dehydrogenase activity was studied in sketetal muscles of the rat, rabbit and frog. The dehydrogenase activity in the skeletal muscles of the rat and rabbit was higher than that of the frog. The enzyme activity was found to depend upon the buffer, being higher in tris-HCl buffer than in triethanolamine buffer.

  11. Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi.

    PubMed

    Cheleski, Juliana; Freitas, Renato F; Wiggers, Helton José; Rocha, Josmar R; de Araújo, Ana Paula Ulian; Montanari, Carlos A

    2011-04-01

    Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352±21 and 272±25 μM, respectively, which were consistent with the values for the untagged enzyme reported in the literature. We have demonstrated by the use of Isothermal Titration Calorimetry (ITC) that this vector modification resulted in activity preserved for a higher period. We also report here the use of response surface methodology (RSM) to determine the region of optimal conditions for enzyme activity. A quadratic model was developed by RSM to describe the enzyme activity in terms of pH and temperature as independent variables. According to the RMS contour plots and variance analysis, the maximum enzyme activity was at 29.1°C and pH 8.6. Above 37°C, the enzyme activity starts to fall, which may be related to previous

  12. Fusion of phospholipid vesicles induced by muscle glyceraldehyde-3-phosphate dehydrogenase in the absence of calcium.

    PubMed

    Morero, R D; Viñals, A L; Bloj, B; Farías, R N

    1985-04-01

    Ca2+-induced fusion of phospholipid vesicles (phosphatidylcholine/phosphatidic acid, 9:1 mol/mol) prepared by ethanolic injection was followed by five different procedures: resonance energy transfer, light scattering, electron microscopy, intermixing of aqueous content, and gel filtration through Sepharose 4-B. The five methods gave concordant results, showing that vesicles containing only 10% phosphatidic acid can be induced to fuse by millimolar concentrations of Ca2+. When the fusing capability of several soluble proteins was assayed, it was found that concanavalin A, bovine serum albumin, ribonuclease, and protease were inactive. On the other hand, lysozyme, L-lactic dehydrogenase, and muscle and yeast glyceraldehyde-3-phosphate dehydrogenase were capable of inducing vesicle fusion. Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle, the most extensively studied protein, proved to be very effective: 0.1 microM was enough to induce complete intermixing of bilayer phospholipid vesicles. Under conditions used in this work, fusion was accompanied by leakage of internal contents. The fusing capability of glyceraldehyde-3-phosphate dehydrogenase was not affected by 5 mM ethylenediaminetetraacetic acid. The Ca2+ concentration in the medium, as determined by atomic absorption spectroscopy, was 5 ppm. Heat-denatured enzyme was incapable of inducing fusion. We conclude that glyceraldehyde-3-phosphate dehydrogenase is a soluble protein inherently endowed with the capability of fusing phospholipid vesicles.

  13. Nonreversible d-Glyceraldehyde 3-Phosphate Dehydrogenase of Plant Tissues 1

    PubMed Central

    Kelly, G. J.; Gibbs, Martin

    1973-01-01

    Preparations of TPN-linked nonreversible d-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.9), free of TPN-linked reversible d-glyceraldehyde 3-phosphate dehydrogenase, have been obtained from green shoots, etiolated shoots, and cotyledons of pea (Pisum sativum), cotyledons of peanut (Arachis hypogea), and leaves of maize (Zea mays). The properties of the enzyme were similar from each of these sources: the Km values for d-glyceraldehyde 3-phosphate and TPN were about 20 μm and 3 μm, respectively. The enzyme activity was inhibited by l-glyceraldehyde 3-phosphate, d-erythrose 4-phosphate, and phosphohydroxypyruvate. Activity was found predominantly in photosynthetic and gluconeogenic tissues of higher plants. A light-induced, phytochrome-mediated increase of enzyme activity in a photosynthetic tissue (pea shoots) was demonstrated. Appearance of enzyme activity in a gluconeogenic tissue (endosperm of castor bean, Ricinus communis) coincided with the conversion of fat to carbohydrate during germination. In photosynthetic tissue, the enzyme is located outside the chloroplast, and at in vivo levels of triose-phosphates and pyridine nucleotides, the activity is probably greater than that of DPN-linked reversible d-glyceraldehyde 3-phosphate dehydrogenase. Several possible roles for the enzyme in plant carbohydrate metabolism are considered. PMID:16658509

  14. EXPRESSION OF THE SPERMATOGENIC CELL-SPECIFIC GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDS) IN RAT TESTIS

    EPA Science Inventory

    The spermatogenic cell-specific variant of glyceraldehyde 3-phosphate dehydrogenase (GAPDS) has been cloned from a rat testis cDNA library and its pattern of expression determined. A 1417 nucleotide cDNA has been found to encode an enzyme with substantial homology to mouse GAPDS...

  15. Widespread occurrence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase among gram-positive bacteria.

    PubMed

    Iddar, Abdelghani; Valverde, Federico; Assobhei, Omar; Serrano, Aurelio; Soukri, Abdelaziz

    2005-12-01

    The non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDHN, NADP+-specific, EC 1.2.1.9) is present in green eukaryotes and some Streptococcus strains. The present report describes the results of activity and immunoblot analyses, which were used to generate the first survey of bacterial GAPDHN distribution in a number of Bacillus, Streptococcus and Clostridium strains. Putative gapN genes were identified after PCR amplification of partial 700-bp sequences using degenerate primers constructed from highly conserved protein regions. Alignment of the amino acid sequences of these fragments with those of known sequences from other eukaryotic and prokaryotic GAPDHNs, demonstrated the presence of conserved residues involved in catalytic activity that are not conserved in aldehyde dehydrogenases, a protein family closely linked to GAPDHNs. The results confirm that the basic structural features of the members of the GAPDHN family have been conserved throughout evolution and that no identity exists with phosphorylating GAPDHs. Furthermore, phylogenetic trees generated from multiple sequence alignments suggested a close relationship between plant and bacterial GAPDHN families.

  16. [The pentose phosphate pathway and NADP-dependent glycerol-3-phosphate dehydrogenase activity in some tissues of albino rat].

    PubMed

    Glushankov, E P; Epifanova, Iu E; Kolotilova, A I

    1976-10-01

    The NADP-dependent glycerol-3-phosphate dehydrogenase activity in liver, heart and skeletal muscle of rat was studied. The activity is found when glyceraldehyde-3-phosphate or ribose-5-phosphate in the presence of ATP are taken as substrates. The data obtained confirm that NADP-dependent glycerol-3-phosphate dehydrogenase exists in skeletal muscle and demonstrate that it is found in heart muscle as well.

  17. Discovery of covalent inhibitors of glyceraldehyde-3-phosphate dehydrogenase, a target for the treatment of malaria.

    PubMed

    Bruno, Stefano; Pinto, Andrea; Paredi, Gianluca; Tamborini, Lucia; De Micheli, Carlo; La Pietra, Valeria; Marinelli, Luciana; Novellino, Ettore; Conti, Paola; Mozzarelli, Andrea

    2014-09-11

    We developed a new class of covalent inhibitors of Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase, a validated target for the treatment of malaria, by screening a small library of 3-bromo-isoxazoline derivatives that inactivate the enzyme through a covalent, selective bond to the catalytic cysteine, as demonstrated by mass spectrometry. Substituents on the isoxazolinic ring modulated the potency up to 20-fold, predominantly due to an electrostatic effect, as assessed by computational analysis. PMID:25137375

  18. Comparative molecular analysis of evolutionarily distant glyceraldehyde-3-phosphate dehydrogenase from Sardina pilchardus and Octopus vulgaris.

    PubMed

    Baibai, Tarik; Oukhattar, Laila; Mountassif, Driss; Assobhei, Omar; Serrano, Aurelio; Soukri, Abdelaziz

    2010-12-01

    The NAD(+)-dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12), which is recognized as a key to central carbon metabolism in glycolysis and gluconeogenesis and as an important allozymic polymorphic biomarker, was purified from muscles of two marine species: the skeletal muscle of Sardina pilchardus Walbaum (Teleost, Clupeida) and the incompressible arm muscle of Octopus vulgaris (Mollusca, Cephalopoda). Comparative biochemical studies have revealed that they differ in their subunit molecular masses and in pI values. Partial cDNA sequences corresponding to an internal region of the GapC genes from Sardina and Octopus were obtained by polymerase chain reaction using degenerate primers designed from highly conserved protein motifs. Alignments of the deduced amino acid sequences were used to establish the 3D structures of the active site of two enzymes as well as the phylogenetic relationships of the sardine and octopus enzymes. These two enzymes are the first two GAPDHs characterized so far from teleost fish and cephalopod, respectively. Interestingly, phylogenetic analyses indicated that the sardina GAPDH is in a cluster with the archetypical enzymes from other vertebrates, while the octopus GAPDH comes together with other molluscan sequences in a distant basal assembly closer to bacterial and fungal orthologs, thus suggesting their different evolutionary scenarios.

  19. Reciprocal Phosphorylation of Yeast Glycerol-3-Phosphate Dehydrogenases in Adaptation to Distinct Types of Stress

    PubMed Central

    Lee, Yong Jae; Jeschke, Grace R.; Roelants, Françoise M.; Thorner, Jeremy

    2012-01-01

    Eukaryotic cells have evolved mechanisms for ensuring growth and survival in the face of stress caused by a fluctuating environment. Saccharomyces cerevisiae has two homologous glycerol-3-phosphate dehydrogenases, Gpd1 and Gpd2, that are required to endure various stresses, including hyperosmotic shock and hypoxia. These enzymes are only partially redundant, and their unique functions were attributed previously to differential transcriptional regulation and localization. We find that Gpd1 and Gpd2 are negatively regulated through phosphorylation by distinct kinases under reciprocal conditions. Gpd2 is phosphorylated by the AMP-activated protein kinase Snf1 to curtail glycerol production when nutrients are limiting. Gpd1, in contrast, is a target of TORC2-dependent kinases Ypk1 and Ypk2. Inactivation of Ypk1 by hyperosmotic shock results in dephosphorylation and activation of Gpd1, accelerating recovery through increased glycerol production. Gpd1 dephosphorylation acts synergistically with its transcriptional upregulation, enabling long-term growth at high osmolarity. Phosphorylation of Gpd1 and Gpd2 by distinct kinases thereby enables rapid adaptation to specific stress conditions. Introduction of phosphorylation motifs targeted by distinct kinases provides a general mechanism for functional specialization of duplicated genes during evolution. PMID:22988299

  20. A glyceraldehyde-3-phosphate dehydrogenase with eubacterial features in the amitochondriate eukaryote, Trichomonas vaginalis.

    PubMed

    Markos, A; Miretsky, A; Müller, M

    1993-12-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), localized in the cytosol of Trichomonas vaginalis, was partially purified. The enzyme is specific for NAD+ and is similar in most of its catalytic properties to glycolytic GAPDHs from other organisms. Its sensitivity to koningic acid is similar to levels observed in GAPDHs from eubacteria and two orders of magnitude lower than those observed for eukaryotic GAPDHs. The complete amino acid sequence of T. vaginalis GAPDH was derived from the N-terminal sequence of the purified protein and the deduced sequence of a cDNA clone. It showed great similarity to other eubacterial and eukaryotic GAPDH sequences. The sequence of the S-loop displayed a eubacterial signature. The overall sequence was more similar to eubacterial sequences than to cytosolic and glycosomal eukaryotic sequences. In phylogenetic trees obtained with distance matrix and parsimony methods T. vaginalis GAPDH clustered with its eubacterial homologs. GAPDHs of other amitochondriate protists, belonging to early branches of the eukaryotic lineage (Giardia lamblia and Entamoeba histolytica--Smith M.W. and Doolittle R.F., unpublished data in GenBank), showed typical eukaryotic signatures and clustered with other eukaryotic sequences, indicating that T. vaginalis GAPDH occupies an anomalous position, possibly due to horizontal gene transfer from a eubacterium.

  1. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase of Candida albicans is a surface antigen.

    PubMed Central

    Gil-Navarro, I; Gil, M L; Casanova, M; O'Connor, J E; Martínez, J P; Gozalbo, D

    1997-01-01

    A lambda gt11 cDNA library from Candida albicans ATCC 26555 was screened by using pooled sera from two patients with systemic candidiasis and five neutropenic patients with high levels of anti-C. albicans immunoglobulin M antibodies. Seven clones were isolated from 60,000 recombinant phages. The most reactive one contained a 0.9-kb cDNA encoding a polypeptide immunoreactive only with sera from patients with systemic candidiasis. The whole gene was isolated from a genomic library by using the cDNA as a probe. The nucleotide sequence of the coding region showed homology (78 to 79%) to the Saccharomyces cerevisiae TDH1 to TDH3 genes coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and their amino acid sequences showed 76% identity; thus, this gene has been named C. albicans TDH1. A rabbit polyclonal antiserum against the purified cytosolic C. albicans GAPDH (polyclonal antibody [PAb] anti-CA-GAPDH) was used to identify the GAPDH in the beta-mercaptoethanol extracts containing cell wall moieties. Indirect immunofluorescence demonstrated the presence of GAPDH at the C. albicans cell surface, particularly on the blastoconidia. Semiquantitative flow cytometry analysis showed the sensitivity of this GAPDH form to trypsin and its resistance to be removed with 2 M NaCl or 2% sodium dodecyl sulfate. The decrease in fluorescence in the presence of soluble GAPDH indicates the specificity of the labelling. In addition, a dose-dependent GAPDH enzymatic activity was detected in intact blastoconidia and germ tube cells. This activity was reduced by pretreatment of the cells with trypsin, formaldehyde, and PAb anti-CA-GAPDH. These observations indicate that an immunogenic, enzymatically active cell wall-associated form of the glycolytic enzyme GAPDH is found at the cell surface of C. albicans cells. PMID:9260938

  2. A novel glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter for expressing transgenes in the halotolerant alga Dunaliella salina.

    PubMed

    Jia, Yanlong; Li, Shenke; Allen, George; Feng, Shuying; Xue, Lexun

    2012-05-01

    A major challenge for efficient transgene expression in Dunaliella salina is to find strong endogenous promoters to drive the transgene expression. In the present study, a novel glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was cloned and used to drive expressions of the bialaphos resistance (bar) gene and of the N-terminal fragment of human canstatin (Can-N). The results showed that the bar gene was transcribed by the GAPDH promoter and integrated into the genome of the transformants of D. salina. Furthermore, the PCR identification, Southern and western blots indicated that Can-N was expressed in transgenic D. salina, demonstrating that the promoter of the D. salina GAPDH gene is suitable for driving expression of heterologous genes in transgenic D. salina.

  3. Catalysis of nitrite generation from nitroglycerin by glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

    PubMed

    Seabra, Amedea B; Ouellet, Marc; Antonic, Marija; Chrétien, Michelle N; English, Ann M

    2013-11-30

    Vascular relaxation to nitroglycerin (glyceryl trinitrate; GTN) requires its bioactivation by mechanisms that remain controversial. We report here that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the release of nitrite from GTN. In assays containing dithiothreitol (DTT) and NAD(+), the GTN reductase activity of purified GAPDH produces nitrite and 1,2-GDN as the major products. A vmax of 2.6nmolmin(-)(1)mg(-)(1) was measured for nitrite production by GAPDH from rabbit muscle and a GTN KM of 1.2mM. Reductive denitration of GTN in the absence of DTT results in dose- and time-dependent inhibition of GAPDH dehydrogenase activity. Disulfiram, a thiol-modifying drug, inhibits both the dehydrogenase and GTN reductase activity of GAPDH, while DTT or tris(2-carboxyethyl)phosphine reverse the GTN-induced inhibition. Incubation of intact human erythrocytes or hemolysates with 2mM GTN for 60min results in 50% inhibition of GAPDH's dehydrogenase activity, indicating that GTN is taken up by these cells and that the dehydrogenase is a target of GTN. Thus, erythrocyte GAPDH may contribute to GTN bioactivation.

  4. Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism

    SciTech Connect

    Yeh, Joanne I.; Chinte, Unmesh; Du, Shoucheng

    2008-04-02

    Sn-glycerol-3-phosphate dehydrogenase (GlpD) is an essential membrane enzyme, functioning at the central junction of respiration, glycolysis, and phospholipid biosynthesis. Its critical role is indicated by the multitiered regulatory mechanisms that stringently controls its expression and function. Once expressed, GlpD activity is regulated through lipid-enzyme interactions in Escherichia coli. Here, we report seven previously undescribed structures of the fully active E. coli GlpD, up to 1.75 {angstrom} resolution. In addition to elucidating the structure of the native enzyme, we have determined the structures of GlpD complexed with substrate analogues phosphoenolpyruvate, glyceric acid 2-phosphate, glyceraldehyde-3-phosphate, and product, dihydroxyacetone phosphate. These structural results reveal conformational states of the enzyme, delineating the residues involved in substrate binding and catalysis at the glycerol-3-phosphate site. Two probable mechanisms for catalyzing the dehydrogenation of glycerol-3-phosphate are envisioned, based on the conformational states of the complexes. To further correlate catalytic dehydrogenation to respiration, we have additionally determined the structures of GlpD bound with ubiquinone analogues menadione and 2-n-heptyl-4-hydroxyquinoline N-oxide, identifying a hydrophobic plateau that is likely the ubiquinone-binding site. These structures illuminate probable mechanisms of catalysis and suggest how GlpD shuttles electrons into the respiratory pathway. Glycerol metabolism has been implicated in insulin signaling and perturbations in glycerol uptake and catabolism are linked to obesity in humans. Homologs of GlpD are found in practically all organisms, from prokaryotes to humans, with >45% consensus protein sequences, signifying that these structural results on the prokaryotic enzyme may be readily applied to the eukaryotic GlpD enzymes.

  5. Role of two different glyceraldehyde-3-phosphate dehydrogenases in controlling the reversible Embden-Meyerhof-Parnas pathway in Thermoproteus tenax: regulation on protein and transcript level.

    PubMed

    Brunner, N A; Siebers, B; Hensel, R

    2001-04-01

    The hyperthermophilic archaeum Thermoproteus tenax uses a variant of the Embden-Meyerhof-Parnas (EMP) pathway as the main route for carbohydrate metabolism. This variant is characterized by a reversible nonallosteric PPi-dependent phosphofructokinase and two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity, phosphate dependence, and allosteric behavior. Although the nonphosphorylating NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN; E.C. 1.2.1.8) fulfills exclusively catabolic purposes, the phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (NADP+-GAPDH; E.C. 1.2.1.13) exhibits anabolic features. The gene encoding the NADP+-GAPDH was cloned, sequenced, and expressed in Escherichia coli. The deduced protein sequence displayed 47%-53% sequence identity to archaeal phosphorylating GAPDHs. The kinetic parameters of the NADP+-GAPDH showed a clear preference for the reductive reaction with a 5-fold-higher specific activity in the reductive reaction as compared to the oxidative reaction and a 20-fold-lower Km for 1,3-bisphosphoglycerate as compared to glyceraldehyde-3-phosphate. Contrary to GAPN, the enzyme is not allosterically regulated. The coding gene overlaps by 1 bp with a preceding open reading frame coding for 3-phosphoglycerate kinase (PGK; E.C. 2.7.2.3). Northern analyses identified mono- and bicistronic messages of both genes in an equimolar ratio. Transcript levels and specific activity of NADP+-GAPDH and PGK were 3- to 4-fold higher under autotrophic conditions as compared to heterotrophic conditions, whereas transcript abundance and specific activity of GAPN remained constant in autotrophically and heterotrophically grown cells. The different regulation of the two counteracting glyceraldehyde-3-phosphate dehydrogenases is discussed with respect to the flux control of the T. tenax-specific EMP variant.

  6. Citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice recapitulate features of human citrin deficiency.

    PubMed

    Saheki, Takeyori; Iijima, Mikio; Li, Meng Xian; Kobayashi, Keiko; Horiuchi, Masahisa; Ushikai, Miharu; Okumura, Fumihiko; Meng, Xiao Jian; Inoue, Ituro; Tajima, Atsushi; Moriyama, Mitsuaki; Eto, Kazuhiro; Kadowaki, Takashi; Sinasac, David S; Tsui, Lap-Chee; Tsuji, Mihoko; Okano, Akira; Kobayashi, Tsuyoshi

    2007-08-24

    Citrin is the liver-type mitochondrial aspartate-glutamate carrier that participates in urea, protein, and nucleotide biosynthetic pathways by supplying aspartate from mitochondria to the cytosol. Citrin also plays a role in transporting cytosolic NADH reducing equivalents into mitochondria as a component of the malate-aspartate shuttle. In humans, loss-of-function mutations in the SLC25A13 gene encoding citrin cause both adult-onset type II citrullinemia and neonatal intrahepatic cholestasis, collectively referred to as human citrin deficiency. Citrin knock-out mice fail to display features of human citrin deficiency. Based on the hypothesis that an enhanced glycerol phosphate shuttle activity may be compensating for the loss of citrin function in the mouse, we have generated mice with a combined disruption of the genes for citrin and mitochondrial glycerol 3-phosphate dehydrogenase. The resulting double knock-out mice demonstrated citrullinemia, hyperammonemia that was further elevated by oral sucrose administration, hypoglycemia, and a fatty liver, all features of human citrin deficiency. An increased hepatic lactate/pyruvate ratio in the double knock-out mice compared with controls was also further elevated by the oral sucrose administration, suggesting that an altered cytosolic NADH/NAD(+) ratio is closely associated with the hyperammonemia observed. Microarray analyses identified over 100 genes that were differentially expressed in the double knock-out mice compared with wild-type controls, revealing genes potentially involved in compensatory or downstream effects of the combined mutations. Together, our data indicate that the more severe phenotype present in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice represents a more accurate model of human citrin deficiency than citrin knock-out mice.

  7. Succination of proteins by fumarate: mechanism of inactivation of glyceraldehyde-3-phosphate dehydrogenase in diabetes.

    PubMed

    Blatnik, Matthew; Thorpe, Suzanne R; Baynes, John W

    2008-04-01

    S-(2-succinyl)cysteine (2SC) is a chemical modification of proteins formed by a Michael addition reaction between the Krebs cycle intermediate, fumarate, and thiol groups in protein--a process known as succination of protein. Succination causes irreversible inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in vitro. GAPDH was immunoprecipitated from muscle of diabetic rats, then analyzed by ultra-performance liquid chromatography-electrospray ionization-mass spectroscopy. Succination of GAPDH was increased in muscle of diabetic rats, and the extent of succination correlated strongly with the decrease in specific activity of the enzyme. We propose that 2SC is a biomarker of mitochondrial and oxidative stress in diabetes and that succination of GAPDH and other thiol proteins may provide the chemical link between glucotoxicity and the pathogenesis of diabetic complications.

  8. Receptor protein kinase FERONIA controls leaf starch accumulation by interacting with glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Yang, Tao; Wang, Long; Li, Chiyu; Liu, Ying; Zhu, Sirui; Qi, Yinyao; Liu, Xuanming; Lin, Qinglu; Luan, Sheng; Yu, Feng

    2015-09-11

    Cell expansion is coordinated by several cues, but available energy is the major factor determining growth. Receptor protein kinase FERONIA (FER) is a master regulator of cell expansion, but the details of its control mechanisms are not clear. Here we show that FER interacts with cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GAPC1 and GAPC2), that catalyzes a key reaction in glycolysis, which contributes to energy production. When there is an FER deficiency, there are corresponding decreases in the enzyme activity of GAPDH and increased amounts of starch. More importantly, gapc1/2 mutants mimic fer4 mutants. These data indicate that FER regulated starch content is an evolutionarily conserved function in plants that connects the cell expansion and energy metabolism pathways.

  9. The sweet side of RNA regulation: glyceraldehyde-3-phosphate dehydrogenase as a noncanonical RNA-binding protein.

    PubMed

    White, Michael R; Garcin, Elsa D

    2016-01-01

    The glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has a vast array of extraglycolytic cellular functions, including interactions with nucleic acids. GAPDH has been implicated in the translocation of transfer RNA (tRNA), the regulation of cellular messenger RNA (mRNA) stability and translation, as well as the regulation of replication and gene expression of many single-stranded RNA viruses. A growing body of evidence supports GAPDH-RNA interactions serving as part of a larger coordination between intermediary metabolism and RNA biogenesis. Despite the established role of GAPDH in nucleic acid regulation, it is still unclear how and where GAPDH binds to its RNA targets, highlighted by the absence of any conserved RNA-binding sequences. This review will summarize our current understanding of GAPDH-mediated regulation of RNA function. WIREs RNA 2016, 7:53-70. doi: 10.1002/wrna.1315 For further resources related to this article, please visit the WIREs website.

  10. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity

    PubMed Central

    Allonso, Diego; Andrade, Iamara S.; Conde, Jonas N.; Coelho, Diego R.; Rocha, Daniele C. P.; da Silva, Manuela L.; Ventura, Gustavo T.

    2015-01-01

    ABSTRACT Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. IMPORTANCE Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the

  11. Glyceraldehyde-3-phosphate dehydrogenase is regulated by ferredoxin-NADP reductase in the diatom Asterionella formosa.

    PubMed

    Mekhalfi, Malika; Puppo, Carine; Avilan, Luisana; Lebrun, Régine; Mansuelle, Pascal; Maberly, Stephen C; Gontero, Brigitte

    2014-07-01

    Diatoms are a widespread and ecologically important group of heterokont algae that contribute c. 20% to global productivity. Previous work has shown that regulation of their key Calvin cycle enzymes differs from that of the Plantae, and that in crude extracts, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can be inhibited by nicotinamide adenine dinucleotide phosphate reduced (NADPH) under oxidizing conditions. The freshwater diatom, Asterionella formosa, was studied using enzyme kinetics, chromatography, surface plasmon resonance, mass spectrometry and sequence analysis to determine the mechanism behind this GAPDH inhibition. GAPDH interacted with ferredoxin-nicotinamide adenine dinucleotide phosphate (NADP) reductase (FNR) from the primary phase of photosynthesis, and the small chloroplast protein, CP12. Sequences of copurified GAPDH and FNR were highly homologous with published sequences. However, the widespread ternary complex among GAPDH, phosphoribulokinase and CP12 was absent. Activity measurements under oxidizing conditions showed that NADPH can inhibit GAPDH-CP12 in the presence of FNR, explaining the earlier observed inhibition within crude extracts. Diatom plastids have a distinctive metabolism, including the lack of the oxidative pentose phosphate pathway, and so cannot produce NADPH in the dark. The observed down-regulation of GAPDH in the dark may allow NADPH to be rerouted towards other reductive processes contributing to their ecological success.

  12. Glyceraldehyde 3-phosphate dehydrogenase-telomere association correlates with redox status in Trypanosoma cruzi.

    PubMed

    Pariona-Llanos, Ricardo; Pavani, Raphael Souza; Reis, Marcelo; Noël, Vincent; Silber, Ariel Mariano; Armelin, Hugo Aguirre; Cano, Maria Isabel Nogueira; Elias, Maria Carolina

    2015-01-01

    Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA. PMID:25775131

  13. Glyceraldehyde 3-Phosphate Dehydrogenase-Telomere Association Correlates with Redox Status in Trypanosoma cruzi

    PubMed Central

    Pariona-Llanos, Ricardo; Pavani, Raphael Souza; Reis, Marcelo; Noël, Vincent; Silber, Ariel Mariano; Armelin, Hugo Aguirre; Cano, Maria Isabel Nogueira; Elias, Maria Carolina

    2015-01-01

    Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA. PMID:25775131

  14. Assisted folding of D-glyceraldehyde-3-phosphate dehydrogenase by trigger factor.

    PubMed Central

    Huang, G. C.; Li, Z. Y.; Zhou, J. M.; Fischer, G.

    2000-01-01

    The Escherichia coli trigger factor is a peptidyl-prolyl cis-trans isomerase that catalyzes proline-limited protein folding extremely well. Here, refolding of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of trigger factor was investigated. The regain of activity of GAPDH was markedly increased by trigger factor after either long- or short-term denaturation, and detectable aggregation of GAPDH intermediates was prevented. In both cases, time courses of refolding of GAPDH were decelerated by trigger factor. The reactivation yield of GAPDH showed a slow down-turn when molar ratios of trigger factor to GAPDH were above 5, due to tight binding between trigger factor and GAPDH intermediates. Such inactive bound GAPDH could be partially rescued from trigger factor by addition of reduced alphaLA as competitor, by further diluting the refolding mixture, or by disrupting hydrophobic interactions in the complexes. A model for trigger factor assisted refolding of GAPDH is proposed. We also suggest that assisted refolding of GAPDH is due mainly to the chaperone function of trigger factor. PMID:10892818

  15. Structural and functional properties of glycerol-3-phosphate dehydrogenase from a mammalian hibernator.

    PubMed

    de la Roche, Marc; Tessier, Shannon N; Storey, Kenneth B

    2012-02-01

    Glycerol-3-phosphate dehydrogenase (G3PDH; E.C.1.1.1.8) was purified from liver and skeletal muscle of black-tailed prairie dogs (Cynomys ludivicianus), a hibernating species. Native and subunit molecular masses of the dimeric enzyme were 77 and 40 kD, respectively, and both tissues contained a single isozyme with a pI of 6.4. Kinetic parameters of purified G3PDH from prairie dog liver and muscle were characterized at 22 and 5 °C and compared with rabbit muscle G3PDH. Substrate affinities for hibernator muscle G3PDH were stable (NAD) or increased significantly (K(m) G3P and DHAP decreased) at low temperature whereas K(m) NAD and DHAP of rabbit G3PDH increased. Prairie dog G3PDH showed greater conservation of K(m) G3P over a wide temperature range as well as greater thermal stability and resistance to chemical denaturation by guanidine hydrochloride than the rabbit enzyme. In addition, using the protein sequence of the hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and bioinformatics tools, the deduced protein structure of G3PDH was compared between heterothermic and homeothermic mammals. Structural and functional characteristics of G3PDH from the hibernating species would support enzyme function over a wide range of core body temperatures over cycles of torpor and arousal. PMID:22180227

  16. Simple method for isolation of glyceraldehyde 3-phosphate dehydrogenase and the improvement of myofibril gel properties.

    PubMed

    Miyaguchi, Yuji; Sakamoto, Taro; Sasaki, Shun; Nakade, Koji; Tanabe, Manabu; Ichinoseki, Satoko; Numata, Masahiro; Kosai, Kiichi

    2011-02-01

    Porcine glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (G3PD) was prepared effectively by a combination of ethylene diamine tetra-acetate (EDTA) pretreatment and affinity purification. After salting out of porcine sarcoplasmic proteins (SP) with ammonium sulfate at 75% saturation, the obtained supernatant (SP-f3) was treated with EDTA, leaving G3PD in the supernatant (G3PD-E) and most other SPs in the precipitate. At that time, the separation of G3PD-E required more than 20 mmol/L EDTA. G3PD-E was then subjected to affinity purification by batchwise method using blue-sepharose CL-6B, and purified G3PD (G3PD-AP) was obtained using 2 mol/L potassium chloride (KCl) as an eluent. Texture analysis showed that the hardness, adhesiveness and gumminess of the myofibril gel at 0.2-mol/L NaCl increased with the addition of G3PD-AP. Scanning electron microscopy revealed that the G3PD-AP reinforced the gel network of the myofibril. However, scanning electron micrograph analysis showed that the network-structure of the gel by the addition of G3PD-AP developed in a different manner from that by adding 0.6 mol/L NaCl. These results showed that glycolytic enzyme, G3PD, contributes to the improvement of the rheological properties of meat products.

  17. Occurrence of a multimeric high-molecular-weight glyceraldehyde-3-phosphate dehydrogenase in human serum.

    PubMed

    Kunjithapatham, Rani; Geschwind, Jean-Francois; Devine, Lauren; Boronina, Tatiana N; O'Meally, Robert N; Cole, Robert N; Torbenson, Michael S; Ganapathy-Kanniappan, Shanmugasundaram

    2015-04-01

    Cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phylogenetically conserved, ubiquitous enzyme that plays an indispensable role in energy metabolism. Although a wealth of information is available on cellular GAPDH, there is a clear paucity of data on its extracellular counterpart (i.e., the secreted or extracellular GAPDH). Here, we show that the extracellular GAPDH in human serum is a multimeric, high-molecular-weight, yet glycolytically active enzyme. The high-molecular-weight multimers of serum GAPDH were identified by immunodetection on one- and two-dimensional gel electrophoresis using multiple antibodies specific for various epitopes of GAPDH. Partial purification of serum GAPDH by DEAE Affigel affinity/ion exchange chromatography further established the multimeric composition of serum GAPDH. In vitro data demonstrated that human cell lines secrete a multimeric, high-molecular-weight enzyme similar to that of serum GAPDH. Furthermore, LC-MS/MS analysis of extracellular GAPDH from human cell lines confirmed the presence of unique peptides of GAPDH in the high-molecular-weight subunits. Furthermore, data from pulse-chase experiments established the presence of high-molecular-weight subunits in the secreted, extracellular GAPDH. Taken together, our findings demonstrate the presence of a high-molecular-weight, enzymatically active secretory GAPDH in human serum that may have a hitherto unknown function in humans.

  18. On the interaction between glyceraldehyde-3-phosphate dehydrogenase and airborne particles: Evidence for electrophilic species

    NASA Astrophysics Data System (ADS)

    Shinyashiki, Masaru; Rodriguez, Chester E.; Di Stefano, Emma W.; Sioutas, Constantinos; Delfino, Ralph J.; Kumagai, Yoshito; Froines, John R.; Cho, Arthur K.

    Many of the adverse health effects of airborne particulate matter (PM) have been attributed to the chemical properties of some of the large number of chemical species present in PM. Some PM component chemicals are capable of generating reactive oxygen species and eliciting a state of oxidative stress. In addition, however, PM can contain chemical species that elicit their effects through covalent bond formation with nucleophilic functions in the cell. In this manuscript, we report the presence of constituents with electrophilic properties in ambient and diesel exhaust particles, demonstrated by their ability to inhibit the thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH is irreversibly inactivated by electrophiles under anaerobic conditions by covalent bond formation. This inactivation can be blocked by the prior addition of a high concentration of dithiothreitol (DTT) as an alternate nucleophile. Addition of DTT after the reaction between the electrophile and GAPDH, however, does not reverse the inactivation. This property has been utilized to develop a procedure that provides a quantitative measure of electrophiles present in samples of ambient particles collected in the Los Angeles Basin and in diesel exhaust particles. The toxicity of electrophiles is the result of irreversible changes in biological molecules; recovery is dependent on resynthesis. If the resynthesis is slow, the irreversible effects can be cumulative and manifest themselves after chronic exposure to low levels of electrophiles.

  19. Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor refined at 2 A resolution.

    PubMed

    Song, S; Li, J; Lin, Z

    1998-07-01

    The crystal structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor, South China sea lobster, was determined and refined at 2 A resolution to an R factor of 17.1% and reasonable stereochemistry. The structure refinement has not altered the overall structure of GAPDH from this lobster species. However, some local changes in conformation and the inclusion of ordered solvent model have resulted in a substantial improvement in the accuracy of the structure. Structure analysis reveals that the two subunits including NAD+ in the asymmetric unit are remarkably similar. The thermal differences between the two subunits found in some regions of the NAD+-binding domain may originate from different crystallographic environments rather than from an inherent molecular asymmetry. In this structure, the side chain of Arg194 does not point toward the active site but forms an ion pair with Asp293 from a neighboring subunit. Structural comparisons with other GAPDH's of known structure reveal that obvious contrast exists between mesophilic and thermophilic GAPDH mainly in the catalytic domain with significant conformational differences in the S-loop, beta7-strand and loop 120-125; the P-axis interface is more conserved than the R- and Q-axis interfaces and the catalytic domain is more conserved than the NAD+-binding domain. Some possible factors affecting the thermostability of this enzyme are tentatively analyzed by comparison with the highly refined structures of thermophilic enzymes.

  20. Isolation and some properties of glycated D-glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle.

    PubMed Central

    He, R Q; Yang, M D; Zheng, X; Zhou, J X

    1995-01-01

    Glycated D-glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from rabbit muscle and human erythrocytes have been investigated. The specific activity of the non-glycated GAPDH from rabbit muscle is approx. 180 units. (One unit is defined as the specific activity required to convert 1 microM of substrate/min per mg of enzyme.) The activity of the glycated enzyme, consisting of two sugars per tetramer, is lower than that of the non-glycated GAPDH. Non-enzymic transamination of the N-termini of glycated GAPDH (gGAPDH) indicates that they are not blocked by glycation. The rate of modification of thiols (Cys-149) with 5,5'-dithiobis-(2-nitrobenzoic acid) was greater for the glycated than the non-glycated enzymes. The rate of modification of amino groups of Lys residues of gGAPDH with o-phthalaldehyde was greater for the non-glycated enzyme. In 0.18 M guanidine-HC1 solution, the emission intensity at 410 nm of a fluorescent NAD+ derivative introduced into the active site decreased to 80%, whereas that of gGAPDH decreased to 50%. This suggests that the glycated sites are near the active site; glycation of the enzyme leads to a change of the microenvironment of Cys-149, alters the conformation of the active site and decreases the activity. Images Figure 1 PMID:7619048

  1. Disruption of NAD+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

    PubMed Central

    Phadke, Manali; Krynetskaia, Natalia; Mishra, Anurag; Barrero, Carlos; Merali, Salim; Gothe, Scott A; Krynetskiy, Evgeny

    2015-01-01

    AIM: To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS: We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding. RESULTS: Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+ (Km = 741 ± 257 μmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION: Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners. PMID:26629320

  2. Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenase regulate metabolic reprogramming of stored red blood cells.

    PubMed

    Reisz, Julie A; Wither, Matthew J; Dzieciatkowska, Monika; Nemkov, Travis; Issaian, Aaron; Yoshida, Tatsuro; Dunham, Andrew J; Hill, Ryan C; Hansen, Kirk C; D'Alessandro, Angelo

    2016-09-22

    Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plays a key regulatory function in glucose oxidation by mediating fluxes through glycolysis or the pentose phosphate pathway (PPP) in an oxidative stress-dependent fashion. Previous studies documented metabolic reprogramming in stored red blood cells (RBCs) and oxidation of GAPDH at functional residues upon exposure to pro-oxidants diamide and H2O2 Here we hypothesize that routine storage of erythrocyte concentrates promotes metabolic modulation of stored RBCs by targeting functional thiol residues of GAPDH. Progressive increases in PPP/glycolysis ratios were determined via metabolic flux analysis after spiking (13)C1,2,3-glucose in erythrocyte concentrates stored in Additive Solution-3 under blood bank conditions for up to 42 days. Proteomics analyses revealed a storage-dependent oxidation of GAPDH at functional Cys152, 156, 247, and His179. Activity loss by oxidation occurred with increasing storage duration and was progressively irreversible. Irreversibly oxidized GAPDH accumulated in stored erythrocyte membranes and supernatants through storage day 42. By combining state-of-the-art ultra-high-pressure liquid chromatography-mass spectrometry metabolic flux analysis with redox and switch-tag proteomics, we identify for the first time ex vivo functionally relevant reversible and irreversible (sulfinic acid; Cys to dehydroalanine) oxidations of GAPDH without exogenous supplementation of excess pro-oxidant compounds in clinically relevant blood products. Oxidative and metabolic lesions, exacerbated by storage under hyperoxic conditions, were ameliorated by hypoxic storage. Storage-dependent reversible oxidation of GAPDH represents a mechanistic adaptation in stored erythrocytes to promote PPP activation and generate reducing equivalents. Removal of irreversibly oxidized, functionally compromised GAPDH identifies enhanced vesiculation as a self-protective mechanism in ex vivo aging erythrocytes.

  3. Diacylglycerol pyrophosphate binds and inhibits the glyceraldehyde-3-phosphate dehydrogenase in barley aleurone.

    PubMed

    Astorquiza, Paula Luján; Usorach, Javier; Racagni, Graciela; Villasuso, Ana Laura

    2016-04-01

    The aleurona cell is a model that allows the study of the antagonistic effect of gibberellic acid (GA) and abscisic acid (ABA). Previous results of our laboratory demonstrated the involvement of phospholipids during the response to ABA and GA. ABA modulates the levels of diacylglycerol, phosphatidic acid and diacylglycerol pyrophosphate (DAG, PA, DGPP) through the activities of phosphatidate phosphatases, phospholipase D, diacylglycerol kinase and phosphatidate kinase (PAP, PLD, DGK and PAK). PA and DGPP are key phospholipids in the response to ABA, since both are capable of modifying the hydrolitic activity of the aleurona. Nevertheless, little is known about the mechanism of action of these phospholipids during the ABA signal. DGPP is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol. The ionization of the pyrophosphate group may be important to allow electrostatic interactions between DGPP and proteins. To understand how DGPP mediates cell functions in barley aleurone, we used a DGPP affinity membrane assay to isolate DGPP-binding proteins from Hordeum vulgare, followed by mass spectrometric sequencing. A cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was identified for being bound to DGPP. To validate our method, the relatively abundant GAPDH was characterized with respect to its lipid-binding properties, by fat western blot. GAPDH antibody interacts with proteins that only bind to DGPP and PA. We also observed that ABA treatment increased GAPDH abundance and enzyme activity. The presence of phospholipids during GAPDH reaction modulated the GAPDH activity in ABA treated aleurone. These data suggest that DGPP binds to GAPDH and this DGPP and GAPDH interaction provides new evidences in the study of DGPP-mediated ABA responses in barley aleurone.

  4. Diacylglycerol pyrophosphate binds and inhibits the glyceraldehyde-3-phosphate dehydrogenase in barley aleurone.

    PubMed

    Astorquiza, Paula Luján; Usorach, Javier; Racagni, Graciela; Villasuso, Ana Laura

    2016-04-01

    The aleurona cell is a model that allows the study of the antagonistic effect of gibberellic acid (GA) and abscisic acid (ABA). Previous results of our laboratory demonstrated the involvement of phospholipids during the response to ABA and GA. ABA modulates the levels of diacylglycerol, phosphatidic acid and diacylglycerol pyrophosphate (DAG, PA, DGPP) through the activities of phosphatidate phosphatases, phospholipase D, diacylglycerol kinase and phosphatidate kinase (PAP, PLD, DGK and PAK). PA and DGPP are key phospholipids in the response to ABA, since both are capable of modifying the hydrolitic activity of the aleurona. Nevertheless, little is known about the mechanism of action of these phospholipids during the ABA signal. DGPP is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol. The ionization of the pyrophosphate group may be important to allow electrostatic interactions between DGPP and proteins. To understand how DGPP mediates cell functions in barley aleurone, we used a DGPP affinity membrane assay to isolate DGPP-binding proteins from Hordeum vulgare, followed by mass spectrometric sequencing. A cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was identified for being bound to DGPP. To validate our method, the relatively abundant GAPDH was characterized with respect to its lipid-binding properties, by fat western blot. GAPDH antibody interacts with proteins that only bind to DGPP and PA. We also observed that ABA treatment increased GAPDH abundance and enzyme activity. The presence of phospholipids during GAPDH reaction modulated the GAPDH activity in ABA treated aleurone. These data suggest that DGPP binds to GAPDH and this DGPP and GAPDH interaction provides new evidences in the study of DGPP-mediated ABA responses in barley aleurone. PMID:26866974

  5. Regulation of Specific Functions of Glial Cells in Somatic Hybrids, II. Control of Inducibility of Glycerol-3-Phosphate Dehydrogenase

    PubMed Central

    Davidson, Richard L.; Benda, Philippe

    1970-01-01

    Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) is induced when glial cells are exposed to hydrocortisone in vitro. In contrast, the enzyme activity in fibroblasts is not affected by the steroid. In an attempt to elucidate the mechanisms controlling inducibility, hybrids between glial cells and fibroblasts were studied. It was found that the activity of the enzyme does not increase when the hybrids are exposed to hydrocortisone. It was also shown that inducibility and the noninduced activity of enzyme are controlled independently. Comparisons of S-100 and glycerol phosphate dehydrogenase activity in the hybrids suggest that all the specialized functions characteristics of glial cells are not coordinately controlled. PMID:4321349

  6. Detection of glyceraldehyde 3-phosphate dehydrogenase messenger RNA using a peptide nucleic acid probe in paraffin-embedded archival specimens.

    PubMed

    Hiroyasu, Makoto; Akatsuka, Shinya; Shirase, Tomoyuki; Toda, Yoshinobu; Hiai, Hiroshi; Toyokuni, Shinya

    2004-04-01

    Although the human genome project has been completed, the functions of many genes remain undetermined. In situ hybridization (ISH) is a key method for identifying cells in which a given messenger RNA is transcribed. Paraffin-embedded specimens remain precious materials for research, but preservation of high-quality RNA in these specimens is not expected unless ample caution was taken during fixation. Peptide nucleic acid (PNA) is a recently developed hybrid molecule with genetic information that has high stability and high affinity to the complementary DNA or RNA. We applied a PNA probe to mRNA ISH of liver specimens obtained by autopsy and embedded in paraffin 28-48 years ago. An 18-mer PNA probe for glyceraldehyde 3-phosphate dehydrogenase was used. Staining was then analyzed in association with morphology by hematoxylin and eosin staining, and with the time between death of the patient and tissue fixation. Notably, specimens fixed with formalin and embedded in paraffin 48 years ago yielded excellent results if the time before fixation was short enough (<8 h). There was a significant inverse correlation between the intensity of ISH staining and the time before fixation. Oligonucleotide PNA probe, albeit at high cost, would increase the value of paraffin-embedded specimens in storage for use in human medical research.

  7. The role of glycerol-3-phosphate dehydrogenase 1 in the progression of fatty liver after acute ethanol administration in mice

    SciTech Connect

    Sato, Tomoki; Morita, Akihito; Mori, Nobuko; Miura, Shinji

    2014-02-21

    Highlights: • Ethanol administration increased GPD1 mRNA expression. • Ethanol administration increased glucose incorporation into TG glycerol moieties. • No increase in hepatic TG levels was observed in ethanol-injected GPD1 null mice. • We propose that GPD1 is required for ethanol-induced TG accumulation in the liver. - Abstract: Acute ethanol consumption leads to the accumulation of triglycerides (TGs) in hepatocytes. The increase in lipogenesis and reduction of fatty acid oxidation are implicated as the mechanisms underlying ethanol-induced hepatic TG accumulation. Although glycerol-3-phosphate (Gro3P), formed by glycerol kinase (GYK) or glycerol-3-phosphate dehydrogenase 1 (GPD1), is also required for TG synthesis, the roles of GYK and GPD1 have been the subject of some debate. In this study, we examine (1) the expression of genes involved in Gro3P production in the liver of C57BL/6J mice in the context of hepatic TG accumulation after acute ethanol intake, and (2) the role of GPD1 in the progression of ethanol-induced fatty liver using GPD1 null mice. As a result, in C57BL/6J mice, ethanol-induced hepatic TG accumulation began within 2 h and was 1.7-fold greater than that observed in the control group after 6 h. The up-regulation of GPD1 began 2 h after administering ethanol, and significantly increased 6 h later with the concomitant escalation in the glycolytic gene expression. The incorporation of {sup 14}C-labelled glucose into TG glycerol moieties increased during the same period. On the other hand, in GPD1 null mice carrying normal GYK activity, no significant increase in hepatic TG level was observed after acute ethanol intake. In conclusion, GPD1 and glycolytic gene expression is up-regulated by ethanol, and GPD1-mediated incorporation of glucose into TG glycerol moieties together with increased lipogenesis, is suggested to play an important role in ethanol-induced hepatic TG accumulation.

  8. Transient Infantile Hypertriglyceridemia, Fatty Liver, and Hepatic Fibrosis Caused by Mutated GPD1, Encoding Glycerol-3-Phosphate Dehydrogenase 1

    PubMed Central

    Basel-Vanagaite, Lina; Zevit, Noam; Zahav, Adi Har; Guo, Liang; Parathath, Saj; Pasmanik-Chor, Metsada; McIntyre, Adam D.; Wang, Jian; Albin-Kaplanski, Adi; Hartman, Corina; Marom, Daphna; Zeharia, Avraham; Badir, Abir; Shoerman, Oded; Simon, Amos J.; Rechavi, Gideon; Shohat, Mordechai; Hegele, Robert A.; Fisher, Edward A.; Shamir, Raanan

    2012-01-01

    The molecular basis for primary hereditary hypertriglyceridemia has been identified in fewer than 5% of cases. Investigation of monogenic dyslipidemias has the potential to expose key metabolic pathways. We describe a hitherto unreported disease in ten individuals manifesting as moderate to severe transient childhood hypertriglyceridemia and fatty liver followed by hepatic fibrosis and the identification of the mutated gene responsible for this condition. We performed SNP array-based homozygosity mapping and found a single large continuous segment of homozygosity on chromosomal region 12q13.12. The candidate region contained 35 genes that are listed in Online Mendelian Inheritance in Man (OMIM) and 27 other genes. We performed candidate gene sequencing and screened both clinically affected individuals (children and adults with hypertriglyceridemia) and also a healthy cohort for mutations in GPD1, which encodes glycerol-3-phosphate dehydrogenase 1. Mutation analysis revealed a homozygous splicing mutation, c.361−1G>C, which resulted in an aberrantly spliced mRNA in the ten affected individuals. This mutation is predicted to result in a truncated protein lacking essential conserved residues, including a functional site responsible for initial substrate recognition. Functional consequences of the mutation were evaluated by measuring intracellular concentrations of cholesterol and triglyceride as well as triglyceride secretion in HepG2 (hepatocellular carcinoma) human cells lines overexpressing normal and mutant GPD1 cDNA. Overexpression of mutant GPD1 in HepG2 cells, in comparison to overexpression of wild-type GPD1, resulted in increased secretion of triglycerides (p = 0.01). This finding supports the pathogenicity of the identified mutation. PMID:22226083

  9. Sulfur mustard induced nuclear translocation of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

    PubMed

    Steinritz, Dirk; Weber, Jana; Balszuweit, Frank; Thiermann, Horst; Schmidt, Annette

    2013-12-01

    Sulfur Mustard (SM) is a vesicant chemical warfare agent, which is acutely toxic to a variety of organ systems including skin, eyes, respiratory system and bone marrow. The underlying molecular pathomechanism was mainly attributed to the alkylating properties of SM. However, recent studies have revealed that cellular responses to SM exposure are of more complex nature and include increased protein expression and protein modifications that can be used as biomarkers. In order to confirm already known biomarkers, to detect potential new ones and to further elucidate the pathomechanism of SM, we conducted large-scale proteomic experiments based on a human keratinocyte cell line (HaCaT) exposed to SM. Surprisingly, our analysis identified glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as one of the up-regulated proteins after exposure of HaCaT cells to SM. In this paper we demonstrate the sulfur mustard induced nuclear translocation of GAPDH in HaCaT cells by 2D gel-electrophoresis (2D GE), immunocytochemistry (ICC), Western Blot (WB) and a combination thereof. 2D GE in combination with MALDI-TOF MS/MS analysis identified GAPDH as an up-regulated protein after SM exposure. Immunocytochemistry revealed a distinct nuclear translocation of GAPDH after exposure to 300μM SM. This finding was confirmed by fractionated WB analysis. 2D GE and subsequent immunoblot staining of GAPDH demonstrated two different spot locations of GAPH (pI 7.0 and pI 8.5) that are related to cytosolic or nuclear GAPDH respectively. After exposure to 300μM SM a significant increase of nuclear GAPDH at pI 8.5 occurred. Nuclear GAPDH has been associated with apoptosis, detection of structural DNA alterations, DNA repair and regulation of genomic integrity and telomere structure. The results of our study add new aspects to the pathophysiology of sulfur mustard toxicity, yet further studies will be necessary to reveal the specific function of nuclear GAPDH in the pathomechanism of sulfur mustard

  10. Negative homotropic cooperativity and affinity heterogeneity: preparation of yeast glyceraldehyde-3-phosphate dehydrogenase with maximal affinity homogeneity.

    PubMed Central

    Gennis, L S

    1976-01-01

    A three-step procedure including affinity chromatography on NAD+-azobenzamidopropyl-Sepharose has been designed for the purification of yeast glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] with maximized specific activity and maximized homogeneity with respect to affinity for the coenzyme, NAD+.Binding isotherms allow the analysis of cooperativity patterns that disclose both the average ligand affinity in the system and the distribution of ligands among the sites, only for systems with complete affinity homogeneity. The presence of affinity heterogeneity, resulting from multiple oligomeric species differing only in their affinity for coenzyme, gives rise to isotherms which falsely manifest apparent negative cooperativity. A method for distinguishing negative homotropic cooperativity from affinity heterogeneity is suggested. PMID:186779

  11. Low-interference washing-free electrochemical immunosensor using glycerol-3-phosphate dehydrogenase as an enzyme label.

    PubMed

    Dutta, Gorachand; Park, Seonhwa; Singh, Amardeep; Seo, Jeongwook; Kim, Sinyoung; Yang, Haesik

    2015-04-01

    In washing-free electrochemical detection, various redox and reactive species cause significant interference. To minimize this interference, we report a washing-free electrochemical immunosensor using flavin adenine dinucleotide (FAD)-dependent glycerol-3-phosphate dehydrogenase (GPDH) and glycerol-3-phosphate (GP) as an enzyme label and its substrate, respectively, because the reaction of FAD-dependent dehydrogenases with dissolved O2 is slow and the level of GP preexisting in blood is low (<0.1 mM). A combination of a low electrocatalytic indium-tin oxide (ITO) electrode and fast electron-mediating Ru(NH3)6(3+) is employed to obtain a high signal-to-background ratio via proximity-dependent electron mediation of Ru(NH3)6(3+) between the ITO electrode and the GPDH label. Electrochemical oxidation of GPDH-generated Ru(NH3)6(2+) is performed at 0.05 V vs Ag/AgCl, at which point the electrochemical interference is very low. When a washing-free immunosensor is applied to cardiac troponin I detection in human serum, the calculated detection limit is approximately 10 pg/mL, indicating that the immunosensor is very sensitive in spite of the use of washing-free detection with a short detection period (10 min for incubation and 100 s for electrochemical measurement). The low-interference washing-free electrochemical immunosensor shows good promise for fast and simple point-of-care testing.

  12. Structural basis for regulation of stability and activity in glyceraldehyde-3-phosphate dehydrogenases. Differential scanning calorimetry and molecular dynamics.

    PubMed

    Makshakova, Olga N; Semenyuk, Pavel I; Kuravsky, Mikhail L; Ermakova, Elena A; Zuev, Yuriy F; Muronetz, Vladimir I

    2015-05-01

    Tissue specific isoforms of human glyceraldehyde-3-phosphate dehydrogenase, somatic (GAPD) and sperm-specific (GAPDS), have been reported to display different levels of both stability and catalytic activity. Here we apply MD simulations to investigate molecular basis of this phenomenon. The protein is a tetramer where each subunit consists of two domains - catalytic and NAD-binding one. We demonstrated key residues responsible for intersubunit and interdomain interactions. Effect of several residues was studied by point mutations. Overall we considered three mutations (Glu96Gln, Glu244Gln and Asp311Asn) disrupting GAPDS-specific salt bridges. Comparison of calculated interaction energies with calorimetric enthalpies confirmed that intersubunit interactions were responsible for enhanced thermostability of GAPDS whereas interdomain interactions had indirect influence on intersubunit contacts. Mutation Asp311Asn was around 10Å far from the active center and corresponded to the closest natural substitution in the isoenzymes. MD simulations revealed that this residue had slight interaction with catalytic residues but influenced the hydrogen bond net and dynamics in active site. These effects can be responsible for a strong influence of this residue on catalytic activity. Overall, our results provide new insight into glyceraldehyde-3-phosphate dehydrogenase structure-function relationships and can be used for the engineering of mutant proteins with modified properties and for development of new inhibitors with indirect influence on the catalytic site. PMID:25869789

  13. Expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

    SciTech Connect

    Elliott, Paul R.; Evans, Daniel; Greenwood, Jacqueline A.; Moody, Peter C. E.

    2008-08-01

    Glyceraldehyde-3-phosphate dehydrogenase A has been cloned, expressed and purified. Apoprotein crystals have been grown which diffracted to 1.75 Å resolution and belonged to space group P2{sub 1}; holo crystals were grown in the presence of NADP, diffracted to 2.6 Å resolution and belonged to space group P3{sub 2}. The classical glycolytic pathway contains an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, with NADP-dependent forms reserved for photosynthetic organisms and archaea. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori is reported; crystals of the protein were grown both in the presence and the absence of NADP.

  14. Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a.

    PubMed

    Fang, M; Jin, A; Zhao, Y; Liu, X

    2016-02-01

    High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy. PMID:26785692

  15. Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a

    PubMed Central

    Fang, M.; Jin, A.; Zhao, Y.; Liu, X.

    2016-01-01

    High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy. PMID:26785692

  16. Glyceraldehyde 3-phosphate dehydrogenase augments the intercellular transmission and toxicity of polyglutamine aggregates in a cell model of Huntington disease.

    PubMed

    Mikhaylova, Elena R; Lazarev, Vladimir F; Nikotina, Alina D; Margulis, Boris A; Guzhova, Irina V

    2016-03-01

    The common feature of Huntington disease is the accumulation of oligomers or aggregates of mutant huntingtin protein (mHTT), which causes the death of a subset of striatal neuronal populations. The cytotoxic species can leave neurons and migrate to other groups of cells penetrating and damaging them in a prion-like manner. We hypothesized that the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), previously shown to elevate the aggregation of mHTT, is associated with an increased efficiency of intercellular propagation of mHTT. GAPDH, on its own or together with polyglutamine species, was shown to be released into the extracellular milieu mainly from dying cells as assessed by a novel enzyme immunoassay, western blotting, and ultrafiltration. The conditioned medium of cells with growing GAPDH-polyQ aggregates was toxic to naïve cells, whereas depletion of the aggregates from the medium lowered this cytotoxicity. The GAPDH component of the aggregates was found to increase their toxicity by two-fold in comparison with polyQ alone. Furthermore, GAPDH-polyQ complexes were shown to penetrate acceptor cells and to increase the capacity of polyQ to prionize its intracellular homolog containing a repeat of 25 glutamine residues. Finally, inhibitors of intracellular transport showed that polyQ-GAPDH complexes, as well as GAPDH itself, penetrated cells using clathrin-mediated endocytosis. This suggested a pivotal role of the enzyme in the intercellular transmission of Huntington disease pathogenicity. In conclusion, GAPDH occurring in complexes with polyglutamine strengthens the prion-like activity and toxicity of the migrating aggregates. Aggregating polygluatmine tracts were shown to release from the cells over-expressing mutant huntingtin in a complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The enzyme enhances the intracellular transport of aggregates to healthy cells, prionization of normal cellular proteins and finally cell death, thus

  17. Evolutionary engineering of a glycerol-3-phosphate dehydrogenase-negative, acetate-reducing Saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations

    PubMed Central

    Guadalupe-Medina, Víctor; Metz, Benjamin; Oud, Bart; van Der Graaf, Charlotte M; Mans, Robert; Pronk, Jack T; van Maris, Antonius J A

    2014-01-01

    Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S. cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd– strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic engineering concept. In this study, laboratory evolution was used to improve osmotolerance of a Gpd– mhpF-expressing S. cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1 M glucose, at a specific growth rate of 0.12 h−1. The evolved strain produced glycerol at low concentrations (0.64 ± 0.33 g l−1). However, these glycerol concentrations were below 10% of those observed with a Gpd+ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth. PMID:24004455

  18. Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways.

    PubMed

    Martínez, Irene; Zhu, Jiangfeng; Lin, Henry; Bennett, George N; San, Ka-Yiu

    2008-11-01

    Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains.

  19. Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways.

    PubMed

    Martínez, Irene; Zhu, Jiangfeng; Lin, Henry; Bennett, George N; San, Ka-Yiu

    2008-11-01

    Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains. PMID:18852061

  20. Adaptation of the glycerol-3-phosphate dehydrogenase Gpd1 to high salinities in the extremely halotolerant Hortaea werneckii and halophilic Wallemia ichthyophaga.

    PubMed

    Lenassi, Metka; Zajc, Janja; Gostinčar, Cene; Gorjan, Alenka; Gunde-Cimerman, Nina; Plemenitaš, Ana

    2011-10-01

    We report the first identification and characterisation of the glycerol-3-phosphate dehydrogenase (GPD) genes from extremely halophilic fungi. The black ascomycetous yeast Hortaea werneckii and the non-melanised basidiomycetous fungus Wallemia ichthyophaga inhabit similar hypersaline environments, yet they have two different strategies of haloadaptation through Gpd1-regulated glycerol synthesis. The extremely halotolerant H. werneckii codes for two salt-inducible GPD1 genes that show similar gene transcription regulation and have 98% amino-acid sequence identity between paralogues; however, they have distinct effects when expressed heterologously in Saccharomyces cerevisiae gpd mutants. Only the HwGpd1B isoform complements the function of Gpd in the gpd1 mutant, whereas none of the Gpd1 isoforms can rescue the salt sensitivity of the gpd1gpd2 double mutant. The obligate halophile W. ichthyophaga codes for only one GPD1 orthologue, the transcription of which is less affected by salt when compared to the H. werneckii homologues. Heterologous expression of WiGPD1 in S. cerevisiae recovers halotolerance of the gpd1 and gpd1gpd2 mutant strains, which is probably due to the overall high amino-acid similarity of the Gpd1 protein in W. ichthyophaga and S. cerevisiae. Phylogenetic analysis of amino-acid sequences reveals that the evolutionary origins of all of these three novel enzymes correspond to the phylogeny of the fungal species from which the genes were identified.

  1. SIRT1 interacts with and protects glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from nuclear translocation: Implications for cell survival after irradiation

    SciTech Connect

    Joo, Hyun-Yoo; Woo, Seon Rang; Shen, Yan-Nan; Yun, Mi Yong; Shin, Hyun-Jin; Park, Eun-Ran; Kim, Su-Hyeon; Park, Jeong-Eun; Ju, Yeun-Jin; Hong, Sung Hee; Hwang, Sang-Gu; Cho, Myung-Haing; Kim, Joon; Lee, Kee-Ho

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer SIRT1 serves to retain GAPDH in the cytosol, preventing GAPDH nuclear translocation. Black-Right-Pointing-Pointer When SIRT1 is depleted, GAPDH translocation occurs even in the absence of stress. Black-Right-Pointing-Pointer Upon irradiation, SIRT1 interacts with GAPDH. Black-Right-Pointing-Pointer SIRT1 prevents irradiation-induced nuclear translocation of GAPDH. Black-Right-Pointing-Pointer SIRT1 presence rather than activity is essential for inhibiting GAPDH translocation. -- Abstract: Upon apoptotic stimulation, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cytosolic enzyme normally active in glycolysis, translocates into the nucleus and activates an apoptotic cascade therein. In the present work, we show that SIRT1 prevents nuclear translocation of GAPDH via interaction with GAPDH. SIRT1 depletion triggered nuclear translocation of cytosolic GAPDH even in the absence of apoptotic stress. Such translocation was not, however, observed when SIRT1 enzymatic activity was inhibited, indicating that SIRT1 protein per se, rather than the deacetylase activity of the protein, is required to inhibit GAPDH translocation. Upon irradiation, SIRT1 prevented irradiation-induced nuclear translocation of GAPDH, accompanied by interaction of SIRT1 and GAPDH. Thus, SIRT1 functions to retain GAPDH in the cytosol, protecting the enzyme from nuclear translocation via interaction with these two proteins. This serves as a mechanism whereby SIRT1 regulates cell survival upon induction of apoptotic stress by means that include irradiation.

  2. Human and pneumococcal cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins are both ligands of human C1q protein.

    PubMed

    Terrasse, Rémi; Tacnet-Delorme, Pascale; Moriscot, Christine; Pérard, Julien; Schoehn, Guy; Vernet, Thierry; Thielens, Nicole M; Di Guilmi, Anne Marie; Frachet, Philippe

    2012-12-14

    C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (K(D) = 0.34-2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response. PMID:23086952

  3. An investigation of the nicotinamide-adenine dinucleotide-induced 'tightening' of the structure of glyceraldehyde 3-phosphate dehydrogenase.

    PubMed Central

    Osborne, H H; Hollaway, M R

    1976-01-01

    An investigation was made of the effect of NAD+ analogues on subunit interactions in yeast and rabbit muscle glyceraldehyde 3-phosphate dehydrogenases by using the subunit exchange (hybridization) method described previously [e.g. see Osborne & Hollaway (1975) Biochem. J. 151, 37-45]. The ligands ATP, ITP, ADP, AMP, cyclic AMP and ADP-ribose like NADH, all caused an apparent weakening of intramolecular subunit interactions, whereas NAD+ caused an apparent increase in the stability of the tetrameric enzyme molecules. A mixture of NMN and AMP, although it did not simulate completely the NAD+-induced 'tightening' of the enzyme structure, did result in a more than 20-fold decrease in the rate of subunit exchange compared with that in the presence of AMP alone. These results show that occupancy of the NMN subsite of the enzyme NAD+-binding site is insufficient in itself to give the marked tightening of the enzyme structure induced by NAD+. The 'tightening' effect is specific in that it seems to require a phosphodiester link between NMN and ADP-ribose. These effects are discussed in terms of the detailed X-ray structure of the lobster holoenzyme [Buehner et al. (1974) J. Mol. Biol. 90, 25-49]. Images PLATE 1 PLATE 2 PMID:183744

  4. An unusual effect of NADP+ on the thermostability of the nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans.

    PubMed

    Arutyunov, Denis; Schmalhausen, Elena; Orlov, Victor; Rahuel-Clermont, Sophie; Nagradova, Natalia; Branlant, Guy; Muronetz, Vladimir

    2013-10-01

    Adiabatic differential scanning calorimetry was used to investigate the effect of NADP+ on the irreversible thermal denaturation of the nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans. The GAPN-NADP+ binary complex showed a strongly decreased thermal stability, with a difference of about 20 °C between the temperatures of the thermal transition peak maxima of the complex and the free protein. This finding was similar to the previously described thermal destabilization of GAPN upon binding of inorganic phosphate to the substrate binding site and can be interpreted as the shift of the equilibrium between 2 conformers of tetrameric GAPN upon addition of the coenzyme. Single amino acid substitution, known to abolish the NADP+ binding, cancelled the calorimetric effect of the coenzyme. GAPN thermal inactivation was considerably decelerated in the presence of NADP+ showing that the apparent change in stability of the active centre can be the opposite to that of the whole protein molecule. NADP+ could also reactivate the inactive GAPN* species, obtained by the heating of the apoenzyme below the thermal denaturation transition temperature. These effects may reflect a mechanism that provides GAPN the sufficient flexibility for the earlier observed profound active site reorganizations required during the catalytic cycle. The elevated thermal stability of the apoenzyme may, in turn, be important for maintaining a constant level of active GAPN--an enzyme that is known to be crucial for the effective supply of the reducing equivalents in S. mutans and its ability to grow under aerobic conditions.

  5. Structural Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Protofibrils Preventing α-Synuclein Oligomeric Species Toxicity*

    PubMed Central

    Ávila, César L.; Torres-Bugeau, Clarisa M.; Barbosa, Leandro R. S.; Sales, Elisa Morandé; Ouidja, Mohand O.; Socías, Sergio B.; Celej, M. Soledad; Raisman-Vozari, Rita; Papy-Garcia, Dulce; Itri, Rosangela; Chehín, Rosana N.

    2014-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that has been associated with neurodegenerative diseases. GAPDH colocalizes with α-synuclein in amyloid aggregates in post-mortem tissue of patients with sporadic Parkinson disease and promotes the formation of Lewy body-like inclusions in cell culture. In a previous work, we showed that glycosaminoglycan-induced GAPDH prefibrillar species accelerate the conversion of α-synuclein to fibrils. However, it remains to be determined whether the interplay among glycosaminoglycans, GAPDH, and α-synuclein has a role in pathological states. Here, we demonstrate that the toxic effect exerted by α-synuclein oligomers in dopaminergic cell culture is abolished in the presence of GAPDH prefibrillar species. Structural analysis of prefibrillar GAPDH performed by small angle x-ray scattering showed a particle compatible with a protofibril. This protofibril is shaped as a cylinder 22 nm long and a cross-section diameter of 12 nm. Using biocomputational techniques, we obtained the first all-atom model of the GAPDH protofibril, which was validated by cross-linking coupled to mass spectrometry experiments. Because GAPDH can be secreted outside the cell where glycosaminoglycans are present, it seems plausible that GAPDH protofibrils could be assembled in the extracellular space kidnapping α-synuclein toxic oligomers. Thus, the role of GAPDH protofibrils in neuronal proteostasis must be considered. The data reported here could open alternative ways in the development of therapeutic strategies against synucleinopathies like Parkinson disease. PMID:24671416

  6. Secreted multifunctional Glyceraldehyde-3-phosphate dehydrogenase sequesters lactoferrin and iron into cells via a non-canonical pathway

    PubMed Central

    Chauhan, Anoop S.; Rawat, Pooja; Malhotra, Himanshu; Sheokand, Navdeep; Kumar, Manoj; Patidar, Anil; Chaudhary, Surbhi; Jakhar, Priyanka; Raje, Chaaya I.; Raje, Manoj

    2015-01-01

    Lactoferrin is a crucial nutritionally important pleiotropic molecule and iron an essential trace metal for all life. The current paradigm is that living organisms have evolved specific membrane anchored receptors along with iron carrier molecules for regulated absorption, transport, storage and mobilization of these vital nutrients. We present evidence for the existence of non-canonical pathway whereby cells actively forage these vital resources from beyond their physical boundaries, by secreting the multifunctional housekeeping enzyme Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) into the extracellular milieu. This effect’s an autocrine/paracrine acquisition of target ligand into the cell. Internalization by this route is extensively favoured even by cells that express surface receptors for lactoferrin and involves urokinase plasminogen activator receptor (uPAR). We also demonstrate the operation of this phenomenon during inflammation, as an arm of the innate immune response where lactoferrin denies iron to invading microorganisms by chelating it and then itself being sequestered into surrounding host cells by GAPDH. PMID:26672975

  7. Purification and properties of glyceraldehyde-3-phosphate dehydrogenase from the skeletal muscle of the hibernating ground squirrel, Ictidomys tridecemlineatus.

