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Sample records for encode carcinogen-metabolizing enzymes

  1. Contributions of Human Enzymes in Carcinogen Metabolism

    PubMed Central

    Rendic, Slobodan; Guengerich, F. Peter

    2012-01-01

    Considerable support exists for roles of metabolism in modulating the carcinogenic properties of chemicals. In particular, many of these compounds are procarcinogens that require activation to electrophilic forms to exert genotoxic effects. We systematically analyzed the existing literature on metabolism of carcinogens by human enzymes, which has been developed largely in the past 25 years. The metabolism and especially bioactivation of carcinogens are dominated by cytochrome P450 enzymes (66% of bioactivations). Within this group, six P450s—1A1, 1A2, 1B1, 2A6, 2E1, and 3A4—accounted for 77% of the P450 activation reactions. The roles of these P450s can be compared with those estimated for drug metabolism and should be considered in issues involving enzyme induction, chemoprevention, molecular epidemiology, inter-individual variations, and risk assessment. PMID:22531028

  2. Bladder cancer risk from the perspective of genetic polymorphisms in the carcinogen metabolizing enzymes.

    PubMed

    Antonova, Olga; Toncheva, Draga; Grigorov, Evgeni

    2015-01-01

    Urinary bladder cancer is a socially significant healthcare problem. A diverse array of aromatic and heterocyclic amines, derived from the chemical and transport industry, diet, and cigarette smoke are considered carcinogens for the bladder. To exert their carcinogenic effect and to initiate the carcinogenic response, the arylamines require a metabolic activation by the host enzymes to chemically reactive compounds. The aim of this article was to review the latest and basic research developments on the role of the polymorphisms in the carcinogen metabolizing enzymes N-acetyltransferase (NAT), Glutathione S-transferases (GST), and Soluble sulfotransferases (SULT), with emphasis on the susceptibility to urinary bladder cancer. A PubMed search was conducted to identify original and review articles containing information about these polymophic variants in different populations and according to their prevalence in bladder cancer patients. We noticed that some genotypes were found to be predisposing and some protective for bladder cancer development. The NAT2 slow genotype, together with GSTM1 null genotype facilitated the development of bladder cancer in almost all ethnic groups. The 213His allele of the SULT1A1 gene which is associated with lower enzyme activity and decreased mutagen activation was reported to protect from bladder cancer in almost all studies. PMID:26854433

  3. Resveratrol modulates drug- and carcinogen-metabolizing enzymes in a healthy volunteer study.

    PubMed

    Chow, H-H Sherry; Garland, Linda L; Hsu, Chiu-Hsieh; Vining, Donna R; Chew, Wade M; Miller, Jessica A; Perloff, Marjorie; Crowell, James A; Alberts, David S

    2010-09-01

    Resveratrol has been shown to exhibit cancer-preventive activities in preclinical studies. We conducted a clinical study to determine the effect of pharmacologic doses of resveratrol on drug- and carcinogen-metabolizing enzymes. Forty-two healthy volunteers underwent baseline assessment of cytochrome P450 (CYP) and phase II detoxification enzymes. CYP1A2, CYP2D6, CYP2C9, and CYP3A4 enzyme activities were measured by the metabolism of caffeine, dextromethorphan, losartan, and buspirone, respectively. Blood lymphocyte glutathione S-transferase (GST) activity and GST-pi level and serum total and direct bilirubin, a surrogate for UDP-glucuronosyl transferase (UGT) 1A1 activity, were measured to assess phase II enzymes. After the baseline evaluation, study participants took 1 g of resveratrol once daily for 4 weeks. Enzyme assessment was repeated upon intervention completion. Resveratrol intervention was found to inhibit the phenotypic indices of CYP3A4, CYP2D6, and CYP2C9 and to induce the phenotypic index of 1A2. Overall, GST and UGT1A1 activities were minimally affected by the intervention, although an induction of GST-pi level and UGT1A1 activity was observed in individuals with low baseline enzyme level/activity. We conclude that resveratrol can modulate enzyme systems involved in carcinogen activation and detoxification, which may be one mechanism by which resveratrol inhibits carcinogenesis. However, pharmacologic doses of resveratrol could potentially lead to increased adverse drug reactions or altered drug efficacy due to inhibition or induction of certain CYPs. Further clinical development of resveratrol for cancer prevention should consider evaluation of lower doses of resveratrol to minimize adverse metabolic drug interactions. PMID:20716633

  4. Influence of certain essential oils on carcinogen-metabolizing enzymes and acid-soluble sulfhydryls in mouse liver.

    PubMed

    Banerjee, S; Sharma, R; Kale, R K; Rao, A R

    1994-01-01

    The influence of essential oils from naturally occurring plant dietary items such as cardamom, celery seed, cumin seed, coriander, ginger, nutmeg, and zanthoxylum on the activities of hepatic carcinogen-metabolizing enzymes (cytochrome P450, aryl hydrocarbon hydroxylase, and glutathione S-transferase) and acid-soluble sulfhydryl level was investigated in Swiss albino mice. Each oil was fed by gavage at 10 microliters/day for 14 days, and then the animals were sacrificed and their hepatic enzyme activities and sulfhydryl levels were evaluated. Only nutmeg and zanthoxylum oils induced cytochrome P450 level significantly (p < 0.05), whereas cardamom oil caused a significant reduction in its activity (p < 0.05). Furthermore, aryl hydrocarbon hydroxylase activity was significantly elevated only by treatment with ginger oil (p < 0.01), whereas nutmeg oil caused a significant reduction in its activity (p < 0.01). The remaining oils did not significantly alter the level of cytochrome P450 and aryl hydrocarbon hydroxylase activity. Glutathione S-transferase activity was significantly elevated in all experimental groups (p < 0.1-p < 0.001) compared with controls. The acid-soluble sulfhydryl was significantly elevated only by the essential oils of cardamom (p < 0.05), nutmeg (p < 0.05), and zanthoxylum (p < 0.01). Our observations suggest that intake of essential oils affects the host enzymes associated with activation and detoxication of xenobiotic compounds, including chemical carcinogens and mutagens. PMID:8072879

  5. Tobacco smoking, polymorphisms in carcinogen metabolism enzyme genes, and risk of localized and advanced prostate cancer: results from the California Collaborative Prostate Cancer Study

    PubMed Central

    Shahabi, Ahva; Corral, Román; Catsburg, Chelsea; Joshi, Amit D; Kim, Andre; Lewinger, Juan Pablo; Koo, Jocelyn; John, Esther M; Ingles, Sue A; Stern, Mariana C

    2014-01-01

    The relationship between tobacco smoking and prostate cancer (PCa) remains inconclusive. This study examined the association between tobacco smoking and PCa risk taking into account polymorphisms in carcinogen metabolism enzyme genes as possible effect modifiers (9 polymorphisms and 1 predicted phenotype from metabolism enzyme genes). The study included cases (n = 761 localized; n = 1199 advanced) and controls (n = 1139) from the multiethnic California Collaborative Case–Control Study of Prostate Cancer. Multivariable conditional logistic regression was performed to evaluate the association between tobacco smoking variables and risk of localized and advanced PCa risk. Being a former smoker, regardless of time of quit smoking, was associated with an increased risk of localized PCa (odds ratio [OR] = 1.3; 95% confidence interval [CI] = 1.0–1.6). Among non-Hispanic Whites, ever smoking was associated with an increased risk of localized PCa (OR = 1.5; 95% CI = 1.1–2.1), whereas current smoking was associated with risk of advanced PCa (OR = 1.4; 95% CI = 1.0–1.9). However, no associations were observed between smoking intensity, duration or pack-year variables, and advanced PCa. No statistically significant trends were seen among Hispanics or African-Americans. The relationship between smoking status and PCa risk was modified by the CYP1A2 rs7662551 polymorphism (P-interaction = 0.008). In conclusion, tobacco smoking was associated with risk of PCa, primarily localized disease among non-Hispanic Whites. This association was modified by a genetic variant in CYP1A2, thus supporting a role for tobacco carcinogens in PCa risk. PMID:25355624

  6. Tobacco smoking, polymorphisms in carcinogen metabolism enzyme genes, and risk of localized and advanced prostate cancer: results from the California Collaborative Prostate Cancer Study.

    PubMed

    Shahabi, Ahva; Corral, Román; Catsburg, Chelsea; Joshi, Amit D; Kim, Andre; Lewinger, Juan Pablo; Koo, Jocelyn; John, Esther M; Ingles, Sue A; Stern, Mariana C

    2014-12-01

    The relationship between tobacco smoking and prostate cancer (PCa) remains inconclusive. This study examined the association between tobacco smoking and PCa risk taking into account polymorphisms in carcinogen metabolism enzyme genes as possible effect modifiers (9 polymorphisms and 1 predicted phenotype from metabolism enzyme genes). The study included cases (n = 761 localized; n = 1199 advanced) and controls (n = 1139) from the multiethnic California Collaborative Case-Control Study of Prostate Cancer. Multivariable conditional logistic regression was performed to evaluate the association between tobacco smoking variables and risk of localized and advanced PCa risk. Being a former smoker, regardless of time of quit smoking, was associated with an increased risk of localized PCa (odds ratio [OR] = 1.3; 95% confidence interval [CI] = 1.0-1.6). Among non-Hispanic Whites, ever smoking was associated with an increased risk of localized PCa (OR = 1.5; 95% CI = 1.1-2.1), whereas current smoking was associated with risk of advanced PCa (OR = 1.4; 95% CI = 1.0-1.9). However, no associations were observed between smoking intensity, duration or pack-year variables, and advanced PCa. No statistically significant trends were seen among Hispanics or African-Americans. The relationship between smoking status and PCa risk was modified by the CYP1A2 rs7662551 polymorphism (P-interaction = 0.008). In conclusion, tobacco smoking was associated with risk of PCa, primarily localized disease among non-Hispanic Whites. This association was modified by a genetic variant in CYP1A2, thus supporting a role for tobacco carcinogens in PCa risk. PMID:25355624

  7. Effect of Siamese cassia leaves on the activities of chemical carcinogen metabolizing enzymes and on mammary gland carcinogenesis in the rat.

    PubMed

    Tepsuwan, A; Kupradinun, P; Kusamran, W R

    1999-07-16

    Male Wistar rats were fed AIN-76 semipurified diet or diet containing 5% ground lyophilized Siamese cassia leaves for 2 weeks before sacrifice. Hepatic S9 fractions were prepared and assayed for the level of cytochrome P450 (P450), the activities of monooxygenase, i.e., aniline hydroxylase (ANH), aminopyrine-N-demethylase (AMD) as well as the capacity to metabolically activate the mutagenicities of aflatoxin B(1) (AFB(1)) and benzo(a)pyrene (B(a)P). In addition, the activities of detoxificating enzymes such as glutathione-S-transferase (GST) and UDP-glucuronyltransferase (UGT) were also measured. It was found that feeding of Siamese cassia leaves significantly reduced the activities of hepatic ANH and AMD as well as the capacity to activate the mutagenicity of AFB(1) towards Salmonella typhimurium TA100, being 31, 73 and 41% of control group, respectively. It also slightly decreased, but not significantly, the capacity to activate the mutagenicity of B(a)P towards S. typhimurium YG1029. On the other hand, however, the activities of both GST and UGT were markedly increased in those animals, being 250 and 220% of control animals. The anticarcinogenic potential of Siamese cassia leaves was also investigated in female Sprague Dawley rats treated with 9,10-dimethyl-1,2-benzanthracene (DMBA). The animals were fed control diet or diet containing ground lyophilized Siamese cassia leaves 2 weeks prior to and 1 week after intragastrically administration of DMBA, and then they were placed on a pellet diet for additional 25 weeks. Interestingly, it was found that feeding of diet containing 2.5 and 4% Siamese cassia leaves resulted in a significant decrease in the multiplicity of mammary gland tumors as well as a slight delay of the onset of tumor development. The incidence of tumors in the group fed 4% Siamese cassia leaves, but not in the 2.5% group, was lowered, although not significantly, than that of control group. The results in the present study therefore demonstrated

  8. Local encoding of computationally designed enzyme activity

    PubMed Central

    Allert, Malin; Dwyer, Mary A.; Hellinga, Homme W.

    2007-01-01

    One aim of computational protein design is to introduce novel enzyme activity into proteins of known structure by predicting mutations that stabilize transition states. Previously we have shown that it is possible to introduce triose phosphate isomerase activity into the ribose-binding protein of Escherichia coli by constructing 17 mutations in the first two layers of residues that surround the wild-type ligand-binding site. Here we report that these mutations can be “transplanted” into a homologous ribose-binding protein, isolated from the hyperthermophilic bacterium Thermoanaerobacter tengcongensis, with retention of catalytic activity, substrate affinity, and reaction pH dependence. The observed 105–106-fold rate enhancement corresponds to 70% of the maximally known transition-state binding energy. The wild-type sequences in these two homologues are almost perfectly conserved in the vicinity of their ribose-binding sites, but diverge significantly at increasing distance from these sites. The results demonstrate that the computationally designed mutations are sufficient to encode the observed enzyme activity, that all the observed activity is locally encoded within the layer of residues directly in contact with the substrate, and that in this case at least 70% of transition state stabilization energy can be achieved using straightforward considerations of stereochemical complementarity between enzyme and reactants. PMID:17196220

  9. Genes Encoding Enzymes Involved in Ethanol Metabolism

    PubMed Central

    Hurley, Thomas D.; Edenberg, Howard J.

    2012-01-01

    The effects of beverage alcohol (ethanol) on the body are determined largely by the rate at which it and its main breakdown product, acetaldehyde, are metabolized after consumption. The main metabolic pathway for ethanol involves the enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Seven different ADHs and three different ALDHs that metabolize ethanol have been identified. The genes encoding these enzymes exist in different variants (i.e., alleles), many of which differ by a single DNA building block (i.e., single nucleotide polymorphisms [SNPs]). Some of these SNPs result in enzymes with altered kinetic properties. For example, certain ADH1B and ADH1C variants that are commonly found in East Asian populations lead to more rapid ethanol breakdown and acetaldehyde accumulation in the body. Because acetaldehyde has harmful effects on the body, people carrying these alleles are less likely to drink and have a lower risk of alcohol dependence. Likewise, an ALDH2 variant with reduced activity results in acetaldehyde buildup and also has a protective effect against alcoholism. In addition to affecting drinking behaviors and risk for alcoholism, ADH and ALDH alleles impact the risk for esophageal cancer. PMID:23134050

  10. Control of steroid, heme, and carcinogen metabolism by nuclear pregnane X receptor and constitutive androstane receptor.

    PubMed

    Xie, Wen; Yeuh, Mei-Fei; Radominska-Pandya, Anna; Saini, Simrat P S; Negishi, Yoichi; Bottroff, Bobbie Sue; Cabrera, Geraldine Y; Tukey, Robert H; Evans, Ronald M

    2003-04-01

    Through a multiplex promoter spanning 218 kb, the phase II UDP-glucuronosyltransferase 1A (UGT1) gene encodes at least eight differently regulated mRNAs whose protein products function as the principal means to eliminate a vast array of steroids, heme metabolites, environmental toxins, and drugs. The orphan nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) were originally identified as sensors able to respond to numerous environmentally derived foreign compounds (xenobiotics) to promote detoxification by phase I cytochrome P450 genes. In this report, we show that both receptors can induce specific UGT1A isoforms including those involved in estrogen, thyroxin, bilirubin, and carcinogen metabolism. Transgenic mice expressing a constitutively active form of human PXR show markedly increased UGT activity toward steroid, heme, and carcinogens, enhanced bilirubin clearance, as well as massively increased steroid clearance. The ability of PXR and constitutive androstane receptor and their ligands to transduce both the phase I and phase II adaptive hepatic response defines a unique transcriptional interface that bridges the ingestion and metabolism of environmental compounds to body physiology. PMID:12644700

  11. Dietary flavonoids: effects on xenobiotic and carcinogen metabolism.

    PubMed

    Moon, Young Jin; Wang, Xiaodong; Morris, Marilyn E

    2006-03-01

    Flavonoids are present in fruits, vegetables and beverages derived from plants (tea, red wine), and in many dietary supplements or herbal remedies including Ginkgo Biloba, Soy Isoflavones, and Milk Thistle. Flavonoids have been described as health-promoting, disease-preventing dietary supplements, and have activity as cancer preventive agents. Additionally, they are extremely safe and associated with low toxicity, making them excellent candidates for chemopreventive agents. The cancer protective effects of flavonoids have been attributed to a wide variety of mechanisms, including modulating enzyme activities resulting in the decreased carcinogenicity of xenobiotics. This review focuses on the flavonoid effects on cytochrome P450 (CYP) enzymes involved in the activation of procarcinogens and phase II enzymes, largely responsible for the detoxification of carcinogens. A number of naturally occurring flavonoids have been shown to modulate the CYP450 system, including the induction of specific CYP isozymes, and the activation or inhibition of these enzymes. Some flavonoids alter CYPs through binding to the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, acting as either AhR agonists or antagonists. Inhibition of CYP enzymes, including CYP 1A1, 1A2, 2E1 and 3A4 by competitive or mechanism-based mechanisms also occurs. Flavones (chrysin, baicalein, and galangin), flavanones (naringenin) and isoflavones (genistein, biochanin A) inhibit the activity of aromatase (CYP19), thus decreasing estrogen biosynthesis and producing antiestrogenic effects, important in breast and prostate cancers. Activation of phase II detoxifying enzymes, such as UDP-glucuronyl transferase, glutathione S-transferase, and quinone reductase by flavonoids results in the detoxification of carcinogens and represents one mechanism of their anticarcinogenic effects. A number of flavonoids including fisetin, galangin, quercetin, kaempferol, and genistein represent potent non

  12. Method for Enzyme Design with Genetically Encoded Unnatural Amino Acids.

    PubMed

    Hu, C; Wang, J

    2016-01-01

    We describe the methodologies for the design of artificial enzymes with genetically encoded unnatural amino acids. Genetically encoded unnatural amino acids offer great promise for constructing artificial enzymes with novel activities. In our studies, the designs of artificial enzyme were divided into two steps. First, we considered the unnatural amino acids and the protein scaffold separately. The scaffold is designed by traditional protein design methods. The unnatural amino acids are inspired by natural structure and organic chemistry methods, and synthesized by either organic chemistry methods or enzymatic conversion. With the increasing number of published unnatural amino acids with various functions, we described an unnatural amino acids toolkit containing metal chelators, redox mediators, and click chemistry reagents. These efforts enable a researcher to search the toolkit for appropriate unnatural amino acids for the study, rather than design and synthesize the unnatural amino acids from the beginning. After the first step, the model enzyme was optimized by computational methods and directed evolution. Lastly, we describe a general method for evolving aminoacyl-tRNA synthetase and expressing unnatural amino acids incorporated into a protein. PMID:27586330

  13. Method for Enzyme Design with Genetically Encoded Unnatural Amino Acids.

    PubMed

    Hu, C; Wang, J

    2016-01-01

    We describe the methodologies for the design of artificial enzymes with genetically encoded unnatural amino acids. Genetically encoded unnatural amino acids offer great promise for constructing artificial enzymes with novel activities. In our studies, the designs of artificial enzyme were divided into two steps. First, we considered the unnatural amino acids and the protein scaffold separately. The scaffold is designed by traditional protein design methods. The unnatural amino acids are inspired by natural structure and organic chemistry methods, and synthesized by either organic chemistry methods or enzymatic conversion. With the increasing number of published unnatural amino acids with various functions, we described an unnatural amino acids toolkit containing metal chelators, redox mediators, and click chemistry reagents. These efforts enable a researcher to search the toolkit for appropriate unnatural amino acids for the study, rather than design and synthesize the unnatural amino acids from the beginning. After the first step, the model enzyme was optimized by computational methods and directed evolution. Lastly, we describe a general method for evolving aminoacyl-tRNA synthetase and expressing unnatural amino acids incorporated into a protein.

  14. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, E.E.; Roessler, P.G.

    1999-07-27

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities. 8 figs.

  15. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, Eric E.; Roessler, Paul G.

    1999-01-01

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities.

  16. Modulation of carcinogen metabolism by nitric oxide-aspirin 2 is associated with suppression of DNA damage and DNA adduct formation.

    PubMed

    MacDonald, Christopher J; Cheng, Robert Y S; Roberts, David D; Wink, David A; Yeh, Grace Chao

    2009-08-14

    Nitric oxide (NO)-donating non-steroidal anti-inflammatory drugs (NSAIDs) represent a promising new class of drugs developed to provide a safer alternative than their conventional NSAID counterparts in chemoprevention. We tested the effects of NO-aspirin 2 on Phase I and Phase II carcinogen-metabolizing enzymes. In HepG2 human hepatoma cells and in LS180 colonic adenocarcinoma cells, NO-aspirin 2 inhibited 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD)-induced cytochrome P450 (CYP) enzyme activity and CYP1A1 and CYP1A2 mRNA expression. These effects were further characterized as being mediated through transcriptional regulation: NO-aspirin 2 inhibited binding of ligand (TCDD)-activated aryl hydrocarbon receptor to the CYP1A1 enhancer sequence; additionally, NO-aspirin 2 suppressed carcinogen-induced expression of CYP1A heterogeneous nuclear RNA. The fate of carcinogen metabolites depends not only on activation by CYP enzymes but also detoxification by Phase II enzymes. Both HepG2 and LS180 cells treated with NO-aspirin 2 showed an increase in glutathione S-transferase-P1 (GST-P1), glutamate-cysteine ligase (GCL), and NAD(P)H:quinone oxidoreductase-1 (NQO1) expression. Compared with two other NO-releasing compounds, diethylenetriamine-NO and the organic nitrate, isosorbide dinitrate, the inhibitory effects of NO-aspirin 2 on TCDD-induced CYP activity and mRNA expression were considerably more potent. Furthermore, aspirin alone had no inhibitory effect on TCDD-induced CYP activity, nor did aspirin up-regulate GCL, GST-P1, or NQO1 expression. Consequent to the effects on carcinogen-metabolizing enzymes, NO-aspirin 2 inhibited [3H]benzo[a]pyrene-DNA adduct formation and DNA damage elicited by TCDD or benzo[a]pyrene. Our results demonstrate that NO-aspirin 2 may be an effective chemopreventive agent by favorably affecting the inhibitory and enhancing effects of Phase I and Phase II carcinogen metabolism, thereby protecting DNA from carcinogenic insult. PMID:19542225

  17. Human Genetic Disorders Caused by Mutations in Genes Encoding Biosynthetic Enzymes for Sulfated Glycosaminoglycans*

    PubMed Central

    Mizumoto, Shuji; Ikegawa, Shiro; Sugahara, Kazuyuki

    2013-01-01

    A number of genetic disorders are caused by mutations in the genes encoding glycosyltransferases and sulfotransferases, enzymes responsible for the synthesis of sulfated glycosaminoglycan (GAG) side chains of proteoglycans, including chondroitin sulfate, dermatan sulfate, and heparan sulfate. The phenotypes of these genetic disorders reflect disturbances in crucial biological functions of GAGs in human. Recent studies have revealed that mutations in genes encoding chondroitin sulfate and dermatan sulfate biosynthetic enzymes cause various disorders of connective tissues. This minireview focuses on growing glycobiological studies of recently described genetic diseases caused by disturbances in biosynthetic enzymes for sulfated GAGs. PMID:23457301

  18. Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

    SciTech Connect

    Mueller, Edith E.; Mayr, Johannes A.; Zimmermann, Franz A.; Feichtinger, Rene G.; Stanger, Olaf; Sperl, Wolfgang; Kofler, Barbara

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer We examined OXPHOS and citrate synthase enzyme activities in HEK293 cells devoid of mtDNA. Black-Right-Pointing-Pointer Enzymes partially encoded by mtDNA show reduced activities. Black-Right-Pointing-Pointer Also the entirely nuclear encoded complex II and citrate synthase exhibit reduced activities. Black-Right-Pointing-Pointer Loss of mtDNA induces a feedback mechanism that downregulates complex II and citrate synthase. -- Abstract: Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complex II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 {rho}{sup 0} cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in {rho}{sup 0} cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.

  19. Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

    SciTech Connect

    Gray, Kevin A.; Zhao, Lishan; Cayouette, Michelle H.

    2015-09-08

    The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

  20. Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

    SciTech Connect

    Gray, Kevin A; Zhao, Lishan; Cayouette, Michelle H

    2015-11-04

    The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

  1. Recombinant plasmids for encoding restriction enzymes DpnI and DpnII of streptococcus pneumontae

    DOEpatents

    Lacks, Sanford A.

    1990-01-01

    Chromosomal DNA cassettes containing genes encoding either the DpnI or DpnII restriction endonucleases from Streptococcus pneumoniae are cloned into a streptococcal vector, pLS101. Large amounts of the restriction enzymes are produced by cells containing the multicopy plasmids, pLS202 and pLS207, and their derivatives pLS201, pLS211, pLS217, pLS251 and pLS252.

  2. Recombinant plasmids for encoding restriction enzymes DpnI and DpnII of Streptococcus pneumontae

    DOEpatents

    Lacks, S.A.

    1990-10-02

    Chromosomal DNA cassettes containing genes encoding either the DpnI or DpnII restriction endonucleases from Streptococcus pneumoniae are cloned into a streptococcal vector, pLS101. Large amounts of the restriction enzymes are produced by cells containing the multicopy plasmids, pLS202 and pLS207, and their derivatives pLS201, pLS211, pLS217, pLS251 and pLS252. 9 figs.

  3. Halloween genes encode P450 enzymes that mediate steroid hormone biosynthesis in Drosophila melanogaster.

    PubMed

    Gilbert, Lawrence I

    2004-02-27

    Mutation of members of the Halloween gene family results in embryonic lethality. We have shown that two of these genes code for enzymes responsible for specific steps in the synthesis of ecdysone, a polyhydroxylated sterol that is the precursor of the major molting hormone of all arthropods, 20-hydroxyecdysone. These two mitochondrial P450 enzymes, coded for by disembodied (dib) (CYP302A1) and shadow (sad) (CYP315A1), are the C22 and C2 hydroxylases, respectively, as shown by transfection of the gene into S2 cells and subsequent biochemical analysis. These are the last two enzymes in the ecdysone biosynthetic pathway. A third enzyme, necessary for the critical conversion of ecdysone to 20-hydroxyecdysone, the 20-monooxygenase, is encoded by shade (shd) (CYP314A1). All three enzymes are mitochondrial although shade has motifs suggesting both mitochondrial and microsomal locations. By tagging these enzymes, their subcellular location has been confirmed by confocal microscopy. Shade is present in several tissues as expected while disembodied and shadow are restricted to the ring gland. The paradigm used should allow us to define the enzymes mediating the entire ecdysteroid biosynthetic pathway. PMID:15026169

  4. Halloween genes encode P450 enzymes that mediate steroid hormone biosynthesis in Drosophila melanogaster.

    PubMed

    Gilbert, Lawrence I

    2004-02-27

    Mutation of members of the Halloween gene family results in embryonic lethality. We have shown that two of these genes code for enzymes responsible for specific steps in the synthesis of ecdysone, a polyhydroxylated sterol that is the precursor of the major molting hormone of all arthropods, 20-hydroxyecdysone. These two mitochondrial P450 enzymes, coded for by disembodied (dib) (CYP302A1) and shadow (sad) (CYP315A1), are the C22 and C2 hydroxylases, respectively, as shown by transfection of the gene into S2 cells and subsequent biochemical analysis. These are the last two enzymes in the ecdysone biosynthetic pathway. A third enzyme, necessary for the critical conversion of ecdysone to 20-hydroxyecdysone, the 20-monooxygenase, is encoded by shade (shd) (CYP314A1). All three enzymes are mitochondrial although shade has motifs suggesting both mitochondrial and microsomal locations. By tagging these enzymes, their subcellular location has been confirmed by confocal microscopy. Shade is present in several tissues as expected while disembodied and shadow are restricted to the ring gland. The paradigm used should allow us to define the enzymes mediating the entire ecdysteroid biosynthetic pathway.

  5. Functional analysis of N-linking oligosaccharyl transferase enzymes encoded by deep-sea vent proteobacteria.

    PubMed

    Mills, Dominic C; Jervis, Adrian J; Abouelhadid, Sherif; Yates, Laura E; Cuccui, Jon; Linton, Dennis; Wren, Brendan W

    2016-04-01

    Bacterial N-linking oligosaccharyl transferases (OTase enzymes) transfer lipid-linked glycans to selected proteins in the periplasm and were first described in the intestinal pathogen Campylobacter jejuni, a member of the ε-proteobacteria-subdivision of bacteria. More recently, orthologues from other ε-proteobacterial Campylobacter and Helicobacter species and a δ-proteobacterium, Desulfovibrio desulfuricans, have been described, suggesting that these two subdivisions of bacteria may be a source of further N-linked protein glycosylation systems. Whole-genome sequencing of both ε- and δ-proteobacteria from deep-sea vent habitats, a rich source of species from these subdivisions, revealed putative ORFs encoding OTase enzymes and associated adjacent glycosyltransferases similar to the C. jejuni N-linked glycosylation locus. We expressed putative OTase ORFs from the deep-sea vent species Nitratiruptor tergarcus, Sulfurovum lithotrophicum and Deferribacter desulfuricans in Escherichia coli and showed that they were able to functionally complement the C. jejuni OTase, CjPglB. The enzymes were shown to possess relaxed glycan specificity, transferring diverse glycan structures and demonstrated different glycosylation sequon specificities. Additionally, a permissive D. desulfuricans acceptor protein was identified, and we provide evidence that the N-linked glycan synthesized by N. tergarcus and S. lithotrophicum contains an acetylated sugar at the reducing end. This work demonstrates that deep-sea vent bacteria encode functional N-glycosylation machineries and are a potential source of biotechnologically important OTase enzymes. PMID:26610891

  6. Functional analysis of N-linking oligosaccharyl transferase enzymes encoded by deep-sea vent proteobacteria

    PubMed Central

    Mills, Dominic C.; Jervis, Adrian J.; Abouelhadid, Sherif; Yates, Laura E.; Cuccui, Jon; Linton, Dennis; Wren, Brendan W.

    2016-01-01

    Bacterial N-linking oligosaccharyl transferases (OTase enzymes) transfer lipid-linked glycans to selected proteins in the periplasm and were first described in the intestinal pathogen Campylobacter jejuni, a member of the ε-proteobacteria-subdivision of bacteria. More recently, orthologues from other ε-proteobacterial Campylobacter and Helicobacter species and a δ-proteobacterium, Desulfovibrio desulfuricans, have been described, suggesting that these two subdivisions of bacteria may be a source of further N-linked protein glycosylation systems. Whole-genome sequencing of both ε- and δ-proteobacteria from deep-sea vent habitats, a rich source of species from these subdivisions, revealed putative ORFs encoding OTase enzymes and associated adjacent glycosyltransferases similar to the C. jejuni N-linked glycosylation locus. We expressed putative OTase ORFs from the deep-sea vent species Nitratiruptor tergarcus, Sulfurovum lithotrophicum and Deferribacter desulfuricans in Escherichia coli and showed they were able to functionally complement the C. jejuni OTase, CjPglB . The enzymes were shown to possess relaxed glycan specificity, transferring diverse glycan structures and demonstrated different glycosylation sequon specificities. Additionally a permissive D. desulfuricans acceptor protein was identified, and we provide evidence that the N-linked glycan synthesised by N. tergarcus and S. lithotrophicum contains an acetylated sugar at the reducing end. This work demonstrates that deep-sea vent bacteria encode functional N-glycosylation machineries and are a potential source of biotechnologically important OTase enzymes. PMID:26610891

  7. Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them

    DOEpatents

    Gray, Kevin A.; Zhao, Lishan; Cayouette, Michelle H.

    2012-01-24

    The invention provides polypeptides having any cellulolytic activity, e.g., a cellulase activity, a endoglucanase, a cellobiohydrolase, a beta-glucosidase, a xylanase, a mannanse, a .beta.-xylosidase, an arabinofuranosidase, and/or an oligomerase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. In one aspect, the invention provides polypeptides having an oligomerase activity, e.g., enzymes that convert recalcitrant soluble oligomers to fermentable sugars in the saccharification of biomass. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

  8. Sequence Analysis of the Gene Encoding Amylosucrase from Neisseria polysaccharea and Characterization of the Recombinant Enzyme

    PubMed Central

    Potocki De Montalk, G.; Remaud-Simeon, M.; Willemot, R. M.; Planchot, V.; Monsan, P.

    1999-01-01

    The Neisseria polysaccharea gene encoding amylosucrase was subcloned and expressed in Escherichia coli. Sequencing revealed that the deduced amino acid sequence differs significantly from that previously published. Comparison of the sequence with that of enzymes of the α-amylase family predicted a (β/α)8-barrel domain. Six of the eight highly conserved regions in amylolytic enzymes are present in amylosucrase. Among them, four constitute the active site in α-amylases. These sites were also conserved in the sequence of glucosyltransferases and dextransucrases. Nevertheless, the evolutionary tree does not show strong homology between them. The amylosucrase was purified by affinity chromatography between fusion protein glutathione S-transferase–amylosucrase and glutathione-Sepharose 4B. The pure enzyme linearly elongated some branched chains of glycogen, to an average degree of polymerization of 75. PMID:9882648

  9. Controlling enzyme inhibition using an expanded set of genetically encoded amino acids.

    PubMed

    Zheng, Shun; Kwon, Inchan

    2013-09-01

    Enzyme inhibition plays an important role in drug development, metabolic pathway regulation, and biocatalysis with product inhibition. When an inhibitor has high structural similarities to the substrate of an enzyme, controlling inhibitor binding without affecting enzyme substrate binding is often challenging and requires fine-tuning of the active site. We hypothesize that an extended set of genetically encoded amino acids can be used to design an enzyme active site that reduces enzyme inhibitor binding without compromising substrate binding. As a model case, we chose murine dihydrofolate reductase (mDHFR), substrate dihydrofolate, and inhibitor methotrexate. Structural models of mDHFR variants containing non-natural amino acids complexed with each ligand were constructed to identify a key residue for inhibitor binding and non-natural amino acids to replace the key residue. Then, we discovered that replacing the key phenylalanine residue with two phenylalanine analogs (p-bromophenylalanine (pBrF) and L-2-naphthylalanine (2Nal)) enhances binding affinity toward the substrate dihydrofolate over the inhibitor by 4.0 and 5.8-fold, respectively. Such an enhanced selectivity is mainly due to a reduced inhibitor binding affinity by 2.1 and 4.3-fold, respectively. The catalytic efficiency of the mDHFR variant containing pBrF is comparable to that of wild-type mDHFR, whereas the mDHFR variant containing 2Nal exhibits a moderate decrease in the catalytic efficiency. The work described here clearly demonstrates the feasibility of selectively controlling enzyme inhibition using an expanded set of genetically encoded amino acids.

  10. Two genes encoding new carotenoid-modifying enzymes in the green sulfur bacterium Chlorobium tepidum.

    PubMed

    Maresca, Julia A; Bryant, Donald A

    2006-09-01

    The green sulfur bacterium Chlorobium tepidum produces chlorobactene as its primary carotenoid. Small amounts of chlorobactene are hydroxylated by the enzyme CrtC and then glucosylated and acylated to produce chlorobactene glucoside laurate. The genes encoding the enzymes responsible for these modifications of chlorobactene, CT1987, and CT0967, have been identified by comparative genomics, and these genes were insertionally inactivated in C. tepidum to verify their predicted function. The gene encoding chlorobactene glucosyltransferase (CT1987) has been named cruC, and the gene encoding chlorobactene lauroyltransferase (CT0967) has been named cruD. Homologs of these genes are found in the genomes of all sequenced green sulfur bacteria and filamentous anoxygenic phototrophs as well as in the genomes of several nonphotosynthetic bacteria that produce similarly modified carotenoids. The other bacteria in which these genes are found are not closely related to green sulfur bacteria or to one another. This suggests that the ability to synthesize modified carotenoids has been a frequently transferred trait.

  11. Nucleotide sequencing and characterization of the genes encoding benzene oxidation enzymes of Pseudomonas putida

    SciTech Connect

    Irie, S.; Doi, S.; Yorifuji, T.; Takagi, M.; Yano, K.

    1987-11-01

    The nucleotide sequence of the genes from Pseudomonas putida encoding oxidation of benzene to catechol was determined. Five open reading frames were found in the sequence. Four corresponding protein molecules were detected by a DNA-directed in vitro translation system. Escherichia coli cells containing the fragment with the four open reading frames transformed benzene to cis-benzene glycol, which is an intermediate of the oxidation of benzene to catechol. The relation between the product of each cistron and the components of the benzene oxidation enzyme system is discussed.

  12. Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

    PubMed Central

    Dougherty, W G; Semler, B L

    1993-01-01

    Many viruses express their genome, or part of their genome, initially as a polyprotein precursor that undergoes proteolytic processing. Molecular genetic analyses of viral gene expression have revealed that many of these processing events are mediated by virus-encoded proteinases. Biochemical activity studies and structural analyses of these viral enzymes reveal that they have remarkable similarities to cellular proteinases. However, the viral proteinases have evolved unique features that permit them to function in a cellular environment. In this article, the current status of plant and animal virus proteinases is described along with their role in the viral replication cycle. The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors. Images PMID:8302216

  13. The Saccharomyces cerevisiae YPR184w gene encodes the glycogen debranching enzyme.

    PubMed

    Teste, M A; Enjalbert, B; Parrou, J L; François, J M

    2000-12-01

    The YPR184w gene encodes a 1536-amino acid protein that is 34-39% identical to the mammal, Drosophila melanogaster and Caenorhabditis elegans glycogen debranching enzyme. The N-terminal part of the protein possesses the four conserved sequences of the alpha-amylase superfamily, while the C-terminal part displays 50% similarity with the C-terminal of other eukaryotic glycogen debranching enzymes. Reliable measurement of alpha-1,4-glucanotransferase and alpha-1, 6-glucosidase activity of the yeast debranching enzyme was determined in strains overexpressing YPR184w. The alpha-1, 4-glucanotransferase activity of a partially purified preparation of debranching enzyme preferentially transferred maltosyl units than maltotriosyl. Deletion of YPR184w prevents glycogen degradation, whereas overexpression had no effect on the rate of glycogen breakdown. In response to stress and growth conditions, the transcriptional control of YPR184w gene, renamed GDB1 (for Glycogen DeBranching gene), is strictly identical to that of other genes involved in glycogen metabolism.

  14. Analysis of a polygalacturonase gene of Ustilago maydis and characterization of the encoded enzyme.

    PubMed

    Castruita-Domínguez, José P; González-Hernández, Sandra E; Polaina, Julio; Flores-Villavicencio, Lérida L; Alvarez-Vargas, Aurelio; Flores-Martínez, Alberto; Ponce-Noyola, Patricia; Leal-Morales, Carlos A

    2014-05-01

    Ustilago maydis is a pathogenic fungus that produces the corn smut. It is a biotrophic parasite that depends on living plant tissues for its proliferation and development. Polygalacturonases are secreted by pathogens to solubilize the plant cell-wall and are required for pathogen virulence. In this paper, we report the isolation of a U. maydis polygalacturonase gene (Pgu1) and the functional and structural characterization of the encoded enzyme. The U. maydis Pgu1 gene is expressed when the fungus is grown in liquid culture media containing different carbon sources. In plant tissue, the expression increased as a function of incubation time. Pgu1 gene expression was detected during plant infection around 10 days post-infection with U. maydis FB-D12 strain in combination with teliospore formation. Synthesis and secretion of active recombinant PGU1 were achieved using Pichia pastoris, the purified enzyme had a optimum temperature of 34 °C, optimum pH of 4.5, a Km of 57.84 g/L for polygalacturonic acid, and a Vmax of 28.9 µg/min mg. Structural models of PGU1 based on homologous enzymes yielded a typical right-handed β-helix fold of pectinolytic enzymes classified in the glycosyl hydrolases family 28, and the U. maydis PGU1 is related with endo rather than exo polygalacturonases.

  15. Down-regulation of the carcinogen-metabolizing enzyme cytochrome P450 1a1 by vanadium.

    PubMed

    Anwar-Mohamed, Anwar; El-Kadi, Ayman O S

    2008-09-01

    Vanadium (V(5+)), a heavy metal contaminant with important toxicological consequences, has received considerable attention as an anticancer agent, although the mechanisms remain unknown. As a first step to investigate these mechanisms, we examined the effect of V(5+) (as ammonium metavanadate, NH(4)VO(3)) on the expression of the aryl hydrocarbon receptor (AhR)-regulated gene: cytochrome P450 1a1 (Cyp1a1) at each step of the AhR signal transduction pathway, using Hepa 1c1c7 cells. Our results showed a significant reduction in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated induction of Cyp1a1 mRNA, protein and activity levels after V(5+) treatments in a dose-dependent manner. Investigation of the effect of coexposure to V(5+) and TCDD at transcriptional levels revealed that V(5+) significantly inhibited TCDD-mediated induction of AhR-dependent luciferase reporter gene expression. Furthermore, despite not affecting the direct activation of the cytosolic AhR by TCDD and subsequently transforming it to a DNA-binding form, V(5+) inhibited the nuclear accumulation of liganded AhR and subsequent formation of the AhR/aryl hydrocarbon nuclear translocator (Arnt)/xenobiotic responsive element (XRE) complex. Importantly, the V(5+)-mediated inhibition of AhR/Arnt/XRE complex formation coincided with a significant decrease in ecto-ATPase activity. Looking at the post-transcriptional and post-translational effects of V(5+) on existing Cyp1a1 mRNA and protein levels, we showed that V(5+) did not affect Cyp1a1 mRNA or protein stability, thus eliminating possible role of V(5+) in modifying Cyp1a1 gene expression through these mechanisms. This study provides the first evidence that V(5+) down-regulates the expression of Cyp1a1 at the transcriptional level through an ATP-dependent mechanism.

  16. 1,10-phenanthroline stabilizes mRNA of the carcinogen-metabolizing enzyme, cytochrome P450 1a1.

    PubMed

    Chou, Mou-Tsy; Chu, Wen-Cheng; Hong, Wei-Fu; Huang, Min-Cong; Liu, Wen-Je; Lin, Shin-Chang; Huang, See-Chang; Chen, Fei-Yun; Hsiao, Wen-Feng; Liu, Yi-Wen; Wu, Jin-Yi; Su, Jyan-Gwo J

    2010-02-01

    1,10-phenanthroline (phen), flufenamic acid, and indomethacin are inhibitors of aldo-keto reductases 1C1 (AKR1C1), but only phen decreased the benzo[a]pyrene (BaP)-induced cytochrome P450 1a1 (Cyp1a1) protein level. Therefore the decrease in the BaP-induced Cyp1a1 protein level was not due to inhibition of Akr1c1, but to phen itself. Phen decreased the BaP-induced Cyp1a1 promoter activity and protein expression, and in contrast, it increased Cyp1a1 mRNA, resulting from an increase in mRNA stability. Phen is also known as a transition metal ion-chelator. Along with the phen study, we also found that Zn(2+), Fe(2+) and Cu(2+) increased Cyp1a1 mRNA and protein stability. Our results show that phen stabilized the mRNA of Cyp1a1, although it decreased cell viability. In addition, Zn(2+) and Fe(2+) highly neutralized phen's suppression of Cyp1a1 protein expression, but they only slightly neutralized phen's promotion of mRNA stability and suppression of cell viability, and had no effect on phen's suppression of promoter activity. Phen's effect on Cyp1a1 expression was reversible, which indicates that phen is non-covalently linked to its target. This report elucidates a new role for phen of stabilizing Cyp1a1 mRNA, and provides information for further studies on mRNA stabilization.

  17. dUTPase: the frequently overlooked enzyme encoded by many retroviruses.

    PubMed

    Hizi, Amnon; Herzig, Eytan

    2015-08-12

    Retroviruses are among the best studied viruses in last decades due to their pivotal involvement in cellular processes and, most importantly, in causing human diseases, most notably-acquired immunodeficiency syndrome (AIDS) that is triggered by human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively). Numerous studied were conducted to understand the involvement of the three cardinal retroviral enzymes, reverse transcriptase, integrase and protease, in the life cycle of the viruses. These studies have led to the development of many inhibitors of these enzymes as anti-retroviral specific drugs that are used for routine treatments of HIV/AIDS patients. Interestingly, a fourth virus-encoded enzyme, the deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is also found in several major retroviral groups. The presence and the importance of this enzyme to the life cycle of retroviruses were usually overlooked by most retrovirologists, although the occurrence of dUTPases, particularly in beta-retroviruses and in non-primate retroviruses, is known for more than 20 years. Only more recently, retroviral dUTPases were brought into the limelight and were shown in several cases to be essential for viral replication. Therefore, it is likely that future studies on this enzyme will advance our knowledge to a level that will allow designing novel, specific and potent anti-dUTPase drugs that are effective in combating retroviral diseases. The aim of this review is to give concise background information on dUTPases in general and to summarize the most relevant data on retroviral dUTPases and their involvement in the replication processes and pathogenicity of the viruses, as well as in possibly-associated human diseases.

  18. 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

  19. Distribution of genes encoding aminoglycoside-modifying enzymes among clinical isolates of methicillin-resistant staphylococci.

    PubMed

    Perumal, N; Murugesan, S; Krishnan, P

    2016-01-01

    The objective of this study was to determine the distribution of genes encoding aminoglycoside-modifying enzymes (AMEs) and staphylococcal cassette chromosome mec (SCCmec) elements among clinical isolates of methicillin-resistant staphylococci (MRS). Antibiotic susceptibility test was done using Kirby-Bauer disk diffusion method. The presence of SCCmec types and AME genes, namely, aac (6')-Ie-aph (2''), aph (3')-IIIa and ant (4')-Ia was determined using two different multiplex polymerase chain reaction. The most encountered AME genes were aac (6')-Ie-aph (2'') (55.4%) followed by aph (3')-IIIa (32.3%) and ant (4')-Ia gene (9%). SCCmec type I (34%) was predominant in this study. In conclusion, the aac (6')-Ie-aph (2'') was the most common AME gene and SCCmec type I was most predominant among the MRS isolates. PMID:27514959

  20. Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes

    PubMed Central

    Scott, Andrew; Noga, Marek J.; de Graaf, Paul; Westerlaken, Ilja; Yildirim, Esengul; Danelon, Christophe

    2016-01-01

    The goal of bottom-up synthetic biology culminates in the assembly of an entire cell from separate biological building blocks. One major challenge resides in the in vitro production and implementation of complex genetic and metabolic pathways that can support essential cellular functions. Here, we show that phospholipid biosynthesis, a multiple-step process involved in cell membrane homeostasis, can be reconstituted starting from the genes encoding for all necessary proteins. A total of eight E. coli enzymes for acyl transfer and headgroup modifications were produced in a cell-free gene expression system and were co-translationally reconstituted in liposomes. Acyl-coenzyme A and glycerol-3-phosphate were used as canonical precursors to generate a variety of important bacterial lipids. Moreover, this study demonstrates that two-step acyl transfer can occur from enzymes synthesized inside vesicles. Besides clear implications for growth and potentially division of a synthetic cell, we postulate that gene-based lipid biosynthesis can become instrumental for ex vivo and protein purification-free production of natural and non-natural lipids. PMID:27711229

  1. Functional genomics and SNP analysis of human genes encoding proline metabolic enzymes

    PubMed Central

    Williams, D. Bart; Zhaorigetu, Siqin; Khalil, Shadi; Wan, Guanghua; Valle, David

    2009-01-01

    Proline metabolism in mammals involves two other amino acids, glutamate and ornithine, and five enzymatic activities, Δ1-pyrroline-5-carboxylate (P5C) reductase (P5CR), proline oxidase, P5C dehydrogenase, P5C synthase and ornithine-δ-aminotransferase (OAT). With the exception of OAT, which catalyzes a reversible reaction, the other 4 enzymes are unidirectional, suggesting that proline metabolism is purpose-driven, tightly regulated, and compartmentalized. In addition, this tri-amino-acid system also links with three other pivotal metabolic systems, namely the TCA cycle, urea cycle, and pentose phosphate pathway. Abnormalities in proline metabolism are relevant in several diseases: six monogenic inborn errors involving metabolism and/or transport of proline and its immediate metabolites have been described. Recent advances in the Human Genome Project, in silico database mining techniques, and research in dissecting the molecular basis of proline metabolism prompted us to utilize functional genomic approaches to analyze human genes which encode proline metabolic enzymes in the context of gene structure, regulation of gene expression, mRNA variants, protein isoforms, and single nucleotide polymorphisms. PMID:18506409

  2. Plant eR Genes That Encode Photorespiratory Enzymes Confer Resistance against Disease

    PubMed Central

    Taler, Dvir; Galperin, Marjana; Benjamin, Ido; Cohen, Yigal; Kenigsbuch, David

    2004-01-01

    Downy mildew caused by the oomycete pathogen Pseudoperonospora cubensis is a devastating foliar disease of cucurbits worldwide. We previously demonstrated that the wild melon line PI 124111F (PI) is highly resistant to all pathotypes of P. cubensis. That resistance was controlled genetically by two partially dominant, complementary loci. Here, we show that unlike other plant disease resistance genes, which confer an ability to resist infection by pathogens expressing corresponding avirulence genes, the resistance of PI to P. cubensis is controlled by enhanced expression of the enzymatic resistance (eR) genes At1 and At2. These constitutively expressed genes encode the photorespiratory peroxisomal enzyme proteins glyoxylate aminotransferases. The low expression of At1 and At2 in susceptible melon lines is regulated mainly at the transcriptional level. This regulation is independent of infection with the pathogen. Transgenic melon plants overexpressing either of these eR genes displayed enhanced activity of glyoxylate aminotransferases and remarkable resistance against P. cubensis. The cloned eR genes provide a new resource for developing downy mildew–resistant melon varieties. PMID:14688292

  3. Identification of the DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus

    SciTech Connect

    Morgan, J.R.; Cohen, L.K.; Roberts, B.E.

    1984-10-01

    The DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus were located on the viral genome. The formation of an enzyme-guanylate covalent intermediate labeled with (alpha-/sup 32/P)GTP allowed the identification of the large subunit of the capping enzyme and was used to monitor the appearance of the enzyme during the infectious cycle. This assay confirmed that after vaccinia infection, a novel 84,000-molecular-weight polypeptide corresponding to the large subunit was rapidly synthesized before viral DNA replication. Hybrid-selected cell-free translation of early viral mRNA established that vaccinia virus encoded a polypeptide identical in molecular weight with the /sup 32/P-labeled 84,000-molecular-weight polypeptide found in vaccinia virions. Like the authentic capping enzyme, this virus-encoded cell-free translation product bound specifically to DNA-cellulose. A comparison of the partial proteolytic digestion fragments generated by V8 protease, chymotrypsin, and trypsin demonstrated that the /sup 32/P-labeled large subunit and the (/sup 35/S)methionine-labeled cell-free translation product were identical. The mRNA encoding the large subunit of the capping enzyme was located 3.1 kilobase pairs to the left of the HindIII D restriction fragment of the vaccinia genome. Furthermore, the mRNA was determined to be 3.0 kilobases in size, and its 5 and 3 termini were precisely located by S1 nuclease analysis.

  4. Enzymes Catalyzing the Early Steps of Clavulanic Acid Biosynthesis Are Encoded by Two Sets of Paralogous Genes in Streptomyces clavuligerus

    PubMed Central

    Jensen, Susan E.; Elder, Kenneth J.; Aidoo, Kwamena A.; Paradkar, Ashish S.

    2000-01-01

    Genes encoding the proteins required for clavulanic acid biosynthesis and for cephamycin biosynthesis are grouped into a “supercluster” in Streptomyces clavuligerus. Nine open reading frames (ORFs) associated with clavulanic acid biosynthesis were located in a 15-kb segment of the supercluster, including six ORFs encoding known biosynthetic enzymes or regulatory proteins, two ORFs that have been reported previously but whose involvement in clavulanic acid biosynthesis is unclear, and one ORF not previously reported. Evidence for the involvement of these ORFs in clavulanic acid production was obtained by generating mutants and showing that all were defective for clavulanic acid production when grown on starch asparagine medium. However, when five of the nine mutants, including mutants defective in known clavulanic acid biosynthetic enzymes, were grown in a soy-based medium, clavulanic acid-producing ability was restored. This ability to produce clavulanic acid when seemingly essential biosynthetic enzymes have been mutated suggests that paralogous genes encoding functionally equivalent proteins exist for each of the five genes but that these paralogues are expressed only in the soy-based medium. The five genes that have paralogues encode proteins involved in the early steps of the pathway common to the biosynthesis of both clavulanic acid and the other clavam metabolites produced by this organism. No evidence was seen for paralogues of the four remaining genes involved in late, clavulanic acid-specific steps in the pathway. PMID:10681345

  5. Regulation of genes encoding cellulolytic enzymes by Pal-PacC signaling in Aspergillus nidulans.

    PubMed

    Kunitake, Emi; Hagiwara, Daisuke; Miyamoto, Kentaro; Kanamaru, Kyoko; Kimura, Makoto; Kobayashi, Tetsuo

    2016-04-01

    Cellulosic biomass represents a valuable potential substitute for fossil-based fuels. As such, there is a strong need to develop efficient biotechnological processes for the enzymatic hydrolysis of cellulosic biomass via the optimization of cellulase production by fungi. Ambient pH is an important factor affecting the industrial production of cellulase. In the present study, we demonstrate that several Aspergillus nidulans genes encoding cellulolytic enzymes are regulated by Pal-PacC-mediated pH signaling, as evidenced by the decreased cellulase productivity of the palC mutant and pacC deletants of A. nidulans. The deletion of pacC was observed to result in delayed induction and decreased expression of the cellulase genes based on time course expression analysis. The genome-wide identification of PacC-regulated genes under cellobiose-induced conditions demonstrated that genes expressed in a PacC-dependent manner included 82 % of ClrB (a transcriptional activator of the cellulase genes)-regulated genes, including orthologs of various transporter and β-glucosidase genes considered to be involved in cellobiose uptake or production of stronger inducer molecules. Together with the significant overlap between ClrB- and PacC-regulated genes, the results suggest that PacC-mediated regulation of the cellulase genes involves not only direct regulation by binding to their promoter regions but also indirect regulation via modulation of the expression of genes involved in ClrB-dependent transcriptional activation. Our findings are expected to contribute to the development of more efficient industrial cellulase production methods.

  6. Genes encoding hub and bottleneck enzymes of the Arabidopsis metabolic network preferentially retain homeologs through whole genome duplication

    PubMed Central

    2010-01-01

    Background Whole genome duplication (WGD) occurs widely in angiosperm evolution. It raises the intriguing question of how interacting networks of genes cope with this dramatic evolutionary event. Results In study of the Arabidopsis metabolic network, we assigned each enzyme (node) with topological centralities (in-degree, out-degree and between-ness) to measure quantitatively their centralities in the network. The Arabidopsis metabolic network is highly modular and separated into 11 interconnected modules, which correspond well to the functional metabolic pathways. The enzymes with higher in-out degree and between-ness (defined as hub and bottleneck enzymes, respectively) tend to be more conserved and preferentially retain homeologs after WGD. Moreover, the simultaneous retention of homeologs encoding enzymes which catalyze consecutive steps in a pathway is highly favored and easily achieved, and enzyme-enzyme interactions contribute to the retention of one-third of WGD enzymes. Conclusions Our analyses indicate that the hub and bottleneck enzymes of metabolic network obtain great benefits from WGD, and this event grants clear evolutionary advantages in adaptation to different environments. PMID:20478072

  7. Rubisco in marine symbiotic dinoflagellates: form II enzymes in eukaryotic oxygenic phototrophs encoded by a nuclear multigene family.