    PubMed

    Bell, Ryan A V; Smith, Jeffrey C; Storey, Kenneth B

    2014-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the skeletal muscle of euthermic and torpid Ictidomys tridecemlineatus was purified to electrophoretic homogeneity using a novel method involving Blue-agarose and Phenyl-agarose chromatography. Kinetic analysis of the enzymes isolated from the two conditions suggested the existence of two structurally distinct proteins, with GAPDH V max being 40-60% less for the enzyme from the torpid condition (in both glycolytic and gluconeogenic directions) as compared to the euthermic enzyme form. Thermal denaturation, in part determined by differential scanning fluorimetry, revealed that purified GAPDH from the torpid animals was significantly more stable that the enzyme from the euthermic condition. Mass spectrometry combined with Western blot analyses of purified GAPDH indicate that the cellular GAPDH population is extensively modified, with posttranslational phosphorylation, acetylation and methylation being detected. Global reduction in GAPDH tyrosine phosphorylation during torpor as well as site specific alterations in methylation sites suggests that that the stable changes observed in kinetic and structural GAPDH properties may be due to posttranslational modification of this enzyme during torpor. Taken together, these results suggest a stable suppression of GAPDH (possibly by some reversible posttranslational modification) during ground squirrel torpor, which likely contributes to the overall reduction in carbohydrate metabolism when these animals switch to lipid fuels during dormancy.

  8. Nuclear translocation and accumulation of glyceraldehyde-3-phosphate dehydrogenase involved in diclazuril-induced apoptosis in Eimeria tenella (E. tenella).

    PubMed

    Wang, Congcong; Han, Chunzhou; Li, Tao; Yang, Dehao; Shen, Xiaojiong; Fan, Yinxin; Xu, Yang; Zheng, Wenli; Fei, Chenzhong; Zhang, Lifang; Xue, Feiqun

    2013-01-01

    In mammalian cells, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) has recently been shown to be implicated in numerous apoptotic paradigms, especially in neuronal apoptosis, and has been demonstrated to play a vital role in some neurodegenerative disorders. However, this phenomenon has not been reported in protists. In the present study, we report for the first time that such a mechanism is involved in diclazuril-induced apoptosis in Eimeria tenella (E. tenella). We found that upon treatment of parasites with diclazuril, the expression levels of GAPDH transcript and protein were significantly increased in second-generation merozoites. Then, we examined the subcellular localization of GAPDH by fluorescence microscopy and Western blot analysis. The results show that a considerable amount of GAPDH protein appeared in the nucleus within diclazuril-treated second-generation merozoites; in contrast, the control group had very low levels of GAPDH in the nucleus. The glycolytic activity of GAPDH was kinetically analyzed in different subcellular fractions. A substantial decrease (48.5%) in glycolytic activity of GAPDH in the nucleus was displayed. Moreover, the activities of caspases-3, -9, and -8 were measured in cell extracts using specific caspase substrates. The data show significant increases in caspase-3 and caspase-9 activities in the diclazuril-treated group.

  9. Improved purification of sn-glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae and its inhibition by ethanol

    SciTech Connect

    Merkel, J.R.; Chen, S.M.; Osinchak, J.; Trumbore, M.

    1986-05-01

    An improved purification procedure yielded a homogeneous preparation of sn-glycerol-3-phosphate dehydrogenase (GPD) from commercially available baker's yeast. The enzyme had an apparent molecular weight of 42,000 by SDS-polyacrylamide gel electrophoresis. This differs from the 31,000 reported earlier on the basis of its elution from a calibrated Sepharose 6B column. When denatured by guanidine (6M) and chromatographed on a Sephadex G-100 column with 6M guanidine in 0.1M phosphate buffer, pH 6.5, containing 0.1M ..beta..-mercaptoethanol, GPD eluted with the approximately 42,000 mw proteins. S. cerevisiae GPD is an NAD-dependent oxidoreductase. With NADH as the variable substrate the GPD-catalyzed reduction of dihydroxacetone phosphate (DHAP) had a K/sub M/ of 0.018 mM and was competitively inhibited by ethanol. With DHAP as the variable substrate and NADH constant GPD catalyzed the reduction with a K/sub M/ of 0.37 mM and was noncompetitively inhibited by ethanol. The calculated K/sub i/ for the non-competitive inhibition was 3.4M. K/sub i/ for the competitive inhibition of NADH by ethanol varied with increasing concentrations of ethanol indicating a more complex mechanism than a truly competitive one.

  10. Impact of glycerol-3-phosphate dehydrogenase on virulence factor production by Pseudomonas aeruginosa.

    PubMed

    Daniels, Jonathan B; Scoffield, Jessica; Woolnough, Jessica L; Silo-Suh, Laura

    2014-12-01

    Pseudomonas aeruginosa establishes life-long chronic infections in the cystic fibrosis (CF) lung by utilizing various adaptation strategies. Some of these strategies include altering metabolic pathways to utilize readily available nutrients present in the host environment. The airway sputum contains various host-derived nutrients that can be utilized by P. aeruginosa, including phosphatidylcholine, a major component of lung surfactant. Pseudomonas aeruginosa can degrade phosphatidylcholine to glycerol and fatty acids to increase the availability of usable carbon sources in the CF lung. In this study, we show that some CF-adapted P. aeruginosa isolates utilize glycerol more efficiently as a carbon source than nonadapted isolates. Furthermore, a mutation in a gene required for glycerol utilization impacts the production of several virulence factors in both acute and chronic isolates of P. aeruginosa. Taken together, the results suggest that interference with this metabolic pathway may have potential therapeutic benefits. PMID:25409940

  11. Two glycerol 3-phosphate dehydrogenase isogenes from Candida versatilis SN-18 play an important role in glycerol biosynthesis under osmotic stress.

    PubMed

    Mizushima, Daiki; Iwata, Hisashi; Ishimaki, Yuki; Ogihara, Jun; Kato, Jun; Kasumi, Takafumi

    2016-05-01

    Two isogenes of glycerol 3-phosphate dehydrogenase (GPD) from Candida versatilis SN-18 were cloned and sequenced. These intronless genes (Cagpd1 and Cagpd2) were both predicted to encode a 378 amino acid polypeptide, and the deduced amino acid sequences mutually showed 76% identity. Interestingly, Cagpd1 and Cagpd2 were located tandemly in a locus of genomic DNA within a 262 bp interval. To our knowledge, this represents a novel instance of isogenic genes relating to glucose metabolism. The stress response element (STRE) was found respectively at -93 to -89 bp upstream of the 5'end of Cagpd1 and -707 to -703 bp upstream of Cagpd2, indicating that these genes are involved in osmotic stress response. In heterologous expression using a gpd1Δgpd2Δ double deletion mutant of Saccharomyces cerevisiae, Cagpd1 and Cagpd2 transformants complemented the function of GPD, with Cagpd2 being much more effective than Cagpd1 in promoting growth and glycerol synthesis. Phylogenetic analysis of the amino acid sequences suggested that Cagpd1p and Cagpd2p are NADP(+)-dependent GPDs (EC 1.1.1.94). However, crude enzyme extract from Cagpd1 and Cagpd2 transformants showed GPD activity with only NAD(+) as cofactor. Hence, both Cagpd1p and Cagpd2p are likely NAD(+)-dependent GPDs (EC 1.1.1.8), similar to GPDs from S. cerevisiae and Candida magnoliae. PMID:26906228

  12. The interactions of 9,10-phenanthrenequinone with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a potential site for toxic actions.

    PubMed

    Rodriguez, Chester E; Fukuto, Jon M; Taguchi, Keiko; Froines, John; Cho, Arthur K

    2005-06-30

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the oxidative phosphorylation of glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate, one of the precursors for glycolytic ATP biosynthesis. The enzyme contains an active site cysteine thiolate, which is critical for its catalytic function. As part of a continuing study of the interactions of quinones with biological systems, we have examined the susceptibility of GAPDH to inactivation by 9,10-phenanthrenequinone (9,10-PQ). In a previous study of quinone toxicity, this quinone, whose actions have been exclusively attributed to reactive oxygen species (ROS) generation, caused a reduction in the glycolytic activity of GAPDH under aerobic and anaerobic conditions, indicating indirect and possible direct actions on this enzyme. In this study, the effects of 9,10-PQ on GAPDH were examined in detail under aerobic and anaerobic conditions so that the role of oxygen could be distinguished from the direct effects of the quinone. The results indicate that, in the presence of the reducing agent DTT, GAPDH inhibition by 9,10-PQ under aerobic conditions was mostly indirect and comparable to the direct actions of exogenously-added H2O2 on this enzyme. GAPDH was also inhibited by 9,10-PQ anaerobically, but in a somewhat more complex manner. This quinone, which is not considered an electrophile, inhibited GAPDH in a time-dependent manner, consistent with irreversible modification and comparable to the electrophilic actions of 1,4-benzoquinone (1,4-BQ). Analysis of the anaerobic inactivation kinetics for the two quinones revealed comparable inactivation rate constants (k(inac)), but a much lower inhibitor binding constant (K(i)) for 1,4-BQ. Protection and thiol titration studies suggest that these quinones bind to the NAD+ binding site and modify the catalytic thiol from this site. Thus, 9,10-PQ inhibits GAPDH by two distinct mechanisms: through ROS generation that results in the oxidization of GAPDH thiols, and by an

  13. Active site cysteine-null glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rescues nitric oxide-induced cell death.

    PubMed

    Kubo, Takeya; Nakajima, Hidemitsu; Nakatsuji, Masatoshi; Itakura, Masanori; Kaneshige, Akihiro; Azuma, Yasu-Taka; Inui, Takashi; Takeuchi, Tadayoshi

    2016-02-29

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a homotetrameric enzyme involved in a key step of glycolysis, also has a role in mediating cell death under nitrosative stress. Our previous reports suggest that nitric oxide-induced intramolecular disulfide-bonding GAPDH aggregation, which occurs through oxidation of the active site cysteine (Cys-152), participates in a mechanism to account for nitric oxide-induced death signaling in some neurodegenerative/neuropsychiatric disorders. Here, we demonstrate a rescue strategy for nitric oxide-induced cell death accompanied by GAPDH aggregation in a mutant with a substitution of Cys-152 to alanine (C152A-GAPDH). Pre-incubation of purified wild-type GAPDH with C152A-GAPDH under exposure to nitric oxide inhibited wild-type GAPDH aggregation in a concentration-dependent manner in vitro. Several lines of structural analysis revealed that C152A-GAPDH extensively interfered with nitric oxide-induced GAPDH-amyloidogenesis. Overexpression of doxycycline-inducible C152A-GAPDH in SH-SY5Y neuroblastoma significantly rescued nitric oxide-induced death, concomitant with the decreased formation of GAPDH aggregates. Further, both co-immunoprecipitation assays and simulation models revealed a heterotetramer composed of one dimer each of wild-type GAPDH and C152A-GAPDH. These results suggest that the C152A-GAPDH mutant acts as a dominant-negative molecule against GAPDH aggregation via the formation of this GAPDH heterotetramer. This study may contribute to a new therapeutic approach utilizing C152A-GAPDH against brain damage in nitrosative stress-related disorders.

  14. Phylogenetically-based variation in the regulation of the Calvin cycle enzymes, phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase, in algae.

    PubMed

    Maberly, Stephen C; Courcelle, Carine; Groben, Rene; Gontero, Brigitte

    2010-03-01

    Aquatic photosynthesis is responsible for about half of the global production and is undertaken by a huge phylogenetic diversity of algae that are poorly studied. The diversity of redox-regulation of phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was investigated in a wide range of algal groups under standard conditions. Redox-regulation of PRK was greatest in chlorophytes, low or absent in a red alga and most chromalveolates, and linked to the number of amino acids between two regulatory cysteine residues. GAPDH regulation was not strongly-related to the different forms of this enzyme and was less variable than for PRK. Addition of recombinant CP12, a protein that forms a complex with PRK and GAPDH, to crude extracts inhibited GAPDH and PRK inversely in the Plantae, but in most chromalveolates had little effect on GAPDH and inhibited or stimulated PRK depending on the species. Patterns of enzyme regulation were used to produce a phylogenetic tree in which cryptophytes and haptophytes, at the base of the chromalveolates, formed a distinct clade. A second clade comprised only chromalveolates. A third clade comprised a mixture of Plantae, an excavate and three chromalveolates: a marine diatom and two others (a xanthophyte and eustigmatophyte) that are distinguished by a low content of chlorophyll c and a lack of fucoxanthin. Regulation of both enzymes was greater in freshwater than in marine taxa, possibly because most freshwaters are more dynamic than oceans. This work highlights the importance of understanding enzyme regulation in diverse algae if their ecology and productivity is to be understood.

  15. A monoclonal antibody that inhibits translation in Sf21 cell lysates is specific for glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Van Meter, Kipp E; Stuart, Melissa K

    2008-11-01

    Monoclonal antibody (Mab) 8B7 was shown in a previous study to inhibit protein translation in lysates of Sf21 cells. The antibody was thought to be specific for a 60-kDa form of elongation factor-1 alpha (EF-1alpha), primarily because the antigen immunoprecipitated by Mab 8B7 cross-reacted with Mab CBP-KK1, an antibody generated to EF-1alpha from Trypanosoma brucei. The purpose of the current study was to investigate further the antigenic specificity of Mab 8B7. The concentration of the 60-kDa antigen relative to total cellular protein proved insufficient for its definitive identification. However, subcellular fractionation of Sf21 cells yielded an additional protein of 37 kDa in the cytosolic and microsomal fractions that was reactive with Mab 8B7. The 37-kDa protein could be easily visualized by colloidal Coomassie Blue G-250 staining as a series of pI 6.9-8.4 spots on two-dimensional gels. Excision of an abundant immunoreactive spot enabled identification of the protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and protein database searching. Subsequent immunoblotting of purified rabbit skeletal muscle GAPDH with Mab 8B7 confirmed the antibody's specificity for GAPDH. Besides the pivotal role GAPDH plays in glycolysis, the enzyme has a number of noncanonical functions, including binding to mRNA and tRNA. The ability of Mab 8B7 to disrupt these lesser-known functions of GAPDH may account for the antibody's inhibitory effect on in vitro translation. PMID:18850593

  16. Expression, purification, crystallization and preliminary X-ray analysis of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

    SciTech Connect

    Elliott, Paul R.; Mohammad, Shabaz; Melrose, Helen J.; Moody, Peter C. E.

    2008-08-01

    Glyceraldehyde-3-phosphate dehydrogenase B from H. pylori has been cloned, expressed, purified and crystallized in the presence of NAD. Crystals of GAPDHB diffracted to 2.8 Å resolution and belonged to space group P6{sub 5}22, with unit-cell parameters a = b = 166.1, c = 253.1 Å. Helicobacter pylori is a dangerous human pathogen that resides in the upper gastrointestinal tract. Little is known about its metabolism and with the onset of antibiotic resistance new treatments are required. In this study, the expression, purification, crystallization and preliminary X-ray diffraction of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from H. pylori are reported.

  17. Inhibition of glyceraldehyde 3-phosphate dehydrogenase by plasma and serum ultrafiltrates due in part to a low-molecular-weight, nonpeptide material.

    PubMed

    Schwartz, P L; Turfus, I M

    1975-05-01

    In an attempt to verify the existence in the blood of a diabetogenic peptide (somantin) derived from growth hormone, ultrafiltrates from plasma and serum from normal and diabetic subjects were prepared. The freeze-dried residues of these ultrafiltrates inhibited glyceraldehyde 3-phosphate dehydrogenase as somantin is claimed to do. However, the behavior of the inhibitory material on gel filtration on Sephadex G-10 indicated a molecular weight well below 700, rather than the considerably larger size claimed for somantin. The inhibitory material did not adsorb to Dowex 50W cation exchange resin at pH 2.5, while over 95 percent of ninhydrin-positive material was retained. Acid hydrolysis of the inhibitory material did not abolish its activity. Because of the presence of this low-molecular-weight, nonpeptide inhibitory material, inhibition of glyceraldehyde 3-phosphate dehydrogenase by a simple ultrafiltrate of plasma or serum is probably not a definitive measure of somantin. PMID:1128227

  18. Comparison of the regulation, metabolic functions, and roles in virulence of the glyceraldehyde-3-phosphate dehydrogenase homologues gapA and gapB in Staphylococcus aureus.

    PubMed

    Purves, Joanne; Cockayne, Alan; Moody, Peter C E; Morrissey, Julie A

    2010-12-01

    The Gram-positive bacterium Staphylococcus aureus contains two glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologues known as GapA and GapB. GapA has been characterized as a functional GAPDH protein, but currently there is no biological evidence for the role of GapB in metabolism in S. aureus. In this study we show through a number of complementary methods that S. aureus GapA is essential for glycolysis while GapB is essential in gluconeogenesis. These proteins are reciprocally regulated in response to glucose concentrations, and both are influenced by the glycolysis regulator protein GapR, which is the first demonstration of the role of this regulator in S. aureus and the first indication that GapR homologues control genes other than those within the glycolytic operon. Furthermore, we show that both GapA and GapB are important in the pathogenesis of S. aureus in a Galleria mellonella model of infection, showing for the first time in any bacteria that both glycolysis and gluconeogenesis have important roles in virulence.

  19. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of glyceraldehyde-3-phosphate dehydrogenase from Streptococcus agalactiae NEM316

    PubMed Central

    Nagarajan, Revathi; Ponnuraj, Karthe

    2014-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme involved in glycolysis. Despite lacking the secretory signal sequence, this cytosolic enzyme has been found localized at the surface of several bacteria and fungi. As a surface protein, GAPDH exhibits various adhesive functions, thereby facilitating colonization and invasion of host tissues. Streptococcus agalactiae, also known as group B streptococcus (GBS), binds onto the host using its surface adhesins and causes sepsis and pneumonia in neonates. GAPDH is one of the surface adhesins of GBS binding to human plasminogen and is a virulent factor associated with host colonization. Although the surface-associated GAPDH has been shown to bind to a variety of host extracellular matrix (ECM) molecules in various bacteria, the molecular mechanism underlying their interaction is not fully understood. To investigate this, structural studies on GAPDH of S. agalactiae were initiated. The gapC gene of S. agalactiae NEM316 encoding GAPDH protein was cloned into pET-28a vector, overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity. The purified protein was crystallized using the hanging-drop vapour-diffusion method. The GAPDH crystals obtained in two different crystallization conditions diffracted to 2.8 and 2.6 Å resolution, belonging to two different space groups P21 and P212121, respectively. The structure was solved by molecular replacement and structure refinement is now in progress. PMID:25005093

  20. The complete sequence of a full length cDNA for human liver glyceraldehyde-3-phosphate dehydrogenase: evidence for multiple mRNA species.

    PubMed Central

    Arcari, P; Martinelli, R; Salvatore, F

    1984-01-01

    A recombinant M13 clone (O42) containing a 65 b.p. cDNA fragment from human fetal liver mRNA coding for glyceraldehyde-3-phosphate dehydrogenase has been identified and it has been used to isolate from a full-length human adult liver cDNA library a recombinant clone, pG1, which has been subcloned in M13 phage and completely sequenced with the chain terminator method. Besides the coding region of 1008 b.p., the cDNA sequence includes 60 nucleotides at the 5'-end and 204 nucleotides at the 3'-end up to the polyA tail. Hybridization of pG1 to human liver total RNA shows only one band about the size of pG1 cDNA. A much stronger hybridization signal was observed using RNA derived from human hepatocarcinoma and kidney carcinoma cell lines. Sequence homology between clone 042 and the homologous region of clone pG1 is 86%. On the other hand, homology among the translated sequences and the known human muscle protein sequence ranges between 77 and 90%; these data demonstrate the existence of more than one gene coding for G3PD. Southern blot of human DNA, digested with several restriction enzymes, also indicate that several homologous sequences are present in the human genome. Images PMID:6096821

  1. Antibodies to inactive conformations of glyceraldehyde-3-phosphate dehydrogenase inactivate the apo- and holoforms of the enzyme.

    PubMed

    Arutiunova, E I; Pleten, A P; Nagradova, N K; Muronetz, V I

    2006-06-01

    Polyclonal antibodies produced after the immunization of a rabbit with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus were used to isolate two types of antibodies interacting with different non-native forms of the antigen. Type I antibodies were purified using Sepharose-bound apo-GAPDH that was treated with glutaraldehyde to stabilize the enzyme in the tetrameric form. Type II antibodies were isolated using immobilized denatured monomers of the enzyme. It was shown that the type I antibodies bound to the native holo- and apoforms of the enzyme with the ratio of one antibody molecule per GAPDH tetramer. While interacting with the native holoenzyme, the type I antibodies induce a time-dependent decrease in its activity by 80-90%. In the case of the apoenzyme, the decrease in the activity constitutes only 25%, this indicating that only one subunit of the tetramer is inactivated. Differential scanning calorimetry experiments showed that the formation of the complex between both forms of the enzyme and the type I antibodies resulted in a shift of the maximum of the thermal capacity curves (T(m) value) to lower temperatures. The extremely stable holoenzyme was affected to the greatest extent, the shift of the T(m) value constituting approximately 20 degrees C. We assume that the formation of the complex between the holo- or apo-GAPDH and the type I antibody results in time-dependent conformational changes in the enzyme molecule. Thus, the antibodies induce the structural rearrangements yielding the conformation that is identical to the structure of the antigen used for the selection of the antibodies (i.e., inactive). The interaction of the antibodies with the apo-GAPDH results in the inactivation of the subunit directly bound to the antibody. Virtually complete inactivation of the holoenzyme by the antibodies is likely due to the transmission of the conformational changes through the intersubunit contacts. The type II antibodies, which

  2. The specific role of plastidial glycolysis in photosynthetic and heterotrophic cells under scrutiny through the study of glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Anoman, Armand Djoro; Flores-Tornero, María; Rosa-Telléz, Sara; Muñoz-Bertomeu, Jesús; Segura, Juan; Ros, Roc

    2016-01-01

    The cellular compartmentalization of metabolic processes is an important feature in plants where the same pathways could be simultaneously active in different compartments. Plant glycolysis occurs in the cytosol and plastids of green and non-green cells in which the requirements of energy and precursors may be completely different. Because of this, the relevance of plastidial glycolysis could be very different depending on the cell type. In the associated study, we investigated the function of plastidial glycolysis in photosynthetic and heterotrophic cells by specifically driving the expression of plastidial glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in a glyceraldehyde-3-phosphate dehydrogenase double mutant background (gapcp1gapcp2). We showed that GAPCp is not functionally significant in photosynthetic cells, while it plays a crucial function in heterotrophic cells. We also showed that (i) GAPCp activity expression in root tips is necessary for primary root growth, (ii) its expression in heterotrophic cells of aerial parts and roots is necessary for plant growth and development, and (iii) GAPCp is an important metabolic connector of carbon and nitrogen metabolism through the phosphorylated pathway of serine biosynthesis (PPSB). We discuss here the role that this pathway could play in the control of plant growth and development. PMID:26953506

  3. Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803.

    PubMed Central

    Valverde, F; Losada, M; Serrano, A

    1997-01-01

    The gap-2 gene, encoding the NAD(P)-dependent D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH2) of the cyanobacterium Synechocystis sp. strain PCC 6803, was cloned by functional complementation of an Escherichia coli gap mutant with a genomic DNA library; this is the first time that this cloning strategy has been used for a GAPDH involved in photosynthetic carbon assimilation. The Synechocystis DNA region able to complement the E. coli gap mutant was narrowed down to 3 kb and fully sequenced. A single complete open reading frame of 1,011 bp encoding a protein of 337 amino acids was found and identified as the putative gap-2 gene identified in the complete genome sequence of this organism. Determination of the transcriptional start point, identification of putative promoter and terminator sites, and orientation of the truncated flanking genes suggested the gap-2 transcript should be monocystronic, a possibility further confirmed by Northern blot studies. Both natural and recombinant homotetrameric GAPDH2s were purified and found to exhibit virtually identical physicochemical and kinetic properties. The recombinant GAPDH2 showed the dual pyridine nucleotide specificity characteristic of the native cyanobacterial enzyme, and similar ratios of NAD- to NADP-dependent activities were found in cell extracts from Synechocystis as well as in those from the complemented E. coli clones. The deduced amino acid sequence of Synechocystis GAPDH2 presented a high degree of identity with sequences of the chloroplastic NADP-dependent enzymes. In agreement with this result, immunoblot analysis using monospecific antibodies raised against GAPDH2 showed the presence of the 38-kDa GAPDH subunit not only in crude extracts from the gap-2-expressing E. coli clones and all cyanobacteria that were tested but also in those from eukaryotic microalgae and plants. Western and Northern blot experiments showed that gap-2 is conspicuously expressed, although at different levels, in Synechocystis

  4. Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells.

    PubMed

    Shyr, Y Y; Hepburn, A G; Widholm, J M

    1992-04-01

    CAR and C1, two carrot (Daucus carota L.) suspension cultures of different genotypes, were subjected to stepwise selection for tolerance to the herbicide glyphosate [(N-phosphonomethyl)glycine]. The specific activity of the target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), as well as the mRNA level and copy number of the structural gene increased with each glyphosate selection step. Therefore, the tolerance to glyphosate is due to stepwise amplification of the EPSPS genes. During the amplification process, DNA rearrangement did not occur within the EPSPS gene of the CAR cell line but did occur during the selection step from 28 to 35 mM glyphosate for the C1 cell line, as determined by Southern hybridization of selected cell DNA following EcoRI restriction endonuclease digestion. Two cell lines derived from a previously selected glyphosate-tolerant cell line (PR), which also had undergone EPSPS gene amplification but have been maintained in glyphosate-free medium for 2 and 5 years, have lost 36 and 100% of the increased EPSPS activity, respectively. Southern blot analysis of these lines confirms that the amplified DNA is relatively stable in the absence of selection. These studies demonstrate that stepwise selection for glyphosate resistance reproducibly produces stepwise amplification of the EPSPS genes. The relative stability of this amplification indicates that the amplified genes are not extrachromosomal.

  5. Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco.

    PubMed

    Ye, G N; Hajdukiewicz, P T; Broyles, D; Rodriguez, D; Xu, C W; Nehra, N; Staub, J M

    2001-02-01

    Plastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the efficacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the efficacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10,000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.

  6. The 2',4'-dihydroxychalcone could be explored to develop new inhibitors against the glycerol-3-phosphate dehydrogenase from Leishmania species.

    PubMed

    Passalacqua, Thais G; Torres, Fábio A E; Nogueira, Camila T; de Almeida, Leticia; Del Cistia, Mayara L; dos Santos, Mariana B; Regasini, Luis O; Graminha, Márcia A S; Marchetto, Reinaldo; Zottis, Aderson

    2015-09-01

    The enzyme glycerol-3-phosphate dehydrogenase (G3PDH) from Leishmania species is considered as an attractive target to design new antileishmanial drugs and a previous in silico study reported on the importance of chalcones to achieve its inhibition. Here, we report the identification of a synthetic chalcone in our in vitro assays with promastigote cells from Leishmania amazonensis, its biological activity in animal models, and docking followed by molecular dynamics simulation to investigate the molecular interactions and structural patterns that are crucial to achieve the inhibition complex between this compound and G3PDH. A molecular fragment of this natural product derivative can provide new inhibitors with increased potency and selectivity. PMID:26169126

  7. Separate physiological roles for two isozymes of pyridine nucleotide-linked glycerol-3-phosphate dehydrogenase in chicken.

    NASA Technical Reports Server (NTRS)

    White, H. B., III; Kaplan, N. O.

    1972-01-01

    The isozymes considered are designated 'liver type' and 'muscle type' based on the tissue of highest concentration. Electrophoretic analysis shows that the liver type is found in small amounts or is undetectable in all tissues studied except liver. The muscle type is found in skeletal muscles and kidney. Presumptive hybrid enzymes occur at low levels in chicken liver and kidney. The tissue distribution of glyceron-3-P dehydrogenase in several birds capable of sustained flight is different than in chicken.

  8. Characterization of Arabidopsis lines deficient in GAPC-1, a cytosolic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Rius, Sebastián P; Casati, Paula; Iglesias, Alberto A; Gomez-Casati, Diego F

    2008-11-01

    Phosphorylating glyceraldehyde-3-P dehydrogenase (GAPC-1) is a highly conserved cytosolic enzyme that catalyzes the conversion of glyceraldehyde-3-P to 1,3-bis-phosphoglycerate; besides its participation in glycolysis, it is thought to be involved in additional cellular functions. To reach an integrative view on the many roles played by this enzyme, we characterized a homozygous gapc-1 null mutant and an as-GAPC1 line of Arabidopsis (Arabidopsis thaliana). Both mutant plant lines show a delay in growth, morphological alterations in siliques, and low seed number. Embryo development was altered, showing abortions and empty embryonic sacs in basal and apical siliques, respectively. The gapc-1 line shows a decrease in ATP levels and reduced respiratory rate. Furthermore, both lines exhibit a decrease in the expression and activity of aconitase and succinate dehydrogenase and reduced levels of pyruvate and several Krebs cycle intermediates, as well as increased reactive oxygen species levels. Transcriptome analysis of the gapc-1 mutants unveils a differential accumulation of transcripts encoding for enzymes involved in carbon partitioning. According to these studies, some enzymes involved in carbon flux decreased (phosphoenolpyruvate carboxylase, NAD-malic enzyme, glucose-6-P dehydrogenase) or increased (NAD-malate dehydrogenase) their activities compared to the wild-type line. Taken together, our data indicate that a deficiency in the cytosolic GAPC activity results in modifications of carbon flux and mitochondrial dysfunction, leading to an alteration of plant and embryo development with decreased number of seeds, indicating that GAPC-1 is essential for normal fertility in Arabidopsis plants. PMID:18820081

  9. The investigation of substrate-induced changes in subunit interactions in glyceraldehyde 3-phosphate dehydrogenases by measurement of the kinetics and thermodynamics of subunit exchange.

    PubMed Central

    Osborne, H H; Hollaway, M R

    1975-01-01

    An investigation was made of changes in subunit interactions in glyceraldehyde 3-phosphate dehydrogenase on binding NAD+, NADH and other substrates by using the previously developed method of measurement of rates and extent of subunit exchange between the rabbit enzyme (R4), yeast enzyme (Y4) and rabbit-yeast hybrid (R2Y2) [Osborne & Hollaway (1974) Biochem. J. 143, 651-662]. The free energy of activation for the conversion of tetramer into dimer for the rabbit enzyme (R4 leads to 2R2) is increased by at least 12kJ/mol in the presence of NAD+. This increase is interpreted in terms of an NAD+-induced 'tightening' of the tetrameric structure probably involving increased interaction at the subunit interfaces across the QR plane of the molecule [see Buehner et al. (1974) J. Mol. Biol. 82, 563-585]. This tightening of the structure only occurs on binding the third NAD+ molecule to a given enzyme molecule. Conversely, binding of NADH causes a decrease in the free energy of activation for the R4 leads to 2R2 and Y4 leads to 2Y2 conversions by at least 10kJ/mol. This is interpreted as a NADH-induced 'loosening' of the structures arising from decreased interactions across the subunit interfaces involving the QR dissociation plane. In the presence of NADH the increase in the rate of subunit exchange is such that it is not possible to separate the hybrid from the other species if electrophoresis is carried out with NADH in the separation media. In the presence of a mixture of NADH and NAD+ the effect of NAD+ on subunit exchange is dominant. The results are discussed in terms of the known co-operativty between binding sites in glyceraldehyde 3-phosphate dehydrogenases. Images PLATE 1(a) PLATE 1(b) PLATE 2(a) PLATE 2(b) PLATE 2(c) PMID:174555

  10. Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates.

    PubMed

    Wang, Jiancai; Xu, Ronghua; Wang, Ruling; Haque, Mohammad Enamul; Liu, Aizhong

    2016-06-01

    The conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACC) is the rate-limiting step in fatty acid biosynthesis. In this study, a gene coding for ACC was isolated and characterized from an oleaginous yeast, Lipomyces starkeyi. Real-time quantitative PCR (qPCR) analysis of L. starkeyi acetyl-CoA carboxylase gene (LsACC1) showed that the expression levels were upregulated with the fast accumulation of lipids. The LsACC1 was co-overexpressed with the glycerol 3-phosphate dehydrogenase gene (GPD1), which regulates lipids biosynthesis by supplying another substrates glycerol 3-phosphate for storage lipid assembly, in the non-oleaginous yeast Saccharomyces cerevisiae. Further, the S. cerevisiae acetyl-CoA carboxylase (ScACC1) was transferred with GPD1 and its function was analyzed in comparison with LsACC1. The results showed that overexpressed LsACC1 and GPD1 resulted in a 63% increase in S. cerevisiae. This study gives new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts.

  11. ATP-driven transhydrogenation and ionization of water in a reconstituted glyceraldehyde-3-phosphate dehydrogenases (phosphorylating and non-phosphorylating) model system.

    PubMed

    Serrano, A; Mateos, M I; Losada, M

    1993-12-30

    In an unbuffered medium, an intense acidification occurs during the oxidation of D-glyceraldehyde-3-phosphate (G3P) to 3-phospho-D-glycerate (PGA) catalyzed by NADP(+)-specific non-phosphorylating G3P dehydrogenase, an enzyme that photosynthetic eukaryotic cells contain exclusively in their cytosol. The true enzymatic character of this proton release is the consequence of the following redox/acid-base reaction: G3P + NADP+ + H2O-->PGA + NADPH + 2H+. When the well-established ATP-dependent reduction of PGA to G3P, catalyzed by PGA kinase and NAD(+)-specific phosphorylating G3P dehydrogenase, was coupled through the intermediate G3P to the above reverse oxidation reaction, a transient alkalinization of the medium followed by its acidification accompanied transhydrogenation from NADH to NADP+. The significance of the observed endergonic transhydrogenation and ionization of water at the expense of the chemical energy of ATP in this reconstituted enzyme system as well as its relevance for the export of reducing power (H-) across the chloroplast membrane and the maintenance of the pH gradient that exists between the stroma and the cytosol are discussed. PMID:8280152

  12. Overexpression of glycerol-3-phosphate acyltransferase gene improves chilling tolerance in tomato.

    PubMed

    Sui, Na; Li, Meng; Zhao, Shi-Jie; Li, Feng; Liang, Hui; Meng, Qing-Wei

    2007-10-01

    A tomato (Lycopersicon esculentum Mill.) glycerol-3-phosphate acyltransferase gene (LeGPAT) was isolated. The deduced amino acid sequence revealed that LeGPAT contained four acyltransferase domains, showing high identities with GPAT in other plant species. A GFP fusion protein of LeGPAT was targeted to chloroplast in cowpea mesophyll protoplast. RNA gel blot showed that the mRNA accumulation of LeGPAT in the wild type (WT) was induced by chilling temperature. Higher expression levels were observed when tomato leaves were exposed to 4 degrees C for 4 h. RNA gel and western blot analysis confirmed that the sense gene LeGPAT was transferred into the tomato genome and overexpressed under the control of 35S-CaMV. Although tomato is classified as a chilling-sensitive plant, LeGPAT exhibited selectivity to 18:1 over 16:0. Overexpression of LeGPAT increased total activity of LeGPAT and cis-unsaturated fatty acids in PG in thylakoid membrane. Chilling treatment induced less ion leakage from the transgenic plants than from the WT. The photosynthetic rate and the maximal photochemical efficiency of PS II (Fv/Fm) in transgenic plants decreased more slowly during chilling stress and recovered faster than in WT under optimal conditions. The oxidizable P700 in both WT and transgenic plants decreased obviously at chilling temperature under low irradiance, but the oxidizable P700 recovered faster in transgenic plants than in the WT. These results indicate that overexpression of LeGPAT increased the levels of PG cis-unsaturated fatty acids in thylakoid membrane, which was beneficial for the recovery of chilling-induced PS I photoinhibition in tomato.

  13. Glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR) and nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN), key enzymes of the respective modified Embden-Meyerhof pathways in the hyperthermophilic crenarchaeota Pyrobaculum aerophilum and Aeropyrum pernix.

    PubMed

    Reher, Matthias; Gebhard, Susanne; Schönheit, Peter

    2007-08-01

    The growth of Pyrobaculum aerophilum on yeast extract and nitrate was stimulated by the addition of maltose. Extracts of maltose/yeast extract/nitrate-grown cells contained all enzyme activities of a modified Embden-Meyerhof (EM) pathway, including ATP-dependent glucokinase, phosphoglucose isomerase, ATP-dependent 6-phosphofructokinase, fructose-1,6-phosphate aldolase, triose-phosphate isomerase, GAPOR, phosphoglycerate mutase, enolase and pyruvate kinase. The activity of GAPOR was stimulated about fourfold by maltose, indicating a role in sugar degradation. GAPOR was purified 200-fold to homogeneity and characterized as a 67 kDa monomeric, extremely thermostable protein. The enzyme showed high specificity for glyceraldehyde-3-phosphate and did not use glyceraldehyde, acetaldehyde or formaldehyde as substrates. By matrix-assisted laser desorption/ionization-time of flight analysis of the purified enzyme, ORF PA1029 was identified as a coding gene, gapor, in the sequenced genome of Pyrobaculum aerophilum. The data indicate that the (micro)aerophilic Pyrobaculum aerophilum contains a functional GAPOR as part of a modified EM pathway. Cells of the strictly aerobic crenarchaeon Aeropyrum pernix also contain enzyme activities of a modified EM pathway similar to that of Pyrobaculum aerophilum, except that a GAPN activity replaces GAPOR activity.

  14. Inactivation of glyceraldehyde-3-phosphate dehydrogenase by a reactive metabolite of acetaminophen and mass spectral characterization of an arylated active site peptide.

    PubMed

    Dietze, E C; Schäfer, A; Omichinski, J G; Nelson, S D

    1997-10-01

    Acetaminophen (4'-hydroxyacetanilide, APAP) is a widely used analgesic and antipyretic drug that can cause hepatic necrosis under some circumstances via cytochrome P450-mediated oxidation to a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Although the mechanism of hepatocellular injury caused by APAP is not fully understood, it is known that NAPQI forms covalent adducts with several hepatocellular proteins. Reported here is the identification of one of these proteins as glyceraldehyde-3-phosphate dehydrogenase [GAPDH, D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12]. Two hours after the administration of hepatotoxic doses of [14C]APAP to mice, at a time prior to overt cell damage, hepatocellular GAPDH activity was significantly decreased concurrent with the formation of a 14C-labeled GAPDH adduct. A nonhepatotoxic regioisomer of APAP, 3'-hydroxyacetanilide (AMAP), was found to decrease GAPDH activity to a lesser extent than APAP, and radiolabel from [14C]AMAP bound to a lesser extent to GAPDH at a time when its overall binding to hepatocellular proteins was almost equivalent to that of APAP. In order to determine the nature of the covalent adduct between GAPDH and APAP, its major reactive and toxic metabolite, NAPQI, was incubated with purified porcine muscle GAPDH. Microsequencing analysis and fast atom bombardment mass spectrometry (FAB-MS) with collision-induced dissociation (CID) were used to characterize one of the adducts as APAP bound to the cysteinyl sulfhydryl group of Cys-149 in the active site peptide of GAPDH. PMID:9348431

  15. Aromatic hydrocarbons upregulate glyceraldehyde-3-phosphate dehydrogenase and induce changes in actin cytoskeleton. Role of the aryl hydrocarbon receptor (AhR).

    PubMed

    Reyes-Hernández, O D; Mejía-García, A; Sánchez-Ocampo, E M; Castro-Muñozledo, F; Hernández-Muñoz, R; Elizondo, G

    2009-12-21

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme involved in several cellular functions including glycolysis, membrane transport, microtubule assembly, DNA replication and repair, nuclear RNA export, apoptosis, and the detection of nitric oxide stress. Therefore, modifications in the regulatory ability and function of GAPDH may alter cellular homeostasis. We report here that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphthoflavone, which are well-known ligands for the aryl hydrocarbon receptor (AhR), increase GAPDH mRNA levels in vivo and in vitro, respectively. These compounds fail to induce GAPDH transcription in an AhR-null mouse model, suggesting that the increase in GAPDH level is dependent upon AhR activation. To analyse the consequences of AhR ligands on GAPDH function, mice were treated with TCDD and the level of liver activity of GAPDH was determined. The results showed that TCDD treatment increased GAPDH activity. On the other hand, treatment of Hepa-1 cells with beta-naphthoflavone leads to an increase in microfilament density when compared to untreated cultures. Collectively, these results suggest that AhR ligands, such as polycyclic hydrocarbons, can modify GAPDH expression and, therefore, have the potential to alter the multiple functions of this enzyme.

  16. Plastidial Glycolytic Glyceraldehyde-3-Phosphate Dehydrogenase Is an Important Determinant in the Carbon and Nitrogen Metabolism of Heterotrophic Cells in Arabidopsis.

    PubMed

    Anoman, Armand D; Muñoz-Bertomeu, Jesús; Rosa-Téllez, Sara; Flores-Tornero, María; Serrano, Ramón; Bueso, Eduardo; Fernie, Alisdair R; Segura, Juan; Ros, Roc

    2015-11-01

    This study functionally characterizes the Arabidopsis (Arabidopsis thaliana) plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in photosynthetic and heterotrophic cells. We expressed the enzyme in gapcp double mutants (gapcp1gapcp2) under the control of photosynthetic (Rubisco small subunit RBCS2B [RBCS]) or heterotrophic (phosphate transporter PHT1.2 [PHT]) cell-specific promoters. Expression of GAPCp1 under the control of RBCS in gapcp1gapcp2 had no significant effect on the metabolite profile or growth in the aerial part (AP). GAPCp1 expression under the control of the PHT promoter clearly affected Arabidopsis development by increasing the number of lateral roots and having a major effect on AP growth and metabolite profile. Our results indicate that GAPCp1 is not functionally important in photosynthetic cells but plays a fundamental role in roots and in heterotrophic cells of the AP. Specifically, GAPCp activity may be required in root meristems and the root cap for normal primary root growth. Transcriptomic and metabolomic analyses indicate that the lack of GAPCp activity affects nitrogen and carbon metabolism as well as mineral nutrition and that glycerate and glutamine are the main metabolites responding to GAPCp activity. Thus, GAPCp could be an important metabolic connector of glycolysis with other pathways, such as the phosphorylated pathway of serine biosynthesis, the ammonium assimilation pathway, or the metabolism of γ-aminobutyrate, which in turn affect plant development. PMID:26134167

  17. A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase.

    PubMed

    Bommareddy, Rajesh Reddy; Chen, Zhen; Rappert, Sugima; Zeng, An-Ping

    2014-09-01

    Engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. In this work, a de novo NADPH generation pathway is proposed by altering the coenzyme specificity of a native NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to NADP, which consequently has the potential to produce additional NADPH in the glycolytic pathway. Specifically, the coenzyme specificity of GAPDH of Corynebacterium glutamicum is systematically manipulated by rational protein design and the effect of the manipulation for cellular metabolism and lysine production is evaluated. By a combinatorial modification of four key residues within the coenzyme binding sites, different GAPDH mutants with varied coenzyme specificity were constructed. While increasing the catalytic efficiency of GAPDH towards NADP enhanced lysine production in all of the tested mutants, the most significant improvement of lysine production (~60%) was achieved with the mutant showing similar preference towards both NAD and NADP. Metabolic flux analysis with (13)C isotope studies confirmed that there was no significant change of flux towards the pentose phosphate pathway and the increased lysine yield was mainly attributed to the NADPH generated by the mutated GAPDH. The present study highlights the importance of protein engineering as a key strategy in de novo pathway design and overproduction of desired products.

  18. Regulation of cyclic electron flow in C₃ plants: differential effects of limiting photosynthesis at ribulose-1,5-bisphosphate carboxylase/oxygenase and glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Livingston, Aaron K; Kanazawa, Atsuko; Cruz, Jeffrey A; Kramer, David M

    2010-11-01

    Cyclic electron flow around photosystem I (CEF1) is thought to augment chloroplast ATP production to meet metabolic needs. Very little is known about the induction and regulation of CEF1. We investigated the effects on CEF1 of antisense suppression of the Calvin-Benson enzymes glyceraldehyde-3-phosphate dehydrogenase (gapR), and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (SSU), in tobacco (Nicotiana tabacum cv. Wisconsin 38). The gapR, but not ssuR, mutants showed substantial increases in CEF1, demonstrating that specific intermediates, rather than slowing of assimilation, induce CEF1. Both types of mutant showed increases in steady-state transthylakoid proton motive force (pmf) and subsequent activation of the photoprotective q(E) response. With gapR, the increased pmf was caused both by up-regulation of CEF1 and down-regulation of the ATP synthase. In ssuR, the increased pmf was attributed entirely to a decrease in ATP synthase activity, as previously seen in wild-type plants when CO₂ levels were decreased. Comparison of major stromal metabolites in gapR, ssuR and hcef1, a mutant with decreased fructose 1,6-bisphosphatase activity, showed that neither the ATP/ADP ratio, nor major Calvin-Benson cycle intermediates can directly account for the activation of CEF1, suggesting that chloroplast redox status or reactive oxygen species regulate CEF1.

  19. Possible role of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase in growth promotion of Arabidopsis seedlings by low levels of selenium.

    PubMed

    Takeda, Toru; Fukui, Yuki

    2015-01-01

    We explored functional significance of selenium (Se) in Arabidopsis physiology. Se at very low concentrations in cultivation exerted a considerable positive effect on Arabidopsis growth with no indication of oxidative stress, whereas Se at higher concentrations significantly suppressed the growth and brought serious oxidative damage. Respiration, ATP levels, and the activity of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (NAD-GAPDH) were enhanced in Arabidopsis grown in the medium containing 1.0 μM Se. Addition of an inhibitor of glutathione (GSH) synthesis to the medium abolished both of the Se-dependent growth promotion and NAD-GAPDH up-regulation. Assay of NAD-GAPDH purified from seedlings subjected to Se interventions raised the possibility of a direct connection between the activity of this enzyme and Arabidopsis growth. These results reveal that trace amounts of Se accelerate Arabidopsis growth, and suggest that this pro-growth effect of Se arises enhancing mitochondrial performance in a GSH-dependent manner, in which NAD-GAPDH may serve as a key regulator.

  20. Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS).

    PubMed

    Sexton, Jonathan Z; Danshina, Polina V; Lamson, David R; Hughes, Mark; House, Alan J; Yeh, Li-An; O'Brien, Deborah A; Williams, Kevin P

    2011-01-01

    Glycolytic isozymes that are restricted to the male germline are potential targets for the development of reversible, non-hormonal male contraceptives. GAPDHS, the sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase, is an essential enzyme for glycolysis making it an attractive target for rational drug design. Toward this goal, we have optimized and validated a high-throughput spectrophotometric assay for GAPDHS in 384-well format. The assay was stable over time and tolerant to DMSO. Whole plate validation experiments yielded Z' values >0.8 indicating a robust assay for HTS. Two compounds were identified and confirmed from a test screen of the Prestwick collection. This assay was used to screen a diverse chemical library and identified fourteen small molecules that modulated the activity of recombinant purified GAPDHS with confirmed IC50 values ranging from 1.8 to 42 µM. These compounds may provide useful scaffolds as molecular tools to probe the role of GAPDHS in sperm motility and long term to develop potent and selective GAPDHS inhibitors leading to novel contraceptive agents. PMID:21760877

  1. A H2 very high frequency capacitively coupled plasma inactivates glyceraldehyde 3-phosphate dehydrogenase(GapDH) more efficiently than UV photons and heat combined

    NASA Astrophysics Data System (ADS)

    Stapelmann, Katharina; Lackmann, Jan-Wilm; Buerger, Ines; Bandow, Julia Elisabeth; Awakowicz, Peter

    2014-02-01

    Plasma sterilization is a promising alternative to commonly used sterilization techniques, because the conventional methods suffer from certain limitations, e.g. incompatibility with heat-sensitive materials, or use of toxic agents. However, plasma-based sterilization mechanisms are not fully understood yet. A low-pressure very high frequency capacitively coupled plasma is used to investigate the impact of a hydrogen discharge on the protein glyceraldehyde 3-phosphate dehydrogenase (GapDH). GapDH is an enzyme of glycolysis. As a part of the central metabolism, it occurs in nearly all organisms from bacteria to humans. The plasma is investigated with absolutely calibrated optical emission spectroscopy in order to identify and to quantify plasma components that can contribute to enzyme inactivation. The contribution of UV photons and heat to GapDH inactivation is investigated separately, and neither seems to be a major factor. In order to investigate the mechanisms of GapDH inactivation by the hydrogen discharge, samples are investigated for etching, induction of amino acid backbone breaks, and chemical modifications. While neither etching nor strand breaks are observed, chemical modifications occur at different amino acid residues of GapDH. Deamidations of asparagines as well as methionine and cysteine oxidations are detected after VHF-CCP treatment. In particular, oxidation of the cysteine in the active centre is known to lead to GapDH inactivation.

  2. DNA vaccine encoding the moonlighting protein Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) leads to partial protection in a mouse model of human filariasis.

    PubMed

    Steisslinger, Vera; Korten, Simone; Brattig, Norbert W; Erttmann, Klaus D

    2015-10-26

    River blindness, caused by the filarial parasite Onchocerca volvulus, is a major socio-economic and public health problem in Sub-Saharan Africa. In January 2015, The Onchocerciasis Vaccine for Africa (TOVA) Initiative has been launched with the aim of providing new tools to complement mass drug administration (MDA) of ivermectin, thereby promoting elimination of onchocerciasis in Africa. In this context we here present Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) as a possible DNA vaccine candidate. We report that in a laboratory model for filariasis, immunization with Ov-GAPDH led to a significant reduction of adult worm load and microfilaraemia in BALB/c mice after challenge infection with the filarial parasite Litomosoides sigmodontis. Mice were either vaccinated with Ov-GAPDH.DNA plasmid (Ov-pGAPDH.DNA) alone or in combination with recombinantly expressed Ov-GAPDH protein (Ov-rGAPDH). During the following challenge infection of immunized and control mice with L. sigmodontis, those formulations which included the DNA plasmid, led to a significant reduction of adult worm loads (up to 57% median reduction) and microfilaraemia (up to 94% reduction) in immunized animals. In a further experiment, immunization with a mixture of four overlapping, synthetic Ov-GAPDH peptides (Ov-GAPDHpept), with alum as adjuvant, did not significantly reduce worm loads. Our results indicate that DNA vaccination with Ov-GAPDH has protective potential against filarial challenge infection in the mouse model. This suggests a transfer of the approach into the cattle Onchocerca ochengi model, where it is possible to investigate the effects of this vaccination in the context of a natural host-parasite relationship. PMID:26320419

  3. Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme.

    PubMed

    Polyakova, Oxana V; Roitel, Olivier; Asryants, Regina A; Poliakov, Alexei A; Branlant, Guy; Muronetz, Vladimir I

    2005-04-01

    We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH-groups modified by 5,5'-dithiobis(2-nitrobenzoate); (2) O-R-type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O-P-type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O-R-type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants K(d) of 0.4 and 0.9 muM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg-ATP. Chemically modified GAPDH and the O-R-type mutant dimers inhibited GroEL-assisted refolding of urea-denatured wild-type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O-R-dimers, the O-P-type mutant dimers neither bound nor affected GroEL-assisted refolding of the wild-type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity. PMID:15741339

  4. Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme

    PubMed Central

    Polyakova, Oxana V.; Roitel, Olivier; Asryants, Regina A.; Poliakov, Alexei A.; Branlant, Guy; Muronetz, Vladimir I.

    2005-01-01

    We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH-groups modified by 5,5′-dithiobis(2-nitrobenzoate); (2) O-R-type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O-P-type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O-R-type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants Kd of 0.4 and 0.9 μM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg-ATP. Chemically modified GAPDH and the O-R-type mutant dimers inhibited GroEL-assisted refolding of urea-denatured wild-type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O-R-dimers, the O-P-type mutant dimers neither bound nor affected GroEL-assisted refolding of the wild-type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity. PMID:15741339

  5. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Protein-Protein Interaction Inhibitor Reveals a Non-catalytic Role for GAPDH Oligomerization in Cell Death.

    PubMed

    Qvit, Nir; Joshi, Amit U; Cunningham, Anna D; Ferreira, Julio C B; Mochly-Rosen, Daria

    2016-06-24

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important glycolytic enzyme, has a non-catalytic (thus a non-canonical) role in inducing mitochondrial elimination under oxidative stress. We recently demonstrated that phosphorylation of GAPDH by δ protein kinase C (δPKC) inhibits this GAPDH-dependent mitochondrial elimination. δPKC phosphorylation of GAPDH correlates with increased cell injury following oxidative stress, suggesting that inhibiting GAPDH phosphorylation should decrease cell injury. Using rational design, we identified pseudo-GAPDH (ψGAPDH) peptide, an inhibitor of δPKC-mediated GAPDH phosphorylation that does not inhibit the phosphorylation of other δPKC substrates. Unexpectedly, ψGAPDH decreased mitochondrial elimination and increased cardiac damage in an animal model of heart attack. Either treatment with ψGAPDH or direct phosphorylation of GAPDH by δPKC decreased GAPDH tetramerization, which corresponded to reduced GAPDH glycolytic activity in vitro and ex vivo Taken together, our study identified the potential mechanism by which oxidative stress inhibits the protective GAPDH-mediated elimination of damaged mitochondria. Our study also identified a pharmacological tool, ψGAPDH peptide, with interesting properties. ψGAPDH peptide is an inhibitor of the interaction between δPKC and GAPDH and of the resulting phosphorylation of GAPDH by δPKC. ψGAPDH peptide is also an inhibitor of GAPDH oligomerization and thus an inhibitor of GAPDH glycolytic activity. Finally, we found that ψGAPDH peptide is an inhibitor of the elimination of damaged mitochondria. We discuss how this unique property of increasing cell damage following oxidative stress suggests a potential use for ψGAPDH peptide-based therapy. PMID:27129213

  6. Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase.

    PubMed

    Howard, Thomas P; Lloyd, Julie C; Raines, Christine A

    2011-07-01

    In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed.

  7. DNA vaccine encoding the moonlighting protein Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) leads to partial protection in a mouse model of human filariasis.

    PubMed

    Steisslinger, Vera; Korten, Simone; Brattig, Norbert W; Erttmann, Klaus D

    2015-10-26

    River blindness, caused by the filarial parasite Onchocerca volvulus, is a major socio-economic and public health problem in Sub-Saharan Africa. In January 2015, The Onchocerciasis Vaccine for Africa (TOVA) Initiative has been launched with the aim of providing new tools to complement mass drug administration (MDA) of ivermectin, thereby promoting elimination of onchocerciasis in Africa. In this context we here present Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) as a possible DNA vaccine candidate. We report that in a laboratory model for filariasis, immunization with Ov-GAPDH led to a significant reduction of adult worm load and microfilaraemia in BALB/c mice after challenge infection with the filarial parasite Litomosoides sigmodontis. Mice were either vaccinated with Ov-GAPDH.DNA plasmid (Ov-pGAPDH.DNA) alone or in combination with recombinantly expressed Ov-GAPDH protein (Ov-rGAPDH). During the following challenge infection of immunized and control mice with L. sigmodontis, those formulations which included the DNA plasmid, led to a significant reduction of adult worm loads (up to 57% median reduction) and microfilaraemia (up to 94% reduction) in immunized animals. In a further experiment, immunization with a mixture of four overlapping, synthetic Ov-GAPDH peptides (Ov-GAPDHpept), with alum as adjuvant, did not significantly reduce worm loads. Our results indicate that DNA vaccination with Ov-GAPDH has protective potential against filarial challenge infection in the mouse model. This suggests a transfer of the approach into the cattle Onchocerca ochengi model, where it is possible to investigate the effects of this vaccination in the context of a natural host-parasite relationship.

  8. Improvement of NADPH bioavailability in Escherichia coli by replacing NAD(+)-dependent glyceraldehyde-3-phosphate dehydrogenase GapA with NADP (+)-dependent GapB from Bacillus subtilis and addition of NAD kinase.

    PubMed

    Wang, Yipeng; San, Ka-Yiu; Bennett, George N

    2013-12-01

    Enzymatic synthesis of some industrially important compounds depends heavily on cofactor NADPH as the reducing agent. This is especially true in the synthesis of chiral compounds that are often used as pharmaceutical intermediates to generate the correct stereochemistry in bioactive products. The high cost and technical difficulty of cofactor regeneration often pose a challenge for such biocatalytic reactions. In this study, to increase NADPH bioavailability, the native NAD(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gapA gene in Escherichia coli was replaced with a NADP(+)-dependent gapB from Bacillus subtilis. To overcome the limitation of NADP(+) availability, E. coli NAD kinase, nadK was also coexpressed with gapB. The recombinant strains were then tested in three reporting systems: biosynthesis of lycopene, oxidation of cyclohexanone with cyclohexanone monooxygenase (CHMO), and an anaerobic system utilizing 2-haloacrylate reductase (CAA43). In all the reporting systems, replacing NAD(+)-dependent GapA activity with NADP(+)-dependent GapB activity increased the synthesis of NADPH-dependent compounds. The increase was more pronounced when NAD kinase was also overexpressed in the case of the one-step reaction catalyzed by CAA43 which approximately doubled the product yield. These results validate this novel approach to improve NADPH bioavailability in E. coli and suggest that the strategy can be applied in E. coli or other bacterium-based production of NADPH-dependent compounds.

  9. The length of the combined 3' untranslated region and poly(A) tail does not control rates of glyceraldehyde-3-phosphate dehydrogenase mRNA translation in three species of parasitic protists.

    PubMed

    ter Kuile, B H; Sallés, F J

    2000-06-01

    Experimental observations suggested that the length of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA 3' end has a role in regulating rates of translation in the parasitic protists Trypanosoma brucei, Leishmania donovani, and Trichomonas vaginalis. Using a PCR assay for poly(A) tail length, we measured the size of the RNA 3' end under different growth conditions in all three species. Our results showed that the combined 3' untranslated region and poly(A) tail of GAPDH mRNA do not vary with different rates of translation.

  10. Differential methylation of the gene encoding myo-inositol 3-phosphate synthase (Isyna1) in rat tissues

    PubMed Central

    Seelan, Ratnam S; Pisano, M Michele; Greene, Robert M; Casanova, Manuel F; Parthasarathy, Ranga N

    2011-01-01

    Aims Myo-inositol levels are frequently altered in several brain disorders. Myo-inositol 3-phosphate synthase, encoded by the Isyna1 gene, catalyzes the synthesis of myo-inositol in cells. Very little is known about the mechanisms regulating Isyna1 expression in brain and other tissues. In this study, we have examined the role of DNA methylation in regulating Isyna1 expression in rat tissues. Materials & methods Transfection analysis using in vitro methylated promoter constructs, Southern blot analysis of genomic DNA from various tissues digested with a methylation-sensitive enzyme and CpG methylation profiling of genomic DNA from different tissues were used to determine differential methylation of Isyna1 in tissues. Transfection analysis using plasmids harboring mutated CpG residues in the 5’-upstream region of Isyna1 was used to identify critical residues mediating promoter activity. Results The −700 bp to −500 bp region (region 1) of Isyna1 exhibited increased methylation in brain cortex compared with other tissues; it also exhibited sex-specific methylation differences between matched male and female brain cortices. Mutation analysis identified one CpG residue in region 1 necessary for promoter activity in neuronal cells. A tissue-specific differentially methylated region (T-DMR) was found to be localized between +450 bp and +650 bp (region 3). This DMR was comparatively highly methylated in spleen, moderately methylated in brain cortex and poorly methylated in testis, consistent with mRNA levels observed in these tissues. Conclusion Rat Isyna1 exhibits tissue-specific DNA methylation. Brain DNA was uniquely methylated in the 5’-upstream region and displayed gender specificity. A T-DMR was identified within the gene body of Isyna1. These findings suggest that Isyna1 is regulated, in part, by DNA methylation and that significant alterations in methylation patterns during development could have a major impact on inositol phosphate synthase expression in

  11. The Aldehyde Dehydrogenase Gene Superfamily Resource Center

    PubMed Central

    2009-01-01

    The website http://www.aldh.org is a publicly available database for nomenclature and functional and molecular sequence information for members of the aldehyde dehydrogenase (ALDH) gene superfamily for animals, plants, fungi and bacteria. The site has organised gene-specific records. It provides synopses of ALDH gene records, marries trivial terms to correct nomenclature and links global accession identifiers with source data. Server-side alignment software characterises the integrity of each sequence relative to the latest genomic assembly and provides identifier-specific detail reports, including a graphical presentation of the transcript's exon - intron structure, its size, coding sequence, genomic strand and locus. Also included are a summary of substrates, inhibitors and enzyme kinetics. The site provides reference lists and is designed to facilitate data mining by interested investigators. PMID:20038501

  12. The FAD-dependent glycerol-3-phosphate dehydrogenase of Giardia duodenalis: an unconventional enzyme that interacts with the g14-3-3 and it is a target of the antitumoral compound NBDHEX

    PubMed Central

    Lalle, Marco; Camerini, Serena; Cecchetti, Serena; Finelli, Renata; Sferra, Gabriella; Müller, Joachim; Ricci, Giorgio; Pozio, Edoardo

    2015-01-01

    The flagellated protozoan Giardia duodenalis is a worldwide parasite causing giardiasis, an acute and chronic diarrheal disease. Metabolism in G. duodenalis has a limited complexity thus making metabolic enzymes ideal targets for drug development. However, only few metabolic pathways (i.e., carbohydrates) have been described so far. Recently, the parasite homolog of the mitochondrial-like glycerol-3-phosphate dehydrogenase (gG3PD) has been identified among the interactors of the g14-3-3 protein. G3PD is involved in glycolysis, electron transport, glycerophospholipids metabolism, and hyperosmotic stress response, and is emerging as promising target in tumor treatment. In this work, we demonstrate that gG3PD is a functional flavoenzyme able to convert glycerol-3-phosphate into dihydroxyacetone phosphate and that its activity and the intracellular glycerol level increase during encystation. Taking advantage of co-immunoprecipitation assays and deletion mutants, we provide evidence that gG3PD and g14-3-3 interact at the trophozoite stage, the intracellular localization of gG3PD is stage dependent and it partially co-localizes with mitosomes during cyst development. Finally, we demonstrate that the gG3PD activity is affected by the antitumoral compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, that results more effective in vitro at killing G. duodenalis trophozoites than the reference drug metronidazole. Overall, our results highlight the involvement of gG3PD in processes crucial for the parasite survival thus proposing this enzyme as target for novel antigiardial interventions. PMID:26082764

  13. Mapping of two ugp genes coding for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia coli.

    PubMed Central

    Schweizer, H; Grussenmeyer, T; Boos, W

    1982-01-01

    Two genes, ugpA and ugpB, coding for a binding protein-dependent sn-glycerol-3-phosphate transport system, were mapped at 75.3 min on the Escherichia coli chromosome. A Tn10 insertion in ugpA resulted in loss of transport activity but still allowed the synthesis of the sn-glycerol-3-phosphate-binding protein. This Tn10 insertion was found to be linked by P1 transduction to pit, aroB, malA, asd, and livH with 2.5, 2.8, 25, 63.5, and 83% cotransduction frequency. An insertion of Mud (Ampr lac) in ugpB resulted in the loss of the binding protein. ugpB is closely linked to ugpA. It is either the structural gene for the binding protein or located proximal to it. The analysis of the crosses allowed the ordering of the markers in the clockwise direction as follows: aroB, malA, asd, ugpA, ugpB, livH, pit. Images PMID:6281238

  14. Transgenic tobacco simultaneously overexpressing glyphosate N-acetyltransferase and 5-enolpyruvylshikimate-3-phosphate synthase are more resistant to glyphosate than those containing one gene.

    PubMed

    Liu, Yunjun; Cao, Gaoyi; Chen, Rongrong; Zhang, Shengxue; Ren, Yuan; Lu, Wei; Wang, Jianhua; Wang, Guoying

    2015-08-01

    5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) and glyphosate N-acetyltransferase (GAT) can detoxify glyphosate by alleviating the suppression of shikimate pathway. In this study, we obtained transgenic tobacco plants overexpressing AM79 aroA, GAT, and both of them, respectively, to evaluate whether overexpression of both genes could confer transgenic plants with higher glyphosate resistance. The transgenic plants harboring GAT or AM79 aroA, respectively, showed good glyphosate resistance. As expected, the hybrid plants containing both GAT and AM79 aroA exhibited improved glyphosate resistance than the transgenic plants overexpressing only a single gene. When grown on media with high concentration of glyphosate, seedlings containing a single gene were severely inhibited, whereas plants expressing both genes were affected less. When transgenic plants grown in the greenhouse were sprayed with glyphosate, less damage was observed for the plants containing both genes. Metabolomics analysis showed that transgenic plants containing two genes could maintain the metabolism balance better than those containing one gene after glyphosate treatment. Glyphosate treatment did not lead to a huge increase of shikimate contents of tobacco leaves in transgenic plants overexpressing two genes, whereas significant increase of shikimate contents in transgenic plants containing only a single gene was observed. These results demonstrated that pyramiding both aroA and GAT in transgenic plants can enhance glyphosate resistance, and this strategy can be used for the development of transgenic glyphosate-resistant crops.

  15. Arabidopsis AtGPAT1, a Member of the Membrane-Bound Glycerol-3-Phosphate Acyltransferase Gene Family, Is Essential for Tapetum Differentiation and Male Fertility

    PubMed Central

    Zheng, Zhifu; Xia, Qun; Dauk, Melanie; Shen, Wenyun; Selvaraj, Gopalan; Zou, Jitao

    2003-01-01

    Membrane-bound glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) mediates the initial step of glycerolipid biosynthesis in the extraplastidic compartments of plant cells. Here, we report the molecular characterization of a novel GPAT gene family from Arabidopsis, designated AtGPAT. The corresponding polypeptides possess transmembrane domains and GPAT activity when expressed heterologously in a yeast lipid mutant. The functional significance of one isoform, AtGPAT1, is the focus of the present study. Disruption of the AtGPAT1 gene causes a massive pollen development arrest, and subsequent introduction of the gene into the mutant plant rescues the phenotype, illustrating a pivotal role for AtGPAT1 in pollen development. Microscopic examinations revealed that the gene lesion results in a perturbed degeneration of the tapetum, which is associated with altered endoplasmic reticulum profiles and reduced secretion. In addition to the sporophytic effect, AtGPAT1 also exerts a gametophytic effect on pollen performance, as the competitive ability of a pollen grain to pollinate is dependent on the presence of an AtGPAT1 gene. Deficiency in AtGPAT1 correlates with several fatty acid composition changes in flower tissues and seeds. Unexpectedly, however, a loss of AtGPAT1 causes no significant change in seed oil content. PMID:12897259

  16. Molecular cloning of gluconobacter oxydans DSM 2003 xylitol dehydrogenase gene

    PubMed Central

    Sadeghi, H. Mir Mohammad; Ahmadi, R.; Aghaabdollahian, S.; Mofid, M.R.; Ghaemi, Y.; Abedi, D.

    2011-01-01

    Due to the widespread applications of xylitol dehydrogenase, an enzyme used for the production of xylitol, the present study was designed for the cloning of xylitol dehydrogenase gene from Glcunobacter oxydans DSM 2003. After extraction of genomic DNA from this bacterium, xylitol dehydrogenase gene was replicated using polymerase chain reaction (PCR). The amplified product was entered into pTZ57R cloning vector by T/A cloning method and transformation was performed by heat shocking of the E. coli XL1-blue competent cells. Following plasmid preparation, the cloned gene was digested out and ligated into the expression vector pET-22b(+). Electrophoresis of PCR product showed a 789 bp band. Recombinant plasmid (rpTZ57R) was then constructed. This plasmid was double digested with XhoI and EcoRI resulting in 800 bp and 2900 bp bands. The obtained insert was ligated into pET-22b(+) vector and its orientation was confirmed with XhoI and BamHI restriction enzymes. In conclusion, in the present study the recombinant expression vector containing xylitol dehydrogenase gene has been constructed and can be used for the production of this enzyme in high quantities. PMID:22110522

  17. Three homologous genes encoding sn-glycerol-3-phosphate acyltransferase 4 exhibit different expression patterns and functional divergence in Brassica napus.

    PubMed

    Chen, Xue; Truksa, Martin; Snyder, Crystal L; El-Mezawy, Aliaa; Shah, Saleh; Weselake, Randall J

    2011-02-01

    Brassica napus is an allotetraploid (AACC) formed from the fusion of two diploid progenitors, Brassica rapa (AA) and Brassica oleracea (CC). Polyploidy and genome-wide rearrangement during the evolution process have resulted in genes that are present as multiple homologs in the B. napus genome. In this study, three B. napus homologous genes encoding endoplasmic reticulum-bound sn-glycerol-3-phosphate acyltransferase 4 (GPAT4) were identified and characterized. Although the three GPAT4 homologs share a high sequence similarity, they exhibit different expression patterns and altered epigenetic features. Heterologous expression in yeast further revealed that the three BnGPAT4 homologs encoded functional GPAT enzymes but with different levels of polypeptide accumulation. Complementation of the Arabidopsis (Arabidopsis thaliana) gpat4 gpat8 double mutant line with individual BnGPAT4 homologs suggested their physiological roles in cuticle formation. Analysis of gpat4 RNA interference lines of B. napus revealed that the BnGPAT4 deficiency resulted in reduced cutin content and altered stomatal structures in leaves. Our results revealed that the BnGPAT4 homologs have evolved into functionally divergent forms and play important roles in cutin synthesis and stomatal development.

  18. Cloning and sequencing of the gene encoding the 72-kilodalton dehydrogenase subunit of alcohol dehydrogenase from Acetobacter aceti.

    PubMed

    Inoue, T; Sunagawa, M; Mori, A; Imai, C; Fukuda, M; Takagi, M; Yano, K

    1989-06-01

    A genomic library of Acetobacter aceti DNA was constructed by using a broad-host-range cosmid vector. Complementation of a spontaneous alcohol dehydrogenase-deficient mutant resulted in the isolation of a plasmid designated pAA701. Subcloning and deletion analysis of pAA701 limited the region that complemented the deficiency in alcohol dehydrogenase activity of the mutant. The nucleotide sequence of this region was determined and showed that this region contained the full structural gene for the 72-kilodalton dehydrogenase subunit of the alcohol dehydrogenase enzyme complex. The predicted amino acid sequence of the gene showed homology with sequences of methanol dehydrogenase structural genes of Paracoccus denitrificans and Methylobacterium organophilum.

  19. Divergent properties and phylogeny of cyanobacterial 5-enol-pyruvyl-shikimate-3-phosphate synthases: evidence for horizontal gene transfer in the Nostocales.

    PubMed

    Forlani, Giuseppe; Bertazzini, Michele; Barillaro, Donatella; Rippka, Rosmarie

    2015-01-01

    As it represents the target of the successful herbicide glyphosate, great attention has been paid to the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase. However, inconsistent results have been reported concerning the sensitivity of the enzyme from cyanobacteria, and consequent inhibitory effects on cyanobacterial growth. The properties of EPSP synthase were investigated in a set of 42 strains representative of the large morphological diversity of these prokaryotes. Publicly available protein sequences were analyzed, and related to enzymatic features. In most cases, the native protein showed an unusual homodimeric composition and a general sensitivity to micromolar doses of glyphosate. By contrast, eight out of 15 Nostocales strains were found to possess a monomeric EPSP synthase, whose activity was inhibited only at concentrations exceeding 1 mM. Sequence analysis showed that these two forms are only distantly related, the latter clustering separately in a clade composed of diverse bacterial phyla. The results are consistent with the occurrence of a horizontal gene transfer event involving an evolutionarily distant organism. Moreover, data suggest that the existence of class I (glyphosate-sensitive) and class II (glyphosate-tolerant) EPSP synthases representing two distinct phylogenetic clades is an oversimplification because of the limited number of analyzed samples. PMID:25229999

  20. Recommended nomenclature for the vertebrate alcohol dehydrogenase gene family.

    PubMed

    Duester, G; Farrés, J; Felder, M R; Holmes, R S; Höög, J O; Parés, X; Plapp, B V; Yin, S J; Jörnvall, H

    1999-08-01

    The alcohol dehydrogenase (ADH) gene family encodes enzymes that metabolize a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Studies on 19 vertebrate animals have identified ADH orthologs across several species, and this has now led to questions of how best to name ADH proteins and genes. Seven distinct classes of vertebrate ADH encoded by non-orthologous genes have been defined based upon sequence homology as well as unique catalytic properties or gene expression patterns. Each class of vertebrate ADH shares <70% sequence identity with other classes of ADH in the same species. Classes may be further divided into multiple closely related isoenzymes sharing >80% sequence identity such as the case for class I ADH where humans have three class I ADH genes, horses have two, and mice have only one. Presented here is a nomenclature that uses the widely accepted vertebrate ADH class system as its basis. It follows the guidelines of human and mouse gene nomenclature committees, which recommend coordinating names across species boundaries and eliminating Roman numerals and Greek symbols. We recommend that enzyme subunits be referred to by the symbol "ADH" (alcohol dehydrogenase) followed by an Arabic number denoting the class; i.e. ADH1 for class I ADH. For genes we recommend the italicized root symbol "ADH" for human and "Adh" for mouse, followed by the appropriate Arabic number for the class; i.e. ADH1 or Adh1 for class I ADH genes. For organisms where multiple species-specific isoenzymes exist within a class, we recommend adding a capital letter after the Arabic number; i.e. ADH1A, ADH1B, and ADH1C for human alpha, beta, and gamma class I ADHs, respectively. This nomenclature will accommodate newly discovered members of the vertebrate ADH family, and will facilitate functional and evolutionary studies. PMID:10424757

  1. Crystal structure of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the ESKAPE pathogen Acinetobacter baumannii.

    PubMed

    Sutton, Kristin A; Breen, Jennifer; Russo, Thomas A; Schultz, L Wayne; Umland, Timothy C

    2016-03-01

    The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the sixth step of the seven-step shikimate pathway. Chorismate, the product of the pathway, is a precursor for the biosynthesis of aromatic amino acids, siderophores and metabolites such as folate, ubiquinone and vitamin K. The shikimate pathway is present in bacteria, fungi, algae, plants and apicomplexan parasites, but is absent in humans. The EPSP synthase enzyme produces 5-enolpyruvylshikimate 3-phosphate and phosphate from phosphoenolpyruvate and shikimate 3-phosphate via a transferase reaction, and is the target of the herbicide glyphosate. The Acinetobacter baumannii gene encoding EPSP synthase, aroA, has previously been demonstrated to be essential during host infection for the growth and survival of this clinically important drug-resistant ESKAPE pathogen. Prephenate dehydrogenase is also encoded by the bifunctional A. baumannii aroA gene, but its activity is dependent upon EPSP synthase since it operates downstream of the shikimate pathway. As part of an effort to evaluate new antimicrobial targets, recombinant A. baumannii EPSP (AbEPSP) synthase, comprising residues Ala301-Gln756 of the aroA gene product, was overexpressed in Escherichia coli, purified and crystallized. The crystal structure, determined to 2.37 Å resolution, is described in the context of a potential antimicrobial target and in comparison to EPSP synthases that are resistant or sensitive to the herbicide glyphosate. PMID:26919521

  2. [Sorbitol-6-Phosphate Dehydrogenase Gene Polymorhism in Malus Mill. (Rosaceae)].

    PubMed

    Boris, K V; Kudryavtsev, A M; Kochieva, E Z

    2015-11-01

    The sorbitol-6-phosphate dehydrogenase gene (S6PDH) sequences of six representatives of the genus Malus, which belong to five different taxonomic sections, were examined for the first time. The exon-intron structure and polymorphism of the nucleotide and amino acid sequences of these genes was characterized. The intraspecific polymorphism of the S6PDH gene was assessed for the first time in 40 Russian and foreign apple (Malus domestica) cultivars. It was demonstrated that the interspecific polymorphism level of the S6PDH coding sequences in the studied. representatives of the genus Malus was 4%, and the intraspecific polymorphism level of M. domestica cultivars was very low, constituting 0.96%. PMID:26845854

  3. [Sorbitol-6-Phosphate Dehydrogenase Gene Polymorhism in Malus Mill. (Rosaceae)].

    PubMed

    Boris, K V; Kudryavtsev, A M; Kochieva, E Z

    2015-11-01

    The sorbitol-6-phosphate dehydrogenase gene (S6PDH) sequences of six representatives of the genus Malus, which belong to five different taxonomic sections, were examined for the first time. The exon-intron structure and polymorphism of the nucleotide and amino acid sequences of these genes was characterized. The intraspecific polymorphism of the S6PDH gene was assessed for the first time in 40 Russian and foreign apple (Malus domestica) cultivars. It was demonstrated that the interspecific polymorphism level of the S6PDH coding sequences in the studied. representatives of the genus Malus was 4%, and the intraspecific polymorphism level of M. domestica cultivars was very low, constituting 0.96%.

  4. Intron-dependent evolution of chicken glyceraldehyde phosphate dehydrogenase gene.

    PubMed

    Stone, E M; Rothblum, K N; Schwartz, R J

    The function of introns in the evolution of genes can be explained in at least two ways: either introns appeared late in evolution and therefore could not have participated in the construction of primordial genes, or RNA splicing and introns existed in the earliest organisms but were lost during the evolution of the modern prokaryotes. The latter alternative allows the possibility of intron participation in the formation of primordial genes before the divergence of modern prokaryotes and eukaryotes. Blake suggested that evidence for intron-facilitated evolution of a gene might be found by comparing the borders of functional protein domains with the placement of introns. We therefore examined glyceraldehyde phosphate dehydrogenase (GAPDH), a glycolytic enzyme, because it is the first protein for which the following data are available: X-ray crystallographic studies demonstrating structurally independent protein 'domains' which were highly conserved during the divergence of prokaryotes and eukaryotes; and a study of genomic organization which mapped introns in the gene. Sequencing of the chicken GAPDH gene revealed 11 introns. We report here that sites of three of the introns (IV, VI and XI) correspond closely with the borders of the NAD-binding, catalytic and helical tail domains of the enzyme, supporting the hypothesis that introns did have a role in the evolution of primitive genes. In addition, other biochemical and structural data were used to construct a model of the intron-mediated assembly of the GAPDH gene that explains the existence of 10 introns.

  5. Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis

    DOEpatents

    Ingram, Lonnie O.; Conway, Tyrrell

    1992-01-01

    The alcohol dehydrogenase II gene from Zymomonas mobilis has been cloned and sequenced. This gene can be expressed at high levels in other organisms to produce acetaldehyde or to convert acetaldehyde to ethanol.

  6. Cloning and Polymorphisms of Yak Lactate Dehydrogenase b Gene

    PubMed Central

    Wang, Guosheng; Zhao, Xingbo; Zhong, Juming; Cao, Meng; He, Qinghua; Liu, Zhengxin; Lin, Yaqiu; Xu, Yaou; Zheng, Yucai

    2013-01-01

    The main objective of this work was to study the unique polymorphisms of the lactate dehydrogenase-1 (LDH1) gene in yak (Bos grunniens). Native polyacrylamide gel electrophoresis revealed three phenotypes of LDH1 (a tetramer of H subunit) in yak heart and longissimus muscle extracts. The corresponding gene, ldhb, encoding H subunits of three LDH1 phenotypes was obtained by RT-PCR. A total of six nucleotide differences were detected in yak ldhb compared with that of cattle, of which five mutations cause amino acid substitutions. Sequence analysis shows that the G896A and C689A, mutations of ldhb gene, result in alterations of differently charged amino acids, and create the three phenotypes (F, M, and S) of yak LDH1. Molecular modeling of the H subunit of LDH indicates that the substituted amino acids are not located within NAD+ or substrate binding sites. PCR-RFLP examination of G896A mutation demonstrated that most LDH1-F samples are actually heterozygote at this site. These results help to elucidate the molecular basis and genetic characteristic of the three unique LDH1 phenotypes in yak. PMID:23739677

  7. Isolation, sequence, and characterization of the Cercospora nicotianae phytoene dehydrogenase gene.

    PubMed Central

    Ehrenshaft, M; Daub, M E

    1994-01-01

    We have cloned and sequenced the Cercospora nicotianae gene for the carotenoid biosynthetic enzyme phytoene dehydrogenase. Analysis of the derived amino acid sequence revealed it has greater than 50% identity with its counterpart in Neurospora crassa and approximately 30% identity with prokaryotic phytoene dehydrogenases and is related, but more distantly, to phytoene dehydrogenases from plants and cyanobacteria. Our analysis confirms that phytoene dehydrogenase proteins fall into two groups: those from plants and cyanobacteria and those from eukaryotic and noncyanobacter prokaryotic microbes. Southern analysis indicated that the C. nicotianae phytoene dehydrogenase gene is present in a single copy. Extraction of beta-carotene, the sole carotenoid accumulated by C. nicotianae, showed that both light- and dark-grown cultures synthesize carotenoids, but higher levels accumulate in the light. Northern (RNA) analysis of poly(A)+ RNA, however, showed no differential accumulation of phytoene dehydrogenase mRNA between light- and dark-grown fungal cultures. Images PMID:8085820

  8. Isolation, sequence, and characterization of the Cercospora nicotianae phytoene dehydrogenase gene.

    PubMed

    Ehrenshaft, M; Daub, M E

    1994-08-01

    We have cloned and sequenced the Cercospora nicotianae gene for the carotenoid biosynthetic enzyme phytoene dehydrogenase. Analysis of the derived amino acid sequence revealed it has greater than 50% identity with its counterpart in Neurospora crassa and approximately 30% identity with prokaryotic phytoene dehydrogenases and is related, but more distantly, to phytoene dehydrogenases from plants and cyanobacteria. Our analysis confirms that phytoene dehydrogenase proteins fall into two groups: those from plants and cyanobacteria and those from eukaryotic and noncyanobacter prokaryotic microbes. Southern analysis indicated that the C. nicotianae phytoene dehydrogenase gene is present in a single copy. Extraction of beta-carotene, the sole carotenoid accumulated by C. nicotianae, showed that both light- and dark-grown cultures synthesize carotenoids, but higher levels accumulate in the light. Northern (RNA) analysis of poly(A)+ RNA, however, showed no differential accumulation of phytoene dehydrogenase mRNA between light- and dark-grown fungal cultures.

  9. Increased IMP dehydrogenase gene expression in solid tumor tissues and tumor cell lines

    SciTech Connect

    Collart, F.R.; Chubb, C.B.; Mirkin, B.L.; Huberman, E.

    1992-07-10

    IMP dehydrogenase, a regulatory enzyme of guanine nucleotide biosynthesis, may play a role in cell proliferation and malignancy. To assess this possibility, we examined IMP dehydrogenase expression in a series of human solid tumor tissues and tumor cell lines in comparison with their normal counterparts. Increased IMP dehydrogenase gene expression was observed in brain tumors relative to normal brain tissue and in sarcoma cells relative to normal fibroblasts. Similarly, in several B- and T-lymphoid leukemia cell lines, elevated levels of IMP dehydrogenase mRNA and cellular enzyme were observed in comparison with the levels in peripheral blood lymphocytes. These results are consistent with an association between increased IMP dehydrogenase expression and either enhanced cell proliferation or malignant transformation.

  10. [Characterization of aldehyde dehydrogenase gene fragment from mung bean Vigna radiata using the polymerase chain reaction].

    PubMed

    Ponomarev, A G; Bubiakina, V V; Tatarinova, T D; Zelenin, S M

    1998-01-01

    Two degenerate oligonucleotide sequence primers and polymerase chain reactions on total DNA have been utilized to clone on 651--bp gene fragment coding the central part of amino acid sequence of an earlier unknown aldehyde dehydrogenase (ALDH) from mung bean. The deduced partial amino acid sequence for this aldehyde dehydrogenase shows about 65% sequence identity to ALDHs of Vibrio cholerae Rhodococcus sp., Alcaligenes eutrophus and about 45% sequence identity to mammalian ALDHs 1 and 2, ALDHs of Aspergillus niger and A, nidulans, the betain aldehyde dehydrogenase from spinach. Alignment of the mung bean aldehyde dehydrogenase partial amino acid sequence with the sequence of 16 NAD(P)(+)-dependent aldehyde dehydrogenases has demonstrated that all strictly conserved amino acid residues and all three conservative regions are identical. PMID:9778740

  11. Simultaneous substitution of Gly96 to Ala and Ala183 to Thr in 5-enolpyruvylshikimate-3-phosphate synthase gene of E. coli (k12) and transformation of rapeseed (Brassica napus L.) in order to make tolerance to glyphosate.

    PubMed

    Kahrizi, Danial; Salmanian, Ali Hatef; Afshari, Afsoon; Moieni, Ahmad; Mousavi, Amir

    2007-01-01

    Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This is a key enzyme in the aromatic amino acid biosynthesis pathway of microorganisms and plants. The manipulation of bacterial EPSPS gene in order to reduce its affinity for glyphosate, followed by its transfer to plants is one of the most effective approaches for the production of glyphosate-tolerant plants. In this study, we chose to focus on amino acid residues glycine96 and alanine183 of the E. coli (k12) EPSPS enzyme. These two amino acids are important residues for glyphosate binding. We used site directed mutagenesis (SDM) to induce point mutations in the E. coli EPSPS gene, in order to convert glycine96 to alanine (Gly96Ala) and alanine183 to threonine (Ala183Thr). After confirming the mutation by sequencing, the altered EPSPS gene was transferred to rapeseed (Brassica napus L.) via Agrobacterium-mediated transformation. The transformed explants were screened in shoot induction medium containing 25 mg L-1 kanamycin. Glyphosate tolerance was assayed in putative transgenic plants. Statistical analysis of data showed that there was a significant difference between the transgenic and control plants. It was observed that transgenic plants were resistant to glyphosate at a concentration of 10 mM whereas the non-transformed control plants were unable to survive 1 mM glyphosate. The presence and copy numbers of the transgene were confirmed with PCR and Southern blotting analysis, respectively.

  12. A second gene for acyl-(acyl-carrier-protein): glycerol-3-phosphate acyltransferase in squash, Cucurbita moschata cv. Shirogikuza(*), codes for an oleate-selective isozyme: molecular cloning and protein purification studies.

    PubMed

    Nishida, I; Sugiura, M; Enju, A; Nakamura, M

    2000-12-01

    A new isogene for acyl-(acyl-carrier-protein):glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) in squash has been cloned and the gene product was identified as oleate-selective GPAT. Using PCR primers that could hybridise with exons for a previously cloned squash GPAT, we obtained two PCR products of different size: one coded for a previously cloned squash GPAT corresponding to non-selective isoforms AT2 and AT3, and the other for a new isozyme, probably the oleate-selective isoform AT1. Full-length amino acid sequences of respective isozymes were deduced from the nucleotide sequences of genomic genes and cDNAs, which were cloned by a series of PCR-based methods. Thus, we designated the new gene CmATS1;1 and the other one CmATS1;2. Genome blot analysis revealed that the squash genome contained the two isogenes at non-allelic loci. AT1-active fractions were partially purified, and three polypeptide bands were identified as being AT1 polypeptides, which exhibited relative molecular masses of 39.5-40.5 kDa, pI values of 6.75-7.15, and oleate selectivity over palmitate. Partial amino-terminal sequences obtained from two of these bands verified that the new isogene codes for AT1 polypeptides.

  13. Identification and molecular characterization of the aco genes encoding the Pelobacter carbinolicus acetoin dehydrogenase enzyme system.

    PubMed Central

    Oppermann, F B; Steinbüchel, A

    1994-01-01

    Use of oligonucleotide probes, which were deduced from the N-terminal sequences of the purified enzyme components, identified the structural genes for the alpha and beta subunits of E1 (acetoin:2,6-dichlorophenolindophenol oxidoreductase), E2 (dihydrolipoamide acetyltransferase), and E3 (dihydrolipoamide dehydrogenase) of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system, which were designated acoA, acoB, acoC, and acoL, respectively. The nucleotide sequences of acoA (979 bp), acoB (1,014 bp), acoC (1,353 bp), and acoL (1,413 bp) as well as of acoS (933 bp), which encodes a protein with an M(r) of 34,421 exhibiting 64.7% amino acid identity to the Escherichia coli lipA gene product, were determined. These genes are clustered on a 6.1-kbp region. Heterologous expression of acoA, acoB, acoC, acoL, and acoS in E. coli was demonstrated. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 34,854), E1 beta (M(r), 36,184), E2 (M(r), 47,281), and E3 (M(r), 49,394) exhibited striking similarities to the amino acid sequences of the components of the Alcaligenes eutrophus acetoin-cleaving system. Homologies of up to 48.7% amino acid identity to the primary structures of the enzyme components of various 2-oxo acid dehydrogenase complexes also were found. In addition, the respective genes of the 2-oxo acid dehydrogenase complexes and of the acetoin dehydrogenase enzyme system were organized very similarly, indicating a close relationship of the P. carbinolicus acetoin dehydrogenase enzyme system to 2-oxo acid dehydrogenase complexes. Images PMID:8110297

  14. Aldehyde Dehydrogenase Gene Superfamily in Populus: Organization and Expression Divergence between Paralogous Gene Pairs

    PubMed Central

    Tian, Feng-Xia; Zang, Jian-Lei; Wang, Tan; Xie, Yu-Li; Zhang, Jin; Hu, Jian-Jun

    2015-01-01

    Aldehyde dehydrogenases (ALDHs) constitute a superfamily of NAD(P)+-dependent enzymes that catalyze the irreversible oxidation of a wide range of reactive aldehydes to their corresponding nontoxic carboxylic acids. ALDHs have been studied in many organisms from bacteria to mammals; however, no systematic analyses incorporating genome organization, gene structure, expression profiles, and cis-acting elements have been conducted in the model tree species Populus trichocarpa thus far. In this study, a comprehensive analysis of the Populus ALDH gene superfamily was performed. A total of 26 Populus ALDH genes were found to be distributed across 12 chromosomes. Genomic organization analysis indicated that purifying selection may have played a pivotal role in the retention and maintenance of PtALDH gene families. The exon-intron organizations of PtALDHs were highly conserved within the same family, suggesting that the members of the same family also may have conserved functionalities. Microarray data and qRT-PCR analysis indicated that most PtALDHs had distinct tissue-specific expression patterns. The specificity of cis-acting elements in the promoter regions of the PtALDHs and the divergence of expression patterns between nine paralogous PtALDH gene pairs suggested that gene duplications may have freed the duplicate genes from the functional constraints. The expression levels of some ALDHs were up- or down-regulated by various abiotic stresses, implying that the products of these genes may be involved in the adaptation of Populus to abiotic stresses. Overall, the data obtained from our investigation contribute to a better understanding of the complexity of the Populus ALDH gene superfamily and provide insights into the function and evolution of ALDH gene families in vascular plants. PMID:25909656

  15. Aldehyde Dehydrogenase Gene Superfamily in Populus: Organization and Expression Divergence between Paralogous Gene Pairs.

    PubMed

    Tian, Feng-Xia; Zang, Jian-Lei; Wang, Tan; Xie, Yu-Li; Zhang, Jin; Hu, Jian-Jun

    2015-01-01

    Aldehyde dehydrogenases (ALDHs) constitute a superfamily of NAD(P)+-dependent enzymes that catalyze the irreversible oxidation of a wide range of reactive aldehydes to their corresponding nontoxic carboxylic acids. ALDHs have been studied in many organisms from bacteria to mammals; however, no systematic analyses incorporating genome organization, gene structure, expression profiles, and cis-acting elements have been conducted in the model tree species Populus trichocarpa thus far. In this study, a comprehensive analysis of the Populus ALDH gene superfamily was performed. A total of 26 Populus ALDH genes were found to be distributed across 12 chromosomes. Genomic organization analysis indicated that purifying selection may have played a pivotal role in the retention and maintenance of PtALDH gene families. The exon-intron organizations of PtALDHs were highly conserved within the same family, suggesting that the members of the same family also may have conserved functionalities. Microarray data and qRT-PCR analysis indicated that most PtALDHs had distinct tissue-specific expression patterns. The specificity of cis-acting elements in the promoter regions of the PtALDHs and the divergence of expression patterns between nine paralogous PtALDH gene pairs suggested that gene duplications may have freed the duplicate genes from the functional constraints. The expression levels of some ALDHs were up- or down-regulated by various abiotic stresses, implying that the products of these genes may be involved in the adaptation of Populus to abiotic stresses. Overall, the data obtained from our investigation contribute to a better understanding of the complexity of the Populus ALDH gene superfamily and provide insights into the function and evolution of ALDH gene families in vascular plants.

  16. Functional Replacement of the Escherichia coli d-(−)-Lactate Dehydrogenase Gene (ldhA) with the l-(+)-Lactate Dehydrogenase Gene (ldhL) from Pediococcus acidilactici†

    PubMed Central

    Zhou, Shengde; Shanmugam, K. T.; Ingram, L. O.

    2003-01-01

    The microbial production of l-(+)-lactic acid is rapidly expanding to allow increased production of polylactic acid (PLA), a renewable, biodegradable plastic. The physical properties of PLA can be tailored for specific applications by controlling the ratio of l-(+) and d-(−) isomers. For most uses of PLA, the l-(+) isomer is more abundant. As an approach to reduce costs associated with biocatalysis (complex nutrients, antibiotics, aeration, product purification, and waste disposal), a recombinant derivative of Escherichia coli W3110 was developed that contains five chromosomal deletions (focA-pflB frdBC adhE ackA ldhA). This strain was constructed from a d-(−)-lactic acid-producing strain, SZ63 (focA-pflB frdBC adhE ackA), by replacing part of the chromosomal ldhA coding region with Pediococcus acidilactici ldhL encoding an l-lactate dehydrogenase. Although the initial strain (SZ79) grew and fermented poorly, a mutant (SZ85) was readily isolated by selecting for improved growth. SZ85 exhibited a 30-fold increase in l-lactate dehydrogenase activity in comparison to SZ79, functionally replacing the native d-lactate dehydrogenase activity. Sequencing revealed mutations in the upstream, coding, and terminator regions of ldhL in SZ85, which are presumed to be responsible for increased l-lactate dehydrogenase activity. SZ85 produced l-lactic acid in M9 mineral salts medium containing glucose or xylose with a yield of 93 to 95%, a purity of 98% (based on total fermentation products), and an optical purity greater than 99%. Unlike other recombinant biocatalysts for l-lactic acid, SZ85 remained prototrophic and is devoid of plasmids and antibiotic resistance genes. PMID:12676706

  17. Metabolic engineering of enhanced glycerol-3-phosphate synthesis to increase lipid production in Synechocystis sp. PCC 6803.

    PubMed

    Wang, Xi; Xiong, Xiaochao; Sa, Na; Roje, Sanja; Chen, Shulin

    2016-07-01

    With the growing attention to global warming and energy sustainability, biosynthesis of lipids by photosynthetic microorganisms has attracted more interest for the production of renewable transportation fuels. Recently, the cyanobacterium Synechocystis sp. PCC 6803 has been widely used for biofuel production through metabolic engineering because of its efficient photosynthesis and well-developed genetic tools. In lipid biosynthesis, glycerol-3-phosphate (G3P) is a key node for both CO2 fixation and lipid metabolism in cyanobacteria. However, few studies have explored the use of G3P synthesis to improve photosynthetic lipid production. In this study, metabolic engineering combined with flux balance analysis (FBA) was conducted to reveal the effect of G3P synthesis on lipid production. Heterologous genes that encoded glycerol-3-phosphate dehydrogenase (GPD) and diacylglycerol acyltransferase (DGAT) were engineered into Synechocystis sp. PCC 6803 to enhance G3P supply and lipid production. The resultant recombinant Synechocystis produced higher levels of lipids without a significant reduction in cell growth. Compared with the wild-type strain, lipid content and productivity of the engineered cyanobacteria increased by up to 36 and 31 %, respectively, under autotrophic conditions. Lipid production under mixotrophic conditions of the engineered cyanobacteria was also investigated. This work demonstrated that enhanced G3P synthesis was an important factor in photosynthetic lipid production and that introducing heterologous GPD and DGAT genes was an effective strategy to increase lipid production in Synechocystis sp. PCC 6803. PMID:27154348

  18. Metabolic engineering of enhanced glycerol-3-phosphate synthesis to increase lipid production in Synechocystis sp. PCC 6803.

    PubMed

    Wang, Xi; Xiong, Xiaochao; Sa, Na; Roje, Sanja; Chen, Shulin

    2016-07-01

    With the growing attention to global warming and energy sustainability, biosynthesis of lipids by photosynthetic microorganisms has attracted more interest for the production of renewable transportation fuels. Recently, the cyanobacterium Synechocystis sp. PCC 6803 has been widely used for biofuel production through metabolic engineering because of its efficient photosynthesis and well-developed genetic tools. In lipid biosynthesis, glycerol-3-phosphate (G3P) is a key node for both CO2 fixation and lipid metabolism in cyanobacteria. However, few studies have explored the use of G3P synthesis to improve photosynthetic lipid production. In this study, metabolic engineering combined with flux balance analysis (FBA) was conducted to reveal the effect of G3P synthesis on lipid production. Heterologous genes that encoded glycerol-3-phosphate dehydrogenase (GPD) and diacylglycerol acyltransferase (DGAT) were engineered into Synechocystis sp. PCC 6803 to enhance G3P supply and lipid production. The resultant recombinant Synechocystis produced higher levels of lipids without a significant reduction in cell growth. Compared with the wild-type strain, lipid content and productivity of the engineered cyanobacteria increased by up to 36 and 31 %, respectively, under autotrophic conditions. Lipid production under mixotrophic conditions of the engineered cyanobacteria was also investigated. This work demonstrated that enhanced G3P synthesis was an important factor in photosynthetic lipid production and that introducing heterologous GPD and DGAT genes was an effective strategy to increase lipid production in Synechocystis sp. PCC 6803.

  19. Gene Expression Variation in Duplicate Lactate dehydrogenase Genes: Do Ecological Species Show Distinct Responses?

    PubMed Central

    Cristescu, Melania E.; Demiri, Bora; Altshuler, Ianina; Crease, Teresa J.

    2014-01-01

    Lactate dehydrogenase (LDH) has been shown to play an important role in adaptation of several aquatic species to different habitats. The genomes of Daphnia pulex, a pond species, and Daphnia pulicaria, a lake inhabitant, encode two L-LDH enzymes, LDHA and LDHB. We estimated relative levels of Ldh gene expression in these two closely related species and their hybrids in four environmental settings, each characterized by one of two temperatures (10°C or 20°C), and one of two concentrations of dissolved oxygen (DO; 6.5–7 mg/l or 2–3 mg/l). We found that levels of LdhA expression were 4 to 48 times higher than LdhB expression (p<0.005) in all three groups (the two parental species and hybrids). Moreover, levels of LdhB expression differed significantly (p<0.05) between D. pulex and D. pulicaria, but neither species differed from the hybrid. Consistently higher expression of LdhA relative to LdhB in both species and the hybrid suggests that the two isozymes could be performing different functions. No significant differences in levels of gene expression were observed among the four combinations of temperature and dissolved oxygen (p>0.1). Given that Daphnia dwell in environments characterized by fluctuating conditions with long periods of low dissolved oxygen concentration, we suggest that these species could employ regulated metabolic depression to survive in such environments. PMID:25080082

  20. The ferredoxin-dependent conversion of glyceraldehyde-3-phosphate in the hyperthermophilic archaeon Pyrococcus furiosus represents a novel site of glycolytic regulation.

    PubMed

    van der Oost, J; Schut, G; Kengen, S W; Hagen, W R; Thomm, M; de Vos, W M

    1998-10-23

    The fermentative conversion of glucose in anaerobic hyperthermophilic Archaea is a variant of the classical Embden-Meyerhof pathway found in Bacteria and Eukarya. A major difference of the archaeal glycolytic pathway concerns the conversion of glyceraldehyde-3-phosphate. In the hyperthermophilic archaeon Pyrococcus furiosus, this reaction is catalyzed by an unique enzyme, glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR). Here, we report the isolation, characterization, and transcriptional analysis of the GAPOR-encoding gene. GAPOR is related to a family of ferredoxin-dependent tungsten enzymes in (hyper)thermophilic Archaea and, in addition, to a hypothetical protein in Escherichia coli. Electron paramagnetic resonance analysis of the purified P. furiosus GAPOR protein confirms the anticipated involvement of tungsten in catalysis. During glycolysis in P. furiosus, GAPOR gene expression is induced, whereas the activity of glyceraldehyde-3-phosphate dehydrogenase is repressed. It is discussed that this unprecedented unidirectional reaction couple in the pyrococcal glycolysis and gluconeogenesis gives rise to a novel site of glycolytic regulation that might be widespread among Archaea.

  1. Alcoholism and alcohol drinking habits predicted from alcohol dehydrogenase genes.

    PubMed

    Tolstrup, Janne Schurmann; Nordestgaard, Børge Grønne; Rasmussen, Søren; Tybjaerg-Hansen, Anne; Grønbaek, Morten

    2008-06-01

    Alcohol drinking habits and alcoholism are partly genetically determined. Alcohol is degraded primarily by alcohol dehydrogenase (ADH) wherein genetic variation that affects the rate of alcohol degradation is found in ADH1B and ADH1C. It is biologically plausible that these variations may be associated with alcohol drinking habits and alcoholism. By genotyping 9080 white men and women from the general population, we found that men and women with ADH1B slow vs fast alcohol degradation drank more alcohol and had a higher risk of everyday drinking, heavy drinking, excessive drinking and of alcoholism. For example, the weekly alcohol intake was 9.8 drinks (95% confidence interval (CI): 9.1-11) among men with the ADH1B.1/1 genotype compared to 7.5 drinks (95% CI: 6.4-8.7) among men with the ADH1B.1/2 genotype, and the odds ratio (OR) for heavy drinking was 3.1 (95% CI: 1.7-5.7) among men with the ADH1B.1/1 genotype compared to men with the ADH1B.1/2 genotype. Furthermore, individuals with ADH1C slow vs fast alcohol degradation had a higher risk of heavy and excessive drinking. For example, the OR for heavy drinking was 1.4 (95% CI: 1.1-1.8) among men with the ADH1C.1/2 genotype and 1.4 (95% CI: 1.0-1.9) among men with the ADH1B.2/2 genotype, compared with men with the ADH1C.1/1 genotype. Results for ADH1B and ADH1C genotypes among men and women were similar. Finally, because slow ADH1B alcohol degradation is found in more than 90% of the white population compared to less than 10% of East Asians, the population attributable risk of heavy drinking and alcoholism by ADH1B.1/1 genotype was 67 and 62% among the white population compared with 9 and 24% among the East Asian population.

  2. Cloning of the rat pyruvate dehydrogenase kinase 4 gene promoter: activation of pyruvate dehydrogenase kinase 4 by the peroxisome proliferator-activated receptor gamma coactivator.

    PubMed

    Ma, Ke; Zhang, Yi; Elam, Marshall B; Cook, George A; Park, Edwards A

    2005-08-19

    The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the metabolism of glucose to acetyl-CoA. Phosphorylation of pyruvate dehydrogenase by the pyruvate dehydrogenase kinases (PDK) inhibits pyruvate dehydrogenase complex activity. There are four PDK isoforms, and expression of PDK4 and PDK2 genes is elevated in starvation and diabetes, allowing glucose to be conserved while fatty acid oxidation is increased. In these studies we have investigated the transcriptional mechanisms by which the expression of the PDK4 gene is increased. The peroxisome proliferator-activated receptor gamma coactivator (PGC-1alpha) stimulates the expression of genes involved in hepatic gluconeogenesis and mitochondrial fatty acid oxidation. We have found that PGC-1alpha will induce the expression of both the PDK2 and PDK4 genes in primary rat hepatocytes and ventricular myocytes. We cloned the promoter for the rat PDK4 gene. Hepatic nuclear factor 4 (HNF4), which activates many genes in the liver, will induce PDK4 expression. Although HNF4 and PGC-1alpha interact to stimulate several genes encoding gluconeogenic enzymes, the induction of PDK4 does not involve interactions of PGC-1alpha with HNF4. Using the chromatin immunoprecipitation assay, we have demonstrated that HNF4 and PGC-1alpha are associated with the PDK4 gene in vivo. Our data suggest that by inducing PDK genes PGC-1alpha will direct pyruvate away from metabolism into acetyl-CoA and toward the formation of oxaloacetate and into the gluconeogenic pathway. PMID:15967803

  3. Problem-Based Test: The Effect of Fibroblast Growth Factor on Gene Expression

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

    This paper shows the results of an experiment in which the effects of fibroblast growth factor (FGF), actinomycin D (Act D; an inhibitor of transcription), and cycloheximide (CHX; an inhibitor of translation) were studied on the expression of two genes: a gene called "Fnk" and the gene coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).…

  4. Gene algD coding for GDPmannose dehydrogenase is transcriptionally activated in mucoid Pseudomonas aeruginosa.

    PubMed Central

    Deretic, V; Gill, J F; Chakrabarty, A M

    1987-01-01

    Transcriptional regulation of alginate biosynthesis by Pseudomonas aeruginosa was studied. A DNA region complementing the alg-5 mutation within the alginate gene cluster was found by RNA-DNA dot blot and Northern hybridization to be transcriptionally activated in mucoid P. aeruginosa. This region was subcloned as a 3.2-kilobase BglII-ClaI DNA fragment on the broad-host-range controlled transcription vector pMMB24, and gene products were analyzed by expression from the tac promoter. A 48-kilodalton polypeptide was detected in extracts of P. aeruginosa and 35S-labeled Escherichia coli maxicells. By using the same expression system, GDPmannose dehydrogenase activity was detected in both P. aeruginosa and E. coli. Thus, gene algD coding for this enzyme was found to be present in the transcriptionally active DNA area. Insertion of the xylE gene within the BglII-ClaI fragment disrupted the induction of the 48-kilodalton polypeptide, GDPmannose dehydrogenase activity, and alg-5 complementing ability. With the algD-xylE transcription fusion, activation of algD gene expression was shown to occur in mucoid P. aeruginosa of different origins. In addition, regulation of the algD promoter activity was demonstrated to be mediated by a diffusible factor. Images PMID:3025179

  5. Deletion of the Bacillus subtilis isocitrate dehydrogenase gene causes a block at stage I of sporulation.

    PubMed Central

    Jin, S; Levin, P A; Matsuno, K; Grossman, A D; Sonenshein, A L

    1997-01-01

    A Bacillus subtilis mutant with a deletion of citC, the gene encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, had a greatly reduced ability to sporulate. Analysis of expression of lacZ fusions to various sporulation gene promoters revealed that in the citC mutant development is probably blocked between stage 0 and stage II. That is, genes expressed very early in sporulation, under the direct control of the Spo0A transcription factor, were induced normally in the citC mutant. However, genes expressed after asymmetric septation (stage II) in wild-type cells were not induced in the citC mutant. Analysis of cell morphology by thin-section electron microscopy and immunofluorescence microscopy showed that the mutant formed axial chromosomal filaments and accumulated rings of FtsZ protein at potential polar division sites but failed to form asymmetric division septa, indicating that sporulation is blocked at stage I. The growth and sporulation defects of the B. subtilis citC mutant were fully overcome by introduction and expression of the Escherichia coli icd gene, encoding an isocitrate dehydrogenase similar to the enzyme from B. subtilis. PMID:9244258

  6. Genomic organization and expression of the human fatty aldehyde dehydrogenase gene (FALDH)

    SciTech Connect

    Rogers, G.R.; Markova, N.G.; Compton, J.G.

    1997-01-15

    Mutations in the fatty aldehyde dehydrogenase (FALDH) gene cause Sjoegren-Larsson syndrome (SLS) - a disease characterized by mental retardation, spasticity, and congenital ichthyosis. To facilitate mutation analysis in SLS and to study the pathogenesis of FALDH deficiency, we have determined the structural organization and characterized expression of the FALDH (proposed designation ALDH10) gene. The gene consists of 10 exons spanning about 30.5 kb. A TATA-less promoter is associated with the major transcription initiation site found to be 258 hp upstream of the ATG codon. The G4C-rich sequences surrounding the transcription initiation site encompassed regulatory elements that interacted with proteins in HeLa nuclear extracts and were able to promote transcription in vitro. FALDH is widely expressed as three transcripts of 2, 3.8, and 4.0 kb, which originate from multiple polyadenylation signals in the 3{prime} UTR. An alternatively spliced mRNA was detected that contains an extra exon and encodes an enzyme that is likely to have altered membrane-binding properties. The FALDH gene lies only 50-85 kb from ALDH3, an aldehyde dehydrogenase gene that has homologous sequence and intron/exon structure. 25 refs., 4 figs., 1 tab.

  7. Molecular phylogeny and evolution of alcohol dehydrogenase (Adh) genes in legumes

    PubMed Central

    Fukuda, Tatsuya; Yokoyama, Jun; Nakamura, Toru; Song, In-Ja; Ito, Takuro; Ochiai, Toshinori; Kanno, Akira; Kameya, Toshiaki; Maki, Masayuki

    2005-01-01

    Background Nuclear genes determine the vast range of phenotypes that are responsible for the adaptive abilities of organisms in nature. Nevertheless, the evolutionary processes that generate the structures and functions of nuclear genes are only now be coming understood. The aim of our study is to isolate the alcohol dehydrogenase (Adh) genes in two distantly related legumes, and use these sequences to examine the molecular evolutionary history of this nuclear gene. Results We isolated the expressed Adh genes from two species of legumes, Sophora flavescens Ait. and Wisteria floribunda DC., by a RT-PCR based approach and found a new Adh locus in addition to homologues of the Adh genes found previously in legumes. To examine the evolution of these genes, we compared the species and gene trees and found gene duplication of the Adh loci in the legumes occurred as an ancient event. Conclusion This is the first report revealing that some legume species have at least two Adh gene loci belonging to separate clades. Phylogenetic analyses suggest that these genes resulted from relatively ancient duplication events. PMID:15836788

  8. The Natural History of Class I Primate Alcohol Dehydrogenases Includes Gene Duplication, Gene Loss, and Gene Conversion

    PubMed Central

    Carrigan, Matthew A.; Uryasev, Oleg; Davis, Ross P.; Zhai, LanMin; Hurley, Thomas D.; Benner, Steven A.

    2012-01-01

    Background Gene duplication is a source of molecular innovation throughout evolution. However, even with massive amounts of genome sequence data, correlating gene duplication with speciation and other events in natural history can be difficult. This is especially true in its most interesting cases, where rapid and multiple duplications are likely to reflect adaptation to rapidly changing environments and life styles. This may be so for Class I of alcohol dehydrogenases (ADH1s), where multiple duplications occurred in primate lineages in Old and New World monkeys (OWMs and NWMs) and hominoids. Methodology/Principal Findings To build a preferred model for the natural history of ADH1s, we determined the sequences of nine new ADH1 genes, finding for the first time multiple paralogs in various prosimians (lemurs, strepsirhines). Database mining then identified novel ADH1 paralogs in both macaque (an OWM) and marmoset (a NWM). These were used with the previously identified human paralogs to resolve controversies relating to dates of duplication and gene conversion in the ADH1 family. Central to these controversies are differences in the topologies of trees generated from exonic (coding) sequences and intronic sequences. Conclusions/Significance We provide evidence that gene conversions are the primary source of difference, using molecular clock dating of duplications and analyses of microinsertions and deletions (micro-indels). The tree topology inferred from intron sequences appear to more correctly represent the natural history of ADH1s, with the ADH1 paralogs in platyrrhines (NWMs) and catarrhines (OWMs and hominoids) having arisen by duplications shortly predating the divergence of OWMs and NWMs. We also conclude that paralogs in lemurs arose independently. Finally, we identify errors in database interpretation as the source of controversies concerning gene conversion. These analyses provide a model for the natural history of ADH1s that posits four ADH1 paralogs in

  9. Cloning and molecular evolution of the aldehyde dehydrogenase 2 gene (Aldh2) in bats (Chiroptera).

    PubMed

    Chen, Yao; Shen, Bin; Zhang, Junpeng; Jones, Gareth; He, Guimei

    2013-02-01

    Old World fruit bats (Pteropodidae) and New World fruit bats (Phyllostomidae) ingest significant quantities of ethanol while foraging. Mitochondrial aldehyde dehydrogenase (ALDH2, encoded by the Aldh2 gene) plays an important role in ethanol metabolism. To test whether the Aldh2 gene has undergone adaptive evolution in frugivorous and nectarivorous bats in relation to ethanol elimination, we sequenced part of the coding region of the gene (1,143 bp, ~73 % coverage) in 14 bat species, including three Old World fruit bats and two New World fruit bats. Our results showed that the Aldh2 coding sequences are highly conserved across all bat species we examined, and no evidence of positive selection was detected in the ancestral branches leading to Old World fruit bats and New World fruit bats. Further research is needed to determine whether other genes involved in ethanol metabolism have been the targets of positive selection in frugivorous and nectarivorous bats.

  10. Molecular cloning and characterization of human pyruvate dehydrogenase. beta. subunit gene

    SciTech Connect

    Koike, Kichiko; Urata, Yoshishige; Koike, Masahiko )

    1990-08-01

    A genomic clone encompassing the entire gene for the human pyruvate dehydrogenase {beta} subunit (PDH{beta}) has been isolated by screening a leukocyte genomic library with a nick-translated human foreskin fibroblast PDH{beta} cDNA probe. The 18-kilobase clone was characterized by restriction enzyme analysis, extensive DNA sequencing, and primer-extension analysis. The PDH{beta} structural gene is composed of 10 exons and 9 introns. All intron-exon splice junctions follow the GT/AG rule. The Alu family was found in introns 2 and 8. The 5{prime} flanking region of the PDH{beta} gene contains a CAAT consensus promoter sequence but no TATA sequence. Primer-extension analysis indicated the PDH{beta} gene transcription start site is an adenine residue located 132 bases upstream from the initiation codon in exon 1.

  11. Chromosomal localization and structure of the human type II IMP dehydrogenase gene

    SciTech Connect

    Glesne, D.; Huberman, E. |; Collart, F.; Varkony, T.; Drabkin, H.

    1994-05-01

    We determined the chromosomal localization and structure of the gene encoding human type II inosine 5{prime}-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205), an enzyme associated with cellular proliferation, malignant transformation, and differentiation. Using polymerase chain reaction (PCR) primers specific for type II IMPDH, we screened a panel of human-Chinese hamster cell somatic hybrids and a separate deletion panel of chromosome 3 hybrids and localized the gene to 3p21.1{yields}p24.2. Two overlapping yeast artificial chromosome clones containing the full gene for type II IMPDH were isolated and a physical map of 117 kb of human genomic DNA in this region of chromosome 3 was constructed. The gene for type II IMPDH was localized and oriented on this map and found to span no more than 12.5 kb.

  12. Histamine dehydrogenase from Rhizobium sp.: gene cloning, expression in Escherichia coli, characterization and application to histamine determination.

    PubMed

    Bakke, Mikio; Sato, Tsuneo; Ichikawa, Keiichi; Nishimura, Ikuko

    2005-09-29

    The gene encoding histamine dehydrogenase in Rhizobium sp. 4--9 has been cloned and overexpressed in Escherichia coli. The coding region of the gene was 2,079 bp and encoded a protein of 693 amino acids with a calculated molecular mass of 76,732 Da. This histamine dehydrogenase was related to histamine dehydrogenase from Nocardioides simplex (54.5% identical), trimethylamine dehydrogenase from Methylophilus methylotrophus (39.3% identical) and dimethylamine dehydrogenase from Hyphomicrobium X (38.1% identical), which have a covalent 6-S-cysteinyl flavin mononucleotide and a [4Fe--4S] cluster as redox cofactors. Sequence alignment and a UV-visible absorption spectrum supported the presence of these cofactors in this histamine dehydrogenase. The investigation of the enzymatic properties suggested that this enzyme exhibited the most excellent substrate specificity toward histamine among all amine oxidases or dehydrogenases found to date. The recombinant enzyme was able to be used for the colorimetric determination of histamine, which gave a linear calibration curve and identical data with conventional methods. PMID:15964650

  13. Genome-Wide Identification and Functional Classification of Tomato (Solanum lycopersicum) Aldehyde Dehydrogenase (ALDH) Gene Superfamily

    PubMed Central

    Lopez-Valverde, Francisco J.; Robles-Bolivar, Paula; Lima-Cabello, Elena; Gachomo, Emma W.; Kotchoni, Simeon O.

    2016-01-01

    Aldehyde dehydrogenases (ALDHs) is a protein superfamily that catalyzes the oxidation of aldehyde molecules into their corresponding non-toxic carboxylic acids, and responding to different environmental stresses, offering promising genetic approaches for improving plant adaptation. The aim of the current study is the functional analysis for systematic identification of S. lycopersicum ALDH gene superfamily. We performed genome-based ALDH genes identification and functional classification, phylogenetic relationship, structure and catalytic domains analysis, and microarray based gene expression. Twenty nine unique tomato ALDH sequences encoding 11 ALDH families were identified, including a unique member of the family 19 ALDH. Phylogenetic analysis revealed 13 groups, with a conserved relationship among ALDH families. Functional structure analysis of ALDH2 showed a catalytic mechanism involving Cys-Glu couple. However, the analysis of ALDH3 showed no functional gene duplication or potential neo-functionalities. Gene expression analysis reveals that particular ALDH genes might respond to wounding stress increasing the expression as ALDH2B7. Overall, this study reveals the complexity of S. lycopersicum ALDH gene superfamily and offers new insights into the structure-functional features and evolution of ALDH gene families in vascular plants. The functional characterization of ALDHs is valuable and promoting molecular breeding in tomato for the improvement of stress tolerance and signaling. PMID:27755582

  14. Estrogen-related receptors stimulate pyruvate dehydrogenase kinase isoform 4 gene expression.

    PubMed

    Zhang, Yi; Ma, Ke; Sadana, Prabodh; Chowdhury, Farhana; Gaillard, Stephanie; Wang, Fang; McDonnell, Donald P; Unterman, Terry G; Elam, Marshall B; Park, Edwards A

    2006-12-29

    The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the oxidation of glucose to acetyl-CoA. Phosphorylation of PDC by the pyruvate dehydrogenase kinases (PDK2 and PDK4) inhibits PDC activity. Expression of the PDK genes is elevated in diabetes, leading to the decreased oxidation of pyruvate to acetyl-CoA. In these studies we have investigated the transcriptional regulation of the PDK4 gene by the estrogen-related receptors (ERRalpha and ERRgamma). The ERRs are orphan nuclear receptors whose physiological roles include the induction of fatty acid oxidation in heart and muscle. Previously, we found that the peroxisome proliferator-activated receptor gamma coactivator (PGC-1alpha) stimulates the expression of PDK4. Here we report that ERRalpha and ERRgamma stimulate the PDK4 gene in hepatoma cells, suggesting a novel role for ERRs in controlling pyruvate metabolism. In addition, both ERR isoforms recruit PGC-1alpha to the PDK4 promoter. Insulin, which decreases the expression of the PDK4 gene, inhibits the induction of PDK4 by ERRalpha and ERRgamma. The forkhead transcription factor (FoxO1) binds the PDK4 gene and contributes to the induction of PDK4 by ERRs and PGC-1alpha. Insulin suppresses PDK4 expression in part through the dissociation of FoxO1 and PGC-1alpha from the PDK4 promoter. Our data demonstrate a key role for the ERRs in the induction of hepatic PDK4 gene expression. PMID:17079227

  15. Mutations in the genes encoding 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase interact to cause cortisone reductase deficiency.

    PubMed

    Draper, Nicole; Walker, Elizabeth A; Bujalska, Iwona J; Tomlinson, Jeremy W; Chalder, Susan M; Arlt, Wiebke; Lavery, Gareth G; Bedendo, Oliver; Ray, David W; Laing, Ian; Malunowicz, Ewa; White, Perrin C; Hewison, Martin; Mason, Philip J; Connell, John M; Shackleton, Cedric H L; Stewart, Paul M

    2003-08-01

    In cortisone reductase deficiency (CRD), activation of cortisone to cortisol does not occur, resulting in adrenocorticotropin-mediated androgen excess and a phenotype resembling polycystic ovary syndrome (PCOS; refs. 1,2). This suggests a defect in the gene HSD11B1 encoding 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), a primary regulator of tissue-specific glucocorticoid bioavailability. We identified intronic mutations in HSD11B1 that resulted in reduced gene transcription in three individuals with CRD. In vivo, 11beta-HSD1 catalyzes the reduction of cortisone to cortisol whereas purified enzyme acts as a dehydrogenase converting cortisol to cortisone. Oxo-reductase activity can be regained using a NADPH-regeneration system and the cytosolic enzyme glucose-6-phosphate dehydrogenase. But the catalytic domain of 11beta-HSD1 faces into the lumen of the endoplasmic reticulum (ER; ref. 6). We hypothesized that endolumenal hexose-6-phosphate dehydrogenase (H6PDH) regenerates NADPH in the ER, thereby influencing directionality of 11beta-HSD1 activity. Mutations in exon 5 of H6PD in individuals with CRD attenuated or abolished H6PDH activity. These individuals have mutations in both HSD11B1 and H6PD in a triallelic digenic model of inheritance, resulting in low 11beta-HSD1 expression and ER NADPH generation with loss of 11beta-HSD1 oxo-reductase activity. CRD defines a new ER-specific redox potential and establishes H6PDH as a potential factor in the pathogenesis of PCOS. PMID:12858176

  16. Three alcohol dehydrogenase genes and one acetyl-CoA synthetase gene are responsible for ethanol utilization in Yarrowia lipolytica.

    PubMed

    Gatter, Michael; Ottlik, Stephanie; Kövesi, Zsolt; Bauer, Benjamin; Matthäus, Falk; Barth, Gerold

    2016-10-01

    The non-conventional yeast Yarrowia lipolytica is able to utilize a wide range of different substrates like glucose, glycerol, ethanol, acetate, proteins and various hydrophobic molecules. Although most metabolic pathways for the utilization of these substrates have been clarified by now, it was not clear whether ethanol is oxidized by alcohol dehydrogenases or by an alternative oxidation system inside the cell. In order to detect the genes that are required for ethanol utilization in Y. lipolytica, eight alcohol dehydrogenase (ADH) genes and one alcohol oxidase gene (FAO1) have been identified and respective deletion strains were tested for their ability to metabolize ethanol. As a result of this, we found that the availability of ADH1, ADH2 or ADH3 is required for ethanol utilization in Y. lipolytica. A strain with deletions in all three genes is lacking the ability to utilize ethanol as sole carbon source. Although Adh2p showed by far the highest enzyme activity in an in vitro assay, the availability of any of the three genes was sufficient to enable a decent growth. In addition to ADH1, ADH2 and ADH3, an acetyl-CoA synthetase encoding gene (ACS1) was found to be essential for ethanol utilization. As Y. lipolytica is a non-fermenting yeast, it is neither able to grow under anaerobic conditions nor to produce ethanol. To investigate whether Y. lipolytica may produce ethanol, the key genes of alcoholic fermentation in S. cerevisiae, ScADH1 and ScPDC1, were overexpressed in an ADH and an ACS1 deletion strain. However, instead of producing ethanol, the respective strains regained the ability to use ethanol as single carbon source and were still not able to grow under anaerobic conditions. PMID:27486067

  17. Three alcohol dehydrogenase genes and one acetyl-CoA synthetase gene are responsible for ethanol utilization in Yarrowia lipolytica.

    PubMed

    Gatter, Michael; Ottlik, Stephanie; Kövesi, Zsolt; Bauer, Benjamin; Matthäus, Falk; Barth, Gerold

    2016-10-01

    The non-conventional yeast Yarrowia lipolytica is able to utilize a wide range of different substrates like glucose, glycerol, ethanol, acetate, proteins and various hydrophobic molecules. Although most metabolic pathways for the utilization of these substrates have been clarified by now, it was not clear whether ethanol is oxidized by alcohol dehydrogenases or by an alternative oxidation system inside the cell. In order to detect the genes that are required for ethanol utilization in Y. lipolytica, eight alcohol dehydrogenase (ADH) genes and one alcohol oxidase gene (FAO1) have been identified and respective deletion strains were tested for their ability to metabolize ethanol. As a result of this, we found that the availability of ADH1, ADH2 or ADH3 is required for ethanol utilization in Y. lipolytica. A strain with deletions in all three genes is lacking the ability to utilize ethanol as sole carbon source. Although Adh2p showed by far the highest enzyme activity in an in vitro assay, the availability of any of the three genes was sufficient to enable a decent growth. In addition to ADH1, ADH2 and ADH3, an acetyl-CoA synthetase encoding gene (ACS1) was found to be essential for ethanol utilization. As Y. lipolytica is a non-fermenting yeast, it is neither able to grow under anaerobic conditions nor to produce ethanol. To investigate whether Y. lipolytica may produce ethanol, the key genes of alcoholic fermentation in S. cerevisiae, ScADH1 and ScPDC1, were overexpressed in an ADH and an ACS1 deletion strain. However, instead of producing ethanol, the respective strains regained the ability to use ethanol as single carbon source and were still not able to grow under anaerobic conditions.

  18. Gene cloning, expression, and characterization of a novel acetaldehyde dehydrogenase from Issatchenkia terricola strain XJ-2.

    PubMed

    Yao, Zhengying; Zhang, Chong; Lu, Fengxia; Bie, Xiaomei; Lu, Zhaoxin

    2012-03-01

    Acetaldehyde is a known mutagen and carcinogen. Active aldehyde dehydrogenase (ALDH) represents an important mechanism for acetaldehyde detoxification. A yeast strain XJ-2 isolated from grape samples was found to produce acetaldehyde dehydrogenase with a high activity of 2.28 U/mg and identified as Issatchenkia terricola. The enzyme activity was validated by oxidizing acetaldehyde to acetate with NAD(+) as coenzyme based on the headspace gas chromatography analysis. A novel acetaldehyde dehydrogenase gene (ist-ALD) was cloned by combining SiteFinding-PCR and self-formed adaptor PCR. The ist-ALD gene comprised an open reading frame of 1,578 bp and encoded a protein of 525 amino acids. The predicted protein of ist-ALD showed the highest identity (73%) to ALDH from Pichia angusta. The ist-ALD gene was expressed in Escherichia coli, and the gene product (ist-ALDH) presented a productivity of 442.3 U/mL cells. The purified ist-ALDH was a homotetramer of 232 kDa consisting of 57 kDa-subunit according to the SDS-PAGE and native PAGE analysis. Ist-ALDH exhibited the optimal activity at pH 9.0 and 40°C, respectively. The activity of ist-ALDH was enhanced by K(+), NH4(+), dithiothreitol, and 2-mercaptoethanol but strongly inhibited by Ag(+), Hg(2+), Cu(2+), and phenylmethyl sulfonylfluoride. In the presence of NAD(+), ist-ALDH could oxidize many aliphatic, aromatic, and heterocyclic aldehydes, preferably acetaldehyde. Kinetic study revealed that ist-ALDH had a k (cat) value of 27.71/s and a k (cat)/K (m) value of 26.80 × 10(3)/(mol s) on acetaldehyde, demonstrating ist-ALDH, a catalytically active enzyme by comparing with other ALDHs. These studies indicated that ist-ALDH was a potential enzymatic product for acetaldehyde detoxification. PMID:21858493

  19. Isolation and characterization of full-length putative alcohol dehydrogenase genes from polygonum minus

    NASA Astrophysics Data System (ADS)

    Hamid, Nur Athirah Abd; Ismail, Ismanizan

    2013-11-01

    Polygonum minus, locally named as Kesum is an aromatic herb which is high in secondary metabolite content. Alcohol dehydrogenase is an important enzyme that catalyzes the reversible oxidation of alcohol and aldehyde with the presence of NAD(P)(H) as co-factor. The main focus of this research is to identify the gene of ADH. The total RNA was extracted from leaves of P. minus which was treated with 150 μM Jasmonic acid. Full-length cDNA sequence of ADH was isolated via rapid amplification cDNA end (RACE). Subsequently, in silico analysis was conducted on the full-length cDNA sequence and PCR was done on genomic DNA to determine the exon and intron organization. Two sequences of ADH, designated as PmADH1 and PmADH2 were successfully isolated. Both sequences have ORF of 801 bp which encode 266 aa residues. Nucleotide sequence comparison of PmADH1 and PmADH2 indicated that both sequences are highly similar at the ORF region but divergent in the 3' untranslated regions (UTR). The amino acid is differ at the 107 residue; PmADH1 contains Gly (G) residue while PmADH2 contains Cys (C) residue. The intron-exon organization pattern of both sequences are also same, with 3 introns and 4 exons. Based on in silico analysis, both sequences contain "classical" short chain alcohol dehydrogenases/reductases ((c) SDRs) conserved domain. The results suggest that both sequences are the members of short chain alcohol dehydrogenase family.

  20. Cloning, characterization, and regulation of the human type II IMP dehydrogenase gene

    SciTech Connect

    Glesne, D.A.; Huberman, E. |

    1997-01-01

    Human type II inosine 5{prime}-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) is the rate-limiting enzyme in de novo guanine nucleotide biosynthesis. Regulated IMPDH activity is associated with cellular proliferation, transformation, and differentiation. The authors cloned and sequenced the entire gene for type II IMPDH and here provide details regarding the organization of the gene and the characterization of its promoter. The gene spans approximately 5 kb and is disrupted by 12 introns. The transcriptional start sites were determined by S1 nuclease mapping to be somewhat heterogeneous but predominated at 102 and 85 nucleotides from the translational initiation codon. Through the use of heterologous gene constructs and transient transfection assays, a minimal promoter from {minus}206 to {minus}85 was defined. This promoter is TATA-less and contains several transcription factor motifs including four potential Sp 1 binding sites. The minimal promoter is GC-rich (69%) and resembles a CpG island. Through the use of gel mobility shift assays, nuclear proteins were shown to specifically interact with this minimal promoter. Stable transfectants were used to demonstrate that the down-regulation of IMPDH gene expression in response to reduced cellular proliferation occurs by a transcriptional mechanism.

  1. Potato tuber cytokinin oxidase/dehydrogenase genes: biochemical properties, activity, and expression during tuber dormancy progression.

    PubMed

    Suttle, Jeffrey C; Huckle, Linda L; Lu, Shunwen; Knauber, Donna C

    2014-03-15

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [(3)H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression.

  2. Potato tuber cytokinin oxidase/dehydrogenase genes: biochemical properties, activity, and expression during tuber dormancy progression.

    PubMed

    Suttle, Jeffrey C; Huckle, Linda L; Lu, Shunwen; Knauber, Donna C

    2014-03-15

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [(3)H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression. PMID:24594397

  3. An amino acid substitution in the pyruvate dehydrogenase E1{alpha} gene, affecting mitochondrial import of the precursor protein

    SciTech Connect

    Takakubo, F.; Thorburn, D.R.; Dahl, H.H.M.

    1995-10-01

    A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1{alpha} deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1{alpha}immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1{alpha} targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1{alpha} sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease. 58 refs., 5 figs., 1 tab.

  4. Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics.

    PubMed

    Brocker, Chad; Vasiliou, Melpomene; Carpenter, Sarah; Carpenter, Christopher; Zhang, Yucheng; Wang, Xiping; Kotchoni, Simeon O; Wood, Andrew J; Kirch, Hans-Hubert; Kopečný, David; Nebert, Daniel W; Vasiliou, Vasilis

    2013-01-01

    In recent years, there has been a significant increase in the number of completely sequenced plant genomes. The comparison of fully sequenced genomes allows for identification of new gene family members, as well as comprehensive analysis of gene family evolution. The aldehyde dehydrogenase (ALDH) gene superfamily comprises a group of enzymes involved in the NAD(+)- or NADP(+)-dependent conversion of various aldehydes to their corresponding carboxylic acids. ALDH enzymes are involved in processing many aldehydes that serve as biogenic intermediates in a wide range of metabolic pathways. In addition, many of these enzymes function as 'aldehyde scavengers' by removing reactive aldehydes generated during the oxidative degradation of lipid membranes, also known as lipid peroxidation. Plants and animals share many ALDH families, and many genes are highly conserved between these two evolutionarily distinct groups. Conversely, both plants and animals also contain unique ALDH genes and families. Herein we carried out genome-wide identification of ALDH genes in a number of plant species-including Arabidopsis thaliana (thale crest), Chlamydomonas reinhardtii (unicellular algae), Oryza sativa (rice), Physcomitrella patens (moss), Vitis vinifera (grapevine) and Zea mays (maize). These data were then combined with previous analysis of Populus trichocarpa (poplar tree), Selaginella moellindorffii (gemmiferous spikemoss), Sorghum bicolor (sorghum) and Volvox carteri (colonial algae) for a comprehensive evolutionary comparison of the plant ALDH superfamily. As a result, newly identified genes can be more easily analyzed and gene names can be assigned according to current nomenclature guidelines; our goal is to clarify previously confusing and conflicting names and classifications that might confound results and prevent accurate comparisons between studies. PMID:23007552

  5. An experimental test for lineage-specific position effects on alcohol dehydrogenase (Adh) genes in Drosophila

    PubMed Central

    Siegal, Mark L.; Hartl, Daniel L.

    1998-01-01

    Independent transgene insertions differ in expression based on their location in the genome; these position effects are of interest because they reflect the influence of genome organization on gene regulation. Position effects also represent potentially insurmountable obstacles to the rigorous functional comparison of homologous genes from different species because (i) quantitative variation in expression of each gene across genomic positions (generalized position effects, or GPEs) may overwhelm differences between the genes of interest, or (ii) divergent genes may be differentially sensitive to position effects, reflecting unique interactions between each gene and its genomic milieu (lineage-specific position effects, or LSPEs). We have investigated both types of position-effect variation by applying our method of transgene coplacement, which allows comparisons of transgenes in the same position in the genome of Drosophila melanogaster. Here we report an experimental test for LSPE in Drosophila. The alcohol dehydrogenase (Adh) genes of D. melanogaster and Drosophila affinidisjuncta differ in both tissue distribution and amounts of ADH activity. Despite this striking regulatory divergence, we found a very high correlation in overall ADH activity between the genes of the two species when placed in the same genomic position as assayed in otherwise Adh-null adults and larvae. These results argue against the influence of LSPE for these sequences, although the effects of GPE are significant. Our new findings validate the coplacement approach and show that it greatly magnifies the power to detect differences in expression between transgenes. Transgene coplacement thus dramatically extends the range of functional and evolutionary questions that can be addressed by transgenic technology. PMID:9861000

  6. Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics

    PubMed Central

    Brocker, Chad; Vasiliou, Melpomene; Carpenter, Sarah; Carpenter, Christopher; Zhang, Yucheng; Wang, Xiping; Kotchoni, Simeon O.; Wood, Andrew J.; Kirch, Hans-Hubert; Kopečný, David; Nebert, Daniel W.

    2012-01-01

    In recent years, there has been a significant increase in the number of completely sequenced plant genomes. The comparison of fully sequenced genomes allows for identification of new gene family members, as well as comprehensive analysis of gene family evolution. The aldehyde dehydrogenase (ALDH) gene superfamily comprises a group of enzymes involved in the NAD+- or NADP+-dependent conversion of various aldehydes to their corresponding carboxylic acids. ALDH enzymes are involved in processing many aldehydes that serve as biogenic intermediates in a wide range of metabolic pathways. In addition, many of these enzymes function as ‘aldehyde scavengers’ by removing reactive aldehydes generated during the oxidative degradation of lipid membranes, also known as lipid peroxidation. Plants and animals share many ALDH families, and many genes are highly conserved between these two evolutionarily distinct groups. Conversely, both plants and animals also contain unique ALDH genes and families. Herein we carried outgenome-wide identification of ALDH genes in a number of plant species—including Arabidopsis thaliana (thale crest), Chlamydomonas reinhardtii (unicellular algae), Oryza sativa (rice), Physcomitrella patens (moss), Vitis vinifera (grapevine) and Zea mays (maize). These data were then combined with previous analysis of Populus trichocarpa (poplar tree), Selaginella moellindorffii (gemmiferous spikemoss), Sorghum bicolor (sorghum) and Volvox carteri (colonial algae) for a comprehensive evolutionary comparison of the plant ALDH superfamily. As a result, newly identified genes can be more easily analyzed and gene names can be assigned according to current nomenclature guidelines; our goal is to clarify previously confusing and conflicting names and classifications that might confound results and prevent accurate comparisons between studies. PMID:23007552

  7. Regulation of the ald gene encoding alanine dehydrogenase by AldR in Mycobacterium smegmatis.

    PubMed

    Jeong, Ji-A; Baek, Eun-Young; Kim, Si Wouk; Choi, Jong-Soon; Oh, Jeong-Il

    2013-08-01

    The regulatory gene aldR was identified 95 bp upstream of the ald gene encoding L-alanine dehydrogenase in Mycobacterium smegmatis. The AldR protein shows sequence similarity to the regulatory proteins of the Lrp/AsnC family. Using an aldR deletion mutant, we demonstrated that AldR serves as both activator and repressor for the regulation of ald gene expression, depending on the presence or absence of L-alanine. The purified AldR protein exists as a homodimer in the absence of L-alanine, while it adopts the quaternary structure of a homohexamer in the presence of L-alanine. The binding affinity of AldR for the ald control region was shown to be increased significantly by L-alanine. Two AldR binding sites (O1 and O2) with the consensus sequence GA-N₂-ATC-N₂-TC and one putative AldR binding site with the sequence GA-N₂-GTT-N₂-TC were identified upstream of the ald gene. Alanine and cysteine were demonstrated to be the effector molecules directly involved in the induction of ald expression. The cellular level of L-alanine was shown to be increased in M. smegmatis cells grown under hypoxic conditions, and the hypoxic induction of ald expression appears to be mediated by AldR, which senses the intracellular level of alanine.

  8. CcpA-dependent regulation of Bacillus subtilis glutamate dehydrogenase gene expression.

    PubMed

    Belitsky, Boris R; Kim, Hyun-Jin; Sonenshein, Abraham L

    2004-06-01

    The Bacillus subtilis rocG gene, encoding catabolic glutamate dehydrogenase, was found to be subject to direct CcpA-dependent glucose repression. The effect of CcpA required the presence of both the HPr and Crh proteins. The primary CcpA binding site was identified by mutational analysis and DNase I footprinting. In the absence of inducers of the Roc pathway, rocG was still expressed at a low level due to readthrough transcription. CcpA-dependent repression of rocG readthrough transcription proved to contribute to the slow growth rate of B. subtilis cells in glucose-glutamate medium. Increased readthrough expression of rocG was shown to be partially responsible for the growth defect of ccpA strains in glucose-ammonium medium.

  9. Integrated Proteomics and Metabolomics of Arabidopsis Acclimation to Gene-Dosage Dependent Perturbation of Isopropylmalate Dehydrogenases

    PubMed Central

    Dufresne, Craig P.; Zhu, Ning; Pang, Qiuying; Chen, Sixue

    2013-01-01

    Maintaining metabolic homeostasis is critical for plant growth and development. Here we report proteome and metabolome changes when the metabolic homeostasis is perturbed due to gene-dosage dependent mutation of Arabidopsis isopropylmalate dehydrogenases (IPMDHs). By integrating complementary quantitative proteomics and metabolomics approaches, we discovered that gradual ablation of the oxidative decarboxylation step in leucine biosynthesis caused imbalance of amino acid homeostasis, redox changes and oxidative stress, increased protein synthesis, as well as a decline in photosynthesis, which led to rearrangement of central metabolism and growth retardation. Disruption of IPMDHs involved in aliphatic glucosinolate biosynthesis led to synchronized increase of both upstream and downstream biosynthetic enzymes, and concomitant repression of the degradation pathway, indicating metabolic regulatory mechanisms in controlling glucosinolate biosynthesis. PMID:23533573

  10. Implication of an Aldehyde Dehydrogenase Gene and a Phosphinothricin N-Acetyltransferase Gene in the Diversity of Pseudomonas cichorii Virulence

    PubMed Central

    Tanaka, Masayuki; Wali, Ullah Md; Nakayashiki, Hitoshi; Fukuda, Tatsuya; Mizumoto, Hiroyuki; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

    2011-01-01

    Pseudomonas cichorii harbors the hrp genes. hrp-mutants lose their virulence on eggplant but not on lettuce. A phosphinothricin N-acetyltransferase gene (pat) is located between hrpL and an aldehyde dehydrogenase gene (aldH) in the genome of P. cichorii. Comparison of nucleotide sequences and composition of the genes among pseudomonads suggests a common ancestor of hrp and pat between P. cichorii strains and P. viridiflava strains harboring the single hrp pathogenicity island. In contrast, phylogenetic diversification of aldH corresponded to species diversification amongst pseudomonads. In this study, the involvement of aldH and pat in P. cichorii virulence was analyzed. An aldH-deleted mutant (ΔaldH) and a pat-deleted mutant (Δpat) lost their virulence on eggplant but not on lettuce. P. cichorii expressed both genes in eggplant leaves, independent of HrpL, the transcriptional activator for the hrp. Inoculation into Asteraceae species susceptible to P. cichorii showed that the involvement of hrp, pat and aldH in P. cichorii virulence is independent of each other and has no relationship with the phylogeny of Asteraceae species based on the nucleotide sequences of ndhF and rbcL. It is thus thought that not only the hrp genes but also pat and aldH are implicated in the diversity of P. cichorii virulence on susceptible host plant species. PMID:24704843

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

    PubMed Central

    Costeas, P A; Chinsky, J M

    2000-01-01

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

  12. Genetics Home Reference: pyruvate dehydrogenase deficiency

    MedlinePlus

    ... control the activity of the complex: pyruvate dehydrogenase phosphatase turns on (activates) the complex, while pyruvate dehydrogenase ... binding protein (the PDHX gene), and pyruvate dehydrogenase phosphatase (the PDP1 gene) have been identified in people ...