    PubMed

    Rowan, R; Whitney, S M; Fowler, A; Yellowlees, D

    1996-03-01

    Genes encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were cloned from dinoflagellate symbionts (Symbiodinium spp) of the giant clam Tridacna gigas and characterized. Strikingly, Symbiodinium Rubisco is completely different from other eukaryotic (form I) Rubiscos: it is a form II enzyme that is approximately 65% identical to Rubisco from Rhodospirillum rubrum (Rubisco forms I and II are approximately 25 to 30% identical); it is nuclear encoded by a multigene family; and the predominantly expressed Rubisco is encoded as a precursor polyprotein. One clone appears to contain a predominantly expressed Rubisco locus (rbcA), as determined by RNA gel blot analysis of Symbiodinium RNA and sequencing of purified Rubisco protein. Another contains an enigmatic locus (rbcG) that exhibits an unprecedented pattern of amino acid replacement but does not appear to be a pseudogene. The expression of rbcG has not been analyzed; it was detected only in the minor of two taxa of Symbiodinium that occur together in T. gigas. This study confirms and describes a previously unrecognized branch of Rubisco's evolution: a eukaryotic form II enzyme that participates in oxygenic photosynthesis and is encoded by a diverse, nuclear multigene family.

  8. Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

    PubMed

    Dong, G; Vieille, C; Zeikus, J G

    1997-09-01

    The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants.

  9. Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature.

    PubMed

    Busk, Peter K; Lange, Mette; Pilgaard, Bo; Lange, Lene

    2014-01-01

    The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls. PMID:25461894

  10. Several Genes Encoding Enzymes with the Same Activity Are Necessary for Aerobic Fungal Degradation of Cellulose in Nature

    PubMed Central

    Busk, Peter K.; Lange, Mette; Pilgaard, Bo; Lange, Lene

    2014-01-01

    The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls. PMID:25461894

  11. A Ti plasmid-encoded enzyme required for degradation of mannopine is functionally homologous to the T-region-encoded enzyme required for synthesis of this opine in crown gall tumors.

    PubMed

    Kim, K S; Chilton, W S; Farrand, S K

    1996-06-01

    The mocC gene encoded by the octopine/mannityl opine-type Ti plasmid pTi15955 is related at the nucleotide sequence level to mas1' encoded by the T region of this plasmid. While Mas1 is required for the synthesis of mannopine (MOP) by crown gall tumor cells, MocC is essential for the utilization of MOP by Agrobacterium spp. A cosmid clone of pTi15955, pYDH208, encodes mocC and confers the utilization of MOP on strain NT1 and on strain UIA5, a derivative of NT1 lacking the 450-kb cryptic plasmid pAtC58. NT1 or UIA5 harboring pYDH208 with an insertion mutation in mocC failed to utilize MOP as the sole carbon source. Plasmid pSa-C, which encodes only mocC, complemented this mutation in both strains. This plasmid also was sufficient to confer utilization of MOP on NT1 but not on UIA5. Computer analysis showed that MocC is related at the amino acid sequence level to members of the short-chain alcohol dehydrogenase family of oxidoreductases. Lysates prepared from Escherichia coli cells expressing mocC contained an enzymatic activity that oxidizes MOP to deoxyfructosyl glutamine (santhopine [SOP]) in the presence of NAD+. The reaction catalyzed by the MOP oxidoreductase is reversible; in the presence of NADH, the enzyme reduced SOP to MOP. The apparent Km values of the enzyme for MOP and SOP were 6.3 and 1.2 mM, respectively. Among analogs of MOP tested, only N-1-(1-deoxy-D-lyxityl)-L-glutamine and N-1-(1-deoxy-D-mannityl)-L-asparagine served as substrates for MOP oxidoreductase. These results indicate that mocC encodes an oxidoreductase that, as an oxidase, is essential for the catabolism of MOP. The reductase activity of this enzyme is precisely the reaction ascribed to its T-region-encoded homolog, Mas1, which is responsible for biosynthesis of mannopine in crown gall tumors.

  12. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    SciTech Connect

    Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

    2014-04-08

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  13. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    DOEpatents

    Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

    2013-07-23

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  14. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    DOEpatents

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Ward, Thomas E.

    2016-03-22

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  15. 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.

  16. Multiple horizontally acquired genes from fungal and prokaryotic donors encode cellulolytic enzymes in the bdelloid rotifer Adineta ricciae.

    PubMed

    Szydlowski, L; Boschetti, C; Crisp, A; Barbosa, E G G; Tunnacliffe, A

    2015-07-25

    The bdelloid rotifer, Adineta ricciae, an anhydrobiotic microinvertebrate, exhibits a high rate of horizontal gene transfer (HGT), with as much as 10% of its transcriptome being of foreign origin. Approximately 80% of these foreign transcripts are involved in metabolic processes, and therefore bdelloids represent a useful model for assessing the contribution of HGT to biochemical diversity. To validate this concept, we focused on cellulose digestion, an unusual activity in animals, which is represented by at least 16 genes encoding cellulolytic enzymes in A. ricciae. These genes have been acquired from a variety of different donor organisms among the bacteria and fungi, demonstrating that bdelloids use diverse genetic resources to construct a novel biochemical pathway. A variable complement of the cellulolytic gene set was found in five other bdelloid species, indicating a dynamic process of gene acquisition, duplication and loss during bdelloid evolution. For example, in A. ricciae, gene duplications have led to the formation of three copies of a gene encoding a GH45 family glycoside hydrolase, at least one of which encodes a functional enzyme; all three of these gene copies are present in a close relative, Adineta vaga, but only one copy was found in each of four Rotaria species. Furthermore, analysis of expression levels of the cellulolytic genes suggests that a bacterial-origin cellobiase is upregulated upon desiccation. In summary, bdelloid rotifers have apparently developed cellulolytic functions by the acquisition and domestication of multiple foreign genes.

  17. Metabolic gene clusters encoding the enzymes of two branches of the 3-oxoadipate pathway in the pathogenic yeast Candida albicans.

    PubMed

    Gérecová, Gabriela; Neboháčová, Martina; Zeman, Igor; Pryszcz, Leszek P; Tomáška, Ľubomír; Gabaldón, Toni; Nosek, Jozef

    2015-05-01

    The pathogenic yeast Candida albicans utilizes hydroxyderivatives of benzene via the catechol and hydroxyhydroquinone branches of the 3-oxoadipate pathway. The genetic basis and evolutionary origin of this catabolic pathway in yeasts are unknown. In this study, we identified C. albicans genes encoding the enzymes involved in the degradation of hydroxybenzenes. We found that the genes coding for core components of the 3-oxoadipate pathway are arranged into two metabolic gene clusters. Our results demonstrate that C. albicans cells cultivated in media containing hydroxybenzene substrates highly induce the transcription of these genes as well as the corresponding enzymatic activities. We also found that C. albicans cells assimilating hydroxybenzenes cope with the oxidative stress by upregulation of cellular antioxidant systems such as alternative oxidase and catalase. Moreover, we investigated the evolution of the enzymes encoded by these clusters and found that most of them share a particularly sparse phylogenetic distribution among Saccharomycotina, which is likely to have been caused by extensive gene loss. We exploited this fact to find co-evolving proteins that are suitable candidates for the missing enzymes of the pathway. PMID:25743787

  18. Identification and expression analysis of castor bean (Ricinus communis) genes encoding enzymes from the triacylglycerol biosynthesis pathway.

    PubMed

    Cagliari, Alexandro; Margis-Pinheiro, Márcia; Loss, Guilherme; Mastroberti, Alexandra Antunes; de Araujo Mariath, Jorge Ernesto; Margis, Rogério

    2010-11-01

    Castor bean (Ricinus communis) oil contains ricinoleic acid-rich triacylglycerols (TAGs). As a result of its physical and chemical properties, castor oil and its derivatives are used for numerous bio-based products. In this study, we survey the Castor Bean Genome Database to report the identification of TAG biosynthesis genes. A set of 26 genes encoding six distinct classes of enzymes involved in TAGs biosynthesis were identified. In silico characterization and sequence analysis allowed the identification of plastidic isoforms of glycerol-3-phosphate acyltransferase and lysophosphatidate acyltransferase enzyme families, involved in the prokaryotic lipid biosynthesis pathway, that form a cluster apart from the cytoplasmic isoforms, involved in the eukaryotic pathway. In addition, two distinct membrane bound diacylglycerol acyltransferase enzymes were identified. Quantitative expression pattern analyses demonstrated variations in gene expressions during castor seed development. A tendency of maximum expression level at the middle of seed development was observed. Our results represent snapshots of global transcriptional activities of genes encompassing six enzyme families involved in castor bean TAG biosynthesis that are present during seed development. These genes represent potential targets for biotechnological approaches to produce nutritionally and industrially desirable oils.

  19. Isolation of DNA encoding sucrase genes from Streptococcus salivarius and partial characterization of the enzymes expressed in Escherichia coli.

    PubMed Central

    Houck, C M; Pear, J R; Elliott, R; Perchorowicz, J T

    1987-01-01

    Restriction enzyme fragments containing two sucrase genes have been isolated from a cosmid library of Streptococcus salivarius DNA. The genes were expressed in Escherichia coli cells, and the properties of both enzymes were studied in partially purified protein extracts from E. coli. One gene encoding an invertase-type sucrase was subcloned on a 2.4-kilobase-pair fragment. The sucrase enzyme had a Km for sucrose of 48 mM and a pH optimum of 6.5. The S. salivarius sucrase clone showed no detectable hybridization to a yeast invertase clone. Two overlapping subclones which had 1 kilobase pair of DNA in common were used to localize a fructosyltransferase gene. The fructosyltransferase had a Km of 93 mM and a pH optimum of 7.0. The product of the fructosyltransferase was a levan. A fructosyltransferase clone from Bacillus subtilis did not hybridize to S. salivarius DNA. The properties of the enzymes were compared with those of previously characterized sucrases. Images PMID:3112128

  20. Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites§

    PubMed Central

    Hunt, Paul; Afonso, Ana; Creasey, Alison; Culleton, Richard; Sidhu, Amar Bir Singh; Logan, John; Valderramos, Stephanie G; McNae, Iain; Cheesman, Sandra; do Rosario, Virgilio; Carter, Richard; Fidock, David A; Cravo, Pedro

    2007-01-01

    Artemisinin- and artesunate-resistant Plasmodium chabaudi mutants, AS-ART and AS-ATN, were previously selected from chloroquine-resistant clones AS-30CQ and AS-15CQ respectively. Now, a genetic cross between AS-ART and the artemisinin-sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS-30CQ and AS-ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS-ATN to artemisinin derivates, the other cannot account solely for the resistance of AS-ART, relative to the responses of its sensitive progenitor AS-30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug-response phenotype was not genetically stable. No mutations in the UBP-1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed. PMID:17581118

  1. Energy Landscapes Encoding Function in Enzymes Investigated Over Broad Time Scales

    NASA Astrophysics Data System (ADS)

    Callender, Robert

    2011-03-01

    The operating hypothesis of much of our current work is that atomic motion, over broad time scales (femtoseconds to milliseconds, the latter being the time scale of most enzyme catalyzed reactions), contributes to enzymic catalysis in proteins. It is clear from our work that specific types of motions are important in binding of ligands to proteins and transition state formation in enzymatic catalysis. Since new experimental and theoretical approaches are needed to understand the dynamical nature of proteins broadly and enzymatic catalysis specifically, we have employed time-resolved ``pump-probe'' spectroscopic techniques because of the sensitivity of these type of approaches to all relevant time scales. And we have also developed and applied new theoretical methods. The talk will focus on how lactate dehydrogenase brings about catalysis based on current experimental and theoretical studies. Work supported by NIH Grant P01GM068036.

  2. Effect of carbamates on mRNA encoding lipid enzymes in hamster flank organs.

    PubMed

    López-Lezama, Juan C; Cabeza, Marisa; Mayorga, Israel; Soriano, Juan; Sainz, Teresita; Bratoeff, Eugene

    2014-05-01

    Flank organs are an androgen-dependent pilosebaceous complex present in male and female hamsters. These organs have been used for the evaluation of antiandrogenic drugs, which could be used for the treatment of androgen-dependent afflictions. This study demonstrated the role of four different steroidal carbamates 7-10 in the expression of mRNAs coding for different enzymes involved in the lipid metabolism in flank organs. To determine the biological effects of compounds 7-10 on the expression of mRNA coding for lipid enzymes, steroids 7-10, testosterone (T), progesterone (P), and/or 7-10 were applied on the flank organs. Later, the mRNA expression for the enzymes was determined by polymerase chain reaction. The binding of 8 and 9 to the progesterone receptor (PR) as well as their effects on the activity of 5α-reductase were also evaluated. Treatments with T, P, and 7-10 increased the mRNA expression for glycerol 3-phosphate acyl transferase (GPAT), β-hydroxy-β-methylglutaryl-CoA synthase (HMG-CoA-S), β-hydroxy-β-methylglutaryl-CoA reductase (HMG-CoA-R), phosphatidylinositol synthase (PI-S), and squalene-synthase (SQ-S). However, the combined treatments with P + 7-10 decreased the expression of GPAT, HMG-CoA-S, and HMG-CoA-R. Expression of mRNA for all enzymes was variable under treatment with T + 7-10. Data showed that these carbamates did not bind to the PR, but inhibited the activity of 5α-reductase. Carbamates 7-10 changed the mRNA expression model induced by T and P in flank organs.

  3. PCR Primers to Study the Diversity of Expressed Fungal Genes Encoding Lignocellulolytic Enzymes in Soils Using High-Throughput Sequencing

    PubMed Central

    Barbi, Florian; Bragalini, Claudia; Vallon, Laurent; Prudent, Elsa; Dubost, Audrey; Fraissinet-Tachet, Laurence; Marmeisse, Roland; Luis, Patricia

    2014-01-01

    Plant biomass degradation in soil is one of the key steps of carbon cycling in terrestrial ecosystems. Fungal saprotrophic communities play an essential role in this process by producing hydrolytic enzymes active on the main components of plant organic matter. Open questions in this field regard the diversity of the species involved, the major biochemical pathways implicated and how these are affected by external factors such as litter quality or climate changes. This can be tackled by environmental genomic approaches involving the systematic sequencing of key enzyme-coding gene families using soil-extracted RNA as material. Such an approach necessitates the design and evaluation of gene family-specific PCR primers producing sequence fragments compatible with high-throughput sequencing approaches. In the present study, we developed and evaluated PCR primers for the specific amplification of fungal CAZy Glycoside Hydrolase gene families GH5 (subfamily 5) and GH11 encoding endo-β-1,4-glucanases and endo-β-1,4-xylanases respectively as well as Basidiomycota class II peroxidases, corresponding to the CAZy Auxiliary Activity family 2 (AA2), active on lignin. These primers were experimentally validated using DNA extracted from a wide range of Ascomycota and Basidiomycota species including 27 with sequenced genomes. Along with the published primers for Glycoside Hydrolase GH7 encoding enzymes active on cellulose, the newly design primers were shown to be compatible with the Illumina MiSeq sequencing technology. Sequences obtained from RNA extracted from beech or spruce forest soils showed a high diversity and were uniformly distributed in gene trees featuring the global diversity of these gene families. This high-throughput sequencing approach using several degenerate primers constitutes a robust method, which allows the simultaneous characterization of the diversity of different fungal transcripts involved in plant organic matter degradation and may lead to the

  4. PCR primers to study the diversity of expressed fungal genes encoding lignocellulolytic enzymes in soils using high-throughput sequencing.

    PubMed

    Barbi, Florian; Bragalini, Claudia; Vallon, Laurent; Prudent, Elsa; Dubost, Audrey; Fraissinet-Tachet, Laurence; Marmeisse, Roland; Luis, Patricia

    2014-01-01

    Plant biomass degradation in soil is one of the key steps of carbon cycling in terrestrial ecosystems. Fungal saprotrophic communities play an essential role in this process by producing hydrolytic enzymes active on the main components of plant organic matter. Open questions in this field regard the diversity of the species involved, the major biochemical pathways implicated and how these are affected by external factors such as litter quality or climate changes. This can be tackled by environmental genomic approaches involving the systematic sequencing of key enzyme-coding gene families using soil-extracted RNA as material. Such an approach necessitates the design and evaluation of gene family-specific PCR primers producing sequence fragments compatible with high-throughput sequencing approaches. In the present study, we developed and evaluated PCR primers for the specific amplification of fungal CAZy Glycoside Hydrolase gene families GH5 (subfamily 5) and GH11 encoding endo-β-1,4-glucanases and endo-β-1,4-xylanases respectively as well as Basidiomycota class II peroxidases, corresponding to the CAZy Auxiliary Activity family 2 (AA2), active on lignin. These primers were experimentally validated using DNA extracted from a wide range of Ascomycota and Basidiomycota species including 27 with sequenced genomes. Along with the published primers for Glycoside Hydrolase GH7 encoding enzymes active on cellulose, the newly design primers were shown to be compatible with the Illumina MiSeq sequencing technology. Sequences obtained from RNA extracted from beech or spruce forest soils showed a high diversity and were uniformly distributed in gene trees featuring the global diversity of these gene families. This high-throughput sequencing approach using several degenerate primers constitutes a robust method, which allows the simultaneous characterization of the diversity of different fungal transcripts involved in plant organic matter degradation and may lead to the

  5. Significance of Polymorphisms and Expression of Enzyme-Encoding Genes Related to Glutathione in Hematopoietic Cancers and Solid Tumors

    PubMed Central

    Zmorzyński, Szymon; Świderska-Kołacz, Grażyna; Koczkodaj, Dorota; Filip, Agata Anna

    2015-01-01

    Antioxidant compounds such as glutathione and its enzymes have become the focus of attention of medical sciences. Glutathione, a specific tripeptide, is involved in many intercellular processes. The glutathione concentration is determined by the number of GAG repeats in gamma-glutamylcysteine synthetase. GAG polymorphisms are associated with an increased risk of schizophrenia, berylliosis, diabetes, lung cancer, and nasopharyngeal tumors. Cancer cells with high glutathione concentration are resistant to chemotherapy treatment. The oxidized form of glutathione is formed by glutathione peroxidases (GPXs). The changes in activity of GPX1, GPX2, and GPX3 isoforms may be associated with the development of cancers, for example, prostate cancer or even colon cancer. Detoxification of glutathione conjugates is possible due to activity of glutathione S-transferases (GSTs). Polymorphisms in GSTM1, GSTP1, and GSTO1 enzymes increase the risk of developing breast cancer and hepatocellular carcinoma. Gamma-glutamyl transpeptidases (GGTs) are responsible for glutathione degradation. Increased activity of GGT correlates with adverse prognosis in patients with breast cancer. Studies on genes encoding glutathione enzymes are continued in order to determine the correlation between DNA polymorphisms in cancer patients. PMID:26682223

  6. Transcriptional regulation of genes encoding ABA metabolism enzymes during the fruit development and dehydration stress of pear 'Gold Nijisseiki'.

    PubMed

    Dai, Shengjie; Li, Ping; Chen, Pei; Li, Qian; Pei, Yuelin; He, Suihuan; Sun, Yufei; Wang, Ya; Kai, Wenbin; Zhao, Bo; Liao, Yalan; Leng, Ping

    2014-09-01

    To investigate the contribution of abscisic acid (ABA) in pear 'Gold Nijisseiki' during fruit ripening and under dehydration stress, two cDNAs (PpNCED1 and PpNCED2) which encode 9-cis-epoxycarotenoid dioxygenase (NCED) (a key enzyme in ABA biosynthesis), two cDNAs (PpCYP707A1 and PpCYP707A2) which encode 8'-hydroxylase (a key enzyme in the oxidative catabolism of ABA), one cDNA (PpACS3) which encodes 1-aminocyclopropane-1-carboxylic acid (ACC), and one cDNA (PpACO1) which encodes ACC oxidase involved in ethylene biosynthesis were cloned from 'Gold Nijisseiki' fruit. In the pulp, peel and seed, expressions of PpNCED1 and PpNCED2 rose in two stages which corresponded with the increase of ABA levels. The expression of PpCYP707A1 dramatically declined after 60-90 days after full bloom (DAFB) in contrast to the changes of ABA levels during this period, while PpCYP707A2 stayed low during the whole development of fruit. Application of exogenous ABA at 100 DAFB increased the soluble sugar content and the ethylene release but significantly decreased the titratable acid and chlorophyll contents in fruits. When fruits harvested at 100 DAFB were stored in the laboratory (25 °C, 50% relative humidity), the ABA content and the expressions of PpNCED1/2 and PpCYP707A1 in the pulp, peel and seed increased significantly, while ethylene reached its highest value after the maximum peak of ABA accompanied with the expressions of PpACS3 and PpACO1. In sum the endogenous ABA may play an important role in the fruit ripening and dehydration of pear 'Gold Nijisseiki' and the ABA level was regulated mainly by the dynamics of PpNCED1, PpNCED2 and PpCYP707A1 at the transcriptional level.

  7. Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.

    PubMed

    Miosic, Silvija; Thill, Jana; Milosevic, Malvina; Gosch, Christian; Pober, Sabrina; Molitor, Christian; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

    2014-01-01

    During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa.

  8. The two endo-β-N-acetylglucosaminidase genes from Arabidopsis thaliana encode cytoplasmic enzymes controlling free N-glycan levels.

    PubMed

    Fischl, Richard M; Stadlmann, Johannes; Grass, Josephine; Altmann, Friedrich; Léonard, Renaud

    2011-10-01

    Endo-β-N-acetylglucosaminidases (ENGases) cleave N-glycans from proteins and/or peptides by hydrolyzing the O-glycosidic linkage between the two core-N-acetylglucosamine (GlcNAc) residues. Although, two homologous genes potentially encoding ENGases have been identified in Arabidopsis thaliana, their respective substrate specificity, their subcellular and their organ specific localization was hitherto unknown. In order to investigate the role of ENGases in this model plant species, we transiently expressed the two A. thaliana genes in Nicotiana benthamiana and determined the substrate specificities, as well as the Km values, of the purified recombinant enzymes. The assumed predominantly cytosolic localisation of both enzymes, here referred to as AtENGase85A and AtENGase85B, was determined by confocal microscopy of plant leaves expressing the respective GFP-fusion constructs. For the individual characterization of the two enzymes expression patterns in planta, single knock-out plants were selected for both genes. Although both enzymes are present in most organs, only AtENGase85A (At5g05460) was expressed in stems and no ENGase activity was detected in siliques. A double knock-out was generated by crossing but-like single knock-out plants-no apparent phenotype was observed. In contrast, in this double knock-out, free N-glycans carrying a single GlcNAc at the reducing end are completely absent and their counterparts with two GlcNAc-visible only at a trace level in wild type-accumulated dramatically.

  9. Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.

    PubMed

    Miosic, Silvija; Thill, Jana; Milosevic, Malvina; Gosch, Christian; Pober, Sabrina; Molitor, Christian; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

    2014-01-01

    During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa. PMID:25393679

  10. Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters.

    PubMed

    de Lima-Morales, Daiana; Chaves-Moreno, Diego; Wos-Oxley, Melissa L; Jáuregui, Ruy; Vilchez-Vargas, Ramiro; Pieper, Dietmar H

    2015-10-16

    Pseudomonas veronii 1YdBTEX2, a benzene and toluene degrader, and Pseudomonas veronii 1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcription PCR.

  11. Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters

    PubMed Central

    de Lima-Morales, Daiana; Chaves-Moreno, Diego; Wos-Oxley, Melissa L.; Jáuregui, Ruy; Vilchez-Vargas, Ramiro

    2015-01-01

    Pseudomonas veronii 1YdBTEX2, a benzene and toluene degrader, and Pseudomonas veronii 1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcription PCR. PMID:26475106

  12. Lipopolysaccharide phosphorylating enzymes encoded in the genomes of Gram-negative bacteria are related to the eukaryotic protein kinases

    PubMed Central

    Krupa, A.; Srinivasan, N.

    2002-01-01

    By means of profile-matching procedures, conservation of functionally important residues, and fold-recognition techniques, we show that two distinct families of lipopolysaccharide kinases encoded in the genomes of Gram-negative bacteria are related to each other and to two distinct classes of proteins, namely eukaryotic protein kinases and right open reading frame (RIO1). Members of one of the lipopolysaccharide kinase families are identified only in pathogenic bacteria. Phosphorylation by these enzymes is relevant in the construction of outer membrane, immune response, and pathogenic virulence. The class of proteins called RIO1, also related to eukaryotic protein kinases and previously known to occur only in archaea and eukaryotes, are now identified in eubacteria as well. It has been suggested here that RIO1 proteins are intermediately related to lipopolysaccharide kinases and eukaryotic protein kinases implying an evolutionary relationship between the three classes of proteins. PMID:12021457

  13. Clusters of genes encoding fructan biosynthesizing enzymes in wheat and barley.

    PubMed

    Huynh, Bao-Lam; Mather, Diane E; Schreiber, Andreas W; Toubia, John; Baumann, Ute; Shoaei, Zahra; Stein, Nils; Ariyadasa, Ruvini; Stangoulis, James C R; Edwards, James; Shirley, Neil; Langridge, Peter; Fleury, Delphine

    2012-10-01

    Fructans are soluble carbohydrates with health benefits and possible roles in plant adaptation. Fructan biosynthetic genes were isolated using comparative genomics and physical mapping followed by BAC sequencing in barley. Genes encoding sucrose:sucrose 1-fructosyltransferase (1-SST), fructan:fructan 1-fructosyltransferase (1-FFT) and sucrose:fructan 6-fructosyltransferase (6-SFT) were clustered together with multiple copies of vacuolar invertase genes and a transposable element on two barley BAC. Intron-exon structures of the genes were similar. Phylogenetic analysis of the fructosyltransferases and invertases in the Poaceae showed that the fructan biosynthetic genes may have evolved from vacuolar invertases. Quantitative real-time PCR was performed using leaf RNA extracted from three wheat cultivars grown under different conditions. The 1-SST, 1-FFT and 6-SFT genes had correlated expression patterns in our wheat experiment and in existing barley transcriptome database. Single nucleotide polymorphism (SNP) markers were developed and successfully mapped to a major QTL region affecting wheat grain fructan accumulation in two independent wheat populations. The alleles controlling high- and low- fructan in parental lines were also found to be associated in fructan production in a diverse set of 128 wheat lines. To the authors' knowledge, this is the first report on the mapping and sequencing of a fructan biosynthetic gene cluster and in particular, the isolation of a novel 1-FFT gene from barley.

  14. Clusters of genes encoding fructan biosynthesizing enzymes in wheat and barley.

    PubMed

    Huynh, Bao-Lam; Mather, Diane E; Schreiber, Andreas W; Toubia, John; Baumann, Ute; Shoaei, Zahra; Stein, Nils; Ariyadasa, Ruvini; Stangoulis, James C R; Edwards, James; Shirley, Neil; Langridge, Peter; Fleury, Delphine

    2012-10-01

    Fructans are soluble carbohydrates with health benefits and possible roles in plant adaptation. Fructan biosynthetic genes were isolated using comparative genomics and physical mapping followed by BAC sequencing in barley. Genes encoding sucrose:sucrose 1-fructosyltransferase (1-SST), fructan:fructan 1-fructosyltransferase (1-FFT) and sucrose:fructan 6-fructosyltransferase (6-SFT) were clustered together with multiple copies of vacuolar invertase genes and a transposable element on two barley BAC. Intron-exon structures of the genes were similar. Phylogenetic analysis of the fructosyltransferases and invertases in the Poaceae showed that the fructan biosynthetic genes may have evolved from vacuolar invertases. Quantitative real-time PCR was performed using leaf RNA extracted from three wheat cultivars grown under different conditions. The 1-SST, 1-FFT and 6-SFT genes had correlated expression patterns in our wheat experiment and in existing barley transcriptome database. Single nucleotide polymorphism (SNP) markers were developed and successfully mapped to a major QTL region affecting wheat grain fructan accumulation in two independent wheat populations. The alleles controlling high- and low- fructan in parental lines were also found to be associated in fructan production in a diverse set of 128 wheat lines. To the authors' knowledge, this is the first report on the mapping and sequencing of a fructan biosynthetic gene cluster and in particular, the isolation of a novel 1-FFT gene from barley. PMID:22864927

  15. Functional Analysis of the Phycomyces carRA Gene Encoding the Enzymes Phytoene Synthase and Lycopene Cyclase

    PubMed Central

    Sanz, Catalina; Velayos, Antonio; Álvarez, María Isabel; Benito, Ernesto P.; Eslava, Arturo P.

    2011-01-01

    Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism. PMID:21858003

  16. Heat maps for intramolecular communication in an RNP enzyme encoding glutamine.

    PubMed

    Rodríguez-Hernández, Annia; Perona, John J

    2011-03-01

    Allosteric signaling within large ribonucleoproteins modulates both catalytic function and biological specificity, but the spatial extent and quantitative magnitudes of long-distance free-energy couplings have yet to be well characterized. Here, we employ pre-steady-state kinetics to generate a comprehensive mapping of intramolecular communication in the glutaminyl-tRNA synthetase:tRNA(Gln) complex. Alanine substitution at 29 positions across the protein-RNA interface reveals distinct coupling amplitudes for glutamine binding and aminoacyl-tRNA formation on the enzyme, respectively, implying the existence of multiple signaling pathways. Structural models suggest that long-range signal propagation from the tRNA anticodon is dynamically driven, whereas shorter pathways are mediated by induced-fit rearrangements. Seven protein contacts with the distal tRNA vertical arm each weaken glutamine binding affinity across distances up to 40 Å, demonstrating that negative allosteric coupling plays a key role in enforcing the selective RNA-amino acid pairing at the heart of the genetic code. PMID:21397189

  17. Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

    PubMed Central

    Vágner, J; Barany, G; Lam, K S; Krchnák, V; Sepetov, N F; Ostrem, J A; Strop, P; Lebl, M

    1996-01-01

    Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads. PMID:8710846

  18. SARS-CoV ORF1b-encoded nonstructural proteins 12-16: replicative enzymes as antiviral targets.

    PubMed

    Subissi, Lorenzo; Imbert, Isabelle; Ferron, François; Collet, Axelle; Coutard, Bruno; Decroly, Etienne; Canard, Bruno

    2014-01-01

    The SARS (severe acute respiratory syndrome) pandemic caused ten years ago by the SARS-coronavirus (SARS-CoV) has stimulated a number of studies on the molecular biology of coronaviruses. This research has provided significant new insight into many mechanisms used by the coronavirus replication-transcription complex (RTC). The RTC directs and coordinates processes in order to replicate and transcribe the coronavirus genome, a single-stranded, positive-sense RNA of outstanding length (∼27-32kilobases). Here, we review the up-to-date knowledge on SARS-CoV replicative enzymes encoded in the ORF1b, i.e., the main RNA-dependent RNA polymerase (nsp12), the helicase/triphosphatase (nsp13), two unusual ribonucleases (nsp14, nsp15) and RNA-cap methyltransferases (nsp14, nsp16). We also review how these enzymes co-operate with other viral co-factors (nsp7, nsp8, and nsp10) to regulate their activity. These last ten years of research on SARS-CoV have considerably contributed to unravel structural and functional details of one of the most fascinating replication/transcription machineries of the RNA virus world. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses". PMID:24269475

  19. Cytokinin affects nuclear- and plastome-encoded energy-converting plastid enzymes.

    PubMed

    Kasten, B; Buck, F; Nuske, J; Reski, R

    1997-01-01

    Cytokinins induce two specific morphological alterations in mosses: (i) the differentiation of a tip-growing cell into a three-faced apical cell (the so-called bud), and (ii) the division of chloroplasts. In a developmental mutant of the moss Physcomitrella patens (Hedw.) B.S.G. (mutant PC22) impeded in both cellular differentiation (bud production) and chloroplast division, addition of cytokinin (N6-delta 2-isopentenyladenine) led to bud production after 3 d in the wild type and after 7 d in the mutant. Hormone induced a division of the mutant macrochloroplasts starting within 24 h and ongoing for 72 h. During this period the abundances of several plastid proteins changed in both genotypes as judged by two-dimensional-protein gel electrophoresis, silver staining and subsequent quantification with novel computer software. Eight of these polypeptides were isolated independently, subjected to microsequencing and thus identified, resulting in the first protein sequence data from a moss. Three polypeptides (24 kDa, 22 kDa, 20 kDa) were found to be homologous to enhancer protein OEE2 of the oxygen-evolving complex, four to represent isoforms of phosphoglycerate kinase (EC 2.7.2.3), and one was identified as the beta-chain of chloroplast ATPase (EC 3.6.1.34). Possible involvement of these key enzymes of the chloroplast energy-conversion machinery in organelle division and in cellular differentiation is discussed. Further sequence information was obtained from both subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Amounts of these polypeptides were not appreciably affected by cytokinin in moss chloroplasts.

  20. Detecting the frequency of aminoglycoside modifying enzyme encoding genes among clinical isolates of methicillin-resistant Staphylococcus aureus

    PubMed Central

    Shokravi, Zahra; Mehrad, Laleh; Ramazani, Ali

    2015-01-01

    Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) plays an important role in causing many serious nosocomial infections. In this study, the antimicrobial susceptibility and the frequency of aminoglycoside modifying enzyme encoding genes among clinical isolates of methicillin-resistant Staphylococcus aureus was investigated from two university hospitals of Zanjan province of Iran. Methods: In this study, the antimicrobial susceptibility of MRSA isolates to various antibiotics was investigated by the disk diffusion method. Multiplex PCR assays were used for the determination of aminoglycoside modifying enzyme (AME) genes and staphylococcal cassette chromosome mec (SCCmec) types in MRSA strains. Results: All 58 MRSA isolates were sensitive to vancomycin. Resistance to penicillin G, oxacilin, gentamicin, erythromycin, clindamycin, kanamycin, and tobramycin was found in 96.4%, 98.3%, 51.7%, 53.4%, 55.2%, 62% and 58.6% of the isolates, respectively. The most prevalent AME genes were aac(6′)/aph(2′′) (48.3 %) followed by ant(4)-Ia (24%). The aph(3′)-Ia gene was the least frequent AME gene among MRSA isolates (19%). Of the 58 tested MRSA isolates, 5 (8.6%) were harboured SCCmec type I, 11 (19%) SCCmec type II, 20 (34.5%) SCCmec type III, 17 (29.3%) SCCmec type IVa, 1 (1.7%) SCCmec type IVb, 2 (3.4%) SCCmec type IVc, 11 (19%) SCCmec type IVd, and, 18 (31%) SCCmec type V. Nineteen isolates were not typeable. Conclusion: In conclusion, the aac (6′)/aph (2′′) was the most common aminoglycoside modifying enzyme gene and SCCmec type II and V were the most frequent types detected in hospital isolates, respectively. PMID:26191502

  1. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  2. Cytochrome P-450 monooxygenase systems in aquatic species: Carcinogen metabolism and biomarkers for carcinogen and pollutant exposure

    SciTech Connect

    Stegeman, J.J. ); Lech, J.J. )

    1991-01-01

    High levels of polynuclear aromatic hydrocarbon (PAH) carcinogens commonly occur in aquatic systems where neoplasms arise in fish and other animals. Enzymes that transform PAHs can act in initiating these diseases and can indicate the contamination of fish by carcinogens and other pollutants. Cytochrome P-450 has similar roles in activating PAH carcinogens in fish and mammalian species. PAHs and many chlorinated hydrocarbons, e.g., polychlorinated biphenyls (PCBs) induce a form of cytochrome P-450 in fish that is the primary catalyst of PAH metabolism. The induction of this P-450 in fish can accelerate the disposition of hydrocarbons but can also enhance the formation of carcinogenic derivatives of PAHs. Invertebrates have lower rates of PAH metabolism than fish. The induction of P-450 forms can indicate the exposure of fish to PAHs, PCBs, and other toxic compounds. This is not restricted to carcinogens. Environmental induction has been detected in fish from contaminated areas by use of catalytic assay, antibodies to fish P-450, and cDNA probes that hybridize with P-450 messenger RNA. Application of these methods can provide sensitive biological monitoring tools that can detect environmental contamination of fish by some carcinogens and tumor promoters. The potential for using P-450 induction to detect direct-acting carcinogens and tumor promoters that are noninducers is limited, although such compounds can be expected to co-occur with pollutants that are inducers.

  3. Molecular identification and interaction assay of the gene (OsUbc13) encoding a ubiquitin-conjugating enzyme in rice*

    PubMed Central

    Wang, Ya; Xu, Meng-yun; Liu, Jian-ping; Wang, Mu-gui; Yin, Hai-qing; Tu, Ju-min

    2014-01-01

    The ubiquitin (Ub)-conjugating enzyme, Ubc13, has been known to be involved in error-free DNA damage tolerance (or post-replication repair) via catalyzing Lys63-linked polyubiquitin chains formation together with a Ubc variant. However, its functions remain largely unknown in plant species, especially in monocotyledons. In this study, we cloned a Ub-conjugating enzyme, OsUbc13, that shares the conserved domain of Ubc with AtUBC13B in Oryza sativa L., which encodes a protein of 153 amino acids; the deduced sequence shares high similarities with other homologs. Real-time quantitative polymerase chain reaction (PCR) indicated that OsUbc13 transcripts could be detected in all tissues examined, and the expression level was higher in palea, pistil, stamen, and leaf, and lower in root, stem, and lemma; the expression of OsUbc13 was induced by low temperature, methylmethane sulfate (MMS), and H2O2, but repressed by mannitol, abscisic acid (ABA), and NaCl. OsUbc13 was probably localized in the plasma and nuclear membranes. About 20 proteins, which are responsible for the positive yeast two-hybrid interaction of OsUbc13, were identified. These include the confirmed OsVDAC (correlated with apoptosis), OsMADS1 (important for development of floral organs), OsB22EL8 (related to reactive oxygen species (ROS) scavenging and DNA protection), and OsCROC-1 (required for formation of Lys63 polyubiquitylation and error-free DNA damage tolerance). The molecular characterization provides a foundation for the functional study of OsUbc13. PMID:25001222

  4. Molecular identification and interaction assay of the gene (OsUbc13) encoding a ubiquitin-conjugating enzyme in rice.

    PubMed

    Wang, Ya; Xu, Meng-yun; Liu, Jian-ping; Wang, Mu-gui; Yin, Hai-qing; Tu, Ju-min

    2014-07-01

    The ubiquitin (Ub)-conjugating enzyme, Ubc13, has been known to be involved in error-free DNA damage tolerance (or post-replication repair) via catalyzing Lys63-linked polyubiquitin chains formation together with a Ubc variant. However, its functions remain largely unknown in plant species, especially in monocotyledons. In this study, we cloned a Ub-conjugating enzyme, OsUbc13, that shares the conserved domain of Ubc with AtUBC13B in Oryza sativa L., which encodes a protein of 153 amino acids; the deduced sequence shares high similarities with other homologs. Real-time quantitative polymerase chain reaction (PCR) indicated that OsUbc13 transcripts could be detected in all tissues examined, and the expression level was higher in palea, pistil, stamen, and leaf, and lower in root, stem, and lemma; the expression of OsUbc13 was induced by low temperature, methylmethane sulfate (MMS), and H(2)O(2), but repressed by mannitol, abscisic acid (ABA), and NaCl. OsUbc13 was probably localized in the plasma and nuclear membranes. About 20 proteins, which are responsible for the positive yeast two-hybrid interaction of OsUbc13, were identified. These include the confirmed OsVDAC (correlated with apoptosis), OsMADS1 (important for development of floral organs), OsB22EL8 (related to reactive oxygen species (ROS) scavenging and DNA protection), and OsCROC-1 (required for formation of Lys63 polyubiquitylation and error-free DNA damage tolerance). The molecular characterization provides a foundation for the functional study of OsUbc13. PMID:25001222

  5. Cytochrome P-450 monooxygenase systems in aquatic species: carcinogen metabolism and biomarkers for carcinogen and pollutant exposure.

    PubMed Central

    Stegeman, J J; Lech, J J

    1991-01-01

    High levels of polynuclear aromatic hydrocarbon (PAH) carcinogens commonly occur in aquatic systems where neoplasms arise in fish and other animals. Enzymes that transform PAHs can act in initiating these diseases and can indicate the contamination of fish by carcinogens and other pollutants. Cytochrome P-450 has similar roles in activating PAH carcinogens in fish and mammalian species. PAHs and many chlorinated hydrocarbons, e.g., polychlorinated biphenyls (PCBs) induce a form of cytochrome P-450 in fish that is the primary catalyst of PAH metabolism. The induction of this P-450 in fish can accelerate the disposition of hydrocarbons, but can also enhance the formation of carcinogenic derivatives of PAHs. Invertebrates have lower rates of PAH metabolism than fish. These rates are not obviously inducible by exposure to PAHs or PCBs. The lower rates of foreign compound metabolism contribute to higher pollutant residue levels in bivalve mollusks (clams, mussels, etc.) than in fish and may limit the involvement of some procarcinogens (requiring activation) in disease processes in invertebrates. The induction of P-450 forms can indicate the exposure of fish to PAHs, PCBs, and other toxic compounds. This is not restricted to carcinogens. Environmental induction has been detected in fish from contaminated areas by use of catalytic assay, antibodies to fish P-450, and cDNA probes that hybridize with P-450 messenger RNA. Application of these methods can provide sensitive biological monitoring tools that can detect environmental contamination of fish by some carcinogens and tumor promoters.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2050047

  6. NUCLEOTIDE SEQUENCING AND TRANSCRIPTIONAL MAPPING OF THE GENES ENCODING BIPHENYL DIOXYGENASE, A MULTICOM- PONENT POLYCHLORINATED-BIPHENYL-DEGRADING ENZYME IN PSEUDOMONAS STRAIN LB400

    EPA Science Inventory

    The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. Six open reading frames were identified, four of which are homologous to the components of toluene dioxy...

  7. Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides.

    PubMed

    Oehlenschlæger, Christian Berg; Løvgreen, Monika Nøhr; Reinauer, Eva; Lehtinen, Emilia; Pind, Marie-Louise Lindberg; Harris, Pernille; Martinussen, Jan; Willemoës, Martin

    2015-05-15

    Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database.

  8. Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides

    PubMed Central

    Oehlenschlæger, Christian Berg; Løvgreen, Monika Nøhr; Reinauer, Eva; Lehtinen, Emilia; Pind, Marie-Louise Lindberg; Martinussen, Jan

    2015-01-01

    Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database. PMID:25746996

  9. [Effect of colchicine and Triton X-100 on expression of the enzyme-encoding genes in nongerminating seeds of sugarbeet (Beta vulgaris L.)].

    PubMed

    Kirikovich, S S; Levites, E V

    2011-01-01

    The expression of the enzyme-coding genes, controlling glucose-phosphate isomerase (GPI), malate dehydrogenase (MDH), and alcohol dehydrogenase (ADH), was examined in nongerminating seeds of sugarbeet after Triton X-100 (TX-100) and colchicine treatment. Two types of changes revealed included modification of the enzymatic loci expression (change of the isozyme electrophoretic mobility) and inactivation of standard profiles. In the MDH and GPI systems, these processes were found to be associated. Complete isozyme modification was accompanied with the disappearance of standard profiles. In the ADH system, the treatment with TX-100 and colchicine gave rise to two independent processes, including silencing of the Adh1 locus and the appearance of the ADH isozymes with abnormal electrophoretic mobility, which were probably the products of the Adh2 locus. It was suggested that the effect of TX-100 and colchicine on the expression of the enzyme-encoding genes examined depended on the intracellular localization of the encoded enzymes.

  10. Analysis of the metatranscriptome of microbial communities of an alkaline hot sulfur spring revealed different gene encoding pathway enzymes associated with energy metabolism.

    PubMed

    Tripathy, Swetaleena; Padhi, Soumesh Kumar; Mohanty, Sriprakash; Samanta, Mrinal; Maiti, Nikhil Kumar

    2016-07-01

    Alkaline sulfur hot springs notable for their specialized and complex ecosystem powered by geothermal energy are abundantly rich in different chemotrophic and phototrophic thermophilic microorganisms. Survival and adaptation of these organisms in the extreme environment is specifically related to energy metabolism. To gain a better understanding of survival mechanism of the organisms in these ecosystems, we determined the different gene encoding enzymes associated with anaerobic pathways of energy metabolism by applying the metatranscriptomics approach. The analysis of the microbial population of hot sulfur spring revealed the presence of both aerobic and anaerobic organisms indicating dual mode of lifestyle of the community members. Proteobacteria (28.1 %) was the most dominant community. A total of 988 reads were associated with energy metabolism, out of which 33.7 % of the reads were assigned to nitrogen, sulfur, and methane metabolism based on KEGG classification. The major lineages of hot spring communities were linked with the anaerobic pathways. Different gene encoding enzymes (hao, nir, nar, cysH, cysI, acs) showed the involvement of microbial members in nitrification, denitrification, dissimilatory sulfate reduction, and methane generation. This study enhances our understanding of important gene encoding enzymes involved in energy metabolism, required for the survival and adaptation of microbial communities in the hot spring.

  11. Analysis of the metatranscriptome of microbial communities of an alkaline hot sulfur spring revealed different gene encoding pathway enzymes associated with energy metabolism.

    PubMed

    Tripathy, Swetaleena; Padhi, Soumesh Kumar; Mohanty, Sriprakash; Samanta, Mrinal; Maiti, Nikhil Kumar

    2016-07-01

    Alkaline sulfur hot springs notable for their specialized and complex ecosystem powered by geothermal energy are abundantly rich in different chemotrophic and phototrophic thermophilic microorganisms. Survival and adaptation of these organisms in the extreme environment is specifically related to energy metabolism. To gain a better understanding of survival mechanism of the organisms in these ecosystems, we determined the different gene encoding enzymes associated with anaerobic pathways of energy metabolism by applying the metatranscriptomics approach. The analysis of the microbial population of hot sulfur spring revealed the presence of both aerobic and anaerobic organisms indicating dual mode of lifestyle of the community members. Proteobacteria (28.1 %) was the most dominant community. A total of 988 reads were associated with energy metabolism, out of which 33.7 % of the reads were assigned to nitrogen, sulfur, and methane metabolism based on KEGG classification. The major lineages of hot spring communities were linked with the anaerobic pathways. Different gene encoding enzymes (hao, nir, nar, cysH, cysI, acs) showed the involvement of microbial members in nitrification, denitrification, dissimilatory sulfate reduction, and methane generation. This study enhances our understanding of important gene encoding enzymes involved in energy metabolism, required for the survival and adaptation of microbial communities in the hot spring. PMID:27290724

  12. Cloning and expression of APE, the cDNA encoding the major human apurinic endonuclease: definition of a family of DNA repair enzymes.

    PubMed Central

    Demple, B; Herman, T; Chen, D S

    1991-01-01

    Abasic (AP) sites are common, potentially mutagenic DNA damages that are attacked by AP endonucleases. The biological roles of these enzymes in metazoans have not been tested. We have cloned the human cDNA (APE) that encodes the main nuclear AP endonuclease. The predicted Ape protein, which contains likely nuclear transport signals, is a member of a family of DNA repair enzymes that includes two bacterial AP endonucleases (ExoA protein of Streptococcus pneumoniae and exonuclease III of Escherichia coli) and Rrp1 protein of Drosophila melanogaster. Purified Ape protein lacks the 3'-exonuclease activity against undamaged DNA that is found in the bacterial and Drosophila enzymes, but the lack of obvious amino acid changes to account for this difference suggests that the various enzyme functions evolved by fine tuning a conserved active site. Expression of the active human enzyme in AP endonuclease-deficient E. coli conferred significant resistance to killing by the DNA-alkylating agent methyl methanesulfonate. The APE cDNA provides a molecular tool for analyzing the role of this central enzyme in maintaining genetic stability in humans. Images PMID:1722334

  13. AtROS1 overexpression provides evidence for epigenetic regulation of genes encoding enzymes of flavonoid biosynthesis and antioxidant pathways during salt stress in transgenic tobacco

    PubMed Central

    Bharti, Poonam; Mahajan, Monika; Vishwakarma, Ajay K.; Bhardwaj, Jyoti; Yadav, Sudesh Kumar

    2015-01-01

    In plants, epigenetic changes have been identified as regulators of developmental events during normal growth as well as environmental stress exposures. Flavonoid biosynthetic and antioxidant pathways play a significant role in plant defence during their exposure to environmental cues. The aim of this study was to unravel whether genes encoding enzymes of flavonoid biosynthetic and antioxidant pathways are under epigenetic regulation, particularly DNA methylation, during salt stress. For this, a repressor of silencing from Arabidopsis, AtROS1, was overexpressed in transgenic tobacco. Generated transgenics were evaluated to examine the influence of AtROS1 on methylation status of promoters as well as on coding regions of genes encoding enzymes of flavonoids biosynthesis and antioxidant pathways. Overexpression of AtROS1 increases the demethylation levels of both promoters as well as coding regions of genes encoding chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, and anthocyanidin synthase of the flavonoid biosynthetic pathway, and glutathione S-transferase, ascorbate peroxidase, glutathione peroxidase, and glutathione reductase of the antioxidant pathway during control conditions. The level of demethylation was further increased at promoters as well as coding regions of these genes during salt-stress conditions. Transgenic tobacco overexpressing AtROS1 showed tolerance to salt stress that could have been due to the higher expression levels of the genes encoding enzymes of the flavonoid biosynthetic and antioxidant pathways. This is the first comprehensive study documenting the epigenetic regulation of flavonoid biosynthetic and antioxidant pathways during salt-stress exposure of plants. PMID:26116024

  14. Two Sets of Paralogous Genes Encode the Enzymes Involved in the Early Stages of Clavulanic Acid and Clavam Metabolite Biosynthesis in Streptomyces clavuligerus

    PubMed Central

    Tahlan, Kapil; Park, Hyeon Ung; Wong, Annie; Beatty, Perrin H.; Jensen, Susan E.

    2004-01-01

    Recently, a second copy of a gene encoding proclavaminate amidinohydrolase (pah1), an enzyme involved in the early stages of clavulanic acid and clavam metabolite biosynthesis in Streptomyces clavuligerus, was identified and isolated. Using Southern analysis, we have now isolated second copies of the genes encoding the carboxyethylarginine synthase (ceaS) and β-lactam synthetase (bls) enzymes. These new paralogues are given the gene designations ceaS1 and bls1 and are located immediately upstream of pah1 on the chromosome. Furthermore, sequence analysis of the region downstream of pah1 revealed a second copy of a gene encoding ornithine acetyltransferase (oat1), thus indicating the presence of a cluster of paralogue genes. ceaS1, bls1, and oat1 display 73, 60, and 63% identities, respectively, at the nucleotide level to the original ceaS2, bls2, and oat2 genes from the clavulanic acid gene cluster. Single mutants defective in ceaS1, bls1, or oat1 were prepared and characterized and were found to be affected to variable degrees in their ability to produce clavulanic acid and clavam metabolites. Double mutants defective in both copies of the genes were also prepared and tested. The ceaS1/ceaS2 and the bls1/bls2 mutant strains were completely blocked in clavulanic acid and clavam metabolite biosynthesis. On the other hand, oat1/oat2 double mutants still produced some clavulanic acid and clavam metabolites. This may be attributed to the presence of the argJ gene in S. clavuligerus, which encodes yet another ornithine acetyltransferase enzyme that may be able to compensate for the lack of OAT1 and -2 in the double mutants. PMID:14982786

  15. An intact SAM-dependent methyltransferase fold is encoded by the human endothelin-converting enzyme-2 gene

    SciTech Connect

    Tempel, W.; Wu, H.; Dombrovsky, L.; Zeng, H.; Loppnau, P.; Zhu, H.; Plotnikov, A.N.; Bochkarev, A.

    2010-08-17

    A recent survey of protein expression patterns in patients with Alzheimer's disease (AD) has identified ece2 (chromosome: 3; Locations: 3q27.1) as the most significantly downregulated gene within the tested group. ece2 encodes endothelin-converting enzyme ECE2, a metalloprotease with a role in neuropeptide processing. Deficiency in the highly homologous ECE1 has earlier been linked to increased levels of AD-related {beta}-amyloid peptide in mice, consistent with a role for ECE in the degradation of that peptide. Initially, ECE2 was presumed to resemble ECE1, in that it comprises a single transmembrane region of {approx}20 residues flanked by a small amino-terminal cytosolic segment and a carboxy-terminal lumenar peptidase domain. The carboxy-terminal domain has significant sequence similarity to both neutral endopeptidase, for which an X-ray structure has been determined, and Kell blood group protein. After their initial discovery, multiple isoforms of ECE1 and ECE2 were discovered, generated by alternative splicing of multiple exons. The originally described ece2 transcript, RefSeq NM{_}174046, contains the amino-terminal cytosolic portion followed by the transmembrane region and peptidase domain (Fig. 1, isoform B). Another ece2 transcript, available from the Mammalian Gene Collection under MGC2408 (Fig. 1, isoform C), RefSeq accession NM{_}032331, is predicted to be translated into a 255 residue peptide with low but detectable sequence similarity to known S-adenosyl-L-methionine (SAM)-dependent methyltransferases (SAM-MTs), such as the hypothetical protein TT1324 from Thermus thermophilis, PDB code 2GS9, which shares 30% amino acid sequence identity with ECE2 over 138 residues of the sequence. Intriguingly, another 'elongated' ece2 transcript (Fig. 1, isoform A) (RefSeq NM{_}014693) contains an amino-terminal portion of the putative SAM-MT domain, the transmembrane domain, and the protease domain. This suggests the possibility for coexistence of the putative SAM

  16. RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica.

    PubMed

    Blackman, Leila M; Cullerne, Darren P; Torreña, Pernelyn; Taylor, Jen; Hardham, Adrienne R

    2015-01-01

    RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1

  17. RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica

    PubMed Central

    Blackman, Leila M.; Cullerne, Darren P.; Torreña, Pernelyn; Taylor, Jen; Hardham, Adrienne R.

    2015-01-01

    RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1

  18. Studying the organization of genes encoding plant cell wall degrading enzymes in Chrysomela tremula provides insights into a leaf beetle genome.

    PubMed

    Pauchet, Y; Saski, C A; Feltus, F A; Luyten, I; Quesneville, H; Heckel, D G

    2014-06-01

    The ability of herbivorous beetles from the superfamilies Chrysomeloidea and Curculionoidea to degrade plant cell wall polysaccharides has only recently begun to be appreciated. The presence of plant cell wall degrading enzymes (PCWDEs) in the beetle's digestive tract makes this degradation possible. Sequences encoding these beetle-derived PCWDEs were originally identified from transcriptomes and strikingly resemble those of saprophytic and phytopathogenic microorganisms, raising questions about their origin; e.g. are they insect- or microorganism-derived? To demonstrate unambiguously that the genes encoding PCWDEs found in beetle transcriptomes are indeed of insect origin, we generated a bacterial artificial chromosome library from the genome of the leaf beetle Chrysomela tremula, containing 18 432 clones with an average size of 143 kb. After hybridizing this library with probes derived from 12 C. tremula PCWDE-encoding genes and sequencing the positive clones, we demonstrated that the latter genes are encoded by the insect's genome and are surrounded by genes possessing orthologues in the genome of Tribolium castaneum as well as in three other beetle genomes. Our analyses showed that although the level of overall synteny between C. tremula and T. castaneum seems high, the degree of microsynteny between both species is relatively low, in contrast to the more closely related Colorado potato beetle. PMID:24456018

  19. Studying the organization of genes encoding plant cell wall degrading enzymes in Chrysomela tremula provides insights into a leaf beetle genome.

    PubMed

    Pauchet, Y; Saski, C A; Feltus, F A; Luyten, I; Quesneville, H; Heckel, D G

    2014-06-01

    The ability of herbivorous beetles from the superfamilies Chrysomeloidea and Curculionoidea to degrade plant cell wall polysaccharides has only recently begun to be appreciated. The presence of plant cell wall degrading enzymes (PCWDEs) in the beetle's digestive tract makes this degradation possible. Sequences encoding these beetle-derived PCWDEs were originally identified from transcriptomes and strikingly resemble those of saprophytic and phytopathogenic microorganisms, raising questions about their origin; e.g. are they insect- or microorganism-derived? To demonstrate unambiguously that the genes encoding PCWDEs found in beetle transcriptomes are indeed of insect origin, we generated a bacterial artificial chromosome library from the genome of the leaf beetle Chrysomela tremula, containing 18 432 clones with an average size of 143 kb. After hybridizing this library with probes derived from 12 C. tremula PCWDE-encoding genes and sequencing the positive clones, we demonstrated that the latter genes are encoded by the insect's genome and are surrounded by genes possessing orthologues in the genome of Tribolium castaneum as well as in three other beetle genomes. Our analyses showed that although the level of overall synteny between C. tremula and T. castaneum seems high, the degree of microsynteny between both species is relatively low, in contrast to the more closely related Colorado potato beetle.