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

  15. The Alcohol Dehydrogenase Gene Family in Melon (Cucumis melo L.): Bioinformatic Analysis and Expression Patterns

    PubMed Central

    Jin, Yazhong; Zhang, Chong; Liu, Wei; Tang, Yufan; Qi, Hongyan; Chen, Hao; Cao, Songxiao

    2016-01-01

    Alcohol dehydrogenases (ADH), encoded by multigene family in plants, play a critical role in plant growth, development, adaptation, fruit ripening and aroma production. Thirteen ADH genes were identified in melon genome, including 12 ADHs and one formaldehyde dehydrogenease (FDH), designated CmADH1-12 and CmFDH1, in which CmADH1 and CmADH2 have been isolated in Cantaloupe. ADH genes shared a lower identity with each other at the protein level and had different intron-exon structure at nucleotide level. No typical signal peptides were found in all CmADHs, and CmADH proteins might locate in the cytoplasm. The phylogenetic tree revealed that 13 ADH genes were divided into three groups respectively, namely long-, medium-, and short-chain ADH subfamily, and CmADH1,3-11, which belongs to the medium-chain ADH subfamily, fell into six medium-chain ADH subgroups. CmADH12 may belong to the long-chain ADH subfamily, while CmFDH1 may be a Class III ADH and serve as an ancestral ADH in melon. Expression profiling revealed that CmADH1, CmADH2, CmADH10 and CmFDH1 were moderately or strongly expressed in different vegetative tissues and fruit at medium and late developmental stages, while CmADH8 and CmADH12 were highly expressed in fruit after 20 days. CmADH3 showed preferential expression in young tissues. CmADH4 only had slight expression in root. Promoter analysis revealed several motifs of CmADH genes involved in the gene expression modulated by various hormones, and the response pattern of CmADH genes to ABA, IAA and ethylene were different. These CmADHs were divided into ethylene-sensitive and –insensitive groups, and the functions of CmADHs were discussed. PMID:27242871

  16. The Alcohol Dehydrogenase Gene Family in Melon (Cucumis melo L.): Bioinformatic Analysis and Expression Patterns.

    PubMed

    Jin, Yazhong; Zhang, Chong; Liu, Wei; Tang, Yufan; Qi, Hongyan; Chen, Hao; Cao, Songxiao

    2016-01-01

    Alcohol dehydrogenases (ADH), encoded by multigene family in plants, play a critical role in plant growth, development, adaptation, fruit ripening and aroma production. Thirteen ADH genes were identified in melon genome, including 12 ADHs and one formaldehyde dehydrogenease (FDH), designated CmADH1-12 and CmFDH1, in which CmADH1 and CmADH2 have been isolated in Cantaloupe. ADH genes shared a lower identity with each other at the protein level and had different intron-exon structure at nucleotide level. No typical signal peptides were found in all CmADHs, and CmADH proteins might locate in the cytoplasm. The phylogenetic tree revealed that 13 ADH genes were divided into three groups respectively, namely long-, medium-, and short-chain ADH subfamily, and CmADH1,3-11, which belongs to the medium-chain ADH subfamily, fell into six medium-chain ADH subgroups. CmADH12 may belong to the long-chain ADH subfamily, while CmFDH1 may be a Class III ADH and serve as an ancestral ADH in melon. Expression profiling revealed that CmADH1, CmADH2, CmADH10 and CmFDH1 were moderately or strongly expressed in different vegetative tissues and fruit at medium and late developmental stages, while CmADH8 and CmADH12 were highly expressed in fruit after 20 days. CmADH3 showed preferential expression in young tissues. CmADH4 only had slight expression in root. Promoter analysis revealed several motifs of CmADH genes involved in the gene expression modulated by various hormones, and the response pattern of CmADH genes to ABA, IAA and ethylene were different. These CmADHs were divided into ethylene-sensitive and -insensitive groups, and the functions of CmADHs were discussed. PMID:27242871

  17. The pyruvate dehydrogenase E1 alpha gene is testosterone and prolactin regulated in prostate epithelial cells.

    PubMed

    Costello, L C; Liu, Y; Zou, J; Franklin, R B

    2000-02-01

    The prostate gland of humans and other animals has the unique function of accumulating and secreting extraordinarily high levels of citrate. The prostate secretory epithelial cells synthesize citrate which, due to a limiting mitochondrial (m-) aconitase, accumulates rather than being oxidized. Thus citrate is essentially an end product of metabolism in prostate. For continued net citrate production, a continual source of oxaloacetate (OAA) and acetyl CoA is required. Glucose via pyruvate oxidation provides the source of Acetyl CoA. In prostate cells, citrate production is regulated by testosterone and/or by prolactin. Both hormones selectively regulate the level and activity of pyruvate dehydrogenase E1 alpha (E1a) in animal prostate cells; thereby regulating the availability of acetyl CoA for citrate synthesis. Studies were conducted to determine if testosterone and prolactin might regulate the expression of the E1a gene in prostate epithelial cells. Prolactin treatment of rat ventral and lateral prostate cells and human PC3 cells increased the levels of E1a mRNA and the rates of transcription of the E1a gene. Testosterone also increased the mRNA level and transcription of E1a in rat ventral prostate cells, and in PC3 cells transfected with androgen receptor. However, testosterone treatment resulted in a repression of E1a gene expression in lateral prostate cells. Evidence is presented which supports the view that prolactin regulation of E1a is mediated via PKC. The rapidity of the effects of both hormones is representative of an immediate-early gene response. To our knowledge this represents the first report in any mammalian cells that, in addition to its constitutive expression in all mammalian cells, the E1a gene is a hormonally-regulated gene in specifically targeted prostate epithelial cells. PMID:10711720

  18. CO dehydrogenase genes found in metagenomic fosmid clones from the deep mediterranean sea.

    PubMed

    Martin-Cuadrado, Ana-Belen; Ghai, Rohit; Gonzaga, Aitor; Rodriguez-Valera, Francisco

    2009-12-01

    The use of carbon monoxide (CO) as a biological energy source is widespread in microbes. In recent years, the role of CO oxidation in superficial ocean waters has been shown to be an important energy supplement for heterotrophs (carboxydovores). The key enzyme CO dehydrogenase was found in both isolates and metagenomes from the ocean's photic zone, where CO is continuously generated by organic matter photolysis. We have also found genes that code for both forms I (low affinity) and II (high affinity) in fosmids from a metagenomic library generated from a 3,000-m depth in the Mediterranean Sea. Analysis of other metagenomic databases indicates that similar genes are also found in the mesopelagic and bathypelagic North Pacific and on the surfaces of this and other oceanic locations (in lower proportions and similarities). The frequency with which this gene was found indicates that this energy-generating metabolism would be at least as important in the bathypelagic habitat as it is in the photic zone. Although there are no data about CO concentrations or origins deep in the ocean, it could have a geothermal origin or be associated with anaerobic metabolism of organic matter. The identities of the microbes that carry out these processes were not established, but they seem to be representatives of either Bacteroidetes or Chloroflexi.

  19. Origin and evolution of a new gene descended from alcohol dehydrogenase in Drosophila.

    PubMed

    Begun, D J

    1997-02-01

    Drosophila alcohol dehydrogenase (Adh) is highly conserved in size, organization, and amino acid sequence. Adh-psi was hypothesized to be a pseudogene derived from an Adh duplication in the repleta group of Drosophila; however, several results from molecular analyses of this gene conflict with currently held notions of molecular evolution. Perhaps the most difficult observations to reconcile with the pseudogene hypothesis are that the hypothetical replacement sites of Adh-psi evolve only slightly more quickly than replacement sites of closely related, functional Adh genes, and that the replacement sites of the pseudogenes evolve considerably more slowly than neighboring silent sites. The data have been presented as a paradox that challenges our understanding of the mechanisms underlying DNA sequence divergence. Here I show that Adh-psi is actually a new, functional gene recently descended from an Adh duplication. This descendant recruited approximately 60 new N-terminal amino acids, is considerably more basic than ADH, and is evolving at a faster rate than Adh. Furthermore, though the descendant is clearly functional, as inferred from molecular evolution and population genetic data, it retains no obvious ADH activity. This probably reflects functional divergence from its Adh ancestor.

  20. The alcohol dehydrogenase gene is nested in the outspread locus of Drosophila melanogaster

    SciTech Connect

    McNabb, S.; Greig, S.; Davis, T.

    1996-06-01

    This report describes the structure and expression of the outspread (osp) gene of Drosophila melanogaster. Previous work showed that chromosomal breakpoints associated with mutations of the osp locus map to both sides of the alcohol dehydrogenase gene (Adh), suggesting that Adh and the adjacent gene Adh{sup r} are nested in osp. We extended a chromosomal walk and mapped additional osp mutations to define the maximum molecular limit of osp as 119 kb. We identified a 6-kb transcript that hybridizes to osp region DNA and is altered or absent in osp mutants. Accumulation of this RNA peaks during embryonic and pupal periods. The osp cDNAs comprise two distinct classes based on alternative splicing patterns. The 5{prime} end of the longest cDNA was extended by PCR amplification. When hybridized to the osp walk, the 5{prime} extension verifies that Adh and Adh{sup r} are nested in osp and shows that osp has a transcription unit of {ge}74 kb. In situ hybridization shows that osp is expressed both maternally and zygotically. In the ovary, osp is transcribed in nurse cells and localized in the oocyte. In embryos, expression is most abundant in the developing visceral and somatic musculature. 55 refs., 11 figs., 1 tab.

  1. Characterization of the gene coding for GDP-mannose dehydrogenase (algD) from Azotobacter vinelandii.

    PubMed Central

    Campos, M; Martínez-Salazar, J M; Lloret, L; Moreno, S; Núñez, C; Espín, G; Soberón-Chávez, G

    1996-01-01

    Azotobacter vinelandii presents a differentiation process leading to the formation of desiccation-resistant cysts. Alginate, the exopolysaccharide produced by this bacterium, has been postulated to have a role in cyst formation. Here, we report the cloning and characterization of the A. vinelandii gene coding for the enzyme GDP-mannose dehydrogenase (algD), which is the key enzyme for alginate synthesis in Pseudomonas aeruginosa. This gene has a high degree of similarity with the algD gene from P. aeruginosa, and similar proteins seem to be involved in algD regulation in both bacteria. We show the existence of two mRNA start sites; one of these sites corresponds to a promoter transcribed by RNA polymerase containing a sigma E subunit. An A. vinelandii algD mutant which is completely impaired in alginate production and which is unable to form desiccation-resistant cells was constructed. The effects of NH4, NO3, and NaCl concentrations on algD transcription for three A. vinelandii strains producing different alginate levels were evaluated. We found a strict correlation between alginate production and algD transcription for the three strains studied; however, the effects on algD transcription under the conditions studied were different for each strain. The nitrogen source regulates algD expression in the wild-type strain. PMID:8606150

  2. Isolation and characterization of the Xanthine dehydrogenase gene of the Mediterranean fruit fly, Ceratitis capitata.

    PubMed Central

    Pitts, R J; Zwiebel, L J

    2001-01-01

    Xanthine dehydrogenase (XDH) is a member of the molybdenum hydroxylase family of enzymes catalyzing the oxidation of hypoxanthine and xanthine to uric acid. The enzyme is also required for the production of one of the major Drosophila eye pigments, drosopterin. The XDH gene has been isolated in many species representing a broad cross section of the major groups of living organisms, including the cDNA encoding XDH from the Mediterranean fruit fly Ceratitis capitata (CcXDH) described here. CcXDH is closely related to other insect XDHs and is able to rescue the phenotype of the Drosophila melanogaster XDH mutant, rosy, in germline transformation experiments. A previously identified medfly mutant, termed rosy, whose phenotype is suggestive of a disruption in XDH function, has been examined for possible mutations in the XDH gene. However, we find no direct evidence that a mutation in the CcXDH gene or that a reduction in the CcXDH enzyme activity is present in rosy medflies. Conclusive studies of the nature of the medfly rosy mutant will require rescue by germline transformation of mutant medflies. PMID:11514452

  3. Regulation of IMP dehydrogenase gene expression by its end products, guanine nucleotides.

    PubMed Central

    Glesne, D A; Collart, F R; Huberman, E

    1991-01-01

    To study the regulation of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanine nucleotide biosynthesis, we examined the effects of nucleosides, nucleotides, nucleotide analogs, or the IMPDH inhibitor mycophenolic acid (MPA) on the steady-state levels of IMPDH mRNA. The results indicated that IMPDH gene expression is regulated inversely by the intracellular level of guanine ribonucleotides. We have shown that treatment with guanosine increased the level of cellular guanine ribonucleotides and subsequently reduced IMPDH steady-state mRNA levels in a time- and dose-dependent manner. Conversely, MPA treatment diminished the level of guanine ribonucleotides and increased IMPDH mRNA levels. Both of these effects on the steady-state level of IMPDH mRNA could be negated by cotreatment with guanosine and MPA. The down regulation of IMPDH gene expression by guanosine or its up regulation by MPA was not due to major changes in transcriptional initiation and elongation or mRNA stability in the cytoplasm but rather was due to alterations in the levels of the IMPDH mRNA in the nucleus. These results suggest that IMPDH gene expression is regulated by a posttranscriptional, nuclear event in response to fluctuations in the intracellular level of guanine ribonucleotides. Images PMID:1717828

  4. Two potential fish glycerol-3-phosphate phosphatases.

    PubMed

    Raymond, James A

    2015-06-01

    Winter-acclimated rainbow smelt (Osmerus mordax Mitchill) produce high levels of glycerol as an antifreeze. A common pathway to glycerol involves the enzyme glycerol-3-phosphate phosphatase (GPP), but no GPP has yet been identified in fish or any other animal. Here, two phosphatases assembled from existing EST libraries (from winter-acclimated smelt and cold-acclimated smelt hepatocytes) were found to resemble a glycerol-associated phosphatase from a glycerol-producing alga, Dunaliella salina, and a recently discovered GPP from a bacterium, Mycobacterium tuberculosis. Recombinant proteins were generated and were found to have GPP activity on the order of a few μMol Pi/mg enzyme/min. The two enzymes have acidic pH optima (~5.5) similar to that previously determined for GPP activity in liver tissue, with about 1/3 of their peak activities at neutral pH. The two enzymes appear to account for the GPP activity of smelt liver, but due to their reduced activities at neutral pH, their contributions to glycerol production in vivo remain unclear. Similar enzymes may be active in a glycerol-producing insect, Dendroctonus ponderosae.

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

    PubMed Central

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

    1994-01-01

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

  6. Cloning of the Arabidopsis and Rice Formaldehyde Dehydrogenase Genes: Implications for the Origin of Plant Adh Enzymes

    PubMed Central

    Dolferus, R.; Osterman, J. C.; Peacock, W. J.; Dennis, E. S.

    1997-01-01

    This article reports the cloning of the genes encoding the Arabidopsis and rice class III ADH enzymes, members of the alcohol dehydrogenase or medium chain reductase/dehydrogenase superfamily of proteins with glutathione-dependent formaldehyde dehydrogenase activity (GSH-FDH). Both genes contain eight introns in exactly the same positions, and these positions are conserved in plant ethanol-active Adh genes (class P). These data provide further evidence that plant class P genes have evolved from class III genes by gene duplication and acquisition of new substrate specificities. The position of introns and similarities in the nucleic acid and amino acid sequences of the different classes of ADH enzymes in plants and humans suggest that plant and animal class III enzymes diverged before they duplicated to give rise to plant and animal ethanol-active ADH enzymes. Plant class P ADH enzymes have gained substrate specificities and evolved promoters with different expression properties, in keeping with their metabolic function as part of the alcohol fermentation pathway. PMID:9215914

  7. Complete knockout of the lactate dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3 down-regulated CHO cells.

    PubMed

    Yip, Shirley S M; Zhou, Meixia; Joly, John; Snedecor, Bradley; Shen, Amy; Crawford, Yongping

    2014-09-01

    Accumulation of high level of lactate can negatively impact cell growth during fed-batch culture process. In this study, we attempted to knockout the lactate dehydrogenase A (LDHA) gene in CHO cells in order to attenuate the lactate level. To prevent the potential deleterious effect of pyruvate accumulation, consequent to LDHA knockout, on cell culture, we chose a pyruvate dehydrogenase kinase 1, 2, and 3 (PDHK1, 2, and 3) knockdown cell line in which to knock out LDHA alleles. Around 3,000 clones were screened to obtain 152 mutants. Only heterozygous mutants were identified. An attempt to knockout the remaining wild-type allele from one such heterozygote yielded only two mutants after screening 567 clones. One had an extra valine. Another evidenced a duplication event, possessing at lease one wild-type and two different frameshifted alleles. Both mutants still retained LDH activity. Together, our data strongly suggest that a complete knockout of LDHA is lethal in CHO cells, despite simultaneous down-regulation of PDHK1, 2, and 3. PMID:24841241

  8. Amplification of the IMP dehydrogenase gene in Chinese hamster cells resistant to mycophenolic acid.

    PubMed Central

    Collart, F R; Huberman, E

    1987-01-01

    The regulation of IMP dehydrogenase (IMPDH) was analyzed in Chinese hamster V79 cell variants that exhibit different degrees of resistance to the cytotoxic effect of mycophenolic acid, a specific inhibitor of IMPDH. Western blot (immunoblot) analysis with an IMPDH antiserum revealed a 14- to 27-fold increase in the amount of enzyme in the mycophenolic acid-resistant cells. The antiserum was also used to screen for a phage containing the IMPDH cDNA sequence from a lambda gt11 expression library. Northern blot (RNA blot) analyses of total cellular and poly(A)+ RNA showed that an IMPDH cDNA probe hybridized to a 2.2-kilobase transcript, the amount of which was associated with increased resistance. Southern blotting with the probe indicated an amplification of the IMPDH gene in the mycophenolic acid-resistant cells. Our findings suggest that the acquired mycophenolic acid resistance of the V79 cell variants is associated with increases in the amount and activity of IMPDH and the number of IMPDH gene copies. Images PMID:2890098

  9. Silencing of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase gene enhances glioma radiosensitivity

    SciTech Connect

    Kim, Sung Youl; Yoo, Young Hyun; Park, Jeen-Woo

    2013-04-05

    Highlights: •Silencing of the IDPm gene enhances IR-induced autophagy in glioma cells. •Autophagy inhibition augmented apoptosis of irradiated glioma cells. •Results offer a redox-active therapeutic strategy for the treatment of cancer. -- Abstract: Reactive oxygen species (ROS) levels are elevated in organisms that have been exposed to ionizing radiation and are protagonists in the induction of cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are primary functions of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDPm) via the supply of NADPH for antioxidant systems. In the present study, we report an autophagic response to ionizing radiation in A172 glioma cells transfected with small interfering RNA (siRNA) targeting the IDPm gene. Autophagy in A172 transfectant cells was associated with enhanced autophagolysosome formation and GFP–LC3 punctuation/aggregation. Furthermore, we found that the inhibition of autophagy by chloroquine augmented apoptotic cell death of irradiated A172 cells transfected with IDPm siRNA. Taken together, our data suggest that autophagy functions as a survival mechanism in A172 cells against ionizing radiation-induced apoptosis and the sensitizing effect of IDPm siRNA and autophagy inhibitor on the ionizing radiation-induced apoptotic cell death of glioma cells offers a novel redox-active therapeutic strategy for the treatment of cancer.

  10. Cloning and sequence analysis of the safflower betaine aldehyde dehydrogenase gene.

    PubMed

    Wang, Y B; Guan, L L; Xu, Y W; Shen, H; Wu, W

    2014-01-01

    In response to salinity or drought stress, many plants accumulate glycine betaine, which is a regulator of osmosis. In plants, the last step in betaine synthesis is catalyzed by betaine aldehyde dehydrogenase (BADH), a nuclear-encoded chloroplastic enzyme. Based on the conserved oligo amino acid residues of the published BADH genes from other higher plant species, a cDNA sequence, designated CtBADH, was isolated from safflower (Carthamus tinctorius L.) using a polymerase chain reaction approach. The clones were 1.7 kb on average, and contained an open reading frame predicting a polypeptide of 503 amino acids with 84.5% identity to that of Helianthus annuus. The deduced amino acid sequence showed a decapeptide, Val-Thr-Leu-Geu-Leu-Gly-Gly-Lys-Ser-Pro and Cys, which is essential for proper functioning of BADH. Phylogenetic analysis indicated that CtBADH grouped with other dicotyledonous plant BADH genes, and subgrouped in the composite family. Prediction of secondary structure and subcellular localization suggested that the protein encoded by CtBADH contains 33 coils, 15 alpha helixes, and 21 beta strands, and most likely targets the chloroplast or mitochondria.

  11. Ectopic Expression of the Chinese Cabbage Malate Dehydrogenase Gene Promotes Growth and Aluminum Resistance in Arabidopsis

    PubMed Central

    Li, Qing-Fei; Zhao, Jing; Zhang, Jing; Dai, Zi-Hui; Zhang, Lu-Gang

    2016-01-01

    Malate dehydrogenases (MDHs) are key metabolic enzymes that play important roles in plant growth and development. In the present study, we isolated the full-length and coding sequences of BraMDH from Chinese cabbage [Brassica campestris L. ssp. pekinensis (Lour) Olsson]. We conducted bioinformatics analysis and a subcellular localization assay, which revealed that the BraMDH gene sequence contained no introns and that BraMDH is localized to the chloroplast. In addition, the expression pattern of BraMDH in Chinese cabbage was investigated, which revealed that BraMDH was heavily expressed in inflorescence apical meristems, as well as the effect of BraMDH overexpression in two homozygous transgenic Arabidopsis lines, which resulted in early bolting and taller inflorescence stems. Furthermore, the fresh and dry weights of aerial tissue from the transgenic Arabidopsis plants were significantly higher than those from the corresponding wild-type plants, as were plant height, the number of rosette leaves, and the number of siliques produced, and the transgenic plants also exhibited stronger aluminum resistance when treated with AlCl3. Therefore, our results suggest that BraMDH has a dramatic effect on plant growth and that the gene is involved in both plant growth and aluminum resistance. PMID:27536317

  12. Succinate dehydrogenase gene mutations are strongly associated with paraganglioma of the organ of Zuckerkandl.

    PubMed

    Lodish, Maya B; Adams, Karen T; Huynh, Thanh T; Prodanov, Tamara; Ling, Alex; Chen, Clara; Shusterman, Suzanne; Jimenez, Camilo; Merino, Maria; Hughes, Marybeth; Cradic, Kendall W; Milosevic, Dragana; Singh, Ravinder J; Stratakis, Constantine A; Pacak, Karel

    2010-09-01

    Organ of Zuckerkandl paragangliomas (PGLs) are rare neuroendocrine tumors that are derived from chromaffin cells located around the origin of the inferior mesenteric artery extending to the level of the aortic bifurcation. Mutations in the genes encoding succinate dehydrogenase subunits (SDH) B, C, and D (SDHx) have been associated with PGLs, but their contribution to PGLs of the organ of Zuckerkandl PGLs is not known. We aimed to describe the clinical presentation of patients with PGLs of the organ of Zuckerkandl and investigate the prevalence of SDHx mutations and other genetic defects among them. The clinical characteristics of 14 patients with PGL of the organ of Zuckerkandl were analyzed retrospectively; their DNA was tested for SDHx mutations and deletions. Eleven out of 14 (79%) patients with PGLs of the organ of Zuckerkandl were found to have mutations in the SDHB (9) or SDHD (2) genes; one patient was found to have the Carney-Stratakis syndrome (CSS), and his PGL was discovered during surgery for gastrointestinal stromal tumor. Our results show that SDHx mutations are prevalent in pediatric and adult PGLs of the organ of Zuckerkandl. Patients with PGLs of the organ of Zuckerkandl should be screened for SDHx mutations and the CSS; in addition, asymptomatic carriers of an SDHx mutation among the relatives of affected patients may benefit from tumor screening for early PGL detection.

  13. Ectopic Expression of the Chinese Cabbage Malate Dehydrogenase Gene Promotes Growth and Aluminum Resistance in Arabidopsis.

    PubMed

    Li, Qing-Fei; Zhao, Jing; Zhang, Jing; Dai, Zi-Hui; Zhang, Lu-Gang

    2016-01-01

    Malate dehydrogenases (MDHs) are key metabolic enzymes that play important roles in plant growth and development. In the present study, we isolated the full-length and coding sequences of BraMDH from Chinese cabbage [Brassica campestris L. ssp. pekinensis (Lour) Olsson]. We conducted bioinformatics analysis and a subcellular localization assay, which revealed that the BraMDH gene sequence contained no introns and that BraMDH is localized to the chloroplast. In addition, the expression pattern of BraMDH in Chinese cabbage was investigated, which revealed that BraMDH was heavily expressed in inflorescence apical meristems, as well as the effect of BraMDH overexpression in two homozygous transgenic Arabidopsis lines, which resulted in early bolting and taller inflorescence stems. Furthermore, the fresh and dry weights of aerial tissue from the transgenic Arabidopsis plants were significantly higher than those from the corresponding wild-type plants, as were plant height, the number of rosette leaves, and the number of siliques produced, and the transgenic plants also exhibited stronger aluminum resistance when treated with AlCl3. Therefore, our results suggest that BraMDH has a dramatic effect on plant growth and that the gene is involved in both plant growth and aluminum resistance. PMID:27536317

  14. Phylogeny and structure of the cinnamyl alcohol dehydrogenase gene family in Brachypodium distachyon.

    PubMed

    Bukh, Christian; Nord-Larsen, Pia Haugaard; Rasmussen, Søren K

    2012-10-01

    Cinnamyl alcohol dehydrogenase (CAD) catalyses the final step of the monolignol biosynthesis, the conversion of cinnamyl aldehydes to alcohols, using NADPH as a cofactor. Seven members of the CAD gene family were identified in the genome of Brachypodium distachyon and five of these were isolated and cloned from genomic DNA. Semi-quantitative reverse-transcription PCR revealed differential expression of the cloned genes, with BdCAD5 being expressed in all tissues and highest in root and stem while BdCAD3 was only expressed in stem and spikes. A phylogenetic analysis of CAD-like proteins placed BdCAD5 on the same branch as bona fide CAD proteins from maize (ZmCAD2), rice (OsCAD2), sorghum (SbCAD2) and Arabidopsis (AtCAD4, 5). The predicted three-dimensional structures of both BdCAD3 and BdCAD5 resemble that of AtCAD5. However, the amino-acid residues in the substrate-binding domains of BdCAD3 and BdCAD5 are distributed symmetrically and BdCAD3 is similar to that of poplar sinapyl alcohol dehydrogenase (PotSAD). BdCAD3 and BdCAD5 expressed and purified from Escherichia coli both showed a temperature optimum of about 50 °C and molar weight of 49 kDa. The optimal pH for the reduction of coniferyl aldehyde were pH 5.2 and 6.2 and the pH for the oxidation of coniferyl alcohol were pH 8 and 9.5, for BdCAD3 and BdCAD5 respectively. Kinetic parameters for conversion of coniferyl aldehyde and coniferyl alcohol showed that BdCAD5 was clearly the most efficient enzyme of the two. These data suggest that BdCAD5 is the main CAD enzyme for lignin biosynthesis and that BdCAD3 has a different role in Brachypodium. All CAD enzymes are cytosolic except for BdCAD4, which has a putative chloroplast signal peptide adding to the diversity of CAD functions. PMID:23028019

  15. In vitro expression of Candida albicans alcohol dehydrogenase genes involved in acetaldehyde metabolism.

    PubMed

    Bakri, M M; Rich, A M; Cannon, R D; Holmes, A R

    2015-02-01

    Alcohol consumption is a risk factor for oral cancer, possibly via its conversion to acetaldehyde, a known carcinogen. The oral commensal yeast Candida albicans may be one of the agents responsible for this conversion intra-orally. The alcohol dehydrogenase (Adh) family of enzymes are involved in acetaldehyde metabolism in yeast but, for C. albicans it is not known which family member is responsible for the conversion of ethanol to acetaldehyde. In this study we determined the expression of mRNAs from three C. albicans Adh genes (CaADH1, CaADH2 and CaCDH3) for cells grown in different culture media at different growth phases by Northern blot analysis and quantitative reverse transcription polymerase chain reaction. CaADH1 was constitutively expressed under all growth conditions but there was differential expression of CaADH2. CaADH3 expression was not detected. To investigate whether CaAdh1p or CaAdh2p can contribute to alcohol catabolism in C. albicans, each gene from the reference strain C. albicans SC5314 was expressed in Saccharomyces cerevisiae. Cell extracts from an CaAdh1p-expressing S. cerevisiae recombinant, but not an CaAdh2p-expressing recombinant, or an empty vector control strain, possessed ethanol-utilizing Adh activity above endogenous S. cerevisiae activity. Furthermore, expression of C. albicans Adh1p in a recombinant S. cerevisiae strain in which the endogenous ScADH2 gene (known to convert ethanol to acetaldehyde in this yeast) had been deleted, conferred an NAD-dependent ethanol-utilizing, and so acetaldehyde-producing, Adh activity. We conclude that CaAdh1p is the enzyme responsible for ethanol use under in vitro growth conditions, and may contribute to the intra-oral production of acetaldehyde.

  16. Gene transcript accumulation and in situ mRNA hybridization of two putative glutamate dehydrogenase genes in etiolated Glycine max seedlings.

    PubMed

    Dimou, M; Tsaniklidis, G; Aivalakis, G; Katinakis, P

    2015-01-01

    Glutamate dehydrogenase (EC 1.4.1.2) is a multimeric enzyme that catalyzes the reversible amination of α-ketoglutarate to form glutamate. We characterized cDNA clones of two Glycine max sequences, GmGDH1 and GmGDH2, that code for putative α- and β-subunits, respectively, of the NADH dependent enzyme. Temporal and spatial gene transcript accumulation studies using semiquantitative RT-PCR and in situ hybridization have shown an overlapping gene transcript accumulation pattern with differences in relative gene transcript accumulation in the organs examined. Detection of NADH-dependent glutamate dehydrogenase activity in situ using a histochemical method showed concordance with the spatial gene transcript accumulation patterns. Our findings suggest that although the two gene transcripts are co-localized in roots of etiolated soybean seedlings, the ratio of the two subunits of the active holoenzyme may vary among tissues.

  17. The Xenopus alcohol dehydrogenase gene family: characterization and comparative analysis incorporating amphibian and reptilian genomes

    PubMed Central

    2014-01-01

    Background The alcohol dehydrogenase (ADH) gene family uniquely illustrates the concept of enzymogenesis. In vertebrates, tandem duplications gave rise to a multiplicity of forms that have been classified in eight enzyme classes, according to primary structure and function. Some of these classes appear to be exclusive of particular organisms, such as the frog ADH8, a unique NADP+-dependent ADH enzyme. This work describes the ADH system of Xenopus, as a model organism, and explores the first amphibian and reptilian genomes released in order to contribute towards a better knowledge of the vertebrate ADH gene family. Results Xenopus cDNA and genomic sequences along with expressed sequence tags (ESTs) were used in phylogenetic analyses and structure-function correlations of amphibian ADHs. Novel ADH sequences identified in the genomes of Anolis carolinensis (anole lizard) and Pelodiscus sinensis (turtle) were also included in these studies. Tissue and stage-specific libraries provided expression data, which has been supported by mRNA detection in Xenopus laevis tissues and regulatory elements in promoter regions. Exon-intron boundaries, position and orientation of ADH genes were deduced from the amphibian and reptilian genome assemblies, thus revealing syntenic regions and gene rearrangements with respect to the human genome. Our results reveal the high complexity of the ADH system in amphibians, with eleven genes, coding for seven enzyme classes in Xenopus tropicalis. Frogs possess the amphibian-specific ADH8 and the novel ADH1-derived forms ADH9 and ADH10. In addition, they exhibit ADH1, ADH2, ADH3 and ADH7, also present in reptiles and birds. Class-specific signatures have been assigned to ADH7, and ancestral ADH2 is predicted to be a mixed-class as the ostrich enzyme, structurally close to mammalian ADH2 but with class-I kinetic properties. Remarkably, many ADH1 and ADH7 forms are observed in the lizard, probably due to lineage-specific duplications. ADH4 is not

  18. Retinoic acid response element in the human alcohol dehydrogenase gene ADH3: implications for regulation of retinoic acid synthesis.

    PubMed Central

    Duester, G; Shean, M L; McBride, M S; Stewart, M J

    1991-01-01

    Retinoic acid regulation of one member of the human class I alcohol dehydrogenase (ADH) gene family was demonstrated, suggesting that the retinol dehydrogenase function of ADH may play a regulatory role in the biosynthetic pathway for retinoic acid. Promoter activity of human ADH3, but not ADH1 or ADH2, was shown to be activated by retinoic acid in transient transfection assays of Hep3B human hepatoma cells. Deletion mapping experiments identified a region in the ADH3 promoter located between -328 and -272 bp which confers retinoic acid activation. This region was also demonstrated to confer retinoic acid responsiveness on the ADH1 and ADH2 genes in heterologous promoter fusions. Within a 34-bp stretch, the ADH3 retinoic acid response element (RARE) contains two TGACC motifs and one TGAAC motif, both of which exist in RAREs controlling other genes. A block mutation of the TGACC sequence located at -289 to -285 bp eliminated the retinoic acid response. As assayed by gel shift DNA binding studies, the RARE region (-328 to -272 bp) of ADH3 bound the human retinoic acid receptor beta (RAR beta) and was competed for by DNA containing a RARE present in the gene encoding RAR beta. Since ADH catalyzes the conversion of retinol to retinal, which can be further converted to retinoic acid by aldehyde dehydrogenase, these results suggest that retinoic acid activation of ADH3 constitutes a positive feedback loop regulating retinoic acid synthesis. Images PMID:1996113

  19. [Polymorphism of the gene for subunit 6 of the NADh dehydrogenase complex (ND6) in ethnic russian population in Russia].

    PubMed

    Kornienko, I V; Vodolazhskiĭ, D I; Mikhalkovich, L S; Pavlichenko, G N; Ivanov, P L

    2003-01-01

    A sample of ethnic Russians of Russia was tested for polymorphism of the NADH dehydrogenase subunit 6 (ND6) gene mapping to the mtDNA region 14,170-14,569. Genetic diversity of ND6 haplotypes was estimated at 0.406, and probability of haplotype random match, at 0.598. Combined with typing the mtDNA control region, analysis of the ND6 gene polymorphism was assumed to improve the reliability of forensic identification. Several point substitutions in the ND6 gene region proved to be associated with particular transitions in the mtDNA control region; the association was characterized with the phi coefficient.

  20. Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast.

    PubMed

    Schifferdecker, Anna Judith; Siurkus, Juozas; Andersen, Mikael Rørdam; Joerck-Ramberg, Dorte; Ling, Zhihao; Zhou, Nerve; Blevins, James E; Sibirny, Andriy A; Piškur, Jure; Ishchuk, Olena P

    2016-04-01

    Dekkera bruxellensis is a non-conventional Crabtree-positive yeast with a good ethanol production capability. Compared to Saccharomyces cerevisiae, its tolerance to acidic pH and its utilization of alternative carbon sources make it a promising organism for producing biofuel. In this study, we developed an auxotrophic transformation system and an expression vector, which enabled the manipulation of D. bruxellensis, thereby improving its fermentative performance. Its gene ADH3, coding for alcohol dehydrogenase, was cloned and overexpressed under the control of the strong and constitutive promoter TEF1. Our recombinant D. bruxellensis strain displayed 1.4 and 1.7 times faster specific glucose consumption rate during aerobic and anaerobic glucose fermentations, respectively; it yielded 1.2 times and 1.5 times more ethanol than did the parental strain under aerobic and anaerobic conditions, respectively. The overexpression of ADH3 in D. bruxellensis also reduced the inhibition of fermentation by anaerobiosis, the "Custer effect". Thus, the fermentative capacity of D. bruxellensis could be further improved by metabolic engineering. PMID:26743658

  1. Transgenic barley overexpressing a cytokinin dehydrogenase gene shows greater tolerance to drought stress.

    PubMed

    Pospíšilová, Hana; Jiskrová, Eva; Vojta, Petr; Mrízová, Katarína; Kokáš, Filip; Čudejková, Mária Majeská; Bergougnoux, Veronique; Plíhal, Ondřej; Klimešová, Jana; Novák, Ondřej; Dzurová, Lenka; Frébort, Ivo; Galuszka, Petr

    2016-09-25

    Together with auxins, cytokinins are the main plant hormones involved in many different physiological processes. Given this knowledge, cytokinin levels can be manipulated by genetic modification in order to improve agronomic parameters of cereals in relation to, for example, morphology, yield, and tolerance to various stresses. The barley (Hordeum vulgare) cultivar Golden Promise was transformed using the cytokinin dehydrogenase 1 gene from Arabidopsis thaliana (AtCKX1) under the control of mild root-specific β-glucosidase promoter from maize. Increased cytokinin degradation activity was observed positively to affect the number and length of lateral roots. The impact on morphology depended upon the recombinant protein's subcellular compartmentation. While assumed cytosolic and vacuolar targeting of AtCKX1 had negligible effect on shoot growth, secretion of AtCKX1 protein to the apoplast had a negative effect on development of the aerial part and yield. Upon the application of severe drought stress, all transgenic genotypes maintained higher water content and showed better growth and yield parameters during revitalization. Higher tolerance to drought stress was most caused by altered root morphology resulting in better dehydration avoidance. PMID:26773738

  2. Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae.

    PubMed

    Satake, Ryoko; Ichiyanagi, Atsushi; Ichikawa, Keiichi; Hirokawa, Kozo; Araki, Yasuko; Yoshimura, Taro; Gomi, Keiko

    2015-11-01

    Glucose dehydrogenase (GDH) is of interest for its potential applications in the field of glucose sensors. To improve the performance of glucose sensors, GDH is required to have strict substrate specificity. A novel flavin adenine dinucleotide (FAD)-dependent GDH was isolated from Mucor prainii NISL0103 and its enzymatic properties were characterized. This FAD-dependent GDH (MpGDH) exhibited high specificity toward glucose. High specificity for glucose was also observed even in the presence of saccharides such as maltose, galactose and xylose. The molecular masses of the glycoforms of GDH ranged from 90 to 130 kDa. After deglycosylation, a single 80 kDa band was observed. The gene encoding MpGDH was cloned and expressed in Aspergillus sojae. The apparent kcat and Km values of recombinant enzyme for glucose were found to be 749.7 s(-1) and 28.3 mM, respectively. The results indicated that the characteristics of MpGDH were suitable for assaying blood glucose levels.

  3. Structure of RNA 3'-phosphate cyclase bound to substrate RNA.

    PubMed

    Desai, Kevin K; Bingman, Craig A; Cheng, Chin L; Phillips, George N; Raines, Ronald T

    2014-10-01

    RNA 3'-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3'-phosphate to form a 2',3'-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA-AMP and RNA(3')pp(5')A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3'-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3'-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3'-phosphate is poised for in-line attack on the P-N bond that links the phosphorous atom of AMP to N(ε) of His307. Thus, we provide the first insights into RNA 3'-phosphate termini recognition and the mechanism of 3'-phosphate activation by an Rtc enzyme.

  4. Molecular characterization of the gene cluster coxMSL encoding the molybdenum-containing carbon monoxide dehydrogenase of Oligotropha carboxidovorans.

    PubMed Central

    Schübel, U; Kraut, M; Mörsdorf, G; Meyer, O

    1995-01-01

    The CO dehydrogenase structural genes (cox) and orf4 are clustered in the transcriptional order coxM--> coxS--> coxL--> orf4 on the 128-kb megaplasmid pHCG3 of the carboxidotroph Oligotropha carboxidovorans OM5. Sequence analysis suggested association of molybdopterin cytosine dinucleotide and flavin adenine dinucleotide with CoxL and of the [2Fe-2S] clusters with CoxS. PMID:7721710

  5. Association between common alcohol dehydrogenase gene (ADH) variants and schizophrenia and autism

    PubMed Central

    Wang, Kesheng; Zhang, Xiang-Yang; Pan, Xinghua; Wang, Guilin; Tan, Yunlong; Zhong, Chunlong; Krystal, John H.; State, Matthew; Zhang, Heping

    2013-01-01

    Humans express at least seven alcohol dehydrogenase (ADH) isoforms that are encoded by ADH gene cluster (ADH7–ADH1C–ADH1B–ADH1A–ADH6–ADH4–ADH5) at chromosome 4. ADHs are key catabolic enzymes for retinol and ethanol. The functional ADH variants (mostly rare) have been implicated in alcoholism risk. In addition to catalyzing the oxidation of retinol and ethanol, ADHs may be involved in the metabolic pathways of several neurotransmitters that are implicated in the neurobiology of neuropsychiatric disorders. In the present study, we comprehensively examined the associations between common ADH variants [minor allele frequency (MAF) >0.05] and 11 neuropsychiatric and neurological disorders. A total of 50,063 subjects in 25 independent cohorts were analyzed. The entire ADH gene cluster was imputed across these 25 cohorts using the same reference panels. Association analyses were conducted, adjusting for multiple comparisons. We found 28 and 15 single nucleotide polymorphisms (SNPs), respectively, that were significantly associated with schizophrenia in African-Americans and autism in European-Americans after correction by false discovery rate (FDR) (q <0.05); and 19 and 6 SNPs, respectively, that were significantly associated with these two disorders after region-wide correction by SNPSpD (8.9 × 10−5 ≤ p ≤ 0.0003 and 2.4 × 10−5 ≤ p ≤ 0.0003, respectively). No variants were significantly associated with the other nine neuropsychiatric disorders, including alcohol dependence. We concluded that common ADH variants conferred risk for both schizophrenia in African-Americans and autism in European-Americans. PMID:23468174

  6. Association between common alcohol dehydrogenase gene (ADH) variants and schizophrenia and autism.

    PubMed

    Zuo, Lingjun; Wang, Kesheng; Zhang, Xiang-Yang; Pan, Xinghua; Wang, Guilin; Tan, Yunlong; Zhong, Chunlong; Krystal, John H; State, Matthew; Zhang, Heping; Luo, Xingguang

    2013-07-01

    Humans express at least seven alcohol dehydrogenase (ADH) isoforms that are encoded by ADH gene cluster (ADH7-ADH1C-ADH1B-ADH1A-ADH6-ADH4-ADH5) at chromosome 4. ADHs are key catabolic enzymes for retinol and ethanol. The functional ADH variants (mostly rare) have been implicated in alcoholism risk. In addition to catalyzing the oxidation of retinol and ethanol, ADHs may be involved in the metabolic pathways of several neurotransmitters that are implicated in the neurobiology of neuropsychiatric disorders. In the present study, we comprehensively examined the associations between common ADH variants [minor allele frequency (MAF) >0.05] and 11 neuropsychiatric and neurological disorders. A total of 50,063 subjects in 25 independent cohorts were analyzed. The entire ADH gene cluster was imputed across these 25 cohorts using the same reference panels. Association analyses were conducted, adjusting for multiple comparisons. We found 28 and 15 single nucleotide polymorphisms (SNPs), respectively, that were significantly associated with schizophrenia in African-Americans and autism in European-Americans after correction by false discovery rate (FDR) (q < 0.05); and 19 and 6 SNPs, respectively, that were significantly associated with these two disorders after region-wide correction by SNPSpD (8.9 × 10(-5) ≤ p ≤ 0.0003 and 2.4 × 10(-5) ≤ p ≤ 0.0003, respectively). No variants were significantly associated with the other nine neuropsychiatric disorders, including alcohol dependence. We concluded that common ADH variants conferred risk for both schizophrenia in African-Americans and autism in European-Americans.

  7. Molecular Characterization of Two Lactate Dehydrogenase Genes with a Novel Structural Organization on the Genome of Lactobacillus sp. Strain MONT4

    PubMed Central

    Weekes, Jennifer; Yüksel, Gülhan Ü.

    2004-01-01

    Two lactate dehydrogenase (ldh) genes from Lactobacillus sp. strain MONT4 were cloned by complementation in Escherichia coli DC1368 (ldh pfl) and were sequenced. The sequence analysis revealed a novel genomic organization of the ldh genes. Subcloning of the individual ldh genes and their Northern blot analyses indicated that the genes are monocistronic. PMID:15466577

  8. Substrate specificity modification of the stromal glycerol-3-phosphate acyltransferase.

    PubMed

    Ferri, S R; Toguri, T

    1997-01-15

    The stromal glycerol-3-phosphate acyltransferases (GPATs; EC 2.3.1.15) from spinach (Spinacia oleracea) and squash (Cucurbita moschata) were expressed in Escherichia coli and their activities with palmitoyl-CoA and oleoyl-CoA compared. The GPAT from squash, a chilling-sensitive plant, was found to have the greatest difference in activities between the two substrates, using palmitoyl-CoA over three times faster than oleoyl-CoA. In contrast, the enzyme from spinach, a chilling-tolerant plant, preferred oleoyl-CoA over palmitoyl-CoA. By using conserved restriction endonuclease sites each of the two genes was divided into three fragments of roughly equal size and recombined to create six different chimeras. All chimeras retained a large portion of their original activity but in most cases the specificity was greatly altered. The central third of the protein was found to contain the structural features which determine substrate specificity of the wild-type GPATs. Two of the chimeras, which have a spinach-derived central region and a squash-derived carboxyl region, were found to have greatly enhanced specificities for 18:1 acyl chains, potentially making them ideal for decreasing the level of saturation of plant membrane lipids through genetic engineering.

  9. Sucrose induces expression of the sorbitol-6-phosphate dehydrogenase gene in source leaves of loquat.

    PubMed

    Suzuki, Yasuo; Dandekar, Abhaya M

    2014-03-01

    Rosaceae fruit trees use sorbitol and sucrose as translocating sugars and the sorbitol-to-sucrose ratio in source leaves determines apple fruit quality. Here, we investigate the effects of sugars on the expression of genes encoding key photosynthetic enzymes, including sorbitol-6-phosphate dehydrogenase (S6PDH, EC 1.1.1.200), sucrose phosphate synthase (SPS, EC 2.4.1.14), and ADP-glucose pyrophosphorylase (ADPGPPase, EC 2.7.7.27) to understand the sugar-signaling mechanism in Rosaceae fruit trees. Mature leaf-petiole cuttings of loquat (Eriobotrya japonica Lindl. cv. Mogi) were supplied with a water, sorbitol or sucrose solution for 2 days at 20°C. The relative levels of the transcripts were analyzed by real-time polymerase chain reaction (PCR). S6PDH transcription was decreased by sorbitol but drastically increased by sucrose. SPS and ADPGPPase large subunit transcription were decreased by sucrose and sorbitol. The simultaneous application of sorbitol and sucrose revealed that S6PDH transcription increased in a dose-dependent manner with sucrose. These results show that both sorbitol and sucrose work as signaling molecules in source organs of Rosaceae fruit trees. These trees have mechanisms to positively keep sorbitol as the dominant translocating sugar, suggesting that sorbitol plays an important role in their survival strategy. Effects of various sugars on S6PDH expression were investigated. Palatinose, a sucrose analog, increased S6PDH transcription much more drastically than sucrose. Mannose and 3-O-methylglucose, glucose analogs, also increased S6PDH transcription; however, glucose did not. Models of sugar signaling in source organs of Rosaceae fruit trees are discussed.

  10. Regulated Expression of Three Alcohol Dehydrogenase Genes in Barley Aleurone Layers 1

    PubMed Central

    Hanson, Andrew D.; Jacobsen, John V.; Zwar, John A.

    1984-01-01

    Three genes specify alcohol dehydrogenase (EC 1.1.1.1.; ADH) enzymes in barley (Hordeum vulgare L.) (Adh 1, Adh 2, and Adh 3). Their polypeptide products (ADH 1, ADH 2, ADH 3) dimerize to give a total of six ADH isozymes which can be resolved by native gel electrophoresis and stained for enzyme activity. Under fully aerobic conditions, aleurone layers of cv Himalaya had a high titer of a single isozyme, the homodimer containing ADH 1 monomers. This isozyme was accumulated by the aleurone tissue during the later part of seed development, and survived seed drying and rehydration. The five other possible ADH isozymes were induced by O2 deficit. The staining of these five isozymes on electrophoretic gels increased progressively in intensity as O2 levels were reduced below 5%, and were most intense at 0% O2. In vivo35S labeling and specific immunoprecipitation of ADH peptides, followed by isoelectric focusing of the ADH peptides in the presence of 8 molar urea (urea-IEF) demonstrated the following. (a) Aleurone layers incubated in air synthesized ADH 1 and a trace of ADH 2; immature layers from developing seeds behaved similarly. (b) At 5% O2, synthesis of ADH 2 increased and ADH 3 appeared. (c) At 2% and 0% O2, the synthesis of all three ADH peptides increased markedly. Cell-free translation of RNA isolated from aleurone layers, followed by immunoprecipitation and urea-IEF of in vitro synthesized ADH peptides, showed that levels of mRNA for all three ADH peptides rose sharply during 1 day of O2 deprivation. Northern hybridizations with a maize Adh 2 cDNA clone established that the clone hybridized with barley mRNA comparable in size to maize Adh 2 mRNA, and that the level of this barley mRNA increased 15- to 20-fold after 1 day at 5% or 2% O2, and about 100-fold after 1 day at 0% O2. We conclude that in aleurone layers, expression of the three barley Adh genes is maximal in the absence of O2, that regulation of mRNA level is likely to be a major controlling factor, and

  11. Dual transcriptional control of the acetaldehyde dehydrogenase gene ald of Corynebacterium glutamicum by RamA and RamB.

    PubMed

    Auchter, Marc; Arndt, Annette; Eikmanns, Bernhard J

    2009-03-10

    Corynebacterium glutamicum has been shown to grow with ethanol as the sole or as additional carbon and energy source and accordingly, to possess both alcohol dehydrogenase and acetaldehyde dehydrogenase (ALDH) activities, which are responsible for the two-step ethanol oxidation to acetate. Here we identify and functionally analyze the C. glutamicum ALDH gene (cg3096, ald), its expression and its regulation. Directed inactivation of the chromosomal ald gene led to the absence of detectable ALDH activity and to the inability to grow on or to utilize ethanol, indicating that the ald gene product is essential for ethanol metabolism and that no ALDH isoenzymes are present in C. glutamicum. Transcriptional analysis revealed that ald from C. glutamicum is monocistronic, that ald transcription is initiated 92 nucleotides upstream of the translational start codon ATG and that ald expression is much lower in the presence of glucose in the growth medium. Further analysis revealed that transcription of the ald gene is under control of the transcriptional regulators RamA and RamB. Both these proteins directly bind to the respective promoter region, RamA is essential for expression and RamB exerts a slightly negative effect on ald expression on all carbon sources tested.

  12. Diverging regulation of pyruvate dehydrogenase kinase isoform gene expression in cultured human muscle cells.

    PubMed

    Abbot, Emily L; McCormack, James G; Reynet, Christine; Hassall, David G; Buchan, Kevin W; Yeaman, Stephen J

    2005-06-01

    The pyruvate dehydrogenase complex occupies a central and strategic position in muscle intermediary metabolism and is primarily regulated by phosphorylation/dephosphorylation. The identification of multiple isoforms of pyruvate dehydrogenase kinase (PDK1-4) and pyruvate dehydrogenase phosphatase (PDP1-2) has raised intriguing new possibilities for chronic pyruvate dehydrogenase complex control. Experiments to date suggest that PDK4 is the major isoenzyme responsible for changes in pyruvate dehydrogenase complex activity in response to various different metabolic conditions. Using a cultured human skeletal muscle cell model system, we found that expression of both PDK2 and PDK4 mRNA is upregulated in response to glucose deprivation and fatty acid supplementation, the effects of which are reversed by insulin treatment. In addition, insulin directly downregulates PDK2 and PDK4 mRNA transcript abundance via a phosphatidylinositol 3-kinase-dependent pathway, which may involve glycogen synthase kinase-3 but does not utilize the mammalian target of rapamycin or mitogen-activated protein kinase signalling pathways. In order to further elucidate the regulation of PDK, the role of the peroxisome proliferators-activated receptors (PPAR) was investigated using highly potent subtype selective agonists. PPARalpha and PPARdelta agonists were found to specifically upregulate PDK4 mRNA expression, whereas PPARgamma activation selectively decreased PDK2 mRNA transcript abundance. PDP1 mRNA expression was unaffected by all conditions analysed. These results suggest that in human muscle, hormonal and nutritional conditions may control PDK2 and PDK4 mRNA expression via a common signalling mechanism. In addition, PPARs appear to independently regulate specific PDK isoform transcipt levels, which are likely to impart important metabolic mediation of fuel utilization by the muscle. PMID:15955060

  13. Sorbitol synthesis by an engineered Lactobacillus casei strain expressing a sorbitol-6-phosphate dehydrogenase gene within the lactose operon.

    PubMed

    Nissen, Lorenzo; Pérez-Martínez, Gaspar; Yebra, María J

    2005-08-01

    Sorbitol is claimed to have important health-promoting effects and Lactobacillus casei is a lactic acid bacterium relevant as probiotic and used as a cheese starter culture. A sorbitol-producing L. casei strain might therefore be of considerable interest in the food industry. A recombinant strain of L. casei was constructed by the integration of a d-sorbitol-6-phosphate dehydrogenase-encoding gene (gutF) in the chromosomal lactose operon (strain BL232). gutF expression in this strain followed the same regulation as that of the lac genes, that is, it was repressed by glucose and induced by lactose. (13)C-nuclear magnetic resonance analysis of supernatants of BL232 resting cells demonstrated that, when pre-grown on lactose, cells were able to synthesize sorbitol from glucose. Inactivation of the l-lactate dehydrogenase gene in BL232 led to an increase in sorbitol production, suggesting that the engineered route provides an alternative pathway for NAD(+) regeneration. PMID:16002237

  14. Metabolism of L-glyceraldehyde 3-phosphate in Escherichia coli

    SciTech Connect

    Kalyananda, M.K.G.S.

    1985-01-01

    E. coli is able to incorporate L-glyceraldehyde and L-glyceraldehyde 3-phosphate into phospholipids, L-(3-/sup 3/H)Glyceraldehyde was synthesized and the purity and the chemical identity of the product were checked by paper chromatography. L-(3-/sup 3/H)Glyceraldehyde 3-phosphate was synthesized from L-(3-/sup 3/H)glyceraldehyde in a reaction catalyzed by glycerokinase. E. coli extract contains a new enzyme activity which catalyzes an NADPH dependent reduction of L-glyceraldehyde 3-phosphate into sn-glycerol 3-phosphate. A procedure, specifically suitable for assaying the reductase activity in the crude extract, was developed. A more convenient spectrophotometric assay method was employed for the purified enzyme. At moderate concentrations sulfhydryl group inhibitors had no effect on the enzyme activity of L-GAP reductase. At 100..mu..M concentration Zn/sup +2/ inhibited the enzyme activity by about 30% while Mn/sup +2/ elevated the activity by about the same margin. Mg/sup +2/, Ca/sup +2/ and Fe/sup +2/ were without effect at this concentration. L-Glyceraldehyde 3-phosphate is known to be bactericidal at 1.25 ..mu..M concentration and the D-enantiomer is without effect. Furthermore, methylglyoxal is known to be bactericidal at or above 0.5 mM concentration. Strains of E. coli resistant to 1 mM methylglyoxal were isolated. The cell extract prepared from the mutant possessed increased capacity to transform methylglyoxal into D-lactate via a glutathione dependent reaction. These mutants were less sensitive to 2.5 mM DL-GAP suggesting that conversion of L-glyceraldehyde 3-phosphate into methylglyoxal may at least partly be responsible for the bactericidal activity of L-GAP.

  15. Essential fructosuria: increased levels of fructose 3-phosphate in erythrocytes.

    PubMed

    Petersen, A; Steinmann, B; Gitzelmann, R

    1992-01-01

    Erythrocytes of 3 adult siblings with essential fructosuria contained 45-200 mumol/l fructose 3-phosphate (Fru-3-P), i.e. 3-15 times the concentration in normal controls. Sorbitol 3-phosphate was also increased, but to a lesser degree. An oral load with 50 g of fructose produced an additional 40 mumol/l increase of erythrocyte Fru-3-P after 5 h. The rate of Fru-3-P formation by red cells in vitro was normal. HbA1 and HbA1c were normal. The suspected pathogenetic role of Fru-3-P in diabetic complications is questioned.

  16. The glycerol-3-phosphate permease GlpT is the only fosfomycin transporter in Pseudomonas aeruginosa.

    PubMed

    Castañeda-García, Alfredo; Rodríguez-Rojas, Alexandro; Guelfo, Javier R; Blázquez, Jesús

    2009-11-01

    Fosfomycin is transported into Escherichia coli via both glycerol-3-phosphate (GlpT) and a hexose phosphate transporter (UhpT). Consequently, the inactivation of either glpT or uhpT confers increased fosfomycin resistance in this species. The inactivation of other genes, including ptsI and cyaA, also confers significant fosfomycin resistance. It has been assumed that identical mechanisms are responsible for fosfomycin transport into Pseudomonas aeruginosa cells. The study of an ordered library of insertion mutants in P. aeruginosa PA14 demonstrated that only insertions in glpT confer significant resistance. To explore the uniqueness of this resistance target in P. aeruginosa, the linkage between fosfomycin resistance and the use of glycerol-3-phosphate was tested. Fosfomycin-resistant (Fos-R) mutants were obtained in LB and minimal medium containing glycerol as the sole carbon source at a frequency of 10(-6). However, no Fos-R mutants grew on plates containing fosfomycin and glycerol-3-phosphate instead of glycerol (mutant frequency, < or = 5 x 10(-11)). In addition, 10 out of 10 independent spontaneous Fos-R mutants, obtained on LB-fosfomycin, harbored mutations in glpT, and in all cases the sensitivity to fosfomycin was recovered upon complementation with the wild-type glpT gene. The analysis of these mutants provides additional insights into the structure-function relationship of glycerol-3-phosphate the transporter in P. aeruginosa. Studies with glucose-6-phosphate and different mutant derivatives strongly suggest that P. aeruginosa lacks a specific transport system for this sugar. Thus, glpT seems to be the only fosfomycin resistance mutational target in P. aeruginosa. The high frequency of Fos-R mutations and their apparent lack of fitness cost suggest that Fos-R variants will be obtained easily in vivo upon the fosfomycin treatment of P. aeruginosa infections.

  17. Cloning and sequencing of the gene cluster encoding two subunits of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenes.

    PubMed

    Tamaki, T; Fukaya, M; Takemura, H; Tayama, K; Okumura, H; Kawamura, Y; Nishiyama, M; Horinouchi, S; Beppu, T

    1991-02-16

    The membrane-bound alcohol dehydrogenase (ADH) from Acetobacter polyoxogenes NBI1028 is composed of a 72 kDa subunit and a 44 kDa cytochrome c subunit. The amino acid sequences of the two regions of the 72 kDa subunit were determined to prepare oligonucleotides for the purpose of amplification of a DNA fragment corresponding to the intermediate region by the polymerase chain reaction. A 0.5 kb DNA fragment thus amplified was used as the probe to clone a 7.0 kb PstI fragment coding for the whole 72 kDa subunit. Nucleotide sequencing and immunoblot analysis revealed that the cloned fragment contained the full structural genes for the 72 kDa and the 44 kDa subunits and they were clustered with the same transcription polarity. The predicted amino acid sequence of the gene for the 72 kDa subunit showed homology with that of the 72 kDa subunit from ADH of A. aceti and those of methanol dehydrogenase from methylotrophic bacteria. The 72 and 44 kDa subunits contained one and three typical haem binding sequences, respectively.

  18. Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

    SciTech Connect

    Li, Yongchao; Tschaplinski, Timothy J; Engle, Nancy L; Hamilton, Choo Yieng; Rodriguez, Jr., Miguel; Liao, James C; Schadt, Christopher Warren; Guss, Adam M; Yang, Yunfeng; Graham, David E

    2012-01-01

    Background: The model bacterium Clostridium cellulolyticum efficiently hydrolyzes crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels. Therefore genetic engineering will likely be required to improve the ethanol yield. Random mutagenesis, plasmid transformation, and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism. Results: The first targeted gene inactivation system was developed for C. cellulolyticum, based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh) and L-malate dehydrogenase (Ccel_0137; mdh) genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products (by molarity), corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four-times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant s TCA pathway. Conclusions: The efficient intron-based gene inactivation system produced the first gene-targeted mutations in C. cellulolyticum. As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in C. cellulolyticum and rapid genetic engineering to

  19. Genome-wide identification and analysis of the aldehyde dehydrogenase (ALDH) gene superfamily in apple (Malus × domestica Borkh.).

    PubMed

    Li, Xiaoqin; Guo, Rongrong; Li, Jun; Singer, Stacy D; Zhang, Yucheng; Yin, Xiangjing; Zheng, Yi; Fan, Chonghui; Wang, Xiping

    2013-10-01

    Aldehyde dehydrogenases (ALDHs) represent a protein superfamily encoding NAD(P)(+)-dependent enzymes that oxidize a wide range of endogenous and exogenous aliphatic and aromatic aldehydes. In plants, they are involved in many biological processes and play a role in the response to environmental stress. In this study, a total of 39 ALDH genes from ten families were identified in the apple (Malus × domestica Borkh.) genome. Synteny analysis of the apple ALDH (MdALDH) genes indicated that segmental and tandem duplications, as well as whole genome duplications, have likely contributed to the expansion and evolution of these gene families in apple. Moreover, synteny analysis between apple and Arabidopsis demonstrated that several MdALDH genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes appeared before the divergence of lineages that led to apple and Arabidopsis. In addition, phylogenetic analysis, as well as comparisons of exon-intron and protein structures, provided further insight into both their evolutionary relationships and their putative functions. Tissue-specific expression analysis of the MdALDH genes demonstrated diverse spatiotemporal expression patterns, while their expression profiles under abiotic stress and various hormone treatments indicated that many MdALDH genes were responsive to high salinity and drought, as well as different plant hormones. This genome-wide identification, as well as characterization of evolutionary relationships and expression profiles, of the apple MdALDH genes will not only be useful for the further analysis of ALDH genes and their roles in stress response, but may also aid in the future improvement of apple stress tolerance.

  20. Thioredoxin-1 Regulates Cellular Heme Insertion by Controlling S-Nitrosation of Glyceraldehyde-3-phosphate Dehydrogenase*

    PubMed Central

    Chakravarti, Ritu; Stuehr, Dennis J.

    2012-01-01

    NO generated by inducible NOS (iNOS) causes buildup of S-nitrosated GAPDH (SNO-GAPDH) in cells, which then inhibits further iNOS maturation by limiting the heme insertion step (Chakravarti, R., Aulak, K. S., Fox, P. L., and Stuehr, D. J. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 18004–18009). We investigated what regulates this process utilizing a slow-release NO donor (NOC-18) and studying changes in cellular SNO-GAPDH levels during and after NO exposure. Culturing macrophage-like cells with NOC-18 during cytokine activation caused buildup of heme-free (apo) iNOS and SNO-GAPDH. Upon NOC-18 removal, the cells quickly recovered their heme insertion capacity in association with rapid SNO-GAPDH denitrosation, implying that these processes are linked. We then altered cell expression of thioredoxin-1 (Trx1) or S-nitrosoglutathione reductase, both of which can function as a protein denitrosylase. Trx1 knockdown increased SNO-GAPDH levels in cells, made heme insertion hypersensitive to NO, and increased the recovery time, whereas Trx1 overexpression greatly diminished SNO-GAPDH buildup and protected heme insertion from NO inhibition. In contrast, knockdown of S-nitrosoglutathione reductase expression had little effect on these parameters. Experiments utilizing C152S GAPDH confirmed that the NO effects are all linked to S-nitrosation of GAPDH at Cys-152. We conclude (i) that NO inhibition of heme insertion and its recovery can be rapid and dynamic processes and are inversely linked to the S-nitrosation of GAPDH and (ii) that the NO sensitivity of heme insertion can vary depending on the Trx1 expression level due to Trx1 acting as an SNO-GAPDH denitrosylase. Together, our results identify a new way that cells regulate heme protein maturation during inflammation. PMID:22457359

  1. The alcohol dehydrogenase gene adh1 is induced in Aspergillus flavus grown on medium conducive to aflatoxin biosynthesis.

    PubMed Central

    Woloshuk, C P; Payne, G A

    1994-01-01

    An Aspergillus flavus cDNA library was screened by differential hybridization to isolate clones corresponding to genes that are actively transcribed under culture conditions conducive to aflatoxin biosynthesis. One clone with a 1.28-kb insert was isolated, and its nucleotide sequence was determined. The nucleotide sequence of this clone had 75% DNA identity to those of the alcohol dehydrogenase genes from Aspergillus nidulans, and the putative polypeptide translated from the cDNA sequence had 82% similarity with the amino acid sequences of the A. nidulans proteins. Thus, this gene has been designated adh1. Southern hybridization analysis of genomic DNA from A. flavus indicated that there was one copy of the adh1 gene. Northern (RNA) hybridization analysis indicated that the adh1 transcript accumulated in culture medium conducive to aflatoxin production and the timing of accumulation of adh1 transcripts was similar to that for aflatoxin. Fusion of the promoter region of adh1 to a beta-glucuronidase reporter gene indicated that accumulation of the adh1 transcript was the result of transcriptional activation. These molecular data support previous physiological evidence that showed the importance of carbohydrate metabolism during aflatoxin biosynthesis. Images PMID:8135521

  2. Disruption of seven hypothetical aryl alcohol dehydrogenase genes from Saccharomyces cerevisiae and construction of a multiple knock-out strain.

    PubMed

    Delneri, D; Gardner, D C; Bruschi, C V; Oliver, S G

    1999-11-01

    By in silicio analysis, we have discovered that there are seven open reading frames (ORFs) in Saccharomyces cerevisiae whose protein products show a high degree of amino acid sequence similarity to the aryl alcohol dehydrogenase (AAD) of the lignin-degrading fungus Phanerochaete chrysosporium. Yeast cultures grown to stationary phase display a significant aryl alcohol dehydrogenase activity by degrading aromatic aldehydes to the corresponding alcohols. To study the biochemical and the biological role of each of the AAD genes, a series of mutant strains carrying deletion of one or more of the AAD-coding sequences was constructed by PCR-mediated gene replacement, using the readily selectable marker kanMX. The correct targeting of the PCR-generated disruption cassette into the genomic locus was verified by analytical PCR and by pulse-field gel electrophoresis (PFGE) followed by Southern blot analysis. Double, triple and quadruple mutant strains were obtained by classical genetic methods, while the construction of the quintuple, sextuple and septuple mutants was achieved by using the marker URA3 from Kluyveromyces lactis, HIS3 from Schizosaccharomyces pombe and TRP1 from S. cerevisiae. None of the knock-out strains revealed any mutant phenotype when tested for the degradation of aromatic aldehydes using both spectrophotometry and high performance liquid chromatography (HPLC). Specific tests for changes in the ergosterol and phospholipids profiles did not reveal any mutant phenotype and mating and sporulation efficiencies were not affected in the septuple deletant. Compared to the wild-type strain, the septuple deletant showed an increased resistance to the anisaldehyde, but there is a possibility that the nutritional markers used for gene replacement are causing this effect.

  3. Eukaryotic aldehyde dehydrogenase (ALDH) genes: human polymorphisms, and recommended nomenclature based on divergent evolution and chromosomal mapping.

    PubMed

    Vasiliou, V; Bairoch, A; Tipton, K F; Nebert, D W

    1999-08-01

    As currently being performed with an increasing number of superfamilies, a standardized gene nomenclature system is proposed here, based on divergent evolution, using multiple alignment analysis of all 86 eukaryotic aldehyde dehydrogenase (ALDH) amino-acid sequences known at this time. The ALDHs represent a superfamily of NAD(P)(+)-dependent enzymes having similar primary structures that oxidize a wide spectrum of endogenous and exogenous aliphatic and aromatic aldehydes. To date, a total of 54 animal, 15 plant, 14 yeast, and three fungal ALDH genes or cDNAs have been sequenced. These ALDHs can be divided into a total of 18 families (comprising 37 subfamilies), and all nonhuman ALDH genes are named here after the established human ALDH genes, when possible. An ALDH protein from one gene family is defined as having approximately < or = 40% amino-acid identity to that from another family. Two members of the same subfamily exhibit approximately > or = 60% amino-acid identity and are expected to be located at the same subchromosomal site. For naming each gene, it is proposed that the root symbol 'ALDH' denoting 'aldehyde dehydrogenase' be followed by an Arabic number representing the family and, when needed, a letter designating the subfamily and an Arabic number denoting the individual gene within the subfamily; all letters are capitalized in all mammals except mouse and fruit fly, e.g. 'human ALDH3A1 (mouse, Drosophila Aldh3a1).' It is suggested that the Human Gene Nomenclature Guidelines (http://++www.gene.ucl.ac.uk/nomenclature/guidelines.h tml) be used for all species other than mouse and Drosophila. Following these guidelines, the gene is italicized, whereas the corresponding cDNA, mRNA, protein or enzyme activity is written with upper-case letters and without italics, e.g. 'human, mouse or Drosophila ALDH3A1 cDNA, mRNA, or activity'. If an orthologous gene between species cannot be identified with certainty, sequential naming of these genes will be carried out

  4. Isolation, characterization, and mapping of gene encoding dihydrolipoyl succinyltransferase (E2k) of human [alpha]-ketoglutarate dehydrogenase complex

    SciTech Connect

    Ali, G.; Cai, Xingang; Sheu, Kwan-Fu R.; Blass, J.P. ); Wasco, W.; Gaston, S.M.; Tanzi, R.E.; Cooper, A.J.L.; Gusella, J.F. ); Szabo, P. )

    1994-03-01

    The authors have isolated and sequenced cDNAs representing the full-length (2987-bp) gene for dihydrolipoyl succinyltransferase (E2k component) of the human [alpha]-ketoglutarate dehydrogenase complex (KHDHC) from a human fetal brain cDNA library. The E2k cDNA was mapped to human chromosome 14 using a somatic cell hybrid panel, and more precisely to band 14q24.3 by in situ hybridization. This cDNA also cross-hybridized to an apparent E2k pseudogene on chromosome 1p31. Northern analysis revealed the E2k gene to be ubiquitously expressed in peripheral tissues and brain. Interestingly, chromosome 14q24.3 has recently been reported to contain gene defects for an early-onset form of familial Alzheimer's disease and for Machado-Joseph disease. Future studies will be necessary to determine whether the E2K gene plays a role in either of these two disorders.

  5. Overexpression of cytokinin dehydrogenase genes in barley (Hordeum vulgare cv. Golden Promise) fundamentally affects morphology and fertility.

    PubMed

    Mrízová, Katarína; Jiskrová, Eva; Vyroubalová, Šárka; Novák, Ondřej; Ohnoutková, Ludmila; Pospíšilová, Hana; Frébort, Ivo; Harwood, Wendy A; Galuszka, Petr

    2013-01-01

    Barley is one of the most important cereal crops grown worldwide. It has numerous applications, but its utility could potentially be extended by genetically manipulating its hormonal balances. To explore some of this potential we identified gene families of cytokinin dehydrogenases (CKX) and isopentenyl transferases, enzymes that respectively irreversibly degrade and synthesize cytokinin (CK) plant hormones, in the raw sequenced barley genome. We then examined their spatial and temporal expression patterns by immunostaining and qPCR. Two CKX-specific antibodies, anti-HvCKX1 and anti-HvCKX9, predominantly detect proteins in the aleurone layer of maturing grains and leaf vasculature, respectively. In addition, two selected CKX genes were used for stable, Agrobacterium tumefaciens-mediated transformation of the barley cultivar Golden Promise. The results show that constitutive overexpression of CKX causes morphological changes in barley plants and prevents their transition to flowering. In all independent transgenic lines roots proliferated more rapidly and root-to-shoot ratios were higher than in wild-type plants. Only one transgenic line, overexpressing CKX under the control of a promoter from a phosphate transporter gene, which is expressed more strongly in root tissue than in aerial parts, yielded progeny. Analysis of several T1-generation plants indicates that plants tend to compensate for effects of the transgene and restore CK homeostasis later during development. Depleted CK levels during early phases of development are restored by down-regulation of endogenous CKX genes and reinforced de novo biosynthesis of CKs.

  6. Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene, XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant.

    PubMed

    Kötter, P; Amore, R; Hollenberg, C P; Ciriacy, M

    1990-12-01

    A P. stipitis cDNA library in lambda gt11 was screened using antisera against P. stipitis xylose reductase and xylitol dehydrogenase, respectively. The resulting cDNA clones served as probes for screening a P. stipitis genomic library. The genomic XYL2 gene was isolated and the nucleotide sequence of the 1089 bp structural gene, and of adjacent non-coding regions, was determined. The XYL2 open-reading frame codes for a protein of 363 amino acids with a predicted molecular mass of 38.5 kDa. The XYL2 gene is actively expressed in S. cerevisiae transformants. S. cerevisiae cells transformed with a plasmid, pRD1, containing both the xylose reductase gene (XYL1) and the xylitol dehydrogenase gene (XYL2), were able to grow on xylose as a sole carbon source. In contrast to aerobic glucose metabolism, S. cerevisiae XYL1-XYL2 transformants utilize xylose almost entirely oxidatively.

  7. Gene structure and chromosomal localization of the human HSD11K gene encoding the kidney (type 2) isozyme of 11{beta}-hydroxysteroid dehydrogenase

    SciTech Connect

    Agarwal, A.K.; Rogerson, F.M.; Mune, T.; White, P.C.

    1995-09-01

    11{beta}-hydroxysteroid dehydrogenase (11{beta}HSD) converts glucocorticoids to inactive products and is thus thought to confer specificity for aldosterone on the type I mineralocorticoid receptor in the kidney. Recent studies indicate the presence of at least two isozymes of 11{beta}HSD. In vitro, the NAD{sup +}-dependent kidney (type 2) isozyme catalyzes 11{beta}-dehydrogenase but not reductase reactions, whereas the NADP{sup +}-dependent liver (type 1) isozyme catalyzes both reactions. We have now characterized the human gene encoding kidney 11{beta}HSD (HSD11K). A bacteriophage P1 clone was isolated after screening a human genomic library by hybridization with sheep HSD11K cDNA. The gene consists of 5 exons spread over 6 kb. The nucleotide binding domain lies in the first exon are GC-rich (80%), suggesting that the gene may be transcriptionally regulated by factors that recognize GC-rich sequences. Fluorescence in situ hybridization of metaphase chromosomes with a positive P1 clone localized the gene to chromosome 16q22. In contrast, the HSD11L (liver isozyme) gene is located on chromosome 1 and contains 6 exons; the coding sequences of these genes are only 21% identical. HSD11K is expressed at high levels in the placenta and kidney of midgestation human fetuses and at lower levels in lung and testes. Different transcriptional start sites are utilized in kidney and placenta. These data should be applicable to genetic analysis of the syndrome of apparent mineralocorticoid excess, which may represent a deficiency of 11{beta}HSD. 25 refs., 5 figs.

  8. Cloning and expression of a putative alcohol dehydrogenase gene of Entamoeba histolytica and its application to immunological examination.

    PubMed Central

    Kimura, A; Hara, Y; Kimoto, T; Okuno, Y; Minekawa, Y; Nakabayashi, T

    1996-01-01

    To clone and express the genes encoding major antigens of Entamoeba histolytica, we constructed a lambda gt11 cDNA library for E. histolytica HM1:IMSS and screened it with pooled sera from patients with amoebiasis. A 1,223-bp cDNA was cloned (clone 1223), and its nucleotide sequence was determined. The amino acid sequence predicted to be encoded by the open reading frame of clone 1223 consisted of 396 residues and showed 32.5 and 32.3% homology to the NADH-dependent butanol dehydrogenases I and II (bdhA and bdhB) of Clostridium acetobutylicum, respectively. In addition, 29 of the 34 consensus positions of bdhA and bdhB were also well conserved in clone 1223. The recombinant protein expressed from clone 1223 had an estimated molecular mass of 43.5 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigenicity and specificity of the recombinant protein were evaluated by an enzyme-linked immunosorbent assay using sera obtained from two clinical groups of patients with amoebiasis and a group of healthy controls. The recombinant protein had potent and specific antigenicity. In all, 53 serum samples (88.3%) from 60 patients with amoebiasis were positive for immunoglobulin G antibody against the recombinant protein, with a mean optical density value of 0.42. In contrast, 53 of 54 healthy control serum samples were negative, with only 1 positive serum sample showing the lower optical density value. These results suggested that clone 1223 is promising in terms of providing a useful antigen for the accurate serodiagnosis of amoebiasis and that the gene encodes a putative alcohol dehydrogenase of E. histolytica. PMID:8705667

  9. [Cloning and expression analysis of glucose-6-phosphate dehydrogenase 1 (G6PDH1) gene from Chimonanthus praecox].

    PubMed

    Wang, Xiao-hui; Liu, Xiao; Gao, Bo-wen; Zhang, Zhong-xiu; Shi, She-po; Tu, Peng-fei

    2015-11-01

    Glucose-6-phosphate dehydrogenase is main regulatory enzyme for pentose phosphate pathway. To amplify the core sequence of G6PDH gene from Chimonanthus praecox, the primers were synthesized, based on the conserved nucleotide sequence of other reported plant G6PDH genes. The specific primers were designed according to the major fragment. The full length cDNA of the G6PDH1 gene was isolated by the 3' and 5' rapid amplification of cDNA ends approach. Transcript levels of G6PDH1 isoform was measured by real-time quantitative RT-PCR in different tissues and in responds to cold treatment. The G6PDH1 subcellular localization, transmembrane domain, three-dimensional structure, and phylogenetic analysis were predicted by different software to analysis the bioinformatics of G6PDH1 protein. The G6PDH1 cDNA sequence was 2 011 bp in length and consisted of 1 551 bp Open Reading Frame (ORF) , encoding a protein of 516 amino acids. Expression analysis results in different tissues showed that G6PDH1 was primarily observed in flowers and roots, as opposed to the leaves and stems. Cold treatment experiments indicated that cold treatment caused a rapid increase in G6PDH1 expression in flowers within 12 h. The full-length cDNA of G6PDH1 and its expression analysis will play an important role for further study on cold stress responses in Ch. praecox. PMID:27071249

  10. Genome-Wide Analysis of Sorbitol Dehydrogenase (SDH) Genes and Their Differential Expression in Two Sand Pear (Pyrus pyrifolia) Fruits

    PubMed Central

    Dai, Meisong; Shi, Zebin; Xu, Changjie

    2015-01-01

    Through RNA-seq of a mixed fruit sample, fourteen expressed sorbitol dehydrogenase (SDH) genes have been identified from sand pear (Pyrus pyrifolia Nakai). Comparative phylogenetic analysis of these PpySDHs with those from other plants supported the closest relationship of sand pear with Chinese white pear (P. bretschneideri). The expression levels varied greatly among members, and the strongest six (PpySDH2, PpySDH4, PpySDH8, PpySDH12, PpySDH13 and PpySDH14) accounted for 96% of total transcript abundance of PpySDHs. Tissue-specific expression of these six members was observed in nine tissues or organs of sand pear, with the greatest abundance found in functional leaf petioles, followed by the flesh of young fruit. Expression patterns of these six PpySDH genes during fruit development were analyzed in two sand pear cultivars, “Cuiguan” and “Cuiyu”. Overall, expression of PpySDHs peaked twice, first at the fruitlet stage and again at or near harvest. The transcript abundance of PpySDHs was higher in “Cuiguan” than in “Cuiyu”, accompanied by a higher content of sugars and higher ratio of fructose to sorbitol maintained in the former cultivar at harvest. In conclusion, it was suggested that multiple members of the SDH gene family are possibly involved in sand pear fruit development and sugar accumulation and may affect both the sugar amount and sugar composition. PMID:26068235

  11. Genome-Wide Analysis of Sorbitol Dehydrogenase (SDH) Genes and Their Differential Expression in Two Sand Pear (Pyrus pyrifolia) Fruits.

    PubMed

    Dai, Meisong; Shi, Zebin; Xu, Changjie

    2015-01-01

    Through RNA-seq of a mixed fruit sample, fourteen expressed sorbitol dehydrogenase (SDH) genes have been identified from sand pear (Pyrus pyrifolia Nakai). Comparative phylogenetic analysis of these PpySDHs with those from other plants supported the closest relationship of sand pear with Chinese white pear (P. bretschneideri). The expression levels varied greatly among members, and the strongest six (PpySDH2, PpySDH4, PpySDH8, PpySDH12, PpySDH13 and PpySDH14) accounted for 96% of total transcript abundance of PpySDHs. Tissue-specific expression of these six members was observed in nine tissues or organs of sand pear, with the greatest abundance found in functional leaf petioles, followed by the flesh of young fruit. Expression patterns of these six PpySDH genes during fruit development were analyzed in two sand pear cultivars, "Cuiguan" and "Cuiyu". Overall, expression of PpySDHs peaked twice, first at the fruitlet stage and again at or near harvest. The transcript abundance of PpySDHs was higher in "Cuiguan" than in "Cuiyu", accompanied by a higher content of sugars and higher ratio of fructose to sorbitol maintained in the former cultivar at harvest. In conclusion, it was suggested that multiple members of the SDH gene family are possibly involved in sand pear fruit development and sugar accumulation and may affect both the sugar amount and sugar composition.

  12. Morphological and metabolic changes in transgenic wheat with altered glycerol-3-phosphate acyltransferase or acyl-acyl carrier protein (ACP) thioesterase activities.

    PubMed

    Edlin, D A; Kille, P; Wilkinson, M D; Jones, H D; Harwood, J L

    2000-12-01

    We have transformed varieties of wheat with a Pisum sativum glycerol-3-phosphate acyltransferase gene, and also with an Arabidopsis thaliana acyl-ACP thioesterase gene. Morphological (growth, organelle development) and metabolic changes (fatty acid labelling of chloroplast and non-chloroplast lipids) have been observed in transgenics with altered gene expression for either enzyme. PMID:11171169

  13. Mutational Analyses of Glucose Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Genes in Pseudomonas fluorescens Reveal Their Effects on Growth and Alginate Production.

    PubMed

    Maleki, Susan; Mærk, Mali; Valla, Svein; Ertesvåg, Helga

    2015-05-15

    The biosynthesis of alginate has been studied extensively due to the importance of this polymer in medicine and industry. Alginate is synthesized from fructose-6-phosphate and thus competes with the central carbon metabolism for this metabolite. The alginate-producing bacterium Pseudomonas fluorescens relies on the Entner-Doudoroff and pentose phosphate pathways for glucose metabolism, and these pathways are also important for the metabolism of fructose and glycerol. In the present study, the impact of key carbohydrate metabolism enzymes on growth and alginate synthesis was investigated in P. fluorescens. Mutants defective in glucose-6-phosphate dehydrogenase isoenzymes (Zwf-1 and Zwf-2) or glucose dehydrogenase (Gcd) were evaluated using media containing glucose, fructose, or glycerol. Zwf-1 was shown to be the most important glucose-6-phosphate dehydrogenase for catabolism. Both Zwf enzymes preferred NADP as a coenzyme, although NAD was also accepted. Only Zwf-2 was active in the presence of 3 mM ATP, and then only with NADP as a coenzyme, indicating an anabolic role for this isoenzyme. Disruption of zwf-1 resulted in increased alginate production when glycerol was used as the carbon source, possibly due to decreased flux through the Entner-Doudoroff pathway rendering more fructose-6-phosphate available for alginate biosynthesis. In alginate-producing cells grown on glucose, disruption of gcd increased both cell numbers and alginate production levels, while this mutation had no positive effect on growth in a non-alginate-producing strain. A possible explanation is that alginate synthesis might function as a sink for surplus hexose phosphates that could otherwise be detrimental to the cell.

  14. Localization of the gene (OGDH) coding for the E1k component of the [alpha]-ketoglutarate dehydrogenase complex to chromosome 7p13-p11. 2

    SciTech Connect

    Szabo, P.; Cai, X.; Ali, G.; Blass, J.P. )

    1994-03-15

    [alpha]-Ketoglutarate dehydrogenase (E1k), also designated oxoglutarate dehydrogenase (OGDH; EC 1.2.4.2), is a component of the enzyme complex that catalyzes the conversion of [alpha]-ketogluterate to succinyl coenzyme A, a critical step in the Krebs tricarboxylic acid cycle. Deficiencies in the activity of this enzyme complex have been observed in brain and peripheral cells of patients with Alzheimer's disease. This finding led the authors to localize the genes for the polypeptides that compose the [alpha]-ketoglutarate dehydrogenase complex (KDGHC). The E1k locus was mapped to chromosome 7p13-p11.2 using a pair of human-rodent somatic cell hybrid panels. A second related sequence, possibly a pseudogene, was identified and mapped to chromosome 10. 16 refs., 1 fig.

  15. Cloning and characterization of a novel betaine aldehyde dehydrogenase gene from Suaeda corniculata.

    PubMed

    Wang, F W; Wang, M L; Guo, C; Wang, N; Li, X W; Chen, H; Dong, Y Y; Chen, X F; Wang, Z M; Li, H Y

    2016-01-01

    Glycine betaine is an important quaternary ammonium compound that is produced in response to several abiotic stresses in many organisms. The synthesis of glycine betaine requires the catalysis of betaine aldehyde dehydrogenase (BADH), which can convert betaine aldehyde into glycine betaine in plants, especially in halotolerant plants. In this study, we isolated the full-length cDNA of BADH from Suaeda corniculata (ScBADH) using reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends. Next, we analyzed the expression profile of ScBADH using real-time PCR. The results showed that ScBADH expression was induced in the roots, stems, and leaves of S. corniculata seedlings under salt and drought stress. Next, ScBADH was overexpressed in Arabidopsis, resulting in the transgenic plants exhibiting enhanced tolerance over wild-type plants under salt and drought stress. We then analyzed the levels of glycine betaine and proline, as well as superoxide dismutase (SOD) activity, during salt stress in WT and transgenic Arabidopsis. The results indicated that overexpression of ScBADH produced more glycine betaine and proline, and increased SOD activity under NaCl treatment. Our results suggest that ScBADH might be a positive regulator in plants during the response to NaCl. PMID:27420939

  16. Target validation of the inosine monophosphate dehydrogenase (IMPDH) gene in Cryptosporidium using Phylomer(®) peptides.

    PubMed

    Jefferies, R; Yang, R; Woh, C K; Weldt, T; Milech, N; Estcourt, A; Armstrong, T; Hopkins, R; Watt, P; Reid, S; Armson, A; Ryan, U M

    2015-01-01

    Cryptosporidiosis, a gastroenteric disease characterised mainly by diarrheal illnesses in humans and mammals is caused by infection with the protozoan parasite Cryptosporidium. Treatment options for cryptosporidiosis are limited, with the current therapeutic nitazoxanide, only partly efficacious in immunocompetent individuals. The parasite lacks de novo purine synthesis, and is exclusively dependant on purine salvage from its host. Inhibition of the inosine 5' monophosphate dehydrogenase (IMPDH), a purine salvage enzyme that is essential for DNA synthesis, thereby offers a potential drug target against this parasite. In the present study, a yeast-two-hybrid system was used to identify Phylomer peptides within a library constructed from the genomes of 25 phylogenetically diverse bacteria that targeted the IMPDH of Cryptosporidium parvum (IMPcp) and Cryptosporidium hominis (IMPch). We identified 38 unique interacting Phylomers, of which, 12 were synthesised and screened against C. parvum in vitro. Two Phylomers exhibited significant growth inhibition (81.2-83.8% inhibition; P < 0.05), one of which consistently exhibited positive interactions with IMPcp and IMPch during primary and recapitulation yeast two-hybrid screening and did not interact with either of the human IMPDH proteins. The present study highlightsthe potential of Phylomer peptides as target validation tools for Cryptosporidium and other organisms and diseases because of their ability to bind with high affinity to target proteins and disrupt function. PMID:25447124

  17. Characterization and regulation of the NADP-linked 7 alpha-hydroxysteroid dehydrogenase gene from Clostridium sordellii.

    PubMed Central

    Coleman, J P; Hudson, L L; Adams, M J

    1994-01-01

    A bile acid-inducible NADP-linked 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) from Clostridium sordellii ATCC 9714 was purified 310-fold by ion-exchange, gel filtration, and dye-ligand affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed one predominant peptide band (30,000 Da). The N-terminal sequence was determined, and the corresponding oligonucleotides were synthesized and used to screen EcoRI and HindIII genomic digests of C. sordellii. Two separate fragments (4,500 bp, EcoRI; 3,200 bp, HindIII) were subsequently cloned by ligation to pUC19 and transformation into Escherichia coli DH5 alpha-MCR. The EcoRI fragment was shown to contain a truncated 7 alpha-HSDH gene, while the HindIII fragment contained the entire coding region. E. coli clones containing the HindIII insert expressed high levels of an NADP-linked 7 alpha-HSDH. Nucleotide sequence analyses suggest that the 7 alpha-HSDH is encoded by a monocistronic transcriptional unit, with DNA sequence elements resembling rho-independent terminators located in both the upstream and downstream flanking regions. The transcriptional start site was located by primer extension analysis. Northern (RNA) blot analysis indicated that induction is mediated at the transcriptional level in response to the presence of bile acid in the growth medium. In addition, growth-phase-dependent expression is observed in uninduced cultures. Analysis of the predicted protein sequence indicates that the enzyme can be classified in the short-chain dehydrogenase group. Images PMID:8050999

  18. A New Bacterial Steroid Degradation Gene Cluster in Comamonas testosteroni TA441 Which Consists of Aromatic-Compound Degradation Genes for Seco-Steroids and 3-Ketosteroid Dehydrogenase Genes

    PubMed Central

    Horinouchi, Masae; Hayashi, Toshiaki; Yamamoto, Takako; Kudo, Toshiaki

    2003-01-01

    In Comamonas testosteroni TA441, testosterone is degraded via aromatization of the A ring, which is cleaved by the meta-cleavage enzyme TesB, and further degraded by TesD, the hydrolase for the product of TesB. TesEFG, encoded downstream of TesD, are probably hydratase, aldolase, and dehydrogenase for degradation of 2-oxohex-4-enoicacid, one of the products of TesD. Here we present a new and unique steroid degradation gene cluster in TA441, which consists of ORF18, ORF17, tesI, tesH, ORF11, ORF12, and tesDEFG. TesH and TesI are 3-ketosteroid-Δ1-dehydrogenase and 3-ketosteroid-Δ4(5α)-dehydrogenase, respectively, which work in the early steps of steroid degradation. ORF17 probably encodes the reductase component of 9α-hydroxylase for 1,4-androstadiene-3,17-dione, which is the product of TesH in testosterone degradation. Gene disruption experiments showed that these genes are necessary for steroid degradation and do not have any isozymes in TA441. By Northern blot analysis, these genes were shown to be induced when TA441 was incubated with steroids (testosterone and cholic acid) but not with aromatic compounds [phenol, biphenyl, and 3-(3-hydroxyphenyl)propionic acid], indicating that these genes function exclusively in steroid degradation. PMID:12902225

  19. A Korean patient with glutaric aciduria type 1 with a novel mutation in the glutaryl CoA dehydrogenase gene.

    PubMed

    Kim, Hee Su; Yu, Hee Joon; Lee, Jeehun; Park, Hyung-Doo; Kim, Ji Hye; Shin, Hyung-Jin; Jin, Dong Kyu; Lee, Munhyang

    2014-01-01

    Mutations in the glutaryl-CoA dehydrogenase gene can result in Glutaric aciduria type 1(GA 1) by accumulation of glutaric acid, 3-hydroxyglutaric acid (3-OH-GA), and glutarylcarnitine (C5DC). GA 1 is characterized by macrocephaly, subdural hemorrhage (SDH), and dystonic movement disorder after acute encephalopathic crisis. We report a Korean patient with GA1 and a novel mutation. A 16-month-old boy presented with SDH, macrocephaly, and developmental delay. In the neurologic examination, the patient had mild axial hypotonia, but otherwise normal neurologic functions. The brain MRI showed large amounts of bilateral SDH and high signal intensity in both basal ganglia and thalamus. Metabolic screening tests detected highly elevated urinary GA levels but 3-OH-glutaric acid was normal. C5DC was 0.94 μM/L (reference range < 0.3 μM/L). The patient had compound heterozygous mutations of the GCDH gene: p.Arg257Gln (c.770G>A) and p.Cys308Arg (c.922T>C). p.Cys308Arg is a novel mutation; reports of p.Arg257Gln were also rare both in Caucasians and Asian populations. In summary, we hereby report one Korean patient with GA1 with clinical, biochemical, and radiologic characteristics confirmed by genetic analysis. PMID:24795062

  20. Pigs fed camelina meal increase hepatic gene expression of cytochrome 8b1, aldehyde dehydrogenase, and thiosulfate transferase

    PubMed Central

    2014-01-01

    Camelina sativa is an oil seed crop which can be grown on marginal lands. Camelina seed oil is rich in omega-3 fatty acids (>35%) and γ-tocopherol but is also high in erucic acid and glucosinolates. Camelina meal, is the by-product after the oil has been extracted. Camelina meal was fed to 28 d old weaned pigs at 3.7% and 7.4% until age 56 d. The camelina meal supplements in the soy based diets, improved feed efficiency but also significantly increased the liver weights. Gene expression analyses of the livers, using intra-species microarrays, identified increased expression of phase 1 and phase 2 drug metabolism enzymes. The porcine versions of the enzymes were confirmed by real time PCR. Cytochrome 8b1 (CYP8B1), aldehyde dehydrogenase 2 (Aldh2), and thiosulfate transferase (TST) were all significantly stimulated. Collectively, these genes implicate the camelina glucosinolate metabolite, methyl-sulfinyldecyl isothiocyanate, as the main xeniobiotic, causing increased hepatic metabolism and increased liver weight. PMID:24383433

  1. Genetic Diversity of Echinococcus granulosus in Southwest China Determined by the Mitochondrial NADH Dehydrogenase Subunit 2 Gene

    PubMed Central

    Wang, Jiahai; Wang, Ning; Hu, Dandan; Zhong, Xiuqin; Wang, Shuxian; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

    2014-01-01

    We evaluated genetic diversity and structure of Echinococcus granulosus by analyzing the complete mitochondrial NADH dehydrogenase subunit 2 (ND2) gene in 51 isolates of E. granulosus sensu stricto metacestodes collected at three locations in this region. We detected 19 haplotypes, which formed a distinct clade with the standard sheep strain (G1). Hence, all 51 isolates were identified as E. granulosus sensu stricto (G1–G3). Genetic relationships among haplotypes were not associated with geographical divisions, and fixation indices (Fst) among sampling localities were low. Hence, regional populations of E. granulosus in the southwest China are not differentiated, as gene flow among them remains high. This information is important for formulating unified region-wide prevention and control measures. We found large negative Fu's Fs and Tajima's D values and a unimodal mismatch distribution, indicating that the population has undergone a demographic expansion. We observed high genetic diversity among the E. granulosus s. s. isolates, indicating that the parasite population in this important bioregion is genetically robust and likely to survive and spread. The data from this study will prove valuable for future studies focusing on improving diagnosis and prevention methods and developing robust control strategies. PMID:24592194

  2. Genetic diversity of Echinococcus granulosus in southwest China determined by the mitochondrial NADH dehydrogenase subunit 2 gene.

    PubMed

    Wang, Jiahai; Wang, Ning; Hu, Dandan; Zhong, Xiuqin; Wang, Shuxian; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

    2014-01-01

    We evaluated genetic diversity and structure of Echinococcus granulosus by analyzing the complete mitochondrial NADH dehydrogenase subunit 2 (ND2) gene in 51 isolates of E. granulosus sensu stricto metacestodes collected at three locations in this region. We detected 19 haplotypes, which formed a distinct clade with the standard sheep strain (G1). Hence, all 51 isolates were identified as E. granulosus sensu stricto (G1-G3). Genetic relationships among haplotypes were not associated with geographical divisions, and fixation indices (Fst) among sampling localities were low. Hence, regional populations of E. granulosus in the southwest China are not differentiated, as gene flow among them remains high. This information is important for formulating unified region-wide prevention and control measures. We found large negative Fu's Fs and Tajima's D values and a unimodal mismatch distribution, indicating that the population has undergone a demographic expansion. We observed high genetic diversity among the E. granulosus s. s. isolates, indicating that the parasite population in this important bioregion is genetically robust and likely to survive and spread. The data from this study will prove valuable for future studies focusing on improving diagnosis and prevention methods and developing robust control strategies.

  3. The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

    PubMed Central

    Saliola, Michele; Bartoccioni, Paola Chiara; De Maria, Ilaria; Lodi, Tiziana; Falcone, Claudio

    2004-01-01

    We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose. PMID:15189981

  4. Deletion of the aceE gene (encoding a component of pyruvate dehydrogenase) attenuates Salmonella enterica serovar Enteritidis.

    PubMed

    Pang, Ervinna; Tien-Lin, Chang; Selvaraj, Madhan; Chang, Jason; Kwang, Jimmy

    2011-10-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major food-borne pathogen. From a transposon insertion mutant library created previously using S. Enteritidis 10/02, one of the mutants was identified to have a 50% lethal dose (LD(50) ) at least 100 times that of the parental strain in young chicks, with an attenuation in a poorly studied gene encoding a component of pyruvate dehydrogenase, namely the aceE gene. Evaluation of the in vitro virulence characteristics of the ΔaceE∷kan mutant revealed that it was less able to invade epithelial cells, less resistant to reactive oxygen intermediate, less able to survive within a chicken macrophage cell line and had a retarded growth rate compared with the parental strain. Young chicks vaccinated with 2 × 10(9) CFU of the ΔaceE∷kan mutant were protected from the subsequent challenge of the parental strain, with the mutant colonized in the liver and spleen in a shorter time than the group infected with the parental strain. In addition, compared with the parental strain, the ΔaceE∷kan mutant did not cause persistent eggshell contamination of vaccinated hens.

  5. Model of early self-replication based on covalent complementarity for a copolymer of glycerate-3-phosphate and glycerol-3-phosphate

    NASA Technical Reports Server (NTRS)

    Weber, Arthur L.

    1989-01-01

    Glyceraldehyde-3-phosphate acts as the substrate in a model of early self-replication of a phosphodiester copolymer of glycerate-3-phosphate and glycerol-3-phosphate. This model of self-replication is based on covalent complementarity in which information transfer is mediated by a single covalent bond, in contrast to multiple weak interactions that establish complementarity in nucleic acid replication. This replication model is connected to contemporary biochemistry through its use of glyceraldehyde-3-phosphate, a central metabolite of glycolysis and photosynthesis.

  6. Heterologous expression of betaine aldehyde dehydrogenase gene from Ammopiptanthus nanus confers high salt and heat tolerance to Escherichia coli.

    PubMed

    Yu, Hao-Qiang; Wang, Ying-Ge; Yong, Tai-Ming; She, Yue-Hui; Fu, Feng-Ling; Li, Wan-Chen

    2014-10-01

    Betaine aldehyde dehydrogenase (BADH) catalyzes the synthesis of glycine betaine, a regulator of osmosis, and therefore BADH is considered to play a significant role in response of plants to abiotic stresses. Here, based on the conserved residues of the deduced amino acid sequences of the homologous BADH genes, we cloned the AnBADH gene from the xerophytic leguminous plant Ammopiptanthus nanus by using reverse transcription PCR and rapid amplification of cDNA ends. The full-length cDNA is 1,868 bp long without intron, and contains an open reading frame of 1512 bp, and 3'- and 5'-untranslated regions of 294 and 62 bp. It encodes a 54.71 kDa protein of 503 amino acids. The deduced amino acid sequence shares high homology, conserved amino acid residues and sequence motifs crucial for the function with the BADHs in other leguminous species. The sequence of the open reading frame was used to construct a prokaryotic expression vector pET32a-AnBADH, and transform Escherichia coli. The transformants expressed the heterologous AnBADH gene under the induction of isopropyl β-D-thiogalactopyranoside, and demonstrated significant enhancement of salt and heat tolerance under the stress conditions of 700 mmol L(-1) NaCl and 55°C high temperature. This result suggests that the AnBADH gene might play a crucial role in adaption of A. nanus to the abiotic stresses, and have the potential to be applied to transgenic operations of commercially important crops for improvement of abiotic tolerance. PMID:25046139

  7. [Cloning of silkworm 3-hydroxyisobutyrate dehydrogenase gene and its expression patterns analysis in simulated weightless environment].

    PubMed

    Tian, Zongcheng; Zhou, Bo; Qian, Airong; Xu, Huiyun; Di, Shengmeng; Zhao, Yunpo; Zhang, Yuping; Liu, Jia; Huang, Yongping; Shang, Peng

    2008-12-01

    The full length cDNA of silkworm hibadh gene was cloned by RT-PCR and RACE (Rapid amplification of cDNA ends) technique. The hibadh gene and its deduced amino acid sequences were analyzed. The tissue distribution of hibadh gene in 5th instar silkworm larvae was tested by RT-PCR. The expression patterns of hibadh gene in simulated weightless environment were analyzed by real time RT-PCR. The results showed that the full length hibadh cDNA sequence was 1074 bp in lenth, including an open read frame of 969 bp encoding the entire coding region of Hibadh (GenBank accession No. EU719652). The deduced amino acid sequence similarities of hibadh between silkworm and Burkholderia ambifaria, Drosophila melanogaster, Apis mellifera, Xenopus tropicalis, Mus musculus, Homo sapiens were 46%, 43%, 48%, 44%, 45%, 45%, respectively. Signal peptide analysis showed that Hibadh was a secretory protein. There wasn't glycosyl-phosphatidyl inositol anchor site in Hibadh amino acid sequence. Molecular weight and isoelectric point of Hibadh were 34.1 kD and 9.14 respectively. The RT-PCR tests indicated that the hibadh gene expressed in head, silk gland, midgut, cuticle, blood, fat body, tuba malpighii of the 5th instar silkworm larvae. There were different expression patterns of hibadh gene during different silkworm embryo period in simulated weightless environment. Simulated weightlessness resulted in the expression of silkworm hibadh gene up regulated 2.3-fold (P < 0.05), up regulated 4.6-fold (P<0.01), down regulated 7.6-fold (P < 0.01), down regulated 2.6-fold (P < 0.05) during apophysis formation period, inverse period, trachea formation period, and whole embryo period, respectively. There was no significant change of hibadh gene expression during other period of silkworm embryo between simulated weightless and control groups. There were different response patterns to simulated weightless environment between hibadh gene and whole body of silkworm. Gene showed much higher sensitivity

  8. The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene, adhE, Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

    PubMed Central

    Lo, Jonathan; Zheng, Tianyong; Hon, Shuen; Olson, Daniel G.

    2015-01-01

    ABSTRACT Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase. IMPORTANCE Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol

  9. 4-dihydrotrisporin-dehydrogenase, an enzyme of the sex hormone pathway of Mucor mucedo: purification, cloning of the corresponding gene, and developmental expression.

    PubMed

    Wetzel, Jana; Scheibner, Olaf; Burmester, Anke; Schimek, Christine; Wöstemeyer, Johannes

    2009-01-01

    The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (-) mating-type-specific enzyme in the pathway from beta-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (-) mating type of Mucor mucedo was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene, TSP2, was isolated. A labeled TSP2 hybridization probe detects a single-copy gene in the genome of M. mucedo. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of M. mucedo. At the enzyme level, activity is found exclusively in the (-) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the TSP2 gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the TSP2 genes in the three sequenced zygomycetous genomes (Phycomyces blakesleeanus, Rhizopus oryzae, and Mucor circinelloides), the closest relative is the Myxococcus xanthus CsgA gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation.

  10. 4-Dihydrotrisporin-Dehydrogenase, an Enzyme of the Sex Hormone Pathway of Mucor mucedo: Purification, Cloning of the Corresponding Gene, and Developmental Expression▿

    PubMed Central

    Wetzel, Jana; Scheibner, Olaf; Burmester, Anke; Schimek, Christine; Wöstemeyer, Johannes

    2009-01-01

    The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (−) mating-type-specific enzyme in the pathway from β-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (−) mating type of Mucor mucedo was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene, TSP2, was isolated. A labeled TSP2 hybridization probe detects a single-copy gene in the genome of M. mucedo. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of M. mucedo. At the enzyme level, activity is found exclusively in the (−) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the TSP2 gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the TSP2 genes in the three sequenced zygomycetous genomes (Phycomyces blakesleeanus, Rhizopus oryzae, and Mucor circinelloides), the closest relative is the Myxococcus xanthus CsgA gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation. PMID:18931040

  11. Functional characterization of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 10/glycerol-3-phosphate acyltransferase isoform 3

    PubMed Central

    Sukumaran, Suja; Barnes, Robert I; Garg, Abhimanyu; Agarwal, Anil K

    2016-01-01

    Synthesis of phospholipids can occur de novo or via remodeling of the existing phospholipids. Synthesis of triglycerides, a form of energy storage in cells, is an end product of these pathways. Several 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs) acylate lysophosphatidic acid (LPA) at the sn-2 (carbon 2) position to produce phosphatidic acid (PA). These enzymes are involved in phospholipids and triglyceride synthesis through an evolutionary conserved process involving serial acylations of glycerol-3-phosphate. We cloned a cDNA predicted to be an AGPAT isoform (AGPAT10). This cDNA has been recently identified as glycerol-3-phosphate-O-acyltransferase isoform 3 (GPAT3). When this AGPAT10/GPAT3 cDNA was expressed in Chinese Hamster ovary cells, the protein product localizes to the endoplasmic reticulum. In vitro enzymatic activity using lysates of human embryonic kidney-293 cells infected with recombinant AGPAT10/GPAT3 adenovirus show that the protein has a robust AGPAT activity with an apparent Vmax of 2 nmol/min per mg protein, but lacks GPAT enzymatic activity. This AGPAT has similar substrate specificities for LPA and acyl-CoA as shown for another known isoform, AGPAT2. We further show that when overexpressed in human Huh-7 cells depleted of endogenous AGPAT activity by sh-RNA-AGPAT2-lentivirus, the protein again demonstrates AGPAT activity. These observations strongly suggest that the cDNA previously identified as GPAT3 has AGPAT activity and thus we prefer to identify this clone as AGPAT10 as well. PMID:19318427

  12. 4-Dihydromethyltrisporate dehydrogenase from Mucor mucedo, an enzyme of the sexual hormone pathway: purification, and cloning of the corresponding gene.

    PubMed

    Czempinski, K; Kruft, V; Wöstemeyer, J; Burmester, A

    1996-09-01

    We have purified the NADP-dependent 4-dihydromethyltrisporate dehydrogenase from the zygomycete Mucor mucedo. The enzyme is involved in the biosynthesis of trisporic acid, the sexual hormone of zygomycetes, which induces the first steps of zygophore development. Protein was obtained from the (-) mating type of M. mucedo after induction with trisporic acid, and purified by gel filtration and affinity chromatography steps. On SDS-PAGE a band with an apparent molecular mass of 33 kDa was ascribed to the enzyme. After transferring onto PVDF membranes the protein was digested with endoprotease Lys-C, and several peptides were sequenced. Oligonucleotides derived from protein sequence data were used for PCR amplification of genomic M. mucedo DNA. The PCR fragment was used as probe for isolation of the corresponding cDNA and complete genomic DNA clones. Comparison of protein and DNA sequence data showed that the cloned fragment corresponded to the purified protein. Search for similarity with protein sequences of the Swiss-Prot database revealed a relationship to enzymes belonging to the aldo/keto reductase superfamily. Southern-blot analysis of genomic DNA with the labelled cloned fragment detected a single-copy gene in both mating types of M. mucedo. PCR with genomic DNA from other zygomycetes gave rise to several fragments. Hybridization analysis with the cloned M. mucedo fragment showed that a fragment of similar length cross-hybridized in Blakeslea trispora (Choanephoraceae) as well as in Parasitella parasitica and Absidia glauca (Mucoraceae). The promoter region of the gene contains DNA elements with similarity to a cAMP-regulated gene of Dictyostelium discoideum.

  13. Molecular cloning, functional analysis of three cinnamyl alcohol dehydrogenase (CAD) genes in the leaves of tea plant, Camellia sinensis.

    PubMed

    Deng, Wei-Wei; Zhang, Ming; Wu, Jian-Qiang; Jiang, Zheng-Zhong; Tang, Lei; Li, Ye-Yun; Wei, Chao-Ling; Jiang, Chang-Jun; Wan, Xiao-Chun

    2013-02-15

    Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) is considered to be a key enzyme in lignin biosynthesis, but little was known about CADs in tea plants (Camellia sinensis). A full-length cDNA sequence (CsCAD2) was isolated by suppressive subtractive hybridization (SSH) in Ectropis oblique feeding-induced tea plants, and another two full-length cDNA sequences (CsCAD1 and CsCAD3) were obtained from a transcriptome obtained by deep sequencing. However, they showed only 20-54% identities. Phylogenetic analysis revealed that they belonged to three different families. DNA gel blotting analysis revealed that two copies of CsCAD1 and CsCAD2 genes existed in tea genome, but CsCAD3 likely had only one copy. Recombinant proteins of these CsCADs were produced in Escherichia coli. The activity of purified recombinant CsCAD2 protein was up to 0.43 μmol min(-1) mg(-1). However, the other two recombinant proteins had lower activities, probably due to incomplete refolding. qRT-PCR analysis indicated that while CsCAD3 was strongly up-regulated in tea plants after E. oblique attack and mechanical damage, CsCAD1 and CsCAD2 showed only moderate or no changes in transcript levels. Treatment of defence-related hormones methyl jasmonate (MeJA) and salicylic acid (SA) elevated the expression of CsCAD1 and CsCAD2, but decreased the transcript abundance of CsCAD3. The transcript levels of CsCAD2 did not change after applying abscisic acid (ABA), whereas CsCAD1 and CsCAD3 were induced. These results suggested that these three CsCAD genes in tea plants may play a role in defense against insects and pathogens and adaptation to abiotic stresses and these genes likely have divergant functions.

  14. Identification and Characterization of the Glucose-6-Phosphate Dehydrogenase Gene Family in the Para Rubber Tree, Hevea brasiliensis

    PubMed Central

    Long, Xiangyu; He, Bin; Fang, Yongjun; Tang, Chaorong

    2016-01-01

    As a key enzyme in the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PDH) provides nicotinamide adenine dinucleotide phosphate (NADPH) and intermediary metabolites for rubber biosynthesis, and plays an important role in plant development and stress responses. In this study, four Hevea brasiliensis (Para rubber tree) G6PDH genes (HbG6PDH1 to 4) were identified and cloned using a genome-wide scanning approach. All four HbG6PDH genes encode functional G6PDH enzymes as shown by heterologous expression in E. coli. Phylogeny analysis and subcellular localization prediction show that HbG6PDH3 is a cytosolic isoform, while the other three genes (HbG6PDH1, 2 and 4) are plastidic isoforms. The subcellular locations of HbG6PDH3 and 4, two latex-abundant isoforms were further verified by transient expression in rice protoplasts. Enzyme activity assay and expression analysis showed HbG6PDH3 and 4 were implicated in PPP during latex regeneration, and to influence rubber production positively in rubber tree. The cytosolic HbG6PDH3 is a predominant isoform in latex, implying a principal role for this isoform in controlling carbon flow and NADPH production in the PPP during latex regeneration. The expression pattern of plastidic HbG6PDH4 correlates well with the degree of tapping panel dryness, a physiological disorder that stops the flow of latex from affected rubber trees. In addition, the four HbG6PDHs responded to temperature and drought stresses in root, bark, and leaves, implicating their roles in maintaining redox balance and defending against oxidative stress. PMID:26941770

  15. Enhanced drought and heat stress tolerance of tobacco plants with ectopically enhanced cytokinin oxidase/dehydrogenase gene expression.

    PubMed

    Macková, Hana; Hronková, Marie; Dobrá, Jana; Turečková, Veronika; Novák, Ondřej; Lubovská, Zuzana; Motyka, Václav; Haisel, Daniel; Hájek, Tomáš; Prášil, Ilja Tom; Gaudinová, Alena; Štorchová, Helena; Ge, Eva; Werner, Tomáš; Schmülling, Thomas; Vanková, Radomíra

    2013-07-01

    Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms.

  16. Isoepoxydon dehydrogenase (idh) gene expression in relation to patulin production by Penicillium expansum under different temperature and atmosphere.

    PubMed

    De Clercq, N; Vlaemynck, G; Van Pamel, E; Van Weyenberg, S; Herman, L; Devlieghere, F; De Meulenaer, B; Van Coillie, E

    2016-03-01

    Penicillium expansum growth and patulin production occur mainly at post-harvest stage during the long-term storage of apples. Low temperature in combination with reduced oxygen concentrations is commonly applied as a control strategy to extend apple shelf life and supply the market throughout the year. Our in vitro study investigated the effect of temperature and atmosphere on expression of the idh gene in relation to the patulin production by P. expansum. The idh gene encodes the isoepoxydon dehydrogenase enzyme, a key enzyme in the patulin biosynthesis pathway. First, a reverse transcription real-time PCR (RT-qPCR) method was optimized to measure accurately the P. expansum idh mRNA levels relative to the mRNA levels of three reference genes (18S, β-tubulin, calmodulin), taking into account important parameters such as PCR inhibition and multiple reference gene stability. Subsequently, two P. expansum field isolates and one reference strain were grown on apple puree agar medium (APAM) under three conditions of temperature and atmosphere: 20 °C - air, 4 °C - air and 4 °C - controlled atmosphere (CA; 3% O2). When P. expansum strains reached a 0.5 and 2.0 cm colony diameter, idh expression and patulin concentrations were determined by means of the developed RT-qPCR and an HPLC-UV method, respectively. The in vitro study showed a clear reduction in patulin production and down-regulation of the idh gene expression when P. expansum was grown under 4 °C - CA. The results suggest that stress (low temperature and oxygen level) caused a delay of the fungal metabolism rather than a complete inhibition of toxin biosynthesis. A good correlation was found between the idh expression and patulin production, corroborating that temperature and atmosphere affected patulin production by acting at the transcriptional level of the idh gene. Finally, a reliable RT-qPCR can be considered as an alternative tool to investigate the effect of control strategies on the toxin formation in

  17. Isoepoxydon dehydrogenase (idh) gene expression in relation to patulin production by Penicillium expansum under different temperature and atmosphere.

    PubMed

    De Clercq, N; Vlaemynck, G; Van Pamel, E; Van Weyenberg, S; Herman, L; Devlieghere, F; De Meulenaer, B; Van Coillie, E

    2016-03-01

    Penicillium expansum growth and patulin production occur mainly at post-harvest stage during the long-term storage of apples. Low temperature in combination with reduced oxygen concentrations is commonly applied as a control strategy to extend apple shelf life and supply the market throughout the year. Our in vitro study investigated the effect of temperature and atmosphere on expression of the idh gene in relation to the patulin production by P. expansum. The idh gene encodes the isoepoxydon dehydrogenase enzyme, a key enzyme in the patulin biosynthesis pathway. First, a reverse transcription real-time PCR (RT-qPCR) method was optimized to measure accurately the P. expansum idh mRNA levels relative to the mRNA levels of three reference genes (18S, β-tubulin, calmodulin), taking into account important parameters such as PCR inhibition and multiple reference gene stability. Subsequently, two P. expansum field isolates and one reference strain were grown on apple puree agar medium (APAM) under three conditions of temperature and atmosphere: 20 °C - air, 4 °C - air and 4 °C - controlled atmosphere (CA; 3% O2). When P. expansum strains reached a 0.5 and 2.0 cm colony diameter, idh expression and patulin concentrations were determined by means of the developed RT-qPCR and an HPLC-UV method, respectively. The in vitro study showed a clear reduction in patulin production and down-regulation of the idh gene expression when P. expansum was grown under 4 °C - CA. The results suggest that stress (low temperature and oxygen level) caused a delay of the fungal metabolism rather than a complete inhibition of toxin biosynthesis. A good correlation was found between the idh expression and patulin production, corroborating that temperature and atmosphere affected patulin production by acting at the transcriptional level of the idh gene. Finally, a reliable RT-qPCR can be considered as an alternative tool to investigate the effect of control strategies on the toxin formation in

  18. Departure from neutrality at the mitochondrial NADH dehydrogenase subunit 2 gene in humans, but not in chimpanzees.

    PubMed Central

    Wise, C A; Sraml, M; Easteal, S

    1998-01-01

    To test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution, nucleotide sequences were determined for the 1041 bp of the NADH dehydrogenase subunit 2 (ND2) gene in 20 geographically diverse humans and 20 common chimpanzees. Contingency tests of neutrality were performed using four mutational categories for the ND2 molecule: synonymous and nonsynonymous mutations in the transmembrane regions, and synonymous and nonsynonymous mutations in the surface regions. The following three topological mutational categories were also used: intraspecific tips, intraspecific interiors, and interspecific fixed differences. The analyses reveal a significantly greater number of nonsynonymous polymorphisms within human transmembrane regions than expected based on interspecific comparisons, and they are inconsistent with a neutral equilibrium model. This pattern of excess nonsynonymous polymorphism is not seen within chimpanzees. Statistical tests of neutrality, such as TAJIMA's D test, and the D and F tests proposed by FU and LI, indicate an excess of low frequency polymorphisms in the human data, but not in the chimpanzee data. This is consistent with recent directional selection, a population bottleneck or background selection of slightly deleterious mutations in human mtDNA samples. The analyses further support the idea that mitochondrial genome evolution is governed by selective forces that have the potential to affect its use as a "neutral" marker in evolutionary and population genetic studies. PMID:9475751

  19. Glu504Lys Single Nucleotide Polymorphism of Aldehyde Dehydrogenase 2 Gene and the Risk of Human Diseases

    PubMed Central

    Zhao, Yan; Wang, Chuancai

    2015-01-01

    Aldehyde dehydrogenase (ALDH) 2 is a mitochondrial enzyme that is known for its important role in oxidation and detoxification of ethanol metabolite acetaldehyde. ALDH2 also metabolizes other reactive aldehydes such as 4-hydroxy-2-nonenal and acrolein. The Glu504Lys single nucleotide polymorphism (SNP) of ALDH2 gene, which is found in approximately 40% of the East Asian populations, causes defect in the enzyme activity of ALDH2, leading to alterations in acetaldehyde metabolism and alcohol-induced “flushing” syndrome. Evidence suggests that ALDH2 Glu504Lys SNP is a potential candidate genetic risk factor for a variety of chronic diseases such as cardiovascular disease, cancer, and late-onset Alzheimer's disease. In addition, the association between ALDH2 Glu504Lys SNP and the development of these chronic diseases appears to be affected by the interaction between the SNP and lifestyle factors such as alcohol consumption as well as by the presence of other genetic variations. PMID:26491656

  20. A new high phenyl lactic acid-yielding Lactobacillus plantarum IMAU10124 and a comparative analysis of lactate dehydrogenase gene.

    PubMed

    Zhang, Xiqing; Zhang, Shuli; Shi, Yan; Shen, Fadi; Wang, Haikuan

    2014-07-01

    Phenyl lactic acid (PLA) has been widely reported as a new natural antimicrobial compound. In this study, 120 Lactobacillus plantarum strains were demonstrated to produce PLA using high-performance liquid chromatography. Lactobacillus plantarum IMAU10124 was screened with a PLA yield of 0.229 g L(-1) . Compared with all previous reports, this is the highest PLA-producing lactic acid bacteria (LAB) when grown in MRS broth without any optimizing conditions. When 3.0 g L(-1) phenyl pyruvic acid (PPA) was added to the medium as substrate, PLA production reached 2.90 g L(-1) , with the highest 96.05% conversion rate. A lowest PLA-yielding L. plantarum IMAU40105 (0.043 g L(-1) ) was also screened. It was shown that the conversion from PPA to PLA by lactic dehydrogenase (LDH) is the key factor in the improvement of PLA production by LAB. Comparing the LDH gene of two strains, four amino acid mutation sites were found in this study in the LDH of L. plantarum IMAU10124.

  1. Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

    PubMed Central

    Henningsen, Brooks M.; Hon, Shuen; Covalla, Sean F.; Sonu, Carolina; Argyros, D. Aaron; Barrett, Trisha F.; Wiswall, Erin; Froehlich, Allan C.

    2015-01-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter−1 acetate during fermentation of 114 g liter−1 glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter−1, this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter−1 and raised the ethanol yield to 7% above the wild-type level. PMID:26386051

  2. Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

    PubMed

    Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

    2015-12-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level.

  3. Improved production of homo-D-lactic acid via xylose fermentation by introduction of xylose assimilation genes and redirection of the phosphoketolase pathway to the pentose phosphate pathway in L-Lactate dehydrogenase gene-deficient Lactobacillus plantarum.

    PubMed

    Okano, Kenji; Yoshida, Shogo; Yamada, Ryosuke; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

    2009-12-01

    The production of optically pure d-lactic acid via xylose fermentation was achieved by using a Lactobacillus plantarum NCIMB 8826 strain whose l-lactate dehydrogenase gene was deficient and whose phosphoketolase genes were replaced with a heterologous transketolase gene. After 60 h of fermentation, 41.2 g/liter of d-lactic acid was produced from 50 g/liter of xylose.

  4. Cloning, sequencing, and functional analysis of the 5'-flanking region of the rat 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase gene.

    PubMed

    Lin, H K; Penning, T M

    1995-09-15

    Rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HSD/DD) is a member of the aldo-keto reductase gene superfamily. It displays high constitutive expression and inactivates circulating steroid hormones and suppresses the formation of polycyclic aromatic hydrocarbon anti- and syn-diol-epoxides (ultimate carcinogens). To elucidate mechanisms responsible for constitutive expression of the 3 alpha-HSD/DD gene a rat genomic library obtained from adult Sprague-Dawley female liver (HaeIII partial digest) was screened, using a probe corresponding to the 5'-end of the cDNA (-15 to +250), and a 15.8-kb genomic clone was isolated. Sequencing revealed that 6.3 kb contained exon 1 (+16 to +138 bp) plus additional introns and exons. The transcription start site (+1) was located by primer extension analysis, and the initiation codon, ATG, was located at +55 bp. The remaining 9.5 kb represented the 5'-flanking region of the rat 3 alpha-HSD/DD gene. A 1.6-kb fragment of this region was sequenced. A TATTTAA sequence (TATA box) was found at 33 bp upstream from the major transcription start site. cis-acting elements responsible for the constitutive expression of the rat 3 alpha-HSD/DD gene were located on the 5'-flanking region by transient transfection of reporter-gene (chloramphenicol acetyl transferase, CAT) constructs into human hepatoma cells (HepG2). CAT assays identified the basal promoter between (-199 and +55 bp), the presence of a proximal enhancer (-498 to -199 bp) which stimulated CAT activity 6-fold, the existence of a powerful silencer (-755 to -498 bp), and a strong distal enhancer (-4.0 to -2.0 kb) which increased CAT activity by 20-40-fold. A computer search of available consensus sequences for trans-acting factors revealed that a cluster of Oct-sites were uniquely located in the silencer region. Using the negative response element (-797 to -498 bp) as a probe and nuclear extracts from HepG2 cells, three bands were identified by gel mobility shift

  5. Sox3 binds to 11β-hydroxysteroid dehydrogenase gene promoter suggesting transcriptional interaction in catfish.

    PubMed

    Rajakumar, Anbazhagan; Senthilkumaran, Balasubramanian

    2016-04-01

    In fishes, the expression of steroidogenic enzyme genes and their related transcription factors (TFs) are critical for the regulation of steroidogenesis and gonadal development. 11-KT is the potent androgen and hence, 11β-hsd, enzyme involved in 11-KT production is important. Regulation of 11β-hsd gene was never studied in any fishes. At first 11β-hsd was cloned and recombinant protein was tested for enzyme activity prior to expression and promoter motif analysis. Expression changes revealed stage- and sex-dependent increase in the ontogenic studies. Further, 11β-hsd expression was higher during spawning phase of reproductive cycle and was found to be gonadotropin inducible both in vivo and in vitro. ∼2kb of 5' upstream region of 11β-hsd, was cloned from catfish genomic DNA library and in silico promoter analysis revealed putative TF binding sites such as Sox3, Wt1, Pax2, Dmrt1 and Ad4BP/SF-1. Luciferase reporter assay using the sequential deletion constructs in human embryonic kidney and Chinese hamster ovary cells revealed considerable promoter activity of the constructs containing Sox3, but not with other motifs largely. Site-directed mutagenesis, Sox3 over expression, electrophoretic mobility shift and chromatin immunoprecipitation assays further substantiated the binding of Sox3 to its corresponding cis-acting element in the upstream promoter motif of 11β-hsd. This is the first report to show that Sox3 binds to the 11β-hsd gene promoter and transactivates to regulate male reproduction in a teleost. PMID:26772480

  6. Inactivation of the 3-phosphoglycerate dehydrogenase gene in mice: changes in gene expression and associated regulatory networks resulting from serine deficiency.

    PubMed

    Furuya, Shigeki; Yoshida, Kazuyuki; Kawakami, Yuriko; Yang, Jyung Hoon; Sayano, Tomoko; Azuma, Norihiro; Tanaka, Hideyuki; Kuhara, Satoru; Hirabayashi, Yoshio

    2008-08-01

    D-3-Phosphoglycerate dehydrogenase (Phgdh) is a necessary enzyme for de novo L-serine biosynthesis. Mutations in the human PHGDH cause serine deficiency disorders characterized by severe neurological symptoms including congenital microcephaly and psychomotor retardation. We showed previously that targeted disruption of Phgdh in mice causes overall growth retardation with severe brain microcephaly and leads to embryonic lethality. Here, amino acid analysis of Phgdh knockout (KO) mouse embryos demonstrates that free serine and glycine concentrations are decreased markedly in head samples, reflecting the metabolic changes of serine deficiency found in human patients. To understand the pathogenesis of serine deficiency disorders at the molecular level, we have exploited this animal model to identify altered gene expression patterns using a microarray technology. Comparative microarray analysis of the Phgdh KO and wild-type head at gestational day 13.5 revealed an upregulation of genes involved in transfer RNA aminoacylation, amino acid metabolism, amino acid transport, transcriptional regulation, and translation, and a downregulation of genes involved in transcription in neuronal progenitors and muscle and cartilage development. A computational network analysis software was used to construct transcriptional regulatory networks operative in the Phgdh KO embryos in vivo. These observations suggest that Phgdh inactivation alters transcriptional programs in several regulatory networks. PMID:18228065

  7. Down-regulation of rat mitochondrial branched-chain 2-oxoacid dehydrogenase kinase gene expression by glucocorticoids.

    PubMed Central

    Huang, Y S; Chuang, D T

    1999-01-01

    The mammalian mitochondrial branched-chain 2-oxoacid dehydrogenase (BCOD) complex is regulated by a reversible phosphorylation (inactivation)/dephosphorylation (activation) cycle. In the present study, the effects of glucocorticoids on the level of BCOD kinase mRNA were investigated in rat hepatoma cell lines (H4IIE and FTO-2B), as well as in the rat. In H4IIE cells, dexamethasone was found to significantly reduce steady-state concentrations of BCOD kinase mRNA after a 48 h culture, and this was correlated with a 2-fold increase in the dephosphorylated form of the BCOD complex. The half-life of the kinase mRNA in H4IIE cells was not affected by dexamethasone treatment. Therefore, the decrease in the steady-state kinase mRNA level resulting from dexamethasone treatment was not caused by changes in mRNA stability, which raised the possibility of regulation at the level of gene transcription. To identify the negative glucocorticoid-responsive element in the kinase promoter, nested deletion constucts in the 3.0 kb promoter region were examined in H4IIE cells cultured in the presence or absence of dexamethasone. No significant differences in promoter activity were observed on either transient or stable transfection. The data showed that the glucocorticoid-responsive element was located outside the 3. 0 kb promoter region. At the physiological level, hepatic BCOD kinase mRNA levels were reduced in rats injected intraperitoneally with dexamethasone. This effect was liver-specific, and was not detected in other tissues. These results suggest that the down-regulation of kinase gene expression by glucocorticoids is mediated through a liver-specific or -enriched transcription factor(s). PMID:10215586

  8. Mutations in PDX1, the human lipoyl-containing component X of the pyruvate dehydrogenase-complex gene on chromosome 11p1, in congenital lactic acidosis.

    PubMed Central

    Aral, B; Benelli, C; Ait-Ghezala, G; Amessou, M; Fouque, F; Maunoury, C; Créau, N; Kamoun, P; Marsac, C

    1997-01-01

    We have identified and sequenced a cDNA that encodes an apparent human orthologue of a yeast protein-X component (ScPDX1) of pyruvate dehydrogenase multienzyme complexes. The new human cDNA that has been referred to as "HsPDX1" cDNA was cloned by use of the "database cloning" strategy and had a 1,506-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 20% identical with that encoded by the yeast PDX1 gene and 40% identical with that encoded by the lipoate acetyltransferase component of the pyruvate dehydrogenase and included a lipoyl-bearing domain that is conserved in some dehydrogenase enzyme complexes. Northern blot analysis demonstrated that the major HsPDX1 mRNA was 2.5 kb in length and was expressed mainly in human skeletal and cardiac muscles but was also present, at low levels, in other tissues. FISH analysis performed with a P1-derived artificial chromosome (PAC)-containing HsPDX1 gene sublocalized the gene to 11p1.3. Molecular investigation of PDX1 deficiency in four patients with neonatal lactic acidemias revealed mutations 78del85 and 965del59 in a homozygous state, and one other patient had no PDX1 mRNA expression. Images Figure 2 Figure 3 Figure 4 PMID:9399911

  9. Genomic clones of Aspergillus nidulans containing alcA, the structural gene for alcohol dehydrogenase and alcR, a regulatory gene for ethanol metabolism.

    PubMed

    Doy, C H; Pateman, J A; Olsen, J E; Kane, H J; Creaser, E H

    1985-04-01

    Our aim was to obtain from Aspergillus nidulans a genomic bank and then clone a region we expected from earlier genetic mapping to contain two closely linked genes, alcA, the structural gene for alcohol dehydrogenase (ADH) and alcR, a positive trans-acting regulatory gene for ethanol metabolism. The expression of alcA is repressed by carbon catabolites. A genomic restriction fragment characteristic of the alcA-alcR region was identified, cloned in pBR322, and used to select from a genomic bank in lambda EMBL3A three overlapping clones covering 24 kb of DNA. Southern genomic analysis of wild-type, alcA and alcR mutants showed that the mutants contained extra DNA at sites near the center of the cloned DNA and are close together, as expected for alcA and alcR. Transcription from the cloned DNA and hybridization with a clone carrying the Saccharomyces cerevisiae gene for ADHI (ADC1) are both confined to the alcA-alcR region. At least one of several species of mature mRNA is about 1 kb, the size required to code for ADH. For all species, carbon catabolite repression overrides control by induction. The overall characteristics of transcription, hybridization to ADC1 and earlier work suggest that alcA consists of a number of exons and/or that the alcA-alcR region represents a cluster of alcA-related genes or sequences.

  10. Three members of the human pyruvate dehydrogenase kinase gene family are direct targets of the peroxisome proliferator-activated receptor beta/delta.

    PubMed

    Degenhardt, Tatjana; Saramäki, Anna; Malinen, Marjo; Rieck, Markus; Väisänen, Sami; Huotari, Anne; Herzig, Karl-Heinz; Müller, Rolf; Carlberg, Carsten

    2007-09-14

    The nuclear receptors peroxisome proliferator-activated receptors (PPARs) are known for their critical role in the metabolic syndrome. Here, we show that they are direct regulators of the family of pyruvate dehydrogenase kinase (PDK) genes, whose products act as metabolic homeostats in sensing hunger and satiety levels in key metabolic tissues by modulating the activity of the pyruvate dehydrogenase complex. Mis-regulation of this tightly controlled network may lead to hyperglycemia. In human embryonal kidney cells we found the mRNA expression of PDK2, PDK3 and PDK4 to be under direct primary control of PPAR ligands, and in normal mouse kidney tissue Pdk2 and Pdk4 are PPAR targets. Both, treatment of HEK cells with PPARbeta/delta-specific siRNA and the genetic disruption of the Pparbeta/delta gene in mouse fibroblasts resulted in reduced expression of Pdk genes and abolition of induction by PPARbeta/delta ligands. These findings suggest that PPARbeta/delta is a key regulator of PDK genes, in particular the PDK4/Pdk4 gene. In silico analysis of the human PDK genes revealed two candidate PPAR response elements in the PDK2 gene, five in the PDK3 gene and two in the PDK4 gene, but none in the PDK1 gene. For seven of these sites we could demonstrate both PPARbeta/delta ligand responsiveness in context of their chromatin region and simultaneous association of PPARbeta/delta with its functional partner proteins, such as retinoidXreceptor, co-activator and mediator proteins and phosphorylated RNA polymerase II. In conclusion, PDK2, PDK3 and PDK4 are primary PPARbeta/delta target genes in humans underlining the importance of the receptor in the control of metabolism. PMID:17669420

  11. Molecular cloning and characterization of a putative proline dehydrogenase gene in the Colorado potato beetle, Leptinotarsa decemlineata.

    PubMed

    Wan, Pin-Jun; Lü, Dong; Guo, Wen-Chao; Ahmat, Tursun; Yang, Lu; Mu, Li-Li; Li, Guo-Qing

    2014-04-01

    Leptinotarsa decemlineata adults exhibit a season-dependent activity. In spring, post-diapause beetles often fly a long distance from overwintering sites to potato fields. In summer and autumn, the flight ability is sharply reduced. Proline is the main energy substrate of L. decemlineata during flight and proline dehydrogenase (ProDH) catalyzes the first step in proline catabolism. Here we identified a putative LdProDH gene; it had three cDNA isoforms which shared the same 5'UTR and coding region, but differed in the lengths of 3'UTRs (515, 1 092 and 1 242 bp for isoforms-1, -2 and -3, respectively). LdProDH encoded a 616 amino acid protein that showed high sequence similarity to ProDH-like proteins from other insect species. LdProDH was expressed in the third and fourth instars larvae and adults, but not in pupae. Dietary ingestion of bacterially expressed LdProDH-dsRNA by adults significantly decreased its messenger RNA (mRNA) level, and caused an elevation of free proline content in the hemolymph. Further observation revealed that three canonical polyadenylation signals (AATAAA) were tandemly located in the 3'UTR of isoform-3. The first, second and third polyadenylation sites gave rise to isoforms-1, -2 and -3, respectively. Analysis of the genomic DNA uncovered that the three isoforms resulted from alternative polyadenylation. The mRNA level of isoform-1, which expressed at low levels in pre-diapause adults, became abundant in post-diapause beetles. It is indicated that the LdProDH expression is fine-tuned through 3'UTR to control proline catabolism for the season-dependent activity of L. decemlineata adults. PMID:23956209

  12. Molecular cloning and characterization of a putative proline dehydrogenase gene in the Colorado potato beetle, Leptinotarsa decemlineata.

    PubMed

    Wan, Pin-Jun; Lü, Dong; Guo, Wen-Chao; Ahmat, Tursun; Yang, Lu; Mu, Li-Li; Li, Guo-Qing

    2014-04-01

    Leptinotarsa decemlineata adults exhibit a season-dependent activity. In spring, post-diapause beetles often fly a long distance from overwintering sites to potato fields. In summer and autumn, the flight ability is sharply reduced. Proline is the main energy substrate of L. decemlineata during flight and proline dehydrogenase (ProDH) catalyzes the first step in proline catabolism. Here we identified a putative LdProDH gene; it had three cDNA isoforms which shared the same 5'UTR and coding region, but differed in the lengths of 3'UTRs (515, 1 092 and 1 242 bp for isoforms-1, -2 and -3, respectively). LdProDH encoded a 616 amino acid protein that showed high sequence similarity to ProDH-like proteins from other insect species. LdProDH was expressed in the third and fourth instars larvae and adults, but not in pupae. Dietary ingestion of bacterially expressed LdProDH-dsRNA by adults significantly decreased its messenger RNA (mRNA) level, and caused an elevation of free proline content in the hemolymph. Further observation revealed that three canonical polyadenylation signals (AATAAA) were tandemly located in the 3'UTR of isoform-3. The first, second and third polyadenylation sites gave rise to isoforms-1, -2 and -3, respectively. Analysis of the genomic DNA uncovered that the three isoforms resulted from alternative polyadenylation. The mRNA level of isoform-1, which expressed at low levels in pre-diapause adults, became abundant in post-diapause beetles. It is indicated that the LdProDH expression is fine-tuned through 3'UTR to control proline catabolism for the season-dependent activity of L. decemlineata adults.

  13. Impact of multiple Alcohol Dehydrogenase gene polymorphisms on risk of laryngeal, esophageal, gastric and colorectal cancers in Chinese Han population

    PubMed Central

    An, Jiaze; Zhao, Junsheng; Zhang, Xiyang; Ding, Rui; Geng, Tingting; Feng, Tian; Jin, Tianbo

    2015-01-01

    Alcohol intake is positively associated with the risk of upper aerodigestive tract (UADT) cancers; but its effect on gastric or colorectal cancer is controversial. Previous study had identified several single nucleotide polymorphisms (SNPs) of Alcohol Dehydrogenase (ADH) genes associated with UADT cancers in European and Japanese populations. We sought to determine if these SNPs associated with laryngeal, esophageal, gastric or colorectal cancer in Chinese population. We conducted a case-control study among 1577 cases and 1013 healthy controls from northwest China. Five SNPs associated with UADT cancers risk were selected from previous genome-wide association studies and genotyped using Sequenom Mass-ARRAY technology. Odds ratios and 95% confidence intervals (CIs) were calculated by unconditional logistic regression adjusting for age and gender. We identified that the minor alleles of rs1789924 and rs971074 were associated with decreased risk of laryngeal cancer (OR = 0.311; 95% CI = 0.161-0.602; P < 0.001) and esophagus cancer (OR = 0.711; 95% CI = 0.526-0.962; P = 0.027) in allelic model analysis, respectively. In the genetic model analysis, we found the “C/T” genotype of rs1789924 was associated with decreased laryngeal cancer risk in codominant model (P = 0.046) and overdominant model (P = 0.013); the “C/T-T/T” genotype of rs1789924 was associated with reduced risk of laryngeal cancer under dominant model (P = 0.013). Additionally, none of the SNPs was associated with gastric or colorectal cancer in our study. Our data shed new light on the association between ADH SNPs and respiratory and digestive tract cancers susceptibility in the Han Chinese population. PMID:26396927

  14. Functional assignment of gene AAC16202.1 from Rhodobacter capsulatus SB1003: new insights into the bacterial SDR sorbitol dehydrogenases family.

    PubMed

    Sola-Carvajal, Agustín; García-García, María Inmaculada; Sánchez-Carrón, Guiomar; García-Carmona, Francisco; Sánchez-Ferrer, Alvaro

    2012-11-01

    Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with over 60,000 non-redundant sequences in the database, many of which need a correct functional assignment. Among them, the gene AAC16202.1 (NCBI) from Rhodobacter capsulatus SB1003 has been assigned in Uniprot both as a sorbitol dehydrogenase (#D5AUY1) and, as an N-acetyl-d-mannosamine dehydrogenase (#O66112), both enzymes being of biotechnological interest. When the gene was overexpressed in Escherichia coli Rosetta (DE3)pLys, the purified enzyme was not active toward N-acetyl-d-mannosamine, whereas it was active toward d-sorbitol and d-fructose. However, the relative activities toward xylitol and l-iditol (0.45 and 6.9%, respectively) were low compared with that toward d-sorbitol. Thus, the enzyme could be considered sorbitol dehydrogenase (SDH) with very low activity toward xylitol, which could increase its biotechnological interest for determining sorbitol without the unspecific cross-determination of added xylitol in food and pharma compositions. The tetrameric enzyme (120 kDa) showed similar catalytic efficiency (2.2 × 10(3) M(-1) s(-1)) to other sorbitol dehydrogenases for d-sorbitol, with an optimum pH of 9.0 and an optimum temperature of 37 °C. The enzyme was also more thermostable than other reported SDH, ammonium sulfate being the best stabilizer in this respect, increasing the melting temperature (T(m)) up to 52.9 °C. The enzyme can also be considered as a new member of the Zn(2+) independent SDH family since no effect on activity was detected in the presence of divalent cations or chelating agents. Finally, its in silico analysis enabled the specific conserved sequence blocks that are the fingerprints of bacterial sorbitol dehydrogenases and mainly located at C-terminal of the protein, to be determined for the first time. This knowledge will facilitate future data curation of present databases and a better functional assignment of newly described

  15. Intracellular glycerol influences resistance to freeze stress in Saccharomyces cerevisiae: analysis of a quadruple mutant in glycerol dehydrogenase genes and glycerol-enriched cells.

    PubMed

    Izawa, Shingo; Sato, Machiko; Yokoigawa, Kumio; Inoue, Yoshiharu

    2004-11-01

    Glycerol is well known as a cryoprotectant similar to trehalose. However, there is little information about the effects of intracellular glycerol on the freeze-thaw stress tolerance of yeast. Through analysis of a quadruple-knockout mutant of glycerol dehydrogenase genes (ara1 Delta gcy1 Delta gre3 Delta ypr1 Delta) in Saccharomyces cerevisiae, we revealed that the decrease in glycerol dehydrogenase activity led to increased levels of intracellular glycerol. We also found that this mutant showed higher tolerance to freeze stress than wild type strain W303-1A. Furthermore, we demonstrated that intracellular-glycerol-enriched cells cultured in glycerol medium acquire tolerance to freeze stress and retain high leavening ability in dough even after frozen storage for 7 days. These results suggest the possibility of using intracellular-glycerol-enriched cells to develop better frozen dough.

  16. Intracellular glycerol influences resistance to freeze stress in Saccharomyces cerevisiae: analysis of a quadruple mutant in glycerol dehydrogenase genes and glycerol-enriched cells.