  20. Expression of Genes Encoding the Enzymes for Glycogen and Trehalose Metabolism in L3 and L4 Larvae of Anisakis simplex

    PubMed Central

    Łopieńska-Biernat, E.; Zaobidna, E. A.; Dmitryjuk, M.

    2015-01-01

    Trehalose and glycogen metabolism plays an important role in supporting life processes in many nematodes, including Anisakis simplex. Nematodes, cosmopolitan helminths parasitizing sea mammals and humans, cause a disease known as anisakiasis. The aim of this study was to investigate the expression of genes encoding the enzymes involved in the metabolism of trehalose and glycogen—trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), glycogen synthase (GS), and glycogen phosphorylase (GP)—in stage L3 and stage L4 larvae of A. simplex. The expression of mRNA all four genes, tps, tpp, gs, and gp, was examined by real-time polymerase chain reaction. The A. simplex ribosomal gene (18S) was used as a reference gene. Enzymatic activity was determined. The expression of trehalose enzyme genes was higher in L3 than in L4 larvae, but an inverse relationship was noted for the expression of gs and gp genes. PMID:26783451

  1. RNase G-dependent degradation of the eno mRNA encoding a glycolysis enzyme enolase in Escherichia coli.

    PubMed

    Kaga, Naoko; Umitsuki, Genryou; Nagai, Kazuo; Wachi, Masaaki

    2002-10-01

    Escherichia coli RNase G, encoded by the rng gene, is involved in the processing of 16S rRNA and degradation of the adhE mRNA encoding a fermentative alcohol dehydrogenase. In a search for the intracellular target RNAs of RNase G other than the 16S rRNA precursor and adhE mRNA, total cellular proteins from rng+ and rng::cat cells were compared by two-dimensional gel electrophoresis. The amount of enolase encoded by the eno gene reproducibly increased two- to three-fold in the rng::cat mutant strain compared with the rng+ parent strain. Rifampicin chase experiments showed that the half-life of the eno mRNA was some 3 times longer in the rng::cat mutant than in the wild type. These results indicate that the eno mRNA was a substrate of RNase G in vivo, in addition to 16S rRNA precursor and adhE mRNA. PMID:12450135

  2. Arabidopsis thaliana contains two differentially expressed 3-hydroxy-3-methylglutaryl-CoA reductase genes, which encode microsomal forms of the enzyme.

    PubMed Central

    Enjuto, M; Balcells, L; Campos, N; Caelles, C; Arró, M; Boronat, A

    1994-01-01

    The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC 1.1.1.34) catalyzes the first rate-limiting step in plant isoprenoid biosynthesis. Arabidopsis thaliana contains two genes, HMG1 and HMG2, that encode HMGR. We have cloned these two genes and analyzed their structure and expression. HMG1 and HMG2 consist of four exons and three small introns that interrupt the coding sequence at equivalent positions. The two genes share sequence similarity in the coding regions but not in the 5'- or 3'-flanking regions. HMG1 mRNA is detected in all tissues, whereas the presence of HMG2 mRNA is restricted to young seedlings, roots, and inflorescences. The similarity between the two encoded proteins (HMGR1 and HMGR2) is restricted to the regions corresponding to the membrane and the catalytic domains. Arabidopsis HMGR2 represents a divergent form of the enzyme that has no counterpart among plant HMGRs characterized so far. By using a coupled in vitro transcription-translation assay, we show that both HMGR1 and HMGR2 are cotranslationally inserted into endoplasmic reticulum-derived microsomal membranes. Our results suggest that the endoplasmic reticulum is the only cell compartment for the targeting of HMGR in Arabidopsis and support the hypothesis that in higher plants the formation of mevalonate occurs solely in the cytosol. Images PMID:8302869

  3. Modulated expression of genes encoding estrogen metabolizing enzymes by G1-phase cyclin-dependent kinases 6 and 4 in human breast cancer cells.

    PubMed

    Jia, Yi; Domenico, Joanne; Swasey, Christina; Wang, Meiqin; Gelfand, Erwin W; Lucas, Joseph J

    2014-01-01

    G1-phase cell cycle defects, such as alterations in cyclin D1 or cyclin-dependent kinase (cdk) levels, are seen in most tumors. For example, increased cyclin D1 and decreased cdk6 levels are seen in many human breast tumors. Overexpression of cdk6 in breast tumor cells in culture has been shown to suppress proliferation, unlike the growth stimulating effects of its close homolog, cdk4. In addition to directly affecting proliferation, alterations in cdk6 or cdk4 levels in breast tumor cells also differentially influence levels of numerous steroid metabolic enzymes (SMEs), including those involved in estrogen metabolism. Overexpression of cdk6 in tumor cell lines having low cdk6 resulted in decreased levels of mRNAs encoding aldo-keto reductase (AKR)1C1, AKR1C2 and AKR1C3, which are hydroxysteroid dehydrogenases (HSDs) involved in steroid hormone metabolism. In contrast, increasing cdk4 dramatically increased these transcript levels, especially those encoding AKR1C3, an enzyme that converts estrone to 17β-estradiol, a change that could result in a pro-estrogenic state favoring tumor growth. Effects on other estrogen metabolizing enzymes, including cytochrome P450 (CYP) 19 aromatase, 17β-HSD2, and CYP1B1 transcripts, were also observed. Interactions of cdk6 and cdk4, but not cyclin D1, with the promoter region of a cdk-regulated gene, 17β-HSD2, were detected. The results uncover a previously unsuspected link between the cell cycle and hormone metabolism and differential roles for cdk6 and cdk4 in a novel mechanism for pre-receptor control of steroid hormone action, with important implications for the origin and treatment of steroid hormone-dependent cancers. PMID:24848372

  4. Differential expression of genes encoding anti-oxidant enzymes in Sydney rock oysters, Saccostrea glomerata (Gould) selected for disease resistance.

    PubMed

    Green, Timothy J; Dixon, Tom J; Devic, Emilie; Adlard, Robert D; Barnes, Andrew C

    2009-05-01

    Sydney rock oysters (Saccostrea glomerata) selectively bred for disease resistance (R) and wild-caught control oysters (W) were exposed to a field infection of disseminating neoplasia. Cumulative mortality of W oysters (31.7%) was significantly greater than R oysters (0.0%) over the 118 days of the experiment. In an attempt to understand the biochemical and molecular pathways involved in disease resistance, differentially expressed sequence tags (ESTs) between R and W S. glomerata hemocytes were identified using the PCR technique, suppression subtractive hybridisation (SSH). Sequencing of 300 clones from two SSH libraries revealed 183 distinct sequences of which 113 shared high similarity to sequences in the public databases. Putative function could be assigned to 64 of the sequences. Expression of nine ESTs homologous to genes previously shown to be involved in bivalve immunity was further studied using quantitative reverse-transcriptase PCR (qRT-PCR). The base-line expression of an extracellular superoxide dismutase (ecSOD) and a small heat shock protein (sHsP) were significantly increased, whilst peroxiredoxin 6 (Prx6) and interferon inhibiting cytokine factor (IK) were significantly decreased in R oysters. From these results it was hypothesised that R oysters would be able to generate the anti-parasitic compound, hydrogen peroxide (H(2)O(2)) faster and to higher concentrations during respiratory burst due to the differential expression of genes for the two anti-oxidant enzymes of ecSOD and Prx6. To investigate this hypothesis, protein extracts from hemolymph were analysed for oxidative burst enzyme activity. Analysis of the cell free hemolymph proteins separated by native-polyacrylamide gel electrophoresis (PAGE) failed to detect true superoxide dismutase (SOD) activity by assaying dismutation of superoxide anion in zymograms. However, the ecSOD enzyme appears to generate hydrogen peroxide, presumably via another process, which is yet to be elucidated. This

  5. A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

    PubMed

    Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

  6. A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

    PubMed Central

    Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions. PMID:8597660

  7. Indirect enzyme-linked immunosorbent assay for detection of immunoglobulin G reactive with a recombinant protein expressed from the gene encoding the 116-kilodalton protein of Mycoplasma pneumoniae.

    PubMed

    Duffy, M F; Whithear, K G; Noormohammadi, A H; Markham, P F; Catton, M; Leydon, J; Browning, G F

    1999-04-01

    Serology remains the method of choice for laboratory diagnosis of Mycoplasma pneumoniae infection. Currently available serological tests employ complex cellular fractions of M. pneumoniae as antigen. To improve the specificity of M. pneumoniae diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned M. pneumoniae gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactivity with patient sera and the most antigenic was further assessed for its serodiagnostic potential by indirect enzyme-linked immunosorbent assay (ELISA). The ELISA based on the recombinant protein was equivalent in sensitivity to the commercial test (Serodia Myco II; Fujirebio Inc.) to which it was compared. Southern and Western blotting data suggested that the recombinant protein derived from the 116-kDa protein of M. pneumoniae could provide a species-specific diagnostic tool, although further assessment is required.

  8. Molecular regulation of sinapate ester metabolism in Brassica napus: expression of genes, properties of the encoded proteins and correlation of enzyme activities with metabolite accumulation.

    PubMed

    Milkowski, Carsten; Baumert, Alfred; Schmidt, Diana; Nehlin, Lilian; Strack, Dieter

    2004-04-01

    Members of the Brassicaceae family accumulate specific sinapate esters, i.e. sinapoylcholine (sinapine), which is considered as a major antinutritive compound in seeds of important crop plants like Brassica napus, and sinapoylmalate, which is implicated in UV-B tolerance in leaves. We have studied the molecular regulation of the sinapate ester metabolism in B. napus, and we describe expression of genes, some properties of the encoded proteins and profiles of the metabolites and enzyme activities. The cloned cDNAs encoding the key enzymes of sinapine biosynthesis, UDP-glucose (UDP-Glc):B. napus sinapate glucosyltransferase (BnSGT1) and sinapoylglucose:B. napus choline sinapoyltransferase (BnSCT), were functionally expressed. BnSGT1 belongs to a subgroup of plant GTs catalysing the formation of 1-O-hydroxycinnamoyl-beta-d-glucoses. BnSCT is another member of serine carboxypeptidase-like (SCPL) family of acyltransferases. The B. napus genome contains at least two SGT and SCT genes, each derived from its progenitors B. oleracea and B. rapa. BnSGT1 and BnSCT activities are subjected to pronounced transcriptional regulation. BnSGT1 transcript level increases throughout early stages of seed development until the early cotyledonary stage, and stays constant in later stages. The highest level of BnSGT1 transcripts is reached in 2-day-old seedlings followed by a dramatic decrease. In contrast, expression of BnSCT is restricted to developing seeds. Regulation of gene expression at the transcript level seems to be responsible for changes of BnSGT1 and BnSCT activities during seed and seedling development of B. napus. Together with sinapine esterase (SCE) and sinapoylglucose:malate sinapoyltransferase (SMT), activities of BnSGT1 and BnSCT show a close correlation with the accumulation kinetics of the corresponding metabolites.

  9. Suppression of 9-cis-Epoxycarotenoid Dioxygenase, Which Encodes a Key Enzyme in Abscisic Acid Biosynthesis, Alters Fruit Texture in Transgenic Tomato1[W][OA

    PubMed Central

    Sun, Liang; Sun, Yufei; Zhang, Mei; Wang, Ling; Ren, Jie; Cui, Mengmeng; Wang, Yanping; Ji, Kai; Li, Ping; Li, Qian; Chen, Pei; Dai, Shengjie; Duan, Chaorui; Wu, Yan; Leng, Ping

    2012-01-01

    Cell wall catabolism during fruit ripening is under complex control and is key for fruit quality and shelf life. To examine the role of abscisic acid (ABA) in tomato (Solanum lycopersicum) fruit ripening, we suppressed SlNCED1, which encodes 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in the biosynthesis of ABA. To suppress SlNCED1 specifically in tomato fruits, and thus avoid the pleiotropic phenotypes associated with ABA deficiency, we used an RNA interference construct driven by the fruit-specific E8 promoter. ABA accumulation and SlNCED1 transcript levels in the transgenic fruit were down-regulated to between 20% and 50% of the levels measured in the control fruit. This significant reduction in NCED activity led to a down-regulation in the transcription of genes encoding major cell wall catabolic enzymes, specifically polygalacturonase (SlPG), pectin methyl esterase (SlPME), β-galactosidase precursor mRNA (SlTBG), xyloglucan endotransglycosylase (SlXET), endo-1,4-β-cellulose (SlCels), and expansin (SlExp). This resulted in an increased accumulation of pectin during ripening. In turn, this led to a significant extension of the shelf life to 15 to 29 d compared with a shelf life of only 7 d for the control fruit and an enhancement of fruit firmness at the mature stage by 30% to 45%. In conclusion, ABA affects cell wall catabolism during tomato fruit ripening via down-regulation of the expression of major catabolic genes (SlPG, SlPME, SlTBG, SlXET, SlCels, and SlExp). PMID:22108525

  10. Identification of ten mevalonate enzyme-encoding genes and their expression in response to juvenile hormone levels in Leptinotarsa decemlineata (Say).

    PubMed

    Li, Qian; Meng, Qing-Wei; Lü, Feng-Gong; Guo, Wen-Chao; Li, Guo-Qing

    2016-06-15

    The mevalonate pathway is responsible for the biosynthesis of many essential molecules important in insect development, reproduction, chemical communication and defense. Based on Leptinotarsa decemlineata transcriptome and genome data, we identified ten genes that encoded acetoacetyl-CoA thiolase (LdAACT1 and LdAACT2), hydroxymethylglutaryl (HMA)-CoA synthase (LdHMGS), HMG-CoA reductase (LdHMGR1 and LdHMGR2), mevalonate kinase (LdMevK), phospho-mevalonate kinase (LdPMK), mevalonate diphosphate decarboxylase (LdMDD), isopentenyl-diphosphate isomerase (LdIDI) and farnesyl pyrophosphate synthetase (LdFPPS). Nine of these genes (except for LdAACT1) were mainly expressed in the larval brain-corpora cardiaca-corpora allata complex, and adult ovary and testis. The 9 genes were transcribed at high levels right after each ecdysis, and at low levels in the mid instar. Therefore, the 9 genes were indicated to be involved in JH biosynthesis. Moreover, knockdown of a JH biosynthesis gene LdJHAMT to lower JH titer significantly downregulated the transcription of the 9 genes. Ingestion of JH to activate JH signaling also significantly suppressed the expression of the 9 genes. It appears that the accumulation of JH precursors in LdJHAMT RNAi larvae and a high JH titer in JH-fed specimens may cause negative feedbacks to repress the expression of the 9 mevalonate enzyme-encoding genes (excluding LdAACT1) to balance the enzyme quantity in L. decemlineata. PMID:26899871

  11. The Variability of Sesquiterpenes Emitted from Two Zea mays Cultivars Is Controlled by Allelic Variation of Two Terpene Synthase Genes Encoding Stereoselective Multiple Product Enzymes

    PubMed Central

    Köllner, Tobias G.; Schnee, Christiane; Gershenzon, Jonathan; Degenhardt, Jörg

    2004-01-01

    The mature leaves and husks of Zea mays release a complex blend of terpene volatiles after anthesis consisting predominantly of bisabolane-, sesquithujane-, and bergamotane-type sesquiterpenes. The varieties B73 and Delprim release the same volatile constituents but in significantly different proportions. To study the molecular genetic and biochemical mechanisms controlling terpene diversity and distribution in these varieties, we isolated the closely related terpene synthase genes terpene synthase4 (tps4) and tps5 from both varieties. The encoded enzymes, TPS4 and TPS5, each formed the same complex mixture of sesquiterpenes from the precursor farnesyl diphosphate but with different proportions of products. These mixtures correspond to the sesquiterpene blends observed in the varieties B73 and Delprim, respectively. The differences in the stereoselectivity of TPS4 and TPS5 are determined by four amino acid substitutions with the most important being a Gly instead of an Ala residue at position 409 at the catalytic site of the enzyme. Although both varieties contain tps4 and tps5 alleles, their differences in terpene composition result from the fact that B73 has only a single functional allele of tps4 and no functional alleles of tps5, whereas Delprim has only a functional allele of tps5 and no functional alleles of tps4. Lack of functionality was shown to be attributable to frame-shift mutations or amino acid substitutions that greatly reduce the activity of their encoded proteins. Therefore, the diversity of sesquiterpenes in these two maize cultivars is strongly influenced by single nucleotide changes in the alleles of two terpene synthase genes. PMID:15075399

  12. Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase.

    PubMed Central

    Llanos, R M; Harris, C J; Hillier, A J; Davidson, B E

    1993-01-01

    The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported. The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively. The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh. Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host. The identity of ldh was established previously by the same approach (R. M. Llanos, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 174:6956-6964, 1992). Each of the genes is preceded by a potential ribosome binding site. The operon is expressed in a 4.1-kb transcript. The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon. The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons. This degree of bias suggests that the operon is highly expressed. The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L. lactis. For this reason, we have called the operon the las (lactic acid synthesis) operon. Images PMID:8478320

  13. Evolutionary diversification and characterization of the eubacterial gene family encoding DXR type II, an alternative isoprenoid biosynthetic enzyme

    PubMed Central

    2013-01-01

    Background Isoprenoids constitute a vast family of natural compounds performing diverse and essential functions in all domains of life. In most eubacteria, isoprenoids are synthesized through the methylerythritol 4-phosphate (MEP) pathway. The production of MEP is usually catalyzed by deoxyxylulose 5-phosphate reductoisomerase (DXR-I) but a few organisms use an alternative DXR-like enzyme (DXR-II). Results Searches through 1498 bacterial complete proteomes detected 130 sequences with similarity to DXR-II. Phylogenetic analysis identified three well-resolved clades: the DXR-II family (clustering 53 sequences including eleven experimentally verified as functional enzymes able to produce MEP), and two previously uncharacterized NAD(P)-dependent oxidoreductase families (designated DLO1 and DLO2 for DXR-II-like oxidoreductases 1 and 2). Our analyses identified amino acid changes critical for the acquisition of DXR-II biochemical function through type-I functional divergence, two of them mapping onto key residues for DXR-II activity. DXR-II showed a markedly discontinuous distribution, which was verified at several levels: taxonomic (being predominantly found in Alphaproteobacteria and Firmicutes), metabolic (being mostly found in bacteria with complete functional MEP pathways with or without DXR-I), and phenotypic (as no biological/phenotypic property was found to be preferentially distributed among DXR-II-containing strains, apart from pathogenicity in animals). By performing a thorough comparative sequence analysis of GC content, 3:1 dinucleotide frequencies, codon usage and codon adaptation indexes (CAI) between DXR-II sequences and their corresponding genomes, we examined the role of horizontal gene transfer (HGT), as opposed to an scenario of massive gene loss, in the evolutionary origin and diversification of the DXR-II subfamily in bacteria. Conclusions Our analyses support a single origin of the DXR-II family through functional divergence, in which constitutes

  14. Analysis of genes encoding the 2,4-dichlorophenoxyacetic acid-degrading enzyme from Sphingomonas agrestis 58-1.

    PubMed

    Shimojo, Mitsuhiro; Kawakami, Mitsuyasu; Amada, Kei

    2009-07-01

    A 2,4-dichlorophenoxy acetic acid (2,4-D)-degrading bacterium, strain 58-1, was newly isolated from soil samples collected in the Fukuoka Prefecture, Japan, and grown on an enrichment culture medium containing 2,4-D as the sole carbon source. Phylogenic analysis identified strain 58-1 as Sphingomonas agrestis. In 2,4-D degraders, classes I, II, and III inherit the tfdA, cadA, and tfdAalpha genes, respectively, and the results from degenerate-PCR indicated that this strain belongs to the class II degraders. A clone that includes the cadA gene homolog of S. agrestis 58-1 was screened from a library by using the PCR amplified fragment as a DNA probe. The cloned fragment was sequenced and found to consist of 5043 nucleotides and include 3 open reading frames (orfs). The orf1, orf2, and orf3 genes encode polypeptides consisting of 412, 448, and 177 amino acids, respectively. The Orf2 product shares a high degree of sequence similarity (92%) with the large subunit of 2,4-D oxygenase from the Bradyrhizobium sp. strain HW13, which belongs to the class III 2,4-D degraders, while the orf3 product shared 63% sequence similarity with the small subunit of 2,4-D oxygenase from the strain HW13. The results of the functional expression analysis using various deletion mutants in Escherichia coli revealed that the expression of both orf2 and orf3 genes, but not orf1, is essential for the conversion of 2,4-D to 2,4-DCP. From these results, we conclude the first isolation of 2,4-D oxygenase genes from a class II 2,4-D degrader.

  15. Arabidopsis CYP94B3 encodes jasmonyl-L-isoleucine 12-hydroxylase, a key enzyme in the oxidative catabolism of jasmonate.

    PubMed

    Kitaoka, Naoki; Matsubara, Takuya; Sato, Michio; Takahashi, Kosaku; Wakuta, Shinji; Kawaide, Hiroshi; Matsui, Hirokazu; Nabeta, Kensuke; Matsuura, Hideyuki

    2011-10-01

    The hormonal action of jasmonate in plants is controlled by the precise balance between its biosynthesis and catabolism. It has been shown that jasmonyl-L-isoleucine (JA-Ile) is the bioactive form involved in the jasmonate-mediated signaling pathway. However, the catabolism of JA-Ile is poorly understood. Although a metabolite, 12-hydroxyJA-Ile, has been characterized, detailed functional studies of the compound and the enzyme that produces it have not been conducted. In this report, the kinetics of wound-induced accumulation of 12-hydroxyJA-Ile in plants were examined, and its involvement in the plant wound response is described. Candidate genes for the catabolic enzyme were narrowed down from 272 Arabidopsis Cyt P450 genes using Arabidopsis mutants. The candidate gene was functionally expressed in Pichia pastoris to reveal that CYP94B3 encodes JA-Ile 12-hydroxylase. Expression analyses demonstrate that expression of CYP94B3 is induced by wounding and shows specific activity toward JA-Ile. Plants grown in medium containing JA-Ile show higher sensitivity to JA-Ile in cyp94b3 mutants than in wild-type plants. These results demonstrate that CYP94B3 plays a major regulatory role in controlling the level of JA-Ile in plants. PMID:21849397

  16. Arabidopsis CYP94B3 encodes jasmonyl-L-isoleucine 12-hydroxylase, a key enzyme in the oxidative catabolism of jasmonate.

    PubMed

    Kitaoka, Naoki; Matsubara, Takuya; Sato, Michio; Takahashi, Kosaku; Wakuta, Shinji; Kawaide, Hiroshi; Matsui, Hirokazu; Nabeta, Kensuke; Matsuura, Hideyuki

    2011-10-01

    The hormonal action of jasmonate in plants is controlled by the precise balance between its biosynthesis and catabolism. It has been shown that jasmonyl-L-isoleucine (JA-Ile) is the bioactive form involved in the jasmonate-mediated signaling pathway. However, the catabolism of JA-Ile is poorly understood. Although a metabolite, 12-hydroxyJA-Ile, has been characterized, detailed functional studies of the compound and the enzyme that produces it have not been conducted. In this report, the kinetics of wound-induced accumulation of 12-hydroxyJA-Ile in plants were examined, and its involvement in the plant wound response is described. Candidate genes for the catabolic enzyme were narrowed down from 272 Arabidopsis Cyt P450 genes using Arabidopsis mutants. The candidate gene was functionally expressed in Pichia pastoris to reveal that CYP94B3 encodes JA-Ile 12-hydroxylase. Expression analyses demonstrate that expression of CYP94B3 is induced by wounding and shows specific activity toward JA-Ile. Plants grown in medium containing JA-Ile show higher sensitivity to JA-Ile in cyp94b3 mutants than in wild-type plants. These results demonstrate that CYP94B3 plays a major regulatory role in controlling the level of JA-Ile in plants.

  17. Operon structure and functional analysis of the genes encoding thermophilic desulfurizing enzymes of Paenibacillus sp. A11-2.

    PubMed

    Ishii, Y; Konishi, J; Okada, H; Hirasawa, K; Onaka, T; Suzuki, M

    2000-04-01

    Paenibacillus A11-2 can efficiently cleave two carbon&bond;sulfur bonds in dibenzothiophene (DBT) and alkyl DBTs, which are refractory by conventional petroleum hydrodesulfurization, to remove sulfur atom at high temperatures. An 8.7-kb DNA fragment containing the genes for the DBT desulfurizing enzymes of A11-2 was cloned in Escherichia coli and characterized. Heterologous expression analysis of the deletion mutants identified three open reading frames that were required for the desulfurization of DBT to 2-hydroxybiphenyl (2-HBP). The three genes were designated tdsA, tdsB, and tdsC (for thermophilic desulfurization). Both the nucleotide sequences and the deduced amino acid sequences show significant homology to dszABC genes of Rhodococcus sp. IGTS8, but there are several local differences between them. Subclone analysis revealed that the product of tdsC oxidizes DBT to DBT-5,5'-dioxide via DBT-5-oxide, the product of tdsA converts DBT-5,5'-dioxide to 2-(2-hydroxyphenyl) benzene sulfinate, and the product of tdsB converts 2-(2-hydroxyphenyl)benzene sulfinate to 2-HBP. Cell-free extracts of a recombinant E. coli harboring all the three desulfurization genes converted DBT to 2-HBP at both 37 and 50 degrees C. In vivo and in vitro exhibition of desulfurization activity of the recombinant genes derived from a Paenibacillus indicates that an E. coli oxidoreductase can be functionally coupled with the monooxygenases of a gram-positive thermophile.

  18. Human adenovirus early region 4 open reading frame 1 genes encode growth-transforming proteins that may be distantly related to dUTP pyrophosphatase enzymes.

    PubMed Central

    Weiss, R S; Lee, S S; Prasad, B V; Javier, R T

    1997-01-01

    An essential oncogenic determinant of subgroup D human adenovirus type 9 (Ad9), which uniquely elicits estrogen-dependent mammary tumors in rats, is encoded by early region 4 open reading frame 1 (E4 ORF1). Whereas Ad9 E4 ORF1 efficiently induces transformed foci on the established rat embryo fibroblast cell line CREF, the related subgroup A Ad12 and subgroup C Ad5 E4 ORF1s do not (R. T. Javier, J. Virol. 68:3917-3924, 1994). In this study, we found that the lack of transforming activity associated with non-subgroup D adenovirus E4 ORF1s in CREF cells correlated with significantly reduced protein levels compared to Ad9 E4 ORF1 in these cells. In the human cell line TE85, however, the non-subgroup D adenovirus E4 ORF1s produced protein levels higher than those seen in CREF cells as well as transforming activities similar to that of Ad9 E4 ORF1, suggesting that all adenovirus E4 ORF1 polypeptides possess comparable cellular growth-transforming activities. In addition, searches for known proteins related to these novel viral transforming proteins revealed that the E4 ORF1 proteins had weak sequence similarity, over the entire length of the E4 ORF1 polypeptides, with a variety of organismal and viral dUTP pyrophosphatase (dUTPase) enzymes. Even though adenovirus E4 ORF1 proteins lacked conserved protein motifs of dUTPase enzymes or detectable enzymatic activity, E4 ORF1 and dUTPase proteins were predicted to possess strikingly similar secondary structure arrangements. It was also established that an avian adenovirus protein, encoded within a genomic location analogous to that of the human adenovirus E4 ORF1s, was a genuine dUTPase enzyme. Although no functional similarity was found for the E4 ORF1 and dUTPase proteins, we propose that human adenovirus E4 ORF1 genes have evolved from an ancestral adenovirus dUTPase and, from this structural framework, developed novel transforming properties. PMID:9032316

  19. A mammalian homolog of the yeast LCB1 encodes a component of serine palmitoyltransferase, the enzyme catalyzing the first step in sphingolipid synthesis.

    PubMed

    Hanada, K; Hara, T; Nishijima, M; Kuge, O; Dickson, R C; Nagiec, M M

    1997-12-19

    Serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzes the initial step dedicated to sphingolipid biosynthesis and is thought to be a key enzyme for regulating cellular sphingolipid content. For SPT activity, the yeast Saccharomyces cerevisiae requires two genes, LCB1 and LCB2. We isolated mammalian LCB1 cDNA homologs from mouse and Chinese hamster ovary (CHO) cells and an LCB2 cDNA homolog from CHO cells. The mammalian LCB1 proteins are predicted to have about 35% amino acid identity to the yeast Lcb1 protein, whereas the CHO LCB2 protein is predicted to have about 40% amino acid identity to the yeast Lcb2 protein. Northern blot analysis of mRNA isolated from various mouse tissues revealed that the tissue distribution of both LCB1 and LCB2 messengers followed a similar pattern. Transfection of an SPT-defective CHO mutant strain with a CHO LCB1-expressing plasmid restored both SPT activity and de novo sphingolipid synthesis to the wild type levels, whereas transfection of the mutant strain with a CHO LCB2-expressing plasmid did not exhibit any recovery effects, indicating that the SPT defect in the mutant cells is specifically complemented by the CHO LCB1 homolog. Furthermore, when the SPT-defective mutant cells were transfected with a plasmid encoding a His6-tagged CHO LCB1 protein, SPT activity bound to a Ni2+-immobilized resin. These results indicate that the CHO LCB1 homolog encodes a component of SPT. PMID:9405408

  20. [Cloning and analysis of cDNA encoding key enzyme gene (dxr) of the non-MVA pathway in Taxus chinensis cells].

    PubMed

    Zheng, Qing-Ping; Yu, Long-Jiang; Liu, Zhi; Li, Mo-Yi; Xiang, Fu; Yang, Qin

    2004-07-01

    Two distinct routes (classical mevalonate pathway and a novel mevalonate-independent pathway) are utilized by plants for the biosynthesis of isopentenyl diphosphate, the universal precursor of isoprenoids (Fig. 1). Present researches indicated that taxol was synthesized mainly via non-mevalonate pathway, but not genetic evidence was showed. The second step in non-mevalonate pathway involves an intramolecular rearrangement and subsequent reduction of deoxyxylulose phosphate to yield 2-C-methyl-D-erythritol-4-phosphate, and 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) with responsibility for this reaction was considered as a key enzyme. As a tool for the isolation of genes in terpenoid biosynthesis in plants, total RNA was prepared from Taxus chinensis suspension cells, a cell type highly specialized for diterpene (taxol). A reverse transcription-PCR strategy based on the design of degenerated oligonucleotides was developed for isolating the gene encoding a gymnosperm homolog of this enzyme from Taxus chinensis. Through sequence analysis by Blast P online, the resulting cDNA showed highly homologous to 1-deoxy-D-xylulose 5-phosphate reductoisomerases, with 95% identification compared with Arabidopsis thaliana (Q9XFS9), 94% with Mentha x piperita (Q9XESO), 80% with Synechococcus elongatus (Q8DK30), 78% with Synechocystis sp. PCC 6803 (Q55663) and Nostoc sp. PCC 7120 (Q8YP49), and 73% with Synechococcus leopoliensis (Q9RKT1). Deduced amino acid sequences were also analyzed by PROSITE, ClustalX (1.81) and Phylio (3.6 alpha), and data present evidence for the existence of this deoxyxyluose phosphate reductoisomerase in Taxus chinensis. This is the first report of the dxr gene cloned from gymnosperm. PMID:15968987

  1. Deletion of a gene cluster encoding pectin degrading enzymes in Caldicellulosiruptor bescii reveals an important role for pectin in plant biomass recalcitrance

    SciTech Connect

    Chung, Daehwan; Pattathil, Sivakumar; Biswal, Ajaya K.; Hahn, Michael G.; Mohnen, Debra; Westpheling, Janet

    2014-10-10

    A major obstacle, and perhaps the most important economic barrier to the effective use of plant biomass for the production of fuels, chemicals, and bioproducts, is our current lack of knowledge of how to efficiently and effectively deconstruct wall polymers for their subsequent use as feedstocks. Plants represent the most desired source of renewable energy and hydrocarbons because they fix CO2, making their use carbon neutral. Their biomass structure, however, is a barrier to deconstruction, and this is often referred to as recalcitrance. Members of the bacterial genus Caldicellulosiruptor have the ability to grow on unpretreated plant biomass and thus provide an assay for plant deconstruction and biomass recalcitrance. Using recently developed genetic tools for manipulation of these bacteria, a deletion of a gene cluster encoding enzymes for pectin degradation was constructed, and the resulting mutant was reduced in its ability to grow on both dicot and grass biomass, but not on soluble sugars. The plant biomass from three phylogenetically diverse plants, Arabidopsis (a herbaceous dicot), switchgrass (a monocot grass), and poplar (a woody dicot), was used in these analyses. These biomass types have cell walls that are significantly different from each other in both structure and composition. While pectin is a relatively minor component of the grass and woody dicot substrates, the reduced growth of the mutant on all three biomass types provides direct evidence that pectin plays an important role in biomass recalcitrance. Glycome profiling of the plant material remaining after growth of the mutant on Arabidopsis biomass compared to the wild-type revealed differences in the rhamnogalacturonan I, homogalacturonan, arabinogalactan, and xylan profiles. In contrast, only minor differences were observed in the glycome profiles of the switchgrass and poplar biomass. In conclusion, the combination of microbial digestion and plant biomass analysis provides a new

  2. Deletion of a gene cluster encoding pectin degrading enzymes in Caldicellulosiruptor bescii reveals an important role for pectin in plant biomass recalcitrance

    DOE PAGESBeta

    Chung, Daehwan; Pattathil, Sivakumar; Biswal, Ajaya K.; Hahn, Michael G.; Mohnen, Debra; Westpheling, Janet

    2014-10-10

    A major obstacle, and perhaps the most important economic barrier to the effective use of plant biomass for the production of fuels, chemicals, and bioproducts, is our current lack of knowledge of how to efficiently and effectively deconstruct wall polymers for their subsequent use as feedstocks. Plants represent the most desired source of renewable energy and hydrocarbons because they fix CO2, making their use carbon neutral. Their biomass structure, however, is a barrier to deconstruction, and this is often referred to as recalcitrance. Members of the bacterial genus Caldicellulosiruptor have the ability to grow on unpretreated plant biomass and thusmore » provide an assay for plant deconstruction and biomass recalcitrance. Using recently developed genetic tools for manipulation of these bacteria, a deletion of a gene cluster encoding enzymes for pectin degradation was constructed, and the resulting mutant was reduced in its ability to grow on both dicot and grass biomass, but not on soluble sugars. The plant biomass from three phylogenetically diverse plants, Arabidopsis (a herbaceous dicot), switchgrass (a monocot grass), and poplar (a woody dicot), was used in these analyses. These biomass types have cell walls that are significantly different from each other in both structure and composition. While pectin is a relatively minor component of the grass and woody dicot substrates, the reduced growth of the mutant on all three biomass types provides direct evidence that pectin plays an important role in biomass recalcitrance. Glycome profiling of the plant material remaining after growth of the mutant on Arabidopsis biomass compared to the wild-type revealed differences in the rhamnogalacturonan I, homogalacturonan, arabinogalactan, and xylan profiles. In contrast, only minor differences were observed in the glycome profiles of the switchgrass and poplar biomass. In conclusion, the combination of microbial digestion and plant biomass analysis provides a new and

  3. Modulation of carcinogen metabolizing cytochromes P450 in rat liver and kidney by cabbage and sauerkraut juices: comparison with the effects of indole-3-carbinol and phenethyl isothiocyanate.

    PubMed

    Szaefer, Hanna; Krajka-Kuźniak, Violetta; Bartoszek, Agnieszka; Baer-Dubowska, Wanda

    2012-08-01

    This study investigated the effect of raw cabbage and sauerkraut juices on the activity and expression of CYP1A1, 1A2, 1B1 and 2B in Wistar rat liver and kidney. The results were compared with those of two commercially available products of glucosinolates degradation: indole-3-carbinol (I3C) and phenethyl isothiocyanate (PEITC). Significant differences in the effect of the cabbage juices as well as I3C and PEITC between the liver and kidney were found. In the liver, both juices decreased the activities of enzymatic markers of CYP1A1 and CYP1A2 after 10 days of the experiment, while in the kidney an enhancement of the activity of these enzymes was observed on days 4 and 10. The increased activity of these enzymes and CYP1A1/1A2 protein level in the liver was found after 30 days of treatment with sauerkraut juice. A similar effect was observed as a result of PEITC treatment. I3C increased the expression and activity of hepatic CYPs at all time points investigated. In conclusion, the present study demonstrates that raw cabbage and sauerkraut juices may affect CYPs involved in the activation of carcinogens/xenobiotics and in this way exert anticarcinogenic activity. The final effects, however, depend on the time-frame of exposure and the type of tissue.

  4. A gene cluster involved in degradation of substituted salicylates via ortho cleavage in Pseudomonas sp. strain MT1 encodes enzymes specifically adapted for transformation of 4-methylcatechol and 3-methylmuconate.

    PubMed

    Cámara, Beatriz; Bielecki, Piotr; Kaminski, Filip; dos Santos, Vitor Martins; Plumeier, Iris; Nikodem, Patricia; Pieper, Dietmar H

    2007-03-01

    Pseudomonas sp. strain MT1 has recently been reported to degrade 4- and 5-chlorosalicylate by a pathway assumed to consist of a patchwork of reactions comprising enzymes of the 3-oxoadipate pathway. Genes encoding the initial steps in the degradation of salicylate and substituted derivatives were now localized and sequenced. One of the gene clusters characterized (sal) showed a novel gene arrangement, with salA, encoding a salicylate 1-hydroxylase, being clustered with salCD genes, encoding muconate cycloisomerase and catechol 1,2-dioxygenase, respectively, and was expressed during growth on salicylate and chlorosalicylate. A second gene cluster (cat), exhibiting the typical catRBCA arrangement of genes of the catechol branch of the 3-oxoadipate pathway in Pseudomonas strains, was expressed during growth on salicylate. Despite their high sequence similarities with isoenzymes encoded by the cat gene cluster, the catechol 1,2-dioxygenase and muconate cycloisomerase encoded by the sal cluster showed unusual kinetic properties. Enzymes were adapted for turnover of 4-chlorocatechol and 3-chloromuconate; however, 4-methylcatechol and 3-methylmuconate were identified as the preferred substrates. Investigation of the substrate spectrum identified 4- and 5-methylsalicylate as growth substrates, which were effectively converted by enzymes of the sal cluster into 4-methylmuconolactone, followed by isomerization to 3-methylmuconolactone. The function of the sal gene cluster is therefore to channel both chlorosubstituted and methylsubstituted salicylates into a catechol ortho cleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways.

  5. The genes BnSCT1 and BnSCT2 from Brassica napus encoding the final enzyme of sinapine biosynthesis: molecular characterization and suppression.

    PubMed

    Weier, Diana; Mittasch, Juliane; Strack, Dieter; Milkowski, Carsten

    2008-01-01

    This study describes the molecular characterization of the genes BnSCT1 and BnSCT2 from oilseed rape (Brassica napus) encoding the enzyme 1-O-sinapoyl-beta-glucose:choline sinapoyltransferase (SCT; EC 2.3.1.91). SCT catalyzes the 1-O-beta-acetal ester-dependent biosynthesis of sinapoylcholine (sinapine), the most abundant phenolic compound in seeds of B. napus. GUS fusion experiments indicated that seed specificity of BnSCT1 expression is caused by an inducible promoter confining transcription to embryo tissues and the aleurone layer. A dsRNAi construct designed to silence seed-specifically the BnSCT1 gene was effective in reducing the sinapine content of Arabidopsis seeds thus defining SCT genes as targets for molecular breeding of low sinapine cultivars of B. napus. Sequence analyses revealed that in the allotetraploid genome of B. napus the gene BnSCT1 represents the C genome homologue from the B. oleracea progenitor whereas BnSCT2 was derived from the Brassica A genome of B. rapa. The BnSCT1 and BnSCT2 loci showed colinearity with the homologous Arabidopsis SNG2 gene locus although the genomic microstructure revealed the deletion of a cluster of three genes and several coding regions in the B. napus genome.

  6. Identification, characterization and developmental expression of Halloween genes encoding P450 enzymes mediating ecdysone biosynthesis in the tobacco hornworm, Manduca sexta.

    PubMed

    Rewitz, Kim F; Rybczynski, Robert; Warren, James T; Gilbert, Lawrence I

    2006-03-01

    The insect molting hormone 20-hydroxyecdysone (20E) plays a central role in regulating gene expression during development and metamorphosis. In many Lepidoptera, the pro-hormone 3-dehydroecdysone (3DE), synthesized from cholesterol in the prothoracic gland, is rapidly converted to ecdysone (E) by a hemolymph reductase, and E is subsequently converted to 20E in various peripheral target tissues. Recently, four Drosophila melanogaster P450 enzymes, encoded by specific Halloween genes, were cloned and functionally characterized as mediating the last hydroxylation steps leading to 20E. We extended this work to the tobacco hornworm Manduca sexta, an established model for endocrinological and developmental studies. cDNA clones were obtained for three Manduca orthologs of CYP306A1 (phantom; phm, the 25-hydroxylase), CYP302A1 (disembodied; dib, the 22-hydroxylase) and CYP315A1 (shadow; sad, the 2-hydroxylase), expressed predominantly in the prothoracic gland during the fifth (final) larval instar and during pupal-adult development, with fifth instar mRNA levels closely paralleling the hemolymph ecdysteroid titer. The data indicate that transcriptional regulation of phm, dib and sad plays a role in the developmentally varying steroidogenic capacities of the prothoracic glands during the fifth instar. The consistent expression of the Halloween genes confirms the importance of the prothoracic glands in pupal-adult development. These studies establish Manduca as an excellent model for examining the regulation of the Halloween genes. PMID:16503480

  7. The genes BnSCT1 and BnSCT2 from Brassica napus encoding the final enzyme of sinapine biosynthesis: molecular characterization and suppression.

    PubMed

    Weier, Diana; Mittasch, Juliane; Strack, Dieter; Milkowski, Carsten

    2008-01-01

    This study describes the molecular characterization of the genes BnSCT1 and BnSCT2 from oilseed rape (Brassica napus) encoding the enzyme 1-O-sinapoyl-beta-glucose:choline sinapoyltransferase (SCT; EC 2.3.1.91). SCT catalyzes the 1-O-beta-acetal ester-dependent biosynthesis of sinapoylcholine (sinapine), the most abundant phenolic compound in seeds of B. napus. GUS fusion experiments indicated that seed specificity of BnSCT1 expression is caused by an inducible promoter confining transcription to embryo tissues and the aleurone layer. A dsRNAi construct designed to silence seed-specifically the BnSCT1 gene was effective in reducing the sinapine content of Arabidopsis seeds thus defining SCT genes as targets for molecular breeding of low sinapine cultivars of B. napus. Sequence analyses revealed that in the allotetraploid genome of B. napus the gene BnSCT1 represents the C genome homologue from the B. oleracea progenitor whereas BnSCT2 was derived from the Brassica A genome of B. rapa. The BnSCT1 and BnSCT2 loci showed colinearity with the homologous Arabidopsis SNG2 gene locus although the genomic microstructure revealed the deletion of a cluster of three genes and several coding regions in the B. napus genome. PMID:17882453

  8. Cloning and expression of the cDNA encoding human fumarylacetoacetate hydrolase, the enzyme deficient in hereditary tyrosinemia: assignment of the gene to chromosome 15.

    PubMed Central

    Phaneuf, D; Labelle, Y; Bérubé, D; Arden, K; Cavenee, W; Gagné, R; Tanguay, R M

    1991-01-01

    Type 1 hereditary tyrosinemia (HT) is an autosomal recessive disease characterized by a deficiency of the enzyme fumarylacetoacetate hydrolase (FAH; E.C.3.7.1.2). We have isolated human FAH cDNA clones by screening a liver cDNA expression library using specific antibodies and plaque hybridization with a rat FAH cDNA probe. A 1,477-bp cDNA was sequenced and shown to code for FAH by an in vitro transcription-translation assay and sequence homology with tryptic fragments of purified FAH. Transient expression of this FAH cDNA in transfected CV-1 mammalian cells resulted in the synthesis of an immunoreactive protein comigrating with purified human liver FAH on SDS-PAGE and having enzymatic activity as shown by the hydrolysis of the natural substrate fumarylacetoacetate. This indicates that the single polypeptide chain encoded by the FAH gene contains all the genetic information required for functional activity, suggesting that the dimer found in vivo is a homodimer. The human FAH cDNA was used as a probe to determine the gene's chromosomal localization using somatic cell hybrids and in situ hybridization. The human FAH gene maps to the long arm of chromosome 15 in the region q23-q25. Images Figure 1 Figure 3 Figure 4 Figure 6 Figure 8 PMID:1998338

  9. Cloning and expression of the cDNA encoding human fumarylacetoacetate hydrolase, the enzyme deficient in hereditary tyrosinemia: assignment of the gene to chromosome 15.

    PubMed

    Phaneuf, D; Labelle, Y; Bérubé, D; Arden, K; Cavenee, W; Gagné, R; Tanguay, R M

    1991-03-01

    Type 1 hereditary tyrosinemia (HT) is an autosomal recessive disease characterized by a deficiency of the enzyme fumarylacetoacetate hydrolase (FAH; E.C.3.7.1.2). We have isolated human FAH cDNA clones by screening a liver cDNA expression library using specific antibodies and plaque hybridization with a rat FAH cDNA probe. A 1,477-bp cDNA was sequenced and shown to code for FAH by an in vitro transcription-translation assay and sequence homology with tryptic fragments of purified FAH. Transient expression of this FAH cDNA in transfected CV-1 mammalian cells resulted in the synthesis of an immunoreactive protein comigrating with purified human liver FAH on SDS-PAGE and having enzymatic activity as shown by the hydrolysis of the natural substrate fumarylacetoacetate. This indicates that the single polypeptide chain encoded by the FAH gene contains all the genetic information required for functional activity, suggesting that the dimer found in vivo is a homodimer. The human FAH cDNA was used as a probe to determine the gene's chromosomal localization using somatic cell hybrids and in situ hybridization. The human FAH gene maps to the long arm of chromosome 15 in the region q23-q25.

  10. Identification, characterization and developmental expression of Halloween genes encoding P450 enzymes mediating ecdysone biosynthesis in the tobacco hornworm, Manduca sexta.

    PubMed

    Rewitz, Kim F; Rybczynski, Robert; Warren, James T; Gilbert, Lawrence I

    2006-03-01

    The insect molting hormone 20-hydroxyecdysone (20E) plays a central role in regulating gene expression during development and metamorphosis. In many Lepidoptera, the pro-hormone 3-dehydroecdysone (3DE), synthesized from cholesterol in the prothoracic gland, is rapidly converted to ecdysone (E) by a hemolymph reductase, and E is subsequently converted to 20E in various peripheral target tissues. Recently, four Drosophila melanogaster P450 enzymes, encoded by specific Halloween genes, were cloned and functionally characterized as mediating the last hydroxylation steps leading to 20E. We extended this work to the tobacco hornworm Manduca sexta, an established model for endocrinological and developmental studies. cDNA clones were obtained for three Manduca orthologs of CYP306A1 (phantom; phm, the 25-hydroxylase), CYP302A1 (disembodied; dib, the 22-hydroxylase) and CYP315A1 (shadow; sad, the 2-hydroxylase), expressed predominantly in the prothoracic gland during the fifth (final) larval instar and during pupal-adult development, with fifth instar mRNA levels closely paralleling the hemolymph ecdysteroid titer. The data indicate that transcriptional regulation of phm, dib and sad plays a role in the developmentally varying steroidogenic capacities of the prothoracic glands during the fifth instar. The consistent expression of the Halloween genes confirms the importance of the prothoracic glands in pupal-adult development. These studies establish Manduca as an excellent model for examining the regulation of the Halloween genes.

  11. Mutations in B3GALT6, which encodes a glycosaminoglycan linker region enzyme, cause a spectrum of skeletal and connective tissue disorders.

    PubMed

    Nakajima, Masahiro; Mizumoto, Shuji; Miyake, Noriko; Kogawa, Ryo; Iida, Aritoshi; Ito, Hironori; Kitoh, Hiroshi; Hirayama, Aya; Mitsubuchi, Hiroshi; Miyazaki, Osamu; Kosaki, Rika; Horikawa, Reiko; Lai, Angeline; Mendoza-Londono, Roberto; Dupuis, Lucie; Chitayat, David; Howard, Andrew; Leal, Gabriela F; Cavalcanti, Denise; Tsurusaki, Yoshinori; Saitsu, Hirotomo; Watanabe, Shigehiko; Lausch, Ekkehart; Unger, Sheila; Bonafé, Luisa; Ohashi, Hirofumi; Superti-Furga, Andrea; Matsumoto, Naomichi; Sugahara, Kazuyuki; Nishimura, Gen; Ikegawa, Shiro

    2013-06-01

    Proteoglycans (PGs) are a major component of the extracellular matrix in many tissues and function as structural and regulatory molecules. PGs are composed of core proteins and glycosaminoglycan (GAG) side chains. The biosynthesis of GAGs starts with the linker region that consists of four sugar residues and is followed by repeating disaccharide units. By exome sequencing, we found that B3GALT6 encoding an enzyme involved in the biosynthesis of the GAG linker region is responsible for a severe skeletal dysplasia, spondyloepimetaphyseal dysplasia with joint laxity type 1 (SEMD-JL1). B3GALT6 loss-of-function mutations were found in individuals with SEMD-JL1 from seven families. In a subsequent candidate gene study based on the phenotypic similarity, we found that B3GALT6 is also responsible for a connective tissue disease, Ehlers-Danlos syndrome (progeroid form). Recessive loss-of-function mutations in B3GALT6 result in a spectrum of disorders affecting a broad range of skeletal and connective tissues characterized by lax skin, muscle hypotonia, joint dislocation, and spinal deformity. The pleiotropic phenotypes of the disorders indicate that B3GALT6 plays a critical role in a wide range of biological processes in various tissues, including skin, bone, cartilage, tendon, and ligament.

  12. Insulin-like growth factor I stimulates degradation of an mRNA transcript encoding the 14 kDa ubiquitin-conjugating enzyme.

    PubMed Central

    Wing, S S; Bedard, N

    1996-01-01

    Upon fasting, the ubiquitin-dependent proteolytic system is activated in skeletal muscle in parallel with the increases in rates of proteolysis. Levels of mRNA encoding the 14 kDa ubiquitin-conjugating enzyme (E2(14K)), which can catalyse the first irreversible reaction in this pathway, rise and fall in parallel with the rates of proteolysis [Wing and Banville (1994) Am.J. Physiol. 267, E39-E48], indicating that the conjugation of ubiquitin to proteins is a regulated step. To characterize the mechanisms of this regulation, we have examined the effects of insulin, insulin-like growth factor I (IGF-I) and des(1-3) insulin-like growth factor I (DES-IGF-I), which does not bind IGF-binding proteins, on E2(14K) mRNA levels in L6 myotubes. Insulin suppressed levels of E2(14K) mRNA with an IC50 of 4 x 10(-9) M, but had no effects on mRNAs encoding polyubiquitin and proteasome subunits C2 and C8, which, like E2(14K), also increase in skeletal muscle upon fasting. Reduction of E2(14K) mRNA levels was more sensitive to IGF-I with an IC50 of approx. 5 x 10(-10) M. During the incubation of these cells for 12 h there was significant secretion of IGF-I-binding proteins into the medium. DES-IGF-I, which has markedly reduced affinity for these binding proteins, was found to potently reduce E2(14K) mRNA levels with an IC50 of 3 x 10(-11) M. DES-IGF-I did not alter rates of transcription of the E2(14K) gene, but enhanced the rate of degradation of the 1.2 kb mRNA transcript. The half-life of the 1.2 kb transcript was approximately one-third that of the 1.8 kb transcript and can explain the more marked regulation of this transcript observed previously. This indicates that the additional 3' non-coding sequence in the 1.8 kb transcript confers stability. These observations suggest that IGF-I is an important regulator of E2(14K) expression and demonstrate, for the first time, stimulation of degradation of a specific mRNA transcript by this hormone, while overall RNA accumulates. PMID

  13. Effect of nitrogen and phosphorus deficiency on transcriptional regulation of genes encoding key enzymes of starch metabolism in duckweed (Landoltia punctata).

    PubMed

    Zhao, Zhao; Shi, Hui-juan; Wang, Mao-lin; Cui, Long; Zhao, Hai; Zhao, Yun

    2015-01-01

    The production of starch by plants influences their use as biofuels. Nitrogen (N) and phosphorus (P) regulate starch gene expression during plant growth and development, yet the role of key enzymes such as ADP-glucose pyrophosphorylase (E.C. 2.7.7.27 AGPase) in starch metabolism during N- and P-deficiency remains unknown. We investigated the effect of N- and P-deficiency on the expression of large (LeAPL1, LeAPL2, and LeAPL3) and small (LeAPS) subunits of AGPase in duckweed (Landoltia punctata) and their correlation with starch content. We first isolated the full-length cDNA encoding LeAPL1 (GenBank Accession No. KJ603244) and LeAPS (GenBank Accession No. KJ603243); they contained open reading frames of 1554 bp (57.7-kDa polypeptide of 517 amino acids) and 1578 bp (57.0 kDa polypeptide of 525 amino acids), respectively. Real-time PCR analysis revealed that LeAPL1 and LeAPL3 were highly expressed during early stages of N-deficiency, while LeAPL2 was only expressed during late stage. However, in response to P-deficiency, LeAPL1 and LeAPL2 were upregulated during early stages and LeAPL3 was primarily expressed in the late stage. Interestingly, LeAPS was highly expressed following N-deficiency during both stages, but was only upregulated in the early stage after P-deficiency. The activities of AGPase and soluble starch synthesis enzyme (SSS EC 2.4.1.21) were positively correlated with changes in starch content. Furthermore, LeAPL3 and LeSSS (SSS gene) were positively correlated with changes in starch content during N-deficiency, while LeAPS and LeSSS were correlated with starch content in response to P-deficiency. These results elevate current knowledge of the molecular mechanisms underlying starch synthesis.

  14. Inactivation of genes encoding subunits of the peripheral and membrane arms of neurospora mitochondrial complex I and effects on enzyme assembly

    SciTech Connect

    Duarte, M.; Videira, A.; Sousa, R.

    1995-03-01

    We have isolated and characterized the nuclear genes encoding the 12.3-kD subunit of the membrane arm and the 29.9-kD subunit of the peripheral arm of complex I from Neurospora crassa. The former gene was known to be located in linkage group I and the latter is now assigned to linkage group IV of the fungal genome. The genes were separately transformed into different N. crassa strains and transformants with duplicated DNA sequences were isolated. Selected transformants were then mated with other strains to generate repeat-induced point mutations in both copies of the genes present in the nucleus of the parental transformant. From the progeny of the cross, we were then able to recover two individual mutants lacking the 12.3- and 29.9-kD proteins in their mitochondria, mutants nuo12.3 and nuo29.9, respectively. Several other subunits of complex I are present in the mutant organelles, although with altered stoichiometries as compared with those in the wild-type strain. Based on the analysis of Triton-solubilized mitochondrial complexes in sucrose gradients, neither mutant is able to fully assemble complex I. Our results indicate that mutant nuo12.3 separately assembles the peripheral arm and most of the membrane arm of the enzyme. Mutant nuo29.9 seems to accumulate the membrane arm of complex I and to be devoid of the peripheral part. This implicates the 29.9-kD protein in an early step of complex I assembly. 47 refs., 7 figs., 1 tab.

  15. Antioxidant-rich leaf extract of Barringtonia racemosa significantly alters the in vitro expression of genes encoding enzymes that are involved in methylglyoxal degradation III

    PubMed Central

    Kong, Kin Weng; Abdul Aziz, Azlina; Razali, Nurhanani; Aminuddin, Norhaniza

    2016-01-01

    Background Barringtonia racemosa is a medicinal plant belonging to the Lecythidaceae family. The water extract of B. racemosa leaf (BLE) has been shown to be rich in polyphenols. Despite the diverse medicinal properties of B. racemosa, information on its major biological effects and the underlying molecular mechanisms are still lacking. Methods In this study, the effect of the antioxidant-rich BLE on gene expression in HepG2 cells was investigated using microarray analysis in order to shed more light on the molecular mechanism associated with the medicinal properties of the plant. Results Microarray analysis showed that a total of 138 genes were significantly altered in response to BLE treatment (p < 0.05) with a fold change difference of at least 1.5. SERPINE1 was the most significantly up-regulated gene at 2.8-fold while HAMP was the most significantly down-regulated gene at 6.5-fold. Ingenuity Pathways Analysis (IPA) revealed that “Cancer, cell death and survival, cellular movement” was the top network affected by the BLE with a score of 44. The top five canonical pathways associated with BLE were Methylglyoxal Degradation III followed by VDR/RXR activation, TR/RXR activation, PXR/RXR activation and gluconeogenesis. The expression of genes that encode for enzymes involved in methylglyoxal degradation (ADH4, AKR1B10 and AKR1C2) and glycolytic process (ENO3, ALDOC and SLC2A1) was significantly regulated. Owing to the Warburg effect, aerobic glycolysis in cancer cells may increase the level of methylglyoxal, a cytotoxic compound. Conclusions BLE has the potential to be developed into a novel chemopreventive agent provided that the cytotoxic effects related to methylglyoxal accumulation are minimized in normal cells that rely on aerobic glycolysis for energy supply.

  16. Antioxidant-rich leaf extract of Barringtonia racemosa significantly alters the in vitro expression of genes encoding enzymes that are involved in methylglyoxal degradation III

    PubMed Central

    Kong, Kin Weng; Abdul Aziz, Azlina; Razali, Nurhanani; Aminuddin, Norhaniza

    2016-01-01

    Background Barringtonia racemosa is a medicinal plant belonging to the Lecythidaceae family. The water extract of B. racemosa leaf (BLE) has been shown to be rich in polyphenols. Despite the diverse medicinal properties of B. racemosa, information on its major biological effects and the underlying molecular mechanisms are still lacking. Methods In this study, the effect of the antioxidant-rich BLE on gene expression in HepG2 cells was investigated using microarray analysis in order to shed more light on the molecular mechanism associated with the medicinal properties of the plant. Results Microarray analysis showed that a total of 138 genes were significantly altered in response to BLE treatment (p < 0.05) with a fold change difference of at least 1.5. SERPINE1 was the most significantly up-regulated gene at 2.8-fold while HAMP was the most significantly down-regulated gene at 6.5-fold. Ingenuity Pathways Analysis (IPA) revealed that “Cancer, cell death and survival, cellular movement” was the top network affected by the BLE with a score of 44. The top five canonical pathways associated with BLE were Methylglyoxal Degradation III followed by VDR/RXR activation, TR/RXR activation, PXR/RXR activation and gluconeogenesis. The expression of genes that encode for enzymes involved in methylglyoxal degradation (ADH4, AKR1B10 and AKR1C2) and glycolytic process (ENO3, ALDOC and SLC2A1) was significantly regulated. Owing to the Warburg effect, aerobic glycolysis in cancer cells may increase the level of methylglyoxal, a cytotoxic compound. Conclusions BLE has the potential to be developed into a novel chemopreventive agent provided that the cytotoxic effects related to methylglyoxal accumulation are minimized in normal cells that rely on aerobic glycolysis for energy supply. PMID:27635343

  17. Effects of Elevated CO2 on Levels of Primary Metabolites and Transcripts of Genes Encoding Respiratory Enzymes and Their Diurnal Patterns in Arabidopsis thaliana: Possible Relationships with Respiratory Rates

    PubMed Central

    Watanabe, Chihiro K.; Sato, Shigeru; Yanagisawa, Shuichi; Uesono, Yukifumi; Terashima, Ichiro; Noguchi, Ko

    2014-01-01

    Elevated CO2 affects plant growth and photosynthesis, which results in changes in plant respiration. However, the mechanisms underlying the responses of plant respiration to elevated CO2 are poorly understood. In this study, we measured diurnal changes in the transcript levels of genes encoding respiratory enzymes, the maximal activities of the enzymes and primary metabolite levels in shoots of Arabidopsis thaliana grown under moderate or elevated CO2 conditions (390 or 780 parts per million by volume CO2, respectively). We examined the relationships between these changes and respiratory rates. Under elevated CO2, the transcript levels of several genes encoding respiratory enzymes increased at the end of the light period, but these increases did not result in changes in the maximal activities of the corresponding enzymes. The levels of some primary metabolites such as starch and sugar phosphates increased under elevated CO2, particularly at the end of the light period. The O2 uptake rate at the end of the dark period was higher under elevated CO2 than under moderate CO2, but higher under moderate CO2 than under elevated CO2 at the end of the light period. These results indicate that the changes in O2 uptake rates are not directly related to changes in maximal enzyme activities and primary metabolite levels. Instead, elevated CO2 may affect anabolic processes that consume respiratory ATP, thereby affecting O2 uptake rates. PMID:24319073

  18. Cloning and nucleotide sequence of the pvdA gene encoding the pyoverdin biosynthetic enzyme L-ornithine N5-oxygenase in Pseudomonas aeruginosa.

    PubMed Central

    Visca, P; Ciervo, A; Orsi, N

    1994-01-01

    The enzyme L-ornithine N5-oxygenase catalyzes the hydroxylation of L-ornithine (L-Orn), which represents an early step in the biosynthesis of the peptidic moiety of the fluorescent siderophore pyoverdin in Pseudomonas aeruginosa. A gene bank of DNA from P. aeruginosa PAO1 (ATCC 15692) was constructed in the broad-host-range cosmid pLAFR3 and mobilized into the L-Orn N5-oxygenase-defective (pvdA) P. aeruginosa mutant PALS124. Screening for fluorescent transconjugants made it possible to identify the trans-complementing cosmid pPV4, which was able to restore pyoverdin synthesis and L-Orn N5-oxygenase activity in the pvdA mutant PALS124. The 17-kb PAO1 DNA insert of pPV4 contained at least two genetic determinants involved in pyoverdin synthesis, i.e., pvdA and pvdC4, as shown by complementation analysis of a set of mutants blocked in different steps of the pyoverdin biosynthetic pathway. Deletion analysis, subcloning, and transposon mutagenesis enabled us to locate the pvdA gene in a minimum DNA fragment of 1.7 kb flanked by two SphI restriction sites. Complementation of the pvdA mutation was under stringent iron control; both pyoverdin synthesis and L-Orn N5-oxygenase activity were undetectable in cells of the trans-complemented mutant which had been grown in the presence of 100 microM FeCl3. The entire nucleotide sequence of the pvdA gene, from which the primary structure of the encoded polypeptide was deduced, was determined. The pvdA structural gene is 1,278 bp; the cloned DNA fragment contains at the 5' end of the gene a putative ribosome-binding site but apparently lacks known promoterlike sequences. The P. aeruginosa L-Orn N5-oxygenase gene codes for a 426-amino-acid peptide with a predicted molecular mass of 47.7 kDa and an isoelectric point of 8.1. The enzyme shows approximately 50% homology with functional analogs, i.e., L-lysine N6-hydroxylase of aerobactin-producing Escherichia coli and L-Orn N5-oxygenase of ferrichrome-producing Ustilago maydis. The pvd

  19. Tobacco carcinogen-metabolizing genes CYP1A1, GSTM1, and GSTT1 polymorphisms and their interaction with tobacco exposure influence the risk of head and neck cancer in Northeast Indian population.

    PubMed

    Choudhury, Javed Hussain; Singh, Seram Anil; Kundu, Sharbadeb; Choudhury, Biswadeep; Talukdar, Fazlur R; Srivasta, Shilpee; Laskar, Ruhina S; Dhar, Bishal; Das, Raima; Laskar, Shaheen; Kumar, Manish; Kapfo, Wetetsho; Mondal, Rosy; Ghosh, Sankar Kumar

    2015-08-01

    Genetic polymorphisms in tobacco-metabolizing genes may modulate the risk of head and neck cancer (HNC). In Northeast India, head and neck cancers and tobacco consumption remains most prevalent. The aim of the study was to investigate the combined effect of cytochrome P450 1A1 (CYP1A1) T3801C, glutathione S-transferases (GSTs) genes polymorphisms and smoking and tobacco-betel quid chewing in the risk of HNC. The study included 420 subjects (180 cases and 240 controls) from Northeast Indian population. Polymorphisms of CYP1A1 T3801C and GST (M1 & T1) were studied by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and multiplex PCR, respectively. Logistic regression (LR) and multifactor dimensionality reduction (MDR) approach were applied for statistical analysis. LR analysis revealed that subjects carrying CYP1A1 TC/CC + GSTM1 null genotypes had 3.52-fold (P < 0.001) increase the risk of head and neck squamous cell carcinoma (HNSCC). Smokers carrying CYP1A1 TC/CC + GSTM1 null and CYP1A1 TC/CC + GSTT1 null genotypes showed significant association with HNC risk (odds ratio [OR] = 6.42; P < 0.001 and 3.86; P = 0.005, respectively). Similarly, tobacco-betel quid chewers carrying CYP1A1 TC/CC + GSTM1 null genotypes also had several fold increased risk of HNC (P < 0.001). In MDR analysis, the best model for HNSCC risk was the four-factor model of tobacco-betel quid chewing, smoking, CYP1A1 TC/CC, and GSTM1 null genotypes (testing balance accuracy [TBA] = 0.6292; cross-validation consistency [CVC] = 9/10 and P < 0.0001). These findings suggest that interaction of combined genotypes of carcinogen-metabolizing genes with environmental factors might modulate susceptibility of HNC in Northeast Indian population.

  20. The Protein Phosphatases of Synechocystis sp. Strain PCC 6803: Open Reading Frames sll1033 and sll1387 Encode Enzymes That Exhibit both Protein-Serine and Protein-Tyrosine Phosphatase Activity In Vitro.

    SciTech Connect

    Li, Ruiliang; Potters, M B.; Shi, Liang; Kennelly, Peter J.

    2005-09-01

    The open reading frames (ORFs) encoding two potential protein-serine/threonine phosphatases from the cyanobacterium Synechocystis sp. strain PCC 6803 were cloned and their protein products expressed in Escherichia coli cells. The product of ORF sll1033, SynPPM3, is a homologue of the PPM family of protein-serine/threonine phosphatases found in all eukaryotes as well as many members of the Bacteria. Surprisingly, the recombinant protein phosphatase dephosphorylated phosphotyrosine- as well as phosphoserine-containing proteins in vitro. While kinetic analyses indicate that the enzyme was more efficient at dephosphorylating the latter, replacement of Asp(608) by asparagine enhanced activity toward a phosphotyrosine-containing protein fourfold. The product of ORF sll1387, SynPPP1, is the sole homolog of the PPP family of protein phosphatases encoded by the genome of Synechocystis sp. strain PCC 6803. Like many other bacterial PPPs, the enzyme dephosphorylated phosphoserine- and phosphotyrosine-containing proteins with comparable efficiencies. However, while previously described PPPs from prokaryotic organisms required the addition of exogenous metal ion cofactors, such as Mg(2+) or Mn(2+), for activity, recombinantly produced SynPPP1 displayed near-maximal activity in the absence of added metals. Inductively coupled plasma mass spectrometry indicated that recombinant SynPPP1 contained significant quantities, 0.32 to 0.44 mol/mole total, of Mg and Mn. In this respect, the cyanobacterial enzyme resembled eukaryotic members of the PPP family, which are metalloproteins. mRNA encoding SynPPP1 or SynPPM3 could be detected in cells grown under many, but not all, environmental conditions.

  1. The Aspergillus niger D-xylulose kinase gene is co-expressed with genes encoding arabinan degrading enzymes, and is essential for growth on D-xylose and L-arabinose.

    PubMed

    vanKuyk, P A; de Groot, M J; Ruijter, G J; de Vries, R P; Visser, J

    2001-10-01

    The Aspergillus niger D-xylulose kinase encoding gene has been cloned by complementation of a strain deficient in D-xylulose kinase activity. Expression of xkiA was observed in the presence of L-arabinose, L-arabitol and D-xylose. Expression of xkiA is not mediated by XLNR, the xylose-dependent positively-acting xylanolytic regulator. Although the expression of xkiA is subject to carbon catabolite repression, the wide domain regulator CREA is not directly involved. The A. niger D-xylulose kinase was purified to homogeneity, and the molecular mass determined using electrospray ionization mass spectrometry agreed with the calculated molecular mass of 62816.6 Da. The activity of XKIA is highly specific for D-xylulose. Kinetic parameters were determined as Km(D-xylulose) = 0.76 mM and Km(ATP) = 0.061 mM. Increased transcript levels of the genes encoding arabinan and xylan degrading enzymes, observed in the xylulose kinase deficient strain, correlate with increased accumulation of L-arabitol and xylitol, respectively. This result supports the suggestion that L-arabitol may be the specific low molecular mass inducer of the genes involved in arabinan degradation. It also suggests a possible role for xylitol in the induction of xylanolytic genes. Conversely, overproduction of XKIA did not reduce the size of the intracellular arabitol and xylitol pools, and therefore had no effect on expression of genes encoding xylan and arabinan degrading enzymes nor on the activity of the enzymes of the catabolic pathway.

  2. The influence of a polymorphism in the gene encoding angiotensin converting enzyme (ACE) on treatment outcomes in late-onset Pompe patients receiving alglucosidase alfa.

    PubMed

    Baek, Rena C; Palmer, Rachel; Pomponio, Robert J; Lu, Yuefeng; Ma, Xiwen; McVie-Wylie, Alison J

    2016-09-01

    Correlations between angiotensin-converting enzyme (ACE) genotype (I/I, I/D, D/D), disease severity at baseline and response to enzyme replacement therapy (ERT) were assessed in the Pompe disease Late-Onset Treatment Study (LOTS). No correlations were observed between ACE genotype and disease severity at baseline. However, D/D patients appeared to have a reduced response to alglucosidase alfa treatment than I/I or I/D patients, suggesting that ACE polymorphisms may influence the response to alglucosidase alfa treatment and warrants further investigation. PMID:27489778

  3. Cloning and expression of a cDNA encoding betanidin 5-O-glucosyltransferase, a betanidin- and flavonoid-specific enzyme with high homology to inducible glucosyltransferases from the Solanaceae.

    PubMed

    Vogt, T; Grimm, R; Strack, D

    1999-09-01

    Based on protein sequence data and RT-PCR, a full length cDNA encoding betanidin 5-O-glucosyltransferase (5-GT) was obtained from a cDNA library of Dorotheanthus bellidiformis (Burm.f.) N.E.Br. (Aizoaceae). 5-GT catalyses the transfer of glucose from UDP-glucose to the 5-hydroxyl group of the chromogenic betanidin. Betanidin and its conjugates, referred to as betacyanins, are characteristic fruit and flower pigments in most members of the Caryophyllales, which fail to synthesise anthocyanins. The 5-GT cDNA displayed homology to previously published glucosyltransferase sequences and exhibited high identity to sequences of several inducible glucosyltransferases of tobacco and tomato (Solanaceae). The open reading frame encodes a polypeptide of 489 amino acids with a calculated molecular mass of 55.24 kDa. The corresponding cDNA was expressed in Escherichia coli. The recombinant protein displayed identical substrate specificity compared to the native enzyme purified from D. bellidiformis cell suspension cultures. In addition to the natural substrate betanidin, ortho-dihydroxylated flavonols and flavones were glycosylated preferentially at the B-ring 4'-hydroxyl group. 5-GT is the first enzyme of betalain biosynthesis in plants, of which the corresponding cDNA has been cloned and expressed. The results are discussed in relation to molecular evolution of plant glucosyl- transferases. PMID:10504573

  4. Crown gall oncogenesis: evidence that a T-DNA gene from the Agrobacterium Ti plasmid pTiA6 encodes an enzyme that catalyzes synthesis of indoleacetic acid.

    PubMed Central

    Thomashow, L S; Reeves, S; Thomashow, M F

    1984-01-01

    Stable incorporation of tumor-inducing (Ti) plasmid sequences, the T-DNA, into the genomes of dicotyledonous plants results in the formation of crown gall tumors. Previous genetic studies have suggested that the products of the genes encoding transcripts 1 and 2, which are encoded by the TL-DNA region of pTiA6, are responsible for inducing the auxin-independent phenotype of crown gall tissues. Here we report the construction of a plasmid, pMTlacT2, which directs the synthesis of the Mr 49,800 polypeptide encoded by the transcript 2 gene. Cell-free extracts prepared from Escherichia coli harboring this plasmid converted indoleacetamide to indoleacetic acid, the natural auxin of plants; extracts prepared from plasmidless strains of E. coli or strains harboring the cloning vehicle pBR322 did not carry out this reaction. We conclude that the transcript 2 gene of pTiA6 codes for an enzyme that participates in auxin biosynthesis, probably an indoleacetamide hydrolase. Images PMID:6089175

  5. Overexpression of erg20 gene encoding farnesyl pyrophosphate synthase has contrasting effects on activity of enzymes of the dolichyl and sterol branches of mevalonate pathway in Trichoderma reesei.

    PubMed

    Piłsyk, Sebastian; Perlińska-Lenart, Urszula; Górka-Nieć, Wioletta; Graczyk, Sebastian; Antosiewicz, Beata; Zembek, Patrycja; Palamarczyk, Grażyna; Kruszewska, Joanna S

    2014-07-10

    The mevalonate pathway is the most diverse metabolic route resulting in the biosynthesis of at least 30,000 isoprenoid compounds, many of which, such as sterols or dolichols, are indispensable for living cells. In the filamentous fungus Trichoderma of major biotechnological interest isoprenoid metabolites are also involved in the biocontrol processes giving the mevalonate pathway an additional significance. On the other hand, little is known about genes coding for enzymes of the mevalonate pathway in Trichoderma. Here, we present cloning and functional analysis of the erg20 gene from Trichoderma reesei coding for farnesyl pyrophosphate (FPP) synthase (EC 2.5.1.10), an enzyme located at the branching point of the mevalonate pathway. Expression of the gene in a thermosensitive erg20-2 mutant of Saccharomyces cerevisiae impaired in the FPP synthase activity suppressed the thermosensitive phenotype. The same gene overexpressed in T. reesei significantly enhanced the FPP synthase activity and also stimulated the activity of cis-prenyltransferase, an enzyme of the dolichyl branch of the mevalonate pathway. Unexpectedly, the activity of squalene synthase from the other, sterol branch, was significantly decreased without, however, affecting ergosterol level.

  6. Cloning and characterization of Vitis vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase, a homologue of the enzyme encoded by the maize Bronze-1 locus that may primarily serve to glucosylate anthocyanidins in vivo.

    PubMed

    Ford, C M; Boss, P K; Hoj, P B

    1998-04-10

    We report here the cloning and optimized expression at 16 degrees C and the characterization of a Vitis vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase, an enzyme responsible for a late step in grapevine anthocyanin biosynthesis. The properties of this and other UDP-glucose:flavonoid 3-O-glucosyltransferases, homologues of the product encoded by the maize Bronze-1 locus, are a matter of conjecture. The availability of a purified recombinant enzyme allowed for the unambiguous determination of the characteristics of a flavonoid 3-O-glucosyltransferase. Kinetic analyses showed that kcat for glucosylation of cyanidin, an anthocyanidin substrate, is 48 times higher than for glucosylation of the flavonol quercetin, whereas Km values are similar for both substrates. Activity toward other classes of substrates is absent. Cu2+ ions strongly inhibit the action of this and other glucosyltransferases; however, we suggest that this phenomenon in large part is due to Cu2+-mediated substrate degradation rather than inhibition of the enzyme. Additional lines of complementary biochemical data also indicated that in the case of V. vinifera, the principal, if not only, role of UDP-glucose:flavonoid 3-O-glucosyltransferases is to glucosylate anthocyanidins in red fruit during ripening. Other glucosyltransferases with a much higher relative activity toward quercetin are suggested to glucosylate flavonols in a distinct spatial and temporal pattern. It should be considered whether gene products homologous to Bronze-1 in some cases more accurately should be referred to as UDP-glucose:anthocyanidin 3-O-glucosyltransferases.

  7. Expression of genes encoding enzymes involved in the one carbon cycle in rat placenta is determined by maternal micronutrients (folic acid, vitamin B12) and omega-3 fatty acids.

    PubMed

    Khot, Vinita; Kale, Anvita; Joshi, Asmita; Chavan-Gautam, Preeti; Joshi, Sadhana

    2014-01-01

    We have reported that folic acid, vitamin B12, and omega-3 fatty acids are interlinked in the one carbon cycle and have implications for fetal programming. Our earlier studies demonstrate that an imbalance in maternal micronutrients influence long chain polyunsaturated fatty acid metabolism and global methylation in rat placenta. We hypothesize that these changes are mediated through micronutrient dependent regulation of enzymes in one carbon cycle. Pregnant dams were assigned to six dietary groups with varying folic acid and vitamin B12 levels. Vitamin B12 deficient groups were supplemented with omega-3 fatty acid. Placental mRNA levels of enzymes, levels of phospholipids, and glutathione were determined. Results suggest that maternal micronutrient imbalance (excess folic acid with vitamin B12 deficiency) leads to lower mRNA levels of methylene tetrahydrofolate reductase (MTHFR) and methionine synthase , but higher cystathionine b-synthase (CBS) and Phosphatidylethanolamine-N-methyltransferase (PEMT) as compared to control. Omega-3 supplementation normalized CBS and MTHFR mRNA levels. Increased placental phosphatidylethanolamine (PE), phosphatidylcholine (PC), in the same group was also observed. Our data suggests that adverse effects of a maternal micronutrient imbalanced diet may be due to differential regulation of key genes encoding enzymes in one carbon cycle and omega-3 supplementation may ameliorate most of these changes.

  8. Functional expression of a cDNA encoding pea (Pisum sativum L.) raffinose synthase, partial purification of the enzyme from maturing seeds, and steady-state kinetic analysis of raffinose synthesis.

    PubMed

    Peterbauer, Thomas; Mach, Lukas; Mucha, Jan; Richter, Andreas

    2002-09-01

    Raffinose (O-alpha- D-galactopyranosyl-(1-->6)- O-alpha- D-glucopyranosyl-(1<-->2)- O-beta- D-fructofuranoside) is a widespread oligosaccharide in plant seeds and other tissues. Raffinose synthase (EC 2.4.1.82) is the key enzyme that channels sucrose into the raffinose oligosaccharide pathway. We here report on the isolation of a cDNA encoding for raffinose synthase from maturing pea ( Pisum sativum L.) seeds. The coding region of the cDNA was expressed in Spodoptera frugiperda Sf21 insect cells. The recombinant enzyme, a protein of glycoside hydrolase family 36, displayed similar kinetic properties to raffinose synthase partially purified from maturing seeds by anion-exchange and size-exclusion chromatography. Apart from the natural galactosyl donor galactinol ( O-alpha- D-galactopyranosyl-(1-->1)- L- myo-inositol), p-nitrophenyl alpha- D-galactopyranoside, an artificial substrate, was utilized as a galactosyl donor. An equilibrium constant of 4.1 was determined for the galactosyl transfer reaction from galactinol to sucrose. Steady-state kinetic analysis suggested that raffinose synthase is a transglycosidase operating by a ping-pong reaction mechanism and may also act as a glycoside hydrolase. The enzyme was strongly inhibited by 1-deoxygalactonojirimycin, a potent inhibitor for alpha-galactosidases (EC 3.2.1.22). The physiological implications of these observations are discussed.

  9. Encoding Dictionaries.

    ERIC Educational Resources Information Center

    Ide, Nancy

    1995-01-01

    Describes problems in devising a Text Encoding Initiative (TEI) encoding format for dictionaries. Asserts that the high degree of structuring and compression of information are among the most complex text types treated in the TEI. Concludes that the source of some TEI problems lies in the design of Standard Generalized Markup Language (SGML). (CFR)

  10. Isolation of a gene encoding a cellulolytic enzyme from swamp buffalo rumen metagenomes and its cloning and expression in Escherichia coli.

    PubMed

    Cheema, Tanzeem Akbar; Jirajaroenrat, Kanya; Sirinarumitr, Theerapol; Rakshit, Sudip K

    2012-01-01

    Ruminants are capable of hydrolyzing lignocellulosic residues to absorbable sugars by virtue of the microbial communities residing in their rumen. However, large sections of such microbial communities are not yet culturable using conventional laboratory techniques. Therefore in the present study, the metagenomic DNA of swamp buffalo (Bubalus bubalis) rumen contents was explored using culture-independent techniques. The consensus regions of glycosyl hydrolase 5 (GH5) family of cellulases were used as primers for PCR amplification. A full-length metagenomic cellulase gene, Umcel5B29, with a complete open reading frame (ORF) of 1611 bp was identified. The similarity search analysis revealed that Umcel5B29 is closely related to the cellulases (73% to 98% similarity) of ruminal unculturable microorganisms, indicating its phylogenetic origin. Further analysis indicated that Umcel5B29 does not contain a carbohydrate binding module (CBM). Subsequently, Umcel5B29 was overexpressed in Escherichia coli. The recombinant enzyme worked optimally at pH 5.5 and 45°C, a condition similar to the buffalo's rumen. However, the enzyme retained more than 70% of its maximal activity after incubation at pH 4-7 and more than 50% maximal activity after incubation at 30-60°C for 30 min. These characteristics render Umcel5B29 as a potential candidate for the bio-stoning process of denim.

  11. The Fus3/Kss1 MAP kinase homolog Amk1 regulates the expression of genes encoding hydrolytic enzymes in Alternaria brassicicola.

    PubMed

    Cho, Yangrae; Cramer, Robert A; Kim, Kwang-Hyung; Davis, Josh; Mitchell, Thomas K; Figuli, Patricia; Pryor, Barry M; Lemasters, Emily; Lawrence, Christopher B

    2007-06-01

    Mitogen-activated protein (MAP) kinases have been shown to be required for virulence in diverse phytopathogenic fungi. To study its role in pathogenicity, we disrupted the Amk1 MAP kinase gene, a homolog of the Fus3/Kss1 MAP kinases in Saccharomyces cerevisiae, in the necrotrophic Brassica pathogen, Alternaria brassicicola. The amk1 disruption mutants showed null pathogenicity on intact host plants. However, amk1 mutants were able to colonize host plants when they were inoculated on a physically damaged host surface, or when they were inoculated along with nutrient supplements. On intact plants, mutants expressed extremely low amounts of several hydrolytic enzyme genes that were induced over 10-fold in the wild-type during infection. These genes were also dramatically induced in the mutants on wounded plants. These results imply a correlation between virulence and the expression level of specific hydrolytic enzyme genes plus the presence of an unidentified pathway controlling these genes in addition to or in conjunction with the Amk1 pathway. PMID:17280842

  12. Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme.

    PubMed Central

    Fox, J D; Kerby, R L; Roberts, G P; Ludden, P W

    1996-01-01

    In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing. PMID:8626276

  13. Pp6-FEH1 encodes an enzyme for degradation of highly polymerized levan and is transcriptionally induced by defoliation in timothy (Phleum pratense L.)

    PubMed Central

    Tamura, Ken-ichi; Sanada, Yasuharu; Tase, Kazuhiro; Komatsu, Toshinori; Yoshida, Midori

    2011-01-01

    The ability of grasses to regrow after defoliation by cutting or grazing is a vital factor in their survival and an important trait when they are used as forage crops. In temperate grass species accumulating fructans, defoliation induces the activity of a fructan exohydrolase (FEH) that degrades fructans to serve as a carbon source for regrowth. Here, a cDNA from timothy was cloned, named Pp6-FEH1, that showed similarity to wheat fructan 6-exohydrolase (6-FEH). The recombinant enzyme expressed in Pichia pastoris completely degraded fructans that were composed mainly of β(2,6)-linked and linear fructans (levan) with a high degree of polymerization (DP) in the crown tissues of timothy. The substrate specificity of Pp6-FEH1 differed from previously characterized enzymes with 6-FEH activity in fructan-accumulating plants: (i) Pp6-FEH1 showed 6-FEH activity against levan (mean DP 20) that was 4-fold higher than against 6-kestotriose (DP 3), indicating that Pp6-FEH1 has a preference for β(2,6)-linked fructans with high DP; (ii) Pp6-FEH1 had significant activity against β(2,1)-linked fructans, but considerably less than against β(2,6)-linked fructans; (iii) Pp6-FEH1 had weak invertase activity, and its 6-FEH activity was inhibited slightly by sucrose. In the stubble of seedlings and in young haplocorms from adult timothy plants, transcripts of Pp6-FEH1 were significantly increased within 3 h of defoliation, followed by an increase in 6-FEH activity and in the degradation of fructans. These results suggest that Pp6-FEH1 plays a role in the degradation of fructans and the mobilization of carbon sources for regrowth after defoliation in timothy. PMID:21317211

  14. Pp6-FEH1 encodes an enzyme for degradation of highly polymerized levan and is transcriptionally induced by defoliation in timothy (Phleum pratense L.).

    PubMed

    Tamura, Ken-ihi; Sanada, Yasuharu; Tase, Kazuhiro; Komatsu, Toshinori; Yoshida, Midori

    2011-06-01

    The ability of grasses to regrow after defoliation by cutting or grazing is a vital factor in their survival and an important trait when they are used as forage crops. In temperate grass species accumulating fructans, defoliation induces the activity of a fructan exohydrolase (FEH) that degrades fructans to serve as a carbon source for regrowth. Here, a cDNA from timothy was cloned, named Pp6-FEH1, that showed similarity to wheat fructan 6-exohydrolase (6-FEH). The recombinant enzyme expressed in Pichia pastoris completely degraded fructans that were composed mainly of β(2,6)-linked and linear fructans (levan) with a high degree of polymerization (DP) in the crown tissues of timothy. The substrate specificity of Pp6-FEH1 differed from previously characterized enzymes with 6-FEH activity in fructan-accumulating plants: (i) Pp6-FEH1 showed 6-FEH activity against levan (mean DP 20) that was 4-fold higher than against 6-kestotriose (DP 3), indicating that Pp6-FEH1 has a preference for β(2,6)-linked fructans with high DP; (ii) Pp6-FEH1 had significant activity against β(2,1)-linked fructans, but considerably less than against β(2,6)-linked fructans; (iii) Pp6-FEH1 had weak invertase activity, and its 6-FEH activity was inhibited slightly by sucrose. In the stubble of seedlings and in young haplocorms from adult timothy plants, transcripts of Pp6-FEH1 were significantly increased within 3 h of defoliation, followed by an increase in 6-FEH activity and in the degradation of fructans. These results suggest that Pp6-FEH1 plays a role in the degradation of fructans and the mobilization of carbon sources for regrowth after defoliation in timothy.

  15. Isolation of the patC gene encoding the cystathionine beta-lyase of Lactobacillus delbrueckii subsp. bulgaricus and molecular analysis of inter-strain variability in enzyme biosynthesis.

    PubMed

    Aubel, Dominique; Germond, Jacques Edouard; Gilbert, Christophe; Atlan, Danièle

    2002-07-01

    The patC gene encoding the cystathionine beta-lyase (CBL) of Lactobacillus delbrueckii subsp. bulgaricus NCDO 1489 was cloned and expressed in Escherichia coli. Overexpression of CBL complemented the methionine auxotrophy of an E. coli metC mutant, demonstrating in vivo that this enzyme functions as a CBL. However, PatC is distinguishable from the MetC CBLs by a low identity in amino acid sequence, a sensitivity to iodoacetic acid, greater thermostability and a lower substrate affinity. Homologues of patC were detected in the 13 Lb. delbrueckii strains studied, but only seven of them showed CBL activity. In constrast to CBL(+) strains, all CBL-deficient strains analysed were auxotrophic for methionine. This supports the hypothesis that CBLs from lactobacilli are probably involved in methionine biosynthesis. Moreover, the results of this study suggest that post-transcriptional mechanisms account for the differences in CBL activities observed between strains of Lb. delbrueckii.

  16. An Outer Membrane Enzyme Encoded by Salmonella typhimurium lpxR That Removes the 3′-Acyloxyacyl Moiety of Lipid A*

    PubMed Central

    Reynolds, C. Michael; Ribeiro, Anthony A.; McGrath, Sara C.; Cotter, Robert J.; Raetz, Christian R. H.; Trent, M. Stephen

    2009-01-01

    The Salmonella and related bacteria modify the structure of the lipid A portion of their lipopolysaccharide in response to environmental stimuli. Some lipid A modifications are required for virulence and resistance to cationic antimicrobial peptides. We now demonstrate that membranes of Salmonella typhimurium contain a novel hydrolase that removes the 3′-acyloxyacyl residue of lipid A in the presence of 5 mm Ca2+. We have identified the gene encoding the S. typhimurium lipid A 3′-O-deacylase, designated lpxR, by screening an ordered S. typhimurium genomic DNA library, harbored in Escherichia coli K-12, for expression of Ca2+-dependent 3′-O-deacylase activity in membranes. LpxR is synthesized with an N-terminal type I signal peptide and is localized to the outer membrane. Mass spectrometry was used to confirm the position of lipid A deacylation in vitro and the release of the intact 3′-acyloxyacyl group. Heterologous expression of lpxR in the E. coli K-12 W3110, which lacks lpxR, resulted in production of significant amounts of 3′-O-deacylated lipid A in growing cultures. Orthologues of LpxR are present in the genomes of E. coli 0157:H7, Yersinia enterocolitica, Helicobacter pylori, and Vibrio cholerae. The function of LpxR is unknown, but it could play a role in pathogenesis because it might modulate the cytokine response of an infected animal. PMID:16704973

  17. Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae.

    PubMed Central

    Berka, R M; Schneider, P; Golightly, E J; Brown, S H; Madden, M; Brown, K M; Halkier, T; Mondorf, K; Xu, F

    1997-01-01

    A genomic DNA segment encoding an extracellular laccase was isolated from the thermophilic fungus Myceliophthora thermophila, and the nucleotide sequence of this gene was determined. The deduced amino acid sequence of M. thermophila laccase (MtL) shows homology to laccases from diverse fungal genera. A vector containing the M. thermophila laccase coding region, under transcriptional control of an Aspergillus oryzae alpha-amylase gene promoter and terminator, was constructed for heterologous expression in A. oryzae. The recombinant laccase expressed in A. oryzae was purified to electrophoretic homogeneity by anion-exchange chromatography. Amino-terminal sequence data suggests that MtL is synthesized as a preproenzyme. The molecular mass was estimated to be approximately 100 to 140 kDa by gel filtration on Sephacryl S-300 and to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that MtL contains 40 to 60% glycosylation. The laccase shows an absorbance spectrum that is typical of blue copper oxidases, with maxima at 276 and 589 nm, and contains 3.9 copper atoms per subunit. With syringaldazine as a substrate, MtL has optimal activity at pH 6.5 and retains nearly 100% of its activity when incubated at 60 degrees C for 20 min. This is the first report of the cloning and heterologous expression of a thermostable laccase. PMID:9251203

  18. TRANSPARENT TESTA10 Encodes a Laccase-Like Enzyme Involved in Oxidative Polymerization of Flavonoids in Arabidopsis Seed CoatW⃞

    PubMed Central

    Pourcel, Lucille; Routaboul, Jean-Marc; Kerhoas, Lucien; Caboche, Michel; Lepiniec, Loïc; Debeaujon, Isabelle

    2005-01-01

    The Arabidopsis thaliana transparent testa10 (tt10) mutant exhibits a delay in developmentally determined browning of the seed coat, also called the testa. Seed coat browning is caused by the oxidation of flavonoids, particularly proanthocyanidins, which are polymers of flavan-3-ol subunits such as epicatechin and catechin. The tt10 mutant seeds accumulate more epicatechin monomers and more soluble proanthocyanidins than wild-type seeds. Moreover, intact testa cells of tt10 cannot trigger H2O2-independent browning in the presence of epicatechin and catechin, in contrast with wild-type cells. UV–visible light detection and mass spectrometry revealed that the major oxidation products obtained with epicatechin alone are yellow dimers called dehydrodiepicatechin A. These products differ from proanthocyanidins in the nature and position of their interflavan linkages. Flavonol composition was also affected in tt10 seeds, which exhibited a higher ratio of quercetin rhamnoside monomers versus dimers than wild-type seeds. We identified the TT10 gene by a candidate gene approach. TT10 encodes a protein with strong similarity to laccase-like polyphenol oxidases. It is expressed essentially in developing testa, where it colocalizes with the flavonoid end products proanthocyanidins and flavonols. Together, these data establish that TT10 is involved in the oxidative polymerization of flavonoids and functions as a laccase-type flavonoid oxidase. PMID:16243908

  19. Characterization of the genes encoding the 3-carboxy-cis,cis-muconate-lactonizing enzymes from the 4-sulfocatechol degradative pathways of Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2.

    PubMed

    Halak, Sad; Basta, Tamara; Bürger, Sibylle; Contzen, Matthias; Stolz, Andreas

    2006-11-01

    Hydrogenophaga intermedia strain S1 and Agrobacterium radiobacter strain S2 form a mixed bacterial culture which degrades sulfanilate (4-aminobenzenesulfonate) by a novel variation of the beta-ketoadipate pathway via 4-sulfocatechol and 3-sulfomuconate. It was previously proposed that the further metabolism of 3-sulfomuconate is catalysed by modified 3-carboxy-cis,cis-muconate-lactonizing enzymes (CMLEs) and that these 'type 2' enzymes were different from the conventional CMLEs ('type 1') from the protocatechuate pathway in their ability to convert 3-sulfomuconate in addition to 3-carboxy-cis,cis-muconate. In the present study the genes for two CMLEs (pcaB2S1 and pcaB2S2) were cloned from H. intermedia S1 and A. radiobacter S2, respectively. In both strains, these genes were located close to the previously identified genes encoding the 4-sulfocatechol-converting enzymes. The gene products of pcaB2S1 and pcaB2S2 were therefore tentatively identified as type 2 enzymes involved in the metabolism of 3-sulfomuconate. The genes were functionally expressed and the gene products were shown to convert 3-carboxy-cis,cis-muconate and 3-sulfomuconate. 4-Carboxymethylene-4-sulfo-but-2-en-olide (4-sulfomuconolactone) was identified by HPLC-MS as the product, which was enzymically formed from 3-sulfomuconate. His-tagged variants of both CMLEs were purified and compared with the CMLE from the protocatechuate pathway of Pseudomonas putida PRS2000 for the conversion of 3-carboxy-cis,cis-muconate and 3-sulfomuconate. The CMLEs from the 4-sulfocatechol pathway converted 3-sulfomuconate with considerably higher activities than 3-carboxy-cis,cis-muconate. Also the CMLE from P. putida converted 3-sulfomuconate, but this enzyme demonstrated a clear preference for 3-carboxy-cis,cis-muconate as substrate. Thus it was demonstrated that in the 4-sulfocatechol pathway, distinct CMLEs are formed, which are specifically adapted for the preferred conversion of sulfonated substrates.

  20. Phylogenetic Relationships and Functional Genes: Distribution of a Gene (mnxG) Encoding a Putative Manganese-Oxidizing Enzyme in Bacillus Species▿

    PubMed Central

    Mayhew, Lisa E.; Swanner, Elizabeth D.; Martin, Andy P.; Templeton, Alexis S.

    2008-01-01

    Several Bacillus and Paenibacillus species were isolated from Fe and Mn oxide minerals precipitating at a deep subsurface oxic-anoxic interface at Henderson Molybdenum Mine, Empire, CO. The isolates were investigated for their Mn(II)-oxidizing potential and interrogated for possession of the mnxG gene, a gene that codes for a putative Mn(II)-oxidizing enzyme in Bacillus species. Seven of eight Bacillus species were capable of Mn(II) oxidation; however, the mnxG gene was detected in only one isolate. Using sequences of known Bacillus species both with and without amplifiable mnxG genes and Henderson Mine isolates, the 16S rRNA and mnxG gene phylogenies were compared to determine if 16S rRNA sequences could be used to predict the presence or absence of an amplifiable mnxG gene within the genomes of the isolates. We discovered a strong correspondence between 16S rRNA sequence similarity and the presence/absence of an amplifiable mnxG gene in the isolates. The data revealed a complex phylogenetic distribution of the mnxG gene in which vertical inheritance and gene loss influence the distribution of the gene among the Bacillus species included in this study. Comparisons of 16S rRNA and functional gene phylogenies can be used as a tool to aid in unraveling the history and dispersal of the mnxG gene within the Bacillus clade. PMID:18849460

  1. Construction and co-expression of plasmid encoding xylitol dehydrogenase and a cofactor regeneration enzyme for the production of xylitol from D-arabitol.

    PubMed

    Zhou, Peng; Li, Sha; Xu, Hong; Feng, Xiaohai; Ouyang, Pingkai

    2012-07-15

    The biotransformation of D-arabitol into xylitol was investigated with focus on the conversion of D-xylulose into xylitol. This critical conversion was accomplished using Escherichia coli to co-express a xylitol dehydrogenase gene from Gluconobacter oxydans and a cofactor regeneration enzyme gene which was a glucose dehydrogenase gene from Bacillus subtilis for system 1 and an alcohol dehydrogenase gene from G. oxydans for system 2. Both systems efficiently converted D-xylulose into xylitol without the addition of expensive NADH. Approximately 26.91 g/L xylitol was obtained from around 30 g/L D-xylulose within system 1 (E. coli Rosetta/Duet-xdh-gdh), with a 92% conversion yield, somewhat higher than that of system 2 (E. coli Rosetta/Duet-xdh-adh, 24.9 g/L, 85.2%). The xylitol yields for both systems were more than 3-fold higher compared to that of the G. oxydans NH-10 cells (7.32 g/L). The total turnover number (TTN), defined as the number of moles of xylitol formed per mole of NAD(+), was 32,100 for system 1 and 17,600 for system 2. Compared with that of G. oxydans NH-10, the TTN increased by 21-fold for system 1 and 11-fold for system 2, hence, the co-expression systems greatly enhanced the NADH supply for the conversion, benefiting the practical synthesis of xylitol.

  2. Variation in AKR1C3, which Encodes the Neuroactive Steroid Synthetic Enzyme 3α-HSD Type 2 (17β-HSD Type 5), Moderates the Subjective Effects of Alcohol

    PubMed Central

    Milivojevic, Verica; Feinn, Richard; Kranzler, Henry R.; Covault, Jonathan

    2014-01-01

    Rationale Animal models suggest that neuroactive steroids contribute to alcohol’s acute effects. We previously reported that a common non-synonymous polymorphism, AKR1C3*2 in the gene encoding the enzyme 3α-HSD2/17β-HSD5 and a synonymous SNP, rs248793, in SRD5A1, which encodes 5α-reductase, were associated with alcohol dependence (AD). Objectives To investigate whether these polymorphisms moderate subjective effects of alcohol in humans and whether AKR1C3*2 affects neuroactive steroid synthesis. Methods 65 Caucasian men (34 lighter and 31 heavier drinkers; mean age 26.2 y) participated in a double-blind laboratory study where they consumed drinks containing no ethanol or 0.8 g/kg of ethanol. Breath alcohol, heart rate (HR), and self-reported alcohol effects were measured at 40-min intervals and genotype was examined as a moderator of alcohol’s effects. Levels of the neuroactive steroid 5α-androstane-3α,17β-diol and its precursors, 3α,5α-androsterone and dihydrotestosterone, were measured at study entry using GC/MS. Results Initially, carriers of the AD-protective AK1C3*2 G-allele had higher levels of 5α-androstane-3α,17β-diol relative to the precursor 3α,5α-androsterone than C-allele homozygotes. AKR1C3*2 G-allele carriers exhibited greater increases in heart rate and stimulant and sedative effects of alcohol than C-allele homozygotes. The genotype effects on sedation were observed only in heavier drinkers. The only effect of the SRD5A1 SNP was to moderate HR. There were no interactive effects of the two SNPs. Conclusions The observed effects of variation in a gene encoding a neuroactive steroid biosynthetic enzyme on the rate of 17p–reduction of androsterone relative to androstanediol and on alcohol’s sedative effects may help to explain the association of AKR1C3*2 with AD. PMID:24838369

  3. FUNCTIONAL EFFECTS OF POLYMORPHISMS IN THE HUMAN GENE ENCODING 11β-HSD1: A SEQUENCE VARIANT AT THE TRANSLATION START OF 11β-HSD1 ALTERS ENZYME LEVELS

    PubMed Central

    Malavasi, Elise L.V.; Kelly, Val; Nath, Nikita; Gambineri, Alessandra; Dakin, Rachel S.; Pagotto, Uberto; Pasquali, Renato; Walker, Brian R.; Chapman, Karen E.

    2014-01-01

    Regeneration of active glucocorticoids within liver and adipose tissue by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) may be of pathophysiological importance in obesity and Metabolic Syndrome and is a therapeutic target in type 2 diabetes. Polymorphisms in HSD11B1, the gene encoding 11β-HSD1, have been associated with metabolic phenotype in humans, including type 2 diabetes and hypertension. Here we have tested the functional consequences of 2 single nucleotide polymorphisms located in contexts that potentially affect tissue levels of 11β-HSD1. We report no effect of allelic variation at rs846910, a polymorphism within the 5′-flanking region of the gene on HSD11B1 promoter activity in vitro. However, compared to the common G allele, the A allele of rs13306421, a polymorphism located 2 nucleotides 5′ to the translation initiation site, gave higher 11β-HSD1 expression and activity in vitro and was translated at higher levels in in vitro translation reactions, possibly associated with a lower frequency of “leaky scanning”. These data suggest that this polymorphism may have direct functional consequences on levels of 11β-HSD1 enzyme activity in vivo. However, the rs13306421 A sequence variant originally reported in other ethnic groups may be of low prevalence as it was not detected in a population of 600 European caucasian women. PMID:19934376

  4. Targeted disruption of the gene encoding the classical nitroreductase enzyme in Salmonella typhimurium Ames test strains TA1535 and TA1538.

    PubMed

    Yamada, M; Espinosa-Aguirre, J J; Watanabe, M; Matsui, K; Sofuni, T; Nohmi, T

    1997-04-14

    The gene encoding the 'classical nitroreductase' (CNR) of Salmonella typhimurium was disrupted. In this manner, cnr null mutant derivatives of strains TA1535 and TA1538 were constructed, and named YG7131 and YG7127, respectively. In both strain backgrounds, cnr gene disruption reduced nitrofurazone-reductase activity. This reduction almost completely eliminated the nitroreductase activity of strain TA1538. In contrast, the nitroreductase activity of strain TA1535 was much higher than that in TA1538. In this background, cnr gene disruption resulted in a reduction in nitroreductase activity by a similar absolute amount as in TA1538, but representing only about one-quarter of the original activity of TA1535. The results suggest that S. typhimurium has originally at least two distinct nitroreductases, one of which is already deficient in strain TA1538; the CNR is present in both TA1535 and TA1538. Also, these two strains (including their derivatives, TA98 and TA100) are not isogenic with regard to nitroreductase activity. After the introduction of plasmid pKM101, the sensitivities of the strains YG7132 and YG7128, the cnr-null mutants of TA98 and TA100, respectively, against several nitro compounds were compared with those of the conventional cnr-deficient strains TA98NR and TA100NR and the wild-type strains TA98 and TA100. The mutagenicities of 2-nitrofluorene and 1-nitropyrene in YG7132 or TA98NR were ten-fold lower than those of the compounds in TA98. Similarly, the mutagenicity of 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide in strain YG7128 or TA100NR was substantially lower than that of the compound in TA100. However, the mutagenicity of 2-nitronaphthalene in YG7128 was between those observed with TA100 and TA100NR, suggesting that a nitroreductase in S. typhimurium other than CNR is involved in the metabolic activation of this compound. The cnr gene of S. typhimurium positively hybridized with DNA at 13 min on the E. coli chromosome where the nfsB and nfnB genes

  5. Structure and transcriptional regulation of the Nat2 gene encoding for the drug-metabolizing enzyme arylamine N-acetyltransferase type 2 in mice.

    PubMed Central

    Boukouvala, Sotiria; Price, Naomi; Plant, Kathryn E; Sim, Edith

    2003-01-01

    Arylamine N-acetyltransferases (NATs) are polymorphic enzymes, well-known for their role in the metabolism of drugs and carcinogens. Mice have three NAT isoenzymes, of which NAT2 is postulated to be involved in endogenous, as well as xenobiotic, metabolism. To understand expression of the murine Nat2 gene, we have analysed its structure and transcriptional regulation. We have accurately mapped the transcription initiation site 6.5 kb upstream of the coding region of the gene, adjacent to a recently described non-coding exon. Transcription was demonstrated to start from this region in embryonic and adult liver, spleen, submaxillary gland, kidney, brain, thymus, lung and placenta, but not in the heart. Database searches and analyses of cDNA by PCR suggested alternative splicing of the single 6.2 kb intron of Nat2, and determined the position of the polyadenylation signal at 0.44 kb downstream of the coding region of the gene. Examination of the 13 kb sequence flanking the coding and non-coding exons of Nat2 revealed a single promoter, located close to the transcription-initiation site, and indicated regions likely to harbour control elements. The Nat2 promoter consists of an atypical TATA box and a Sp1 [SV40 (simian virus 40) protein 1] box identical with that found in many housekeeping gene promoters. Activity of the Nat2 promoter was severely reduced by deletion or mutation of either of these two elements, whereas the region of the Sp1 box bound cellular protein and resisted DNase I digestion. Finally, the ability of the promoter region to bind cellular protein was reduced by competition with oligonucleotides bearing the Sp1 consensus sequence. PMID:12904181

  6. Starch phosphorylation in potato tubers is influenced by allelic variation in the genes encoding glucan water dikinase, starch branching enzymes I and II, and starch synthase III

    PubMed Central

    Carpenter, Margaret A.; Joyce, Nigel I.; Genet, Russell A.; Cooper, Rebecca D.; Murray, Sarah R.; Noble, Alasdair D.; Butler, Ruth C.; Timmerman-Vaughan, Gail M.

    2015-01-01

    Starch phosphorylation is an important aspect of plant metabolism due to its role in starch degradation. Moreover, the degree of phosphorylation of starch determines its physicochemical properties and is therefore relevant for industrial uses of starch. Currently, starch is chemically phosphorylated to increase viscosity and paste stability. Potato cultivars with elevated starch phosphorylation would make this process unnecessary, thereby bestowing economic and environmental benefits. Starch phosphorylation is a complex trait which has been previously shown by antisense gene repression to be influenced by a number of genes including those involved in starch synthesis and degradation. We have used an association mapping approach to discover genetic markers associated with the degree of starch phosphorylation. A diverse collection of 193 potato lines was grown in replicated field trials, and the levels of starch phosphorylation at the C6 and C3 positions of the glucosyl residues were determined by mass spectrometry of hydrolyzed starch from tubers. In addition, the potato lines were genotyped by amplicon sequencing and microsatellite analysis, focusing on candidate genes known to be involved in starch synthesis. As potato is an autotetraploid, genotyping included determination of allele dosage. Significant associations (p < 0.001) were found with SNPs in the glucan water dikinase (GWD), starch branching enzyme I (SBEI) and the starch synthase III (SSIII) genes, and with a SSR allele in the SBEII gene. SNPs in the GWD gene were associated with C6 phosphorylation, whereas polymorphisms in the SBEI and SBEII genes were associated with both C6 and C3 phosphorylation and the SNP in the SSIII gene was associated with C3 phosphorylation. These allelic variants have potential as genetic markers for starch phosphorylation in potato. PMID:25806042

  7. Bisphenol A-Associated Alterations in the Expression and Epigenetic Regulation of Genes Encoding Xenobiotic Metabolizing Enzymes in Human Fetal Liver

    PubMed Central

    Nahar, Muna S.; Kim, Jung H.; Sartor, Maureen A.; Dolinoy, Dana C.

    2014-01-01

    Alterations in xenobiotic metabolizing enzyme (XME) expression across the life course, along with genetic, nutritional, and environmental regulation, can influence how organisms respond to toxic insults. In this study, we investigated the hypothesis that in utero exposure to the endocrine active compound, bisphenol A (BPA), influences expression and epigenetic regulation of phase I and II XME genes during development. Using healthy 1st to 2nd trimester human fetal liver specimens quantified for internal BPA levels, we examined XME gene expression using PCR Array (n =8) and RNA-sequencing (n =12) platforms. Of the greater than 160 XME genes assayed, 2 phase I and 12 phase II genes exhibited significantly reduced expression with higher BPA levels, including isoforms from the carboxylesterase, catechol O-methyltransferase, glutathione S-transferase, sulfotransferase, and UDP-glucuronosyltransferase families. When the promoters of these candidate genes were evaluated in silico, putative binding sites for the E-twenty-six (ETS) and activator protein1 (AP1) related transcription factor families were identified and unique to 97% of all candidate transcripts. Interestingly, many ETS binding sites contain cytosine-guanine dinucleotides (CpGs) within their consensus sequences. Thus, quantitative analysis of CpG methylation of three candidate genes was conducted across n =50 samples. Higher BPA levels were associated with increased site-specific methylation at COMT (P <0.005) and increased average methylation at SULT2A1 (P <0.020) promoters. While toxicological studies have traditionally focused on high-dose effects and hormonal receptor mediated regulation, our findings suggest the importance of low-dose effects and nonclassical mechanisms of endocrine disruption during development. PMID:24214726

  8. Bioinformatic evaluation of L-arginine catabolic pathways in 24 cyanobacteria and transcriptional analysis of genes encoding enzymes of L-arginine catabolism in the cyanobacterium Synechocystis sp. PCC 6803

    PubMed Central

    Schriek, Sarah; Rückert, Christian; Staiger, Dorothee; Pistorius, Elfriede K; Michel, Klaus-Peter

    2007-01-01

    five different L-arginine-degrading pathways are present in the investigated cyanobacterial species. In Synechocystis sp. PCC 6803 an L-arginine deiminase pathway and an L-arginine oxidase/dehydrogenase pathway represent the major pathways, while the L-arginine decarboxylase pathway most likely only functions in polyamine biosynthesis. The transcripts encoding the enzymes of the two major pathways were constitutively expressed with the exception of the transcript for the carbamate kinase, which was substantially up-regulated in cells grown with L-arginine. PMID:18045455

  9. SUMO-conjugating enzyme (Sce) and FK506-binding protein (FKBP) encoding rice (Oryza sativa L.) genes: genome-wide analysis, expression studies and evidence for their involvement in abiotic stress response.

    PubMed

    Nigam, Neha; Singh, Amanjot; Sahi, Chandan; Chandramouli, Anupama; Grover, Anil

    2008-04-01

    We report an in-depth characterization of two major stress proteins namely SUMO-conjugating enzyme (Sce) and peptidyl prolyl cis-trans isomerase (PPIase) in rice (Oryza sativa L.). Sce mediates addition of SUMO group to various cell proteins, through process referred to as SUMOylation. Rice nuclear genome has two putative genes encoding the Sce protein (OsSce1 and OsSce2). PCR-amplified full-length OsSce1 cDNA functionally complemented the growth defect in yeast cells lacking the equivalent Ubc9 protein (ScDeltaubc9). RT-PCR analysis showed that transcript levels of OsSce1 and OsSce2 in rice seedlings were regulated by temperature stress. OsSce1 protein was localized to the nucleus in onion epidermal cells as evidenced by the transient GFP expression analysis following micro-projectile gun-based shooting of an OsSce1-GFP fusion construct. PPIase proteins assist molecular chaperones in reactions associated with protein folding and protein transport across membrane. There are 23 putative genes encoding for FK506-binding proteins (FKBPs; specific class of PPIase) in rice genome. OsFKBP20 cDNA was isolated as a stress-inducible EST clone. Largest ORF of 561 bases in OsFKBP20 showed characteristic FK506-binding domain at N-terminus and a coiled-coil motif at C-terminus. RNA expression analysis indicated that OsFKBP20 transcript is heat-inducible. OsFKBP20 over-expression in yeast endowed capacity of high temperature tolerance to yeast cells. Yeast two-hybrid analysis showed that OsSce1 protein physically interacts with the OsFKBP20 protein. It is thus proposed that OsSce1 and OsFKBP20 proteins in concert mediate the stress response of rice plants. PMID:18219493

  10. Apd1(+), a Gene Required for Red Pigment Formation in Ade6 Mutants of Schizosaccharomyces Pombe, Encodes an Enzyme Required for Glutathione Biosynthesis: A Role for Glutathione and a Glutathione-Conjugate Pump

    PubMed Central

    Chaudhuri, B.; Ingavale, S.; Bachhawat, A. K.