    PubMed

    Izawa, Shingo; Sato, Machiko; Yokoigawa, Kumio; Inoue, Yoshiharu

    2004-11-01

    Glycerol is well known as a cryoprotectant similar to trehalose. However, there is little information about the effects of intracellular glycerol on the freeze-thaw stress tolerance of yeast. Through analysis of a quadruple-knockout mutant of glycerol dehydrogenase genes (ara1 Delta gcy1 Delta gre3 Delta ypr1 Delta) in Saccharomyces cerevisiae, we revealed that the decrease in glycerol dehydrogenase activity led to increased levels of intracellular glycerol. We also found that this mutant showed higher tolerance to freeze stress than wild type strain W303-1A. Furthermore, we demonstrated that intracellular-glycerol-enriched cells cultured in glycerol medium acquire tolerance to freeze stress and retain high leavening ability in dough even after frozen storage for 7 days. These results suggest the possibility of using intracellular-glycerol-enriched cells to develop better frozen dough. PMID:15127164

  17. Homo-D-lactic acid fermentation from arabinose by redirection of the phosphoketolase pathway to the pentose phosphate pathway in L-lactate dehydrogenase gene-deficient Lactobacillus plantarum.

    PubMed

    Okano, Kenji; Yoshida, Shogo; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

    2009-08-01

    Optically pure d-lactic acid fermentation from arabinose was achieved by using the Lactobacillus plantarum NCIMB 8826 strain whose l-lactate dehydrogenase gene was deficient and whose phosphoketolase gene was substituted with a heterologous transketolase gene. After 27 h of fermentation, 38.6 g/liter of d-lactic acid was produced from 50 g/liter of arabinose.

  18. Efficient production of acetoin in Saccharomyces cerevisiae by disruption of 2,3-butanediol dehydrogenase and expression of NADH oxidase

    PubMed Central

    Bae, Sang-Jeong; Kim, Sujin; Hahn, Ji-Sook

    2016-01-01

    Acetoin is widely used in food and cosmetic industry as taste and fragrance enhancer. For acetoin production in this study, Saccharomyces cerevisiae JHY605 was used as a host strain, where the production of ethanol and glycerol was largely eliminated by deleting five alcohol dehydrogenase genes (ADH1, ADH2, ADH3, ADH4, and ADH5) and two glycerol 3-phosphate dehydrogenase genes (GPD1 and GPD2). To improve acetoin production, acetoin biosynthetic genes from Bacillus subtilis encoding α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD) were overexpressed, and BDH1 encoding butanediol dehydrogenase, which converts acetoin to 2,3-butanediol, was deleted. Furthermore, by NAD+ regeneration through overexpression of water-forming NADH oxidase (NoxE) from Lactococcus lactis, the cofactor imbalance generated during the acetoin production from glucose was successfully relieved. As a result, in fed-batch fermentation, the engineered strain JHY617-SDN produced 100.1 g/L acetoin with a yield of 0.44 g/g glucose. PMID:27279026

  19. Efficient production of acetoin in Saccharomyces cerevisiae by disruption of 2,3-butanediol dehydrogenase and expression of NADH oxidase.

    PubMed

    Bae, Sang-Jeong; Kim, Sujin; Hahn, Ji-Sook

    2016-01-01

    Acetoin is widely used in food and cosmetic industry as taste and fragrance enhancer. For acetoin production in this study, Saccharomyces cerevisiae JHY605 was used as a host strain, where the production of ethanol and glycerol was largely eliminated by deleting five alcohol dehydrogenase genes (ADH1, ADH2, ADH3, ADH4, and ADH5) and two glycerol 3-phosphate dehydrogenase genes (GPD1 and GPD2). To improve acetoin production, acetoin biosynthetic genes from Bacillus subtilis encoding α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD) were overexpressed, and BDH1 encoding butanediol dehydrogenase, which converts acetoin to 2,3-butanediol, was deleted. Furthermore, by NAD(+) regeneration through overexpression of water-forming NADH oxidase (NoxE) from Lactococcus lactis, the cofactor imbalance generated during the acetoin production from glucose was successfully relieved. As a result, in fed-batch fermentation, the engineered strain JHY617-SDN produced 100.1 g/L acetoin with a yield of 0.44 g/g glucose. PMID:27279026

  20. Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II.

    PubMed Central

    Ohta, K; Beall, D S; Mejia, J P; Shanmugam, K T; Ingram, L O

    1991-01-01

    Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high level of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose to ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA). PMID:2059047

  1. Molecular cloning and characterization of two inducible NAD⁺-adh genes encoding NAD⁺-dependent alcohol dehydrogenases from Acetobacter pasteurianus SKU1108.

    PubMed

    Masud, Uraiwan; Matsushita, Kazunobu; Theeragool, Gunjana

    2011-11-01

    The cytosolic NAD⁺-dependent alcohol dehydrogenases (NAD⁺-ADHs) are induced in the quinoprotein ADH-(PQQ-ADH) defective Acetobacter pasteurianus SKU1108 mutant during growth in an ethanol medium. The adhI and adhII genes, which encode NAD⁺-ADH I and ADH II, respectively, of this strain have been cloned and characterized. Sequence analyses have revealed that the adhI gene consists of 1029 bp coding for 342 amino acids, which share 99.71% identity with the same protein from A. pasteurianus IFO 3283. Conversely, the adhII gene is composed of 762 bp encoding for a polypeptide of 253 amino acids, which exhibit 99.60% identity with the A. pasteurianus IFO 3283 protein. ADH I is a member of the group I Zn-dependent long-chain ADHs, while the ADH II belongs to the group II short-chain dehydrogenase/reductase NAD⁺-ADHs. The NAD⁺-adh gene disruptants exhibited a growth reduction when grown in an ethanol medium. In Escherichia coli, ethanol induced adhI and adhII promoter activities by approximately 1.5 and 2.0 times, respectively, and the promoter activity of the adhII gene exceeded that of the adhI gene by approximately 3.5 times. The possible promoter regions of the adhI and adhII genes are located at approximately 81-105 bp and 74-92 bp, respectively, from their respective ATG start codons. Their repressor regions might be located in proximity to these promoters and may repress gene expression in the wild-type, where the membrane-bound ADH effectively functions.

  2. Characterization of a Novel Intestinal Glycerol-3-phosphate Acyltransferase Pathway and Its Role in Lipid Homeostasis.

    PubMed

    Khatun, Irani; Clark, Ronald W; Vera, Nicholas B; Kou, Kou; Erion, Derek M; Coskran, Timothy; Bobrowski, Walter F; Okerberg, Carlin; Goodwin, Bryan

    2016-02-01

    Dietary triglycerides (TG) are absorbed by the enterocytes of the small intestine after luminal hydrolysis into monacylglycerol and fatty acids. Before secretion on chylomicrons, these lipids are reesterified into TG, primarily through the monoacylglycerol pathway. However, targeted deletion of the primary murine monoacylglycerol acyltransferase does not quantitatively affect lipid absorption, suggesting the existence of alternative pathways. Therefore, we investigated the role of the glycerol 3-phosphate pathway in dietary lipid absorption. The expression of glycerol-3-phosphate acyltransferase (GPAT3) was examined throughout the small intestine. To evaluate the role for GPAT3 in lipid absorption, mice harboring a disrupted GPAT3 gene (Gpat3(-/-)) were subjected to an oral lipid challenge and fed a Western-type diet to characterize the role in lipid and cholesterol homeostasis. Additional mechanistic studies were performed in primary enterocytes. GPAT3 was abundantly expressed in the apical surface of enterocytes in the small intestine. After an oral lipid bolus, Gpat3(-/-) mice exhibited attenuated plasma TG excursion and accumulated lipid in the enterocytes. Electron microscopy studies revealed a lack of lipids in the lamina propria and intercellular space in Gpat3(-/-) mice. Gpat3(-/-) enterocytes displayed a compensatory increase in the synthesis of phospholipid and cholesteryl ester. When fed a Western-type diet, hepatic TG and cholesteryl ester accumulation was significantly higher in Gpat3(-/-) mice compared with the wild-type mice accompanied by elevated levels of alanine aminotransferase, a marker of liver injury. Dysregulation of bile acid metabolism was also evident in Gpat3-null mice. These studies identify GPAT3 as a novel enzyme involved in intestinal lipid metabolism.

  3. A novel 5-enolpyruvylshikimate-3-phosphate synthase from Rahnella aquatilis with significantly reduced glyphosate sensitivity.

    PubMed

    Peng, Ri-He; Tian, Yong-Sheng; Xiong, Ai-Sheng; Zhao, Wei; Fu, Xiao-Yan; Han, Hong-Juan; Chen, Chen; Jin, Xiao-Fen; Yao, Quan-Hong

    2012-01-01

    The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroA(R. aquatilis), was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroA(E. coli)), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroA(R. aquatilis) were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroA(E. coli). To probe the sites contributing to increased tolerance to glyphosate, mutant R. aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R. aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious. PMID:22870190

  4. Characterization of a Novel Intestinal Glycerol-3-phosphate Acyltransferase Pathway and Its Role in Lipid Homeostasis.

    PubMed

    Khatun, Irani; Clark, Ronald W; Vera, Nicholas B; Kou, Kou; Erion, Derek M; Coskran, Timothy; Bobrowski, Walter F; Okerberg, Carlin; Goodwin, Bryan

    2016-02-01

    Dietary triglycerides (TG) are absorbed by the enterocytes of the small intestine after luminal hydrolysis into monacylglycerol and fatty acids. Before secretion on chylomicrons, these lipids are reesterified into TG, primarily through the monoacylglycerol pathway. However, targeted deletion of the primary murine monoacylglycerol acyltransferase does not quantitatively affect lipid absorption, suggesting the existence of alternative pathways. Therefore, we investigated the role of the glycerol 3-phosphate pathway in dietary lipid absorption. The expression of glycerol-3-phosphate acyltransferase (GPAT3) was examined throughout the small intestine. To evaluate the role for GPAT3 in lipid absorption, mice harboring a disrupted GPAT3 gene (Gpat3(-/-)) were subjected to an oral lipid challenge and fed a Western-type diet to characterize the role in lipid and cholesterol homeostasis. Additional mechanistic studies were performed in primary enterocytes. GPAT3 was abundantly expressed in the apical surface of enterocytes in the small intestine. After an oral lipid bolus, Gpat3(-/-) mice exhibited attenuated plasma TG excursion and accumulated lipid in the enterocytes. Electron microscopy studies revealed a lack of lipids in the lamina propria and intercellular space in Gpat3(-/-) mice. Gpat3(-/-) enterocytes displayed a compensatory increase in the synthesis of phospholipid and cholesteryl ester. When fed a Western-type diet, hepatic TG and cholesteryl ester accumulation was significantly higher in Gpat3(-/-) mice compared with the wild-type mice accompanied by elevated levels of alanine aminotransferase, a marker of liver injury. Dysregulation of bile acid metabolism was also evident in Gpat3-null mice. These studies identify GPAT3 as a novel enzyme involved in intestinal lipid metabolism. PMID:26644473

  5. A novel 5-enolpyruvylshikimate-3-phosphate synthase from Rahnella aquatilis with significantly reduced glyphosate sensitivity.

    PubMed

    Peng, Ri-He; Tian, Yong-Sheng; Xiong, Ai-Sheng; Zhao, Wei; Fu, Xiao-Yan; Han, Hong-Juan; Chen, Chen; Jin, Xiao-Fen; Yao, Quan-Hong

    2012-01-01

    The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroA(R. aquatilis), was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroA(E. coli)), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroA(R. aquatilis) were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroA(E. coli). To probe the sites contributing to increased tolerance to glyphosate, mutant R. aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R. aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious.

  6. Promoter Methylation Status of Breast Cancer Susceptibility Gene 1 and 17 Beta Hydroxysteroid Dehydrogenase Type 1 Gene in Sporadic Breast Cancer Patients

    PubMed Central

    Hosny, Marwa M.; Sabek, Nagwan A.; El-Abaseri, Taghrid B.; Hassan, Fathalla M.; Farrag, Sherif H.

    2016-01-01

    Epigenetic modifications are involved in breast carcinogenesis. Identifying genes that are epigenetically silenced via methylation could select target patients for diagnostic as well as therapeutic potential. We assessed promoter methylation of breast cancer susceptibility gene 1 (BRCA1) and 17 Beta Hydroxysteroid Dehydrogenase Type 1 (17βHSD-1) in normal and cancer breast tissues of forty sporadic breast cancer (BC) cases using restriction enzyme based methylation-specific PCR (REMS-PCR). In cancerous tissues, BRCA1 and 17βHSD-1 were methylated in 42.5% and 97.5%, respectively, while normal tissues had 35% and 95% methylation, respectively. BRCA1 methylation in normal tissues was 12.2-fold more likely to associate with methylation in cancer tissues (p < 0.001). It correlated significantly with increased age at menopause, mitosis, the negative status of Her2, and the molecular subtype “luminal A” (p = 0.048, p = 0.042, p = 0.007, and p = 0.049, resp.). Methylation of BRCA1 and 17βHSD-1 related to luminal A subtype of breast cancer. Since a small proportion of normal breast epithelial cells had BRCA1 methylation, our preliminary findings suggest that methylation of BRCA1 may be involved in breast tumors initiation and progression; therefore, it could be used as a biomarker for the early detection of sporadic breast cancer. Methylation of 17βHSD-1 in normal and cancer tissue could save patients the long term use of adjuvant antiestrogen therapies. PMID:27413552

  7. Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus.

    PubMed

    Kondo, K; Beppu, T; Horinouchi, S

    1995-09-01

    The membrane-bound alcohol dehydrogenase (ADH) of Acetobacter pasteurianus NCI1452 consists of three different subunits, a 78-kDa dehydrogenase subunit, a 48-kDa cytochrome c subunit, and a 20-kDa subunit of unknown function. For elucidation of the function of the smallest subunit, this gene was cloned from this strain by the oligonucleotide-probing method, and its nucleotide sequence was determined. Comparison of the deduced amino acid sequence and the NH2-terminal sequence determined for the purified protein indicated that the smallest subunit contained a typical signal peptide of 28 amino acids, as did the larger two subunits. This gene complemented the ADH activity of a mutant strain which had lost the smallest subunit. Disruption of this gene on the chromosome resulted in loss of ADH activity in Acetobacter aceti, indicating that the smallest subunit was essential for ADH activity. Immunoblot analyses of cell lysates prepared from various ADH mutants suggested that the smallest subunit was concerned with the stability of the 78-kDa subunit and functioned as a molecular coupler of the 78-kDa subunit to the 48-kDa subunit on the cytoplasmic membrane.

  8. Expression of the murine retinol dehydrogenase 10 (Rdh10) gene correlates with many sites of retinoid signalling during embryogenesis and organ differentiation.

    PubMed

    Cammas, Laura; Romand, Raymond; Fraulob, Valérie; Mura, Carole; Dollé, Pascal

    2007-10-01

    Retinoic acid acts as a signalling molecule regulating many developmental events in vertebrates. As this molecule directly influences gene expression by activating nuclear receptors, its patterns of synthesis have to be tightly regulated, and it is well established that at least three retinaldehyde dehydrogenases (RALDHs) are involved in such tissue-specific synthesis. Whereas embryos from oviparous species can obtain retinaldehyde by metabolizing carotenoids stored in the yolk, placental embryos rely on retinol transferred from the maternal circulation. Here, we show that the gene encoding one of the murine retinol dehydrogenases, Rdh10, is expressed according to complex profiles both during early embryogenesis and organ differentiation. Many of its expression sites correlate with regions of active retinoid signalling and Raldh gene expression, especially with Raldh2 in the early presomitic and somitic mesoderm, retrocardiac and posterior branchial arch region, or later in the pleural mesothelium and kidney cortical region. Rdh10 also shows cell-type and/or regional specificity during development of the palate, teeth, and olfactory system. During limb bud development, it may participate in retinoic acid production in proximal/posterior cells, and eventually in interdigital mesenchyme. These data implicate the retinol to retinaldehyde conversion as the first step in the tissue-specific regulation of retinoic acid synthesis, at least in mammalian embryos.

  9. Characterization of two host specific genes, mannose sensitive hemagglutinin (mshA) and uridyl phosphate dehydrogenase (UDPDH) that are involved in the Vibrio fischeri-Euprymna tasmanica mutualism

    PubMed Central

    Ariyakumar, D.S.; Nishiguchi, M.K.

    2009-01-01

    While much has been known about the mutualistic associations between the sepiolid squid Euprymna tasmanica and the luminescent bacterium, Vibrio fischeri, less is known about the connectivity between the microscopic and molecular basis of initial attachment and persistence in the light organ. Here, we examine the possible effects of two symbiotic genes on specificity and biofilm formation of V. fischeri in squid light organs. Uridine diphosphate-dehydrogenase (UDPDH) and mannose-sensitive hemagglutinin (mshA) mutants were generated in V. fischeri to determine whether each gene has an effect on host colonization, specificity, and biofilm formation. Both squid light organ colonization assays and transmission electron microscopy (TEM) confirmed differences in host colonization between wild-type and mutant strains, and also demonstrated the importance of both UDPDH and mshA gene expression for successful light organ colonization, and furthers our understanding of the genetic factors playing important roles in this environmentally transmitted symbiosis. PMID:19686342

  10. A coniferyl aldehyde dehydrogenase gene from Pseudomonas sp. strain HR199 enhances the conversion of coniferyl aldehyde by Saccharomyces cerevisiae.

    PubMed

    Adeboye, Peter Temitope; Olsson, Lisbeth; Bettiga, Maurizio

    2016-07-01

    The conversion of coniferyl aldehyde to cinnamic acids by Saccharomyces cerevisiae under aerobic growth conditions was previously observed. Bacteria such as Pseudomonas have been shown to harbor specialized enzymes for converting coniferyl aldehyde but no comparable enzymes have been identified in S. cerevisiae. CALDH from Pseudomonas was expressed in S. cerevisiae. An acetaldehyde dehydrogenase (Ald5) was also hypothesized to be actively involved in the conversion of coniferyl aldehyde under aerobic growth conditions in S. cerevisiae. In a second S. cerevisiae strain, the acetaldehyde dehydrogenase (ALD5) was deleted. A prototrophic control strain was also engineered. The engineered S. cerevisiae strains were cultivated in the presence of 1.1mM coniferyl aldehyde under aerobic condition in bioreactors. The results confirmed that expression of CALDH increased endogenous conversion of coniferyl aldehyde in S. cerevisiae and ALD5 is actively involved with the conversion of coniferyl aldehyde in S. cerevisiae. PMID:27070284

  11. Construction of a brewer's yeast having alpha-acetolactate decarboxylase gene from Acetobacter aceti ssp. xylinum integrated in the genome.

    PubMed

    Yamano, S; Kondo, K; Tanaka, J; Inoue, T

    1994-02-14

    alpha-Acetolactate decarboxylase (ALDC) gene from Acetobacter aceti ssp. xylinum has several possible initiation codons in the N-terminus. To determine the initiation codon of the ALDC giving the highest expression levels, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was linked just upstream of each possible initiation codon. The ALDC whose translation starts 130 bp downstream from the first ATG codon had the highest activity in yeast cells. When expression levels of the ALDC gene were compared using three strong yeast promoters of glycolytic genes, alcohol dehydrogenase I (ADC1), phosphoglycerate kinase (PGK) and GPD, the GPD promoter was the strongest. The ALDC gene was integrated in a ribosomal RNA gene of a brewer's yeast by co-transformation with an expression plasmid of G418-resistance gene. The laboratory-scale growth test confirmed that the total diacetyl concentration was reduced in wort.

  12. Streptomyces coelicolor XdhR is a direct target of (p)ppGpp that controls expression of genes encoding xanthine dehydrogenase to promote purine salvage.

    PubMed

    Sivapragasam, Smitha; Grove, Anne

    2016-05-01

    The gene encoding Streptomyces coelicolor xanthine dehydrogenase regulator (XdhR) is divergently oriented from xdhABC, which encodes xanthine dehydrogenase (Xdh). Xdh is required for purine salvage pathways. XdhR was previously shown to repress xdhABC expression. We show that XdhR binds the xdhABC-xdhR intergenic region with high affinity (Kd ∼ 0.5 nM). DNaseI footprinting reveals that this complex formation corresponds to XdhR binding the xdhR gene promoter at two adjacent sites; at higher protein concentrations, protection expands to a region that overlaps the transcriptional and translational start sites of xdhABC. While substrates for Xdh have little effect on DNA binding, GTP and ppGpp dissociate the DNA-XdhR complex. Progression of cells to stationary phase, a condition associated with increased (p)ppGpp production, leads to elevated xdhB expression; in contrast, inhibition of Xdh by allopurinol results in xdhB repression. We propose that XdhR is a direct target of (p)ppGpp, and that expression of xdhABC is upregulated during the stringent response to promote purine salvage pathways, maintain GTP homeostasis and ensure continued (p)ppGpp synthesis. During exponential phase growth, basal levels of xdhABC expression may be achieved by GTP serving as a lower-affinity XdhR ligand.

  13. The multifunctional isopropyl alcohol dehydrogenase of Phytomonas sp. could be the result of a horizontal gene transfer from a bacterium to the trypanosomatid lineage.

    PubMed

    Molinas, Sara M; Altabe, Silvia G; Opperdoes, Fred R; Rider, Mark H; Michels, Paul A M; Uttaro, Antonio D

    2003-09-19

    Isopropyl alcohol dehydrogenase (iPDH) is a dimeric mitochondrial alcohol dehydrogenase (ADH), so far detected within the Trypanosomatidae only in the genus Phytomonas. The cloning, sequencing, and heterologous expression of the two gene alleles of the enzyme revealed that it is a zinc-dependent medium-chain ADH. Both polypeptides have 361 amino acids. A mitochondrial targeting sequence was identified. The mature proteins each have 348 amino acids and a calculated molecular mass of 37 kDa. They differ only in one amino acid, which can explain the three isoenzymes and their respective isoelectric points previously found. A phylogenetic analysis locates iPDH within a cluster with fermentative ADHs from bacteria, sharing 74% similarity and 60% identity with Ralstonia eutropha ADH. The characterization of the two bacterially expressed Phytomonas enzymes and the comparison of their kinetic properties with those of the wild-type iPDH and of the R. eutropha ADH strongly support the idea of a horizontal gene transfer event from a bacterium to a trypanosomatid to explain the origin of the iPDH in Phytomonas. Phytomonas iPDH and R. eutropha ADH are able to use a wide range of substrates with similar Km values such as primary and secondary alcohols, diols, and aldehydes, as well as ketones such as acetone, diacetyl, and acetoin. We speculate that, as for R. eutropha ADH, Phytomonas iPDH acts as a safety valve for the release of excess reducing power. PMID:12853449

  14. Pharmacological activation of the pyruvate dehydrogenase complex reduces statin-mediated upregulation of FOXO gene targets and protects against statin myopathy in rodents.

    PubMed

    Mallinson, Joanne E; Constantin-Teodosiu, Dumitru; Glaves, Philip D; Martin, Elizabeth A; Davies, Wendy J; Westwood, F Russell; Sidaway, James E; Greenhaff, Paul L

    2012-12-15

    We previously reported that statin myopathy is associated with impaired carbohydrate (CHO) oxidation in fast-twitch rodent skeletal muscle, which we hypothesised occurred as a result of forkhead box protein O1 (FOXO1) mediated upregulation of pyruvate dehydrogenase kinase-4 (PDK4) gene transcription. Upregulation of FOXO gene targets known to regulate proteasomal and lysosomal muscle protein breakdown was also evident. We hypothesised that increasing CHO oxidation in vivo, using the pyruvate dehydrogenase complex (PDC) activator, dichloroacetate (DCA), would blunt activation of FOXO gene targets and reduce statin myopathy. Female Wistar Hanover rats were dosed daily for 12 days (oral gavage) with either vehicle (control, 0.5% w/v hydroxypropyl-methylcellulose 0.1% w/v polysorbate-80; n = 9), 88 mg( )kg(-1) day(-1) simvastatin (n = 8), 88 mg( )kg(-1) day(-1) simvastatin + 30 mg kg(-1) day(-1) DCA (n = 9) or 88 mg kg(-1) day(-1) simvastatin + 40 mg kg(-1) day(-1) DCA (n = 9). Compared with control, simvastatin reduced body mass gain and food intake, increased muscle fibre necrosis, plasma creatine kinase levels, muscle PDK4, muscle atrophy F-box (MAFbx) and cathepsin-L mRNA expression, increased PDK4 protein expression, and proteasome and cathepsin-L activity, and reduced muscle PDC activity. Simvastatin with DCA maintained body mass gain and food intake, abrogated the myopathy, decreased muscle PDK4 mRNA and protein, MAFbx and cathepsin-L mRNA, increased activity of PDC and reduced proteasome activity compared with simvastatin. PDC activation abolished statin myopathy in rodent skeletal muscle, which occurred at least in part via inhibition of FOXO-mediated transcription of genes regulating muscle CHO utilisation and protein breakdown.

  15. The Role of Placental 11-Beta Hydroxysteroid Dehydrogenase Type 1 and Type 2 Methylation on Gene Expression and Infant Birth Weight.

    PubMed

    Green, Benjamin B; Armstrong, David A; Lesseur, Corina; Paquette, Alison G; Guerin, Dylan J; Kwan, Lauren E; Marsit, Carmen J

    2015-06-01

    Maternal stress has been linked to infant birth weight outcomes, which itself may be associated with health later in life. The placenta acts as a master regulator for the fetal environment, mediating intrauterine exposures to stress through the activity of genes regulating glucocorticoids, including the 11beta-hydroxysteroid dehydrogenase (HSD11B) type 1 and 2 genes, and so we hypothesized that variation in these genes will be associated with infant birth weight. We investigated DNA methylation levels at six sites across the two genes, as well as mRNA expression for each, and the relationship to infant birth weight. Logistic regressions correcting for potential confounding factors revealed a significant association between methylation at a single CpG site within HSD11B1 and being born large for gestational age. In addition, our analysis identified correlations between methylation and gene expression, including sex-specific transcriptional regulation of HSD11B2. Our work is one of the first comprehensive views of DNA methylation and expression in the placenta for both HSD11B types 1 and 2, linking epigenetic alterations with the regulation of fetal stress and birth weight outcomes. PMID:25788665

  16. Taraxerone enhances alcohol oxidation via increases of alcohol dehyderogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities and gene expressions.

    PubMed

    Sung, Chang-Keun; Kim, Seung-Mi; Oh, Chang-Jin; Yang, Sun-A; Han, Byung-Hee; Mo, Eun-Kyoung

    2012-07-01

    The present study, taraxerone (d-friedoolean-14-en-3-one) was isolated from Sedum sarmentosum with purity 96.383%, and its enhancing effects on alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities were determined: EC(50) values were 512.42 ± 3.12 and 500.16 ± 3.23 μM for ADH and ALDH, respectively. In order to obtain more information on taraxerone related with the alcohol metabolism, 40% ethanol (5 mL/kg body weight) with 0.5-1mM of taraxerone were administered to mice. The plasma alcohol and acetaldehyde concentrations of taraxerone-treated groups were significantly lowered than those of the control group (p<0.01): approximately 20-67% and 7-57% lowered for plasma alcohol and acetaldehyde, respectively. Compare to the control group, the ADH and ALDH expressions in the liver tissues were abruptly increased in the taraxerone-treated groups after ethanol exposure. In addition, taraxerone prevented catalase, superoxide dismutase, and reduced glutathione concentrations from the decrease induced by ethanol administration with the concentration dependent manner.

  17. Negative regulation of phosphatidylinositol 3-phosphate levels in early-to-late endosome conversion

    PubMed Central

    Liu, Kai; Jian, Youli; Sun, Xiaojuan; Yang, Chengkui; Gao, Zhiyang; Zhang, Zhili; Liu, Xuezhao; Li, Yang; Xu, Jing; Jing, Yudong; Mitani, Shohei; He, Sudan

    2016-01-01

    Phosphatidylinositol 3-phosphate (PtdIns3P) plays a central role in endosome fusion, recycling, sorting, and early-to-late endosome conversion, but the mechanisms that determine how the correct endosomal PtdIns3P level is achieved remain largely elusive. Here we identify two new factors, SORF-1 and SORF-2, as essential PtdIns3P regulators in Caenorhabditis elegans. Loss of sorf-1 or sorf-2 leads to greatly elevated endosomal PtdIns3P, which drives excessive fusion of early endosomes. sorf-1 and sorf-2 function coordinately with Rab switching genes to inhibit synthesis of PtdIns3P, allowing its turnover for endosome conversion. SORF-1 and SORF-2 act in a complex with BEC-1/Beclin1, and their loss causes elevated activity of the phosphatidylinositol 3-kinase (PI3K) complex. In mammalian cells, inactivation of WDR91 and WDR81, the homologs of SORF-1 and SORF-2, induces Beclin1-dependent enlargement of PtdIns3P-enriched endosomes and defective degradation of epidermal growth factor receptor. WDR91 and WDR81 interact with Beclin1 and inhibit PI3K complex activity. These findings reveal a conserved mechanism that controls appropriate PtdIns3P levels in early-to-late endosome conversion. PMID:26783301

  18. Rat brain myo-inositol 3-phosphate synthase is a phosphoprotein.

    PubMed

    Parthasarathy, R N; Lakshmanan, J; Thangavel, M; Seelan, R S; Stagner, J I; Janckila, A J; Vadnal, R E; Casanova, M F; Parthasarathy, L K

    2013-06-01

    The therapeutic effects of lithium in bipolar disorder are poorly understood. Lithium decreases free inositol levels by inhibiting inositol monophosphatase 1 and myo-inositol 3-phosphate synthase (IPS). In this study, we demonstrate for the first time that IPS can be phosphorylated. This was evident when purified rat IPS was dephosphorylated by lambda protein phosphatase and analyzed by phospho-specific ProQ-Diamond staining and Western blot analysis. These techniques demonstrated a mobility shift consistent with IPS being phosphorylated. Mass spectral analysis revealed that Serine-524 (S524), which resides in the hinge region derived from exon 11 of the gene, is the site for phosphorylation. Further, an antibody generated against a synthetic peptide of IPS containing monophosphorylated-S524, was able to discriminate the phosphorylated and non-phosphorylated forms of IPS. The phosphoprotein is found in the brain and testis, but not in the intestine. The intestinal IPS isoform lacks the peptide bearing S524, and hence, cannot be phosphorylated. Evidences suggest that IPS is monophosphorylated at S524 and that the removal of this phosphate does not alter its enzymatic activity. These observations suggest a novel function for IPS in brain and other tissues. Future studies should resolve the functional role of phospho-IPS in brain inositol signaling.

  19. Phosphorylation regulates myo-inositol-3-phosphate synthase: a novel regulatory mechanism of inositol biosynthesis.

    PubMed

    Deranieh, Rania M; He, Quan; Caruso, Joseph A; Greenberg, Miriam L

    2013-09-13

    myo-Inositol-3-phosphate synthase (MIPS) plays a crucial role in inositol homeostasis. Transcription of the coding gene INO1 is highly regulated. However, regulation of the enzyme is not well defined. We previously showed that MIPS is indirectly inhibited by valproate, suggesting that the enzyme is post-translationally regulated. Using (32)Pi labeling and phosphoamino acid analysis, we show that yeast MIPS is a phosphoprotein. Mass spectrometry analysis identified five phosphosites, three of which are conserved in the human MIPS. Analysis of phosphorylation-deficient and phosphomimetic site mutants indicated that the three conserved sites in yeast (Ser-184, Ser-296, and Ser-374) and humans (Ser-177, Ser-279, and Ser-357) affect MIPS activity. Both S296A and S296D yeast mutants and S177A and S177D human mutants exhibited decreased enzymatic activity, suggesting that a serine residue is critical at that location. The phosphomimetic mutations S184D (human S279D) and S374D (human S357D) but not the phosphodeficient mutations decreased activity, suggesting that phosphorylation of these two sites is inhibitory. The double mutation S184A/S374A caused an increase in MIPS activity, conferred a growth advantage, and partially rescued sensitivity to valproate. Our findings identify a novel mechanism of regulation of inositol synthesis by phosphorylation of MIPS.

  20. Phosphatidylinositol-3-phosphate is light-regulated and essential for survival in retinal rods

    PubMed Central

    He, Feng; Agosto, Melina A.; Anastassov, Ivan A.; Tse, Dennis Y.; Wu, Samuel M.; Wensel, Theodore G.

    2016-01-01

    Phosphoinositides play important roles in numerous intracellular membrane pathways. Little is known about the regulation or function of these lipids in rod photoreceptor cells, which have highly active membrane dynamics. Using new assays with femtomole sensitivity, we determined that whereas levels of phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate were below detection limits, phosphatidylinositol-3-phosphate (PI(3)P) levels in rod inner/outer segments increased more than 30-fold after light exposure. This increase was blocked in a rod-specific knockout of the PI-3 kinase Vps34, resulting in failure of endosomal and autophagy-related membranes to fuse with lysosomes, and accumulation of abnormal membrane structures. At early ages, rods displayed normal morphology, rhodopsin trafficking, and light responses, but underwent progressive neurodegeneration with eventual loss of both rods and cones by twelve weeks. The degeneration is considerably faster than in rod knockouts of autophagy genes, indicating defects in endosome recycling or other PI(3)P-dependent membrane trafficking pathways are also essential for rod survival. PMID:27245220

  1. Identification of a Second Two-Component Signal Transduction System That Controls Fosfomycin Tolerance and Glycerol-3-Phosphate Uptake

    PubMed Central

    Kurabayashi, Kumiko; Hirakawa, Yuko; Tanimoto, Koichi; Tomita, Haruyoshi

    2014-01-01

    Particular interest in fosfomycin has resurfaced because it is a highly beneficial antibiotic for the treatment of refractory infectious diseases caused by pathogens that are resistant to other commonly used antibiotics. The biological cost to cells of resistance to fosfomycin because of chromosomal mutation is high. We previously found that a bacterial two-component system, CpxAR, induces fosfomycin tolerance in enterohemorrhagic Escherichia coli (EHEC) O157:H7. This mechanism does not rely on irreversible genetic modification and allows EHEC to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin. Here we show that another two-component system, TorSRT, which was originally characterized as a regulatory system for anaerobic respiration utilizing trimethylamine-N-oxide (TMAO), also induces fosfomycin tolerance. Activation of the Tor regulatory pathway by overexpression of torR, which encodes the response regulator, or addition of TMAO increased fosfomycin tolerance in EHEC. We also show that phosphorylated TorR directly represses the expression of glpT, a gene that encodes a symporter of fosfomycin and glycerol-3-phosphate, and activation of the TorR protein results in the reduced uptake of fosfomycin by cells. However, cells in which the Tor pathway was activated had an impaired growth phenotype when cultured with glycerol-3-phosphate as a carbon substrate. These observations suggest that the TorSRT pathway is the second two-component system to reversibly control fosfomycin tolerance and glycerol-3-phosphate uptake in EHEC, and this may be beneficial for bacteria by alleviating the biological cost. We expect that this mechanism could be a potential target to enhance the utility of fosfomycin as chemotherapy against multidrug-resistant pathogens. PMID:25512306

  2. GAPDH gene diversity in spirochetes: a paradigm for genetic promiscuity.

    PubMed

    Figge, R M; Cerff, R

    2001-12-01

    In this study we have determined gap sequences from nine different spirochetes. Phylogenetic analyses of these sequences in the context of all other available eubacterial and a selection of eukaryotic Gap sequences demonstrated that the eubacterial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene diversity encompasses at least five highly distinct gene families. Within these gene families, spirochetes show an extreme degree of sequence divergence that is probably the result of several lateral gene transfer events between spirochetes and other eubacterial phyla, and early gene duplications in the eubacterial ancestor. A Gap1 sequence from the syphilis spirochete Treponema pallidum has recently been shown to be closely related to GapC sequences from Euglenozoa. Here we demonstrate that several other spirochetal species are part of this cluster, supporting the conclusion that an interkingdom gene transfer from spirochetes to Euglenozoa must have occurred. Furthermore, we provide evidence that the GAPDH genes present in the protists Parabasalia may also be of spirochetal descent.

  3. The plastid ndh genes code for an NADH-specific dehydrogenase: isolation of a complex I analogue from pea thylakoid membranes.

    PubMed

    Sazanov, L A; Burrows, P A; Nixon, P J

    1998-02-01

    The plastid genomes of several plants contain ndh genes-homologues of genes encoding subunits of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I, involved in respiration in mitochondria and eubacteria. From sequence similarities with these genes, the ndh gene products have been suggested to form a large protein complex (Ndh complex); however, the structure and function of this complex remains to be established. Herein we report the isolation of the Ndh complex from the chloroplasts of the higher plant Pisum sativum. The purification procedure involved selective solubilization of the thylakoid membrane with dodecyl maltoside, followed by two anion-exchange chromatography steps and one size-exclusion chromatography step. The isolated Ndh complex has an apparent total molecular mass of approximately 550 kDa and according to SDS/PAGE consists of at least 16 subunits including NdhA, NdhI, NdhJ, NdhK, and NdhH, which were identified by N-terminal sequencing and immunoblotting. The Ndh complex showed an NADH- and deamino-NADH-specific dehydrogenase activity, characteristic of complex I, when either ferricyanide or the quinones menadione and duroquinone were used as electron acceptors. This study describes the isolation of the chloroplast analogue of the respiratory complex I and provides direct evidence for the function of the plastid Ndh complex as an NADH:plastoquinone oxidoreductase. Our results are compatible with a dual role for the Ndh complex in the chlororespiratory and cyclic photophosphorylation pathways.

  4. Identification of formate dehydrogenase-specific mRNA species and nucleotide sequence of the fdhC gene of Methanobacterium formicicum.

    PubMed Central

    White, W B; Ferry, J G

    1992-01-01

    The overlapping fdhA and fdhB genes of Methanobacterium formicicum, which encode the alpha and beta subunits, respectively, of formate dehydrogenase, were cotranscribed as part of an approximately 4.5-kb transcript. An additional gene (fdhC) upstream of fdhA was cotranscribed with fdhA and fdhB. The deduced amino acid sequence suggested that fdhC has the potential to encode a hydrophobic polypeptide with a calculated molecular weight of 29,417. A hydropathy plot of the hypothetical polypeptide indicated several potential membrane-spanning regions. The putative fdhC gene product had 28% identity with the deduced amino acid sequence of the nirC gene from Salmonella typhimurium. Northern (RNA) blot analyses and primer extension assays located a transcription start site 268 bp upstream of the initiation codon of fdhC. A sequence identical to the consensus promoter sequence for methanogenic organisms was situated between -35 and -25 bp from the proposed transcription start site. In addition to the 4.5-kb transcript, Northern blot analyses detected a 1.1-kb transcript with an fdhC-specific probe and a 3.4-kb transcript with either an fdhA- or fdhB-specific probe. The levels of all three transcripts were significantly greater in cells grown in media supplemented with molybdate. Images PMID:1378430

  5. Defects in the HSD11 gene encoding 11[beta]-hydroxysteriod dehydrogenase are not found in patients with apparent mineralocorticoid excess or 11-oxoreductase deficiency

    SciTech Connect

    Nikkila, H.; White, P.C. ); Tannin, G.M. ); New, M.I.; Taylor, N.F. ); Kalaitzoglou, G.; Monder, C. )

    1993-09-01

    The syndrome of apparent mineralocorticoid excess (AME) is a form of low renin hypertension that is thought to be caused by congenital deficiency of 11[beta]-hydroxysteroid dehydrogenase (11HSD) activity. This enzyme converts cortisol to cortisone and apparently prevents cortisol from acting as a ligand for the mineralocorticoid (type I) receptor. It also catalyzes the reverse oxoreductase (cortisone to cortisol) reaction. Four patients with AME and the parents of the first patient described (now deceased) were analyzed for mutations in the cloned HSD11 gene encoding an 11HSD enzyme. A patient with suspected cortisone reductase deficiency was also studied. No gross deletions or rearrangements in the HSD11 gene were apparent on hybridizations of blot of genomic DNA. Direct sequencing of polymerase chain reaction-amplified fragments corresponding to the coding sequences, intronexon junctions, and proximal untranslated regions of this gene revealed no mutations. AME may involve mutations in a gene for another enzyme with 11HSD activity or perhaps another cortisol metabolizing enzyme. 48 refs., 2 figs., 2 tabs.

  6. The Agaricus bisporus pruA gene encodes a cytosolic delta 1-pyrroline-5-carboxylate dehydrogenase which is expressed in fruit bodies but not in gill tissue.

    PubMed Central

    Schaap, P J; Müller, Y; Sonnenberg, A S; van Griensven, L J; Visser, J

    1997-01-01

    A fortuitously cloned 3'-truncated cDNA encoding the Agaricus bisporus delta 1-pyrroline-5-carboxylate dehydrogenase was used to characterize the complete gene. The gene would encode a cytosolic polypeptide of 546 amino acids, and the basidiomycetous gene was evenly expressed in various parts of the mushroom except for the gills. No expression was detected in compost-grown mycelium. The steady-state mRNA level of the gene in the vegetative phase was determined on simple synthetic media and was two- to threefold higher with ammonium or proline as the sole nitrogen source compared to glutamate as the sole nitrogen source. Moreover, the steady-state mRNA level was not markedly influenced by addition of ammonium phosphate to proline- or glutamate-utilizing cultures. The results suggest that ammonium and the amino acids proline and glutamate are equally preferred nitrogen sources in this organism and are consistent with previous observations of H. M Kalisz, D.A. Wood, and D. Moore (Trans. Br. Mycol. Soc. 88:221-227, 1987) that A. bisporus continues to degrade protein and secrete ammonium even if ammonium and glucose are present in the culture medium. PMID:8979339

  7. Expression, purification, enzymatic characterization and crystallization of glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi, the first one from phylum Percolozoa.

    PubMed

    Machado, Agnes Thiane Pereira; Silva, Marcio; Iulek, Jorge

    2016-11-01

    Naegleria gruberi had its genome sequenced by Fritz-Laylin and collaborators in 2010. It is not pathogenic, but has characteristics similar to those of Naegleria fowleri, opportunistic pathogen that can cause fatal encephalitis in humans. N. gruberi genome has contributed to a better understanding of the primitive eukaryotic metabolism and revealed the complexity of several metabolic pathways. In this paper we describe the expression, purification, enzyme characterization and crystallization of N. gruberi GAPDH, the first one for an organism belonging to phylum Percolozoa. The results indicated that 10 mM, 8.0 and 25 °C are the optimum arsenate concentration, pH and temperature, respectively. The enzyme presents allosteric positive cooperativity for substrates NAD(+) and G3P as indicated by the Hill coefficients. The phylogenetic proximity between N. fowleri and N. gruberi suggests that contributions from the study of the latter might provide information to assist the search for treatments of Primary Amebic Meningoencephalitis, especially, in this work, taking into account that GAPDH is identified as a therapeutic target. PMID:27426132

  8. Expression, purification, enzymatic characterization and crystallization of glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi, the first one from phylum Percolozoa.

    PubMed

    Machado, Agnes Thiane Pereira; Silva, Marcio; Iulek, Jorge

    2016-11-01

    Naegleria gruberi had its genome sequenced by Fritz-Laylin and collaborators in 2010. It is not pathogenic, but has characteristics similar to those of Naegleria fowleri, opportunistic pathogen that can cause fatal encephalitis in humans. N. gruberi genome has contributed to a better understanding of the primitive eukaryotic metabolism and revealed the complexity of several metabolic pathways. In this paper we describe the expression, purification, enzyme characterization and crystallization of N. gruberi GAPDH, the first one for an organism belonging to phylum Percolozoa. The results indicated that 10 mM, 8.0 and 25 °C are the optimum arsenate concentration, pH and temperature, respectively. The enzyme presents allosteric positive cooperativity for substrates NAD(+) and G3P as indicated by the Hill coefficients. The phylogenetic proximity between N. fowleri and N. gruberi suggests that contributions from the study of the latter might provide information to assist the search for treatments of Primary Amebic Meningoencephalitis, especially, in this work, taking into account that GAPDH is identified as a therapeutic target.

  9. Assignment of the human dihydropyrimidine dehydrogenase gene (DPYD) to chromosome region 1p22 by fluorescence in situ hybridization

    SciTech Connect

    Takai, Setsuo; Fernandez-Salguero, Pedro; Kimura, Shioko

    1994-12-01

    Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the three-step pathway of uracil and thymine catabolism leading to the formation of {beta}-alanine and {beta}-aminobutyric acid, respectively. Several studies have demonstrated the importance of DPD in cancer patients, particularly in those lacking or having only low levels of activity. Patients exhibiting severe toxicity when administered 5-fluorouracil were shown to have low DPD activity. Studies of affected families demonstrated that the deficiency was inherited in an autosomal recessive pattern. DPD deficiency is one of several inherited disorders of pyrimidine metabolism, clinically termed thymine-uracil-uria. 14 refs., 1 fig.

  10. Bioinformatics based structural characterization of glucose dehydrogenase (gdh) gene and growth promoting activity of Leclercia sp. QAU-66

    PubMed Central

    Naveed, Muhammad; Ahmed, Iftikhar; Khalid, Nauman; Mumtaz, Abdul Samad

    2014-01-01

    Glucose dehydrogenase (GDH; EC 1.1. 5.2) is the member of quinoproteins group that use the redox cofactor pyrroloquinoline quinoine, calcium ions and glucose as substrate for its activity. In present study, Leclercia sp. QAU-66, isolated from rhizosphere of Vigna mungo, was characterized for phosphate solubilization and the role of GDH in plant growth promotion of Phaseolus vulgaris. The strain QAU-66 had ability to solubilize phosphorus and significantly (p ≤ 0.05) promoted the shoot and root lengths of Phaseolus vulgaris. The structural determination of GDH protein was carried out using bioinformatics tools like Pfam, InterProScan, I-TASSER and COFACTOR. These tools predicted the structural based functional homology of pyrroloquinoline quinone domains in GDH. GDH of Leclercia sp. QAU-66 is one of the main factor that involved in plant growth promotion and provides a solid background for further research in plant growth promoting activities. PMID:25242947

  11. Disruption of the pdhB Pyruvate Dehydrogenase Gene Affects Colony Morphology, In Vitro Growth and Cell Invasiveness of Mycoplasma agalactiae

    PubMed Central

    Hegde, Shivanand; Rosengarten, Renate; Chopra-Dewasthaly, Rohini

    2015-01-01

    The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates on Mycoplasma agalactiae, which causes significant economic losses through its contribution to contagious agalactia in small ruminants by as yet unknown mechanisms. This lack of knowledge is primarily due to its fastidious growth requirements and the scarcity of genetic tools available for its manipulation and analysis. Transposon mutagenesis of M. agalactiae type strain PG2 resulted in several disruptions throughout the genome. A mutant defective in growth in vitro was found to have a transposon insertion in the pdhB gene, which encodes a component of the pyruvate dehydrogenase complex. This growth difference was quite significant during the actively dividing logarithmic phase but a gradual recovery was observed as the cells approached stationary phase. The mutant also exhibited a different and smaller colony morphology compared to the wild type strain PG2. For complementation, pdhAB was cloned downstream of a strong vpma promoter and upstream of a lacZ reporter gene in a newly constructed complementation vector. When transformed with this vector the pdhB mutant recovered its normal growth and colony morphology. Interestingly, the pdhB mutant also had significantly reduced invasiveness in HeLa cells, as revealed by double immunofluorescence staining. This deficiency was recovered in the complemented strain, which had invasiveness comparable to that of PG2. Taken together, these data indicate that pyruvate dehydrogenase might be an important player in infection with and colonization by M. agalactiae. PMID:25799063

  12. A Functional myo-Inositol Dehydrogenase Gene Is Required for Efficient Nitrogen Fixation and Competitiveness of Sinorhizobium fredii USDA191 To Nodulate Soybean (Glycine max [L.] Merr.)

    PubMed Central

    Jiang, Guoqiao; Krishnan, Ammulu Hari; Kim, Yong-Woong; Wacek, Thomas J.; Krishnan, Hari B.

    2001-01-01

    Inositol derivative compounds provide a nutrient source for soil bacteria that possess the ability to degrade such compounds. Rhizobium strains that are capable of utilizing certain inositol derivatives are better colonizers of their host plants. We have cloned and determined the nucleotide sequence of the myo-inositol dehydrogenase gene (idhA) of Sinorhizobium fredii USDA191, the first enzyme responsible for inositol catabolism. The deduced IdhA protein has a molecular mass of 34,648 Da and shows significant sequence similarity with protein sequences of Sinorhizobium meliloti IdhA and MocA; Bacillus subtilis IolG, YrbE, and YucG; and Streptomyces griseus StrI. S. fredii USDA191 idhA mutants revealed no detectable myo-inositol dehydrogenase activity and failed to grow on myo-inositol as a sole carbon source. Northern blot analysis and idhA-lacZ fusion expression studies indicate that idhA is inducible by myo-inositol. S. fredii USDA191 idhA mutant was drastically affected in its ability to reduce nitrogen and revealed deteriorating bacteroids inside the nodules. The number of bacteria recovered from such nodules was about threefold lower than the number of bacteria isolated from nodules initiated by S. fredii USDA191. In addition, the idhA mutant was also severely affected in its ability to compete with the wild-type strain in nodulating soybean. Under competitive conditions, nodules induced on soybean roots were predominantly occupied by the parent strain, even when the idhA mutant was applied at a 10-fold numerical advantage. Thus, we conclude that a functional idhA gene is required for efficient nitrogen fixation and for competitive nodulation of soybeans by S. fredii USDA191. PMID:11274120

  13. Cloning, heterologous expression and biochemical characterization of plastidial sn-glycerol-3-phosphate acyltransferase from Helianthus annuus.

    PubMed

    Payá-Milans, Miriam; Venegas-Calerón, Mónica; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2015-03-01

    The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyzes the first step of glycerolipid assembly within the stroma of the chloroplast. In the present study, the sunflower (Helianthus annuus, L.) stromal GPAT was cloned, sequenced and characterized. We identified a single ORF of 1344base pairs that encoded a GPAT sharing strong sequence homology with the plastidial GPAT from Arabidopsis thaliana (ATS1, At1g32200). Gene expression studies showed that the highest transcript levels occurred in green tissues in which chloroplasts are abundant. The corresponding mature protein was heterologously overexpressed in Escherichia coli for purification and biochemical characterization. In vitro assays using radiolabelled acyl-ACPs and glycerol-3-phosphate as substrates revealed a strong preference for oleic versus palmitic acid, and weak activity towards stearic acid. The positional fatty acid composition of relevant chloroplast phospholipids from sunflower leaves did not reflect the in vitro GPAT specificity, suggesting a more complex scenario with mixed substrates at different concentrations, competition with other acyl-ACP consuming enzymatic reactions, etc. In summary, this study has confirmed the affinity of this enzyme which would partly explain the resistance to cold temperatures observed in sunflower plants.

  14. A proposal for nomenclature of aldehyde dehydrogenases in Saccharomyces cerevisiae and characterization of the stress-inducible ALD2 and ALD3 genes.

    PubMed

    Navarro-Aviño, J P; Prasad, R; Miralles, V J; Benito, R M; Serrano, R

    1999-07-01

    The complete sequencing of the genome of Saccharomyces cerevisiae indicated that this organism contains five genes encoding aldehyde dehydrogenases. YOR374w and YER073w correspond to the mitochondrial isoforms and we propose as gene names ALD4 and ALD5, respectively. YPL061w has been described as the cytoplasmic constitutive isoform and named ALD6. We characterize here the tandem-repeated ORFs YMR170c and YMR169c as the cytoplasmic stress-inducible isoforms, with gene names ALD2 and ALD3, respectively. The expression of ALD2 and ALD3 is dependent on the general-stress transcription factors Msn2,4 but independent of the HOG MAP kinase pathway. ALD3 is induced by a variety of stresses, including osmotic shock, heat shock, glucose exhaustion, oxidative stress and drugs. ALD2 is only induced by osmotic stress and glucose exhaustion. A double null mutant, ald2 ald3, exhibited unchanged sensitivity to any of the above stresses. The only phenotype detected in this mutant was a reduced growth rate in ethanol medium as compared to the wild type. PMID:10407263

  15. The enhancement of tolerance to salt and cold stresses by modifying the redox state and salicylic acid content via the cytosolic malate dehydrogenase gene in transgenic apple plants.

    PubMed

    Wang, Qing-Jie; Sun, Hong; Dong, Qing-Long; Sun, Tian-Yu; Jin, Zhong-Xin; Hao, Yu-Jin; Yao, Yu-Xin

    2016-10-01

    In this study, we characterized the role of an apple cytosolic malate dehydrogenase gene (MdcyMDH) in the tolerance to salt and cold stresses and investigated its regulation mechanism in stress tolerance. The MdcyMDH transcript was induced by mild cold and salt treatments, and MdcyMDH-overexpressing apple plants possessed improved cold and salt tolerance compared to wild-type (WT) plants. A digital gene expression tag profiling analysis revealed that MdcyMDH overexpression largely altered some biological processes, including hormone signal transduction, photosynthesis, citrate cycle and oxidation-reduction. Further experiments verified that MdcyMDH overexpression modified the mitochondrial and chloroplast metabolisms and elevated the level of reducing power, primarily caused by increased ascorbate and glutathione, as well as the increased ratios of ascorbate/dehydroascorbate and glutathione/glutathione disulphide, under normal and especially stress conditions. Concurrently, the transgenic plants produced a high H2 O2 content, but a low O2·- production rate was observed compared to the WT plants. On the other hand, the transgenic plants accumulated more free and total salicylic acid (SA) than the WT plants under normal and stress conditions. Taken together, MdcyMDH conferred the transgenic apple plants a higher stress tolerance by producing more reductive redox states and increasing the SA level; MdcyMDH could serve as a target gene to genetically engineer salt- and cold-tolerant trees. PMID:26923485

  16. Byssochlamys nivea with patulin-producing capability has an isoepoxydon dehydrogenase gene (idh) with sequence homology to Penicillium expansum and P. griseofulvum.

    PubMed

    Dombrink-Kurtzman, Mary Ann; Engberg, Amy E

    2006-09-01

    Nucleotide sequences of the isoepoxydon dehydrogenase gene (idh) for eight strains of Byssochlamys nivea were determined by constructing GenomeWalker libraries. A striking finding was that all eight strains of B. nivea examined had identical nucleotide sequences, including those of the two introns present. The length of intron 2 was nearly three times the size of introns in strains of Penicillium expansum and P. griseofulvum, but intron 1 was comparable in size to the number of nucleotides present in introns 1 and 2 of P. expansum and P. griseofulvum. A high degree of amino acid homology (88%) existed for the idh genes of the strains of B. nivea when compared with sequences of P. expansum and P. griseofulvum. There were many nucleotide differences present, but they did not affect the amino acid sequence because they were present in the third position. The identity of the B. nivea isolates was confirmed by sequencing the ITS/partial LSU (28 S) rDNA genes. Four B. nivea strains were analysed for production of patulin, a mycotoxin found primarily in apple juice and other fruit products. The B. nivea strains produced patulin in amounts comparable to P. expansum strains. Interest in the genus Byssochlamys is related to the ability of its ascospores to survive pasteurization and cause spoilage of heat-processed fruit products worldwide.

  17. Experimentally Increased Codon Bias in the Drosophila Adh Gene Leads to an Increase in Larval, But Not Adult, Alcohol Dehydrogenase Activity

    PubMed Central

    Hense, Winfried; Anderson, Nathan; Hutter, Stephan; Stephan, Wolfgang; Parsch, John; Carlini, David B.

    2010-01-01

    Although most amino acids can be encoded by more than one codon, the synonymous codons are not used with equal frequency. This phenomenon is known as codon bias and appears to be a universal feature of genomes. The translational selection hypothesis posits that the use of optimal codons, which match the most abundant species of isoaccepting tRNAs, results in increased translational efficiency and accuracy. Previous work demonstrated that the experimental reduction of codon bias in the Drosophila alcohol dehydrogenase (Adh) gene led to a significant decrease in ADH protein expression. In this study we performed the converse experiment: we replaced seven suboptimal leucine codons that occur naturally in the Drosophila melanogaster Adh gene with the optimal codon. We then compared the in vivo ADH activities imparted by the wild-type and mutant alleles. The introduction of optimal leucine codons led to an increase in ADH activity in third-instar larvae. In adult flies, however, the introduction of optimal codons led to a decrease in ADH activity. There is no evidence that other selectively constrained features of the Adh gene, or its rate of transcription, were altered by the synonymous replacements. These results are consistent with translational selection for codon bias being stronger in the larval stage and suggest that there may be a selective conflict over optimal codon usage between different developmental stages. PMID:19966063

  18. Experimentally increased codon bias in the Drosophila Adh gene leads to an increase in larval, but not adult, alcohol dehydrogenase activity.

    PubMed

    Hense, Winfried; Anderson, Nathan; Hutter, Stephan; Stephan, Wolfgang; Parsch, John; Carlini, David B

    2010-02-01

    Although most amino acids can be encoded by more than one codon, the synonymous codons are not used with equal frequency. This phenomenon is known as codon bias and appears to be a universal feature of genomes. The translational selection hypothesis posits that the use of optimal codons, which match the most abundant species of isoaccepting tRNAs, results in increased translational efficiency and accuracy. Previous work demonstrated that the experimental reduction of codon bias in the Drosophila alcohol dehydrogenase (Adh) gene led to a significant decrease in ADH protein expression. In this study we performed the converse experiment: we replaced seven suboptimal leucine codons that occur naturally in the Drosophila melanogaster Adh gene with the optimal codon. We then compared the in vivo ADH activities imparted by the wild-type and mutant alleles. The introduction of optimal leucine codons led to an increase in ADH activity in third-instar larvae. In adult flies, however, the introduction of optimal codons led to a decrease in ADH activity. There is no evidence that other selectively constrained features of the Adh gene, or its rate of transcription, were altered by the synonymous replacements. These results are consistent with translational selection for codon bias being stronger in the larval stage and suggest that there may be a selective conflict over optimal codon usage between different developmental stages.

  19. Experimental Horizontal Gene Transfer of Methylamine Dehydrogenase Mimics Prevalent Exchange in Nature and Overcomes the Methylamine Growth Constraints Posed by the Sub-Optimal N-Methylglutamate Pathway

    PubMed Central

    Nayak, Dipti D.; Marx, Christopher J.

    2015-01-01

    Methylamine plays an important role in the global carbon and nitrogen budget; microorganisms that grow on reduced single carbon compounds, methylotrophs, serve as a major biological sink for methylamine in aerobic environments. Two non-orthologous, functionally degenerate routes for methylamine oxidation have been studied in methylotrophic Proteobacteria: Methylamine dehydrogenase and the N-methylglutamate pathway. Recent work suggests the N-methylglutamate (NMG) pathway may be more common in nature than the well-studied methylamine dehydrogenase (MaDH, encoded by the mau gene cluster). However, the distribution of these pathways across methylotrophs has never been analyzed. Furthermore, even though horizontal gene transfer (HGT) is commonly invoked as a means to transfer these pathways between strains, the physiological barriers to doing so have not been investigated. We found that the NMG pathway is both more abundant and more universally distributed across methylotrophic Proteobacteria compared to MaDH, which displays a patchy distribution and has clearly been transmitted by HGT even amongst very closely related strains. This trend was especially prominent in well-characterized strains of the Methylobacterium extroquens species, which also display significant phenotypic variability during methylamine growth. Strains like Methylobacterium extorquens PA1 that only encode the NMG pathway grew on methylamine at least five-fold slower than strains like Methylobacterium extorquens AM1 that also possess the mau gene cluster. By mimicking a HGT event through the introduction of the M. extorquens AM1 mau gene cluster into the PA1 genome, the resulting strain instantaneously achieved a 4.5-fold increase in growth rate on methylamine and a 11-fold increase in fitness on methylamine, which even surpassed the fitness of M. extorquens AM1. In contrast, when three replicate populations of wild type M. extorquens PA1 were evolved on methylamine as the sole carbon and energy

  20. Gene expression patterns in the hippocampus during the development and aging of Glud1 (Glutamate Dehydrogenase 1) transgenic and wild type mice

    PubMed Central

    2014-01-01

    Background Extraneuronal levels of the neurotransmitter glutamate in brain rise during aging. This is thought to lead to synaptic dysfunction and neuronal injury or death. To study the effects of glutamate hyperactivity in brain, we created transgenic (Tg) mice in which the gene for glutamate dehydrogenase (Glud1) is over-expressed in neurons and in which such overexpression leads to excess synaptic release of glutamate. In this study, we analyzed whole genome expression in the hippocampus, a region important for learning and memory, of 10 day to 20 month old Glud1 and wild type (wt) mice. Results During development, maturation and aging, both Tg and wt exhibited decreases in the expression of genes related to neurogenesis, neuronal migration, growth, and process elongation, and increases in genes related to neuro-inflammation, voltage-gated channel activity, and regulation of synaptic transmission. Categories of genes that were differentially expressed in Tg vs. wt during development were: synaptic function, cytoskeleton, protein ubiquitination, and mitochondria; and, those differentially expressed during aging were: synaptic function, vesicle transport, calcium signaling, protein kinase activity, cytoskeleton, neuron projection, mitochondria, and protein ubiquitination. Overall, the effects of Glud1 overexpression on the hippocampus transcriptome were greater in the mature and aged than the young. Conclusions Glutamate hyperactivity caused gene expression changes in the hippocampus at all ages. Some of these changes may result in premature brain aging. The identification of these genomic expression differences is important in understanding the effects of glutamate dysregulation on neuronal function during aging or in neurodegenerative diseases. PMID:24593767

  1. Evidence for lateral transfer of genes encoding ferredoxins, nitroreductases, NADH oxidase, and alcohol dehydrogenase 3 from anaerobic prokaryotes to Giardia lamblia and Entamoeba histolytica.

    PubMed

    Nixon, Julie E J; Wang, Amy; Field, Jessica; Morrison, Hilary G; McArthur, Andrew G; Sogin, Mitchell L; Loftus, Brendan J; Samuelson, John

    2002-04-01

    Giardia lamblia and Entamoeba histolytica are amitochondriate, microaerophilic protists which use fermentation enzymes like those of bacteria to survive anaerobic conditions within the intestinal lumen. Genes encoding fermentation enzymes and related electron transport peptides (e.g., ferredoxins) in giardia organisms and amebae are hypothesized to be derived from either an ancient anaerobic eukaryote (amitochondriate fossil hypothesis), a mitochondrial endosymbiont (hydrogen hypothesis), or anaerobic bacteria (lateral transfer hypothesis). The goals here were to complete the molecular characterization of giardial and amebic fermentation enzymes and to determine the origins of the genes encoding them, when possible. A putative giardia [2Fe-2S]ferredoxin which had a hypothetical organelle-targeting sequence at its N terminus showed similarity to mitochondrial ferredoxins and the hydrogenosomal ferredoxin of Trichomonas vaginalis (another luminal protist). However, phylogenetic trees were star shaped, with weak bootstrap support, so we were unable to confirm or rule out the endosymbiotic origin of the giardia [2Fe-2S]ferredoxin gene. Putative giardial and amebic 6-kDa ferredoxins, ferredoxin-nitroreductase fusion proteins, and oxygen-insensitive nitroreductases each tentatively supported the lateral transfer hypothesis. Although there were not enough sequences to perform meaningful phylogenetic analyses, the unique common occurrence of these peptides and enzymes in giardia organisms, amebae, and the few anaerobic prokaryotes suggests the possibility of lateral transfer. In contrast, there was more robust phylogenetic evidence for the lateral transfer of G. lamblia genes encoding an NADH oxidase from a gram-positive coccus and a microbial group 3 alcohol dehydrogenase from thermoanaerobic prokaryotes. In further support of lateral transfer, the G. lamblia NADH oxidase and adh3 genes appeared to have an evolutionary history distinct from those of E. histolytica.

  2. Pyruvate dehydrogenase kinase 2 and 4 gene deficiency attenuates nociceptive behaviors in a mouse model of acute inflammatory pain.

    PubMed

    Jha, Mithilesh Kumar; Rahman, Md Habibur; Park, Dong Ho; Kook, Hyun; Lee, In-Kyu; Lee, Won-Ha; Suk, Kyoungho

    2016-09-01

    Pyruvate dehydrogenase (PDH) kinases (PDKs) 1-4, expressed in peripheral and central tissues, regulate the activity of the PDH complex (PDC). The PDC is an important mitochondrial gatekeeping enzyme that controls cellular metabolism. The role of PDKs in diverse neurological disorders, including neurometabolic aberrations and neurodegeneration, has been described. Implications for a role of PDKs in inflammation and neurometabolic coupling led us to investigate the effect of genetic ablation of PDK2/4 on nociception in a mouse model of acute inflammatory pain. Deficiency in Pdk2 and/or Pdk4 in mice led to attenuation of formalin-induced nociceptive behaviors (flinching, licking, biting, or lifting of the injected paw). Likewise, the pharmacological inhibition of PDKs substantially diminished the nociceptive responses in the second phase of the formalin test. Furthermore, formalin-provoked paw edema formation and mechanical and thermal hypersensitivities were significantly reduced in Pdk2/4-deficient mice. Formalin-driven neutrophil recruitment at the site of inflammation, spinal glial activation, and neuronal sensitization were substantially lessened in the second or late phase of the formalin test in Pdk2/4-deficient animals. Overall, our results suggest that PDK2/4 can be a potential target for the development of pharmacotherapy for the treatment of acute inflammatory pain. © 2016 Wiley Periodicals, Inc. PMID:26931482

  3. Fructose metabolism in the human erythrocyte. Phosphorylation to fructose 3-phosphate.

    PubMed Central

    Petersen, A; Kappler, F; Szwergold, B S; Brown, T R

    1992-01-01

    In human erythrocytes, the first step in the metabolism of fructose is generally thought to be phosphorylation to fructose 6-phosphate catalysed by hexokinase. In variance with this assumption, we show here that fructose in these cells is metabolized primarily to fructose 3-phosphate by a specific 3-phosphokinase. This process has an overall estimated Km of 30 mM with respect to extracellular fructose and an apparent Vmax. of 0.6 mumol/h per ml. At a fixed concentration of fructose in the medium, the accumulation of fructose 3-phosphate was linearly dependent on the duration of incubation up to 5 h and was not affected by glucose. Once accumulated, fructose 3-phosphate appears to be degraded and/or relatively slowly metabolized, decreasing by only approximately 30% after a 12 h incubation in a fructose-free medium. PMID:1599419

  4. Molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II of Acinetobacter calcoaceticus.

    PubMed Central

    Gillooly, D J; Robertson, A G; Fewson, C A

    1998-01-01

    The nucleotide sequences of xylB and xylC from Acinetobacter calcoaceticus, the genes encoding benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II, were determined. The complete nucleotide sequence indicates that these two genes form part of an operon and this was supported by heterologous expression and physiological studies. Benzaldehyde dehydrogenase II is a 51654 Da protein with 484 amino acids per subunit and it is typical of other prokaryotic and eukaryotic aldehyde dehydrogenases. Benzyl alcohol dehydrogenase has a subunit Mr of 38923 consisting of 370 amino acids, it stereospecifically transfers the proR hydride of NADH, and it is a member of the family of zinc-dependent long-chain alcohol dehydrogenases. The enzyme appears to be more similar to animal and higher-plant alcohol dehydrogenases than it is to most other microbial alcohol dehydrogenases. Residue His-51 of zinc-dependent alcohol dehydrogenases is thought to be necessary as a general base for catalysis in this category of alcohol dehydrogenases. However, this residue was found to be replaced in benzyl alcohol dehydrogenase from A. calcoaceticus by an isoleucine, and the introduction of a histidine residue in this position did not alter the kinetic coefficients, pH optimum or substrate specificity of the enzyme. Other workers have shown that His-51 is also absent from the TOL-plasmid-encoded benzyl alcohol dehydrogenase of Pseudomonas putida and so these two closely related enzymes presumably have a catalytic mechanism that differs from that of the archetypal zinc-dependent alcohol dehydrogenases. PMID:9494109

  5. Polymorphic sites in the African population detected by sequence analysis of the glucose-6-phosphate dehydrogenase gene outline the evolution of the variants A and A-.

    PubMed Central

    Vulliamy, T J; Othman, A; Town, M; Nathwani, A; Falusi, A G; Mason, P J; Luzzatto, L

    1991-01-01

    The human X chromosome-linked gene encoding glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) is known to be highly polymorphic from the biochemical characterization of enzyme variants. The variant A (with enzyme activity in the normal range) and the variant A- (associated with enzyme deficiency) each have a frequency of about 0.2 in several African populations. Two restriction fragment length polymorphisms have also been found in people of African descent, but not in other populations, whereas a silent mutation has been shown to be polymorphic in Mediterranean, Middle Eastern, African, and Indian populations. We report now on two additional polymorphisms that we have detected by sequence analysis, one in intron 7 and one in intron 8. The analysis of 54 African male subjects for the seven polymorphic sites, clustered within 3 kilobases of the G6PD gene, has revealed only 7 of the 128 possible haplotypes, indicating marked linkage disequilibrium. These data have enabled us to suggest an evolutionary pathway for the different mutations, with only a single ambiguity. The mutation underlying the A variant is the most ancient and the mutation underlying the A- variant is the most recent. Since it seems reasonable that the A- allele is subject to positive selection by malaria, whereas the other alleles are neutral, G6PD may lend itself to the analysis of the role of random genetic drift and selection in determining allele frequencies within a single genetic locus in human populations. Images PMID:1924316

  6. Significant improvement of stress tolerance in tobacco plants by overexpressing a stress-responsive aldehyde dehydrogenase gene from maize (Zea mays).

    PubMed

    Huang, Weizao; Ma, Xinrong; Wang, Qilin; Gao, Yongfeng; Xue, Ying; Niu, Xiangli; Yu, Guirong; Liu, Yongsheng

    2008-11-01

    Aldehyde dehydrogenases (ALDHs) play a central role in detoxification processes of aldehydes generated in plants when exposed to the stressed conditions. In order to identify genes required for the stresses responses in the grass crop Zea mays, an ALDH (ZmALDH22A1) gene was isolated and characterized. ZmALDH22A1 belongs to the family ALDH22 that is currently known only in plants. The ZmALDH22A1 encodes a protein of 593 amino acids that shares high identity with the orthologs from Saccharum officinarum (95%), Oryza sativa (89%), Triticum aestivum (87%) and Arabidopsis thaliana (77%), respectively. Real-time PCR analysis indicates that ZmALDH22A1 is expressed differentially in different tissues. Various elevated levels of ZmALDH22A1 expression have been detected when the seedling roots exposed to abiotic stresses including dehydration, high salinity and abscisic acid (ABA). Tomato stable transformation of construct expressing the ZmALDH22A1 signal peptide fused with yellow fluorescent protein (YFP) driven by the CaMV35S-promoter reveals that the fusion protein is targeted to plastid. Transgenic tobacco plants overexpressing ZmALDH22A1 shows elevated stresses tolerance. Stresses tolerance in transgenic plants is accompanied by a reduction of malondialdehyde (MDA) derived from cellular lipid peroxidation.