    1997-01-01

    Mutants in the adenine biosynthetic pathway of yeasts (ade1 and ade2 of Saccharomyces cerevisiae, ade6 and ade7 of Schizosaccharomyces pombe) accumulate an intense red pigment in their vacuoles when grown under adenine-limiting conditions. The precise events that determine the formation of the pigment are however, still unknown. We have begun a genetic investigation into the nature and cause of pigmentation of ade6 mutants of S. pombe and have discovered that one of these pigmentation defective mutants, apd1 (adenine pigmentation defective), is a strict glutathione auxotroph. The gene apd1(+) was found to encode the first enzyme in glutathione biosynthesis, γ-glutamylcysteine synthetase, gcs1(+). This gene when expressed in the mutant could confer both glutathione prototrophy and the characteristic red pigmentation, and disruption of the gene led to a loss in both phenotypes. Supplementation of glutathione in the medium, however, could only restore growth but not the pigmentation because the cells were unable to achieve sufficient intracellular levels of glutathione. Disruption of the second enzyme in glutathione biosynthesis, glutathione synthetase, gsh2(+), also led to glutathione auxotrophy, but only a partial defect in pigment formation. A reevaluation of the major amino acids previously reported to be present in the pigment indicated that the pigment is probably a glutathione conjugate. The ability of vanadate to inhibit pigment formation indicated that the conjugate was transported into the vacuole through a glutathione-conjugate pump. This was further confirmed using strains of S. cerevisiae bearing disruptions in the recently identified glutathione-conjugate pump, YCF1, where a significant reduction in pigment formation was observed. The pump of S. pombe is distinct from the previously identified vacuolar pump, hmt1p, for transporting cadystin peptides into vacuoles of S. pombe. PMID:9017391

  11. Differentially expressed myo-inositol monophosphatase gene (CaIMP) in chickpea (Cicer arietinum L.) encodes a lithium-sensitive phosphatase enzyme with broad substrate specificity and improves seed germination and seedling growth under abiotic stresses.

    PubMed

    Saxena, Saurabh C; Salvi, Prafull; Kaur, Harmeet; Verma, Pooja; Petla, Bhanu Prakash; Rao, Venkateswara; Kamble, Nitin; Majee, Manoj

    2013-12-01

    myo-Inositol monophosphatase (IMP) is an essential enzyme in the myo-inositol metabolic pathway where it primarily dephosphorylates myo-inositol 1-phosphate to maintain the cellular inositol pool which is important for many metabolic and signalling pathways in plants. The stress-induced increased accumulation of inositol has been reported in a few plants including chickpea; however, the role and regulation of IMP is not well defined in response to stress. In this work, it has been shown that IMP activity is distributed in all organs in chickpea and was noticeably enhanced during environmental stresses. Subsequently, using degenerate oligonucleotides and RACE strategy, a full-length IMP cDNA (CaIMP) was cloned and sequenced. Biochemical study revealed that CaIMP encodes a lithium-sensitive phosphatase enzyme with broad substrate specificity, although maximum activity was observed with the myo-inositol 1-phosphate and l-galactose 1-phosphate substrates. Transcript analysis revealed that CaIMP is differentially expressed and regulated in different organs, stresses and phytohormones. Complementation analysis in Arabidopsis further confirmed the role of CaIMP in l-galactose 1-phosphate and myo-inositol 1-phosphate hydrolysis and its participation in myo-inositol and ascorbate biosynthesis. Moreover, Arabidopsis transgenic plants over-expressing CaIMP exhibited improved tolerance to stress during seed germination and seedling growth, while the VTC4/IMP loss-of-function mutants exhibited sensitivity to stress. Collectively, CaIMP links various metabolic pathways and plays an important role in improving seed germination and seedling growth, particularly under stressful environments.

  12. Differentially expressed myo-inositol monophosphatase gene (CaIMP) in chickpea (Cicer arietinum L.) encodes a lithium-sensitive phosphatase enzyme with broad substrate specificity and improves seed germination and seedling growth under abiotic stresses.

    PubMed

    Saxena, Saurabh C; Salvi, Prafull; Kaur, Harmeet; Verma, Pooja; Petla, Bhanu Prakash; Rao, Venkateswara; Kamble, Nitin; Majee, Manoj

    2013-12-01

    myo-Inositol monophosphatase (IMP) is an essential enzyme in the myo-inositol metabolic pathway where it primarily dephosphorylates myo-inositol 1-phosphate to maintain the cellular inositol pool which is important for many metabolic and signalling pathways in plants. The stress-induced increased accumulation of inositol has been reported in a few plants including chickpea; however, the role and regulation of IMP is not well defined in response to stress. In this work, it has been shown that IMP activity is distributed in all organs in chickpea and was noticeably enhanced during environmental stresses. Subsequently, using degenerate oligonucleotides and RACE strategy, a full-length IMP cDNA (CaIMP) was cloned and sequenced. Biochemical study revealed that CaIMP encodes a lithium-sensitive phosphatase enzyme with broad substrate specificity, although maximum activity was observed with the myo-inositol 1-phosphate and l-galactose 1-phosphate substrates. Transcript analysis revealed that CaIMP is differentially expressed and regulated in different organs, stresses and phytohormones. Complementation analysis in Arabidopsis further confirmed the role of CaIMP in l-galactose 1-phosphate and myo-inositol 1-phosphate hydrolysis and its participation in myo-inositol and ascorbate biosynthesis. Moreover, Arabidopsis transgenic plants over-expressing CaIMP exhibited improved tolerance to stress during seed germination and seedling growth, while the VTC4/IMP loss-of-function mutants exhibited sensitivity to stress. Collectively, CaIMP links various metabolic pathways and plays an important role in improving seed germination and seedling growth, particularly under stressful environments. PMID:24123252

  13. Polymorphisms in genes encoding antioxidant enzymes (SOD2, CAT, GPx, TXNRD, SEPP1, SEP15 and SELS) and risk of chronic kidney disease in Japanese - cross-sectional data from the J-MICC study.

    PubMed

    Hishida, Asahi; Okada, Rieko; Naito, Mariko; Morita, Emi; Wakai, Kenji; Hamajima, Nobuyuki; Hosono, Satoyo; Nanri, Hinako; Turin, Tanvir Chowdhury; Suzuki, Sadao; Kuwabara, Kazuyo; Mikami, Haruo; Budhathoki, Sanjeev; Watanabe, Isao; Arisawa, Kokichi; Kubo, Michiaki; Tanaka, Hideo

    2013-07-01

    Chronic kidney disease (CKD) is well known as a strong risk factor for both of end-stage renal disease and cardiovascular disease. To clarify the association of polymorphisms in the genes encoding antioxidant enzymes (SOD2, CAT, GPx, TXNRD, SEPP1, SEP15 and SELS) with the risk of CKD in Japanese, we examined this association using the cross-sectional data of Japan Multi-Institutional Collaborative Cohort (J-MICC) Study. The subjects were 3,285 men and women, aged 35-69 years, selected from J-MICC Study participants for whom genotyping were conducted by multiplex polymerase chain reaction-based Invader assay. The prevalence of CKD was determined for CKD stages 3-5 (eGFR <60 ml/min/1.73 m(2)). When those with CAT C-262T C/C were defined as reference, those with CAT C-262T C/T demonstrated the OR for CKD of 0.67 (95% CI 0.43-1.06) with the marginally significant trend for decreased odds ratio with increasing numbers of T allele (p = 0.070). There were no significant associations between the other polymorphisms with CKD risk. The present study found a marginally significant trend of the decreased risk of CKD with increasing numbers of T allele of CAT, which may suggest the possibility of personalized risk estimation of this life-limiting disease in the near future.

  14. Expression of the Saccharomyces cerevisiae DNA repair gene RAD6 that encodes a ubiquitin conjugating enzyme, increases in response to DNA damage and in meiosis but remains constant during the mitotic cell cycle.

    PubMed

    Madura, K; Prakash, S; Prakash, L

    1990-02-25

    The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating (E2) enzyme and is required for the repair of damaged DNA, mutagenesis, and sporulation. Here, we report our studies on the regulation of RAD6 gene expression after UV damage, during the mitotic cell cycle, in meiosis, and following heat shock and starvation. RAD6 mRNA levels became elevated in cells exposed to UV light, and at all UV doses the increase in mRNA levels was rapid and occurred within 30 min after exposure to UV. RAD6 mRNA levels also increased in sporulating MATa/MAT alpha cells, and the period of maximal accumulation of RAD6 mRNA during meiosis is coincident with the time during which recombination occurs. However, RAD6 mRNA levels showed no periodic fluctuation in the mitotic cell cycle, were not elevated upon heat shock, and fell in cells in the stationary phase of growth. These observations suggest that RAD6 activity is required throughout the cell cycle rather than being restricted to a specific stage, and that during meiosis, high levels of RAD6 activity may be needed at a stage coincident with genetic recombination. The observation that RAD6 transcription is not induced by heat and starvation, treatments that activate stress responses, suggests that the primary role of RAD6 is in the repair of damaged DNA rather than in adapting cells to stress situations.

  15. The Role of Oxidative Stress in Diabetic Neuropathy: Generation of Free Radical Species in the Glycation Reaction and Gene Polymorphisms Encoding Antioxidant Enzymes to Genetic Susceptibility to Diabetic Neuropathy in Population of Type I Diabetic Patients.

    PubMed

    Babizhayev, Mark A; Strokov, Igor A; Nosikov, Valery V; Savel'yeva, Ekaterina L; Sitnikov, Vladimir F; Yegorov, Yegor E; Lankin, Vadim Z

    2015-04-01

    pathway are detoxified by the glyoxalase system with reduced glutathione as co-factor. The concentration of reduced glutathione may be decreased by oxidative stress and by decreased in situ glutathione reductase activity in diabetes mellitus. Genetic variations within the antioxidant genes therefore could be implicated in the pathogenesis of DN. In this work, the supporting data about the association between the -262T > C polymorphism of the catalase (CAT) gene and DN were shown. The -262TT genotype of the CAT gene was significantly associated with higher erythrocyte catalase activity in blood of DN patients compared to the -262CC genotype (17.8 ± 2.7 × 10(4) IU/g Hb vs. 13.5 ± 3.2 × 10(4) IU/g Hb, P = 0.0022). The role of these factors in the development of diabetic complications and the prospective prevention of DN by supplementation in formulations of transglycating imidazole-containing peptide-based antioxidants (non-hydrolyzed carnosine, carcinine, n-acetylcarcinine) scavenging ROS in the glycation reaction, modifying the activity of enzymic and non-enzymic antioxidant defenses that participate in metabolic processes with ability of controlling at transcriptional levels the differential expression of several genes encoding antioxidant enzymes inherent to DN in Type I Diabetic patients, now deserve investigation. PMID:25427889

  16. The effect of cloudy apple juice on hepatic and mammary gland phase I and II enzymes induced by DMBA in female Sprague-Dawley rats.

    PubMed

    Szaefer, Hanna; Krajka-Kuźniak, Violetta; Ignatowicz, Ewa; Adamska, Teresa; Markowski, Jarosław; Baer-Dubowska, Wanda

    2014-10-01

    Apples abundant in phenolic compounds show a variety of biological activities that may contribute to health beneficial effects against cardiovascular diseases, diabetes, obesity and cancer. We investigated the effect of cloudy apple juice (CAJ) on the hepatic and mammary gland carcinogen metabolizing enzymes, DNA damage and liver injury, altered by 7,12-dimethylbenz[a]anthracene (DMBA). Sprague-Dawley female rats were gavaged with CAJ (10 ml/kg b.w.) for 28 consecutive days. DMBA was administered i.p. on the 27th and the 28th days. In the liver, feeding with CAJ decreased the activities of CYP1A1 and 1A2 and increased phase II enzymes. The activities of all enzymes tested were enhanced in the animals treated with DMBA alone and in combination with CAJ. The most significant changes in the level of the hepatic enzymes tested were observed for GST alpha and NQO1. In mammary gland CAJ induced an increase in the level of GST mu and GST pi, while DMBA and CAJ combined administration elevated GST pi only. This may be beneficial as GST pi is involved in the DMBA detoxification. Additionally, pretreatment with CAJ reduced the level of most of the blood biochemical liver and kidney markers elevated as a result of DMBA treatment. These findings indicate that CAJ may interfere with enzyme system involved in carcinogen metabolism. However, this effect seems to be dependent on tissue and carcinogen and is moderately effective in the case of DMBA. Moreover, CAJ can also provide some protection against the liver and kidney damage.

  17. Enzyme catalysis: Evolution made easy

    NASA Astrophysics Data System (ADS)

    Wee, Eugene J. H.; Trau, Matt

    2014-09-01

    Directed evolution is a powerful tool for the development of improved enzyme catalysts. Now, a method that enables an enzyme, its encoding DNA and a fluorescent reaction product to be encapsulated in a gel bead enables the application of directed evolution in an ultra-high-throughput format.

  18. Chromosomal location of two human genes encoding tetrahydrobiopterin-metabolizing enzymes: 6-pyruvoyl-tetrahydropterin synthase maps to 11q22. 3-q23. 3, and pterin-4[alpha]-carbinolamine dehydratase maps to 10q22

    SciTech Connect

    Thoeny, B.; Heizmann, C.W. ); Mattei, M.G. )

    1994-01-15

    Tetrahydrobiopterin (BH[sub 4]) is the redox cofactor for the aromatic amino acid hydroxylases such as phenylalanine hydroxylase. At least five enzymes are known to be involved in BH[sub 4] biosynthesis and regeneration. A deficiency in several of the BH[sub 4] metabolic enzymes causes variant types of hyperphenylalaninemias in man. Recently, the authors cloned and expressed the human cDNAs for two of the BH[sub 4] enzymes, the 6-pyruvoyl-tetrahydropterin synthase and the pterin-4[alpha]-carbinolamine dehydratase (gene symbols PTS and PCD/DCOH, respectively). In this report, they localized the two genes on the human chromsomes by in situ hybridization. The PTS gene was mapped to the chromosomal region 11q22.3-q23.3, and the PCD/DCOH gene was mapped to the 10q22 band of the genome. 18 refs., 2 figs.

  19. ENCODE data at the ENCODE portal.

    PubMed

    Sloan, Cricket A; Chan, Esther T; Davidson, Jean M; Malladi, Venkat S; Strattan, J Seth; Hitz, Benjamin C; Gabdank, Idan; Narayanan, Aditi K; Ho, Marcus; Lee, Brian T; Rowe, Laurence D; Dreszer, Timothy R; Roe, Greg; Podduturi, Nikhil R; Tanaka, Forrest; Hong, Eurie L; Cherry, J Michael

    2016-01-01

    The Encyclopedia of DNA Elements (ENCODE) Project is in its third phase of creating a comprehensive catalog of functional elements in the human genome. This phase of the project includes an expansion of assays that measure diverse RNA populations, identify proteins that interact with RNA and DNA, probe regions of DNA hypersensitivity, and measure levels of DNA methylation in a wide range of cell and tissue types to identify putative regulatory elements. To date, results for almost 5000 experiments have been released for use by the scientific community. These data are available for searching, visualization and download at the new ENCODE Portal (www.encodeproject.org). The revamped ENCODE Portal provides new ways to browse and search the ENCODE data based on the metadata that describe the assays as well as summaries of the assays that focus on data provenance. In addition, it is a flexible platform that allows integration of genomic data from multiple projects. The portal experience was designed to improve access to ENCODE data by relying on metadata that allow reusability and reproducibility of the experiments.

  20. A R2R3-MYB transcription factor, GmMYB12B2, affects the expression levels of flavonoid biosynthesis genes encoding key enzymes in transgenic Arabidopsis plants.

    PubMed

    Li, Xiao-Wei; Li, Jing-Wen; Zhai, Ying; Zhao, Yan; Zhao, Xu; Zhang, Hai-Jun; Su, Lian-Tai; Wang, Ying; Wang, Qing-Yu

    2013-12-10

    Isoflavones play diverse roles in plant-microbe interactions and are potentially important for human nutrition and health. To study the regulation of isoflavonoid synthesis in soybean, the R2R3-MYB transcription factor GmMYB12B2 was isolated and characterized. Yeast expression experiments demonstrated that GmMYB12B2 showed transcriptional activity. GmMYB12B2 was localized in the nucleus when it was transiently expressed in onion epidermal cells. Real-time quantitative PCR analysis revealed that GmMYB12B2 transcription was increased in roots and mature seeds compared with other organs. The gene expression level in immature embryos was consistent with the accumulation of isoflavones. CHS8 is a key enzyme in plant flavonoid biosynthesis. Transient expression experiments in soybean calli demonstrated that CHS8 was regulated by GmMYB12B2 and produced more fluorescence. The expression levels of some key enzymes in flavonoid biosynthesis were examined in transgenic Arabidopsis lines. The results showed that the expression levels of PAL1, CHS and FLS in transgenic plants were significantly higher than those in wild type plants. However, the expression level of DFR was lower, and the expression levels of CHI, F3H and F3'H were the same in all lines. GmMYB12B2 expression caused a constitutive increase in the accumulation of flavonoids in transgenic Arabidopsis lines compared with wild type plants. PMID:24060295

  1. A R2R3-MYB transcription factor, GmMYB12B2, affects the expression levels of flavonoid biosynthesis genes encoding key enzymes in transgenic Arabidopsis plants.

    PubMed

    Li, Xiao-Wei; Li, Jing-Wen; Zhai, Ying; Zhao, Yan; Zhao, Xu; Zhang, Hai-Jun; Su, Lian-Tai; Wang, Ying; Wang, Qing-Yu

    2013-12-10

    Isoflavones play diverse roles in plant-microbe interactions and are potentially important for human nutrition and health. To study the regulation of isoflavonoid synthesis in soybean, the R2R3-MYB transcription factor GmMYB12B2 was isolated and characterized. Yeast expression experiments demonstrated that GmMYB12B2 showed transcriptional activity. GmMYB12B2 was localized in the nucleus when it was transiently expressed in onion epidermal cells. Real-time quantitative PCR analysis revealed that GmMYB12B2 transcription was increased in roots and mature seeds compared with other organs. The gene expression level in immature embryos was consistent with the accumulation of isoflavones. CHS8 is a key enzyme in plant flavonoid biosynthesis. Transient expression experiments in soybean calli demonstrated that CHS8 was regulated by GmMYB12B2 and produced more fluorescence. The expression levels of some key enzymes in flavonoid biosynthesis were examined in transgenic Arabidopsis lines. The results showed that the expression levels of PAL1, CHS and FLS in transgenic plants were significantly higher than those in wild type plants. However, the expression level of DFR was lower, and the expression levels of CHI, F3H and F3'H were the same in all lines. GmMYB12B2 expression caused a constitutive increase in the accumulation of flavonoids in transgenic Arabidopsis lines compared with wild type plants.

  2. Inducible expression of the chromosomal cdiA from Citrobacter diversus NF85, encoding an ambler class A beta-lactamase, is under similar genetic control to the chromosomal ampC, encoding an ambler class C enzyme, from Citrobacter freundii OS60.

    PubMed

    Jones, M E; Bennett, P M

    1995-01-01

    This study aimed to characterize the molecular basis of beta-lactamase induction in Citrobacter diversus. The chromosomal beta-lactamase encoding region from C. diversus, strain NF85, was cloned and expressed in Escherichia coli. The cloned region was sequenced and open-reading frames encoding a class A beta-lactamase, designated cdiA, and a putative LysR-type transcriptional regulator protein, divergently transcribed from the beta-lactamase gene and designated cdiR, were identified. The nucleotide sequence of the NF85 cdiA was identical to that of the published C. diversus ULA27 ampC sequence. A putative helix-turn-helix DNA-binding motif was located at the N-terminus of CdiR, and homology with enterobacterial AmpR proteins was noted. CdiR was demonstrated to bind to the C. diversus cdiAR intergenic region but not to the C. freundii ampCR intergenic region. A putative CdiR binding motif was identified in the cdiAR intergenic region. The cloned cdiAR region was inducible in E. coli strains SNO3 and HfrH. The inducible phenotype was dependent on the E. coli ampD and ampG gene products. We conclude that the molecular basis of inducible cdiA expression in C. diversus is similar to that of C. freundii ampC.

  3. The dddP gene, encoding a novel enzyme that converts dimethylsulfoniopropionate into dimethyl sulfide, is widespread in ocean metagenomes and marine bacteria and also occurs in some Ascomycete fungi.

    PubMed

    Todd, J D; Curson, A R J; Dupont, C L; Nicholson, P; Johnston, A W B

    2009-06-01

    The marine alphaproteobacterium Roseovarius nubinhibens ISM can produce the gas dimethyl sulfide (DMS) from dimethylsulfoniopropionate (DMSP), a widespread secondary metabolite that occurs in many phytoplankton. Roseovarius possesses a novel gene, termed dddP, which when cloned, confers on Escherichia coli the ability to produce DMS. The DddP polypeptide is in the large family of M24 metallopeptidases and is wholly different from two other enzymes, DddD and DddL, which were previously shown to generate DMS from dimethylsulfoniopropionate. Close homologues of DddP occur in other alphaproteobacteria and more surprisingly, in some Ascomycete fungi. These were the biotechnologically important Aspergillus oryzae and the plant pathogen, Fusarium graminearum. The dddP gene is abundant in the bacterial metagenomic sequences in the Global Ocean Sampling Expedition. Thus, dddP has several novel features and is widely dispersed, both taxonomically and geographically.

  4. ABA-Mediated Inhibition of Germination Is Related to the Inhibition of Genes Encoding Cell-Wall Biosynthetic and Architecture: Modifying Enzymes and Structural Proteins in Medicago truncatula Embryo Axis

    PubMed Central

    Gimeno-Gilles, Christine; Lelièvre, Eric; Viau, Laure; Malik-Ghulam, Mustafa; Ricoult, Claudie; Niebel, Andreas; Leduc, Nathalie; Limami, Anis M.

    2009-01-01

    Radicle emergence and reserves mobilization are two distinct programmes that are thought to control germination. Both programs are influenced by abscissic acid (ABA) but how this hormone controls seed germination is still poorly known. Phenotypic and microscopic observations of the embryo axis of Medicago truncatula during germination in mitotic inhibition condition triggered by 10 μM oryzalin showed that cell division was not required to allow radicle emergence. A suppressive subtractive hybridization showed that more than 10% of up-regulated genes in the embryo axis encoded proteins related to cell-wall biosynthesis. The expression of α-expansins, pectin-esterase, xylogucan-endotransglycosidase, cellulose synthase, and extensins was monitored in the embryo axis of seeds germinated on water, constant and transitory ABA. These genes were overexpressed before completion of germination in the control and strongly inhibited by ABA. The expression was re-established in the ABA transitory-treatment after the seeds were transferred back on water and proceeded to germination. This proves these genes as contributors to the completion of germination and strengthen the idea that cell-wall loosening and remodeling in relation to cell expansion in the embryo axis is a determinant feature in germination. Our results also showed that ABA controls germination through the control of radicle emergence, namely by inhibiting cell-wall loosening and expansion. PMID:19529818

  5. Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Moe, Owen; Cornelius, Richard

    1988-01-01

    Conveys an appreciation of enzyme kinetic analysis by using a practical and intuitive approach. Discusses enzyme assays, kinetic models and rate laws, the kinetic constants (V, velocity, and Km, Michaels constant), evaluation of V and Km from experimental data, and enzyme inhibition. (CW)

  6. Determination of lipolytic enzyme activities.

    PubMed

    Jaeger, Karl-Erich; Kovacic, Filip

    2014-01-01

    Pseudomonas aeruginosa is a versatile human opportunistic pathogen that produces and secretes an arsenal of enzymes, proteins and small molecules many of which serve as virulence factors. Notably, about 40 % of P. aeruginosa genes code for proteins of unknown function, among them more than 80 encoding putative, but still unknown lipolytic enzymes. This group of hydrolases (EC 3.1.1) is known already for decades, but only recently, several of these enzymes have attracted attention as potential virulence factors. Reliable and reproducible enzymatic activity assays are crucial to determine their physiological function and particularly assess their contribution to pathogenicity. As a consequence of the unique biochemical properties of lipids resulting in the formation of micellar structures in water, the reproducible preparation of substrate emulsions is strongly dependent on the method used. Furthermore, the physicochemical properties of the respective substrate emulsion may drastically affect the activities of the tested lipolytic enzymes. Here, we describe common methods for the activity determination of lipase, esterase, phospholipase, and lysophospholipase. These methods cover lipolytic activity assays carried out in vitro, with cell extracts or separated subcellular compartments and with purified enzymes. We have attempted to describe standardized protocols, allowing the determination and comparison of enzymatic activities of lipolytic enzymes from different sources. These methods should also encourage the Pseudomonas community to address the wealth of still unexplored lipolytic enzymes encoded and produced by P. aeruginosa.

  7. Miniaturised optical encoder

    NASA Astrophysics Data System (ADS)

    Carr, John; Desmulliez, Marc P. Y.; Weston, Nick; McKendrick, David; Cunningham, Graeme; McFarland, Geoff; Meredith, Wyn; McKee, Andrew; Langton, Conrad; Eddie, Iain

    2008-08-01

    Optical encoders are pervasive in many sectors of industry including metrology, motion systems, electronics, medical, scanning/ printing, scientific instruments, space research and specialist machine tools. The precision of automated manufacture and assembly has been revolutionised by the adoption of optical diffractive measurement methods. Today's optical encoders comprise discrete components: light source(s), reference and analyser gratings, and a photodiode array that utilise diffractive optic methods to achieve high resolution. However the critical alignment requirements between the optical gratings and to the photodiode array, the bulky nature of the encoder devices and subsequent packaging mean that optical encoders can be prohibitively expensive for many applications and unsuitable for others. We report here on the design, manufacture and test of a miniaturised optical encoder to be used in precision measurement systems. Microsystems manufacturing techniques facilitate the monolithic integration of the traditional encoder components onto a single compound semiconductor chip, radically reducing the size, cost and set-up time. Fabrication of the gratings at the wafer level, by standard photo-lithography, allows for the simultaneous alignment of many devices in a single process step. This development coupled with a unique photodiode configuration not only provides increased performance but also significantly improves the alignment tolerances in both manufacture and set-up. A National Research and Development Corporation type optical encoder chip has been successfully demonstrated under test conditions on both amplitude and phase scales with pitches of 20 micron, 8 micron and 4 micron, showing significantly relaxed alignment tolerances with signal-to-noise ratios greater than 60:1. Various reference mark schemes have also been investigated. Results are presented here.

  8. Polarization encoded color camera.

    PubMed

    Schonbrun, Ethan; Möller, Guðfríður; Di Caprio, Giuseppe

    2014-03-15

    Digital cameras would be colorblind if they did not have pixelated color filters integrated into their image sensors. Integration of conventional fixed filters, however, comes at the expense of an inability to modify the camera's spectral properties. Instead, we demonstrate a micropolarizer-based camera that can reconfigure its spectral response. Color is encoded into a linear polarization state by a chiral dispersive element and then read out in a single exposure. The polarization encoded color camera is capable of capturing three-color images at wavelengths spanning the visible to the near infrared. PMID:24690806

  9. Plasmids encoding therapeutic agents

    DOEpatents

    Keener, William K.

    2007-08-07

    Plasmids encoding anti-HIV and anti-anthrax therapeutic agents are disclosed. Plasmid pWKK-500 encodes a fusion protein containing DP178 as a targeting moiety, the ricin A chain, an HIV protease cleavable linker, and a truncated ricin B chain. N-terminal extensions of the fusion protein include the maltose binding protein and a Factor Xa protease site. C-terminal extensions include a hydrophobic linker, an L domain motif peptide, a KDEL ER retention signal, another Factor Xa protease site, an out-of-frame buforin II coding sequence, the lacZ.alpha. peptide, and a polyhistidine tag. More than twenty derivatives of plasmid pWKK-500 are described. Plasmids pWKK-700 and pWKK-800 are similar to pWKK-500 wherein the DP178-encoding sequence is substituted by RANTES- and SDF-1-encoding sequences, respectively. Plasmid pWKK-900 is similar to pWKK-500 wherein the HIV protease cleavable linker is substituted by a lethal factor (LF) peptide-cleavable linker.

  10. Video Time Encoding Machines

    PubMed Central

    Lazar, Aurel A.; Pnevmatikakis, Eftychios A.

    2013-01-01

    We investigate architectures for time encoding and time decoding of visual stimuli such as natural and synthetic video streams (movies, animation). The architecture for time encoding is akin to models of the early visual system. It consists of a bank of filters in cascade with single-input multi-output neural circuits. Neuron firing is based on either a threshold-and-fire or an integrate-and-fire spiking mechanism with feedback. We show that analog information is represented by the neural circuits as projections on a set of band-limited functions determined by the spike sequence. Under Nyquist-type and frame conditions, the encoded signal can be recovered from these projections with arbitrary precision. For the video time encoding machine architecture, we demonstrate that band-limited video streams of finite energy can be faithfully recovered from the spike trains and provide a stable algorithm for perfect recovery. The key condition for recovery calls for the number of neurons in the population to be above a threshold value. PMID:21296708

  11. Video time encoding machines.

    PubMed

    Lazar, Aurel A; Pnevmatikakis, Eftychios A

    2011-03-01

    We investigate architectures for time encoding and time decoding of visual stimuli such as natural and synthetic video streams (movies, animation). The architecture for time encoding is akin to models of the early visual system. It consists of a bank of filters in cascade with single-input multi-output neural circuits. Neuron firing is based on either a threshold-and-fire or an integrate-and-fire spiking mechanism with feedback. We show that analog information is represented by the neural circuits as projections on a set of band-limited functions determined by the spike sequence. Under Nyquist-type and frame conditions, the encoded signal can be recovered from these projections with arbitrary precision. For the video time encoding machine architecture, we demonstrate that band-limited video streams of finite energy can be faithfully recovered from the spike trains and provide a stable algorithm for perfect recovery. The key condition for recovery calls for the number of neurons in the population to be above a threshold value.

  12. Time-Encoded Imagers.

    SciTech Connect

    Marleau, Peter; Brubaker, Erik

    2014-11-01

    This report provides a short overview of the DNN R&D funded project, Time-Encoded Imagers. The project began in FY11 and concluded in FY14. The Project Description below provides the overall motivation and objectives for the project as well as a summary of programmatic direction. It is followed by a short description of each task and the resulting deliverables.

  13. Organization and control of genes encoding catabolic enzymes in Rhizobiaceae

    SciTech Connect

    Parke, D.; Ornston, L.N.

    1993-03-01

    Rhizobiaceae, a diverse bacterial group comprising rhizobia and agrobacteria, symbiotic partnership with plants form nitrogen-fixing nodules on plant roots or are plant pathogens. Phenolic compounds produced by plants serve as inducers of rhizobial nodulation genes and agrobacterial virulence genes reflect their capacity to utilize numerous aromatics, including phenolics, as a source of carbon and energy. In many microbes the aerobic degradation of numerous aromatic compounds to tricarboxylic acid cycle intermediates is achieved by the [beta]-ketoadipate pathway. Our initial studies focused on the organization and regulation of the ketoadipate pathway in Agrobacterium tumefaciens. We have cloned, identified and characterized a novel regulatory gene that modulates expression of an adjacent pca (protocatechuate) structural gene, pcaD. Regulation of pcaD is mediated by the regulatory gene, termed pcaQ, in concert with the intermediate [beta]-carboxy-cis,cis-muconate. [beta]-carboxy-cis,cismuconate is an unstable chemical, not marketed commercially, and it is unlikely to permeate Escherichia coli cells if supplied in media. Because of these factors, characterization of pcaQ in E. coli required an in vivo delivery system for [beta]-carboxycis,cis-muconate. This was accomplished by designing an E. coli strain that expressed an Acinetobacter calcoaceticus pcaA gene for conversion of protocatechuate to [beta]-carboxy-cis,cis-muconate.

  14. Enzyme complexity in intermediary metabolism.

    PubMed

    Van Schaftingen, Emile; Veiga-da-Cunha, Maria; Linster, Carole L

    2015-07-01

    A good appraisal of the function of enzymes is essential for the understanding of inborn errors of metabolism. However, it is clear now that the 'one gene, one enzyme, one catalytic function' rule oversimplifies the actual situation. Genes often encode several related proteins, which may differ in their subcellular localisation, regulation or function. Furthermore, enzymes often show several catalytic activities. In some cases, this is because they are multifunctional, possessing two or more different active sites that catalyse different, physiologically related reactions. In enzymes with broad specificity or in multispecificity enzymes, a single type of catalytic site performs the same reaction on different physiological substrates at similar rates. Enzymes that act physiologically in only one reaction often show nonetheless substrate promiscuity: they act at low rates on compounds that resemble their physiological substrate(s), thus forming non-classical metabolites, which are in some cases eliminated by metabolite repair. In addition to their catalytic role, enzymes may have moonlighting functions, i.e. non-catalytic functions that are most often not related with their catalytic activity. Deficiency in such functions may participate in the phenotype of inborn errors of metabolism. Evolution has also made that some enzymes have lost their catalytic activity to become allosteric proteins.

  15. Food Enzymes

    ERIC Educational Resources Information Center

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  16. Zinc Enzymes.

    ERIC Educational Resources Information Center

    Bertini, I.; And Others

    1985-01-01

    Discusses the role of zinc in various enzymes concerned with hydration, hydrolysis, and redox reactions. The binding of zinc to protein residues, properties of noncatalytic zinc(II) and catalytic zinc, and the reactions catalyzed by zinc are among the topics considered. (JN)

  17. The Text Encoding Initiative: Flexible and Extensible Document Encoding.

    ERIC Educational Resources Information Center

    Barnard, David T.; Ide, Nancy M.

    1997-01-01

    The Text Encoding Initiative (TEI), an international collaboration aimed at producing a common encoding scheme for complex texts, examines the requirement for generality versus the requirement to handle specialized text types. Discusses how documents and users tax the limits of fixed schemes requiring flexible extensible encoding to support…

  18. Disorders of phonological encoding.

    PubMed

    Butterworth, B

    1992-03-01

    Studies of phonological disturbances in aphasic speech are reviewed. It is argued that failure to test for error consistency in individual patients makes it generally improper to draw inferences about specific disorders of phonological encoding. A minimalist interpretation of available data on phonological errors is therefore proposed that involves variable loss of information in transmission between processing subsystems. Proposals for systematic loss or corruption of phonological information in lexical representations or in translation subsystems is shown to be inadequately grounded. The review concludes with some simple methodological prescriptions for future research.

  19. Enzyme Kinetics

    PubMed Central

    Lam, C. F.; Priest, D. G.

    1972-01-01

    One of the most generally applicable algorithms for the derivation of steady-state rate equations for complex enzyme reaction mechanisms is that of King and Altman. Several modifications of this algorithm have been suggested; however, each requires the generation of numerous valid and invalid patterns and the subsequent elimination of those that are invalid. A method is presented, employing topological theory of linear graphs, for the systematic generation of only those patterns which are valid. This method is readily adaptable to use on a digital computer. An independent method for the calculation of the number of valid patterns is also presented. This calculation can be used to substantiate the accuracy of the patterns obtained. This calculation is also adaptable to computerization. Examples are included to demonstrate both the generation of patterns and the calculation of their number for specific enzyme mechanisms. PMID:5016111

  20. Alkylating enzymes.

    PubMed

    Wessjohann, Ludger A; Keim, Jeanette; Weigel, Benjamin; Dippe, Martin

    2013-04-01

    Chemospecific and regiospecific modifications of natural products by methyl, prenyl, or C-glycosyl moieties are a challenging and cumbersome task in organic synthesis. Because of the availability of an increasing number of stable and selective transferases and cofactor regeneration processes, enzyme-assisted strategies turn out to be promising alternatives to classical synthesis. Two categories of alkylating enzymes become increasingly relevant for applications: firstly prenyltransferases and terpene synthases (including terpene cyclases), which are used in the production of terpenoids such as artemisinin, or meroterpenoids like alkylated phenolics and indoles, and secondly methyltransferases, which modify flavonoids and alkaloids to yield products with a specific methylation pattern such as 7-O-methylaromadendrin and scopolamine.

  1. Rotary encoding device

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B. (Inventor)

    1993-01-01

    A device for position encoding of a rotating shaft in which a polygonal mirror having a number of facets is mounted to the shaft and a light beam is directed towards the facets is presented. The facets of the polygonal mirror reflect the light beam such that a light spot is created on a linear array detector. An analog-to-digital converter is connected to the linear array detector for reading the position of the spot on the linear array detector. A microprocessor with memory is connected to the analog-to-digital converter to hold and manipulate the data provided by the analog-to-digital converter on the position of the spot and to compute the position of the shaft based upon the data from the analog-to-digital converter.

  2. Time encoded radiation imaging

    DOEpatents

    Marleau, Peter; Brubaker, Erik; Kiff, Scott

    2014-10-21

    The various technologies presented herein relate to detecting nuclear material at a large stand-off distance. An imaging system is presented which can detect nuclear material by utilizing time encoded imaging relating to maximum and minimum radiation particle counts rates. The imaging system is integrated with a data acquisition system that can utilize variations in photon pulse shape to discriminate between neutron and gamma-ray interactions. Modulation in the detected neutron count rates as a function of the angular orientation of the detector due to attenuation of neighboring detectors is utilized to reconstruct the neutron source distribution over 360 degrees around the imaging system. Neutrons (e.g., fast neutrons) and/or gamma-rays are incident upon scintillation material in the imager, the photons generated by the scintillation material are converted to electrical energy from which the respective neutrons/gamma rays can be determined and, accordingly, a direction to, and the location of, a radiation source identified.

  3. Linear encoding device

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B. (Inventor)

    1993-01-01

    A Linear Motion Encoding device for measuring the linear motion of a moving object is disclosed in which a light source is mounted on the moving object and a position sensitive detector such as an array photodetector is mounted on a nearby stationary object. The light source emits a light beam directed towards the array photodetector such that a light spot is created on the array. An analog-to-digital converter, connected to the array photodetector is used for reading the position of the spot on the array photodetector. A microprocessor and memory is connected to the analog-to-digital converter to hold and manipulate data provided by the analog-to-digital converter on the position of the spot and to compute the linear displacement of the moving object based upon the data from the analog-to-digital converter.

  4. Characterization of a baculovirus-encoded RNA 5'-triphosphatase.

    PubMed

    Gross, C H; Shuman, S

    1998-09-01

    Autographa californica nuclear polyhedrosis virus (AcNPV) encodes a 168-amino-acid polypeptide that contains the signature motif of the superfamily of protein phosphatases that act via a covalent cysteinyl phosphate intermediate. The sequence of the AcNPV phosphatase is similar to that of the RNA triphosphatase domain of the metazoan cellular mRNA capping enzyme. Here, we show that the purified recombinant AcNPV protein is an RNA 5'-triphosphatase that hydrolyzes the gamma-phosphate of triphosphate-terminated poly(A); it also hydrolyzes ATP to ADP and GTP to GDP. The phosphatase sediments as two discrete components in a glycerol gradient: a 9.5S oligomer and 2.5S putative monomer. The 2.5S form of the enzyme releases 32Pi from 1 microM gamma-32P-labeled triphosphate-terminated poly(A) with a turnover number of 52 min-1 and converts ATP to ADP with Vmax of 8 min-1 and Km of 25 microM ATP. The 9.5S oligomeric form of the enzyme displays an initial pre-steady-state burst of ADP and Pi formation, which is proportional to and stoichiometric with the enzyme, followed by a slower steady-state rate of product formation (approximately 1/10 of the steady-state rate of the 2.5S enzyme). We surmise that the oligomeric enzyme is subject to a rate-limiting step other than reaction chemistry and that this step is either distinct from or slower than the rate-limiting step for the 2.5S enzyme. Replacing the presumptive active site nucleophile Cys-119 by alanine abrogates RNA triphosphatase and ATPase activity. Our findings raise the possibility that baculoviruses encode enzymes that cap the 5' ends of viral transcripts synthesized at late times postinfection by a virus-encoded RNA polymerase. PMID:9696798

  5. Primary enzyme quantitation

    DOEpatents

    Saunders, G.C.

    1982-03-04

    The disclosure relates to the quantitation of a primary enzyme concentration by utilizing a substrate for the primary enzyme labeled with a second enzyme which is an indicator enzyme. Enzyme catalysis of the substrate occurs and results in release of the indicator enzyme in an amount directly proportional to the amount of primary enzyme present. By quantifying the free indicator enzyme one determines the amount of primary enzyme present.

  6. Space vehicle onboard command encoder

    NASA Technical Reports Server (NTRS)

    1975-01-01

    A flexible onboard encoder system was designed for the space shuttle. The following areas were covered: (1) implementation of the encoder design into hardware to demonstrate the various encoding algorithms/code formats, (2) modulation techniques in a single hardware package to maintain comparable reliability and link integrity of the existing link systems and to integrate the various techniques into a single design using current technology. The primary function of the command encoder is to accept input commands, generated either locally onboard the space shuttle or remotely from the ground, format and encode the commands in accordance with the payload input requirements and appropriately modulate a subcarrier for transmission by the baseband RF modulator. The following information was provided: command encoder system design, brassboard hardware design, test set hardware and system packaging, and software.

  7. N-Consecutive-Phase Encoder

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Lee, Ho-Kyoung; Weber, Charles

    1995-01-01

    N-consecutive-phase encoder (NCPE) is conceptual encoder for generating alphabet of N consecutive full-response continuous-phase-modulation (CPM) signals. Enables use of binary preencoder of higher rate than used with simple continuous-phase encoder (CPE). NCPE makes possible to achieve power efficiencies and bandwidth efficiencies greater than conventional trellis coders with continuous-phase frequency-shift keying (CPFSK).

  8. Identification and in vitro characterization of a Marek’s disease virus encoded ribonucleotide reductase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s disease virus (MDV) encodes a ribonucleotide reductase (RR), a key regulatory enzyme in the DNA synthesis pathway. The gene coding for the RR of MDV is located in the unique long (UL) region of the genome. The large subunit is encoded by UL39 (RR1) and is predicted to comprise 860 amino acid...

  9. Prosodic Encoding in Silent Reading.

    ERIC Educational Resources Information Center

    Wilkenfeld, Deborah

    In silent reading, short-memory tasks, such as semantic and syntactic processing, require a stage of phonetic encoding between visual representation and the actual extraction of meaning, and this encoding includes prosodic as well as segmental features. To test for this suprasegmental coding, an experiment was conducted in which subjects were…

  10. Information encoder/decoder using chaotic systems

    DOEpatents

    Miller, Samuel Lee; Miller, William Michael; McWhorter, Paul Jackson

    1997-01-01

    The present invention discloses a chaotic system-based information encoder and decoder that operates according to a relationship defining a chaotic system. Encoder input signals modify the dynamics of the chaotic system comprising the encoder. The modifications result in chaotic, encoder output signals that contain the encoder input signals encoded within them. The encoder output signals are then capable of secure transmissions using conventional transmission techniques. A decoder receives the encoder output signals (i.e., decoder input signals) and inverts the dynamics of the encoding system to directly reconstruct the original encoder input signals.

  11. Information encoder/decoder using chaotic systems

    DOEpatents

    Miller, S.L.; Miller, W.M.; McWhorter, P.J.

    1997-10-21

    The present invention discloses a chaotic system-based information encoder and decoder that operates according to a relationship defining a chaotic system. Encoder input signals modify the dynamics of the chaotic system comprising the encoder. The modifications result in chaotic, encoder output signals that contain the encoder input signals encoded within them. The encoder output signals are then capable of secure transmissions using conventional transmission techniques. A decoder receives the encoder output signals (i.e., decoder input signals) and inverts the dynamics of the encoding system to directly reconstruct the original encoder input signals. 32 figs.

  12. Multiple transcripts encode glucose 6-phosphate dehydrogenase in the southern cattle tick, Rhipicephalus (Boophilus) microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glucose 6-phosphate dehydrogenase (G6PDH) is an enzyme that plays a critical role in the production of NADPH. Here we describe the identification of four transcripts (G6PDH-A, -B, -C, and -D) that putatively encode the enzyme in the southern cattle tick, Rhipicephalus (Boophilus) microplus. The geno...

  13. DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis

    PubMed Central

    2015-01-01

    The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the “structure elucidation problem”: the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS’s utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 104 molecules/bead and sequencing allowed for elucidation of each compound’s synthetic history. We applied DESPS to the combinatorial synthesis of a 75 645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and

  14. DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis.

    PubMed

    MacConnell, Andrew B; McEnaney, Patrick J; Cavett, Valerie J; Paegel, Brian M

    2015-09-14

    The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the "structure elucidation problem": the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS's utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 10(4) molecules/bead and sequencing allowed for elucidation of each compound's synthetic history. We applied DESPS to the combinatorial synthesis of a 75,645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR

  15. PNA-encoded chemical libraries.

    PubMed

    Zambaldo, Claudio; Barluenga, Sofia; Winssinger, Nicolas

    2015-06-01

    Peptide nucleic acid (PNA)-encoded chemical libraries along with DNA-encoded libraries have provided a powerful new paradigm for library synthesis and ligand discovery. PNA-encoding stands out for its compatibility with standard solid phase synthesis and the technology has been used to prepare libraries of peptides, heterocycles and glycoconjugates. Different screening formats have now been reported including selection-based and microarray-based methods that have yielded specific ligands against diverse target classes including membrane receptors, lectins and challenging targets such as Hsp70.

  16. Two digital video encoder circuits

    NASA Astrophysics Data System (ADS)

    Eldon, John A.

    1992-11-01

    Central to `multimedia' image processing is the desire to encode computer graphics data into a standard television signal, complete with line, field, and color subcarrier synchronizing information. The numerous incompatibilities between television and computer display standards render this operation far less trivial than it sounds to anyone who hasn't worked with both types of signals. To simplify the task of encoding computer graphics signals into standard NTSC (North America and Japan) or PAL (most of Europe) television format for display, broadcast, or recording, TRW LSI Products Inc. has introduced the two newest members of it multimedia integrated circuit family, the TMC22090 and TMC22190 digital video encoders.

  17. Crystal Structure of a Virus-Encoded Putative Glycosyltransferase

    SciTech Connect

    Xiang, Ye; Baxa, Ulrich; Zhang, Ying; Steven, Alasdair C.; Lewis, Gentry L.; Van Etten, James L.; Rossmann, Michael G.

    2010-11-22

    The chloroviruses (family Phycodnaviridae), unlike most viruses, encode some, if not most, of the enzymes involved in the glycosylation of their structural proteins. Annotation of the gene product B736L from chlorovirus NY-2A suggests that it is a glycosyltransferase. The structure of the recombinantly expressed B736L protein was determined by X-ray crystallography to 2.3-{angstrom} resolution, and the protein was shown to have two nucleotide-binding folds like other glycosyltransferase type B enzymes. This is the second structure of a chlorovirus-encoded glycosyltransferase and the first structure of a chlorovirus type B enzyme to be determined. B736L is a retaining enzyme and belongs to glycosyltransferase family 4. The donor substrate was identified as GDP-mannose by isothermal titration calorimetry and was shown to bind into the cleft between the two domains in the protein. The active form of the enzyme is probably a dimer in which the active centers are separated by about 40 {angstrom}.

  18. Serial position encoding of signs.

    PubMed

    Miozzo, Michele; Petrova, Anna; Fischer-Baum, Simon; Peressotti, Francesca

    2016-09-01

    Reduced short-term memory (STM) capacity has been reported for sign as compared to speech when items have to be recalled in a specific order. This difference has been attributed to a more precise and efficient serial position encoding in verbal STM (used for speech) than visuo-spatial STM (used for sign). We tested in the present investigation whether the reduced STM capacity with signs stems from a lack of positional encoding available in verbal STM. Error analyses reported in prior studies have revealed that positions are defined in verbal STM by distance from both the start and the end of the sequence (both-edges positional encoding scheme). Our analyses of the errors made by deaf participants with finger-spelled letters revealed that the both-edges positional encoding scheme underlies the STM representation of signs. These results indicate that the cause of the STM disadvantage is not the type of positional encoding but rather the difficulties in binding an item in visuo-spatial STM to its specific position in the sequence. Both-edges positional encoding scheme could be specific of sign, since it has not been found in visuo-spatial STM tasks conducted with hearing participants. PMID:27244095

  19. Multidimensionally encoded magnetic resonance imaging.

    PubMed

    Lin, Fa-Hsuan

    2013-07-01

    Magnetic resonance imaging (MRI) typically achieves spatial encoding by measuring the projection of a q-dimensional object over q-dimensional spatial bases created by linear spatial encoding magnetic fields (SEMs). Recently, imaging strategies using nonlinear SEMs have demonstrated potential advantages for reconstructing images with higher spatiotemporal resolution and reducing peripheral nerve stimulation. In practice, nonlinear SEMs and linear SEMs can be used jointly to further improve the image reconstruction performance. Here, we propose the multidimensionally encoded (MDE) MRI to map a q-dimensional object onto a p-dimensional encoding space where p > q. MDE MRI is a theoretical framework linking imaging strategies using linear and nonlinear SEMs. Using a system of eight surface SEM coils with an eight-channel radiofrequency coil array, we demonstrate the five-dimensional MDE MRI for a two-dimensional object as a further generalization of PatLoc imaging and O-space imaging. We also present a method of optimizing spatial bases in MDE MRI. Results show that MDE MRI with a higher dimensional encoding space can reconstruct images more efficiently and with a smaller reconstruction error when the k-space sampling distribution and the number of samples are controlled.

  20. Fly Photoreceptors Encode Phase Congruency

    PubMed Central

    Friederich, Uwe; Billings, Stephen A.; Hardie, Roger C.; Juusola, Mikko; Coca, Daniel

    2016-01-01

    More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli. PMID:27336733

  1. Fly Photoreceptors Encode Phase Congruency.

    PubMed

    Friederich, Uwe; Billings, Stephen A; Hardie, Roger C; Juusola, Mikko; Coca, Daniel

    2016-01-01

    More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli. PMID:27336733

  2. Synaptic encoding of temporal contiguity

    PubMed Central

    Ostojic, Srdjan; Fusi, Stefano

    2013-01-01

    Often we need to perform tasks in an environment that changes stochastically. In these situations it is important to learn the statistics of sequences of events in order to predict the future and the outcome of our actions. The statistical description of many of these sequences can be reduced to the set of probabilities that a particular event follows another event (temporal contiguity). Under these conditions, it is important to encode and store in our memory these transition probabilities. Here we show that for a large class of synaptic plasticity models, the distribution of synaptic strengths encodes transitions probabilities. Specifically, when the synaptic dynamics depend on pairs of contiguous events and the synapses can remember multiple instances of the transitions, then the average synaptic weights are a monotonic function of the transition probabilities. The synaptic weights converge to the distribution encoding the probabilities also when the correlations between consecutive synaptic modifications are considered. We studied how this distribution depends on the number of synaptic states for a specific model of a multi-state synapse with hard bounds. In the case of bistable synapses, the average synaptic weights are a smooth function of the transition probabilities and the accuracy of the encoding depends on the learning rate. As the number of synaptic states increases, the average synaptic weights become a step function of the transition probabilities. We finally show that the information stored in the synaptic weights can be read out by a simple rate-based neural network. Our study shows that synapses encode transition probabilities under general assumptions and this indicates that temporal contiguity is likely to be encoded and harnessed in almost every neural circuit in the brain. PMID:23641210

  3. Biocatalytic Single Enzyme Nanoparticles

    SciTech Connect

    Grate, Jay W.; Kim, Jungbae

    2004-03-31

    As an innovative way of enzyme stabilization, we recently developed a new enzyme composite of nano-meter scale that we call "single-enzyme nanoparticles (SENs)" (9). Each enzyme molecule is surrounded with a porous composite organic/inorganic network of less than a few nanometers think. This approach represents a new type of enzyme-containing nanostructure. In experiments with perotease (chymotrypsin, CT), the activity of single enzyme nanoparticle form of the enzyme was greatly stabilized compared to the free form, without imposing a serious mass transfer limitation of substrates. In this chapter we will describe the synthesis, characterization and catalytic activity of the new SENs.

  4. Divergence and Convergence in Enzyme Evolution*

    PubMed Central

    Galperin, Michael Y.; Koonin, Eugene V.

    2012-01-01

    Comparative analysis of the sequences of enzymes encoded in a variety of prokaryotic and eukaryotic genomes reveals convergence and divergence at several levels. Functional convergence can be inferred when structurally distinct and hence non-homologous enzymes show the ability to catalyze the same biochemical reaction. In contrast, as a result of functional diversification, many structurally similar enzyme molecules act on substantially distinct substrates and catalyze diverse biochemical reactions. Here, we present updates on the ATP-grasp, alkaline phosphatase, cupin, HD hydrolase, and N-terminal nucleophile (Ntn) hydrolase enzyme superfamilies and discuss the patterns of sequence and structural conservation and diversity within these superfamilies. Typically, enzymes within a superfamily possess common sequence motifs and key active site residues, as well as (predicted) reaction mechanisms. These observations suggest that the strained conformation (the entatic state) of the active site, which is responsible for the substrate binding and formation of the transition complex, tends to be conserved within enzyme superfamilies. The subsequent fate of the transition complex is not necessarily conserved and depends on the details of the structures of the enzyme and the substrate. This variability of reaction outcomes limits the ability of sequence analysis to predict the exact enzymatic activities of newly sequenced gene products. Nevertheless, sequence-based (super)family assignments and generic functional predictions, even if imprecise, provide valuable leads for experimental studies and remain the best approach to the functional annotation of uncharacterized proteins from new genomes. PMID:22069324

  5. How Infants Encode Spatial Extent

    ERIC Educational Resources Information Center

    Duffy, Sean; Huttenlocher, Janellen; Levine, Susan; Duffy, Renee

    2005-01-01

    This study explores how infants encode an object's spatial extent. We habituated 6.5-month-old infants to a dowel inside a container and then tested whether they dishabituate to a change in absolute size when the relation between dowel and container is held constant (by altering the size of both container and dowel) and when the relation changes…

  6. Encoding Standards for Linguistic Corpora.

    ERIC Educational Resources Information Center

    Ide, Nancy

    The demand for extensive reusability of large language text collections for natural languages processing research requires development of standardized encoding formats. Such formats must be capable of representing different kinds of information across the spectrum of text types and languages, capable of representing different levels of…

  7. Insolubilization process increases enzyme stability

    NASA Technical Reports Server (NTRS)

    Billingham, J.; Lyn, J.

    1971-01-01

    Enzymes complexed with polymeric matrices contain properties suggesting application to enzyme-controlled reactions. Stability of insolubilized enzyme derivatives is markedly greater than that of soluble enzymes and physical form of insolubilized enzymes is useful in column and batch processes.

  8. Molecular evolution of nitrogen assimilatory enzymes in marine prasinophytes.

    PubMed

    Ghoshroy, Sohini; Robertson, Deborah L

    2015-01-01

    Nitrogen assimilation is a highly regulated process requiring metabolic coordination of enzymes and pathways in the cytosol, chloroplast, and mitochondria. Previous studies of prasinophyte genomes revealed that genes encoding nitrate and ammonium transporters have a complex evolutionary history involving both vertical and horizontal transmission. Here we examine the evolutionary history of well-conserved nitrogen-assimilating enzymes to determine if a similar complex history is observed. Phylogenetic analyses suggest that genes encoding glutamine synthetase (GS) III in the prasinophytes evolved by horizontal gene transfer from a member of the heterokonts. In contrast, genes encoding GSIIE, a canonical vascular plant and green algal enzyme, were found in the Micromonas genomes but have been lost from Ostreococcus. Phylogenetic analyses placed the Micromonas GSIIs in a larger chlorophyte/vascular plant clade; a similar topology was observed for ferredoxin-dependent nitrite reductase (Fd-NiR), indicating the genes encoding GSII and Fd-NiR in these prasinophytes evolved via vertical transmission. Our results show that genes encoding the nitrogen-assimilating enzymes in Micromonas and Ostreococcus have been differentially lost and as well as recruited from different evolutionary lineages, suggesting that the regulation of nitrogen assimilation in prasinophytes will differ from other green algae.

  9. Isolation and characterization of cDNAs encoding imidazoleglycerolphosphate dehydratase from Arabidopsis thaliana.

    PubMed Central

    Tada, S; Volrath, S; Guyer, D; Scheidegger, A; Ryals, J; Ohta, D; Ward, E

    1994-01-01

    cDNA clones encoding imidazoleglycerolphosphate dehydratase (IGPD; EC 4.2.1.19) from Arabidopsis thaliana were isolated by complementation of a bacterial auxotroph. The predicted primary translation product shared significant identity with the corresponding sequences from bacteria and fungi. As in yeast, the plant enzyme is monofunctional, lacking the histidinol phosphatase activity present in the Escherichia coli protein. IGPD mRNA was present in major organs at all developmental stages assayed. The Arabidopsis genome appears to contain two genes encoding this enzyme, based on DNA gel blot and polymerase chain reaction analysis. PMID:8066131

  10. Monolithic-integrated microlaser encoder.

    PubMed

    Sawada, R; Higurashi, E; Ito, T; Ohguchi, O; Tsubamoto, M

    1999-11-20

    We have developed an extremely small integrated microencoder whose sides are less than 1 mm long. It is 1/100 the size of conventional encoders. This microencoder consists of a laser diode, monolithic photodiodes, and fluorinated polyimide waveguides with total internal reflection mirrors. The instrument can measure the relative displacement between a grating scale and the encoder with a resolution of the order of 0.01 microm; it can also determine the direction in which the scale is moving. By using the two beams that were emitted from the two etched mirrors of the laser diode, by monolithic integration of the waveguide and photodiodes, and by fabrication of a step at the edge of the waveguide, we were able to eliminate conventional bulky optical components such as the beam splitter, the quarter-wavelength plate, bulky mirrors, and bulky photodetectors. PMID:18324228

  11. Encoding information into precipitation structures

    NASA Astrophysics Data System (ADS)

    Martens, Kirsten; Bena, Ioana; Droz, Michel; Rácz, Zoltan

    2008-12-01

    Material design at submicron scales would be profoundly affected if the formation of precipitation patterns could be easily controlled. It would allow the direct building of bulk structures, in contrast to traditional techniques which consist of removing material in order to create patterns. Here, we discuss an extension of our recent proposal of using electrical currents to control precipitation bands which emerge in the wake of reaction fronts in A+ + B- → C reaction-diffusion processes. Our main result, based on simulating the reaction-diffusion-precipitation equations, is that the dynamics of the charged agents can be guided by an appropriately designed time-dependent electric current so that, in addition to the control of the band spacing, the width of the precipitation bands can also be tuned. This makes straightforward the encoding of information into precipitation patterns and, as an amusing example, we demonstrate the feasibility by showing how to encode a musical rhythm.

  12. Expression, function and regulation of mouse cytochrome P450 enzymes: comparison with human P450 enzymes.

    PubMed

    Hrycay, E G; Bandiera, S M

    2009-12-01

    The present review focuses on the expression, function and regulation of mouse cytochrome P450 (Cyp) enzymes. Information compiled for mouse Cyp enzymes is compared with data collected for human CYP enzymes. To date, approximately 40 pairs of orthologous mouse-human CYP genes have been identified that encode enzymes performing similar metabolic functions. Recent knowledge concerning the tissue expression of mouse Cyp enzymes from families 1 to 51 is summarized. The catalytic activities of microsomal, mitochondrial and recombinant mouse Cyp enzymes are discussed and their involvement in the metabolism of exogenous and endogenous compounds is highlighted. The role of nuclear receptors, such as the aryl hydrocarbon receptor, constitutive androstane receptor and pregnane X receptor, in regulating the expression of mouse Cyp enzymes is examined. Targeted disruption of selected Cyp genes has generated numerous Cyp null mouse lines used to decipher the role of Cyp enzymes in metabolic, toxicological and biological processes. In conclusion, the laboratory mouse is an indispensable model for exploring human CYP-mediated activities.

  13. Genetic diversity of genes encoding OKP and LEN beta-lactamases produced by clinical Klebsiella pneumoniae strains in Portugal.

    PubMed

    Mendonça, Nuno; Ferreira, Eugénia; Caniça, Manuela

    2009-03-01

    Of the 308 clinical Klebsiella pneumoniae strains collected in 21 Portuguese health institutions, 11 encoded for LEN and 9 for OKP enzymes; of these, 15 were new enzymes. Ninety-one percent of LEN and all OKP producer strains were resistant to amoxicillin. We demonstrate that these beta-lactamase were highly diverse.

  14. Four transcripts encode glucose 6-phosphate dehydrogenase (G6PDH) in the Southern cattle tick, Rhipicephalus (Boophilus) microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glucose 6-phosphate dehydrogenase (G6PDH) is an enzyme that plays a critical role in the production of NADPH. Here we describe the characterization of four transcripts (G6PDH-A, -B, -C, and -D) that putatively encode the enzyme in the southern cattle tick, Rhipicephalus (Boophilus) microplus. The ...

  15. Vector Encoding in Biochemical Networks

    NASA Astrophysics Data System (ADS)

    Potter, Garrett; Sun, Bo

    Encoding of environmental cues via biochemical signaling pathways is of vital importance in the transmission of information for cells in a network. The current literature assumes a single cell state is used to encode information, however, recent research suggests the optimal strategy utilizes a vector of cell states sampled at various time points. To elucidate the optimal sampling strategy for vector encoding, we take an information theoretic approach and determine the mutual information of the calcium signaling dynamics obtained from fibroblast cells perturbed with different concentrations of ATP. Specifically, we analyze the sampling strategies under the cases of fixed and non-fixed vector dimension as well as the efficiency of these strategies. Our results show that sampling with greater frequency is optimal in the case of non-fixed vector dimension but that, in general, a lower sampling frequency is best from both a fixed vector dimension and efficiency standpoint. Further, we find the use of a simple modified Ornstein-Uhlenbeck process as a model qualitatively captures many of our experimental results suggesting that sampling in biochemical networks is based on a few basic components.

  16. Lessons from modENCODE.

    PubMed

    Brown, James B; Celniker, Susan E

    2015-01-01

    The modENCODE (Model Organism Encyclopedia of DNA Elements) Consortium aimed to map functional elements-including transcripts, chromatin marks, regulatory factor binding sites, and origins of DNA replication-in the model organisms Drosophila melanogaster and Caenorhabditis elegans. During its five-year span, the consortium conducted more than 2,000 genome-wide assays in developmentally staged animals, dissected tissues, and homogeneous cell lines. Analysis of these data sets provided foundational insights into genome, epigenome, and transcriptome structure and the evolutionary turnover of regulatory pathways. These studies facilitated a comparative analysis with similar data types produced by the ENCODE Consortium for human cells. Genome organization differs drastically in these distant species, and yet quantitative relationships among chromatin state, transcription, and cotranscriptional RNA processing are deeply conserved. Of the many biological discoveries of the modENCODE Consortium, we highlight insights that emerged from integrative studies. We focus on operational and scientific lessons that may aid future projects of similar scale or aims in other, emerging model systems. PMID:26133010

  17. Assays for investigating deSUMOylation enzymes.

    PubMed

    Madu, Ikenna G; Chen, Yuan

    2012-07-01

    Post-translational modifications by the SUMO (Small Ubiquitin-like MOdifier) family of proteins are recently discovered essential regulatory mechanisms. All SUMO proteins are synthesized as larger precursors that are matured by SUMO-specific proteases, known as SENPs, which remove several C-terminal amino acids of SUMO to expose the Gly-Gly motif. SENPs also remove SUMO modifications from target proteins, making this modification highly dynamic. At least six deSUMOylation enzymes, all of which are encoded by essential genes, have been identified in mammals. SENP1 has been shown to play an important role in the development of prostate cancer and in angiogenesis. This unit describes and discusses methods for characterizing the deSUMOylation enzymes. These assays enable the identification of inhibitors of these enzymes and investigation of their mechanism of inhibition in order to develop research tools and future therapeutics.

  18. Developments in Enzyme Technology.

    ERIC Educational Resources Information Center

    Chaplin, M. F.

    1984-01-01

    Enzyme technology has a well-established industrial base, with applications that have survived competition. The most prominent applications of enzymes in biotechnology are examined with an explanation of some theoretical background. Topics include extending an enzyme's useful life, partition and diffusion, industrial uses, and therapeutic uses.…

  19. Chloroplast and Cytoplasmic Enzymes

    PubMed Central

    Anderson, Louise E.; Advani, Vimal R.

    1970-01-01

    Three pea (Pisum sativum) leaf chloroplast enzymes—triose phosphate isomerase, glyceric acid 3-phosphate kinase, and fructose 1,6-diphosphate aldolase—have been separated from the corresponding cytoplasmic enzymes by isoelectric focusing. These three enzymes of the reductive pentose phosphate cycle are therefore distinct proteins, not identical with the analogous enzymes of the Embden-Meyerhof-Parnas pathway. PMID:16657347

  20. The optional E. coli prr locus encodes a latent form of phage T4-induced anticodon nuclease.

    PubMed Central

    Levitz, R; Chapman, D; Amitsur, M; Green, R; Snyder, L; Kaufmann, G

    1990-01-01

    The optional Escherichia coli prr locus restricts phage T4 mutants lacking polynucleotide kinase or RNA ligase. Underlying this restriction is the specific manifestation of the T4-induced anticodon nuclease, an enzyme which triggers the cleavage-ligation of the host tRNALys. We report here the molecular cloning, nucleotide sequence and mutational analysis of prr-associated DNA. The results indicate that prr encodes a latent form of anticodon nuclease consisting of a core enzyme and cognate masking agents. They suggest that the T4-encoded factors of anticodon nuclease counteract the prr-encoded masking agents, thus activating the latent enzyme. The encoding of a tRNA cleavage-ligation pathway by two separate genetic systems which cohabitate E. coli may provide a clue to the evolution of RNA splicing mechanisms mediated by proteins. Images Fig.3. Fig.4. PMID:1691706

  1. The average enzyme principle.

    PubMed

    Reznik, Ed; Chaudhary, Osman; Segrè, Daniel

    2013-09-01

    The Michaelis-Menten equation for an irreversible enzymatic reaction depends linearly on the enzyme concentration. Even if the enzyme concentration changes in time, this linearity implies that the amount of substrate depleted during a given time interval depends only on the average enzyme concentration. Here, we use a time re-scaling approach to generalize this result to a broad category of multi-reaction systems, whose constituent enzymes have the same dependence on time, e.g. they belong to the same regulon. This "average enzyme principle" provides a natural methodology for jointly studying metabolism and its regulation.

  2. Novel optical encoder for harsh environments

    NASA Astrophysics Data System (ADS)

    Kress, Bernard; Mueller, Ulrich; Brac-de-la-Perriere, Vincent

    2014-09-01

    We are presenting a new optical encoder architecture for shaft encoding, both in incremental and absolute modes. This encoder is based on a diffractive optics technology platform. We have developed various disk based rotary diffractive encoders previously. This encoder is different in the way it is not a disk composed of successive gratings or computer generated holograms, but rather composed of a single element placed on the shaft. It is thus best suited for hollow shaft or end of shaft applications such as in encoder controlled electrical motors. This new architecture aims at solving some of the problems encountered with previous implementations of diffractive encoders such as disk wobble, disk to shaft centering and also encoding in harsh environments.

  3. Profiling the orphan enzymes.

    PubMed

    Sorokina, Maria; Stam, Mark; Médigue, Claudine; Lespinet, Olivier; Vallenet, David

    2014-06-06

    The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called "orphan enzymes". The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to "local orphan enzymes" that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new activities.

  4. Time Course of Grammatical Encoding in Agrammatism

    ERIC Educational Resources Information Center

    Lee, Jiyeon

    2011-01-01

    Producing a sentence involves encoding a preverbal message into a grammatical structure by retrieving lexical items and integrating them into a functional (semantic-to-grammatical) structure. Individuals with agrammatism are impaired in this grammatical encoding process. However, it is unclear what aspect of grammatical encoding is impaired and…

  5. Plastid-localized amino acid biosynthetic pathways of Plantae are predominantly composed of non-cyanobacterial enzymes

    PubMed Central

    Reyes-Prieto, Adrian; Moustafa, Ahmed

    2012-01-01

    Studies of photosynthetic eukaryotes have revealed that the evolution of plastids from cyanobacteria involved the recruitment of non-cyanobacterial proteins. Our phylogenetic survey of >100 Arabidopsis nuclear-encoded plastid enzymes involved in amino acid biosynthesis identified only 21 unambiguous cyanobacterial-derived proteins. Some of the several non-cyanobacterial plastid enzymes have a shared phylogenetic origin in the three Plantae lineages. We hypothesize that during the evolution of plastids some enzymes encoded in the host nuclear genome were mistargeted into the plastid. Then, the activity of those foreign enzymes was sustained by both the plastid metabolites and interactions with the native cyanobacterial enzymes. Some of the novel enzymatic activities were favored by selective compartmentation of additional complementary enzymes. The mosaic phylogenetic composition of the plastid amino acid biosynthetic pathways and the reduced number of plastid-encoded proteins of non-cyanobacterial origin suggest that enzyme recruitment underlies the recompartmentation of metabolic routes during the evolution of plastids. PMID:23233874

  6. Enzyme kinetics of conjugating enzymes: PAPS sulfotransferase.

    PubMed

    James, Margaret O

    2014-01-01

    The sulfotransferase (SULT) enzymes catalyze the formation of sulfate esters or sulfamates from substrates that contain hydroxy or amine groups, utilizing 3'-phosphoadenosyl-5'-phosphosulfate (PAPS) as the donor of the sulfonic group. The rate of product formation depends on the concentrations of PAPS and substrate as well as the sulfotransferase enzyme; thus, if PAPS is held constant while varying substrate concentration (or vice versa), the kinetic constants derived are apparent constants. When studied over a narrow range of substrate concentrations, classic Michaelis-Menten kinetics can be observed with many SULT enzymes and most substrates. Some SULT enzymes exhibit positive or negative cooperativity during conversion of substrate to product, and the kinetics fit the Hill plot. A characteristic feature of most sulfotransferase-catalyzed reactions is that, when studied over a wide range of substrate concentrations, the rate of product formation initially increases as substrate concentration increases, then decreases at high substrate concentrations, i.e., they exhibit substrate inhibition or partial substrate inhibition. This chapter gives an introduction to sulfotransferases, including a historical note, the nomenclature, a description of the function of SULTs with different types of substrates, presentation of examples of enzyme kinetics with SULTs, and a discussion of what is known about mechanisms of substrate inhibition in the sulfotransferases.

  7. Enzymes for improved biomass conversion

    DOEpatents

    Brunecky, Roman; Himmel, Michael E.

    2016-02-02

    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  8. Catalyzed enzyme electrodes

    SciTech Connect

    Zawodzinski, T.A.; Wilson, M.S.; Rishpon, J.; Gottesfeld, S.

    1992-12-31

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid, polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  9. Catalyzed enzyme electrodes

    SciTech Connect

    Zawodzinski, T.A.; Wilson, M.S.; Rishpon, J.; Gottesfeld, S.

    1993-07-13

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  10. Catalyzed enzyme electrodes

    DOEpatents

    Zawodzinski, Thomas A.; Wilson, Mahlon S.; Rishpon, Judith; Gottesfeld, Shimshon

    1993-01-01

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  11. Rational enzyme redesign

    SciTech Connect

    Ornstein, R.L.

    1994-05-01

    Protein engineering is first a means of elucidating structure-function relations in an enzyme, and second, a means of changing a protein to make it serve a different, but generally related, purpose. In principle, one may change the functional characteristics of an enzyme by altering its substrate specificity, kinetics, optimum range of activity, and chemical mechanism. Obviously one cannot make all possible combinations of amino acid changes for even the smallest enzyme, so the essential question is which changes to make. The intent of rational protein/enzyme redesign is to alter a protein/enzyme in a timely and premeditated fashion. This article provides an outline of the process of rational enzyme redesign.

  12. Molecular mechanisms for protein-encoded inheritance

    SciTech Connect

    Wiltzius, Jed J.W.; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R.; Apostol, Marcin I.; Goldschmidt, Lukasz; Soriaga, Angela B.; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David

    2009-12-01

    In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of {beta}-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct {beta}-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.

  13. Adenylate-forming enzymes.

    PubMed

    Schmelz, Stefan; Naismith, James H

    2009-12-01

    Thioesters, amides, and esters are common chemical building blocks in a wide array of natural products. The formation of these bonds can be catalyzed in a variety of ways. For chemists, the use of an activating group is a common strategy and adenylate enzymes are exemplars of this approach. Adenylating enzymes activate the otherwise unreactive carboxylic acid by transforming the normal hydroxyl leaving group into adenosine monophosphate. Recently there have been a number of studies of such enzymes and in this review we suggest a new classification scheme. The review highlights the diversity in enzyme fold, active site architecture, and metal coordination that has evolved to catalyze this particular reaction. PMID:19836944

  14. Engineering Genetically Encoded FRET Sensors

    PubMed Central

    Lindenburg, Laurens; Merkx, Maarten

    2014-01-01

    Förster Resonance Energy Transfer (FRET) between two fluorescent proteins can be exploited to create fully genetically encoded and thus subcellularly targetable sensors. FRET sensors report changes in energy transfer between a donor and an acceptor fluorescent protein that occur when an attached sensor domain undergoes a change in conformation in response to ligand binding. The design of sensitive FRET sensors remains challenging as there are few generally applicable design rules and each sensor must be optimized anew. In this review we discuss various strategies that address this shortcoming, including rational design approaches that exploit self-associating fluorescent domains and the directed evolution of FRET sensors using high-throughput screening. PMID:24991940

  15. DNA topoisomerase VIII: a novel subfamily of type IIB topoisomerases encoded by free or integrated plasmids in Archaea and Bacteria.

    PubMed

    Gadelle, Danièle; Krupovic, Mart; Raymann, Kasie; Mayer, Claudine; Forterre, Patrick

    2014-07-01

    Type II DNA topoisomerases are divided into two families, IIA and IIB. Types IIA and IIB enzymes share homologous B subunits encompassing the ATP-binding site, but have non-homologous A subunits catalyzing DNA cleavage. Type IIA topoisomerases are ubiquitous in Bacteria and Eukarya, whereas members of the IIB family are mostly present in Archaea and plants. Here, we report the detection of genes encoding type IIB enzymes in which the A and B subunits are fused into a single polypeptide. These proteins are encoded in several bacterial genomes, two bacterial plasmids and one archaeal plasmid. They form a monophyletic group that is very divergent from archaeal and eukaryotic type IIB enzymes (DNA topoisomerase VI). We propose to classify them into a new subfamily, denoted DNA topoisomerase VIII. Bacterial genes encoding a topoisomerase VIII are present within integrated mobile elements, most likely derived from conjugative plasmids. Purified topoisomerase VIII encoded by the plasmid pPPM1a from Paenibacillus polymyxa M1 had ATP-dependent relaxation and decatenation activities. In contrast, the enzyme encoded by mobile elements integrated into the genome of Ammonifex degensii exhibited DNA cleavage activity producing a full-length linear plasmid and that from Microscilla marina exhibited ATP-independent relaxation activity. Topoisomerases VIII, the smallest known type IIB enzymes, could be new promising models for structural and mechanistic studies.

  16. Carbohydrate-related enzymes of important Phytophthora plant pathogens.

    PubMed

    Brouwer, Henk; Coutinho, Pedro M; Henrissat, Bernard; de Vries, Ronald P

    2014-11-01

    Carbohydrate-Active enZymes (CAZymes) form particularly interesting targets to study in plant pathogens. Despite the fact that many CAZymes are pathogenicity factors, oomycete CAZymes have received significantly less attention than effectors in the literature. Here we present an analysis of the CAZymes present in the Phytophthora infestans, Ph. ramorum, Ph. sojae and Pythium ultimum genomes compared to growth of these species on a range of different carbon sources. Growth on these carbon sources indicates that the size of enzyme families involved in degradation of cell-wall related substrates like cellulose, xylan and pectin is not always a good predictor of growth on these substrates. While a capacity to degrade xylan and cellulose exists the products are not fully saccharified and used as a carbon source. The Phytophthora genomes encode larger CAZyme sets when compared to Py. ultimum, and encode putative cutinases, GH12 xyloglucanases and GH10 xylanases that are missing in the Py. ultimum genome. Phytophthora spp. also encode a larger number of enzyme families and genes involved in pectin degradation. No loss or gain of complete enzyme families was found between the Phytophthora genomes, but there are some marked differences in the size of some enzyme families.

  17. Enzyme technology: an overview.

    PubMed

    van Beilen, Jan B; Li, Zhi

    2002-08-01

    Enzymes are being used in numerous new applications in the food, feed, agriculture, paper, leather, and textiles industries, resulting in significant cost reductions. At the same time, rapid technological developments are now stimulating the chemistry and pharma industries to embrace enzyme technology, a trend strengthened by concerns regarding health, energy, raw materials, and the environment.

  18. NMDA receptors and memory encoding.

    PubMed

    Morris, Richard G M

    2013-11-01

    It is humbling to think that 30 years have passed since the paper by Collingridge, Kehl and McLennan showing that one of Jeff Watkins most interesting compounds, R-2-amino-5-phosphonopentanoate (d-AP5), blocked the induction of long-term potentiation in vitro at synapses from area CA3 of the hippocampus to CA1 without apparent effect on baseline synaptic transmission (Collingridge et al., 1983). This dissociation was one of the key triggers for an explosion of interest in glutamate receptors, and much has been discovered since that collectively contributes to our contemporary understanding of glutamatergic synapses - their biophysics and subunit composition, of the agonists and antagonists acting on them, and their diverse functions in different networks of the brain and spinal cord. It can be fairly said that Collingridge et al.'s (1983) observation was the stimulus that has led, on the one hand, to structural biological work at the atomic scale describing the key features of NMDA receptors that enables their coincidence function to happen; and, on the other, to work with whole animals investigating the contributions that calcium signalling via this receptor can have on rhythmical activities controlled by spinal circuits, memory encoding in the hippocampus (the topic of this article), visual cortical plasticity, sensitization in pain, and other functions. In this article, I lay out how my then interest in long-term potentiation (LTP) as a model of memory enabled me to recognise the importance of Collingridge et al.'s discovery - and how I and my colleagues endeavoured to take things forward in the area of learning and memory. This is in some respects a personal story, and I tell it as such. The idea that NMDA receptor activation is essential for memory encoding, though not for storage, took time to develop and to be accepted. Along the way, there have been confusions, challenges, and surprises surrounding the idea that activation of NMDA receptors can

  19. Gene encoding plant asparagine synthetase

    DOEpatents

    Coruzzi, Gloria M.; Tsai, Fong-Ying

    1993-10-26

    The identification and cloning of the gene(s) for plant asparagine synthetase (AS), an important enzyme involved in the formation of asparagine, a major nitrogen transport compound of higher plants is described. Expression vectors constructed with the AS coding sequence may be utilized to produce plant AS; to engineer herbicide resistant plants, salt/drought tolerant plants or pathogen resistant plants; as a dominant selectable marker; or to select for novel herbicides or compounds useful as agents that synchronize plant cells in culture. The promoter for plant AS, which directs high levels of gene expression and is induced in an organ specific manner and by darkness, is also described. The AS promoter may be used to direct the expression of heterologous coding sequences in appropriate hosts.

  20. Cotton cellulose: enzyme adsorption and enzymic hydrolysis

    SciTech Connect

    Beltrame, P.L.; Carniti, P.; Focher, B.; Marzetti, A.; Cattaneo, M.

    1982-01-01

    The adsorption of a crude cellulase complex from Trichoderma viride on variously pretreated cotton cellulose samples was studied in the framework of the Langmuir approach at 2-8 degrees. The saturation amount of adsorbed enzyme was related to the susceptibility of the substrates to hydrolysis. In every case the adsorption process was faster by 2-3 orders of magnitude than the hydrolysis step to give end products. For ZnCl/sub 2/-treated cotton cellulose the Langmuir parameters correlated fairly well with the value of the Michaelis constant, measured for its enzymic hydrolysis, and the adsorptive complex was indistinguishable from the complex of the Michaelis-Menten model for the hydrolysis.

  1. Unconscious relational encoding depends on hippocampus

    PubMed Central

    Duss, Simone B.; Reber, Thomas P.; Hänggi, Jürgen; Schwab, Simon; Wiest, Roland; Müri, René M.; Brugger, Peter; Gutbrod, Klemens

    2014-01-01

    Textbooks divide between human memory systems based on consciousness. Hippocampus is thought to support only conscious encoding, while neocortex supports both conscious and unconscious encoding. We tested whether processing modes, not consciousness, divide between memory systems in three neuroimaging experiments with 11 amnesic patients (mean age = 45.55 years, standard deviation = 8.74, range = 23–60) and 11 matched healthy control subjects. Examined processing modes were single item versus relational encoding with only relational encoding hypothesized to depend on hippocampus. Participants encoded and later retrieved either single words or new relations between words. Consciousness of encoding was excluded by subliminal (invisible) word presentation. Amnesic patients and controls performed equally well on the single item task activating prefrontal cortex. But only the controls succeeded on the relational task activating the hippocampus, while amnesic patients failed as a group. Hence, unconscious relational encoding, but not unconscious single item encoding, depended on hippocampus. Yet, three patients performed normally on unconscious relational encoding in spite of amnesia capitalizing on spared hippocampal tissue and connections to language cortex. This pattern of results suggests that processing modes divide between memory systems, while consciousness divides between levels of function within a memory system. PMID:25273998

  2. Encoders for block-circulant LDPC codes

    NASA Technical Reports Server (NTRS)

    Andrews, Kenneth; Dolinar, Sam; Thorpe, Jeremy

    2005-01-01

    In this paper, we present two encoding methods for block-circulant LDPC codes. The first is an iterative encoding method based on the erasure decoding algorithm, and the computations required are well organized due to the block-circulant structure of the parity check matrix. The second method uses block-circulant generator matrices, and the encoders are very similar to those for recursive convolutional codes. Some encoders of the second type have been implemented in a small Field Programmable Gate Array (FPGA) and operate at 100 Msymbols/second.

  3. Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

    PubMed Central

    Vieille, Claire; Zeikus, Gregory J.

    2001-01-01

    Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described. PMID:11238984

  4. How to achieve high-level expression of microbial enzymes

    PubMed Central

    Liu, Long; Yang, Haiquan; Shin, Hyun-dong; Chen, Rachel R.; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-01-01

    Microbial enzymes have been used in a large number of fields, such as chemical, agricultural and biopharmaceutical industries. The enzyme production rate and yield are the main factors to consider when choosing the appropriate expression system for the production of recombinant proteins. Recombinant enzymes have been expressed in bacteria (e.g., Escherichia coli, Bacillus and lactic acid bacteria), filamentous fungi (e.g., Aspergillus) and yeasts (e.g., Pichia pastoris). The favorable and very advantageous characteristics of these species have resulted in an increasing number of biotechnological applications. Bacterial hosts (e.g., E. coli) can be used to quickly and easily overexpress recombinant enzymes; however, bacterial systems cannot express very large proteins and proteins that require post-translational modifications. The main bacterial expression hosts, with the exception of lactic acid bacteria and filamentous fungi, can produce several toxins which are not compatible with the expression of recombinant enzymes in food and drugs. However, due to the multiplicity of the physiological impacts arising from high-level expression of genes encoding the enzymes and expression hosts, the goal of overproduction can hardly be achieved, and therefore, the yield of recombinant enzymes is limited. In this review, the recent strategies used for the high-level expression of microbial enzymes in the hosts mentioned above are summarized and the prospects are also discussed. We hope this review will contribute to the development of the enzyme-related research field. PMID:23686280

  5. Evaluation of GOES encoder lamps

    NASA Technical Reports Server (NTRS)

    Viehmann, W.; Helmold, N.

    1983-01-01

    Aging characteristics and life expectancies of flight quality, tungsten filament, encoder lamps are similar to those of 'commercial' grade gas filled lamps of similar construction, filament material and filament temperature. The aging and final failure by filament burnout are caused by single crystal growth over large portions of the filament with the concomitant development of facets and notches resulting in reduction of cross section and mechanical weakening of the filament. The life expectancy of presently produced lamps is about one year at their nominal operating voltage of five volts dc. At 4.5 volts, it is about two years. These life times are considerably shorter, and the degradation rates of lamp current and light flux are considerably higher, than were observed in the laboratory and in orbit on lamps of the same type manufactured more than a decade ago. It is speculated that the filaments of these earlier lamps contained a crystallization retarding dopant, possibly thorium oxide. To obtain the desired life expectancy of or = to four years in present lamps, operating voltages of or = to four volts dc would be required.

  6. Chemotactic separation of enzymes.

    PubMed

    Dey, Krishna Kanti; Das, Sambeeta; Poyton, Matthew F; Sengupta, Samudra; Butler, Peter J; Cremer, Paul S; Sen, Ayusman

    2014-12-23

    We demonstrate a procedure for the separation of enzymes based on their chemotactic response toward an imposed substrate concentration gradient. The separation is observed within a two-inlet, five-outlet microfluidic network, designed to allow mixtures of active (ones that catalyze substrate turnover) and inactive (ones that do not catalyze substrate turnover) enzymes, labeled with different fluorophores, to flow through one of the inlets. Substrate solution prepared in phosphate buffer was introduced through the other inlet of the device at the same flow rate. The steady-state concentration profiles of the enzymes were obtained at specific positions within the outlets of the microchannel using fluorescence microscopy. In the presence of a substrate concentration gradient, active enzyme molecules migrated preferentially toward the substrate channel. The excess migration of the active enzyme molecules was quantified in terms of an enrichment coefficient. Experiments were carried out with different pairs of enzymes. Coupling the physics of laminar flow of liquid and molecular diffusion, multiphysics simulations were carried out to estimate the extent of the chemotactic separation. Our results show that, with appropriate microfluidic arrangement, molecular chemotaxis leads to spontaneous separation of active enzyme molecules from their inactive counterparts of similar charge and size.

  7. Biofilm-degrading enzymes from Lysobacter gummosus

    PubMed Central

    Gökçen, Anke; Vilcinskas, Andreas; Wiesner, Jochen

    2014-01-01

    Biofilm-degrading enzymes could be used for the gentle cleaning of industrial and medical devices and the manufacture of biofilm-resistant materials. We therefore investigated 20 species and strains of the bacterial genus Lysobacter for their ability to degrade experimental biofilms formed by Staphylococcus epidermidis, a common nosocomial pathogen typically associated with device-related infections. The highest biofilm-degradation activity was achieved by L. gummosus. The corresponding enzymes were identified by sequencing the L. gummosus genome. Partial purification of the biofilm-degrading activity from an extract of extracellular material followed by peptide mass fingerprinting resulted in the identification of two peptidases (α-lytic protease and β-lytic metalloendopeptidase) that were predicted to degrade bacterial cell walls. In addition, we identified two isoforms of a lysyl endopeptidase and an enzyme similar to metalloproteases from Vibrio spp. Potential peptidoglycan-binding C-terminal fragments of two OmpA-like proteins also co-purified with the biofilm-degrading activity. The L. gummosus genome was found to encode five isoenzymes of α-lytic protease and three isoenzymes of lysyl endopeptidase. These results indicated that the extracellular digestion of biofilms by L. gummosus depends on multiple bacteriolytic and proteolytic enzymes, which could now be exploited for biofilm control. PMID:24518560

  8. Aspergillus Enzymes Involved in Degradation of Plant Cell Wall Polysaccharides

    PubMed Central

    de Vries, Ronald P.; Visser, Jaap

    2001-01-01

    Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a more and more attractive alternative to chemical and mechanical processes. Over the past 15 years, much progress has been made in elucidating the structural characteristics of these polysaccharides and in characterizing the enzymes involved in their degradation and the genes of biotechnologically relevant microorganisms encoding these enzymes. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. This review summarizes our current knowledge of the cell wall polysaccharide-degrading enzymes from aspergilli and the genes by which they are encoded. PMID:11729262

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

    PubMed

    Szuplewski, S; Terracol, R

    2001-08-01

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

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

    PubMed Central

    Szuplewski, S; Terracol, R

    2001-01-01

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

  11. Assessment of the pectin degrading enzyme network of Aspergillus niger by functional genomics.

    PubMed

    Martens-Uzunova, Elena S; Schaap, Peter J

    2009-03-01

    The saprobic fungus Aspergillus niger is an efficient producer of a suite of extracellular enzymes involved in carbohydrate modification and degradation. Genome mining has resulted in the prediction of at least 39 genes encoding enzymes involved in the depolymerisation of the backbone of pectin. Additional genes,encoding enzymatic activities required for the degradation of the arabinan and arabinogalactan sidechains were predicted as well. DNA microarray analysis was used to study the condition-dependent expression of these genes, and to generate insights in possible synergistic interactions between the individual members of the pectin degrading enzyme network. For this purpose, A. niger was grown on sugarbeet pectin and on galacturonic acid, rhamnose and xylose, the main monomeric sugar constituents of pectin. An analysis of the corresponding transcriptomes revealed expression of 46 genes encoding pectinolytic enzymes. Their transcriptional profiles are discussed in detail and a cascade model of pectin degradation is proposed.

  12. Commercial production of microbial enzymes

    SciTech Connect

    Munro, I.G.

    1985-01-01

    The advantages and uses of industrially produced microbial enzymes are described. The processes involved in the production of these enzymes, cultivation techniques, enzyme extraction, enzyme purification and immobilization are outlined. Both the history of enzyme technology and its future development are discussed.

  13. Microdiversity of extracellular enzyme genes among sequenced prokaryotic genomes

    PubMed Central

    Zimmerman, Amy E; Martiny, Adam C; Allison, Steven D

    2013-01-01

    Understanding the relationship between prokaryotic traits and phylogeny is important for predicting and modeling ecological processes. Microbial extracellular enzymes have a pivotal role in nutrient cycling and the decomposition of organic matter, yet little is known about the phylogenetic distribution of genes encoding these enzymes. In this study, we analyzed 3058 annotated prokaryotic genomes to determine which taxa have the genetic potential to produce alkaline phosphatase, chitinase and β-N-acetyl-glucosaminidase enzymes. We then evaluated the relationship between the genetic potential for enzyme production and 16S rRNA phylogeny using the consenTRAIT algorithm, which calculated the phylogenetic depth and corresponding 16S rRNA sequence identity of clades of potential enzyme producers. Nearly half (49.2%) of the genomes analyzed were found to be capable of extracellular enzyme production, and these were non-randomly distributed across most prokaryotic phyla. On average, clades of potential enzyme-producing organisms had a maximum phylogenetic depth of 0.008004–0.009780, though individual clades varied broadly in both size and depth. These values correspond to a minimum 16S rRNA sequence identity of 98.04–98.40%. The distribution pattern we found is an indication of microdiversity, the occurrence of ecologically or physiologically distinct populations within phylogenetically related groups. Additionally, we found positive correlations among the genes encoding different extracellular enzymes. Our results suggest that the capacity to produce extracellular enzymes varies at relatively fine-scale phylogenetic resolution. This variation is consistent with other traits that require a small number of genes and provides insight into the relationship between taxonomy and traits that may be useful for predicting ecological function. PMID:23303371

  14. RNA as an Enzyme.

    ERIC Educational Resources Information Center

    Cech, Thomas R.

    1986-01-01

    Reviews current findings that explain RNA's function as an enzyme in addition to being an informational molecule. Highlights recent research efforts and notes changes in the information base on RNA activity. Includes models and diagrams of RNA activity. (ML)

  15. Enzymes in Analytical Chemistry.

    ERIC Educational Resources Information Center

    Fishman, Myer M.

    1980-01-01

    Presents tabular information concerning recent research in the field of enzymes in analytic chemistry, with methods, substrate or reaction catalyzed, assay, comments and references listed. The table refers to 128 references. Also listed are 13 general citations. (CS)

  16. Transposition of the gene encoding a TEM-12 extended-spectrum beta-lactamase.

    PubMed Central

    Heritage, J; Hawkey, P M; Todd, N; Lewis, I J

    1992-01-01

    An isolate of Klebsiella oxytoca from the blood culture of a child with leukemia was found to produce two beta-lactamases, at least one of which conferred resistance to ceftazidime. Genes encoding both enzymes were located on a single self-transmissible 100-kb plasmid, pOZ201. This plasmid was introduced into Escherichia coli UB5201 (pACYC184), and the gene encoding one beta-lactamase was transposed onto plasmid pACYC184 by exploiting a gene dosage effect. The transposable gene was found to encode a TEM-12 enzyme as determined by nucleotide sequencing. This gene was subsequently transposed onto plasmid pUB307. The transposable element encoding the TEM-12 enzyme has been designated Tn841. Both plasmids pACYC184::Tn841 and pUB307::Tn841 were shown to encode a beta-lactamase with the same isoelectric point and substrate profile as the TEM-12 beta-lactamase. Transposon Tn841, at approximately 7 kb, is larger than TnA (4.8 kb) and transposes at a lower frequency. Although it produced a resolvase which can complement the resolvase of Tn3, its transposase function was not able to complement the transposition of a TnA element which lacked transposase. The occurrence of a gene encoding an extended-spectrum beta-lactamase on a transposable element in a clinically significant bacterium is potentially a cause for concern for the spread of resistance to the extended-spectrum cephalosporins. PMID:1329636

  17. Congruity of Encoding in Children's Redintegrative Memory.

    ERIC Educational Resources Information Center

    Hall, Donald M.; Geis, Mary Fulcher

    The mnemonic consequences of semantic, acoustic, and orthographic encoding and the relationships between encoding and retrieval cues were investigated in an incidental-learning experiment involving 24 first-, third-, and fifth-grade pupils. Each child was asked one orienting question for each of 18 words; the questions differed in the type of…

  18. Pseudochromatic encoding fractional Fourier transform rainbow hologram

    NASA Astrophysics Data System (ADS)

    Guo, Yongkang; Huang, Qizhong; Du, Jinglei

    1998-08-01

    The FRTH is presented in this paper and its properties are discussed. Then we make a pseudo chromatic encoding fractional Fourier transform rainbow hologram, based on its specialty in its reconstruction and that the encoding color has relationship with the order of the reconstruction FRT system, a new type of anti-counterfeiting hologram is introduced.

  19. DNA encoding a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-08-15

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  20. Experiments in encoding multilevel images as quadtrees

    NASA Technical Reports Server (NTRS)

    Lansing, Donald L.

    1987-01-01

    Image storage requirements for several encoding methods are investigated and the use of quadtrees with multigray level or multicolor images are explored. The results of encoding a variety of images having up to 256 gray levels using three schemes (full raster, runlength and quadtree) are presented. Although there is considerable literature on the use of quadtrees to store and manipulate binary images, their application to multilevel images is relatively undeveloped. The potential advantage of quadtree encoding is that an entire area with a uniform gray level may be encoded as a unit. A pointerless quadtree encoding scheme is described. Data are presented on the size of the quadtree required to encode selected images and on the relative storage requirements of the three encoding schemes. A segmentation scheme based on the statistical variation of gray levels within a quadtree quadrant is described. This parametric scheme may be used to control the storage required by an encoded image and to preprocess a scene for feature identification. Several sets of black and white and pseudocolor images obtained by varying the segmentation parameter are shown.

  1. The Acquisition of Syntactically Encoded Evidentiality

    ERIC Educational Resources Information Center

    Rett, Jessica; Hyams, Nina

    2014-01-01

    This article presents several empirical studies of syntactically encoded evidentiality in English. The first part of our study consists of an adult online experiment that confirms claims in Asudeh & Toivonen (2012) that raised Perception Verb Similatives (PVSs; e.g. "John looks like he is sick") encode direct evidentiality. We then…

  2. Overproduction of ligninolytic enzymes

    SciTech Connect

    Elisashvili, Vladimir; Kachlishvili, Eva; Torok, Tamas

    2014-06-17

    Methods, compositions, and systems for overproducing ligninolytic enzymes from the basidiomycetous fungus are described herein. As described, the method can include incubating a fungal strain of Cerrena unicolor IBB 303 in a fermentation system having growth medium which includes lignocellulosic material and then cultivating the fungal strain in the fermentation system under conditions wherein the fungus expresses the ligninolytic enzymes. In some cases, the lignocellulosic material is mandarin peel, ethanol production residue, walnut pericarp, wheat bran, wheat straw, or banana peel.

  3. RNA-modifying enzymes.

    PubMed

    Ferré-D'Amaré, Adrian R

    2003-02-01

    A bewildering number of post-transcriptional modifications are introduced into cellular RNAs by enzymes that are often conserved among archaea, bacteria and eukaryotes. The modifications range from those with well-understood functions, such as tRNA aminoacylation, to widespread but more mysterious ones, such as pseudouridylation. Recent structure determinations have included two types of RNA nucleobase modifying enzyme: pseudouridine synthases and tRNA guanine transglycosylases.

  4. Measurement of enzyme activity.

    PubMed

    Harris, T K; Keshwani, M M

    2009-01-01

    To study and understand the nature of living cells, scientists have continually employed traditional biochemical techniques aimed to fractionate and characterize a designated network of macromolecular components required to carry out a particular cellular function. At the most rudimentary level, cellular functions ultimately entail rapid chemical transformations that otherwise would not occur in the physiological environment of the cell. The term enzyme is used to singularly designate a macromolecular gene product that specifically and greatly enhances the rate of a chemical transformation. Purification and characterization of individual and collective groups of enzymes has been and will remain essential toward advancement of the molecular biological sciences; and developing and utilizing enzyme reaction assays is central to this mission. First, basic kinetic principles are described for understanding chemical reaction rates and the catalytic effects of enzymes on such rates. Then, a number of methods are described for measuring enzyme-catalyzed reaction rates, which mainly differ with regard to techniques used to detect and quantify concentration changes of given reactants or products. Finally, short commentary is given toward formulation of reaction mixtures used to measure enzyme activity. Whereas a comprehensive treatment of enzymatic reaction assays is not within the scope of this chapter, the very core principles that are presented should enable new researchers to better understand the logic and utility of any given enzymatic assay that becomes of interest.

  5. Understanding the importance of protein structure to nature's routes for divergent evolution in TIM barrel enzymes.

    PubMed

    Wise, Eric L; Rayment, Ivan

    2004-03-01

    It is widely agreed that new enzymes evolve from existing ones through the duplication of genes encoding existing enzymes followed by sequence divergence. While evolution is an inherently random process, studies of divergently related enzymes have shown that the evolution of new enzymes follows one of three general routes in which the substrate specificity, reaction mechanism, or active site architecture of the progenitor enzyme is reused in the new enzyme. Recent developments in structural biology relating to divergently related (beta/alpha)8 enzymes have brought new insight into these processes and have revealed that conserved structural elements play an important role in divergent evolution. These studies have shown that, although evolution occurs as a series of random mutations, stable folds such as the (beta/alpha)8 barrel and structural features of the active sites of enzymes are frequently reused in evolution and adapted for new catalytic purposes.

  6. A model for visual memory encoding.

    PubMed

    Nenert, Rodolphe; Allendorfer, Jane B; Szaflarski, Jerzy P

    2014-01-01

    Memory encoding engages multiple concurrent and sequential processes. While the individual processes involved in successful encoding have been examined in many studies, a sequence of events and the importance of modules associated with memory encoding has not been established. For this reason, we sought to perform a comprehensive examination of the network for memory encoding using data driven methods and to determine the directionality of the information flow in order to build a viable model of visual memory encoding. Forty healthy controls ages 19-59 performed a visual scene encoding task. FMRI data were preprocessed using SPM8 and then processed using independent component analysis (ICA) with the reliability of the identified components confirmed using ICASSO as implemented in GIFT. The directionality of the information flow was examined using Granger causality analyses (GCA). All participants performed the fMRI task well above the chance level (>90% correct on both active and control conditions) and the post-fMRI testing recall revealed correct memory encoding at 86.33 ± 5.83%. ICA identified involvement of components of five different networks in the process of memory encoding, and the GCA allowed for the directionality of the information flow to be assessed, from visual cortex via ventral stream to the attention network and then to the default mode network (DMN). Two additional networks involved in this process were the cerebellar and the auditory-insular network. This study provides evidence that successful visual memory encoding is dependent on multiple modules that are part of other networks that are only indirectly related to the main process. This model may help to identify the node(s) of the network that are affected by a specific disease processes and explain the presence of memory encoding difficulties in patients in whom focal or global network dysfunction exists.

  7. A model for visual memory encoding.

    PubMed

    Nenert, Rodolphe; Allendorfer, Jane B; Szaflarski, Jerzy P

    2014-01-01

    Memory encoding engages multiple concurrent and sequential processes. While the individual processes involved in successful encoding have been examined in many studies, a sequence of events and the importance of modules associated with memory encoding has not been established. For this reason, we sought to perform a comprehensive examination of the network for memory encoding using data driven methods and to determine the directionality of the information flow in order to build a viable model of visual memory encoding. Forty healthy controls ages 19-59 performed a visual scene encoding task. FMRI data were preprocessed using SPM8 and then processed using independent component analysis (ICA) with the reliability of the identified components confirmed using ICASSO as implemented in GIFT. The directionality of the information flow was examined using Granger causality analyses (GCA). All participants performed the fMRI task well above the chance level (>90% correct on both active and control conditions) and the post-fMRI testing recall revealed correct memory encoding at 86.33 ± 5.83%. ICA identified involvement of components of five different networks in the process of memory encoding, and the GCA allowed for the directionality of the information flow to be assessed, from visual cortex via ventral stream to the attention network and then to the default mode network (DMN). Two additional networks involved in this process were the cerebellar and the auditory-insular network. This study provides evidence that successful visual memory encoding is dependent on multiple modules that are part of other networks that are only indirectly related to the main process. This model may help to identify the node(s) of the network that are affected by a specific disease processes and explain the presence of memory encoding difficulties in patients in whom focal or global network dysfunction exists. PMID:25272154

  8. Cellulases, nucleic acids encoding them and methods for making and using them

    DOEpatents

    Blum, David; Gemsch Cuenca, Joslin; Dycaico, Mark

    2013-04-23

    This invention relates to molecular and cellular biology and biochemistry. In one aspect, the invention provides polypeptides having cellulase activity, e.g., endoglucanase, cellobiohydrolase, mannanase and/or .beta.-glucosidase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides cellulase activity, e.g., endoglucanase, cellobiohydrolase, mannanase and/or .beta.-glucosidase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts.

  9. Campylobacter jejuni gene cj0511 encodes a serine peptidase essential for colonisation

    PubMed Central

    Karlyshev, A.V.; Thacker, G.; Jones, M.A.; Clements, M.O.; Wren, B.W.

    2014-01-01

    According to MEROPS peptidase database, Campylobacter species encode 64 predicted peptidases. However, proteolytic properties of only a few of these proteins have been confirmed experimentally. In this study we identified and characterised a Campylobacter jejuni gene cj0511 encoding a novel peptidase. The proteolytic activity associated with this enzyme was demonstrated in cell lysates. Moreover, enzymatic studies conducted with a purified protein confirmed a prediction of it being a serine peptidase. Furthermore, cj0511 mutant was found to be severely attenuated in chicken colonisation model, suggesting a role of the Cj0511 protein in infection. PMID:24918062

  10. Marek’s disease virus encoded ribonucleotide reductase large subunit is essential for in vivo replication and plays a critical role in viral pathogenesis.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s disease virus encodes a ribonucleotide reductase (RR) that consists of two subunits namely RR1 and RR2, both of which associate to form an active holoenzyme and both subunits are necessary for enzyme activity. It is an essential enzyme for the conversion of ribonucleotides to deoxyribonucleo...

  11. Chemical and genomic evolution of enzyme-catalyzed reaction networks.

    PubMed

    Kanehisa, Minoru

    2013-09-01

    There is a tendency that a unit of enzyme genes in an operon-like structure in the prokaryotic genome encodes enzymes that catalyze a series of consecutive reactions in a metabolic pathway. Our recent analysis shows that this and other genomic units correspond to chemical units reflecting chemical logic of organic reactions. From all known metabolic pathways in the KEGG database we identified chemical units, called reaction modules, as the conserved sequences of chemical structure transformation patterns of small molecules. The extracted patterns suggest co-evolution of genomic units and chemical units. While the core of the metabolic network may have evolved with mechanisms involving individual enzymes and reactions, its extension may have been driven by modular units of enzymes and reactions.

  12. The nature of chemical innovation: new enzymes by evolution.

    PubMed

    Arnold, Frances H

    2015-11-01

    I describe how we direct the evolution of non-natural enzyme activities, using chemical intuition and information on structure and mechanism to guide us to the most promising reaction/enzyme systems. With synthetic reagents to generate new reactive intermediates and just a few amino acid substitutions to tune the active site, a cytochrome P450 can catalyze a variety of carbene and nitrene transfer reactions. The cyclopropanation, N-H insertion, C-H amination, sulfimidation, and aziridination reactions now demonstrated are all well known in chemical catalysis but have no counterparts in nature. The new enzymes are fully genetically encoded, assemble and function inside of cells, and can be optimized for different substrates, activities, and selectivities. We are learning how to use nature's innovation mechanisms to marry some of the synthetic chemists' favorite transformations with the exquisite selectivity and tunability of enzymes.

  13. Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae.

    PubMed

    Sánchez-López, Juan Francisco; González-Ibarra, Joaquín; Álvarez-Vargas, Aurelio; Milewski, Slawomir; Villagómez-Castro, Julio César; Cano-Canchola, Carmen; López-Romero, Everardo

    2015-06-01

    Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme.

  14. Aminoglycoside Modifying Enzymes

    PubMed Central

    Ramirez, Maria S.; Tolmasky, Marcelo E.

    2010-01-01

    Aminoglycosides have been an essential component of the armamentarium in the treatment of life-threatening infections. Unfortunately, their efficacy has been reduced by the surge and dissemination of resistance. In some cases the levels of resistance reached the point that rendered them virtually useless. Among many known mechanisms of resistance to aminoglycosides, enzymatic modification is the most prevalent in the clinical setting. Aminoglycoside modifying enzymes catalyze the modification at different −OH or −NH2 groups of the 2-deoxystreptamine nucleus or the sugar moieties and can be nucleotidyltranferases, phosphotransferases, or acetyltransferases. The number of aminoglycoside modifying enzymes identified to date as well as the genetic environments where the coding genes are located is impressive and there is virtually no bacteria that is unable to support enzymatic resistance to aminoglycosides. Aside from the development of new aminoglycosides refractory to as many as possible modifying enzymes there are currently two main strategies being pursued to overcome the action of aminoglycoside modifying enzymes. Their successful development would extend the useful life of existing antibiotics that have proven effective in the treatment of infections. These strategies consist of the development of inhibitors of the enzymatic action or of the expression of the modifying enzymes. PMID:20833577

  15. Red cell enzymes.

    PubMed

    Paniker, N V

    1975-03-01

    As compared to other cells of the body, the mammalian red cell has one of the simplest structural organizations. As a result, this cell has been extensively used in studies involving the structure, function, and integrity of cell membranes as well as cytoplasmic events. Additionally, the metabolic activities of the red blood cell are also relatively simple. During the past quarter century or so, an ocean of knowledge has been gathered on various aspects of red cell metabolism and function. The fields of enzymes, hemoglobin, membrane, and metabolic products comprise the major portion of this knowledge. These advances have made valuable contributions to biochemistry and medicine. Despite these favorable aspects of this simple, anucleated cell, it must be conceded that our knowledge about the red cell is far from complete. We are still in the dark concerning the mechanism involved in several aspects of its membrane, hemoglobin, enzymes, and a large number of other constituents. For example, a large number of enzymes with known catalytic activity but with unknown function have eluded investigators despite active pursuit. This review will be a consolidation of our present knowledge of human red cell enzymes, with particular reference to their usefulness in the diagnosis and therapy of disease. Owing to the multitude of publications by prominent investigators on each of the approximately 50 enzymes discussed in this review, it was impossible to cite a majority of them.

  16. Random-walk enzymes

    NASA Astrophysics Data System (ADS)

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C →U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  17. Random-walk enzymes.

    PubMed

    Mak, Chi H; Pham, Phuong; Afif, Samir A; Goodman, Myron F

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C→U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  18. Random-walk enzymes

    PubMed Central

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-01-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C → U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics. PMID:26465508

  19. Random-walk enzymes.

    PubMed

    Mak, Chi H; Pham, Phuong; Afif, Samir A; Goodman, Myron F

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C→U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics. PMID:26465508

  20. The evolution of metabolic enzymes in Plasmodium and trypanosomatids as compared to Saccharomyces and Schizosaccharomyces.

    PubMed

    Palenchar, Peter M; Palenchar, Jennifer B

    2012-07-01

    Understanding how the biological connectivity of genes and gene products affects evolution is an important aspect of understanding evolution. Genes encoding enzymes are frequently used to carry out such analyses. Interestingly, studies have shown that connectivity in the metabolic networks in parasitic protists, including Plasmodium falciparum and Trypanosoma brucei, have been substantially altered as compared to free living eukaryotes, such as Saccharomyces cerevisiae. Herein, we have determined K(a) values, which are a measure of the non-synonymous substitution rate, and used them to examine the differences between the evolution of genes in T. brucei, P. falciparum, S. cerevisiae, and Schizosaccharomyces pombe. All four organisms share similar traits with respect to the evolution of genes encoding metabolic enzymes. First, genes encoding metabolic enzymes have lower K(a) values than genes encoding non-metabolic proteins. In addition, perturbations of the metabolic network appear to have limited affects on the genes encoding enzymes near the perturbation. In most cases, there is a negative relationship between connectivity in the metabolic network of the gene product and the K(a) value for the gene, i.e. examining how much constraint there is on gene evolution when it is connected to many other genes. In addition, we find that the K(a) values of orthologs encoding for metabolic enzymes in each organism are significantly correlated, indicating similar patterns of non-synonymous substitutions. In total, our results indicate that the evolution of genes encoding metabolic enzymes do not tend to be greatly affected by changes in the metabolic network.

  1. Programmable Pulse-Position-Modulation Encoder

    NASA Technical Reports Server (NTRS)

    Zhu, David; Farr, William

    2006-01-01

    A programmable pulse-position-modulation (PPM) encoder has been designed for use in testing an optical communication link. The encoder includes a programmable state machine and an electronic code book that can be updated to accommodate different PPM coding schemes. The encoder includes a field-programmable gate array (FPGA) that is programmed to step through the stored state machine and code book and that drives a custom high-speed serializer circuit board that is capable of generating subnanosecond pulses. The stored state machine and code book can be updated by means of a simple text interface through the serial port of a personal computer.

  2. Pulse Vector-Excitation Speech Encoder

    NASA Technical Reports Server (NTRS)

    Davidson, Grant; Gersho, Allen

    1989-01-01

    Proposed pulse vector-excitation speech encoder (PVXC) encodes analog speech signals into digital representation for transmission or storage at rates below 5 kilobits per second. Produces high quality of reconstructed speech, but with less computation than required by comparable speech-encoding systems. Has some characteristics of multipulse linear predictive coding (MPLPC) and of code-excited linear prediction (CELP). System uses mathematical model of vocal tract in conjunction with set of excitation vectors and perceptually-based error criterion to synthesize natural-sounding speech.

  3. Testis-specific expression of a functional retroposon encoding glucose-6-phosphate dehydrogenase in the mouse

    SciTech Connect

    Hendriksen, P.J.M. |; Hoogerbrugge, J.W.; Baarends, W.M.