  7. Increased inosine 5{prime}-monophosphate dehydrogenase gene expression in replicating cells: A response to growth factors, not to changes in cell cycle parameters

    SciTech Connect

    Tsutani, Hiroshi; Collart, F.R.; Glesne, D.A.; Huberman, E. |

    1997-07-01

    The authors have analyzed levels of inosine 5{prime}-monophosphate dehydrogenase (IMPDH; E.C. 1.1.1.205) type II mRNA levels in a human melanoma cell line, SK-MEL-131, and a Chinese hamster ovary cell line synchronously progressing through the cell cycle following treatment with aphidicolin. Following release from the aphidicolin block at the G{sub 1}-S phase boundary, the type II IMPDH gene was found to be constitutively expressed at a similar level during all stages of the cell cycle. To analyze growth regulation, as opposed to cell cycle regulation, stable SK-MEL-131 transfectants that express a type II IMPDH-promoted heterologous construct were assayed following deprivation of serum growth factors and after restimulation with fresh serum. Serum deprivation resulted in down-regulation of both steady state type II IMPDH mRNA levels and promoter activity, while restimulation with serum resulted in up-regulation of these parameters. These findings support the conclusion that the increase in IMPDH type II gene expression in replicating cells is mainly due to growth factor regulation rather than changes in cell cycle parameters and that this regulation is mediated primarily by a transcriptional mechanism. The increased level of IMPDH expression and activity found in many tumors may therefore also be due to a transcriptionally mediated response to growth factors.

  8. The mitochondrial malate dehydrogenase 1 gene GhmMDH1 is involved in plant and root growth under phosphorus deficiency conditions in cotton.

    PubMed

    Wang, Zhi-An; Li, Qing; Ge, Xiao-Yang; Yang, Chun-Lin; Luo, Xiao-Li; Zhang, An-Hong; Xiao, Juan-Li; Tian, Ying-Chuan; Xia, Gui-Xian; Chen, Xiao-Ying; Li, Fu-Guang; Wu, Jia-He

    2015-07-16

    Cotton, an important commercial crop, is cultivated for its natural fibers, and requires an adequate supply of soil nutrients, including phosphorus, for its growth. Soil phosporus exists primarily in insoluble forms. We isolated a mitochondrial malate dehydrogenase (MDH) gene, designated as GhmMDH1, from Gossypium hirsutum L. to assess its effect in enhancing P availability and absorption. An enzyme kinetic assay showed that the recombinant GhmMDH1 possesses the capacity to catalyze the interconversion of oxaloacetate and malate. The malate contents in the roots, leaves and root exudates was significantly higher in GhmMDH1-overexpressing plants and lower in knockdown plants compared with the wild-type control. Knockdown of GhmMDH1 gene resulted in increased respiration rate and reduced biomass whilst overexpression of GhmMDH1 gave rise to decreased respiration rate and higher biomass in the transgenic plants. When cultured in medium containing only insoluble phosphorus, Al-phosphorus, Fe-phosphorus, or Ca-phosphorus, GhmMDH1-overexpressing plants produced significantly longer roots and had a higher biomass and P content than WT plants, however, knockdown plants showed the opposite results for these traits. Collectively, our results show that GhmMDH1 is involved in plant and root growth under phosphorus deficiency conditions in cotton, owing to its functions in leaf respiration and P acquisition.

  9. Gene ercA, encoding a putative iron-containing alcohol dehydrogenase, is involved in regulation of ethanol utilization in Pseudomonas aeruginosa.

    PubMed

    Hempel, Niels; Görisch, Helmut; Mern, Demissew S

    2013-09-01

    Several two-component regulatory systems are known to be involved in the signal transduction pathway of the ethanol oxidation system in Pseudomonas aeruginosa ATCC 17933. These sensor kinases and response regulators are organized in a hierarchical manner. In addition, a cytoplasmic putative iron-containing alcohol dehydrogenase (Fe-ADH) encoded by ercA (PA1991) has been identified to play an essential role in this regulatory network. The gene ercA (PA1991) is located next to ercS, which encodes a sensor kinase. Inactivation of ercA (PA1991) by insertion of a kanamycin resistance cassette created mutant NH1. NH1 showed poor growth on various alcohols. On ethanol, NH1 grew only with an extremely extended lag phase. During the induction period on ethanol, transcription of structural genes exa and pqqABCDEH, encoding components of initial ethanol oxidation in P. aeruginosa, was drastically reduced in NH1, which indicates the regulatory function of ercA (PA1991). However, transcription in the extremely delayed logarithmic growth phase was comparable to that in the wild type. To date, the involvement of an Fe-ADH in signal transduction processes has not been reported.

  10. Cloning of the Staphylococcus aureus ddh gene encoding NAD+-dependent D-lactate dehydrogenase and insertional inactivation in a glycopeptide-resistant isolate.

    PubMed Central

    Boyle-Vavra, S; de Jonge, B L; Ebert, C C; Daum, R S

    1997-01-01

    The mechanism of low-level glycopeptide resistance among staphylococci is not known. A cytoplasmic protein, provisionally called Ddh (W. M. Milewski, S. Boyle-Vavra, B. Moreira, C. C. Ebert, and R. S. Daum, Antimicrob. Agents Chemother. 40:166-172, 1996), and the RNA transcript that contains the ddh gene, which encodes Ddh, are present in increased amounts in a vancomycin-resistant isolate, 523k, compared with the susceptible parent isolate, 523. Sequence analysis had previously revealed that Ddh is related to NAD+-dependent D-lactate dehydrogenase (D-nLDH) and VanH. This latter protein is essential for high-level glycopeptide resistance in Enterococcus faecium and Enterococcus faecalis by synthesizing the D-lactate needed for biosynthesis of D-lactate-terminating peptidoglycan precursors with low affinity for vancomycin. We now provide the direct evidence that the ddh gene product is Staphylococcus aureus D-nLDH and hereafter refer to the protein as D-nLDH. However, overproduction of this protein in isolate 523k did not result in production of D-lactate-containing peptidoglycan precursors, and susceptibility testing of ddh mutants of 523k demonstrated that S. aureus D-nLDH is not necessary for glycopeptide resistance in this isolate. We conclude that the mechanism of glycopeptide resistance in this isolate is distinct from that in enterococci. PMID:9352927

  11. Coexpression of the pyrroloquinoline quinone and glucose dehydrogenase genes from Serratia marcescens CTM 50650 conferred high mineral phosphate-solubilizing ability to Escherichia coli.

    PubMed

    Ben Farhat, Mounira; Fourati, Amin; Chouayekh, Hichem

    2013-08-01

    The genes gdh and pqqABCDE encoding glucose dehydrogenase and its pyrroloquinoline quinone cofactor were cloned from the mineral phosphate-solubilizing (MPS) bacterium Serratia marcescens CTM 50650. We investigated, for the first time, the impact of their coexpression in Escherichia coli on MPS ability. The production of recombinant PQQGDH conferred high MPS activity to the engineered E. coli. In fact, the amounts of soluble phosphorus (P) produced from tricalcium phosphate, hydroxyapatite, and Gafsa rock phosphate (GRP) were 574, 426, and 217 mg/L, respectively. In an attempt to increase the soluble P concentration, the E. coli strain coexpressing the gdh and pqqABCDE genes was immobilized in agar, calcium alginate, and k-carrageenan and was then further applied in a repeated batch (six batches) fermentation process to solubilize GRP. Compared to other encapsulated systems, alginate cell beads were noted to yield the highest concentration of soluble P, which attained 300 mg/L/batch. MPS efficiency was maximal in the presence of 5 and 40 g/L of GRP and glucose, respectively. PMID:23737304

  12. Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L.

    PubMed

    Kobayashi, Keiichi; Hattori, Takasumi; Hayashi, Rie; Kirimura, Kohtaro

    2014-01-01

    In the tricarboxylic acid (TCA) cycle, NADP(+)-specific isocitrate dehydrogenase (NADP(+)-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP(+) as a cofactor. We constructed an NADP(+)-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP(+)-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP(+)-ICDH activity. Therefore, NADP(+)-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.

  13. The mitochondrial malate dehydrogenase 1 gene GhmMDH1 is involved in plant and root growth under phosphorus deficiency conditions in cotton

    PubMed Central

    Wang, Zhi-An; Li, Qing; Ge, Xiao-Yang; Yang, Chun-Lin; Luo, Xiao-Li; Zhang, An-Hong; Xiao, Juan-Li; Tian, Ying-Chuan; Xia, Gui-Xian; Chen, Xiao-Ying; Li, Fu-Guang; Wu, Jia-He

    2015-01-01

    Cotton, an important commercial crop, is cultivated for its natural fibers, and requires an adequate supply of soil nutrients, including phosphorus, for its growth. Soil phosporus exists primarily in insoluble forms. We isolated a mitochondrial malate dehydrogenase (MDH) gene, designated as GhmMDH1, from Gossypium hirsutum L. to assess its effect in enhancing P availability and absorption. An enzyme kinetic assay showed that the recombinant GhmMDH1 possesses the capacity to catalyze the interconversion of oxaloacetate and malate. The malate contents in the roots, leaves and root exudates was significantly higher in GhmMDH1-overexpressing plants and lower in knockdown plants compared with the wild-type control. Knockdown of GhmMDH1 gene resulted in increased respiration rate and reduced biomass whilst overexpression of GhmMDH1 gave rise to decreased respiration rate and higher biomass in the transgenic plants. When cultured in medium containing only insoluble phosphorus, Al-phosphorus, Fe-phosphorus, or Ca-phosphorus, GhmMDH1-overexpressing plants produced significantly longer roots and had a higher biomass and P content than WT plants, however, knockdown plants showed the opposite results for these traits. Collectively, our results show that GhmMDH1 is involved in plant and root growth under phosphorus deficiency conditions in cotton, owing to its functions in leaf respiration and P acquisition. PMID:26179843

  14. Deletion of the Glucose-6-Phosphate Dehydrogenase Gene KlZWF1 Affects both Fermentative and Respiratory Metabolism in Kluyveromyces lactis▿

    PubMed Central

    Saliola, Michele; Scappucci, Gina; De Maria, Ilaria; Lodi, Tiziana; Mancini, Patrizia; Falcone, Claudio

    2007-01-01

    In Kluyveromyces lactis, the pentose phosphate pathway is an alternative route for the dissimilation of glucose. The first enzyme of the pathway is the glucose-6-phosphate dehydrogenase (G6PDH), encoded by KlZWF1. We isolated this gene and examined its role. Like ZWF1 of Saccharomyces cerevisiae, KlZWF1 was constitutively expressed, and its deletion led to increased sensitivity to hydrogen peroxide on glucose, but unlike the case for S. cerevisiae, the Klzwf1Δ strain had a reduced biomass yield on fermentative carbon sources as well as on lactate and glycerol. In addition, the reduced yield on glucose was associated with low ethanol production and decreased oxygen consumption, indicating that this gene is required for both fermentation and respiration. On ethanol, however, the mutant showed an increased biomass yield. Moreover, on this substrate, wild-type cells showed an additional band of activity that might correspond to a dimeric form of G6PDH. The partial dimerization of the G6PDH tetramer on ethanol suggested the production of an NADPH excess that was negative for biomass yield. PMID:17085636

  15. Caenorhabditis elegans expressing the Saccharomyces cerevisiae NADH alternative dehydrogenase Ndi1p, as a tool to identify new genes involved in complex I related diseases

    PubMed Central

    Cossard, Raynald; Esposito, Michela; Sellem, Carole H.; Pitayu, Laras; Vasnier, Christelle; Delahodde, Agnès; Dassa, Emmanuel P.

    2015-01-01

    Isolated complex I deficiencies are one of the most commonly observed biochemical features in patients suffering from mitochondrial disorders. In the majority of these clinical cases the molecular bases of the diseases remain unknown suggesting the involvement of unidentified factors that are critical for complex I function. The Saccharomyces cerevisiae NDI1 gene, encoding the mitochondrial internal NADH dehydrogenase was previously shown to complement a complex I deficient strain in Caenorhabditis elegans with notable improvements in reproduction and whole organism respiration. These features indicate that Ndi1p can functionally integrate the respiratory chain, allowing complex I deficiency complementation. Taking into account the Ndi1p ability to bypass complex I, we evaluate the possibility to extend the range of defects/mutations causing complex I deficiencies that can be alleviated by NDI1 expression. We report here that NDI1 expressing animals unexpectedly exhibit a slightly shortened lifespan, a reduction in the progeny, and a depletion of the mitochondrial genome. However, Ndi1p is expressed and targeted to the mitochondria as a functional protein that confers rotenone resistance to those animals without affecting their respiration rate and ATP content. We show that the severe embryonic lethality level caused by the RNAi knockdowns of complex I structural subunit encoding genes (e.g., NDUFV1, NDUFS1, NDUFS6, NDUFS8, or GRIM-19 human orthologs) in wild type animals is significantly reduced in the Ndi1p expressing worm. All together these results open up the perspective to identify new genes involved in complex I function, assembly, or regulation by screening an RNAi library of genes leading to embryonic lethality that should be rescued by NDI1 expression. PMID:26124772

  16. Plastid-Expressed Betaine Aldehyde Dehydrogenase Gene in Carrot Cultured Cells, Roots, and Leaves Confers Enhanced Salt Tolerance1

    PubMed Central

    Kumar, Shashi; Dhingra, Amit; Daniell, Henry

    2004-01-01

    Salinity is one of the major factors that limits geographical distribution of plants and adversely affects crop productivity and quality. We report here high-level expression of betaine aldehyde dehydrogenase (BADH) in cultured cells, roots, and leaves of carrot (Daucus carota) via plastid genetic engineering. Homoplasmic transgenic plants exhibiting high levels of salt tolerance were regenerated from bombarded cell cultures via somatic embryogenesis. Transformation efficiency of carrot somatic embryos was very high, with one transgenic event per approximately seven bombarded plates under optimal conditions. In vitro transgenic carrot cells transformed with the badh transgene were visually green in color when compared to untransformed carrot cells, and this offered a visual selection for transgenic lines. BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7-fold more, and accumulated 50- to 54-fold more betaine (93–101 μmol g−1 dry weight of β-Ala betaine and Gly betaine) than untransformed cells grown in liquid medium containing 100 mm NaCl. Transgenic carrot plants expressing BADH grew in the presence of high concentrations of NaCl (up to 400 mm), the highest level of salt tolerance reported so far among genetically modified crop plants. BADH expression was 74.8% in non-green edible parts (carrots) containing chromoplasts, and 53% in proplastids of cultured cells when compared to chloroplasts (100%) in leaves. Demonstration of plastid transformation via somatic embryogenesis utilizing non-green tissues as recipients of foreign DNA for the first time overcomes two of the major obstacles in extending this technology to important crop plants. PMID:15347789

  17. Cloning and overexpression of an NADH-dependent alcohol dehydrogenase gene from Candida maris involved in (R)-selective reduction of 5-acetylfuro[2,3-c]pyridine.

    PubMed

    Kawano, Shigeru; Yano, Miho; Hasegawa, Junzo; Yasohara, Yoshihiko

    2011-01-01

    5-((R)-1-Hydroxyethyl)-furo[2,3-c]pyridine ((R)-FPH) is a useful chiral building block in the synthesis of pharmaceuticals. An NADH-dependent alcohol dehydrogenase (AFPDH) isolated from Candida maris catalyzed the reduction of 5-acetylfuro[2,3-c]pyridine (AFP) to (R)-FPH with 100% enantiomeric excess. The gene encoding AFPDH was cloned and sequenced. The AFPDH gene comprises 762 bp and encodes a polypeptide of 27,230 Da. The deduced amino acid sequence showed a high degree of similarity to those of other members of the short-chain alcohol dehydrogenase superfamily. The AFPDH gene was overexpressed in Escherichia coli under the control of the lac promoter. One L of the cultured broth of an E. coli transformant coexpressing AFPDH and the glucose dehydrogenase (GDH) gene reduced 250 g of AFP to (R)-FPH in an organic solvent two-phase system. Under coupling with NADH regeneration using 2-propanol, 1 L of the cultured broth of an E. coli transformant expressing the AFPDH gene reduced 150 g of AFP to (R)-FPH. The optical purity of the (R)-FPH formed was 100% enantiomeric excess under both reaction conditions.

  18. Glucose-6-phosphate dehydrogenase

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a type of ...

  19. A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance in Escherichia coli and tobacco plants.

    PubMed

    Cao, Gaoyi; Liu, Yunjun; Zhang, Shengxue; Yang, Xuewen; Chen, Rongrong; Zhang, Yuwen; Lu, Wei; Liu, Yan; Wang, Jianhua; Lin, Min; Wang, Guoying

    2012-01-01

    A key enzyme in the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the primary target of the broad-spectrum herbicide glyphosate. Identification of new aroA genes coding for EPSPS with a high level of glyphosate tolerance is essential for the development of glyphosate-tolerant crops. In the present study, the glyphosate tolerance of five bacterial aroA genes was evaluated in the E. coli aroA-defective strain ER2799 and in transgenic tobacco plants. All five aroA genes could complement the aroA-defective strain ER2799, and AM79 aroA showed the highest glyphosate tolerance. Although glyphosate treatment inhibited the growth of both WT and transgenic tobacco plants, transgenic plants expressing AM79 aroA tolerated higher concentration of glyphosate and had a higher fresh weight and survival rate than plants expressing other aroA genes. When treated with high concentration of glyphosate, lower shikimate content was detected in the leaves of transgenic plants expressing AM79 aroA than transgenic plants expressing other aroA genes. These results suggest that AM79 aroA could be a good candidate for the development of transgenic glyphosate-tolerant crops.

  20. LplR, a Repressor Belonging to the TetR Family, Regulates Expression of the l-Pantoyl Lactone Dehydrogenase Gene in Rhodococcus erythropolis

    PubMed Central

    Si, Dayong; Urano, Nobuyuki; Shimizu, Sakayu

    2012-01-01

    The l-pantoyl lactone (l-PL) dehydrogenase (LPLDH) gene (lpldh) has been cloned from Rhodococcus erythropolis AKU2103, and addition of 1,2-propanediol (1,2-PD) was shown to be required for lpldh expression in this strain. In this study, based on an exploration of the nucleotide sequence around lpldh, a TetR-like regulator gene, which we designated lplR, was found upstream of lpldh, and three putative open reading frames existed between the two genes. Disruption of lplR led to 22.8 times higher lpldh expression, even without 1,2-PD induction, than that in wild-type R. erythropolis AKU2103 without 1,2-PD addition. Introduction of a multicopy vector carrying lplR (multi-lplR) into the wild-type and ΔlplR strains led to no detectable LPLDH activity even in the presence of 1,2-PD. The results of an electrophoretic mobility shift assay revealed that purified LplR bound to a 6-bp inverted-repeat sequence located in the promoter/operator region of the operon containing lpldh. These results indicated that LplR is a negative regulator in lpldh expression. Based on the clarification of the expression mechanism of lpldh, recombinant cells showing high LPLDH activity were constructed and used as a catalyst for the conversion of l-PL to ketopantoyl lactone. Finally, a promising production process of d-PL from dl-PL was constructed. PMID:22941082

  1. Glycerol-3-phosphate is a critical mobile inducer of systemic immunity in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glycerol-3-phosphate (G3P) is an important metabolite that contributes to the growth and disease-related physiologies of prokaryotes, plants, animals and humans alike. Here we show that G3P serves as the inducer of an important form of broad-spectrum immunity in plants, termed systemic acquired resi...

  2. Chemical and enzymatic methodologies for the synthesis of enantiomerically pure glyceraldehyde 3-phosphates.

    PubMed

    Gauss, Dominik; Schoenenberger, Bernhard; Wohlgemuth, Roland

    2014-05-01

    Glyceraldehyde 3-phosphates are important intermediates of many central metabolic pathways in a large number of living organisms. d-Glyceraldehyde 3-phosphate (d-GAP) is a key intermediate during glycolysis and can as well be found in a variety of other metabolic pathways. The opposite enantiomer, l-glyceraldehyde 3-phosphate (l-GAP), has been found in a few exciting new pathways. Here, improved syntheses of enantiomerically pure glyceraldehyde 3-phosphates are reported. While d-GAP was synthesized by periodate cleavage of d-fructose 6-phosphate, l-GAP was obtained by enzymatic phosphorylation of l-glyceraldehyde. (1)H- and (31)P NMR spectroscopy was applied in order to examine pH-dependent behavior of GAP over time and to identify potential degradation products. It was found that GAP is stable in acidic aqueous solution below pH 4. At pH 7, methylglyoxal is formed, whereas under alkaline conditions, the formation of lactic acid could be observed.

  3. Opossum alcohol dehydrogenases: Sequences, structures, phylogeny and evolution: evidence for the tandem location of ADH genes on opossum chromosome 5.

    PubMed

    Holmes, Roger S

    2009-03-16

    BLAT (BLAST-Like Alignment Tool) analyses and interrogations of the recently published opossum genome were undertaken using previously reported rat ADH amino acid sequences. Evidence is presented for six opossum ADH genes localized on chromosome 5 and organized in a comparable ADH gene cluster to that reported for human and rat ADH genes. The predicted amino acid sequences and secondary structures for the opossum ADH subunits and the intron-exon boundaries for opossum ADH genes showed a high degree of similarity with other mammalian ADHs, and four opossum ADH classes were identified, namely ADH1, ADH3, ADH6 and ADH4 (for which three genes were observed: ADH4A, ADH4B and ADH4C). Previous biochemical analyses of opossum ADHs have reported the tissue distribution and properties for these enzymes: ADH1, the major liver enzyme; ADH3, widely distributed in opossum tissues with similar kinetic properties to mammalian class 3 ADHs; and ADH4, for which several forms were localized in extrahepatic tissues, especially in the digestive system and in the eye. These ADHs are likely to perform similar functions to those reported for other mammalian ADHs in the metabolism of ingested and endogenous alcohols and aldehydes. Phylogenetic analyses examined opossum, human, rat, chicken and cod ADHs, and supported the proposed designation of opossum ADHs as class I (ADH1), class III (ADH3), class IV (ADH4A, ADH4B and ADH4C) and class VI (ADH6). Percentage substitution rates were examined for ADHs during vertebrate evolution which indicated that ADH3 is evolving at a much slower rate to that of the other ADH classes.

  4. Mutations in the IDH2 gene encoding the catalytic subunit of the yeast NAD+-dependent isocitrate dehydrogenase can be suppressed by mutations in the CIT1 gene encoding citrate synthase and other genes of oxidative metabolism.

    PubMed

    Gadde, D M; McCammon, M T

    1997-08-01

    During a screen for respiration competent yeast mutants that were unable to grow with acetate as a carbon source, two idh2 cit1 double mutants were identified. These strains were defective in the catalytic subunit of the NAD(+)-dependent isocitrate dehydrogenase and citrate synthase of the tricarboxylic acid (TCA) cycle. The strains harboring the idh2 alleles from these strains had two unusual phenotypes. First, their growth on many nonfermentable carbon sources was much poorer than strains containing other idh2 mutations. Second, the poor growth phenotype could be suppressed by the presence of mutations in CIT1 and other genes encoding oxidative functions. Spontaneous suppressor mutants that restore fast growth on glycerol medium to strains harboring two idh2 alleles were isolated, and a large percentage of the suppressor mutations have been identified within the CIT1 gene and at several other loci. Elevated levels of several TCA cycle proteins were observed in these idh2 mutants that were not observed in the presence of suppressing cit1 mutations. Citrate and isocitrate concentrations were also elevated in the idh2 mutants, but probably not to toxic levels. Five idh2 alleles were sequenced to understand the defects of the two classes of mutations. Sequence analysis indicated that the poor growth phenotype was caused by the loss of Idh2p protein. Similarly, eight cit1 alleles were sequenced to understand their characteristics as glycerol suppressors of idh2. These and other studies indicate that any mutation within CIT1 was capable of suppressing the idh2 mutations. Several models to explain these interactions are discussed.

  5. Enhanced production of epsilon-caprolactone by overexpression of NADPH-regenerating glucose 6-phosphate dehydrogenase in recombinant Escherichia coli harboring cyclohexanone monooxygenase gene.

    PubMed

    Lee, Won-Heong; Park, Jin-Byung; Park, Kyungmoon; Kim, Myoung-Dong; Seo, Jin-Ho

    2007-08-01

    Whole-cell conversion of cyclohexanone to epsilon-caprolactone was attempted by recombinant Escherichia coli BL21(DE3) expressing cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871. High concentrations of cyclohexanone and epsilon-caprolactone reduced CHMO-mediated bioconversion of cyclohexanone to epsilon-caprolactone in the resting recombinant E. coli cells. Metabolically active cells were employed by adopting a fed-batch culture to improve the production of epsilon-caprolactone from cyclohexanone. A glucose-limited fed-batch Baeyer-Villiger oxidation where a cyclohexanone level was maintained less than 6 g/l resulted in a maximum epsilon-caprolactone concentration of 11.0 g/l. The maximum epsilon-caprolactone concentration was improved further to 15.3 g/l by coexpression of glucose-6-phosphate dehydrogenase, an NADPH-generating enzyme encoded by the zwf gene which corresponded to a 39% enhancement in epsilon-caprolactone concentration compared with the control experiment performed under the same conditions.

  6. Comparison of the gene expression of aspartate beta-D-semialdehyde dehydrogenase at elevated hydrostatic pressure in deep-sea bacteria.

    PubMed

    Kato, C; Smorawinska, M; Li, L; Horikoshi, K

    1997-04-01

    Aspartate beta-D-semialdehyde dehydrogenase genes (asd) were cloned and sequenced from a deep-sea-adapted strictly barophilic bacterium, Shewanella sp. strain DB6705, and a moderately barophilic bacterium, Shewanella sp. strain DSS12. The determined asd sequences of these two strains were very similar, and the identity of the deduced amino acids sequences was 96.2%. The 5'-ends of the asd mRNA from both strains were localized at corresponding sites by primer extension analysis, and two transcriptional starting points, which differed by only 1 base, were detected. In strain DB6705, a pressure-regulated transcript was mainly observed, whereas in strain DSS12, a pressure-tolerant transcript was observed together with the pressure-regulated transcript. Western-blotting analysis showed that the ASD protein was expressed under higher pressure conditions in DB6705, and under all pressure conditions tested in DSS12, as reflected in the primer extension results. Our findings suggest that asd expression controlled by pressure is one of the important mechanisms involved in the adaptation of microorganisms to the deep-sea environment. PMID:9163523

  7. Rapid detection of medium chain acyl-CoA dehydrogenase gene mutations by non-radioactive, single strand conformation polymorphism minigels.

    PubMed Central

    Iolascon, A; Parrella, T; Perrotta, S; Guardamagna, O; Coates, P M; Sartore, M; Surrey, S; Fortina, P

    1994-01-01

    Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is a common inherited metabolic disorder affecting fatty acid beta oxidation. Identification of carriers is important since the disease can be fatal and is readily treatable once diagnosed. Twelve molecular defects have been identified in the MCAD gene; however, a single highly prevalent mutation, A985G, accounts for > 90% of mutant alleles in the white population. In order to facilitate the molecular diagnosis of MCAD deficiency, oligonucleotide primers were designed to amplify the exon regions encompassing the 12 mutations enzymatically, and PCR products were then screened with a single strand conformation polymorphism (SSCP) based method. Minigels were used allowing much faster run times, and silver staining was used after gel electrophoresis to eliminate the need for radioisotopic labelling strategies. Our non-radioactive, minigel SSCP approach showed that normals can be readily distinguished from heterozygotes and homozygotes for all three of the 12 known MCAD mutations which were detected in our sampling of 48 persons. In addition, each band pattern is characteristic for a specific mutation, including those mapping in the same PCR product like A985G and T1124C. When necessary, the molecular defect was confirmed using either restriction enzyme digestion of PCR products or by direct DNA sequence analysis or both. This rapid, non-radioactive approach can become routine for molecular diagnosis of MCAD deficiency and other genetic disorders. Images PMID:7966191

  8. Suitability of Commonly Used Housekeeping Genes in Gene Expression Studies for Space Radiation Research

    NASA Astrophysics Data System (ADS)

    Arenz, A.; Hellweg, C. E.; Bogner, S.; Lau, P.; Baumstark-Khan, C.

    Research on the effects of ionizing radiation exposure involves the use of real-time reverse transcription polymerase chain reaction qRT-PCR for measuring changes in gene expression Several variables needs to be controlled for gene expression analysis -- different amounts of starting material between the samples variations in enzymatic efficiencies of the reverse transcription step and differences in RNA integrity Normalization of the obtained data to an invariant endogenous control gene reference gene is the elementary step in relative quantification strategy There is a strong correlation between the quality of the normalized data and the stability of the reference gene itself This is especially relevant when the samples have been obtained after exposure to radiation qualities inducing different amounts and kinds of damage leading to a cell cycle delay or even to a cell cycle block In order to determine suitable reference genes as internal controls in qRT-PCR assays after exposure to ionizing radiation we studied the gene expression levels of commonly used reference genes in A549 lung cancer cells Expression levels obtained for human beta actin ACTB human beta-2-microglobulin B2M human glyceraldehyde-3-phosphate dehydrogenase GAPDH human porphobilinogen deaminase PBGD human 18S ribosomal RNA 18S rRNA human glucose-6-phosphate dehydrogenase G6PDH human hypoxanthine phosphoribosyl transferase HPRT human ubiquitin C UBC human transferrin TFRC

  9. Tandem amplification of a chromosomal segment harboring 5-enolpyruvylshikimate-3-phosphate synthase locus confers glyphosate resistance in Kochia scoparia.

    PubMed

    Jugulam, Mithila; Niehues, Kindsey; Godar, Amar S; Koo, Dal-Hoe; Danilova, Tatiana; Friebe, Bernd; Sehgal, Sunish; Varanasi, Vijay K; Wiersma, Andrew; Westra, Philip; Stahlman, Phillip W; Gill, Bikram S

    2014-11-01

    Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations.

  10. Tandem amplification of a chromosomal segment harboring 5-enolpyruvylshikimate-3-phosphate synthase locus confers glyphosate resistance in Kochia scoparia.

    PubMed

    Jugulam, Mithila; Niehues, Kindsey; Godar, Amar S; Koo, Dal-Hoe; Danilova, Tatiana; Friebe, Bernd; Sehgal, Sunish; Varanasi, Vijay K; Wiersma, Andrew; Westra, Philip; Stahlman, Phillip W; Gill, Bikram S

    2014-11-01

    Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations. PMID:25037215

  11. Birth of Archaeal Cells: Molecular Phylogenetic Analyses of G1P Dehydrogenase, G3P Dehydrogenases, and Glycerol Kinase Suggest Derived Features of Archaeal Membranes Having G1P Polar Lipids

    PubMed Central

    2016-01-01

    Bacteria and Eukarya have cell membranes with sn-glycerol-3-phosphate (G3P), whereas archaeal membranes contain sn-glycerol-1-phosphate (G1P). Determining the time at which cells with either G3P-lipid membranes or G1P-lipid membranes appeared is important for understanding the early evolution of terrestrial life. To clarify this issue, we reconstructed molecular phylogenetic trees of G1PDH (G1P dehydrogenase; EgsA/AraM) which is responsible for G1P synthesis and G3PDHs (G3P dehydrogenase; GpsA and GlpA/GlpD) and glycerol kinase (GlpK) which is responsible for G3P synthesis. Together with the distribution of these protein-encoding genes among archaeal and bacterial groups, our phylogenetic analyses suggested that GlpA/GlpD in the Commonote (the last universal common ancestor of all extant life with a cellular form, Commonote commonote) acquired EgsA (G1PDH) from the archaeal common ancestor (Commonote archaea) and acquired GpsA and GlpK from a bacterial common ancestor (Commonote bacteria). In our scenario based on this study, the Commonote probably possessed a G3P-lipid membrane synthesized enzymatically, after which the archaeal lineage acquired G1PDH followed by the replacement of a G3P-lipid membrane with a G1P-lipid membrane. PMID:27774041

  12. Dihydropyrimidine Dehydrogenase Is a Prognostic Marker for Mesenchymal Stem Cell-Mediated Cytosine Deaminase Gene and 5-Fluorocytosine Prodrug Therapy for the Treatment of Recurrent Gliomas

    PubMed Central

    Chung, Taemoon; Na, Juri; Kim, Young-il; Chang, Da-Young; Kim, Young Il; Kim, Hyeonjin; Moon, Ho Eun; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key; Kim, Sung-Soo; Suh-Kim, Haeyoung; Paek, Sun Ha; Youn, Hyewon

    2016-01-01

    We investigated a therapeutic strategy for recurrent malignant gliomas using mesenchymal stem cells (MSC), expressing cytosine deaminase (CD), and prodrug 5-Fluorocytosine (5-FC) as a more specific and less toxic option. MSCs are emerging as a novel cell therapeutic agent with a cancer-targeting property, and CD is considered a promising enzyme in cancer gene therapy which can convert non-toxic 5-FC to toxic 5-Fluorouracil (5-FU). Therefore, use of prodrug 5-FC can minimize normal cell toxicity. Analyses of microarrays revealed that targeting DNA damage and its repair is a selectable option for gliomas after the standard chemo/radio-therapy. 5-FU is the most frequently used anti-cancer drug, which induces DNA breaks. Because dihydropyrimidine dehydrogenase (DPD) was reported to be involved in 5-FU metabolism to block DNA damage, we compared the survival rate with 5-FU treatment and the level of DPD expression in 15 different glioma cell lines. DPD-deficient cells showed higher sensitivity to 5-FU, and the regulation of DPD level by either siRNA or overexpression was directly related to the 5-FU sensitivity. For MSC/CD with 5-FC therapy, DPD-deficient cells such as U87MG, GBM28, and GBM37 showed higher sensitivity compared to DPD-high U373 cells. Effective inhibition of tumor growth was also observed in an orthotopic mouse model using DPD- deficient U87MG, indicating that DPD gene expression is indeed closely related to the efficacy of MSC/CD-mediated 5-FC therapy. Our results suggested that DPD can be used as a biomarker for selecting glioma patients who may possibly benefit from this therapy. PMID:27446484

  13. Dihydropyrimidine Dehydrogenase Is a Prognostic Marker for Mesenchymal Stem Cell-Mediated Cytosine Deaminase Gene and 5-Fluorocytosine Prodrug Therapy for the Treatment of Recurrent Gliomas.

    PubMed

    Chung, Taemoon; Na, Juri; Kim, Young-Il; Chang, Da-Young; Kim, Young Il; Kim, Hyeonjin; Moon, Ho Eun; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key; Kim, Sung-Soo; Suh-Kim, Haeyoung; Paek, Sun Ha; Youn, Hyewon

    2016-01-01

    We investigated a therapeutic strategy for recurrent malignant gliomas using mesenchymal stem cells (MSC), expressing cytosine deaminase (CD), and prodrug 5-Fluorocytosine (5-FC) as a more specific and less toxic option. MSCs are emerging as a novel cell therapeutic agent with a cancer-targeting property, and CD is considered a promising enzyme in cancer gene therapy which can convert non-toxic 5-FC to toxic 5-Fluorouracil (5-FU). Therefore, use of prodrug 5-FC can minimize normal cell toxicity. Analyses of microarrays revealed that targeting DNA damage and its repair is a selectable option for gliomas after the standard chemo/radio-therapy. 5-FU is the most frequently used anti-cancer drug, which induces DNA breaks. Because dihydropyrimidine dehydrogenase (DPD) was reported to be involved in 5-FU metabolism to block DNA damage, we compared the survival rate with 5-FU treatment and the level of DPD expression in 15 different glioma cell lines. DPD-deficient cells showed higher sensitivity to 5-FU, and the regulation of DPD level by either siRNA or overexpression was directly related to the 5-FU sensitivity. For MSC/CD with 5-FC therapy, DPD-deficient cells such as U87MG, GBM28, and GBM37 showed higher sensitivity compared to DPD-high U373 cells. Effective inhibition of tumor growth was also observed in an orthotopic mouse model using DPD- deficient U87MG, indicating that DPD gene expression is indeed closely related to the efficacy of MSC/CD-mediated 5-FC therapy. Our results suggested that DPD can be used as a biomarker for selecting glioma patients who may possibly benefit from this therapy. PMID:27446484

  14. Insulin, CCAAT/Enhancer-Binding Proteins and Lactate Regulate the Human 11β-Hydroxysteroid Dehydrogenase Type 2 Gene Expression in Colon Cancer Cell Lines

    PubMed Central

    Alikhani-Koupaei, Rasoul; Ignatova, Irena D.; Guettinger, Andreas; Frey, Felix J.; Frey, Brigitte M.

    2014-01-01

    11β-Hydroxysteroid dehydrogenases (11beta-HSD) modulate mineralocorticoid receptor transactivation by glucocorticoids and regulate access to the glucocorticoid receptor. The isozyme 11beta-HSD2 is selectively expressed in mineralocorticoid target tissues and its activity is reduced in various disease states with abnormal sodium retention and hypertension, including the apparent mineralocorticoid excess. As 50% of patients with essential hypertension are insulin resistant and hyperinsulinemic, we hypothesized that insulin downregulates the 11beta-HSD2 activity. In the present study we show that insulin reduced the 11beta-HSD2 activity in cancer colon cell lines (HCT116, SW620 and HT-29) at the transcriptional level, in a time and dose dependent manner. The downregulation was reversible and required new protein synthesis. Pathway analysis using mRNA profiling revealed that insulin treatment modified the expression of the transcription factor family C/EBPs (CCAAT/enhancer-binding proteins) but also of glycolysis related enzymes. Western blot and real time PCR confirmed an upregulation of C/EBP beta isoforms (LAP and LIP) with a more pronounced increase in the inhibitory isoform LIP. EMSA and reporter gene assays demonstrated the role of C/EBP beta isoforms in HSD11B2 gene expression regulation. In addition, secretion of lactate, a byproduct of glycolysis, was shown to mediate insulin-dependent HSD11B2 downregulation. In summary, we demonstrate that insulin downregulates HSD11B2 through increased LIP expression and augmented lactate secretion. Such mechanisms are of interest and potential significance for sodium reabsorption in the colon. PMID:25133511

  15. The polymorphism in acetaldehyde dehydrogenase 2 gene, causing a substitution of Glu > Lys(504), is not associated with coronary atherosclerosis severity in Han Chinese.

    PubMed

    Xu, Feng; Chen, Yu-Guo; Geng, Yong-Jian; Zhang, He; Jiang, Chun-Xiao; Sun, Yi; Li, Rui-Jian; Sagar, Madi Bidya; Xue, Li; Zhang, Yun

    2007-11-01

    Alcohol consumption has an important effect on coronary atherosclerotic heart disease (CAD). Acetaldehyde dehydrogenase 2 (ALDH2) is a key enzyme in alcohol metabolism. A G-to-A missense mutation of ALDH2 gene, which causes a Glu > Lys(504) substitution, was recently shown to be associated with carotid atherosclerosis; however, its relationship with coronary atherosclerosis has not been well studied. We, therefore, investigated this relationship in Han Chinese. There are two ALDH2 alleles (1 and 2) and their combination: 1/1 (GG, typical homozygote), 1/2 (GA, heterozygote) and 2/2 (AA, atypical homozygote) in the population. Successive Han Chinese, including 89 with myocardial infarction (MI) and 142 with unstable angina, were recruited, and underwent coronary angiography and gene sequencing. Coronary atherosclerosis severity was expressed by the number of lesioned coronary arteries (>or=50% diameter stenosis) and Gensini score, calculated based on the luminal narrowing degree and its geographic importance, as assessed by angiography. Based on their ALDH2 genotypes, the 231 patients were divided into wild-type (1/1, n = 145) and mutation groups (1/2 and 2/2, n = 86). There were no significant differences in basic clinical data between the two groups; however, the mutation group had significantly higher rates of diabetes mellitus and MI, and lower prevalence of alcohol consumption than wild-type group. Yet, the two groups were not significantly different in coronary atherosclerosis severity. Multiple regression analysis has shown that the ALDH2 genotype 1/2 or 2/2 is an independent risk factor for MI, but is not associated with coronary atherosclerosis severity in Han Chinese. PMID:17984618

  16. Fluorimetric analysis of the binding characteristics of 5-enolpyruvylshikimate-3-phosphate synthase with substrates in Dunaliella salina.

    PubMed

    Cao, Yu; Xu, Hui; Xie, Li; Yi, Yi; Yu, Yingpeng; Feng, Shunli; Qiao, Dairong; Cao, Yi

    2014-09-01

    A general model of the catalytic mechanism for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) has already been proposed. But whether shikimate-3-phosphate (S3P) alone can cause EPSPs' conformation changes, and whether the binding site of phosphoenolpyruvate (PEP) and glyphosate is the same are still in debate. In this paper, DsaroA gene amplified and cloned from Dunaliella salina (our laboratory's early study) was used for DsEPSPs expression and purification. Then the DsEPSP conformation changes as it bind with different substrates were detected by fluorimetry. The results show that we obtained the DsEPSPs by prokaryotic expression and purification, and the S3P binding with DsEPSPs alone cannot cause DsEPSPs to form "close" conformation directly. However, when S3P exits, DsEPSPs did have a trend to change to the "close" conformation. Then the "close" conformation can be formed completely with the addition of phosphoenolpyruvate (PEP) or glyphosate. The inorganic phosphorus can help S3P to induce two domains of DsEPSPs to form "close" conformation. Besides, when DsEPSPs binds with S3P, in 295 nm, only the intensity of emission peak decreases, however, in 280 nm, not only the peak intensity reduces but also the blue-shift phenomenon takes place. The reason for blue-shift phenomenon was the distribution of aromatic amino acids in EPSPs. EPSPs is a good target for novel antibiotics and herbicides, because of shikimic acid pathway is only present in plants and microorganisms, completely absent in mammals, fish, birds, reptiles, and insects. The results demonstrate that the binding of substrates to EPSPs causes a conformational change from an open form to a closed form, that might be important for designing of novel antimicrobial and herbicidal agents that block closure of the enzyme.

  17. With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies

    PubMed Central

    Chapman, Joanne R.; Waldenström, Jonas

    2015-01-01

    The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies. PMID:26555275

  18. With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.

    PubMed

    Chapman, Joanne R; Waldenström, Jonas

    2015-01-01

    The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

  19. The relationship between third-codon position nucleotide content, codon bias, mRNA secondary structure and gene expression in the drosophilid alcohol dehydrogenase genes Adh and Adhr.

    PubMed Central

    Carlini, D B; Chen, Y; Stephan, W

    2001-01-01

    To gain insights into the relationship between codon bias, mRNA secondary structure, third-codon position nucleotide distribution, and gene expression, we predicted secondary structures in two related drosophilid genes, Adh and Adhr, which differ in degree of codon bias and level of gene expression. Individual structural elements (helices) were inferred using the comparative method. For each gene, four types of randomization simulations were performed to maintain/remove codon bias and/or to maintain or alter third-codon position nucleotide composition (N3). In the weakly expressed, weakly biased gene Adhr, the potential for secondary structure formation was found to be much stronger than in the highly expressed, highly biased gene Adh. This is consistent with the observation of approximately equal G and C percentages in Adhr ( approximately 31% across species), whereas in Adh the N3 distribution is shifted toward C (42% across species). Perturbing the N3 distribution to approximately equal amounts of A, G, C, and T increases the potential for secondary structure formation in Adh, but decreases it in Adhr. On the other hand, simulations that reduce codon bias without changing N3 content indicate that codon bias per se has only a weak effect on the formation of secondary structures. These results suggest that, for these two drosophilid genes, secondary structure is a relatively independent, negative regulator of gene expression. Whereas the degree of codon bias is positively correlated with level of gene expression, strong individual secondary structural elements may be selected for to retard mRNA translation and to decrease gene expression. PMID:11606539

  20. Identification and characterization of a novel translational repressor of the steroid-inducible 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase gene in Comamonas testosteroni.

    PubMed

    Xiong, Guangming; Martin, Hans-Jörg; Maser, Edmund

    2003-11-28

    Comamonas testosteroni 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3 alpha-HSD/CR) is a key enzyme in the degradation of steroid compounds in soil and may therefore play a significant role in the bioremediation of hormonally active compounds in the environment. The enzyme is also involved in the degradation of the steroid antibiotic fusidic acid. In addition, 3 alpha-HSD/CR mediates the carbonyl reduction of non-steroidal aldehydes and ketones. Because the gene of 3 alpha-HSD/CR (hsdA) is inducible by steroids, we were interested in the mode of its molecular regulation. Recently, we could identify the first molecular determinant in procaryotic steroid signaling, i.e. a repressor protein (RepA), which acts as a negative regulator by binding to upstream operator sequences of hsdA, thereby blocking hsdA transcription. In this work, we identified and cloned a second novel regulator gene that we named repB. The gene locates 932 bp downstream from hsdA on the C. testosteroni chromosome with an orientation opposite to that of hsdA. The open reading frame of repB consists of 237 bp and translates into a protein of 78 amino acids that was found to act as a repressor that regulates hsdA expression on the translational level. Northern blot analysis, UV-cross linking, gel-shift assays, and competition experiments proved that RepB binds to a 16-nucleotide sequence downstream of AUG at the 5' end of the 3 alpha-HSD/CR mRNA, thereby blocking hsdA translation. Testosterone, on the other hand, was shown to specifically bind to RepB, thereby yielding the release of RepB from the 3 alpha-HSD/CR mRNA such that hsdA translation could proceed. Data bank searches with the RepB primary structure yielded a 46.2% identity to the regulator of nucleoside diphosphate kinase, a formerly unknown protein from Escherichia coli that can restore a growth defect in alginate production in Pseudomonas aeruginosa. In conclusion, the induction of hsdA by steroids in fact is a derepression

  1. The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.

    PubMed Central

    Zammit, V A; Newsholme, E A

    1976-01-01

    Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is

  2. Biochemical genetics of opossum aldehyde dehydrogenase 3: evidence for three ALDH3A-like genes and an ALDH3B-like gene.

    PubMed

    Holmes, Roger S

    2010-04-01

    Mammalian ALDH3 isozymes participate in peroxidic and fatty aldehyde metabolism, and in anterior eye tissue UV-filtration. BLAT analyses were undertaken of the opossum genome using rat ALDH3A1, ALDH3A2, ALDH3B1, and ALDH3B2 amino acid sequences. Two predicted opossum ALDH3A1-like genes and an ALDH3A2-like gene were observed on chromosome 2, as well as an ALDH3B-like gene, which showed similar intron-exon boundaries with other mammalian ALDH3-like genes. Opossum ALDH3 subunit sequences and structures were highly conserved, including residues previously shown to be involved in catalysis and coenzyme binding for rat ALDH3A1. Eleven glycine residues were conserved for all of the opossum ALDH3-like sequences examined, including two glycine residues previously located within the stem of the rat ALDH3A1 active site funnel. Phylogeny studies of human, rat, opossum, and chicken ALDH3-like sequences indicated that the common ancestor for ALDH3A- and ALDH3B-like genes predates the appearance of birds during vertebrate evolution.

  3. Mutation by DNA shuffling of 5-enolpyruvylshikimate-3-phosphate synthase from Malus domestica for improved glyphosate resistance.

    PubMed

    Tian, Yong-Sheng; Xu, Jing; Peng, Ri-He; Xiong, Ai-Sheng; Xu, Hu; Zhao, Wei; Fu, Xiao-Yan; Han, Hong-Juan; Yao, Quan-Hong

    2013-09-01

    A new 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate-tolerant plants. However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate-resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site-directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single-site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate-resistant crops.

  4. [The expression of the sperm-specific lactate dehydrogenase gene Ldh-c in plateau pika (Ochotona curzoniae) cardiac muscle and its effect on the anaerobic glycolysis].

    PubMed

    Li, Xiao; Wei, Lian; Wang, Yang; Xu, Li-Na; Wei, Lin-Na; Wei, Deng-Bang

    2015-06-25

    The plateau pika (Ochotona curzoniae) has a strong adaptability to hypoxic plateau environment. We found that the sperm-specific lactate dehydrogenase (LDH-C4) gene Ldh-c expressed in plateau pika cardiac muscle. In order to shed light on the effect of LDH-C4 on the anaerobic glycolysis in plateau pika cardiac muscle, 20 pikas were randomly divided into the inhibitor group and the control group, and the sample size of each group was 10. The pikas of inhibitor group were injected with 1 mL 1 mol/L N-isopropyl oxamate, a specific LDH-C4 inhibitor, in biceps femoris muscle of hind legs, each leg with 500 μL. The pikas of control group were injected with the same volume of normal saline (0.9% NaCl). The mRNA and protein expression levels of Ldh-c gene in plateau pika cardiac muscle were determined by real-time PCR and Western blot. The activities of LDH, and the contents of lactate (LD) and ATP in cardiac muscle were compared between the inhibitor group and the control group. The results showed that 1) the expression levels of Ldh-c mRNA and protein were 0.47 ± 0.06 and 0.68 ± 0.08, respectively; 2) 30 min after injection of 1 mL 1 mol/L N-isopropyl oxamate in biceps femoris muscle, the concentration of N-isopropyl oxamate in blood was 0.08 mmol/L; 3) in cardiac muscle of the inhibitor group and the control group, the LDH activities were (6.18 ± 0.48) U/mg and (9.08 ± 0.58) U/mg, the contents of LD were (0.21 ± 0.03) mmol/g and (0.26 ± 0.04) mmol/g, and the contents of ATP were (4.40 ± 0.69) nmol/mg and (6.18 ± 0.73) nmol/mg (P < 0.01); 5) the inhibition rates of N-isopropyl oxamate to LDH, LD and ATP were 31.98%, 20.90% and 28.70%, respectively. The results suggest that Ldh-c expresses in cardiac muscle of plateau pika, and the pika cardiac muscle may get at least 28% ATP for its activities by LDH-C4 catalyzed anaerobic glycolysis, which reduces the dependence on oxygen and enhances the adaptation to the hypoxic environments.

  5. Novel rkp Gene Clusters of Sinorhizobium meliloti Involved in Capsular Polysaccharide Production and Invasion of the Symbiotic Nodule: the rkpK Gene Encodes a UDP-Glucose Dehydrogenase

    PubMed Central

    Kereszt, Attila; Kiss, Ernő; Reuhs, Bradley L.; Carlson, Russell W.; Kondorosi, Ádám; Putnoky, Péter

    1998-01-01

    The production of exopolysaccharide (EPS) was shown to be required for the infection process by rhizobia that induce the formation of indeterminate nodules on the roots of leguminous host plants. In Sinorhizobium meliloti (also known as Rhizobium meliloti) Rm41, a capsular polysaccharide (KPS) analogous to the group II K antigens of Escherichia coli can replace EPS during symbiotic nodule development and serve as an attachment site for the strain-specific bacteriophage φ16-3. The rkpA to -J genes in the chromosomal rkp-1 region code for proteins that are involved in the synthesis, modification, and transfer of an as-yet-unknown lipophilic molecule which might function as a specific lipid carrier during KPS biosynthesis. Here we report that with a phage φ16-3-resistant population obtained after random Tn5 mutagenesis, we have identified novel mutants impaired in KPS production by genetic complementation and biochemical studies. The mutations represent two novel loci, designated the rkp-2 and rkp-3 regions, which are required for the synthesis of rhizobial KPS. The rkp-2 region harbors two open reading frames (ORFs) organized in monocistronic transcription units. Although both genes are required for normal lipopolysaccharide production, only the second one, designated rkpK, is involved in the synthesis of KPS. We have demonstrated that RkpK possesses UDP-glucose dehydrogenase activity, while the protein product of ORF1 might function as a UDP-glucuronic acid epimerase. PMID:9765575

  6. Evolution of the adhE gene product of Escherichia coli from a functional reductase to a dehydrogenase. Genetic and biochemical studies of the mutant proteins.

    PubMed

    Membrillo-Hernandez, J; Echave, P; Cabiscol, E; Tamarit, J; Ros, J; Lin, E C

    2000-10-27

    The multifunctional AdhE protein of Escherichia coli (encoded by the adhE gene) physiologically catalyzes the sequential reduction of acetyl-CoA to acetaldehyde and then to ethanol under fermentative conditions. The NH(2)-terminal region of the AdhE protein is highly homologous to aldehyde:NAD(+) oxidoreductases, whereas the COOH-terminal region is homologous to a family of Fe(2+)-dependent ethanol:NAD(+) oxidoreductases. This fusion protein also functions as a pyruvate formate lyase deactivase. E. coli cannot grow aerobically on ethanol as the sole carbon and energy source because of inadequate rate of adhE transcription and the vulnerability of the AdhE protein to metal-catalyzed oxidation. In this study, we characterized 16 independent two-step mutants with acquired and improved aerobic growth ability on ethanol. The AdhE proteins in these mutants catalyzed the sequential oxidation of ethanol to acetaldehyde and to acetyl-CoA. All first stage mutants grew on ethanol with a doubling time of about 240 min. Sequence analysis of a randomly chosen mutant revealed an Ala-267 --> Thr substitution in the acetaldehyde:NAD(+) oxidoreductase domain of AdhE. All second stage mutants grew on ethanol with a doubling time of about 90 min, and all of them produced an AdhE(A267T/E568K). Purified AdhE(A267T) and AdhE(A267T/E568K) showed highly elevated acetaldehyde dehydrogenase activities. It therefore appears that when AdhE catalyzes the two sequential reactions in the counter-physiological direction, acetaldehyde dehydrogenation is the rate-limiting step. Both mutant proteins were more thermosensitive than the wild-type protein, but AdhE(A267T/E568K) was more thermal stable than AdhE(A267T). Since both mutant enzymes exhibited similar kinetic properties, the second mutation probably conferred an increased growth rate on ethanol by stabilizing AdhE(A267T). PMID:10922373

  7. An EPSP synthase inhibitor joining shikimate 3-phosphate with glyphosate: synthesis and ligand binding studies.

    PubMed

    Marzabadi, M R; Gruys, K J; Pansegrau, P D; Walker, M C; Yuen, H K; Sikorski, J A

    1996-04-01

    A novel EPSP synthase inhibitor 4 has been designed and synthesized to probe the configurational details of glyphosate recognition in its herbicidal ternary complex with enzyme and shikimate 3-phosphate (S3P). A kinetic evaluation of the new 3-dephospho analog 12, as well as calorimetric and (31)P NMR spectroscopic studies of enzyme-bound 4, now provides a more precise quantitative definition for the molecular interactions of 4 with this enzyme. The very poor binding, relative to 4, displayed by the 3-dephospho analog 12 is indicative that 4 has a specific interaction with the S3P site. A comparison of Ki(calc) for 12 versus the Ki(app) for 4 indicates that the 3-phosphate group in 4 contributes about 4.8 kcal/mol to binding. This compares well with the 5.2 kcal/mol which the 3-phosphate group in S3P contributes to binding. Isothermal titration calorimetry demonstrates that 4 binds to free enzyme with an observed Kd of 0.53 +/- 0.04 microM. As such, 4 binds only 3-fold weaker than glyphosate and about 150-fold better than N-methylglyphosate. Consequently, 4 represents the most potent N-alkylglyphosate derivative identified to date. However, the resulting thermodynamic binding parameters clearly demonstrate that the formation of EPSPS x 4 is entropy driven like S3P. The binding characteristics of 4 are fully consistent with a primary interaction localized at the S3P subsite. Furthermore, (31)P NMR studies of enzyme-bound 4 confirm the expected interaction at the shikimate 3-phosphate site. However, the chemical shift observed for the phosphonate signal of EPSPS x 4 is in the opposite direction than that observed previously when glyphosate binds with enzyme and S3P. Therefore, when 4 occupies the S3P binding site, there is incomplete overlap at the glyphosate phosphonate subsite. As a glyphosate analog inhibitor, the potency of 4 most likely arises from predominant interactions which occur outside the normal glyphosate binding site. Consequently, 4 is best described

  8. The choice of reference genes for assessing gene expression in sugarcane under salinity and drought stresses

    PubMed Central

    Guo, Jinlong; Ling, Hui; Wu, Qibin; Xu, Liping; Que, Youxiong

    2014-01-01

    Sugarcane (Saccharum spp. hybrids) is a world-wide cash crop for sugar and biofuel in tropical and subtropical regions and suffers serious losses in cane yield and sugar content under salinity and drought stresses. Although real-time quantitative PCR has a numerous advantage in the expression quantification of stress-related genes for the elaboration of the corresponding molecular mechanism in sugarcane, the variation happened across the process of gene expression quantification should be normalized and monitored by introducing one or several reference genes. To validate suitable reference genes or gene sets for sugarcane gene expression normalization, 13 candidate reference genes have been tested across 12 NaCl- and PEG-treated sugarcane samples for four sugarcane genotypes using four commonly used systematic statistical algorithms termed geNorm, BestKeeper, NormFinder and the deltaCt method. The results demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and eukaryotic elongation factor 1-alpha (eEF-1a) were identified as suitable reference genes for gene expression normalization under salinity/drought-treatment in sugarcane. Moreover, the expression analyses of SuSK and 6PGDH further validated that a combination of clathrin adaptor complex (CAC) and cullin (CUL) as reference should be better for gene expression normalization. These results can facilitate the future research on gene expression in sugarcane under salinity and drought stresses. PMID:25391499

  9. The choice of reference genes for assessing gene expression in sugarcane under salinity and drought stresses.

    PubMed

    Guo, Jinlong; Ling, Hui; Wu, Qibin; Xu, Liping; Que, Youxiong

    2014-01-01

    Sugarcane (Saccharum spp. hybrids) is a world-wide cash crop for sugar and biofuel in tropical and subtropical regions and suffers serious losses in cane yield and sugar content under salinity and drought stresses. Although real-time quantitative PCR has a numerous advantage in the expression quantification of stress-related genes for the elaboration of the corresponding molecular mechanism in sugarcane, the variation happened across the process of gene expression quantification should be normalized and monitored by introducing one or several reference genes. To validate suitable reference genes or gene sets for sugarcane gene expression normalization, 13 candidate reference genes have been tested across 12 NaCl- and PEG-treated sugarcane samples for four sugarcane genotypes using four commonly used systematic statistical algorithms termed geNorm, BestKeeper, NormFinder and the deltaCt method. The results demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and eukaryotic elongation factor 1-alpha (eEF-1a) were identified as suitable reference genes for gene expression normalization under salinity/drought-treatment in sugarcane. Moreover, the expression analyses of SuSK and 6PGDH further validated that a combination of clathrin adaptor complex (CAC) and cullin (CUL) as reference should be better for gene expression normalization. These results can facilitate the future research on gene expression in sugarcane under salinity and drought stresses. PMID:25391499

  10. The choice of reference genes for assessing gene expression in sugarcane under salinity and drought stresses.

    PubMed

    Guo, Jinlong; Ling, Hui; Wu, Qibin; Xu, Liping; Que, Youxiong

    2014-11-13

    Sugarcane (Saccharum spp. hybrids) is a world-wide cash crop for sugar and biofuel in tropical and subtropical regions and suffers serious losses in cane yield and sugar content under salinity and drought stresses. Although real-time quantitative PCR has a numerous advantage in the expression quantification of stress-related genes for the elaboration of the corresponding molecular mechanism in sugarcane, the variation happened across the process of gene expression quantification should be normalized and monitored by introducing one or several reference genes. To validate suitable reference genes or gene sets for sugarcane gene expression normalization, 13 candidate reference genes have been tested across 12 NaCl- and PEG-treated sugarcane samples for four sugarcane genotypes using four commonly used systematic statistical algorithms termed geNorm, BestKeeper, NormFinder and the deltaCt method. The results demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and eukaryotic elongation factor 1-alpha (eEF-1a) were identified as suitable reference genes for gene expression normalization under salinity/drought-treatment in sugarcane. Moreover, the expression analyses of SuSK and 6PGDH further validated that a combination of clathrin adaptor complex (CAC) and cullin (CUL) as reference should be better for gene expression normalization. These results can facilitate the future research on gene expression in sugarcane under salinity and drought stresses.

  11. Characterization and site-directed mutagenesis of a novel class II 5-enopyruvylshikimate-3-phosphate (EPSP) synthase from the deep-sea bacterium Alcanivorax sp. L27.

    PubMed

    Zhang, Yi; Yi, Licong; Lin, Yongjun; Zhang, Lili; Shao, Zongze; Liu, Ziduo

    2014-09-01

    The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroAA.sp. A phylogenetic analysis revealed that AroAA.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The KM for PEP and IC50 [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroAA.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC50 [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroAA.sp. The specific activity of the wild-type AroAA.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity.

  12. Characterization and site-directed mutagenesis of a novel class II 5-enopyruvylshikimate-3-phosphate (EPSP) synthase from the deep-sea bacterium Alcanivorax sp. L27.

    PubMed

    Zhang, Yi; Yi, Licong; Lin, Yongjun; Zhang, Lili; Shao, Zongze; Liu, Ziduo

    2014-09-01

    The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroAA.sp. A phylogenetic analysis revealed that AroAA.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The KM for PEP and IC50 [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroAA.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC50 [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroAA.sp. The specific activity of the wild-type AroAA.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity. PMID:25039062

  13. Metabolic engineering of an ATP-neutral Embden-Meyerhof-Parnas pathway in Corynebacterium glutamicum: growth restoration by an adaptive point mutation in NADH dehydrogenase.

    PubMed

    Komati Reddy, Gajendar; Lindner, Steffen N; Wendisch, Volker F

    2015-03-01

    Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154-7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH

  14. Plant Formate Dehydrogenase

    SciTech Connect

    John Markwell

    2005-01-10

    The research in this study identified formate dehydrogenase, an enzyme that plays a metabolic role on the periphery of one-carbon metabolism, has an unusual localization in Arabidopsis thaliana and that the enzyme has an unusual kinetic plasticity. These properties make it possible that this enzyme could be engineered to attempt to engineer plants with an improved photosynthetic efficiency. We have produced transgenic Arabidopsis and tobacco plants with increased expression of the formate dehydrogenase enzyme to initiate further studies.

  15. Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway.

    PubMed

    Zhang, Jia; Zhang, Biao; Wang, Dongmei; Gao, Xiaolian; Sun, Lianhong; Hong, Jiong

    2015-09-01

    Conversion of xylose to ethanol by yeasts is a challenge because of the redox imbalances under oxygen-limited conditions. The thermotolerant yeast Kluyveromyces marxianus grows well with xylose as a carbon source at elevated temperatures, but its xylose fermentation ability is weak. In this study, a combination of the NADPH-preferring xylose reductase (XR) from Neurospora crassa and the NADP(+)-preferring xylitol dehydrogenase (XDH) mutant from Scheffersomyces stipitis (Pichia stipitis) was constructed. The xylose fermentation ability and redox balance of the recombinant strains were improved significantly by over-expression of several downstream genes. The intracellular concentrations of coenzymes and the reduced coenzyme/oxidized coenzyme ratio increased significantly in these metabolic strains. The byproducts, such as glycerol and acetic acid, were significantly reduced by the disruption of glycerol-3-phosphate dehydrogenase (GPD1). The resulting engineered K. marxianus YZJ088 strain produced 44.95 g/L ethanol from 118.39 g/L xylose with a productivity of 2.49 g/L/h at 42 °C. Additionally, YZJ088 realized glucose and xylose co-fermentation and produced 51.43 g/L ethanol from a mixture of 103.97 g/L xylose and 40.96 g/L glucose with a productivity of 2.14 g/L/h at 42 °C. These promising results validate the YZJ088 strain as an excellent producer of ethanol from xylose through the synthetic xylose assimilation pathway. PMID:26253204

  16. Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway.

    PubMed

    Zhang, Jia; Zhang, Biao; Wang, Dongmei; Gao, Xiaolian; Sun, Lianhong; Hong, Jiong

    2015-09-01

    Conversion of xylose to ethanol by yeasts is a challenge because of the redox imbalances under oxygen-limited conditions. The thermotolerant yeast Kluyveromyces marxianus grows well with xylose as a carbon source at elevated temperatures, but its xylose fermentation ability is weak. In this study, a combination of the NADPH-preferring xylose reductase (XR) from Neurospora crassa and the NADP(+)-preferring xylitol dehydrogenase (XDH) mutant from Scheffersomyces stipitis (Pichia stipitis) was constructed. The xylose fermentation ability and redox balance of the recombinant strains were improved significantly by over-expression of several downstream genes. The intracellular concentrations of coenzymes and the reduced coenzyme/oxidized coenzyme ratio increased significantly in these metabolic strains. The byproducts, such as glycerol and acetic acid, were significantly reduced by the disruption of glycerol-3-phosphate dehydrogenase (GPD1). The resulting engineered K. marxianus YZJ088 strain produced 44.95 g/L ethanol from 118.39 g/L xylose with a productivity of 2.49 g/L/h at 42 °C. Additionally, YZJ088 realized glucose and xylose co-fermentation and produced 51.43 g/L ethanol from a mixture of 103.97 g/L xylose and 40.96 g/L glucose with a productivity of 2.14 g/L/h at 42 °C. These promising results validate the YZJ088 strain as an excellent producer of ethanol from xylose through the synthetic xylose assimilation pathway.

  17. Role of intragenic binding of cAMP responsive protein (CRP) in regulation of the succinate dehydrogenase genes Rv0249c-Rv0247c in TB complex mycobacteria

    PubMed Central

    Knapp, Gwendowlyn S.; Lyubetskaya, Anna; Peterson, Matthew W.; Gomes, Antonio L.C.; Ma, Zhuo; Galagan, James E.; McDonough, Kathleen A.

    2015-01-01

    Bacterial pathogens adapt to changing environments within their hosts, and the signaling molecule adenosine 3′, 5′-cyclic monophosphate (cAMP) facilitates this process. In this study, we characterized in vivo DNA binding and gene regulation by the cAMP-responsive protein CRP in M. bovis BCG as a model for tuberculosis (TB)-complex bacteria. Chromatin immunoprecipitation followed by deep-sequencing (ChIP-seq) showed that CRP associates with ∼900 DNA binding regions, most of which occur within genes. The most highly enriched binding region was upstream of a putative copper transporter gene (ctpB), and crp-deleted bacteria showed increased sensitivity to copper toxicity. Detailed mutational analysis of four CRP binding sites upstream of the virulence-associated Rv0249c-Rv0247c succinate dehydrogenase genes demonstrated that CRP directly regulates Rv0249c-Rv0247c expression from two promoters, one of which requires sequences intragenic to Rv0250c for maximum expression. The high percentage of intragenic CRP binding sites and our demonstration that these intragenic DNA sequences significantly contribute to biologically relevant gene expression greatly expand the genome space that must be considered for gene regulatory analyses in mycobacteria. These findings also have practical implications for an important bacterial pathogen in which identification of mutations that affect expression of drug target-related genes is widely used for rapid drug resistance screening. PMID:25940627

  18. Pharmacological glycerol-3-phosphate acyltransferase inhibition decreases food intake and adiposity and increases insulin sensitivity in diet-induced obesity.

    PubMed

    Kuhajda, Francis P; Aja, Susan; Tu, Yajun; Han, Wan Fang; Medghalchi, Susan M; El Meskini, Rajaa; Landree, Leslie E; Peterson, Jonathan M; Daniels, Khadija; Wong, Kody; Wydysh, Edward A; Townsend, Craig A; Ronnett, Gabriele V

    2011-07-01

    Storage of excess calories as triglycerides is central to obesity and its associated disorders. Glycerol-3-phosphate acyltransferases (GPATs) catalyze the initial step in acylglyceride syntheses, including triglyceride synthesis. We utilized a novel small-molecule GPAT inhibitor, FSG67, to investigate metabolic consequences of systemic pharmacological GPAT inhibition in lean and diet-induced obese (DIO) mice. FSG67 administered intraperitoneally decreased body weight and energy intake, without producing conditioned taste aversion. Daily FSG67 (5 mg/kg, 15.3 μmol/kg) produced gradual 12% weight loss in DIO mice beyond that due to transient 9- to 10-day hypophagia (6% weight loss in pair-fed controls). Continued FSG67 maintained the weight loss despite return to baseline energy intake. Weight was lost specifically from fat mass. Indirect calorimetry showed partial protection by FSG67 against decreased rates of oxygen consumption seen with hypophagia. Despite low respiratory exchange ratio due to a high-fat diet, FSG67-treated mice showed further decreased respiratory exchange ratio, beyond pair-fed controls, indicating enhanced fat oxidation. Chronic FSG67 increased glucose tolerance and insulin sensitivity in DIO mice. Chronic FSG67 decreased gene expression for lipogenic enzymes in white adipose tissue and liver and decreased lipid accumulation in white adipose, brown adipose, and liver tissues without signs of damage. RT-PCR showed decreased gene expression for orexigenic hypothalamic neuropeptides AgRP or NPY after acute and chronic systemic FSG67. FSG67 given intracerebroventricularly (100 and 320 nmol icv) produced 24-h weight loss and feeding suppression, indicating contributions from direct central nervous system sites of action. Together, these data point to GPAT as a new potential therapeutic target for the management of obesity and its comorbidities. PMID:21490364

  19. AB073. Mutations in the type II 3β-hydroxysteroid dehydrogenase gene caused primary adrenal insufficiency & 46,XY disorders of sex development

    PubMed Central

    Dung, Vu Chi; Thao, Bui Phuong; Khanh, Nguyen Ngoc; Ngoc, Can Thi Bich; Morel, Yves

    2015-01-01

    Congenital adrenal hyperplasia (CAH) is one of the most common inherited metabolic disorders. It includes a group of autosomal recessive disorders caused by the deficiency of one of the enzymes involved in one of the various steps of adrenal steroid synthesis. 3β-hydroxysteroid dehydrogenase (3β-HSD) deficiency is a rare cause of CAH caused by inactivating mutations in the HSD3B2 gene. Most mutations are located within domains regarded crucial for enzyme function. Our aim is to describe phenotype and to identify mutations of HSD3B2 in two classic β-HSD deficient patients belonging to two apparently unrelated pedigrees. This is a case series study. Family history and clinical manifestations were described. Genomic DNA from these patients was extracted using standard procedures from the peripheral blood leukocytes. Mutation analysis of HSD3B2 was performed using polymerase chain reaction (PCR) and DNA direct sequencing. Vietnamese 46,XY newborn referred at 2.5th month of life with salt loss associated with hyponatremia (123 nmol/L) and hyperpigmentation. The testes were palpable in the scrotum but associated with a severe hypospadias (micropenis 0.5 cm; posterior). At 4 months of age, a second adrenal crisis has occurred with hyponatremia 127 nmol/L and increased 17OH-Progesterone (26.8 ng/mL) in this 46,XY DSD. This clinical and biological data associated with a sibling with female phenotype deceased at 18 months old after adrenal crisis (1st occurred at 7 days of life) suggest the diagnosis of 3β-HSD deficiency. The sequencing of HSD3B2 confirms the diagnosis because he is homozygous for a missense mutation, pAla161Pro. This mutation affects an amino acid conserved in all species and is located in one two alpha-helix involved in the dimerization of the two sub-units of the enzyme. The changing from Alanine to proline could break the alpha-helix. The same mutation has been found in the other Vietnamese family. The 46,XY newborn referred at 3th month of life with

  20. Atomic-level characterization of transport cycle thermodynamics in the glycerol-3-phosphate:phosphate antiporter

    PubMed Central

    Moradi, Mahmoud; Enkavi, Giray; Tajkhorshid, Emad

    2015-01-01

    Membrane transporters actively translocate their substrate by undergoing large-scale structural transitions between inward- (IF) and outward-facing (OF) states (‘alternating-access' mechanism). Despite extensive structural studies, atomic-level mechanistic details of such structural transitions, and as importantly, their coupling to chemical events supplying the energy, remain amongst the most elusive aspects of the function of these proteins. Here we present a quantitative, atomic-level description of the functional thermodynamic cycle for the glycerol-3-phosphate:phosphate antiporter GlpT by using a novel approach in reconstructing the free energy landscape governing the IF↔OF transition along a cyclic transition pathway involving both apo and substrate-bound states. Our results provide a fully atomic description of the complete transport process, offering a structural model for the alternating-access mechanism and substantiating the close coupling between global structural transitions and local chemical events. PMID:26417850

  1. Crystallization and preliminary X-ray analysis of the glycerol-3-phosphate 1-acyltransferase from squash (Cucurbita moschata).