    1997-05-01

    The X-chromosomal gene glucose-6-phosphate dehydrogenase (G6pd) is known to be expressed in most cell types of mammalian species. In the mouse, we have detected a novel gene, designated G6pd-2, encoding a G6PD isoenzyme. G6pd-2 does not contain introns and appears to represent a retroposed gene. This gene is uniquely transcribed in postmeiotic spermatogenic cells in which the X-encoded G6pd gene is not transcribed. Expression of the G6pd-2 sequence in a bacterial system showed that the encoded product is an active enzyme. Zymogramic analysis demonstrated that recombinant G6PD-2, but not recombinant G6PD-1 (the X-chromosome-encoded G6PD), formed tetramers under reducing conditions. Under the same conditions, G6PD tetramers were also found in extracts of spermatids and spermatozoa, indicating the presence of G6pd-2-encoded isoenzyme in these cell types. G6pd-2 is one of the very few known expressed retroposons encoding a functional protein, and the presence of this gene is probably related to X chromosome inactivation during spermatogenesis. 62 refs., 7 figs.

  4. Giant virus Megavirus chilensis encodes the biosynthetic pathway for uncommon acetamido sugars.

    PubMed

    Piacente, Francesco; De Castro, Cristina; Jeudy, Sandra; Molinaro, Antonio; Salis, Annalisa; Damonte, Gianluca; Bernardi, Cinzia; Abergel, Chantal; Tonetti, Michela G

    2014-08-29

    Giant viruses mimicking microbes, by the sizes of their particles and the heavily glycosylated fibrils surrounding their capsids, infect Acanthamoeba sp., which are ubiquitous unicellular eukaryotes. The glycans on fibrils are produced by virally encoded enzymes, organized in gene clusters. Like Mimivirus, Megavirus glycans are mainly composed of virally synthesized N-acetylglucosamine (GlcNAc). They also contain N-acetylrhamnosamine (RhaNAc), a rare sugar; the enzymes involved in its synthesis are encoded by a gene cluster specific to Megavirus close relatives. We combined activity assays on two enzymes of the pathway with mass spectrometry and NMR studies to characterize their specificities. Mg534 is a 4,6-dehydratase 5-epimerase; its three-dimensional structure suggests that it belongs to a third subfamily of inverting dehydratases. Mg535, next in the pathway, is a bifunctional 3-epimerase 4-reductase. The sequential activity of the two enzymes leads to the formation of UDP-l-RhaNAc. This study is another example of giant viruses performing their glycan synthesis using enzymes different from their cellular counterparts, raising again the question of the origin of these pathways.

  5. EcoR124I: from Plasmid-Encoded Restriction-Modification System to Nanodevice

    PubMed Central

    Youell, James; Firman, Keith

    2008-01-01

    Plasmid R124 was first described in 1972 as being a new member of incompatibility group IncFIV, yet early physical investigations of plasmid DNA showed that this type of classification was more complex than first imagined. Throughout the history of the study of this plasmid, there have been many unexpected observations. Therefore, in this review, we describe the history of our understanding of this plasmid and the type I restriction-modification (R-M) system that it encodes, which will allow an opportunity to correct errors, or misunderstandings, that have arisen in the literature. We also describe the characterization of the R-M enzyme EcoR124I and describe the unusual properties of both type I R-M enzymes and EcoR124I in particular. As we approached the 21st century, we began to see the potential of the EcoR124I R-M enzyme as a useful molecular motor, and this leads to a description of recent work that has shown that the R-M enzyme can be used as a nanoactuator. Therefore, this is a history that takes us from a plasmid isolated from (presumably) an infected source to the potential use of the plasmid-encoded R-M enzyme in bionanotechnology. PMID:18535150

  6. Cloning and characterization of oah, the gene encoding oxaloacetate hydrolase in Aspergillus niger.

    PubMed

    Pedersen, H; Hjort, C; Nielsen, J

    2000-03-01

    The enzyme oxaloacetate hydrolase (EC 3.7.1.1), which is involved in oxalate formation, was purified from Aspergillus niger. The native enzyme has a molecular mass of 360-440 kDa, and the denatured enzyme has a molecular mass of 39 kDa, as determined by gel electrophoresis. Enzyme activity is maximal at pH 7.0 and 45 degrees C. The fraction containing the enzyme activity contained at least five proteins. The N-terminal amino acid sequences of four of these proteins were determined. The amino acid sequences were aligned with EST sequences from A. niger, and an EST sequence that showed 100% identity to all four sequences was identified. Using this EST sequence the gene encoding oxaloacetate hydrolase (oah) was cloned by inverse PCR. It consists of an ORF of 1227 bp with two introns of 92 and 112 bp, respectively. The gene encodes a protein of 341 amino acids with a molecular mass of 37 kDa. Under the growth conditions tested, the highest oah expression was found for growth on acetate as carbon source. The gene was expressed only at pH values higher than 4.0.

  7. Pancreatic enzyme pharmacotherapy.

    PubMed

    Ferrone, Marcus; Raimondo, Massimo; Scolapio, James S

    2007-06-01

    Supplemental pancreatic enzyme preparations are provided to patients with conditions of pancreatic exocrine deficiency such as chronic pancreatitis and cystic fibrosis. These patients frequently experience steatorrhea, which occurs from inadequate fat absorption. The delivery of sufficient enzyme concentrations into the duodenal lumen simultaneously with meals can reduce nutrient malabsorption, improve the symptoms of steatorrhea, and in some cases alleviate the pain associated with chronic pancreatitis. Current clinical practices dictate administration of lipase 25,000-40,000 units/meal by using pH-sensitive pancrelipase microspheres, along with dosage increases, compliance checks, and differential diagnosis in cases of treatment failure. Despite the large number of specialty enzyme replacements available commercially, many patients remain dissatisfied with standard therapy, and future developments are needed to optimize treatment in these individuals.

  8. Application of a hierarchical enzyme classification method reveals the role of gut microbiome in human metabolism

    PubMed Central

    2015-01-01

    Background Enzymes are known as the molecular machines that drive the metabolism of an organism; hence identification of the full enzyme complement of an organism is essential to build the metabolic blueprint of that species as well as to understand the interplay of multiple species in an ecosystem. Experimental characterization of the enzymatic reactions of all enzymes in a genome is a tedious and expensive task. The problem is more pronounced in the metagenomic samples where even the species are not adequately cultured or characterized. Enzymes encoded by the gut microbiota play an essential role in the host metabolism; thus, warranting the need to accurately identify and annotate the full enzyme complements of species in the genomic and metagenomic projects. To fulfill this need, we develop and apply a method called ECemble, an ensemble approach to identify enzymes and enzyme classes and study the human gut metabolic pathways. Results ECemble method uses an ensemble of machine-learning methods to accurately model and predict enzymes from protein sequences and also identifies the enzyme classes and subclasses at the finest resolution. A tenfold cross-validation result shows accuracy between 97 and 99% at different levels in the hierarchy of enzyme classification, which is superior to comparable methods. We applied ECemble to predict the entire complements of enzymes from ten sequenced proteomes including the human proteome. We also applied this method to predict enzymes encoded by the human gut microbiome from gut metagenomic samples, and to study the role played by the microbe-derived enzymes in the human metabolism. After mapping the known and predicted enzymes to canonical human pathways, we identified 48 pathways that have at least one bacteria-encoded enzyme, which demonstrates the complementary role of gut microbiome in human gut metabolism. These pathways are primarily involved in metabolizing dietary nutrients such as carbohydrates, amino acids, lipids

  9. Human cytoplasmic actin proteins are encoded by a multigene family

    SciTech Connect

    Engel, J.; Gunning, P.; Kedes, L.

    1982-06-01

    The authors characterized nine human actin genes that they isolated from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and ..cap alpha..-, ..beta..-, and ..gamma..-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria they show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken ..beta..-actin cDNA. They conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.

  10. Phage-encoded Serine Integrases and Other Large Serine Recombinases.

    PubMed

    Smith, Margaret C M

    2015-08-01

    The large serine recombinases (LSRs) are a family of enzymes, encoded in temperate phage genomes or on mobile elements, that precisely cut and recombine DNA in a highly controllable and predictable way. In phage integration, the LSRs act at specific sites, the attP site in the phage and the attB site in the host chromosome, where cleavage and strand exchange leads to the integrated prophage flanked by the recombinant sites attL and attR. The prophage can excise by recombination between attL and attR but this requires a phage-encoded accessory protein, the recombination directionality factor (RDF). Although the LSRs can bind specifically to all the recombination sites, only specific integrase-bound sites can pair in a synaptic complex prior to strand exchange. Recent structural information has led to a breakthrough in our understanding of the mechanism of the LSRs, notably how the LSRs bind to their substrates and how LSRs display this site-selectivity. We also understand that the RDFs exercise control over the LSRs by protein-protein interactions. Other recent work with the LSRs have contributed to our understanding of how all serine recombinases undergo strand exchange subunit rotation, facilitated by surfaces that resemble a molecular bearing.

  11. Structure and regulated expression of genes encoding fructose biphosphate aldolase in Trypanosoma brucei.

    PubMed Central

    Clayton, C E

    1985-01-01

    Low stringency hybridisation with a rabbit aldolase cDNA was used to select cDNA clones encoding fructose biphosphate aldolase in Trypanosoma brucei. A clone which is almost full length encodes a protein of 41 027 daltons which has 50% identity with rabbit aldolase A and slightly lower homology with B-type aldolases. The homologous mRNA is at least 6-fold more abundant in bloodstream trypomastigotes than in procyclic forms, as expected from measurements of enzyme activity. Genomic mapping results indicate that trypanosomes have four copies of the aldolase gene arranged as two copies of a tandem repeat. The protein has a short N-terminal extension (relative to other known aldolases) which could be involved in the glycosomal localisation of the enzyme. Images Fig. 1. Fig. 5. PMID:2998772

  12. An adaptive error-resilient video encoder

    NASA Astrophysics Data System (ADS)

    Cheng, Liang; El Zarki, Magda

    2003-06-01

    When designing an encoder for a real-time video application over a wireless channel, we must take into consideration the unpredictable fluctuation of the quality of the channel and its impact on the transmitted video data. This uncertainty motivates the development of an adaptive video encoding mechanism that can compensate for the infidelity caused either by data loss and/or by the post-processing (error concealment) at the decoder. In this paper, we first explore the major factors that cause quality degradation. We then propose an adaptive progressive replenishment algorithm for a packet loss rate (PLR) feedback enabled system. Assuming the availability of a feedback channel, we discuss a video quality assessment method, which allows the encoder to be aware of the decoder-side perceptual quality. Finally, we present a novel dual-feedback mechanism that guarantees an acceptable level of quality at the receiver side with modest increase in the complexity of the encoder.

  13. Cellobiohydrolase variants and polynucleotides encoding same

    SciTech Connect

    Wogulis, Mark

    2014-10-14

    The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

  14. Cellobiohydrolase variants and polynucleotides encoding the same

    SciTech Connect

    Wogulis, Mark

    2014-09-09

    The present invention relates to variants of a parent cellobiohydrolase. The present invention also relates to polynucleotides encoding the cellobiohydrolase variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the cellobiohydrolase variants.

  15. Cellobiohydrolase variants and polynucleotides encoding same

    DOEpatents

    Wogulis, Mark

    2013-09-24

    The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

  16. Encoding and reinstatement of threat: recognition potentials.

    PubMed

    Weymar, Mathias; Bradley, Margaret M; Hamm, Alfons O; Lang, Peter J

    2014-01-01

    On a recognition test, stimuli originally encoded in the context of shock threat show an enhanced late parietal positivity during later recognition compared to stimuli encoded during safety, particularly for emotionally arousing stimuli. The present study investigated whether this ERP old/new effect is further influenced when a threat context is reinstated during the recognition test. ERPs were measured in a yes-no recognition test for words rated high or low in emotional arousal that were encoded and recognized in the context of cues that signaled threat of shock or safety. Correct recognition of words encoded under threat, irrespective of reinstatement, was associated with an enhanced old-new ERP difference (500-700ms; centro-parietal), and this difference was only reliable for emotionally arousing words. Taken together, the data suggest that information processed in a stressful context are associated with better recollection on later recognition, an effect that was not modulated by reinstating the stressful context at retrieval.

  17. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  18. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

  19. Neurally Encoding Time for Olfactory Navigation

    PubMed Central

    Park, In Jun; Hein, Andrew M.; Bobkov, Yuriy V.; Reidenbach, Matthew A.; Ache, Barry W.; Principe, Jose C.

    2016-01-01

    Accurately encoding time is one of the fundamental challenges faced by the nervous system in mediating behavior. We recently reported that some animals have a specialized population of rhythmically active neurons in their olfactory organs with the potential to peripherally encode temporal information about odor encounters. If these neurons do indeed encode the timing of odor arrivals, it should be possible to demonstrate that this capacity has some functional significance. Here we show how this sensory input can profoundly influence an animal’s ability to locate the source of odor cues in realistic turbulent environments—a common task faced by species that rely on olfactory cues for navigation. Using detailed data from a turbulent plume created in the laboratory, we reconstruct the spatiotemporal behavior of a real odor field. We use recurrence theory to show that information about position relative to the source of the odor plume is embedded in the timing between odor pulses. Then, using a parameterized computational model, we show how an animal can use populations of rhythmically active neurons to capture and encode this temporal information in real time, and use it to efficiently navigate to an odor source. Our results demonstrate that the capacity to accurately encode temporal information about sensory cues may be crucial for efficient olfactory navigation. More generally, our results suggest a mechanism for extracting and encoding temporal information from the sensory environment that could have broad utility for neural information processing. PMID:26730727

  20. [The ENCODE project and functional genomics studies].

    PubMed

    Ding, Nan; Qu, Hongzhu; Fang, Xiangdong

    2014-03-01

    Upon the completion of the Human Genome Project, scientists have been trying to interpret the underlying genomic code for human biology. Since 2003, National Human Genome Research Institute (NHGRI) has invested nearly $0.3 billion and gathered over 440 scientists from more than 32 institutions in the United States, China, United Kingdom, Japan, Spain and Singapore to initiate the Encyclopedia of DNA Elements (ENCODE) project, aiming to identify and analyze all regulatory elements in the human genome. Taking advantage of the development of next-generation sequencing technologies and continuous improvement of experimental methods, ENCODE had made remarkable achievements: identified methylation and histone modification of DNA sequences and their regulatory effects on gene expression through altering chromatin structures, categorized binding sites of various transcription factors and constructed their regulatory networks, further revised and updated database for pseudogenes and non-coding RNA, and identified SNPs in regulatory sequences associated with diseases. These findings help to comprehensively understand information embedded in gene and genome sequences, the function of regulatory elements as well as the molecular mechanism underlying the transcriptional regulation by noncoding regions, and provide extensive data resource for life sciences, particularly for translational medicine. We re-viewed the contributions of high-throughput sequencing platform development and bioinformatical technology improve-ment to the ENCODE project, the association between epigenetics studies and the ENCODE project, and the major achievement of the ENCODE project. We also provided our prospective on the role of the ENCODE project in promoting the development of basic and clinical medicine.

  1. Neurally Encoding Time for Olfactory Navigation.

    PubMed

    Park, In Jun; Hein, Andrew M; Bobkov, Yuriy V; Reidenbach, Matthew A; Ache, Barry W; Principe, Jose C

    2016-01-01

    Accurately encoding time is one of the fundamental challenges faced by the nervous system in mediating behavior. We recently reported that some animals have a specialized population of rhythmically active neurons in their olfactory organs with the potential to peripherally encode temporal information about odor encounters. If these neurons do indeed encode the timing of odor arrivals, it should be possible to demonstrate that this capacity has some functional significance. Here we show how this sensory input can profoundly influence an animal's ability to locate the source of odor cues in realistic turbulent environments-a common task faced by species that rely on olfactory cues for navigation. Using detailed data from a turbulent plume created in the laboratory, we reconstruct the spatiotemporal behavior of a real odor field. We use recurrence theory to show that information about position relative to the source of the odor plume is embedded in the timing between odor pulses. Then, using a parameterized computational model, we show how an animal can use populations of rhythmically active neurons to capture and encode this temporal information in real time, and use it to efficiently navigate to an odor source. Our results demonstrate that the capacity to accurately encode temporal information about sensory cues may be crucial for efficient olfactory navigation. More generally, our results suggest a mechanism for extracting and encoding temporal information from the sensory environment that could have broad utility for neural information processing. PMID:26730727

  2. Enzymes in cancer

    PubMed Central

    Ohnuma, T.; Bergel, F.; Bray, R. C.

    1967-01-01

    1. A procedure for partial purification of asparaginase from chicken liver is presented. 2. The bulk of the enzyme is located in the soluble fraction of chicken liver. 3. Molecular weights of chicken-liver asparaginase and of the guinea-pig serum enzyme, estimated by gel filtration, were 306000 and 210000 respectively. The Michaelis constants (Km) at 37° and pH8·5 were 6·0×10−5m and 7·2×10−5m respectively. 4. At 50° the chicken-liver enzyme was moderately stable, some activity being lost by aggregation; in dilute electrolyte solutions the activity rapidly diminished. 5. The anti-lymphoma effect of guinea-pig serum in mice carrying the 6C3HED tumour was confirmed. Chicken-liver asparaginase also showed an effect but in this case the enzyme preparation had to be administered repeatedly. 6. Guinea-pig serum asparaginase was stable for several days in mouse blood, after intraperitoneal injection, whereas chicken-liver asparaginase rapidly disappeared. 7. Aspartic acid β-hydrazide was shown to be a competitive inhibitor of chicken-liver asparaginase with Ki approx. 5·6×10−4m. In mice it produced an anti-lymphoma effect, as reported previously. PMID:6033763

  3. Toying with Enzyme Catalysis.

    ERIC Educational Resources Information Center

    Richards, Debbie

    1998-01-01

    Describes a set of manipulatives that are used to establish a secure understanding of the concepts related to the environmental factors that affect the activities of enzymes. Includes a description of the model components and procedures for construction of the model. (DDR)

  4. Photoperiodism and Enzyme Activity

    PubMed Central

    Queiroz, Orlando; Morel, Claudine

    1974-01-01

    Metabolic readjustments after a change from long days to short days appear, in Kalanchoe blossfeldiana, to be achieved through the operation of two main mechanisms: variation in enzyme capacity, and circadian rhythmicity. After a lag time, capacity in phosphoenolpyruvate carboxylase and capacity in aspartate aminotransferase increase exponentially and appear to be allometrically linked during 50 to 60 short days; then a sudden fall takes place in the activity of the former. Malic enzyme and alanine aminotransferase behave differently. Thus, the operation of the two sections of the pathway (before and after the malate step) give rise to a continuously changing functional compartmentation in the pathway. Circadian rhythmicity, on the other hand, produces time compartmentation through phase shifts and variation in amplitude, independently for each enzyme. These characteristics suggest that the operation of a so-called biological clock would be involved. We propose the hypothesis that feedback regulation would be more accurate and efficient when applied to an already oscillating, clock-controlled enzyme system. PMID:16658749

  5. Monitoring enzyme kinetic behavior of enzyme-quantum dot bioconjugates

    NASA Astrophysics Data System (ADS)

    Claussen, Jonathan C.; Walper, Scott A.; Susumu, Kimihiro; Ancona, Mario G.; Medintz, Igor L.

    2014-05-01

    Luminescent semiconductor nanocrystals or quantum dots (QDs) hold tremendous promise for in vivo biosensing, cellular imaging, theranostics, and smart molecular sensing probes due to their small size and favorable photonic properties such as resistance to photobleaching, size-tunable PL, and large effective Stokes shifts. Herein, we demonstrate how QD-based bioconjugates can be used to enhance enzyme kinetics. Enzyme-substrate kinetics are analyzed for solutions containing both alkaline phosphatase enzymes and QDs with enzyme-to- QD molar ratios of 2, 12, and 24 as well as for a solution containing the same concentration of enzymes but without QDs. The enzyme kinetic paramters Vmax, KM, and Kcat/KM are extracted from the enzyme progress curves via the Lineweaver-Burk plot. Results demonstrate an approximate increase in enzyme efficiency of 5 - 8% for enzymes immobilized on the QD versus free in solution without QD immobilization.

  6. The other face of restriction: modification-dependent enzymes.

    PubMed

    Loenen, Wil A M; Raleigh, Elisabeth A

    2014-01-01

    The 1952 observation of host-induced non-hereditary variation in bacteriophages by Salvador Luria and Mary Human led to the discovery in the 1960s of modifying enzymes that glucosylate hydroxymethylcytosine in T-even phages and of genes encoding corresponding host activities that restrict non-glucosylated phage DNA: rglA and rglB (restricts glucoseless phage). In the 1980's, appreciation of the biological scope of these activities was dramatically expanded with the demonstration that plant and animal DNA was also sensitive to restriction in cloning experiments. The rgl genes were renamed mcrA and mcrBC (modified cytosine restriction). The new class of modification-dependent restriction enzymes was named Type IV, as distinct from the familiar modification-blocked Types I-III. A third Escherichia coli enzyme, mrr (modified DNA rejection and restriction) recognizes both methylcytosine and methyladenine. In recent years, the universe of modification-dependent enzymes has expanded greatly. Technical advances allow use of Type IV enzymes to study epigenetic mechanisms in mammals and plants. Type IV enzymes recognize modified DNA with low sequence selectivity and have emerged many times independently during evolution. Here, we review biochemical and structural data on these proteins, the resurgent interest in Type IV enzymes as tools for epigenetic research and the evolutionary pressures on these systems.

  7. Bioinformatic Characterization of Glycyl Radical Enzyme-Associated Bacterial Microcompartments

    PubMed Central

    Zarzycki, Jan; Erbilgin, Onur

    2015-01-01

    Bacterial microcompartments (BMCs) are proteinaceous organelles encapsulating enzymes that catalyze sequential reactions of metabolic pathways. BMCs are phylogenetically widespread; however, only a few BMCs have been experimentally characterized. Among them are the carboxysomes and the propanediol- and ethanolamine-utilizing microcompartments, which play diverse metabolic and ecological roles. The substrate of a BMC is defined by its signature enzyme. In catabolic BMCs, this enzyme typically generates an aldehyde. Recently, it was shown that the most prevalent signature enzymes encoded by BMC loci are glycyl radical enzymes, yet little is known about the function of these BMCs. Here we characterize the glycyl radical enzyme-associated microcompartment (GRM) loci using a combination of bioinformatic analyses and active-site and structural modeling to show that the GRMs comprise five subtypes. We predict distinct functions for the GRMs, including the degradation of choline, propanediol, and fuculose phosphate. This is the first family of BMCs for which identification of the signature enzyme is insufficient for predicting function. The distinct GRM functions are also reflected in differences in shell composition and apparently different assembly pathways. The GRMs are the counterparts of the vitamin B12-dependent propanediol- and ethanolamine-utilizing BMCs, which are frequently associated with virulence. This study provides a comprehensive foundation for experimental investigations of the diverse roles of GRMs. Understanding this plasticity of function within a single BMC family, including characterization of differences in permeability and assembly, can inform approaches to BMC bioengineering and the design of therapeutics. PMID:26407889

  8. The Moderately Efficient Enzyme: Futile Encounters and Enzyme Floppiness.

    PubMed

    Bar-Even, Arren; Milo, Ron; Noor, Elad; Tawfik, Dan S

    2015-08-18

    The pioneering model of Henri, Michaelis, and Menten was based on the fast equilibrium assumption: the substrate binds its enzyme reversibly, and substrate dissociation is much faster than product formation. Here, we examine this assumption from a somewhat different point of view, asking what fraction of enzyme-substrate complexes are futile, i.e., result in dissociation rather than product formation. In Knowles' notion of a "perfect" enzyme, all encounters of the enzyme with its substrate result in conversion to product. Thus, the perfect enzyme's catalytic efficiency, kcat/KM, is constrained by only the diffusion on-rate, and the fraction of futile encounters (defined as φ) approaches zero. The available data on >1000 different enzymes suggest that for ≥90% of enzymes φ > 0.99 and for the "average enzyme" φ ≥ 0.9999; namely, <1 of 10(4) encounters is productive. Thus, the "fast equilibrium" assumption holds for the vast majority of enzymes. We discuss possible molecular origins for the dominance of futile encounters, including the coexistence of multiple sub-states of an enzyme's active site (enzyme floppiness) and/or its substrate. Floppiness relates to the inherent flexibility of proteins, but also to conflicting demands, or trade-offs, between rate acceleration (the rate-determining chemical step) and catalytic turnover, or between turnover rate and accuracy. The study of futile encounters and active-site floppiness may contribute to a better understanding of enzyme catalysis, enzyme evolution, and improved enzyme design.

  9. Peroxisomal bifunctional enzyme deficiency.

    PubMed Central

    Watkins, P A; Chen, W W; Harris, C J; Hoefler, G; Hoefler, S; Blake, D C; Balfe, A; Kelley, R I; Moser, A B; Beard, M E

    1989-01-01

    Peroxisomal function was evaluated in a male infant with clinical features of neonatal adrenoleukodystrophy. Very long chain fatty acid levels were elevated in both plasma and fibroblasts, and beta-oxidation of very long chain fatty acids in cultured fibroblasts was significantly impaired. Although the level of the bile acid intermediate trihydroxycoprostanoic acid was slightly elevated in plasma, phytanic acid and L-pipecolic acid levels were normal, as was plasmalogen synthesis in cultured fibroblasts. The latter three parameters distinguish this case from classical neonatal adrenoleukodystrophy. In addition, electron microscopy and catalase subcellular distribution studies revealed that, in contrast to neonatal adrenoleukodystrophy, peroxisomes were present in the patient's tissues. Immunoblot studies of peroxisomal beta-oxidation enzymes revealed that the bifunctional enzyme (enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase) was deficient in postmortem liver samples, whereas acyl-CoA oxidase and the mature form of beta-ketothiolase were present. Density gradient centrifugation of fibroblast homogenates confirmed that intact peroxisomes were present. Immunoblots of fibroblasts peroxisomal fractions showed that they contained acyl-CoA oxidase and beta-ketothiolase, but bifunctional enzyme was not detected. Northern analysis, however, revealed that mRNA coding for the bifunctional enzyme was present in the patient's fibroblasts. These results indicate that the primary biochemical defect in this patient is a deficiency of peroxisomal bifunctional enzyme. It is of interest that the phenotype of this patient resembled neonatal adrenoleukodystrophy and would not have been distinguished from this disorder by clinical study alone. Images PMID:2921319

  10. The Enzyme Function Initiative.

    PubMed

    Gerlt, John A; Allen, Karen N; Almo, Steven C; Armstrong, Richard N; Babbitt, Patricia C; Cronan, John E; Dunaway-Mariano, Debra; Imker, Heidi J; Jacobson, Matthew P; Minor, Wladek; Poulter, C Dale; Raushel, Frank M; Sali, Andrej; Shoichet, Brian K; Sweedler, Jonathan V

    2011-11-22

    The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic, we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include (1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation), (2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia, (3) computational and bioinformatic tools for using the strategy, (4) provision of experimental protocols and/or reagents for enzyme production and characterization, and (5) dissemination of data via the EFI's Website, http://enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal, and pharmaceutical efforts.

  11. Methods for the isolation of genes encoding novel PHB cycle enzymes from complex microbial communities.

    PubMed

    Nordeste, Ricardo F; Trainer, Maria A; Charles, Trevor C

    2010-01-01

    Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bioplastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti allows for the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates finding functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.

  12. Bacteriophage-encoded lytic enzymes control growth of contaminating Lactobacillus found in fuel ethanol fermentations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Reduced yields of ethanol due to bacterial contamination in fermentation cultures weakens the economics of biofuel production. Lactic acid bacteria are considered the most problematic, and surveys of commercial fuel ethanol facilities have found that species of Lactobacillus are predomin...

  13. Cloning and Expression Analysis of MEP Pathway Enzyme-encoding Genes in Osmanthus fragrans

    PubMed Central

    Xu, Chen; Li, Huogeng; Yang, Xiulian; Gu, Chunsun; Mu, Hongna; Yue, Yuanzheng; Wang, Lianggui

    2016-01-01

    The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway is responsible for the biosynthesis of many crucial secondary metabolites, such as carotenoids, monoterpenes, plastoquinone, and tocopherols. In this study, we isolated and identified 10 MEP pathway genes in the important aromatic plant sweet osmanthus (Osmanthus fragrans). Multiple sequence alignments revealed that 10 MEP pathway genes shared high identities with other reported proteins. The genes showed distinctive expression profiles in various tissues, or at different flower stages and diel time points. The qRT-PCR results demonstrated that these genes were highly expressed in inflorescences, which suggested a tissue-specific transcript pattern. Our results also showed that OfDXS1, OfDXS2, and OfHDR1 had a clear diurnal oscillation pattern. The isolation and expression analysis provides a strong foundation for further research on the MEP pathway involved in gene function and molecular evolution, and improves our understanding of the molecular mechanism underlying this pathway in plants. PMID:27690108

  14. Organization and control of genes encoding catabolic enzymes in Rhizobiaceae. Progress report, March 1993

    SciTech Connect

    Parke, D.; Ornston, L.N.

    1993-03-01

    Rhizobiaceae, a diverse bacterial group comprising rhizobia and agrobacteria, symbiotic partnership with plants form nitrogen-fixing nodules on plant roots or are plant pathogens. Phenolic compounds produced by plants serve as inducers of rhizobial nodulation genes and agrobacterial virulence genes reflect their capacity to utilize numerous aromatics, including phenolics, as a source of carbon and energy. In many microbes the aerobic degradation of numerous aromatic compounds to tricarboxylic acid cycle intermediates is achieved by the {beta}-ketoadipate pathway. Our initial studies focused on the organization and regulation of the ketoadipate pathway in Agrobacterium tumefaciens. We have cloned, identified and characterized a novel regulatory gene that modulates expression of an adjacent pca (protocatechuate) structural gene, pcaD. Regulation of pcaD is mediated by the regulatory gene, termed pcaQ, in concert with the intermediate {beta}-carboxy-cis,cis-muconate. {beta}-carboxy-cis,cismuconate is an unstable chemical, not marketed commercially, and it is unlikely to permeate Escherichia coli cells if supplied in media. Because of these factors, characterization of pcaQ in E. coli required an in vivo delivery system for {beta}-carboxycis,cis-muconate. This was accomplished by designing an E. coli strain that expressed an Acinetobacter calcoaceticus pcaA gene for conversion of protocatechuate to {beta}-carboxy-cis,cis-muconate.

  15. The swi4+ gene of Schizosaccharomyces pombe encodes a homologue of mismatch repair enzymes.

    PubMed Central

    Fleck, O; Michael, H; Heim, L

    1992-01-01

    The swi4+ gene of Schizosaccharomyces pombe is involved in termination of copy-synthesis during mating-type switching. The gene was cloned by functional complementation of a swi4 mutant transformed with a genomic library. Determination of the nucleotide sequence revealed an open reading frame of 2979 nucleotides which is interrupted by a 68 bp long intron. The putative Swi4 protein shows homology to Duc-1 (human), Rep-3 (mouse), HexA (Streptococcus pneumoniae) and MutS (Salmonella typhimurium). The prokaryotic proteins are known as essential components involved in mismatch repair. A strain with a disrupted swi4+ gene was constructed and analysed with respect to the switching process. As in swi4 mutants duplications occur in the mating-type region of the swi4 (null) strain, reducing the efficiency of switching. Images PMID:1317550

  16. Methods for the isolation of genes encoding novel PHB cycle enzymes from complex microbial communities.

    PubMed

    Nordeste, Ricardo F; Trainer, Maria A; Charles, Trevor C

    2010-01-01

    Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bioplastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti allows for the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates finding functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries. PMID:20830568

  17. Coordinate regulation of two genes encoding gluconeogenic enzymes by the trans-dominant locus Tse-1.

    PubMed Central

    Lem, J; Chin, A C; Thayer, M J; Leach, R J; Fournier, R E

    1988-01-01

    Tissue-specific extinguisher-1 (Tse-1) is a mouse genetic locus that can repress liver-specific tyrosine aminotransferase gene expression in trans. To search for other Tse-1-responsive genes, hepatoma microcell hybrids retaining mouse chromosome 11 or human chromosome 17, containing murine Tse-1 and human TSE1, respectively, were screened for expression of liver-specific mRNAs. While most liver gene activity was unaffected in such hybrids, phosphoenolpyruvate carboxykinase and tyrosine aminotransferase gene expression was coordinately repressed in these clones. Extinction of both genes was apparently mediated by a single genetic locus that resides on human chromosome 17. Images PMID:2902627

  18. Identification and characterization of kraken, a gene encoding a putative hydrolytic enzyme in Drosophila melanogaster.

    PubMed

    Edwin Chan, H Y; Harris, S J; O'Kane, C J

    1998-11-19

    Kraken, a novel Drosophila gene isolated from a 4-8-h-old Drosophila embryo cDNA library, shows homology to a family of serine hydrolases whose common feature is that they all catalyse breakage of substrates with a carbonyl-containing group. It is a single-copy gene with at least two introns and maps to position 21D on polytene chromosomes. kraken is a member of a conserved gene family. Messenger RNA of kraken is expressed ubiquitously in early embryogenesis. Later, it is concentrated in the foregut and the posterior midgut primordium. Towards the end of embryogenesis, expression of kraken is confined to the gastric caeca. During the third-instar larval stage, kraken is expressed at low levels in the gastric caeca and parts of the gut, and at higher levels in the fat body. We suggest a role for Kraken in detoxification and digestion during embryogenesis and larval development. PMID:9831651

  19. Regulation of 4CL, encoding 4-coumarate: coenzyme A ligase, expression in kenaf under diverse stress conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We cloned the full length 4CL ortholog encoding 4-coumarate: coenzymeA ligase from kenaf (Hibiscus cannabiuns) using degenerate primers and RACE (rapid amplification of cDNA ends) systems. The 4CL is a key regulatory enzyme of the phenylpropanoid pathway that regulates the activation of cinnamic ac...

  20. Next generation chemical proteomic tools for rapid enzyme profiling.

    PubMed

    Uttamchandani, Mahesh; Lu, Candy H S; Yao, Shao Q

    2009-08-18

    Sequencing of the human genome provided a wealth of information about the genomic blueprint of a cell. But genes do not tell the entire story of life and living processes; identifying the roles of enzymes and mapping out their interactions is also crucial. Enzymes catalyze virtually every cellular process and metabolic exchange. They not only are instrumental in sustaining life but also are required for its regulation and diversification. Diseases such as cancer can be caused by minor changes in enzyme activities. In addition, the unique enzymes of pathogenic organisms are ripe targets for combating infections. Consequently, nearly one-third of all current drug targets are enzymes. An estimated 18-29% of eukaryotic genes encode enzymes, but only a limited proportion of enzymes have thus far been characterized. Therefore, little is understood about the physiological roles, substrate specificity, and downstream targets of the vast majority of these important proteins. A key step toward the biological characterization of enzymes, as well as their adoption as drug targets, is the development of global solutions that bridge the gap in understanding these proteins and their interactions. We herein present technological advances that facilitate the study of enzymes and their properties in a high-throughput manner. Over the years, our group has introduced and developed a variety of such enabling platforms for many classes of enzymes, including kinases, phosphatases, and proteases. For each of these different types of enzymes, specific design considerations are required to develop the appropriate chemical tools to characterize each class. These tools include activity-based probes and chemical compound libraries, which are rapidly assembled using efficient combinatorial synthesis or "click chemistry" strategies. The resulting molecular assortments may then be screened against the target enzymes in high-throughput using microplates or microarrays. These techniques offer

  1. An encyclopedia of mouse DNA elements (Mouse ENCODE).

    PubMed

    Stamatoyannopoulos, John A; Snyder, Michael; Hardison, Ross; Ren, Bing; Gingeras, Thomas; Gilbert, David M; Groudine, Mark; Bender, Michael; Kaul, Rajinder; Canfield, Theresa; Giste, Erica; Johnson, Audra; Zhang, Mia; Balasundaram, Gayathri; Byron, Rachel; Roach, Vaughan; Sabo, Peter J; Sandstrom, Richard; Stehling, A Sandra; Thurman, Robert E; Weissman, Sherman M; Cayting, Philip; Hariharan, Manoj; Lian, Jin; Cheng, Yong; Landt, Stephen G; Ma, Zhihai; Wold, Barbara J; Dekker, Job; Crawford, Gregory E; Keller, Cheryl A; Wu, Weisheng; Morrissey, Christopher; Kumar, Swathi A; Mishra, Tejaswini; Jain, Deepti; Byrska-Bishop, Marta; Blankenberg, Daniel; Lajoie, Bryan R; Jain, Gaurav; Sanyal, Amartya; Chen, Kaun-Bei; Denas, Olgert; Taylor, James; Blobel, Gerd A; Weiss, Mitchell J; Pimkin, Max; Deng, Wulan; Marinov, Georgi K; Williams, Brian A; Fisher-Aylor, Katherine I; Desalvo, Gilberto; Kiralusha, Anthony; Trout, Diane; Amrhein, Henry; Mortazavi, Ali; Edsall, Lee; McCleary, David; Kuan, Samantha; Shen, Yin; Yue, Feng; Ye, Zhen; Davis, Carrie A; Zaleski, Chris; Jha, Sonali; Xue, Chenghai; Dobin, Alex; Lin, Wei; Fastuca, Meagan; Wang, Huaien; Guigo, Roderic; Djebali, Sarah; Lagarde, Julien; Ryba, Tyrone; Sasaki, Takayo; Malladi, Venkat S; Cline, Melissa S; Kirkup, Vanessa M; Learned, Katrina; Rosenbloom, Kate R; Kent, W James; Feingold, Elise A; Good, Peter J; Pazin, Michael; Lowdon, Rebecca F; Adams, Leslie B

    2012-08-13

    To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

  2. Multichannel compressive sensing MRI using noiselet encoding.

    PubMed

    Pawar, Kamlesh; Egan, Gary; Zhang, Jingxin

    2015-01-01

    The incoherence between measurement and sparsifying transform matrices and the restricted isometry property (RIP) of measurement matrix are two of the key factors in determining the performance of compressive sensing (CS). In CS-MRI, the randomly under-sampled Fourier matrix is used as the measurement matrix and the wavelet transform is usually used as sparsifying transform matrix. However, the incoherence between the randomly under-sampled Fourier matrix and the wavelet matrix is not optimal, which can deteriorate the performance of CS-MRI. Using the mathematical result that noiselets are maximally incoherent with wavelets, this paper introduces the noiselet unitary bases as the measurement matrix to improve the incoherence and RIP in CS-MRI. Based on an empirical RIP analysis that compares the multichannel noiselet and multichannel Fourier measurement matrices in CS-MRI, we propose a multichannel compressive sensing (MCS) framework to take the advantage of multichannel data acquisition used in MRI scanners. Simulations are presented in the MCS framework to compare the performance of noiselet encoding reconstructions and Fourier encoding reconstructions at different acceleration factors. The comparisons indicate that multichannel noiselet measurement matrix has better RIP than that of its Fourier counterpart, and that noiselet encoded MCS-MRI outperforms Fourier encoded MCS-MRI in preserving image resolution and can achieve higher acceleration factors. To demonstrate the feasibility of the proposed noiselet encoding scheme, a pulse sequences with tailored spatially selective RF excitation pulses was designed and implemented on a 3T scanner to acquire the data in the noiselet domain from a phantom and a human brain. The results indicate that noislet encoding preserves image resolution better than Fouirer encoding. PMID:25965548

  3. Multichannel compressive sensing MRI using noiselet encoding.

    PubMed

    Pawar, Kamlesh; Egan, Gary; Zhang, Jingxin

    2015-01-01

    The incoherence between measurement and sparsifying transform matrices and the restricted isometry property (RIP) of measurement matrix are two of the key factors in determining the performance of compressive sensing (CS). In CS-MRI, the randomly under-sampled Fourier matrix is used as the measurement matrix and the wavelet transform is usually used as sparsifying transform matrix. However, the incoherence between the randomly under-sampled Fourier matrix and the wavelet matrix is not optimal, which can deteriorate the performance of CS-MRI. Using the mathematical result that noiselets are maximally incoherent with wavelets, this paper introduces the noiselet unitary bases as the measurement matrix to improve the incoherence and RIP in CS-MRI. Based on an empirical RIP analysis that compares the multichannel noiselet and multichannel Fourier measurement matrices in CS-MRI, we propose a multichannel compressive sensing (MCS) framework to take the advantage of multichannel data acquisition used in MRI scanners. Simulations are presented in the MCS framework to compare the performance of noiselet encoding reconstructions and Fourier encoding reconstructions at different acceleration factors. The comparisons indicate that multichannel noiselet measurement matrix has better RIP than that of its Fourier counterpart, and that noiselet encoded MCS-MRI outperforms Fourier encoded MCS-MRI in preserving image resolution and can achieve higher acceleration factors. To demonstrate the feasibility of the proposed noiselet encoding scheme, a pulse sequences with tailored spatially selective RF excitation pulses was designed and implemented on a 3T scanner to acquire the data in the noiselet domain from a phantom and a human brain. The results indicate that noislet encoding preserves image resolution better than Fouirer encoding.

  4. Multichannel Compressive Sensing MRI Using Noiselet Encoding

    PubMed Central

    Pawar, Kamlesh; Egan, Gary; Zhang, Jingxin

    2015-01-01

    The incoherence between measurement and sparsifying transform matrices and the restricted isometry property (RIP) of measurement matrix are two of the key factors in determining the performance of compressive sensing (CS). In CS-MRI, the randomly under-sampled Fourier matrix is used as the measurement matrix and the wavelet transform is usually used as sparsifying transform matrix. However, the incoherence between the randomly under-sampled Fourier matrix and the wavelet matrix is not optimal, which can deteriorate the performance of CS-MRI. Using the mathematical result that noiselets are maximally incoherent with wavelets, this paper introduces the noiselet unitary bases as the measurement matrix to improve the incoherence and RIP in CS-MRI. Based on an empirical RIP analysis that compares the multichannel noiselet and multichannel Fourier measurement matrices in CS-MRI, we propose a multichannel compressive sensing (MCS) framework to take the advantage of multichannel data acquisition used in MRI scanners. Simulations are presented in the MCS framework to compare the performance of noiselet encoding reconstructions and Fourier encoding reconstructions at different acceleration factors. The comparisons indicate that multichannel noiselet measurement matrix has better RIP than that of its Fourier counterpart, and that noiselet encoded MCS-MRI outperforms Fourier encoded MCS-MRI in preserving image resolution and can achieve higher acceleration factors. To demonstrate the feasibility of the proposed noiselet encoding scheme, a pulse sequences with tailored spatially selective RF excitation pulses was designed and implemented on a 3T scanner to acquire the data in the noiselet domain from a phantom and a human brain. The results indicate that noislet encoding preserves image resolution better than Fouirer encoding. PMID:25965548

  5. Multi-dimensionally encoded magnetic resonance imaging

    PubMed Central

    Lin, Fa-Hsuan

    2013-01-01

    Magnetic resonance imaging typically achieves spatial encoding by measuring the projection of a q-dimensional object over q-dimensional spatial bases created by linear spatial encoding magnetic fields (SEMs). Recently, imaging strategies using nonlinear SEMs have demonstrated potential advantages for reconstructing images with higher spatiotemporal resolution and reducing peripheral nerve stimulation. In practice, nonlinear SEMs and linear SEMs can be used jointly to further improve the image reconstruction performance. Here we propose the multi-dimensionally encoded (MDE) MRI to map a q-dimensional object onto a p-dimensional encoding space where p > q. MDE MRI is a theoretical framework linking imaging strategies using linear and nonlinear SEMs. Using a system of eight surface SEM coils with an eight-channel RF coil array, we demonstrate the five-dimensional MDE MRI for a two-dimensional object as a further generalization of PatLoc imaging and O-space imaging. We also present a method of optimizing spatial bases in MDE MRI. Results show that MDE MRI with a higher dimensional encoding space can reconstruct images more efficiently and with a smaller reconstruction error when the k-space sampling distribution and the number of samples are controlled. PMID:22926830

  6. Cloud-based uniform ChIP-Seq processing tools for modENCODE and ENCODE

    PubMed Central

    2013-01-01

    Background Funded by the National Institutes of Health (NIH), the aim of the Model Organism ENCyclopedia of DNA Elements (modENCODE) project is to provide the biological research community with a comprehensive encyclopedia of functional genomic elements for both model organisms C. elegans (worm) and D. melanogaster (fly). With a total size of just under 10 terabytes of data collected and released to the public, one of the challenges faced by researchers is to extract biologically meaningful knowledge from this large data set. While the basic quality control, pre-processing, and analysis of the data has already been performed by members of the modENCODE consortium, many researchers will wish to reinterpret the data set using modifications and enhancements of the original protocols, or combine modENCODE data with other data sets. Unfortunately this can be a time consuming and logistically challenging proposition. Results In recognition of this challenge, the modENCODE DCC has released uniform computing resources for analyzing modENCODE data on Galaxy (https://github.com/modENCODE-DCC/Galaxy), on the public Amazon Cloud (http://aws.amazon.com), and on the private Bionimbus Cloud for genomic research (http://www.bionimbus.org). In particular, we have released Galaxy workflows for interpreting ChIP-seq data which use the same quality control (QC) and peak calling standards adopted by the modENCODE and ENCODE communities. For convenience of use, we have created Amazon and Bionimbus Cloud machine images containing Galaxy along with all the modENCODE data, software and other dependencies. Conclusions Using these resources provides a framework for running consistent and reproducible analyses on modENCODE data, ultimately allowing researchers to use more of their time using modENCODE data, and less time moving it around. PMID:23875683

  7. Pyrimidine biosynthetic enzymes of Salmonella typhimurium, repressed specifically by growth in the presence of cytidine.

    PubMed Central

    Kelln, R A; Kinahan, J J; Foltermann, K F; O'Donovan, G A

    1975-01-01

    The repressive effects of exogenous cytidine on growing cells was examined in a specially constructed strain in which the pool sizes of endogenous uridine 5'-diphosphate and uridine 5'-triphosphate cannot be varied by the addition of uracil and/or uridine to the medium. Five enzymes of the pyrimidine biosynthetic pathway and one enzyme of the arginine biosynthetic pathway were assayed from cells grown under a variety of conditions. Cytidine repressed the synthesis of dihydroorotase (encoded by pyrC), dihydroorotate dehydrogenase (encoded by pyrD), and ornithine transcarbamylase (encoded by argI). Moreover, aspartate transcarbamylase (encoded by pyrB) became further derepressed upon cytidine addition, whereas no change occurred in the levels of the last two enzymes (encoded by pyrE and pyrF) of the pyrimidine pathway. Quantitative nucleotide pool determinations have provided evidence that any individual ribo- or deoxyribonucleoside mono-, di-, or triphosphate of cytosine or uracil is not a repressing metabolite for the pyrimidine biosynthetic enzymes. Other nucleotide derivatives or ratios must be considered. PMID:1102530

  8. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1997-11-25

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups. 19 figs.

  9. Enzyme Molar Fractions: A Powerful Tool for Understanding Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Serra, Juan L.; And Others

    1986-01-01

    Deduces the relationship between reduced velocity and molar fractions for productive enzyme complexes; obtains the mathematical expression of molar fractions for an enzyme with two specific binding sites per molecule; and proposes a useful plot to follow the dependence of enzyme molar fractions with the concentration of one of its ligands. (JN)

  10. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, Matthew R.; Bednarski, Mark D.; Gruber, Patrick R.

    1997-01-01

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups.

  11. An information theoretic characterisation of auditory encoding.

    PubMed

    Overath, Tobias; Cusack, Rhodri; Kumar, Sukhbinder; von Kriegstein, Katharina; Warren, Jason D; Grube, Manon; Carlyon, Robert P; Griffiths, Timothy D

    2007-10-23

    The entropy metric derived from information theory provides a means to quantify the amount of information transmitted in acoustic streams like speech or music. By systematically varying the entropy of pitch sequences, we sought brain areas where neural activity and energetic demands increase as a function of entropy. Such a relationship is predicted to occur in an efficient encoding mechanism that uses less computational resource when less information is present in the signal: we specifically tested the hypothesis that such a relationship is present in the planum temporale (PT). In two convergent functional MRI studies, we demonstrated this relationship in PT for encoding, while furthermore showing that a distributed fronto-parietal network for retrieval of acoustic information is independent of entropy. The results establish PT as an efficient neural engine that demands less computational resource to encode redundant signals than those with high information content. PMID:17958472

  12. Feature encoding for color image segmentation

    NASA Astrophysics Data System (ADS)

    Li, Ning; Li, Youfu

    2001-09-01

    An approach for color image segmentation is proposed based on the contributions of color features to segmentation rather than the choice of a particular color space. It is different from the pervious methods where SOFM is used for construct the feature encoding so that the feature-encoding can self-organize the effective features for different color images. Fuzzy clustering is applied for the final segmentation when the well-suited color features and the initial parameter are available. The proposed method has been applied in segmenting different types of color images and the experimental results show that it outperforms the classical clustering method. Our study shows that the feature encoding approach offers great promise in automating and optimizing color image segmentation.

  13. Adaptive delta modulation systems for video encoding

    NASA Technical Reports Server (NTRS)

    Lei, T.-L. R.; Scheinberg, N.; Schilling, D. L.

    1977-01-01

    This paper describes several adaptive delta modulators designed to encode video signals. One- and two-dimensional ADM algorithms are discussed and compared. Results are shown for bit rates of 2 bits/pixel, 1 bit/pixel and 0.5 bits/pixel. Pictures showing the difference between the encoded-decoded pictures and the original pictures are presented. Results are also presented to illustrate the effect of channel errors on the reconstructed picture. A two-dimensional ADM using interframe encoding is also presented. This system operates at the rate of 2 bits/pixel and produces excellent quality pictures when there is little motion. We also describe and illustrate the effect of large amounts of motion on the reconstructed picture.

  14. Structure and strategy in encoding simplified graphs

    NASA Technical Reports Server (NTRS)

    Schiano, Diane J.; Tversky, Barbara

    1992-01-01

    Tversky and Schiano (1989) found a systematic bias toward the 45-deg line in memory for the slopes of identical lines when embedded in graphs, but not in maps, suggesting the use of a cognitive reference frame specifically for encoding meaningful graphs. The present experiments explore this issue further using the linear configurations alone as stimuli. Experiments 1 and 2 demonstrate that perception and immediate memory for the slope of a test line within orthogonal 'axes' are predictable from purely structural considerations. In Experiments 3 and 4, subjects were instructed to use a diagonal-reference strategy in viewing the stimuli, which were described as 'graphs' only in Experiment 3. Results for both studies showed the diagonal bias previously found only for graphs. This pattern provides converging evidence for the diagonal as a cognitive reference frame in encoding linear graphs, and demonstrates that even in highly simplified displays, strategic factors can produce encoding biases not predictable solely from stimulus structure alone.

  15. Interoperability in encoded quantum repeater networks

    NASA Astrophysics Data System (ADS)

    Nagayama, Shota; Choi, Byung-Soo; Devitt, Simon; Suzuki, Shigeya; Van Meter, Rodney

    2016-04-01

    The future of quantum repeater networking will require interoperability between various error-correcting codes. A few specific code conversions and even a generalized method are known, however, no detailed analysis of these techniques in the context of quantum networking has been performed. In this paper we analyze a generalized procedure to create Bell pairs encoded heterogeneously between two separate codes used often in error-corrected quantum repeater network designs. We begin with a physical Bell pair and then encode each qubit in a different error-correcting code, using entanglement purification to increase the fidelity. We investigate three separate protocols for preparing the purified encoded Bell pair. We calculate the error probability of those schemes between the Steane [[7,1,3

  16. Quantum repeater with continuous variable encoding

    NASA Astrophysics Data System (ADS)

    Li, Linshu; Albert, Victor V.; Michael, Marios; Muralidharan, Sreraman; Zou, Changling; Jiang, Liang

    2016-05-01

    Quantum communication enables faithful quantum state transfer between different parties and protocols for cryptographic purposes. However, quantum communication over long distances (>1000km) remains challenging due to optical channel attenuation. This calls for investigation on developing novel encoding schemes that correct photon loss errors efficiently. In this talk, we introduce the generalization of multi-component Schrödinger cat states and propose to encode quantum information in these cat states for ultrafast quantum repeaters. We detail the quantum error correction procedures at each repeater station and characterize the performance of this novel encoding scheme given practical imperfections, such as coupling loss. A comparison with other quantum error correcting codes for bosonic modes will be discussed.

  17. Sulfite oxidizing enzymes

    PubMed Central

    Feng, Changjian; Tollin, Gordon; Enemark, John H.

    2007-01-01

    Sulfite oxidizing enzymes are essential mononuclear molybdenum (Mo) proteins involved in sulfur metabolism of animals, plants and bacteria. There are three such enzymes presently known: (1) sulfite oxidase (SO) in animals, (2) SO in plants, and (3) sulfite dehydrogenase (SDH) in bacteria. X-ray crystal structures of enzymes from all three sources (chicken SO, Arabidopsis thaliana SO, and Starkeya novella SDH) show nearly identical square pyramidal coordination around the Mo atom, even though the overall structures of the proteins and the presence of additional cofactors vary. This structural information provides a molecular basis for studying the role of specific amino acids in catalysis. Animal SO catalyzes the final step in the degradation of sulfur-containing amino acids and is critical in detoxifying excess sulfite. Human SO deficiency is a fatal genetic disorder that leads to early death, and impaired SO activity is implicated in sulfite neurotoxicity. Animal SO and bacterial SDH contain both Mo and heme domains, whereas plant SO only has the Mo domain. Intraprotein electron transfer (IET) between the Mo and Fe centers in animal SO and bacterial SDH is a key step in the catalysis, which can be studied by laser flash photolysis in the presence of deazariboflavin. IET studies on animal SO and bacterial SDH clearly demonstrate the similarities and differences between these two types of sulfite oxidizing enzymes. Conformational change is involved in the IET of animal SO, in which electrostatic interactions may play a major role in guiding the docking of the heme domain to the Mo domain prior to electron transfer. In contrast, IET measurements for SDH demonstrate that IET occurs directly through the protein medium, which is distinctly different from that in animal SO. Point mutations in human SO can result in significantly impaired IET or no IET, thus rationalizing their fatal effects. The recent developments in our understanding of sulfite oxidizing enzyme

  18. A Run-Length Encoding Approach for Path Analysis of C. elegans Search Behavior

    PubMed Central

    Kim, Hongkyun; Furst, Jacob

    2016-01-01

    The nematode Caenorhabditis elegans explores the environment using a combination of different movement patterns, which include straight movement, reversal, and turns. We propose to quantify C. elegans movement behavior using a computer vision approach based on run-length encoding of step-length data. In this approach, the path of C. elegans is encoded as a string of characters, where each character represents a path segment of a specific type of movement. With these encoded string data, we perform k-means cluster analysis to distinguish movement behaviors resulting from different genotypes and food availability. We found that shallow and sharp turns are the most critical factors in distinguishing the differences among the movement behaviors. To validate our approach, we examined the movement behavior of tph-1 mutants that lack an enzyme responsible for serotonin biosynthesis. A k-means cluster analysis with the path string-encoded data showed that tph-1 movement behavior on food is similar to that of wild-type animals off food. We suggest that this run-length encoding approach is applicable to trajectory data in animal or human mobility data. PMID:27462364

  19. A Run-Length Encoding Approach for Path Analysis of C. elegans Search Behavior.

    PubMed

    Huang, Li; Kim, Hongkyun; Furst, Jacob; Raicu, Daniela

    2016-01-01

    The nematode Caenorhabditis elegans explores the environment using a combination of different movement patterns, which include straight movement, reversal, and turns. We propose to quantify C. elegans movement behavior using a computer vision approach based on run-length encoding of step-length data. In this approach, the path of C. elegans is encoded as a string of characters, where each character represents a path segment of a specific type of movement. With these encoded string data, we perform k-means cluster analysis to distinguish movement behaviors resulting from different genotypes and food availability. We found that shallow and sharp turns are the most critical factors in distinguishing the differences among the movement behaviors. To validate our approach, we examined the movement behavior of tph-1 mutants that lack an enzyme responsible for serotonin biosynthesis. A k-means cluster analysis with the path string-encoded data showed that tph-1 movement behavior on food is similar to that of wild-type animals off food. We suggest that this run-length encoding approach is applicable to trajectory data in animal or human mobility data. PMID:27462364

  20. Encoding many qubits in a rotor

    SciTech Connect

    Raynal, Philippe; Kalev, Amir; Suzuki, Jun; Englert, Berthold-Georg

    2010-05-15

    We propose a scheme for encoding many qubits in a single rotor, that is, a continuous and periodic degree of freedom. A key feature of this scheme is its ability to manipulate and entangle the encoded qubits with a single operation on the system. We also show, using quantum error-correcting codes, how to protect the qubits against small errors in angular position and momentum which may affect the rotor. We then discuss the feasibility of this scheme and suggest several candidates for its implementation. The proposed scheme is immediately generalizable to qudits of any finite dimension.