    PubMed

    Turnbull, A P; Rafferty, J B; Sedelnikova, S E; Slabas, A R; Schierer, T P; Kroon, J T; Nishida, I; Murata, N; Simon, J W; Rice, D W

    2001-03-01

    Glycerol-3-phosphate 1-acyltransferase (E.C. 2.3.1.15; G3PAT) catalyses the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acylCoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol 3-phosphate. Crystals of squash G3PAT have been obtained by the hanging-drop method of vapour diffusion using PEG 4000 as the precipitant. These crystals are most likely to belong to space group P2(1)2(1)2(1), with approximate unit-cell parameters a = 61.1, b = 65.1, c = 103.3 A, alpha = beta = gamma = 90 degrees and a monomer in the asymmetric unit. X-ray diffraction data to 1.9 A resolution have been collected in-house using a MAR 345 imaging-plate system.

  2. Glycerol-3-phosphate Acyltransferase Isoform-4 (GPAT4) Limits Oxidation of Exogenous Fatty Acids in Brown Adipocytes*

    PubMed Central

    Cooper, Daniel E.; Grevengoed, Trisha J.; Klett, Eric L.; Coleman, Rosalind A.

    2015-01-01

    Glycerol-3-phosphate acyltransferase-4 (GPAT4) null pups grew poorly during the suckling period and, as adults, were protected from high fat diet-induced obesity. To determine why Gpat4−/− mice failed to gain weight during these two periods of high fat feeding, we examined energy metabolism. Compared with controls, the metabolic rate of Gpat4−/− mice fed a 45% fat diet was 12% higher. Core body temperature was 1 ºC higher after high fat feeding. Food intake, fat absorption, and activity were similar in both genotypes. Impaired weight gain in Gpat4−/− mice did not result from increased heat loss, because both cold tolerance and response to a β3-adrenergic agonist were similar in both genotypes. Because GPAT4 comprises 65% of the total GPAT activity in brown adipose tissue (BAT), we characterized BAT function. A 45% fat diet increased the Gpat4−/− BAT expression of peroxisome proliferator-activated receptor α (PPAR) target genes, Cpt1α, Pgc1α, and Ucp1, and BAT mitochondria oxidized oleate and pyruvate at higher rates than controls, suggesting that fatty acid signaling and flux through the TCA cycle were enhanced. To assess the role of GPAT4 directly, neonatal BAT preadipocytes were differentiated to adipocytes. Compared with controls, Gpat4−/− brown adipocytes incorporated 33% less fatty acid into triacylglycerol and 46% more into the pathway of β-oxidation. The increased oxidation rate was due solely to an increase in the oxidation of exogenous fatty acids. These data suggest that in the absence of cold exposure, GPAT4 limits excessive fatty acid oxidation and the detrimental induction of a hypermetabolic state. PMID:25918168

  3. Molecular characterization of Fasciola hepatica and phylogenetic analysis based on mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I and cytochrome oxidase subunit I) genes from the North-East of Iran

    PubMed Central

    Reaghi, Saber; Haghighi, Ali; Harandi, Majid Fasihi; Spotin, Adel; Arzamani, Kourosh; Rouhani, Soheila

    2016-01-01

    Aim: Fascioliasis is one of the most zoonotic diseases with global extension. As the epidemiological distribution of Fasciola may lead to various genetic patterns of the parasite, the aim of this study is to identify Fasciola hepatica based on spermatogenesis, and phylogenetic analysis using mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I [ND1] and cytochrome oxidase subunit I) gene marker. Materials and Methods: In this study, 90 F. hepatica collected from 30 cattle at slaughterhouse located in three different geographical locations in the North-East of Iran were evaluated based on spermatogenetic ability and internal transcribed spacer 1 gene restriction fragment length polymorphism pattern. Genetic diversity and phylogenetic relationship using mtDNA gene marker for the isolates from the North-East of Iran, and other countries were then analyzed. Results: Partial sequences of mtDNA showed eight haplotypes in both genes. The phylogenic analysis using neighbor joining as well as maximum likelihood methods showed similar topologies of trees. Pairwise fixation index between different F. hepatica populations calculated from the nucleotide data set of ND1 gene are statistically significant and show the genetic difference. Conclusion: F. hepatica found in this region of Iran has different genetic structures through the other Fasciola populations in the world. PMID:27733809

  4. A point mutation in the putative TATA box, detected in nondiseased individuals and patients with hereditary breast cancer, decreases promoter activity of the 17{beta}-hydroxysteroid dehydrogenase type 1 gene 2 (EDH17B2) in vitro

    SciTech Connect

    Peltoketo, H.; Piao, Y.; Isomaa, V.

    1994-09-01

    EDH17B2, the gene encoding 17{beta}-hydroxysteroid dehydrogenase type 1, has been suggested as a candidate for the familial breast cancer gene, BRCA1, located on 17q12-q21. We analyzed the promoter region of EDH17B2 in DNA from 20 control individuals and 40 patients with familial breast cancer. Two frequent (designated vI and vIII) and two rare (vII and vIV) nucleotide variations were present in both the breast cancer patients and the controls, except the alteration vII, which was found only in one patient. Although the data do not support the identification of EDH17B2 as the BRCA1 gene, it is of interest that point mutation vIV (A {yields} C) was located in the putative TATA box of the EDH17B2 gene. Reporter gene analysis showed that the mutation vIV decreases EDH17B2 promoter activity by an average of 45% in in vitro assays, suggesting that nucleotide A at position -27 is significant for efficient transcription. 12 refs., 2 figs., 1 tab.

  5. Differential levels of glutamate dehydrogenase 1 (GLUD1) in Balb/c and C57BL/6 mice and the effects of overexpression of the Glud1 gene on glutamate release in striatum

    PubMed Central

    Hascup, Kevin N; Bao, Xiaodong; Hascup, Erin R; Hui, Dongwei; Xu, Wenhao; Pomerleau, Francois; Huettl, Peter; Michaelis, Mary L; Michaelis, Elias K; Gerhardt, Greg A

    2011-01-01

    We have previously shown that overexpression of the Glud1 (glutamate dehydrogenase 1) gene in neurons of C57BL/6 mice results in increased depolarization-induced glutamate release that eventually leads to selective neuronal injury and cell loss by 12 months of age. However, it is known that isogenic lines of Tg (transgenic) mice produced through back-crossing with one strain may differ in their phenotypic characteristics from those produced using another inbred mouse strain. Therefore, we decided to introduce the Glud1 transgene into the Balb/c strain that has endogenously lower levels of GLUD1 (glutamate dehydrogenase 1) enzyme activity in the brain as compared with C57BL/6. Using an enzyme-based MEA (microelectrode array) that is selective for measuring glutamate in vivo, we measured depolarization-induced glutamate release. Within a discrete layer of the striatum, glutamate release was significantly increased in Balb/c Tg mice compared with wt (wild-type) littermates. Furthermore, Balb/c mice released approx. 50–60% of the amount of glutamate compared with C57BL/6 mice. This is similar to the lower levels of endogenous GLUD1 protein in Balb/c compared with C57BL/6 mice. The development of these Glud1-overexpressing mice may allow for the exploration of key molecular events produced by chronic exposure of neurons to moderate, transient increases in glutamate release, a process hypothesized to occur in neurodegenerative disorders. PMID:21446915

  6. Stereoselective synthesis of (R)-phenylephrine using recombinant Escherichia coli cells expressing a novel short-chain dehydrogenase/reductase gene from Serratia marcescens BCRC 10948.

    PubMed

    Peng, Guan-Jhih; Kuan, Yi-Chia; Chou, Hsiao-Yi; Fu, Tze-Kai; Lin, Jia-Shin; Hsu, Wen-Hwei; Yang, Ming-Te

    2014-01-20

    (R)-Phenylephrine [(R)-PE] is an α1-adrenergic receptor agonist and is widely used as a nasal decongestant to treat the common cold without the side effects of other ephedrine adrenergic drugs. We identified a short-chain dehydrogenase/reductase (SM_SDR) from Serratia marcescens BCRC 10948 that was able to convert 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) into (R)-PE. The SM_SDR used NADPH and NADH as cofactors with specific activities of 17.35±0.71 and 5.57±0.07mU/mg protein, respectively, at 30°C and pH 7.0, thereby indicating that this enzyme could be categorized as an NADPH-preferring short-chain dehydrogenase/reductase. Escherichia coli strain BL21 (DE3) expressing SM_SDR could convert HPMAE into (R)-PE with more than 99% enantiomeric excess. The productivity and conversion yield were 0.57mmolPE/lh and 51.06%, respectively, using 10mM HPMAE. Fructose was the most effective carbon source for the conversion of HPMAE to (R)-PE.

  7. Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions.

    PubMed

    Yamamoto, Shogo; Gunji, Wataru; Suzuki, Hiroaki; Toda, Hiroshi; Suda, Masako; Jojima, Toru; Inui, Masayuki; Yukawa, Hideaki

    2012-06-01

    We previously reported that Corynebacterium glutamicum strain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159-165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD(+) ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses.

  8. Validation of housekeeping genes as an internal control for gene expression studies in Giardia lamblia using quantitative real-time PCR.

    PubMed

    Marcial-Quino, Jaime; Fierro, Francisco; De la Mora-De la Mora, Ignacio; Enríquez-Flores, Sergio; Gómez-Manzo, Saúl; Vanoye-Carlo, America; Garcia-Torres, Itzhel; Sierra-Palacios, Edgar; Reyes-Vivas, Horacio

    2016-04-25

    The analysis of transcript levels of specific genes is important for understanding transcriptional regulation and for the characterization of gene function. Real-time quantitative reverse transcriptase PCR (RT-qPCR) has become a powerful tool to quantify gene expression. The objective of this study was to identify reliable housekeeping genes in Giardia lamblia. Twelve genes were selected for this purpose, and their expression was analyzed in the wild type WB strain and in two strains with resistance to nitazoxanide (NTZ) and metronidazole (MTZ), respectively. RefFinder software analysis showed that the expression of the genes is different in the three strains. The integrated data from the four analyses showed that the NADH oxidase (NADH) and aldolase (ALD) genes were the most steadily expressed genes, whereas the glyceraldehyde-3-phosphate dehydrogenase gene was the most unstable. Additionally, the relative expression of seven genes were quantified in the NTZ- and MTZ-resistant strains by RT-qPCR, using the aldolase gene as the internal control, and the results showed a consistent differential pattern of expression in both strains. The housekeeping genes found in this work will facilitate the analysis of mRNA expression levels of other genes of interest in G. lamblia.

  9. Cloning and expression of the gene encoding alpha-acetolactate decarboxylase from Acetobacter aceti ssp. xylinum in brewer's yeast.

    PubMed

    Yamano, S; Tanaka, J; Inoue, T

    1994-02-14

    Acetobacter aceti ssp. xylinum genomic library was constructed using cosmid pJB8 in Escherichia coli. The gene encoding alpha-acetolactate decarboxylase (ALDC) was isolated from the library by direct measurement of ALDC activity. The ALDC gene was expressed by its own promoter in E. coli. The nucleotide sequence was determined, and an open reading frame which may encode a protein composed of 304 amino acids with a molecular weight of 33,747 was found. A brewer's yeast was transformed with the YEp-type plasmid containing the ALDC gene placed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The laboratory-scale growth test confirmed that the total diacetyl concentration was considerably reduced by the transformant. The analysis of the wort indicates that the Acetobacter ALDC reduces the concentration of diacetyl more effectively than that of 2,3-pentanedione.

  10. Affinity chromatography of nicotinamide-adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide.

    PubMed

    Barry, S; O'Carra, P

    1973-12-01

    1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD(+) through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD(+) (probably through the 8 position of the adenine residue) to a number of different spacer-arm-agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD(+) derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD(+). Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD(+)-binding site of this enzyme. Problems

  11. Modeling of glycerol-3-phosphate transporter suggests a potential 'tilt' mechanism involved in its function.

    PubMed

    Tsigelny, Igor F; Greenberg, Jerry; Kouznetsova, Valentina; Nigam, Sanjay K

    2008-10-01

    Many major facilitator superfamily (MFS) transporters have similar 12-transmembrane alpha-helical topologies with two six-helix halves connected by a long loop. In humans, these transporters participate in key physiological processes and are also, as in the case of members of the organic anion transporter (OAT) family, of pharmaceutical interest. Recently, crystal structures of two bacterial representatives of the MFS family--the glycerol-3-phosphate transporter (GlpT) and lac-permease (LacY)--have been solved and, because of assumptions regarding the high structural conservation of this family, there is hope that the results can be applied to mammalian transporters as well. Based on crystallography, it has been suggested that a major conformational "switching" mechanism accounts for ligand transport by MFS proteins. This conformational switch would then allow periodic changes in the overall transporter configuration, resulting in its cyclic opening to the periplasm or cytoplasm. Following this lead, we have modeled a possible "switch" mechanism in GlpT, using the concept of rotation of protein domains as in the DynDom program17 and membranephilic constraints predicted by the MAPAS program.(23) We found that the minima of energies of intersubunit interactions support two alternate positions consistent with their transport properties. Thus, for GlpT, a "tilt" of 9 degrees -10 degrees rotation had the most favorable energetics of electrostatic interaction between the two halves of the transporter; moreover, this confirmation was sufficient to suggest transport of the ligand across the membrane. We conducted steered molecular dynamics simulations of the GlpT-ligand system to explore how glycerol-3-phosphate would be handled by the "tilted" structure, and obtained results generally consistent with experimental mutagenesis data. While biochemical data remain most consistent with a single-site alternating access model, our results raise the possibility that, while the

  12. Stably expressed housekeeping genes across developmental stages in the two-spotted spider mite, Tetranychus urticae.

    PubMed

    Yang, Chunxiao; Pan, Huipeng; Liu, Yong; Zhou, Xuguo

    2015-01-01

    Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring mRNA expression. To facilitate gene expression studies and obtain more accurate qRT-PCR analysis, normalization relative to stable housekeeping genes is mandatory. In this study, ten housekeeping genes, including beta-actin (Actin) , elongation factor 1 α (EF1A) , glyceralde hyde-3-phosphate dehydrogenase (GAPDH) , ribosomal protein L13 (RPL13) , ribosomal protein 49 (RP49) , α-tubulin (Tubulin) , vacuolar-type H+-ATPase (v-ATPase) , succinate dehydrogenase subunit A (SDHA) , 28S ribosomal RNA (28S) , and 18S ribosomal RNA (18S) from the two-spotted spider mite, Tetranychus urticae, were selected as the candidate reference genes. Four algorithms, geNorm, Normfinder, BestKeeper, and the ΔCt method, were used to evaluate the performance of these candidates as endogenous controls across different developmental stages. In addition, RefFinder, which integrates the above-mentioned software tools, provided the overall ranking of the stability/suitability of these candidate reference genes. Among them, PRL13 and v-ATPase were the two most stable housekeeping genes across different developmental stages. This work is the first step toward establishing a standardized qRT-PCR analysis in T. urticae following the MIQE guideline. With the recent release of the T. urticae genome, results from this study provide a critical piece for the subsequent genomics and functional genomics research in this emerging model system.

  13. Reference gene for primary culture of prostate cancer cells.

    PubMed

    Souza, Aline Francielle Damo; Brum, Ilma Simoni; Neto, Brasil Silva; Berger, Milton; Branchini, Gisele

    2013-04-01

    Selection of reference genes to normalize mRNA levels between samples is critical for gene expression studies because their expression can vary depending on the tissues or cells used and the experimental conditions. We performed ten cell cultures from samples of prostate cancer. Cells were divided into three groups: control (with no transfection protocol), cells transfected with siRNA specific to knockdown the androgen receptor and cells transfected with inespecific siRNAs. After 24 h, mRNA was extracted and gene expression was analyzed by Real-time qPCR. Nine candidates to reference genes for gene expression studies in this model were analyzed (aminolevulinate, delta-, synthase 1 (ALAS1); beta-actin (ACTB); beta-2-microglobulin (B2M); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine phosphoribosyltransferase 1 (HPRT1); succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA); TATA box binding protein (TBP); ubiquitin C (UBC); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Expression stability was calculated NormFinder algorithm to find the most stable genes. NormFinder calculated SDHA as the most stable gene and the gene with the lowest intergroup and intragroup variation, and indicated GAPDH and SDHA as the best combination of two genes for the purpose of normalization. Androgen receptor mRNA expression was evaluated after normalization by each candidate gene and showed statistical difference in the transfected group compared to control group only when normalized by combination of GAPDH and SDHA. Based on the algorithm analysis, the combination of SDHA and GAPDH should be used to normalize target genes mRNA levels in primary culture of prostate cancer cells submitted to transfection with siRNAs.

  14. Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol-3-phosphate-binding proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The internalization of oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors’ cell entry receptor, phosphatidylinositol-3-phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants ...

  15. Aldehyde dehydrogenase (ALDH) 2 associates with oxidation of methoxyacetaldehyde; in vitro analysis with liver subcellular fraction derived from human and Aldh2 gene targeting mouse.

    PubMed

    Kitagawa, K; Kawamoto, T; Kunugita, N; Tsukiyama, T; Okamoto, K; Yoshida, A; Nakayama, K; Nakayama, K

    2000-07-01

    A principal pathway of 2-methoxyethanol (ME) metabolism is to the toxic oxidative product, methoxyacetaldehyde (MALD). To assess the role of aldehyde dehydrogenase (ALDH) in MALD metabolism, in vitro MALD oxidation was examined with liver subcellular fractions from Japanese subjects who carried three different ALDH2 genotypes and Aldh2 knockout mice, which were generated in this study. The activity was distributed in mitochondrial fractions of ALDH2*1/*1 and wild type (Aldh2+/+) mice but not ALDH2*1/*2, *2/*2 subjects or Aldh2 homozygous mutant (Aldh2-/-) mice. These data suggest that ALDH2 is a key enzyme for MALD oxidation and ME susceptibility may be influenced by the ALDH2 genotype. PMID:10913633

  16. Effects of cell volume regulating osmolytes on glycerol 3-phosphate binding to triosephosphate isomerase.

    PubMed

    Gulotta, Miriam; Qiu, Linlin; Desamero, Ruel; Rösgen, Jörg; Bolen, D Wayne; Callender, Robert

    2007-09-01

    During cell volume regulation, intracellular concentration changes occur in both inorganic and organic osmolytes in order to balance the extracellular osmotic stress and maintain cell volume homeostasis. Generally, salt and urea increase the Km's of enzymes and trimethylamine N-oxide (TMAO) counteracts these effects by decreasing Km's. The hypothesis to account for these effects is that urea and salt shift the native state ensemble of the enzyme toward conformers that are substrate-binding incompetent (BI), while TMAO shifts the ensemble toward binding competent (BC) species. Km's are often complex assemblies of rate constants involving several elementary steps in catalysis, so to better understand osmolyte effects we have focused on a single elementary event, substrate binding. We test the conformational shift hypothesis by evaluating the effects of salt, urea, and TMAO on the mechanism of binding glycerol 3-phosphate, a substrate analogue, to yeast triosephosphate isomerase. Temperature-jump kinetic measurements promote a mechanism consistent with osmolyte-induced shifts in the [BI]/[BC] ratio of enzyme conformers. Importantly, salt significantly affects the binding constant through its effect on the activity coefficients of substrate, enzyme, and enzyme-substrate complex, and it is likely that TMAO and urea affect activity coefficients as well. Results indicate that the conformational shift hypothesis alone does not account for the effects of osmolytes on Km's. PMID:17696453

  17. The Inositol-3-Phosphate Synthase Biosynthetic Enzyme Has Distinct Catalytic and Metabolic Roles

    PubMed Central

    Frej, Anna D.; Clark, Jonathan; Le Roy, Caroline I.; Lilla, Sergio; Thomason, Peter A.; Otto, Grant P.; Churchill, Grant; Insall, Robert H.; Claus, Sandrine P.; Hawkins, Phillip; Stephens, Len

    2016-01-01

    Inositol levels, maintained by the biosynthetic enzyme inositol-3-phosphate synthase (Ino1), are altered in a range of disorders, including bipolar disorder and Alzheimer's disease. To date, most inositol studies have focused on the molecular and cellular effects of inositol depletion without considering Ino1 levels. Here we employ a simple eukaryote, Dictyostelium discoideum, to demonstrate distinct effects of loss of Ino1 and inositol depletion. We show that loss of Ino1 results in an inositol auxotrophy that can be rescued only partially by exogenous inositol. Removal of inositol supplementation from the ino1− mutant resulted in a rapid 56% reduction in inositol levels, triggering the induction of autophagy, reduced cytokinesis, and substrate adhesion. Inositol depletion also caused a dramatic generalized decrease in phosphoinositide levels that was rescued by inositol supplementation. However, loss of Ino1 triggered broad metabolic changes consistent with the induction of a catabolic state that was not rescued by inositol supplementation. These data suggest a metabolic role for Ino1 that is independent of inositol biosynthesis. To characterize this role, an Ino1 binding partner containing SEL1L1 domains (Q54IX5) and having homology to mammalian macromolecular complex adaptor proteins was identified. Our findings therefore identify a new role for Ino1, independent of inositol biosynthesis, with broad effects on cell metabolism. PMID:26951199

  18. NADP-dehydrogenases from pepper fruits: effect of maturation.

    PubMed

    Mateos, Rosa M; Bonilla-Valverde, Daniel; del Río, Luis A; Palma, José M; Corpas, Francisco J

    2009-02-01

    NADPH is an important molecule in the redox balance of the cell. Pepper fruits are the second worldwide consumable vegetables and exhibit different phenotypes after maturation. In this paper, two pepper cultivars were studied: Vergasa whose fruits shift from green to red after maturation, and Biela that shifts to yellow. Using fresh fruits from the same plants of the two cultivars at distinct maturation stages, the activity and gene expression of the main NADPH-generating dehydrogenases was studied. The activity analysis of the main NADP-dehydrogenases, glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-isocitrate dehydrogenase (NADP-ICDH) and NADP-malic enzyme (NADP-ME), showed that, except for the G6PDH, all the activities were enhanced (54-100%) in the mature pepper fruits from both cultivars (red or yellow) with respect to green pepper fruits. The content of NADPH and NADP in the mature fruits of both cultivars showed a noteworthy increase with respect to green fruits. For the transcript analysis, a partial cDNA of each NADP-dehydrogenase was obtained, and the NADP-ME was the only NADP-dehydrogenase that showed a significant induction. The increase in the content of NADPH in mature fruits because of the enhanced activity of NADP-dehydrogenases suggests that these NADPH-generating enzymes could be involved in the maturation of pepper fruits.

  19. Multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase causing excessive acetaldehyde production from ethanol by oral streptococci.

    PubMed

    Pavlova, Sylvia I; Jin, Ling; Gasparovich, Stephen R; Tao, Lin

    2013-07-01

    Ethanol consumption and poor oral hygiene are risk factors for oral and oesophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analysed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild-type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius and S. sanguinis showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci.

  20. Selection of Reference Genes for Gene Expression Normalization in Peucedanum praeruptorum Dunn under Abiotic Stresses, Hormone Treatments and Different Tissues

    PubMed Central

    Zhao, Yucheng; Luo, Jun; Xu, Sheng; Wang, Wei; Liu, Tingting; Han, Chao; Chen, Yijun; Kong, Lingyi

    2016-01-01

    Peucedanum praeruptorum Dunn is one of the main traditional Chinese medicines producing coumarins and plenty of literatures are focused on the biosynthesis of coumarins. Quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used method in studying the biosynthesis pathway and the selection of reference genes plays a crucial role in accurate normalization. To facilitate biosynthesis study of coumarins, twelve candidate reference genes were selected from the transcriptome database of P. praeruptorum according to previous studies. Then, BestKeeper, geNoFrm and NormFinder were used for selecting stably expressed reference genes in different tissues and under various stress treatments. The results indicated that, among the twelve candidate reference genes, the SAND family protein (SAND), actin 2 (ACT2), ubiquitin-conjugating enzyme 9 (UBC9), protein phosphatase 2A gene (PP2A) and polypyrimidine tract-binding protein (PTBP1) were the most stable reference genes under different experimental treatments, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin beta-6 (TUB6) were the least stable genes. In addition, the suitability of SAND, TIP41-like protein (TIP41), UBC9, ACT2, TUB6 and their combination as reference genes were confirmed by normalizing the expression of 1-aminocyclopropane-1-carboxylate oxidase (ACO) in different treatments. This work is the first survey of the stability of reference genes in P. praeruptorum and provides guidelines to obtain more accurate qRT-PCR results in P. praeruptorum and other plant species. PMID:27022972

  1. Selection of Reference Genes for Gene Expression Normalization in Peucedanum praeruptorum Dunn under Abiotic Stresses, Hormone Treatments and Different Tissues.

    PubMed

    Zhao, Yucheng; Luo, Jun; Xu, Sheng; Wang, Wei; Liu, Tingting; Han, Chao; Chen, Yijun; Kong, Lingyi

    2016-01-01

    Peucedanum praeruptorum Dunn is one of the main traditional Chinese medicines producing coumarins and plenty of literatures are focused on the biosynthesis of coumarins. Quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used method in studying the biosynthesis pathway and the selection of reference genes plays a crucial role in accurate normalization. To facilitate biosynthesis study of coumarins, twelve candidate reference genes were selected from the transcriptome database of P. praeruptorum according to previous studies. Then, BestKeeper, geNoFrm and NormFinder were used for selecting stably expressed reference genes in different tissues and under various stress treatments. The results indicated that, among the twelve candidate reference genes, the SAND family protein (SAND), actin 2 (ACT2), ubiquitin-conjugating enzyme 9 (UBC9), protein phosphatase 2A gene (PP2A) and polypyrimidine tract-binding protein (PTBP1) were the most stable reference genes under different experimental treatments, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin beta-6 (TUB6) were the least stable genes. In addition, the suitability of SAND, TIP41-like protein (TIP41), UBC9, ACT2, TUB6 and their combination as reference genes were confirmed by normalizing the expression of 1-aminocyclopropane-1-carboxylate oxidase (ACO) in different treatments. This work is the first survey of the stability of reference genes in P. praeruptorum and provides guidelines to obtain more accurate qRT-PCR results in P. praeruptorum and other plant species.

  2. Evaluation of reference genes for gene expression studies in radish (Raphanus sativus L.) using quantitative real-time PCR.

    PubMed

    Xu, Yuanyuan; Zhu, Xianwen; Gong, Yiqin; Xu, Liang; Wang, Yan; Liu, Liwang

    2012-08-01

    Real-time quantitative reverse transcription PCR (RT-qPCR) is a rapid and reliable method for gene expression studies. Normalization based on reference genes can increase the reliability of this technique; however, recent studies have shown that almost no single reference gene is universal for all possible experimental conditions. In this study, eight frequently used reference genes were investigated, including Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Actin2/7 (ACT), Tubulin alpha-5 (TUA), Tubulin beta-1 (TUB), 18S ribosomal RNA (18SrRNA), RNA polymerase-II transcription factor (RPII), Elongation factor 1-b (EF-1b) and Translation elongation factor 2 (TEF2). Expression stability of candidate reference genes was examined across 27 radish samples, representing a range of tissue types, cultivars, photoperiodic and vernalization treatments, and developmental stages. The eight genes in these sample pools displayed a wide range of Ct values and were variably expressed. Two statistical software packages, geNorm and NormFinder showed that TEF2, RPII and ACT appeared to be relatively stable and therefore the most suitable for use as reference genes. These results facilitate selection of desirable reference genes for accurate gene expression studies in radish. PMID:22771808

  3. Selection of reliable reference genes for gene expression studies in Clonostachys rosea 67-1 under sclerotial induction.

    PubMed

    Sun, Zhan-Bin; Li, Shi-Dong; Sun, Man-Hong

    2015-07-01

    Reference genes are important to precisely quantify gene expression by real-time PCR. In order to identify stable and reliable expressed genes in mycoparasite Clonostachys rosea in different modes of nutrition, seven commonly used housekeeping genes, 18S rRNA, actin, β-tubulin, elongation factor 1, ubiquitin, ubiquitin-conjugating enzyme and glyceraldehyde-3-phosphate dehydrogenase, from the effective biocontrol isolate C. rosea 67-1 were tested for their expression under sclerotial induction and during vegetative growth on PDA medium. Analysis by three software programs showed that differences existed among the candidates. Elongation factor 1 was most stable; the M value in geNorm, SD value in Bestkeeper and stability value in Normfinder analysis were 0.405, 0.450 and 0.442, respectively, indicating that the gene elongation factor 1 could be used to normalize gene expression in C. rosea in both vegetative growth and parasitic process. By using elongation factor 1, the expression of a serine protease gene, sep, in different conditions was assessed, which was consistent with the transcriptomic data. This research provides an effective method to quantitate expression changes of target genes in C. rosea, and will assist in further investigation of parasitism-related genes of this fungus.

  4. Ectopic expression of myo-inositol 3-phosphate synthase induces a wide range of metabolic changes and confers salt tolerance in rice.

    PubMed

    Kusuda, Hiroki; Koga, Wataru; Kusano, Miyako; Oikawa, Akira; Saito, Kazuki; Hirai, Masami Yokota; Yoshida, Kaoru T

    2015-03-01

    Salt stress is an important factor that limits crop production worldwide. The salt tolerance of plants is a complex biological process mediated by changes in gene expression and metabolite composition. The enzyme myo-inositol 3-phosphate synthase (MIPS; EC 5.5.1.4) catalyzes the first step of myo-inositol biosynthesis, and overexpression of the MIPS gene enhances salt stress tolerance in several plant species. In this study, we performed metabolite profiling of both MIPS-overexpressing and wild-type rice. The enhanced salt stress tolerance of MIPS-overexpressing plants was clear based on growth and the metabolites under salt stress. We found that constitutive overexpression of the rice MIPS gene resulted in a wide range of metabolic changes. This study demonstrates for the first time that overexpression of the MIPS gene increases various metabolites responsible for protecting plants from abiotic stress. Activation of both basal metabolism, such as glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, and inositol metabolism is induced in MIPS-overexpressing plants. We discuss the relationship between the metabolic changes and the improved salt tolerance observed in transgenic rice.

  5. [Agrobacterium-mediated sunflower transformation (Helianthus annuus L.) in vitro and in Planta using strain of LBA4404 harboring binary vector pBi2E with dsRNA-suppressor proline dehydrogenase gene].

    PubMed

    Tishchenko, E N; Komisarenko, A G; Mikhal'skaia, S I; Sergeeva, L E; Adamenko, N I; Morgun, B V; Kochetov, A V

    2014-01-01

    To estimate the efficiency of proline dehydrogenase gene suppression towards increasing of sunflower (Helianthus annuus L.) tolerance level to water deficit and salinity, we employed strain LBA4404 harboring pBi2E with double-stranded RNA-suppressor, which were prepared on basis arabidopsis ProDH1 gene. The techniques of Agrobacterium-mediated transformation in vitro and in planta during fertilization sunflower have been proposed. There was shown the genotype-depended integration of T-DNA in sunflower genome. PCR-analysis showed that ProDH1 presents in genome of inbred lines transformed in planta, as well as in T1- and T2-generations. In trans-genic regenerants the essential accumulation of free L-proline during early stages of in vitro cultivation under normal conditions was shown. There was established the essential accumulation of free proline in transgenic regenerants during cultivation under lethal stress pressure (0.4 M mannitol and 2.0% sea water salts) and its decline upon the recovery period. These data are declared about effectiveness of suppression of sunflower ProDH and gene participation in processes connected with osmotolerance.

  6. Site-Directed Mutagenesis from Arg195 to His of a Microalgal Putatively Chloroplastidial Glycerol-3-Phosphate Acyltransferase Causes an Increase in Phospholipid Levels in Yeast

    PubMed Central

    Ouyang, Long-Ling; Li, Hui; Yan, Xiao-Jun; Xu, Ji-Lin; Zhou, Zhi-Gang

    2016-01-01

    To analyze the contribution of glycerol-3-phosphate acyltransferase (GPAT) to the first acylation of glycerol-3-phosphate (G-3-P), the present study focused on a functional analysis of the GPAT gene from Lobosphaera incisa (designated as LiGPAT). A full-length cDNA of LiGPAT consisting of a 1,305-bp ORF, a 1,652-bp 5′-UTR, and a 354-bp 3′-UTR, was cloned. The ORF encoded a 434-amino acid peptide, of which 63 residues at the N-terminus defined a chloroplast transit peptide. Multiple sequence alignment and phylogeny analysis of GPAT homologs provided the convincible bioinformatics evidence that LiGPAT was localized to chloroplasts. Considering the conservation of His among the G-3-P binding sites from chloroplastidial GPATs and the substitution of His by Arg at position 195 in the LiGPAT mature protein (designated mLiGPAT), we established the heterologous expression of either mLiGPAT or its mutant (Arg195His) (sdmLiGPAT) in the GPAT-deficient yeast mutant gat1Δ. Lipid profile analyses of these transgenic yeasts not only validated the acylation function of LiGPAT but also indicated that the site-directed mutagenesis from Arg195 to His led to an increase in the phospholipid level in yeast. Semi-quantitative analysis of mLiGPAT and sdmLiGPAT, together with the structural superimposition of their G-3-P binding sites, indicated that the increased enzymatic activity was caused by the enlarged accessible surface of the phosphate group binding pocket when Arg195 was mutated to His. Thus, the potential of genetic manipulation of GPAT to increase the glycerolipid level in L. incisa and other microalgae would be of great interest. PMID:27014309

  7. Carnosine prevents glyceraldehyde 3-phosphate-mediated inhibition of aspartate aminotransferase.

    PubMed

    Swearengin, T A; Fitzgerald, C; Seidler, N W

    1999-08-01

    Post-mitotic tissues, such as the heart, exhibit high concentrations (20 mM) of carnosine (beta-alanyl-l-histidine). Carnosine may have aldehyde scavenging properties. We tested this hypothesis by examining its protective effects against inhibition of enzyme activity by glyceraldehyde 3-phosphate (Glyc3P). Glyc3P is a potentially toxic triose; Glyc3P inhibits the cardiac aspartate aminotransferase (cAAT) by non-enzymatic glycosylation (or glycation) of the protein. cAAT requires pyridoxal 5-phosphate (PyP) for catalysis. We observed that carnosine (20 mM) completely prevents the inhibition of cAAT activity by Glyc3P (5 mM) after brief incubation (30 min at 37 degrees C). After a prolonged incubation (3.25 h) of cAAT with Glyc3P (0.5 mM) at 37 degrees C, the protection by carnosine (20 mM) persisted but PyP availability was affected. In the absence of PyP from the assay medium, cAAT activities (plus Glyc3P) were 95 +/- 18.2 micromol/min per mg protein (mean +/- SD), minus carnosine and 100 +/- 2.4, plus carnosine; control activity was 172 +/- 3.9. When PyP (1.0 microM) was included in the assay medium, cAAT activities (plus Glyc3P) were 93 +/- 14.8, minus carnosine and 151 +/- 16.8, plus carnosine, P < 0. 001; control activity was 180 +/- 17.7. These data, which showed carnosine moderating the effects of both Glyc3P and PyP, suggest that carnosine may be an endogenous aldehyde scavenger.

  8. Glyphosate inhibition of 5-enolpyruvylshikimate 3-phosphate synthease from suspension-cultured cells of Nicotiana silvestris

    SciTech Connect

    Rubin, J.L.; Gaines, C.G.; Jensen, R.A.

    1984-07-01

    Treatment of isogenic suspension-cultured cells of Nicotiana silvestris Speg, et Comes with glyphosate (N-(phosphonomethyl)glycine) led to elevated levels of intracellular shikimate (364-fold increase by 1.0 millimolar glyphosate). In the presence of glyphosate, it is likely that most molecules of shikimate originate from the action of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase-Mn since this isozyme, in contrast to the DAHP synthase-Co isozyme, is insensitive to inhibition by glyphosate. 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19) from N. silvestris was sensitive to micromolar concentrations of glyphosate and possessed a single inhibitor binding site. Rigorous kinetic studies of EPSP synthase required resolution from the multiple phosphatase activities present in crude extracts, a result achieved by ion-exchange column chromatography. Although EPSP synthase exhibited a broad pH profile (50% of maximal activity between pH 6.2 and 8.5), sensitivity to glyphosate increased dramatically with increasing pH within this range. In accordance with these data and the pK/sub a/ values of glyphosate, it is likely that the ionic form of glyphosate inhibiting EPSP synthase is COO/sup -/CH/sub 2/NH/sub 2//sup +/CH/sub 2/PO/sub 3//sup 2 -/, and that a completely ionized phosphono group is essential for inhibition. At pH 7.0, inhibition was competitive with respect to phosphoenolpyruvate (K/sub i/ = 1.25 micromolar) and uncompetitive with respect to shikimate-3-P (K/sub i/ = 18.3 micromolar). All data were consistent with a mechanism of inhibition in which glyphosate competes with PEP for binding to an (enzyme:shikimate-3-P) complex and ultimately forms the dead-end complex of (enzyme:shikimate-3-P:glyphosate). 36 references, 8 figures, 1 table.

  9. A new compound heterozygous frameshift mutation in the type II 3{beta}-hydroxysteroid dehydrogenase 3{beta}-HSD gene causes salt-wasting 3{beta}-HSD deficiency congenital adrenal hyperplasia

    SciTech Connect

    Zhang, L.; Sakkal-Alkaddour, S.; Chang, Ying T.; Yang, Xiaojiang; Songya Pang

    1996-01-01

    We report a new compound heterozygous frameshift mutation in the type II 3{Beta}-hydroxysteroid dehydrogenase (3{beta}-HSD) gene in a Pakistanian female child with the salt-wasting form of 3{Beta}-HSD deficiency congenital adrenal hyperplasia. The etiology for her congenital adrenal hyperplasia was not defined. Although the family history suggested possible 3{beta}-HSd deficiency disorder, suppressed adrenal function caused by excess glucocorticoid therapy in this child at 7 yr of age did not allow hormonal diagnosis. To confirm 3{beta}-HSD deficiency, we sequenced the type II 3{beta}-HSD gene in the patient, her family, and the parents of her deceased paternal cousins. The type II 3{beta}-HSD gene region of a putative promotor, exons I, II, III, and IV, and exon-intron boundaries were amplified by PCR and sequenced in all subjects. The DNA sequence of the child revealed a single nucleotide deletion at codon 318 [ACA(Thr){r_arrow}AA] in exon IV in one allele, and two nucleotide deletions at codon 273 [AAA(Lys){r_arrow}A] in exon IV in the other allele. The remaining gene sequences were normal. The codon 318 mutation was found in one allele from the father, brother, and parents of the deceased paternal cousins. The codon 273 mutation was found in one allele of the mother and a sister. These findings confirmed inherited 3{beta}-HSD deficiency in the child caused by the compound heterozygous type II 3{beta}-HSD gene mutation. Both codons at codons 279 and 367, respectively, are predicted to result in an altered and truncated type II 3{beta}-HSD protein, thereby causing salt-wasting 3{beta}-HSD deficiency in the patient. 21 refs., 2 figs., 1 tab.

  10. Selection of suitable reference genes for quantitative gene expression studies in milk somatic cells of lactating cows (Bos indicus).

    PubMed

    Varshney, N; Mohanty, A K; Kumar, S; Kaushik, J K; Dang, A K; Mukesh, M; Mishra, B P; Kataria, R; Kimothi, S P; Mukhopadhyay, T K; Malakar, D; Prakash, B S; Grover, S; Batish, V K

    2012-06-01

    We assessed the suitability of 9 internal control genes (ICG) in milk somatic cells of lactating cows to find suitable reference genes for use in quantitative PCR (qPCR). Eighteen multiparous lactating Sahiwal cows were used, 6 in each of 3 lactation stages: early (25 ± 5 d in milk), mid (160 ± 15 d in milk), and late (275 ± 25 d in milk) lactation. Nine candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), protein phosphatase 1 regulatory subunit 11 (PPP1R11), β-actin (ACTB), β-2 microglobulin (B2M), 40S ribosomal protein S15a (RPS15A), ubiquitously expressed transcript (UXT), mitochondrial GTPase 1 (MTG1), 18S rRNA (RN18S1), and ubiquitin (UBC)] were evaluated. Three genes, β-casein (CSN2), lactoferrin (LTF), and cathelicidin (CAMP) were chosen as target genes. Very high amplification was observed in 7 ICG and very low level amplification was observed in 2 ICG (UXT and MTG1). Thus, UXT and MTG1 were excluded from further analysis. The qPCR data were analyzed by 2 software packages, geNorm and NormFinder, to determine suitable reference genes, based on their stability and expression. Overall, PPP1R11, ACTB, UBC, and GAPDH were stably expressed among all candidate reference genes. Therefore, these genes could be used as ICG for normalization of qPCR data in milk somatic cells through lactation.

  11. Rapid and Reliable Detection of Glucose-6-Phosphate Dehydrogenase (G6PD) Gene Mutations in Han Chinese Using High-Resolution Melting Analysis

    PubMed Central

    Yan, Jing-bin; Xu, Hong-ping; Xiong, Can; Ren, Zhao-rui; Tian, Guo-li; Zeng, Fanyi; Huang, Shu-zhen

    2010-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked inherited disease, is one of the most common enzymopathies and affects over 400 million people worldwide. In China at least 21 distinct point mutations have been identified so far. In this study high-resolution melting (HRM) analysis was used to screen for G6PD mutations in 260 unrelated Han Chinese individuals, and the rapidity and reliability of this method was investigated. The mutants were readily differentiated by using HRM analysis, which produced distinct melting curves for each tested mutation. Interestingly, G1388A and G1376T, the two most common variants accounting for 50% to 60% of G6PD deficiency mutations in the Chinese population, could be differentiated in a single reaction. Further, two G6PD mutations not previously reported in the Chinese population were identified in this study. One of these mutations, designated “G6PD Jiangxi G1340T,” involved a G1340T substitution in exon 11, predicting a Gly447Val change in the protein. The other mutation involved a C406T substitution in exon 5. The frequencies of the common polymorphism site C1311T/IVS (intervening sequence) XI t93c between patients with G6PD and healthy volunteers were not significantly different. Thus, HRM analysis will be a useful alternative for screening G6PD mutations. PMID:20203002

  12. Rapid and reliable detection of glucose-6-phosphate dehydrogenase (G6PD) gene mutations in Han Chinese using high-resolution melting analysis.

    PubMed

    Yan, Jing-bin; Xu, Hong-ping; Xiong, Can; Ren, Zhao-rui; Tian, Guo-li; Zeng, Fanyi; Huang, Shu-zhen

    2010-05-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked inherited disease, is one of the most common enzymopathies and affects over 400 million people worldwide. In China at least 21 distinct point mutations have been identified so far. In this study high-resolution melting (HRM) analysis was used to screen for G6PD mutations in 260 unrelated Han Chinese individuals, and the rapidity and reliability of this method was investigated. The mutants were readily differentiated by using HRM analysis, which produced distinct melting curves for each tested mutation. Interestingly, G1388A and G1376T, the two most common variants accounting for 50% to 60% of G6PD deficiency mutations in the Chinese population, could be differentiated in a single reaction. Further, two G6PD mutations not previously reported in the Chinese population were identified in this study. One of these mutations, designated "G6PD Jiangxi G1340T," involved a G1340T substitution in exon 11, predicting a Gly447Val change in the protein. The other mutation involved a C406T substitution in exon 5. The frequencies of the common polymorphism site C1311T/IVS (intervening sequence) XI t93c between patients with G6PD and healthy volunteers were not significantly different. Thus, HRM analysis will be a useful alternative for screening G6PD mutations. PMID:20203002

  13. Deletion of a conserved regulatory element in the Drosophila Adh gene leads to increased alcohol dehydrogenase activity but also delays development.

    PubMed Central

    Parsch, J; Russell, J A; Beerman, I; Hartl, D L; Stephan, W

    2000-01-01

    In vivo levels of enzymatic activity may be increased through either structural or regulatory changes. Here we use Drosophila melanogaster alcohol dehydrogenase (ADH) in an experimental test for selective differences between these two mechanisms. The well-known ADH-Slow (S)/Fast (F) amino acid replacement leads to a twofold increase in activity by increasing the catalytic efficiency of the enzyme. Disruption of a highly conserved, negative regulatory element in the Adh 3' UTR also leads to a twofold increase in activity, although this is achieved by increasing in vivo Adh mRNA and protein concentrations. These two changes appear to be under different types of selection, with positive selection favoring the amino acid replacement and purifying selection maintaining the 3' UTR sequence. Using transgenic experiments we show that deletion of the conserved 3' UTR element increases adult and larval Adh expression in both the ADH-F and ADH-S genetic backgrounds. However, the 3' UTR deletion also leads to a significant increase in developmental time in both backgrounds. ADH allozyme type has no detectable effect on development. These results demonstrate a negative fitness effect associated with Adh overexpression. This provides a mechanism whereby natural selection can discriminate between alternative pathways of increasing enzymatic activity. PMID:10978287

  14. Identification and mRNA expression of two 17β-hydroxysteroid dehydrogenase genes in the marine mussel Mytilus galloprovincialis following exposure to endocrine disrupting chemicals.

    PubMed

    Zhang, Yingying; Wang, Qing; Ji, Yinglu; Zhang, Qian; Wu, Huifeng; Xie, Jia; Zhao, Jianmin

    2014-05-01

    17β-Hydroxysteroid dehydrogenases (17β-HSDs) are multifunctional enzymes involved in the metabolism of steroids, fatty acids, retinoids and bile acid. In this study, two novel types of 17β-HSDs (named as MgHsd17b10 and MgHsd17b12) were cloned from Mytilus galloprovincialis by using rapid amplification of cDNA ends (RACE) approaches. Sequence analysis showed that MgHsd17b10 and MgHsd17b12 encoded a polypeptide of 259 and 325 amino acids, respectively. Phylogenetic analysis revealed that MgHsd17b10 and MgHsd17b12 were evolutionarily clustered with other invertebrate 17β-HSD type 10 and 17β-HSD type 12 homologues. The MgHsd17b10 and MgHsd17b12 transcripts could be detected in all examined tissues with higher expression levels in digestive glands and gonad. After exposed to endocrine disrupting chemicals (Bisphenol A or 2,2',4,4'-tetrabromodiphenyl ether), the expression of MgHsd17b10 and MgHsd17b12 transcripts was both down-regulated in digestive glands. These findings suggest that MgHsd17b10 and MgHsd17b12 perhaps play an important role in the endocrine regulation of M. galloprovincialis.

  15. Use of transmitochondrial cybrids to assign a complex I defect to the mitochondrial DNA-encoded NADH dehydrogenase subunit 6 gene mutation at nucleotide pair 14459 that causes Leber hereditary optic neuropathy and dystonia.

    PubMed Central

    Jun, A S; Trounce, I A; Brown, M D; Shoffner, J M; Wallace, D C

    1996-01-01

    A heteroplasmic G-to-A transition at nucleotide pair (np) 14459 within the mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit 6 (ND6) gene has been identified as the cause of Leber hereditary optic neuropathy (LHON) and/or pediatric-onset dystonia in three unrelated families. This ND6 np 14459 mutation changes a moderately conserved alanine to a valine at amino acid position 72 of the ND6 protein. Enzymologic analysis of mitochondrial NADH dehydrogenase (complex I) with submitochondrial particles isolated from Epstein-Barr virus-transformed lymphoblasts revealed a 60% reduction (P < 0.005) of complex I-specific activity in patient cell lines compared with controls, with no differences in enzymatic activity for complexes II plus III, III and IV. This biochemical defect was assigned to the ND6 np 14459 mutation by using transmitochondrial cybrids in which patient Epstein-Barr virus-transformed lymphoblast cell lines were enucleated and the cytoplasts were fused to a mtDNA-deficient (p 0) lymphoblastoid recipient cell line. Cybrids harboring the np 14459 mutation exhibited a 39% reduction (p < 0.02) in complex I-specific activity relative to wild-type cybrid lines but normal activity for the other complexes. Kinetic analysis of the np 14459 mutant complex I revealed that the Vmax of the enzyme was reduced while the Km remained the same as that of wild type. Furthermore, specific activity was inhibited by increasing concentrations of the reduced coenzyme Q analog decylubiquinol. These observations suggest that the np 14459 mutation may alter the coenzyme Q-binding site of complex I. PMID:8622678

  16. A novel 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide.

    PubMed

    Wang, Wei; Xia, Hui; Yang, Xiao; Xu, Ting; Si, Hong Jiang; Cai, Xing Xing; Wang, Feng; Su, Jun; Snow, Allison A; Lu, Bao-Rong

    2014-04-01

    Understanding evolutionary interactions among crops and weeds can facilitate effective weed management. For example, gene flow from crops to their wild or weedy relatives can lead to rapid evolution in recipient populations. In rice (Oryza sativa), transgenic herbicide resistance is expected to spread to conspecific weedy rice (Oryza sativa f. spontanea) via hybridization. Here, we studied fitness effects of transgenic over-expression of a native 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene developed to confer glyphosate resistance in rice. Controlling for genetic background, we examined physiological traits and field performance of crop-weed hybrid lineages that segregated for the presence or absence of this novel epsps transgene. Surprisingly, we found that transgenic F2 crop-weed hybrids produced 48-125% more seeds per plant than nontransgenic controls in monoculture- and mixed-planting designs without glyphosate application. Transgenic plants also had greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and per cent seed germination compared with nontransgenic controls. Our findings suggest that over-expression of a native rice epsps gene can lead to fitness advantages, even without exposure to glyphosate. We hypothesize that over-expressed epsps may be useful to breeders and, if deployed, could result in fitness benefits in weedy relatives following transgene introgression.

  17. Reconstructed Ancestral Myo-Inositol-3-Phosphate Synthases Indicate That Ancestors of the Thermococcales and Thermotoga Species Were More Thermophilic than Their Descendants

    PubMed Central

    Butzin, Nicholas C.; Lapierre, Pascal; Green, Anna G.; Swithers, Kristen S.; Gogarten, J. Peter; Noll, Kenneth M.

    2013-01-01

    The bacterial genomes of Thermotoga species show evidence of significant interdomain horizontal gene transfer from the Archaea. Members of this genus acquired many genes from the Thermococcales, which grow at higher temperatures than Thermotoga species. In order to study the functional history of an interdomain horizontally acquired gene we used ancestral sequence reconstruction to examine the thermal characteristics of reconstructed ancestral proteins of the Thermotoga lineage and its archaeal donors. Several ancestral sequence reconstruction methods were used to determine the possible sequences of the ancestral Thermotoga and Archaea myo-inositol-3-phosphate synthase (MIPS). These sequences were predicted to be more thermostable than the extant proteins using an established sequence composition method. We verified these computational predictions by measuring the activities and thermostabilities of purified proteins from the Thermotoga and the Thermococcales species, and eight ancestral reconstructed proteins. We found that the ancestral proteins from both the archaeal donor and the Thermotoga most recent common ancestor recipient were more thermostable than their descendants. We show that there is a correlation between the thermostability of MIPS protein and the optimal growth temperature (OGT) of its host, which suggests that the OGT of the ancestors of these species of Archaea and the Thermotoga grew at higher OGTs than their descendants. PMID:24391933

  18. A novel 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide

    PubMed Central

    Wang, Wei; Xia, Hui; Yang, Xiao; Xu, Ting; Si, Hong Jiang; Cai, Xing Xing; Wang, Feng; Su, Jun; Snow, Allison A; Lu, Bao-Rong

    2014-01-01

    Understanding evolutionary interactions among crops and weeds can facilitate effective weed management. For example, gene flow from crops to their wild or weedy relatives can lead to rapid evolution in recipient populations. In rice (Oryza sativa), transgenic herbicide resistance is expected to spread to conspecific weedy rice (Oryza sativa f. spontanea) via hybridization. Here, we studied fitness effects of transgenic over-expression of a native 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene developed to confer glyphosate resistance in rice. Controlling for genetic background, we examined physiological traits and field performance of crop–weed hybrid lineages that segregated for the presence or absence of this novel epsps transgene. Surprisingly, we found that transgenic F2 crop–weed hybrids produced 48–125% more seeds per plant than nontransgenic controls in monoculture- and mixed-planting designs without glyphosate application. Transgenic plants also had greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and per cent seed germination compared with nontransgenic controls. Our findings suggest that over-expression of a native rice epsps gene can lead to fitness advantages, even without exposure to glyphosate. We hypothesize that over-expressed epsps may be useful to breeders and, if deployed, could result in fitness benefits in weedy relatives following transgene introgression. PMID:23905647

  19. Kinetic mechanism and order of substrate binding for sn-glycerol-3-phosphate acyltransferase from squash (Cucurbita moschata).

    PubMed

    Hayman, Matthew W; Fawcett, Tony; Slabas, Antoni R

    2002-03-13

    sn-Glycerol-3-phosphate acyltransferase (G3PAT, EC 2.3.1.15), a component of glycerolipid biosynthesis, is an important enzyme in chilling sensitivity in plants. The three-dimensional structure of the enzyme from squash (Cucurbita moschata), without bound substrate, has been determined [Turnbull et al. (2001) Acta Crystallogr. D 57, 451-453; Turnbull et al. (2001) Structure 9, 347-353]. Here we report the kinetic mechanism of plastidial G3PAT from squash and the order of substrate binding using acyl-acyl carrier protein (acyl-ACP) substrates. The reaction proceeds via a compulsory-ordered ternary complex with acyl-ACP binding before glycerol-3-phosphate. We have also determined that the reaction will proceed with C(4:0)-CoA, C(6:0)-CoA and C(12:0)-ACP substrates, allowing a wider choice of acyl groups for future co-crystallisation studies.

  20. Change in ATP-binding cassette B1/19, glutamine synthetase and alcohol dehydrogenase gene expression during root elongation in Betula pendula Roth and Alnus glutinosa L. Gaertn in response to leachate and leonardite humic substances.

    PubMed

    Tahiri, Abdelghani; Delporte, Fabienne; Muhovski, Yordan; Ongena, Marc; Thonart, Philippe; Druart, Philippe

    2016-01-01

    Humic substances (HS) are complex and heterogeneous compounds of humified organic matter resulting from the chemical and microbiological decomposition of organic residues. HS have a positive effect on plant growth and development by improving soil structure and fertility. They have long been recognized as plant growth-promoting substances, particularly with regard to influencing nutrient uptake, root growth and architecture. The biochemical and molecular mechanisms through which HS influence plant physiology are not well understood. This study evaluated the bioactivity of landfill leachate and leonardite HS on alder (Alnus glutinosa L. Gaertn) and birch (Betula pendula Roth) during root elongation in vitro. Changes in root development were studied in relation to auxin, carbon and nitrogen metabolisms, as well as to the stress adaptive response. The cDNA fragments of putative genes encoding two ATP-binding cassette (ABC) transporters (ABCB1 and ABCB19) belonging to the B subfamily of plant ABC auxin transporters were cloned and sequenced. Molecular data indicate that HS and their humic acid (HA) fractions induce root growth by influencing polar auxin transport (PAT), as illustrated by the modulation of the ABCB transporter transcript levels (ABCB1 and ABCB19). There were also changes in alcohol dehydrogenase (ADH) and glutamine synthetase (GS) gene transcript levels in response to HS exposure. These findings confirmed that humic matter affects plant growth and development through various metabolic pathways, including hormonal, carbon and nitrogen metabolisms and stress response or signalization.

  1. Gene structure and mutations of glutaryl-coenzyme A dehydrogenase: Impaired association of enzyme subunits that is due to an A421V substitution causes glutaric acidemia type I in the Amish

    SciTech Connect

    Biery, B.J.; Stein, D.E.; Goodman, S.I.

    1996-11-01

    The structure of the human glutaryl coenzyme A dehydrogenase (GCD) gene was determined to contain 11 exons and to span {approximately}7 kb. Fibroblast DNA from 64 unrelated glutaric academia type I (GA1) patients was screened for mutations by PCR amplification and analysis of SSCP. Fragments with altered electrophoretic mobility were subcloned and sequenced to detect mutations that caused GA1. This report describes the structure of the GCD gene, as well as point mutations and polymorphisms found in 7 of its 11 exons. Several mutations were found in more than one patient, but no one prevalent mutation was detected in the general population. As expected from pedigree analysis, a single mutant allele causes GA1 in the Old Order Amish of Lancaster County, Pennsylvania. Several mutations have been expressed in Escherichia coli, and all produce diminished enzyme activity. Reduced activity in GCD encoded by the A421V mutation in the Amish may be due to impaired association of enzyme subunits. 13 refs., 5 figs., 3 tabs.

  2. Efficient production of optically pure D-lactic acid from raw corn starch by using a genetically modified L-lactate dehydrogenase gene-deficient and alpha-amylase-secreting Lactobacillus plantarum strain.

    PubMed

    Okano, Kenji; Zhang, Qiao; Shinkawa, Satoru; Yoshida, Shogo; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-01-01

    In order to achieve direct and efficient fermentation of optically pure D-lactic acid from raw corn starch, we constructed L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 alpha-amylase (AmyA). The resulting strain produced only D-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct D-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct D-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct D-lactic acid fermentation from raw starch.

  3. Change in ATP-binding cassette B1/19, glutamine synthetase and alcohol dehydrogenase gene expression during root elongation in Betula pendula Roth and Alnus glutinosa L. Gaertn in response to leachate and leonardite humic substances.

    PubMed

    Tahiri, Abdelghani; Delporte, Fabienne; Muhovski, Yordan; Ongena, Marc; Thonart, Philippe; Druart, Philippe

    2016-01-01

    Humic substances (HS) are complex and heterogeneous compounds of humified organic matter resulting from the chemical and microbiological decomposition of organic residues. HS have a positive effect on plant growth and development by improving soil structure and fertility. They have long been recognized as plant growth-promoting substances, particularly with regard to influencing nutrient uptake, root growth and architecture. The biochemical and molecular mechanisms through which HS influence plant physiology are not well understood. This study evaluated the bioactivity of landfill leachate and leonardite HS on alder (Alnus glutinosa L. Gaertn) and birch (Betula pendula Roth) during root elongation in vitro. Changes in root development were studied in relation to auxin, carbon and nitrogen metabolisms, as well as to the stress adaptive response. The cDNA fragments of putative genes encoding two ATP-binding cassette (ABC) transporters (ABCB1 and ABCB19) belonging to the B subfamily of plant ABC auxin transporters were cloned and sequenced. Molecular data indicate that HS and their humic acid (HA) fractions induce root growth by influencing polar auxin transport (PAT), as illustrated by the modulation of the ABCB transporter transcript levels (ABCB1 and ABCB19). There were also changes in alcohol dehydrogenase (ADH) and glutamine synthetase (GS) gene transcript levels in response to HS exposure. These findings confirmed that humic matter affects plant growth and development through various metabolic pathways, including hormonal, carbon and nitrogen metabolisms and stress response or signalization. PMID:26595095

  4. Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR.

    PubMed

    Zampieri, Denise; Nora, Luísa C; Basso, Vanessa; Camassola, Marli; Dillon, Aldo J P

    2014-08-01

    Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a methodology that facilitates the quantification of mRNA expression in a given sample. Analysis of relative gene expression by qRT-PCR requires normalization of the data using a reference gene that is expressed at a similar level in all evaluated conditions. Determining an internal control gene is essential for gene expression studies. Gene expression studies in filamentous fungi frequently use the β-actin gene (actb), β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase as reference genes because they are known to have consistent expression levels. Until now, no study has been performed to select an internal control gene for the filamentous fungal species Penicillium echinulatum. The aim of this study was to evaluate and validate internal control genes to enable the study of gene expression in P. echinulatum using qRT-PCR. P. echinulatum strain S1M29 was grown in conditions to either induce (cellulose and sugar cane bagasse) or repress (glucose) gene expression to analyze 23 candidate normalization genes for stable expression. Two software programs, BestKeeper and geNorm, were used to assess the expression of the candidate normalization genes. The results indicate that the actb reference gene is more stably expressed in P. echinulatum. This is the first report in the literature that determines a normalization gene for this fungus. From the results obtained, we recommend the use of the P. echinulatum actb gene as an endogenous control for gene expression studies of cellulases and hemicellulases by qRT-PCR. PMID:24509829

  5. Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR.

    PubMed

    Zampieri, Denise; Nora, Luísa C; Basso, Vanessa; Camassola, Marli; Dillon, Aldo J P

    2014-08-01

    Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a methodology that facilitates the quantification of mRNA expression in a given sample. Analysis of relative gene expression by qRT-PCR requires normalization of the data using a reference gene that is expressed at a similar level in all evaluated conditions. Determining an internal control gene is essential for gene expression studies. Gene expression studies in filamentous fungi frequently use the β-actin gene (actb), β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase as reference genes because they are known to have consistent expression levels. Until now, no study has been performed to select an internal control gene for the filamentous fungal species Penicillium echinulatum. The aim of this study was to evaluate and validate internal control genes to enable the study of gene expression in P. echinulatum using qRT-PCR. P. echinulatum strain S1M29 was grown in conditions to either induce (cellulose and sugar cane bagasse) or repress (glucose) gene expression to analyze 23 candidate normalization genes for stable expression. Two software programs, BestKeeper and geNorm, were used to assess the expression of the candidate normalization genes. The results indicate that the actb reference gene is more stably expressed in P. echinulatum. This is the first report in the literature that determines a normalization gene for this fungus. From the results obtained, we recommend the use of the P. echinulatum actb gene as an endogenous control for gene expression studies of cellulases and hemicellulases by qRT-PCR.

  6. The essential function of genes for a hydratase and an aldehyde dehydrogenase for growth of Pseudomonas sp. strain Chol1 with the steroid compound cholate indicates an aldolytic reaction step for deacetylation of the side chain.

    PubMed

    Holert, Johannes; Jagmann, Nina; Philipp, Bodo

    2013-08-01

    In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C5 acyl side chain of the C24 steroid compound cholate involves the C22 intermediate 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C24 steroid (22E)-7α,12α-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt, resulted in the accumulation of 7α,12α,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A- and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD(+) as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage.

  7. The First Case of Severe Takotsubo Cardiomyopathy Associated with 5-Fluorouracil in a Patient with Abnormalities of Both Dihydropyrimidine Dehydrogenase (DPYD) and Thymidylate Synthase (TYMS) Genes

    PubMed Central

    Smith, Melissa; Maloney, Antonio

    2016-01-01

    5-Fluorouracil (5-FU) is the backbone of the chemotherapy regimens approved for treatment of many malignancies, especially colorectal cancer (CRC). The incidence of cardiotoxicity associated with 5-FU ranges between 1.5% to 18% and is most commonly manifested as anginal symptoms. Cardiomyopathy is very rarely reported with 5-FU and capecitabine. A 35-year-old Caucasian male with T3, N1, M0 rectal cancer after the initial neoadjuvant chemoradiation with 5FU/LV followed by surgical abdominoperineal resection (APR), began mFOLFOX6 in the adjuvant setting. Following the first treatment, he developed severe cardiomyopathy, with a drop in ejection fraction (EF) to 19% from normal. The cardiac workup showed no ischemic or other etiologies to explain this cardiac event. He was a nonsmoker and only occasionally drank alcohol. He had no previous or family history of heart disease and had normal cholesterol level. He was treated for severe congestive heart failure (CHF). When the patient presented to us for second opinion, we decided to examine him for dihydropyrimidine dehydrogenase (DPD) deficiency and thymidylate synthase (TYMS) polymorphism. The patient was found to be heterozygous for the c.85T>C mutation, resulting in reduced DPYD enzymatic activity and homozygous for TYMS 5’TSER genotype 2R/2R *f. Our group first identified and reported P453L (1358C>T) type DPYD germline mutation in a patient who developed 5-FU induced cardiotoxicity. In this paper, we describe the first case of cardiomyopathy related to DPD deficiency and homozygous polymorphism of TYMS in a patient with colon cancer following 5-FU containing regimen. Fluorouracil-related cardiomyopathy has to be anticipated and treated to prevent the serious consequence of cardiac dysfunction. The prospective testing for DPD deficiency in patients might prevent DPD-deficient patients from severe toxicity or even death, and therefore the development of a unified screening method is warranted. PMID:27752409

  8. Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

    PubMed

    Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

    2015-03-01

    The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes.

  9. Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

    PubMed

    Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

    2015-03-01

    The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes. PMID:25542170

  10. Population study of 1311 C/T polymorphism of Glucose 6 Phosphate Dehydrogenase gene in Pakistan – an analysis of 715 X-chromosomes

    PubMed Central

    Moiz, Bushra; Nasir, Amna; Moatter, Tariq; Naqvi, Zulfiqar Ali; Khurshid, Mohammad

    2009-01-01

    Background Nucleotide 1311 polymorphism at exon 11 of G6PD gene is widely prevalent in various populations of the world. The aim of the study was to evaluate 1311 polymorphism in subjects carrying G6PD Mediterranean gene and in general population living in Pakistan. Results Patients already known to be G6PD deficient were tested for 563C-T (G6PD Mediterranean) and 1311 C-T mutation through RFLP based PCR and gene sequencing. A control group not known to be G6PD deficient was tested for 1311C/T only. C-T transition at nt 1311 was detected in 60/234 X-chromosomes with 563 C-T mutation (gene frequency of 0.26) while in 130 of normal 402 X-chromosomes (gene frequency of 0.32). Conclusion We conclude that 1311 T is a frequent polymorphism both in general populations and in subjects with G6PD Mediterranean gene in Pakistan. The prevalence is higher compared to most of the populations of the world. The present study will help in understanding genetic basis of G6PD deficiency in Pakistani population and in developing ancestral links of its various ethnic groups. PMID:19640310

  11. Quantitative analysis of laminin 5 gene expression in human keratinocytes.

    PubMed

    Akutsu, Nobuko; Amano, Satoshi; Nishiyama, Toshio

    2005-05-01

    To examine the expression of laminin 5 genes (LAMA3, LAMB3, and LAMC2) encoding the three polypeptide chains alpha3, beta3, and gamma2, respectively, in human keratinocytes, we developed novel quantitative polymerase chain reaction (PCR) methods utilizing Thermus aquaticus DNA polymerase, specific primers, and fluorescein-labeled probes with the ABI PRISM 7700 sequence detector system. Gene expression levels of LAMA3, LAMB3, and LAMC2 and glyceraldehyde-3-phosphate dehydrogenase were quantitated reproducibly and sensitively in the range from 1 x 10(2) to 1 x 10(8) gene copies. Basal gene expression level of LAMB3 was about one-tenth of that of LAMA3 or LAMC2 in human keratinocytes, although there was no clear difference among immunoprecipitated protein levels of alpha3, beta3, and gamma2 synthesized in radio-labeled keratinocytes. Human serum augmented gene expressions of LAMA3, LAMB3, and LAMC2 in human keratinocytes to almost the same extent, and this was associated with an increase of the laminin 5 protein content measured by a specific sandwich enzyme-linked immunosorbent assay. These results demonstrate that the absolute mRNA levels generated from the laminin 5 genes do not determine the translated protein levels of the laminin 5 chains in keratinocytes, and indicate that the expression of the laminin 5 genes may be controlled by common regulation mechanisms. PMID:15854126

  12. Cyanobacterial NADPH dehydrogenase complexes

    SciTech Connect

    Ogawa, Teruo; Mi, Hualing

    2007-07-01

    Cyanobacteria possess functionally distinct multiple NADPH dehydrogenase (NDH-1) complexes that are essential to CO2 uptake, photosystem-1 cyclic electron transport and respiration. The unique nature of cyanobacterial NDH-1 complexes is the presence of subunits involved in CO2 uptake. Other than CO2 uptake, chloroplastic NDH-1 complex has similar role as cyanobacterial NDH-1 complexes in photosystem-1 cyclic electron transport and respiration (chlororespiration). In this mini-review we focus on the structure and function of cyanobacterial NDH-1 complexes and their phylogeny. The function of chloroplastic NDH-1 complex and characteristics of plants defective in NDH-1 are also described forcomparison.

  13. The Glycerol-3-Phosphate Acyltransferase TbGAT is Dispensable for Viability and the Synthesis of Glycerolipids in Trypanosoma brucei.

    PubMed

    Patel, Nipul; Pirani, Karim A; Zhu, Tongtong; Cheung-See-Kit, Melanie; Lee, Sungsu; Chen, Daniel G; Zufferey, Rachel

    2016-09-01

    Glycerolipids are the main constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans. Importantly, they occur as a structural component of the glycosylphosphatidylinositol lipid anchor of the abundant cell surface glycoproteins procyclin in procyclic forms and variant surface glycoprotein in bloodstream form, that play crucial roles for the development of the parasite in the insect vector and the mammalian host, respectively. The present work reports the characterization of the glycerol-3-phosphate acyltransferase TbGAT that initiates the biosynthesis of ester glycerolipids. TbGAT restored glycerol-3-phosphate acyltransferase activity when expressed in a Leishmania major deletion strain lacking this activity and exhibited preference for medium length, unsaturated fatty acyl-CoAs. TbGAT localized to the endoplasmic reticulum membrane with its N-terminal domain facing the cytosol. Despite that a TbGAT null mutant in T. brucei procyclic forms lacked glycerol-3-phosphate acyltransferase activity, it remained viable and exhibited similar growth rate as the wild type. TbGAT was dispensable for the biosynthesis of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and GPI-anchored protein procyclin