  1. Vector Adaptive/Predictive Encoding Of Speech

    NASA Technical Reports Server (NTRS)

    Chen, Juin-Hwey; Gersho, Allen

    1989-01-01

    Vector adaptive/predictive technique for digital encoding of speech signals yields decoded speech of very good quality after transmission at coding rate of 9.6 kb/s and of reasonably good quality at 4.8 kb/s. Requires 3 to 4 million multiplications and additions per second. Combines advantages of adaptive/predictive coding, and code-excited linear prediction, yielding speech of high quality but requires 600 million multiplications and additions per second at encoding rate of 4.8 kb/s. Vector adaptive/predictive coding technique bridges gaps in performance and complexity between adaptive/predictive coding and code-excited linear prediction.

  2. Preparing encoded states in an oscillator

    SciTech Connect

    Travaglione, B.C.; Milburn, G.J.

    2002-11-01

    Recently a scheme has been proposed for constructing quantum error-correcting codes that embed a finite-dimensional code space in the infinite-dimensional Hilbert space of a system described by continuous quantum variables. One of the difficult steps in this scheme is the preparation of the encoded states. We show how these states can be generated by coupling a continuous quantum variable to a single qubit. An ion trap quantum computer provides a natural setting for a continuous system coupled to a qubit. We discuss how encoded states may be generated in an ion trap.

  3. Treating Wastewater With Immobilized Enzymes

    NASA Technical Reports Server (NTRS)

    Jolly, Clifford D.

    1991-01-01

    Experiments show enzymes are immobilized on supporting materials to make biocatalyst beds for treatment of wastewater. With suitable combination of enzymes, concentrations of various inorganic and organic contaminants, including ammonia and urea, reduced significantly.

  4. Development and application of a suite of polysaccharide-degrading enzymes for analyzing plant cell walls

    PubMed Central

    Bauer, Stefan; Vasu, Prasanna; Persson, Staffan; Mort, Andrew J.; Somerville, Chris R.

    2006-01-01

    To facilitate analysis of plant cell wall polysaccharide structure and composition, we cloned 74 genes encoding polysaccharide-degrading enzymes from Aspergillus nidulans, Aspergillus fumigatus, and Neurospora crassa and expressed the genes as secreted proteins with C-terminal Myc and 6× His tags. Most of the recombinant enzymes were active in enzyme assays, and optima for pH and temperature were established. A subset of the enzymes was used to fragment polysaccharides from the irregular xylem 9 (irx9) mutant of Arabidopsis. The analysis revealed a decrease in the abundance of xylan in the mutant, indicating that the IRX9 gene, which encodes a putative family 43 glycosyltransferase, is required for xylan synthesis. PMID:16844780

  5. The Catalytic Function of Enzymes.

    ERIC Educational Resources Information Center

    Splittgerber, Allan G.

    1985-01-01

    Discusses: structure of the enzyme molecule; active site; reaction mechanism; transition state; factors affecting enzyme reaction rates, concentration of enzyme; concentration of substrate; product concentration; temperature effects and pH effects; factors causing a lowering of activation energy; proximity and orientation effects; substrate strain…

  6. Protein Crystal Malic Enzyme

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  7. Retrieval during Learning Facilitates Subsequent Memory Encoding

    ERIC Educational Resources Information Center

    Pastotter, Bernhard; Schicker, Sabine; Niedernhuber, Julia; Bauml, Karl-Heinz T.

    2011-01-01

    In multiple-list learning, retrieval during learning has been suggested to improve recall of the single lists by enhancing list discrimination and, at test, reducing interference. Using electrophysiological, oscillatory measures of brain activity, we examined to what extent retrieval during learning facilitates list encoding. Subjects studied 5…

  8. Encoded Archival Description as a Halfway Technology

    ERIC Educational Resources Information Center

    Dow, Elizabeth H.

    2009-01-01

    In the mid 1990s, Encoded Archival Description (EAD) appeared as a revolutionary technology for publishing archival finding aids on the Web. The author explores whether or not, given the advent of Web 2.0, the archival community should abandon EAD and look for something to replace it. (Contains 18 notes.)

  9. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, P.G.; Ohlrogge, J.B.

    1996-09-24

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

  10. Encoding of Others' Beliefs without Overt Instruction

    ERIC Educational Resources Information Center

    Cohen, Adam S.; German, Tamsin C.

    2009-01-01

    Under what conditions do people automatically encode and track the mental states of others? A recent investigation showed that when subjects are instructed to track the location of an object but are not instructed to track a belief about that location in a non-verbal false-belief task, they respond more slowly to questions about an agent's belief,…

  11. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... levels of the mark or space frequencies. (9) Attention Signal generator. The encoder must provide an attention signal that complies with the following: (i) Tone Frequencies. The audio tones shall have... period of not less than 8 nor longer than 25 seconds. NOTE: Prior to July 1, 1995, the Attention...

  12. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... or space frequencies. (9) Attention Signal generator. The encoder must provide an attention signal...) Inadvertent activation. The switch used for initiating the automatic generation of the simultaneous tones... provided with a visual and/or aural indicator which clearly shows that the Attention Signal is...

  13. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... levels of the mark or space frequencies. (9) Attention Signal generator. The encoder must provide an attention signal that complies with the following: (i) Tone Frequencies. The audio tones shall have... period of not less than 8 nor longer than 25 seconds. NOTE: Prior to July 1, 1995, the Attention...

  14. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... or space frequencies. (9) Attention Signal generator. The encoder must provide an attention signal...) Inadvertent activation. The switch used for initiating the automatic generation of the simultaneous tones... provided with a visual and/or aural indicator which clearly shows that the Attention Signal is...

  15. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... or space frequencies. (9) Attention Signal generator. The encoder must provide an attention signal...) Inadvertent activation. The switch used for initiating the automatic generation of the simultaneous tones... provided with a visual and/or aural indicator which clearly shows that the Attention Signal is...

  16. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, Paul G.; Ohlrogge, John B.

    1996-01-01

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives thereof which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides.

  17. Encoding attentional states during visuomotor adaptation

    PubMed Central

    Im, Hee Yeon; Bédard, Patrick; Song, Joo-Hyun

    2015-01-01

    We recently showed that visuomotor adaptation acquired under attentional distraction is better recalled under a similar level of distraction compared to no distraction. This paradoxical effect suggests that attentional state (e.g., divided or undivided) is encoded as an internal context during visuomotor learning and should be reinstated for successful recall (Song & Bédard, 2015). To investigate if there is a critical temporal window for encoding attentional state in visuomotor memory, we manipulated whether participants performed the secondary attention-demanding task concurrently in the early or late phase of visuomotor learning. Recall performance was enhanced when the attentional states between recall and the early phase of visuomotor learning were consistent. However, it reverted to untrained levels when tested under the attentional state of the late-phase learning. This suggests that attentional state is primarily encoded during the early phase of learning before motor errors decrease and reach an asymptote. Furthermore, we demonstrate that when divided and undivided attentional states were mixed during visuomotor adaptation, only divided attention was encoded as an internal cue for memory retrieval. Therefore, a single attentional state appears to be primarily integrated with visuomotor memory while motor error reduction is in progress during learning. PMID:26114683

  18. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, John; Piddington, Chris S.; Kovacevich, Brian R.; Young, Kevin D.; Denome, Sylvia A.

    1994-01-01

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

  19. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, J.; Piddington, C.S.; Kovacevich, B.R.; Young, K.D.; Denome, S.A.

    1994-10-18

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous. 13 figs.

  20. Encoders for block-circulant LDPC codes

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush (Inventor); Abbasfar, Aliazam (Inventor); Jones, Christopher R. (Inventor); Dolinar, Samuel J. (Inventor); Thorpe, Jeremy C. (Inventor); Andrews, Kenneth S. (Inventor); Yao, Kung (Inventor)

    2009-01-01

    Methods and apparatus to encode message input symbols in accordance with an accumulate-repeat-accumulate code with repetition three or four are disclosed. Block circulant matrices are used. A first method and apparatus make use of the block-circulant structure of the parity check matrix. A second method and apparatus use block-circulant generator matrices.

  1. Typicality as a Dimension of Encoding

    ERIC Educational Resources Information Center

    Keller, Dennis; Kellas, George

    1978-01-01

    The salience of encoding attributes in instances of differing levels of category membership was examined using the release from proactive interference (PI) task with college students. Results are discussed in terms of providing converging evidence for Rosch's (1973,1975) theory of semantic category structure. (Editor/RK)

  2. Encoding and Retrieval During Bimanual Rhythmic Coordination

    ERIC Educational Resources Information Center

    Shockley, Kevin; Turvey, Michael T.

    2005-01-01

    In 2 experiments, bimanual 1:1 rhythmic coordination was performed concurrently with encoding or retrieval of word lists. Effects of divided attention (DA) on coordination were indexed by changes in mean relative phase and recurrence measures of shared activity between the 2 limbs. Effects of DA on memory were indexed by deficits in recall…

  3. Encoding and Retrieval in Visual Memory Tasks

    ERIC Educational Resources Information Center

    Frost, Nancy

    1972-01-01

    It was concluded that pictures are encoded differently depending on task expectation. Parallel access of visual and semantic memory codes occurs; but when recognition is expected, a visual cue provides faster access, and when expecting recall, verbal access is more efficient. (Author)

  4. Young Children's Automatic Encoding of Social Categories

    ERIC Educational Resources Information Center

    Weisman, Kara; Johnson, Marissa V.; Shutts, Kristin

    2015-01-01

    The present research investigated young children's automatic encoding of two social categories that are highly relevant to adults: gender and race. Three- to 6-year-old participants learned facts about unfamiliar target children who varied in either gender or race and were asked to remember which facts went with which targets. When participants…

  5. How Attention Modulates Encoding of Dynamic Stimuli

    PubMed Central

    Oren, Noga; Shapira-Lichter, Irit; Lerner, Yulia; Tarrasch, Ricardo; Hendler, Talma; Giladi, Nir; Ash, Elissa L.

    2016-01-01

    When encoding a real-life, continuous stimulus, the same neural circuits support processing and integration of prior as well as new incoming information. This ongoing interplay is modulated by attention, and is evident in regions such as the prefrontal cortex section of the task positive network (TPN), and in the posterior cingulate cortex (PCC), a hub of the default mode network (DMN). Yet the exact nature of such modulation is still unclear. To investigate this issue, we utilized an fMRI task that employed movies as the encoded stimuli and manipulated attentional load via an easy or hard secondary task that was performed simultaneously with encoding. Results showed increased intersubject correlation (inter-SC) levels when encoding movies in a condition of high, as compared to low attentional load. This was evident in bilateral ventrolateral and dorsomedial prefrontal cortices and the dorsal PCC (dPCC). These regions became more attuned to the combination of the movie and the secondary task as the attentional demand of the latter increased. Activation analyses revealed that at higher load the prefrontal TPN regions were more activated, whereas the dPCC was more deactivated. Attentional load also influenced connectivity within and between the networks. At high load the dPCC was anti-correlated to the prefrontal regions, which were more functionally coherent amongst themselves. Finally and critically, greater inter-SC in the dPCC at high load during encoding predicted lower memory strength when that information was retrieved. This association between inter-SC levels and memory strength suggest that as attentional demands increased, the dPCC was more attuned to the secondary task at the expense of the encoded stimulus, thus weakening memory for the encoded stimulus. Together, our findings show that attentional load modulated the function of core TPN and DMN regions. Furthermore, the observed relationship between memory strength and the modulation of the dPCC points

  6. JPEG 2000 Encoding with Perceptual Distortion Control

    NASA Technical Reports Server (NTRS)

    Watson, Andrew B.; Liu, Zhen; Karam, Lina J.

    2008-01-01

    An alternative approach has been devised for encoding image data in compliance with JPEG 2000, the most recent still-image data-compression standard of the Joint Photographic Experts Group. Heretofore, JPEG 2000 encoding has been implemented by several related schemes classified as rate-based distortion-minimization encoding. In each of these schemes, the end user specifies a desired bit rate and the encoding algorithm strives to attain that rate while minimizing a mean squared error (MSE). While rate-based distortion minimization is appropriate for transmitting data over a limited-bandwidth channel, it is not the best approach for applications in which the perceptual quality of reconstructed images is a major consideration. A better approach for such applications is the present alternative one, denoted perceptual distortion control, in which the encoding algorithm strives to compress data to the lowest bit rate that yields at least a specified level of perceptual image quality. Some additional background information on JPEG 2000 is prerequisite to a meaningful summary of JPEG encoding with perceptual distortion control. The JPEG 2000 encoding process includes two subprocesses known as tier-1 and tier-2 coding. In order to minimize the MSE for the desired bit rate, a rate-distortion- optimization subprocess is introduced between the tier-1 and tier-2 subprocesses. In tier-1 coding, each coding block is independently bit-plane coded from the most-significant-bit (MSB) plane to the least-significant-bit (LSB) plane, using three coding passes (except for the MSB plane, which is coded using only one "clean up" coding pass). For M bit planes, this subprocess involves a total number of (3M - 2) coding passes. An embedded bit stream is then generated for each coding block. Information on the reduction in distortion and the increase in the bit rate associated with each coding pass is collected. This information is then used in a rate-control procedure to determine the

  7. Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3', 3'-cGAMP).

    PubMed

    Hallberg, Zachary F; Wang, Xin C; Wright, Todd A; Nan, Beiyan; Ad, Omer; Yeo, Jongchan; Hammond, Ming C

    2016-02-16

    Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3', 3'-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.

  8. Anion-π Enzymes

    PubMed Central

    2016-01-01

    In this report, we introduce artificial enzymes that operate with anion-π interactions, an interaction that is essentially new to nature. The possibility to stabilize anionic intermediates and transition states on an π-acidic surface has been recently demonstrated, using the addition of malonate half thioesters to enolate acceptors as a biologically relevant example. The best chiral anion-π catalysts operate with an addition/decarboxylation ratio of 4:1, but without any stereoselectivity. To catalyze this important but intrinsically disfavored reaction stereoselectively, a series of anion-π catalysts was equipped with biotin and screened against a collection of streptavidin mutants. With the best hit, the S112Y mutant, the reaction occurred with 95% ee and complete suppression of the intrinsically favored side product from decarboxylation. This performance of anion-π enzymes rivals, if not exceeds, that of the best conventional organocatalysts. Inhibition of the S112Y mutant by nitrate but not by bulky anions supports that contributions from anion-π interactions exist and matter, also within proteins. In agreement with docking results, K121 is shown to be essential, presumably to lower the pKa of the tertiary amine catalyst to operate at the optimum pH around 3, that is below the pKa of the substrate. Most importantly, increasing enantioselectivity with different mutants always coincides with increasing rates and conversion, i.e., selective transition-state stabilization. PMID:27413782

  9. Anion-π Enzymes.

    PubMed

    Cotelle, Yoann; Lebrun, Vincent; Sakai, Naomi; Ward, Thomas R; Matile, Stefan

    2016-06-22

    In this report, we introduce artificial enzymes that operate with anion-π interactions, an interaction that is essentially new to nature. The possibility to stabilize anionic intermediates and transition states on an π-acidic surface has been recently demonstrated, using the addition of malonate half thioesters to enolate acceptors as a biologically relevant example. The best chiral anion-π catalysts operate with an addition/decarboxylation ratio of 4:1, but without any stereoselectivity. To catalyze this important but intrinsically disfavored reaction stereoselectively, a series of anion-π catalysts was equipped with biotin and screened against a collection of streptavidin mutants. With the best hit, the S112Y mutant, the reaction occurred with 95% ee and complete suppression of the intrinsically favored side product from decarboxylation. This performance of anion-π enzymes rivals, if not exceeds, that of the best conventional organocatalysts. Inhibition of the S112Y mutant by nitrate but not by bulky anions supports that contributions from anion-π interactions exist and matter, also within proteins. In agreement with docking results, K121 is shown to be essential, presumably to lower the pK a of the tertiary amine catalyst to operate at the optimum pH around 3, that is below the pK a of the substrate. Most importantly, increasing enantioselectivity with different mutants always coincides with increasing rates and conversion, i.e., selective transition-state stabilization. PMID:27413782

  10. Characterising Complex Enzyme Reaction Data

    PubMed Central

    Rahman, Syed Asad; Thornton, Janet M.

    2016-01-01

    The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC) number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG). Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution. PMID:26840640

  11. Industrial use of immobilized enzymes.

    PubMed

    DiCosimo, Robert; McAuliffe, Joseph; Poulose, Ayrookaran J; Bohlmann, Gregory

    2013-08-01

    Although many methods for enzyme immobilization have been described in patents and publications, relatively few processes employing immobilized enzymes have been successfully commercialized. The cost of most industrial enzymes is often only a minor component in overall process economics, and in these instances, the additional costs associated with enzyme immobilization are often not justified. More commonly the benefit realized from enzyme immobilization relates to the process advantages that an immobilized catalyst offers, for example, enabling continuous production, improved stability and the absence of the biocatalyst in the product stream. The development and attributes of several established and emerging industrial applications for immobilized enzymes, including high-fructose corn syrup production, pectin hydrolysis, debittering of fruit juices, interesterification of food fats and oils, biodiesel production, and carbon dioxide capture are reviewed herein, highlighting factors that define the advantages of enzyme immobilization. PMID:23436023

  12. Characterising Complex Enzyme Reaction Data.

    PubMed

    Dönertaş, Handan Melike; Martínez Cuesta, Sergio; Rahman, Syed Asad; Thornton, Janet M

    2016-01-01

    The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC) number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG). Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution.

  13. Industrial use of immobilized enzymes.

    PubMed

    DiCosimo, Robert; McAuliffe, Joseph; Poulose, Ayrookaran J; Bohlmann, Gregory

    2013-08-01

    Although many methods for enzyme immobilization have been described in patents and publications, relatively few processes employing immobilized enzymes have been successfully commercialized. The cost of most industrial enzymes is often only a minor component in overall process economics, and in these instances, the additional costs associated with enzyme immobilization are often not justified. More commonly the benefit realized from enzyme immobilization relates to the process advantages that an immobilized catalyst offers, for example, enabling continuous production, improved stability and the absence of the biocatalyst in the product stream. The development and attributes of several established and emerging industrial applications for immobilized enzymes, including high-fructose corn syrup production, pectin hydrolysis, debittering of fruit juices, interesterification of food fats and oils, biodiesel production, and carbon dioxide capture are reviewed herein, highlighting factors that define the advantages of enzyme immobilization.

  14. Leishmania donovani Encodes a Functional Selenocysteinyl-tRNA Synthase.

    PubMed

    Manhas, Reetika; Gowri, Venkatraman Subramanian; Madhubala, Rentala

    2016-01-15

    The synthesis of selenocysteine, the 21st amino acid, occurs on its transfer RNA (tRNA), tRNA(Sec). tRNA(Sec) is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase (PSTK) in eukaryotes. The selenium donor, selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase. Selenocysteinyl-tRNA synthase (SepSecS) then uses the O-phosphoseryl-tRNA(Sec) and selenophosphate to form Sec-tRNA(Sec) in eukaryotes. Here, we report the characterization of selenocysteinyl-tRNA synthase from Leishmania donovani. Kinetoplastid SepSecS enzymes are phylogenetically closer to worm SepSecS. LdSepSecS was found to exist as a tetramer. Leishmania SepSecS enzyme was found to be active and able to complement the ΔselA deletion in Escherichia coli JS1 strain only in the presence of archaeal PSTK, indicating the conserved nature of the PSTK-SepSecS pathway. LdSepSecS was found to localize in the cytoplasm of the parasite. Gene deletion studies indicate that Leishmania SepSecS is dispensable for the parasite survival. The parasite was found to encode three selenoproteins, which were only expressed in the presence of SepSecS. Selenoproteins of L. donovani are not required for the growth of the promastigotes. Auranofin, a known inhibitor of selenoprotein synthesis showed the same sensitivity toward the wild-type and null mutants suggesting its effect is not through binding to selenoproteins. The three-dimensional structural comparison indicates that human and Leishmania homologs are structurally highly similar but their association modes leading to tetramerization seem different. PMID:26586914

  15. Evolution of Enzyme Kinetic Mechanisms.

    PubMed

    Ulusu, Nuriye Nuray

    2015-06-01

    This review paper discusses the reciprocal kinetic behaviours of enzymes and the evolution of structure-function dichotomy. Kinetic mechanisms have evolved in response to alterations in ecological and metabolic conditions. The kinetic mechanisms of single-substrate mono-substrate enzyme reactions are easier to understand and much simpler than those of bi-bi substrate enzyme reactions. The increasing complexities of kinetic mechanisms, as well as the increasing number of enzyme subunits, can be used to shed light on the evolution of kinetic mechanisms. Enzymes with heterogeneous kinetic mechanisms attempt to achieve specific products to subsist. In many organisms, kinetic mechanisms have evolved to aid survival in response to changing environmental factors. Enzyme promiscuity is defined as adaptation to changing environmental conditions, such as the introduction of a toxin or a new carbon source. Enzyme promiscuity is defined as adaptation to changing environmental conditions, such as the introduction of a toxin or a new carbon source. Enzymes with broad substrate specificity and promiscuous properties are believed to be more evolved than single-substrate enzymes. This group of enzymes can adapt to changing environmental substrate conditions and adjust catalysing mechanisms according to the substrate's properties, and their kinetic mechanisms have evolved in response to substrate variability.

  16. Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

    PubMed

    Kalinowska, Kamila; Nagel, Marie-Kristin; Isono, Erika

    2016-01-01

    Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

  17. Temporal information encoding in dynamic memristive devices

    NASA Astrophysics Data System (ADS)

    Ma, Wen; Chen, Lin; Du, Chao; Lu, Wei D.

    2015-11-01

    We show temporal and frequency information can be effectively encoded in memristive devices with inherent short-term dynamics. Ag/Ag2S/Pd based memristive devices with low programming voltage (˜100 mV) were fabricated and tested. At weak programming conditions, the devices exhibit inherent decay due to spontaneous diffusion of the Ag atoms. When the devices were subjected to pulse train inputs emulating different spiking patterns, the switching probability distribution function diverges from the standard Poisson distribution and evolves according to the input pattern. The experimentally observed switching probability distributions and the associated cumulative probability functions can be well-explained using a model accounting for the short-term decay effects. Such devices offer an intriguing opportunity to directly encode neural signals for neural information storage and analysis.

  18. Storing data encoded DNA in living organisms

    DOEpatents

    Wong; Pak C. , Wong; Kwong K. , Foote; Harlan P.

    2006-06-06

    Current technologies allow the generation of artificial DNA molecules and/or the ability to alter the DNA sequences of existing DNA molecules. With a careful coding scheme and arrangement, it is possible to encode important information as an artificial DNA strand and store it in a living host safely and permanently. This inventive technology can be used to identify origins and protect R&D investments. It can also be used in environmental research to track generations of organisms and observe the ecological impact of pollutants. Today, there are microorganisms that can survive under extreme conditions. As well, it is advantageous to consider multicellular organisms as hosts for stored information. These living organisms can provide as memory housing and protection for stored data or information. The present invention provides well for data storage in a living organism wherein at least one DNA sequence is encoded to represent data and incorporated into a living organism.

  19. Genetically encoded fluorescent sensors of membrane potential

    PubMed Central

    Baker, B. J.; Mutoh, H.; Dimitrov, D.; Akemann, W.; Perron, A.; Iwamoto, Y.; Jin, L.; Cohen, L. B.; Isacoff, E. Y.; Pieribone, V. A.; Hughes, T.; Knöpfel, T.

    2009-01-01

    Imaging activity of neurons in intact brain tissue was conceived several decades ago and, after many years of development, voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome the drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a genetically encoded sensor could in principle be expressed specifically in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression. Here we critically review the current status of these developments. PMID:18679801

  20. Asymmetric synthesis using chiral-encoded metal.

    PubMed

    Yutthalekha, Thittaya; Wattanakit, Chularat; Lapeyre, Veronique; Nokbin, Somkiat; Warakulwit, Chompunuch; Limtrakul, Jumras; Kuhn, Alexander

    2016-08-26

    The synthesis of chiral compounds is of crucial importance in many areas of society and science, including medicine, biology, chemistry, biotechnology and agriculture. Thus, there is a fundamental interest in developing new approaches for the selective production of enantiomers. Here we report the use of mesoporous metal structures with encoded geometric chiral information for inducing asymmetry in the electrochemical synthesis of mandelic acid as a model molecule. The chiral-encoded mesoporous metal, obtained by the electrochemical reduction of platinum salts in the presence of a liquid crystal phase and the chiral template molecule, perfectly retains the chiral information after removal of the template. Starting from a prochiral compound we demonstrate enantiomeric excess of the (R)-enantiomer when using (R)-imprinted electrodes and vice versa for the (S)-imprinted ones. Moreover, changing the amount of chiral cavities in the material allows tuning the enantioselectivity.

  1. Nucleic acid compositions and the encoding proteins

    SciTech Connect

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  2. Asymmetric synthesis using chiral-encoded metal.

    PubMed

    Yutthalekha, Thittaya; Wattanakit, Chularat; Lapeyre, Veronique; Nokbin, Somkiat; Warakulwit, Chompunuch; Limtrakul, Jumras; Kuhn, Alexander

    2016-01-01

    The synthesis of chiral compounds is of crucial importance in many areas of society and science, including medicine, biology, chemistry, biotechnology and agriculture. Thus, there is a fundamental interest in developing new approaches for the selective production of enantiomers. Here we report the use of mesoporous metal structures with encoded geometric chiral information for inducing asymmetry in the electrochemical synthesis of mandelic acid as a model molecule. The chiral-encoded mesoporous metal, obtained by the electrochemical reduction of platinum salts in the presence of a liquid crystal phase and the chiral template molecule, perfectly retains the chiral information after removal of the template. Starting from a prochiral compound we demonstrate enantiomeric excess of the (R)-enantiomer when using (R)-imprinted electrodes and vice versa for the (S)-imprinted ones. Moreover, changing the amount of chiral cavities in the material allows tuning the enantioselectivity. PMID:27562028

  3. DNA-Encoded Dynamic Combinatorial Chemical Libraries.

    PubMed

    Reddavide, Francesco V; Lin, Weilin; Lehnert, Sarah; Zhang, Yixin

    2015-06-26

    Dynamic combinatorial chemistry (DCC) explores the thermodynamic equilibrium of reversible reactions. Its application in the discovery of protein binders is largely limited by difficulties in the analysis of complex reaction mixtures. DNA-encoded chemical library (DECL) technology allows the selection of binders from a mixture of up to billions of different compounds; however, experimental results often show low a signal-to-noise ratio and poor correlation between enrichment factor and binding affinity. Herein we describe the design and application of DNA-encoded dynamic combinatorial chemical libraries (EDCCLs). Our experiments have shown that the EDCCL approach can be used not only to convert monovalent binders into high-affinity bivalent binders, but also to cause remarkably enhanced enrichment of potent bivalent binders by driving their in situ synthesis. We also demonstrate the application of EDCCLs in DNA-templated chemical reactions.

  4. Genetically Encoded Voltage Indicators in Circulation Research

    PubMed Central

    Kaestner, Lars; Tian, Qinghai; Kaiser, Elisabeth; Xian, Wenying; Müller, Andreas; Oberhofer, Martin; Ruppenthal, Sandra; Sinnecker, Daniel; Tsutsui, Hidekazu; Miyawaki, Atsushi; Moretti, Alessandra; Lipp, Peter

    2015-01-01

    Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided. PMID:26370981

  5. Asymmetric synthesis using chiral-encoded metal

    PubMed Central

    Yutthalekha, Thittaya; Wattanakit, Chularat; Lapeyre, Veronique; Nokbin, Somkiat; Warakulwit, Chompunuch; Limtrakul, Jumras; Kuhn, Alexander

    2016-01-01

    The synthesis of chiral compounds is of crucial importance in many areas of society and science, including medicine, biology, chemistry, biotechnology and agriculture. Thus, there is a fundamental interest in developing new approaches for the selective production of enantiomers. Here we report the use of mesoporous metal structures with encoded geometric chiral information for inducing asymmetry in the electrochemical synthesis of mandelic acid as a model molecule. The chiral-encoded mesoporous metal, obtained by the electrochemical reduction of platinum salts in the presence of a liquid crystal phase and the chiral template molecule, perfectly retains the chiral information after removal of the template. Starting from a prochiral compound we demonstrate enantiomeric excess of the (R)-enantiomer when using (R)-imprinted electrodes and vice versa for the (S)-imprinted ones. Moreover, changing the amount of chiral cavities in the material allows tuning the enantioselectivity. PMID:27562028

  6. Genetically Encoded Voltage Indicators in Circulation Research.

    PubMed

    Kaestner, Lars; Tian, Qinghai; Kaiser, Elisabeth; Xian, Wenying; Müller, Andreas; Oberhofer, Martin; Ruppenthal, Sandra; Sinnecker, Daniel; Tsutsui, Hidekazu; Miyawaki, Atsushi; Moretti, Alessandra; Lipp, Peter

    2015-09-08

    Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided.

  7. Asymmetric synthesis using chiral-encoded metal

    NASA Astrophysics Data System (ADS)

    Yutthalekha, Thittaya; Wattanakit, Chularat; Lapeyre, Veronique; Nokbin, Somkiat; Warakulwit, Chompunuch; Limtrakul, Jumras; Kuhn, Alexander

    2016-08-01

    The synthesis of chiral compounds is of crucial importance in many areas of society and science, including medicine, biology, chemistry, biotechnology and agriculture. Thus, there is a fundamental interest in developing new approaches for the selective production of enantiomers. Here we report the use of mesoporous metal structures with encoded geometric chiral information for inducing asymmetry in the electrochemical synthesis of mandelic acid as a model molecule. The chiral-encoded mesoporous metal, obtained by the electrochemical reduction of platinum salts in the presence of a liquid crystal phase and the chiral template molecule, perfectly retains the chiral information after removal of the template. Starting from a prochiral compound we demonstrate enantiomeric excess of the (R)-enantiomer when using (R)-imprinted electrodes and vice versa for the (S)-imprinted ones. Moreover, changing the amount of chiral cavities in the material allows tuning the enantioselectivity.

  8. Temporal information encoding in dynamic memristive devices

    SciTech Connect

    Ma, Wen; Chen, Lin; Du, Chao; Lu, Wei D.

    2015-11-09

    We show temporal and frequency information can be effectively encoded in memristive devices with inherent short-term dynamics. Ag/Ag{sub 2}S/Pd based memristive devices with low programming voltage (∼100 mV) were fabricated and tested. At weak programming conditions, the devices exhibit inherent decay due to spontaneous diffusion of the Ag atoms. When the devices were subjected to pulse train inputs emulating different spiking patterns, the switching probability distribution function diverges from the standard Poisson distribution and evolves according to the input pattern. The experimentally observed switching probability distributions and the associated cumulative probability functions can be well-explained using a model accounting for the short-term decay effects. Such devices offer an intriguing opportunity to directly encode neural signals for neural information storage and analysis.

  9. Mapping and Serodiagnostic Application of a Dominant Epitope within the Human Herpesvirus 8 ORF 65-Encoded Protein

    PubMed Central

    Pau, Chou-Pong; Lam, Lee L.; Spira, Thomas J.; Black, Jodi B.; Stewart, John A.; Pellett, Philip E.; Respess, Richard A.

    1998-01-01

    A dominant epitope within the human herpesvirus 8 (HHV8) ORF 65-encoded protein was mapped to an 8-amino-acid (aa) sequence (RKPPSGKK [aa 162 to 169]) by an amino acid replacement method. Using a 14-aa peptide (P4) encompassing this epitope as the antigen, we developed an enzyme immunoassay for HHV8 antibodies. The presence of P4 antibodies in a panel of 61 human serum specimens was highly correlated with biopsy-confirmed Kaposi’s sarcoma. The homologous Epstein-Barr virus peptide derived from BFBR3-encoded protein did not interfere with the assay, suggesting that P4 is specific for HHV8. PMID:9620379

  10. Human Lung Angiotensin Converting Enzyme

    PubMed Central

    Friedland, Joan; Silverstein, Emanuel; Drooker, Martin; Setton, Charlotte

    1981-01-01

    To enable its immunohistologic localization, angiotensin converting enzyme (EC 3.4.15.1) from human lung was solubilized by trypsinization and purified ∼2,660-fold to apparent homogeneity from a washed lung particulate fraction. The specific activity of pure enzyme was estimated to be 117 μmol/min per mg protein with the substrate hippuryl-l-histidyl-l-leucine. Consistent with previously described lung enzyme studies, catalytic activity was strongly inhibited by EDTA, O-phenanthroline, SQ 20,881, and SQ 14,225 and increased by CoCl2. SQ 20,881 was a somewhat more potent inhibitor than SQ 14,225, unlike rabbit lung enzyme. The Michaelis constant (Km) with hippuryl-l-histidyl-l-leucine was 1.6 mM. The molecular weight was estimated at 150,000 from sucrose density gradient centrifugation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single polypeptide chain estimated at 130,000 daltons. Rabbit antibody to human lung enzyme was prepared by parenteral administration of pure angiotensin-converting enzyme in Freund's adjuvant. Rabbit antibody to human lung angiotensin-converting enzyme appeared to crossreact weakly with the rabbit enzyme and strongly inhibited the catalytic activity of the enzymes from human serum, lung, and lymph node. The specificity of the rabbit antibody and purity of the final human lung enzyme preparation was suggested by the single precipitin lines obtained by radial double immunodiffusion, and by the coincidence of enzyme catalytic activity and immunoreactivity on polyacrylamide gel electrophoresis, with both relatively pure and highly impure enzymes. Generally applicable sensitive analysis of acrylamide gels for immunoreactivity (and subsequently for any other activity) by use of intact gel slices in radial double immunodiffusion was devised. Human lung enzyme was very tightly bound to and catalytically active on anti-human enzyme antibody covalently bound to Sepharose 4B, and could not be readily dissociated without

  11. An Encoding of XQuery in Prolog

    NASA Astrophysics Data System (ADS)

    Almendros-Jiménez, Jesús M.

    In this paper we describe the implementation of (a subset of) the XQuery language using logic programming (in particular, by means of Prolog). Such implementation has been developed using the Prolog interpreter SWI-Prolog. XML files are handled by means of the XML Library of SWI-Prolog. XPath/XQuery are encoded by means of Prolog rules. Such Prolog rules are executed in order to obtain the answer of the query.

  12. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, J.D.; Scott-Craig, J.S.

    1999-10-26

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is presented. The deduced amino acid sequence is provided. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with vectors and seeds from the plants.

  13. Population Encoding With Hodgkin–Huxley Neurons

    PubMed Central

    Lazar, Aurel A.

    2013-01-01

    The recovery of (weak) stimuli encoded with a population of Hodgkin–Huxley neurons is investigated. In the absence of a stimulus, the Hodgkin–Huxley neurons are assumed to be tonically spiking. The methodology employed calls for 1) finding an input–output (I/O) equivalent description of the Hodgkin–Huxley neuron and 2) devising a recovery algorithm for stimuli encoded with the I/O equivalent neuron(s). A Hodgkin–Huxley neuron with multiplicative coupling is I/O equivalent with an Integrate-and-Fire neuron with a variable threshold sequence. For bandlimited stimuli a perfect recovery of the stimulus can be achieved provided that a Nyquist-type rate condition is satisfied. A Hodgkin–Huxley neuron with additive coupling and deterministic conductances is first-order I/O equivalent with a Project-Integrate-and-Fire neuron that integrates a projection of the stimulus on the phase response curve. The stimulus recovery is formulated as a spline interpolation problem in the space of finite length bounded energy signals. A Hodgkin–Huxley neuron with additive coupling and stochastic conductances is shown to be first-order I/O equivalent with a Project-Integrate-and-Fire neuron with random thresholds. For stimuli modeled as elements of Sobolev spaces the reconstruction algorithm minimizes a regularized quadratic optimality criterion. Finally, all previous recovery results of stimuli encoded with Hodgkin–Huxley neurons with multiplicative and additive coupling, and deterministic and stochastic conductances are extended to stimuli encoded with a population of Hodgkin–Huxley neurons. PMID:24194625

  14. Host cell capable of producing enzymes useful for degradation of lignocellulosic material

    SciTech Connect

    Los, Alrik Pieter; Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Damveld, Robbertus Antonius

    2015-08-18

    The invention relates to a host cell comprising at least four different heterologous polynucleotides chosen from the group of polynucleotides encoding cellulases, hemicellulases and pectinases, wherein the host cell is capable of producing the at least four different enzymes chosen from the group of cellulases, hemicellulases and pectinases, wherein the host cell is a filamentous fungus and is capable of secretion of the at least four different enzymes. This host cell can suitably be used for the production of an enzyme composition that can be used in a process for the saccharification of cellulosic material.

  15. The pentafunctional arom enzyme of Saccharomyces cerevisiae is a mosaic of monofunctional domains.

    PubMed Central

    Duncan, K; Edwards, R M; Coggins, J R

    1987-01-01

    The nucleotide sequence of the Saccharomyces cerevisiae ARO1 gene which encodes the arom multifunctional enzyme has been determined. The protein sequence deduced for the pentafunctional arom polypeptide is 1588 amino acids in length and has a calculated Mr of 174555. Functional regions within the polypeptide chain have been identified by comparison with the sequences of the five monofunctional Escherichia coli enzymes whose activities correspond with those of the arom multifunctional enzyme. The observed homologies demonstrate that the arom polypeptide is a mosaic of functional domains and are consistent with the hypothesis that the ARO1 gene evolved by the linking of ancestral E. coli-like genes. PMID:2825635

  16. Role and specificity of plasmid RP4-encoded DNA primase in bacterial conjugation.

    PubMed Central

    Merryweather, A; Barth, P T; Wilkins, B M

    1986-01-01

    The role of the DNA primase of IncP plasmids was examined with a derivative of RP4 containing Tn7 in the primase gene (pri). The mutant was defective in mediating bacterial conjugation, with the deficiency varying according to the bacterial strains used as donors and recipients. Complementation tests involving recombinant plasmids carrying cloned fragments of RP4 indicated that the primase acts to promote some event in the recipient cell after DNA transfer and that this requirement can be satisfied by plasmid primase made in the donor cell. It is proposed that the enzyme or its products or both are transmitted to the recipient cell during conjugation, and the role of the enzyme in the conjugative processing of RP4 is discussed. Specificity of plasmid primases was assessed with derivatives of RP4 and the IncI1 plasmid ColIb-P9, which is known to encode a DNA primase active in conjugation. When supplied in the donor cell, neither of the primases encoded by these plasmids substituted effectively in the nonhomologous conjugation system. Since ColIb primase provided in the recipient cell acted weakly on transferred RP4 DNA, it is suggested that the specificity of these enzymes reflects their inability to be transmitted via the conjugation apparatus of the nonhomologous plasmid. PMID:3522540

  17. Identification and Analysis of a Gene from Calendula officinalis Encoding a Fatty Acid Conjugase

    PubMed Central

    Qiu, Xiao; Reed, Darwin W.; Hong, Haiping; MacKenzie, Samuel L.; Covello, Patrick S.

    2001-01-01

    Two homologous cDNAs, CoFad2 and CoFac2, were isolated from a Calendula officinalis developing seed by a polymerase chain reaction-based cloning strategy. Both sequences share similarity to FAD2 desaturases and FAD2-related enzymes. In C. officinalis plants CoFad2 was expressed in all tissues tested, whereas CoFac2 expression was specific to developing seeds. Expression of CoFad2 cDNA in yeast (Saccharomyces cerevisiae) indicated it encodes a Δ12 desaturase that introduces a double bond at the 12 position of 16:1(9Z) and 18:1(9Z). Expression of CoFac2 in yeast revealed that the encoded enzyme acts as a fatty acid conjugase converting 18:2(9Z, 12Z) to calendic acid 18:3(8E, 10E, 12Z). The enzyme also has weak activity on the mono-unsaturates 16:1(9Z) and 18:1(9Z) producing compounds with the properties of 8,10 conjugated dienes. PMID:11161042

  18. Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida.

    PubMed Central

    Parales, R E; Harwood, C S

    1992-01-01

    beta-Ketoadipate:succinyl-coenzyme A transferase (beta-ketoadipate:succinyl-CoA transferase) (EC 2.8.3.6) carries out the penultimate step in the conversion of benzoate and 4-hydroxybenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the beta-ketoadipate pathway. This report describes the characterization of a DNA fragment from Pseudomonas putida that encodes this enzyme. The fragment complemented mutants defective in the synthesis of the CoA transferase, and two proteins of sizes appropriate to encode the two nonidentical subunits of the enzyme were produced in Escherichia coli when the fragment was placed under the control of a phage T7 promoter. DNA sequence analysis revealed two open reading frames, designated pcaI and pcaJ, that were separated by 8 bp, suggesting that they may comprise an operon. A comparison of the deduced amino acid sequence of the P. putida CoA transferase genes with the sequences of two other bacterial CoA transferases and that of succinyl-CoA:3-ketoacid CoA transferase from pig heart suggests that the homodimeric structure of the mammalian enzyme may have resulted from a gene fusion of the bacterial alpha and beta subunit genes during evolution. Conserved functional groups important to the catalytic activity of CoA transferases were also identified. Images PMID:1624453

  19. Encoded libraries of chemically modified peptides.

    PubMed

    Heinis, Christian; Winter, Greg

    2015-06-01

    The use of powerful technologies for generating and screening DNA-encoded protein libraries has helped drive the development of proteins as pharmaceutical ligands. However the development of peptides as pharmaceutical ligands has been more limited. Although encoded peptide libraries are typically several orders of magnitude larger than classical chemical libraries, can be more readily screened, and can give rise to higher affinity ligands, their use as pharmaceutical ligands is limited by their intrinsic properties. Two of the intrinsic limitations include the rotational flexibility of the peptide backbone and the limited number (20) of natural amino acids. However these limitations can be overcome by use of chemical modification. For example, the libraries can be modified to introduce topological constraints such as cyclization linkers, or to introduce new chemical entities such as small molecule ligands, fluorophores and photo-switchable compounds. This article reviews the chemistry involved, the properties of the peptide ligands, and the new opportunities offered by chemical modification of DNA-encoded peptide libraries.

  20. Absolute Position Encoders With Vertical Image Binning

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B.

    2005-01-01

    Improved optoelectronic patternrecognition encoders that measure rotary and linear 1-dimensional positions at conversion rates (numbers of readings per unit time) exceeding 20 kHz have been invented. Heretofore, optoelectronic pattern-recognition absoluteposition encoders have been limited to conversion rates <15 Hz -- too low for emerging industrial applications in which conversion rates ranging from 1 kHz to as much as 100 kHz are required. The high conversion rates of the improved encoders are made possible, in part, by use of vertically compressible or binnable (as described below) scale patterns in combination with modified readout sequences of the image sensors [charge-coupled devices (CCDs)] used to read the scale patterns. The modified readout sequences and the processing of the images thus read out are amenable to implementation by use of modern, high-speed, ultra-compact microprocessors and digital signal processors or field-programmable gate arrays. This combination of improvements makes it possible to greatly increase conversion rates through substantial reductions in all three components of conversion time: exposure time, image-readout time, and image-processing time.

  1. Neural signals encoding shifts in beliefs

    PubMed Central

    Schwartenbeck, Philipp; FitzGerald, Thomas H.B.; Dolan, Ray

    2016-01-01

    Dopamine is implicated in a diverse range of cognitive functions including cognitive flexibility, task switching, signalling novel or unexpected stimuli as well as advance information. There is also longstanding line of thought that links dopamine with belief formation and, crucially, aberrant belief formation in psychosis. Integrating these strands of evidence would suggest that dopamine plays a central role in belief updating and more specifically in encoding of meaningful information content in observations. The precise nature of this relationship has remained unclear. To directly address this question we developed a paradigm that allowed us to decompose two distinct types of information content, information-theoretic surprise that reflects the unexpectedness of an observation, and epistemic value that induces shifts in beliefs or, more formally, Bayesian surprise. Using functional magnetic-resonance imaging in humans we show that dopamine-rich midbrain regions encode shifts in beliefs whereas surprise is encoded in prefrontal regions, including the pre-supplementary motor area and dorsal cingulate cortex. By linking putative dopaminergic activity to belief updating these data provide a link to false belief formation that characterises hyperdopaminergic states associated with idiopathic and drug induced psychosis. PMID:26520774

  2. Dual-channel spectrally encoded endoscopic probe

    PubMed Central

    Engel, Guy; Genish, Hadar; Rosenbluh, Michael; Yelin, Dvir

    2012-01-01

    High quality imaging through sub-millimeter endoscopic probes provides clinicians with valuable diagnostics capabilities in hard to reach locations within the body. Spectrally encoded endoscopy (SEE) has been shown promising for such task; however, challenging probe fabrication and high speckle noise had prevented its testing in in vivo studies. Here we demonstrate a novel miniature SEE probe which incorporates some of the recent progress in spectrally encoded technology into a compact and robust endoscopic system. A high-quality miniature diffraction grating was fabricated using automated femtosecond laser cutting from a large bulk grating. Using one spectrally encoded channel for imaging and a separate channel for incoherent illumination, the new system has large depth of field, negligible back reflections and well controlled speckle noise which depends on the core diameter of the illumination fiber. Moreover, by using a larger imaging channel, higher groove density grating, shorter wavelength and broader spectrum, the new endoscopic system now allow significant improvements in almost all imaging parameter compared to previous systems, through an ultra-miniature endoscopic probe. PMID:22876349

  3. Virus-encoded microRNAs

    PubMed Central

    Grundhoff, Adam; Sullivan, Christopher S.

    2011-01-01

    microRNAs (miRNAs) are the subject of enormous interest. They are small non-coding RNAs that play a regulatory role in numerous and diverse cellular processes such as immune function, apoptosis and tumorigenesis. Several virus families have been shown to encode miRNAs, and an appreciation for their roles in the viral infectious cycle continues to grow. Despite the identification of numerous (>225) viral miRNAs, an in depth functional understanding of most virus-encoded miRNAs is lacking. Here we focus on a few viral miRNAs with well-defined functions. We use these examples to extrapolate general themes of viral miRNA activities including autoregulation of gene expression, avoidance of host defenses, and a likely important role in maintaining latent and persistent infections. We hypothesize that although the molecular mechanisms and machinery are similar, the majority of viral miRNAs may utilize a target strategy that differs from host miRNAs. That is, many viral miRNAs may have evolved to regulate viral-encoded transcripts or networks of host genes that are unique to viral miRNAs. Included in this latter category are a likely abundant class of viral miRNAs that may regulate only one or a few principal host genes. Key steps forward for the field are discussed, including the need for additional functional studies that utilize surgical viral miRNA mutants combined with relevant models of infection. PMID:21277611

  4. Nucleic acids encoding human trithorax protein

    DOEpatents

    Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

    2001-01-01

    In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

  5. Encoding and decoding messages with chaotic lasers

    SciTech Connect

    Alsing, P.M.; Gavrielides, A.; Kovanis, V.; Roy, R.; Thornburg, K.S. Jr.

    1997-12-01

    We investigate the structure of the strange attractor of a chaotic loss-modulated solid-state laser utilizing return maps based on a combination of intensity maxima and interspike intervals, as opposed to those utilizing Poincar{acute e} sections defined by the intensity maxima of the laser ({dot I}=0,{umlt I}{lt}0) alone. We find both experimentally and numerically that a simple, intrinsic relationship exists between an intensity maximum and the pair of preceding and succeeding interspike intervals. In addition, we numerically investigate encoding messages on the output of a chaotic transmitter laser and its subsequent decoding by a similar receiver laser. By exploiting the relationship between the intensity maxima and the interspike intervals, we demonstrate that the method utilized to encode the message is vital to the system{close_quote}s ability to hide the signal from unwanted deciphering. In this work alternative methods are studied in order to encode messages by modulating the magnitude of pumping of the transmitter laser and also by driving its loss modulation with more than one frequency. {copyright} {ital 1997} {ital The American Physical Society}

  6. Enzyme molecules in solitary confinement.

    PubMed

    Liebherr, Raphaela B; Gorris, Hans H

    2014-09-12

    Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities.

  7. Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme.

    PubMed

    Levati, Elisabetta; Sartini, Sara; Bolchi, Angelo; Ottonello, Simone; Montanini, Barbara

    2016-01-01

    Moonlighting proteins, including metabolic enzymes acting as transcription factors (TF), are present in a variety of organisms but have not been described in higher fungi so far. In a previous genome-wide analysis of the TF repertoire of the plant-symbiotic fungus Tuber melanosporum, we identified various enzymes, including the sulfur-assimilation enzyme phosphoadenosine-phosphosulfate reductase (PAPS-red), as potential transcriptional activators. A functional analysis performed in the yeast Saccharomyces cerevisiae, now demonstrates that a specific variant of this enzyme, PAPS-red A, localizes to the nucleus and is capable of transcriptional activation. TF moonlighting, which is not present in the other enzyme variant (PAPS-red B) encoded by the T. melanosporum genome, relies on a transplantable C-terminal polypeptide containing an alternating hydrophobic/hydrophilic amino acid motif. A similar moonlighting activity was demonstrated for six additional proteins, suggesting that multitasking is a relatively frequent event. PAPS-red A is sulfur-state-responsive and highly expressed, especially in fruitbodies, and likely acts as a recruiter of transcription components involved in S-metabolism gene network activation. PAPS-red B, instead, is expressed at low levels and localizes to a highly methylated and silenced region of the genome, hinting at an evolutionary mechanism based on gene duplication, followed by epigenetic silencing of this non-moonlighting gene variant. PMID:27121330

  8. Robust enzyme design: bioinformatic tools for improved protein stability.

    PubMed

    Suplatov, Dmitry; Voevodin, Vladimir; Švedas, Vytas

    2015-03-01

    The ability of proteins and enzymes to maintain a functionally active conformation under adverse environmental conditions is an important feature of biocatalysts, vaccines, and biopharmaceutical proteins. From an evolutionary perspective, robust stability of proteins improves their biological fitness and allows for further optimization. Viewed from an industrial perspective, enzyme stability is crucial for the practical application of enzymes under the required reaction conditions. In this review, we analyze bioinformatic-driven strategies that are used to predict structural changes that can be applied to wild type proteins in order to produce more stable variants. The most commonly employed techniques can be classified into stochastic approaches, empirical or systematic rational design strategies, and design of chimeric proteins. We conclude that bioinformatic analysis can be efficiently used to study large protein superfamilies systematically as well as to predict particular structural changes which increase enzyme stability. Evolution has created a diversity of protein properties that are encoded in genomic sequences and structural data. Bioinformatics has the power to uncover this evolutionary code and provide a reproducible selection of hotspots - key residues to be mutated in order to produce more stable and functionally diverse proteins and enzymes. Further development of systematic bioinformatic procedures is needed to organize and analyze sequences and structures of proteins within large superfamilies and to link them to function, as well as to provide knowledge-based predictions for experimental evaluation.

  9. The mammalian molybdenum enzymes of mARC.

    PubMed

    Ott, Gudrun; Havemeyer, Antje; Clement, Bernd

    2015-03-01

    The "mitochondrial amidoxime reducing component" (mARC) is the most recently discovered molybdenum-containing enzyme in mammals. All mammalian genomes studied to date contain two mARC genes: MARC1 and MARC2. The proteins encoded by these genes are mARC-1 and mARC-2 and represent the simplest form of eukaryotic molybdenum enzymes, only binding the molybdenum cofactor. In the presence of NADH, mARC proteins exert N-reductive activity together with the two electron transport proteins cytochrome b5 type B and NADH cytochrome b5 reductase. This enzyme system is capable of reducing a great variety of N-hydroxylated substrates. It plays a decisive role in the activation of prodrugs containing an amidoxime structure, and in detoxification pathways, e.g., of N-hydroxylated purine and pyrimidine bases. It belongs to a group of drug metabolism enzymes, in particular as a counterpart of P450 formed N-oxygenated metabolites. Its physiological relevance, on the other hand, is largely unknown. The aim of this article is to summarize our current knowledge of these proteins with a special focus on the mammalian enzymes and their N-reductive activity.

  10. Evolution of an Enzyme from a Noncatalytic Nucleic Acid Sequence.

    PubMed

    Gysbers, Rachel; Tram, Kha; Gu, Jimmy; Li, Yingfu

    2015-01-01

    The mechanism by which enzymes arose from both abiotic and biological worlds remains an unsolved natural mystery. We postulate that an enzyme can emerge from any sequence of any functional polymer under permissive evolutionary conditions. To support this premise, we have arbitrarily chosen a 50-nucleotide DNA fragment encoding for the Bos taurus (cattle) albumin mRNA and subjected it to test-tube evolution to derive a catalytic DNA (DNAzyme) with RNA-cleavage activity. After only a few weeks, a DNAzyme with significant catalytic activity has surfaced. Sequence comparison reveals that seven nucleotides are responsible for the conversion of the noncatalytic sequence into the enzyme. Deep sequencing analysis of DNA pools along the evolution trajectory has identified individual mutations as the progressive drivers of the molecular evolution. Our findings demonstrate that an enzyme can indeed arise from a sequence of a functional polymer via permissive molecular evolution, a mechanism that may have been exploited by nature for the creation of the enormous repertoire of enzymes in the biological world today. PMID:26091540

  11. Dual enzyme electrochemical coding for detecting DNA hybridization.

    PubMed

    Wang, Joseph; Kawde, Abdel-Nasser; Musameh, Mustafa; Rivas, Gustavo

    2002-10-01

    Enzyme-based hybridization assays for the simultaneous electrochemical measurements of two DNA targets are described. Two encoding enzymes, alkaline phosphatase and beta-galactosidase, are used to differentiate the signals of two DNA targets in connection to chronopotentiometric measurements of their electroactive phenol and alpha-naphthol products. These products yield well-defined and resolved peaks at +0.31 V (alpha-naphthol) and +0.63 V (phenol) at the graphite working electrode (vs. Ag/AgCl reference). The position and size of these peaks reflect the identity and level of the corresponding target. The dual target detection capability is coupled to the amplification feature of enzyme tags (to yield fmol detection limits) and with an efficient magnetic removal of non-hybridized nucleic acids. Proper attention is given to the choice of the substrates (for attaining well resolved peaks), to the activity of the enzymes (for obtaining similar sensitivities), and to the selection of the enzymes (for minimizing cross interferences). The new bioassay is illustrated for the simultaneous detection of two DNA sequences related to the BCRA1 breast-cancer gene in a single sample in connection to magnetic beads bearing the corresponding oligonucleotide probes. Prospects for electrochemical coding of multiple DNA targets are discussed.

  12. Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme

    PubMed Central

    Levati, Elisabetta; Sartini, Sara; Bolchi, Angelo; Ottonello, Simone; Montanini, Barbara

    2016-01-01

    Moonlighting proteins, including metabolic enzymes acting as transcription factors (TF), are present in a variety of organisms but have not been described in higher fungi so far. In a previous genome-wide analysis of the TF repertoire of the plant-symbiotic fungus Tuber melanosporum, we identified various enzymes, including the sulfur-assimilation enzyme phosphoadenosine-phosphosulfate reductase (PAPS-red), as potential transcriptional activators. A functional analysis performed in the yeast Saccharomyces cerevisiae, now demonstrates that a specific variant of this enzyme, PAPS-red A, localizes to the nucleus and is capable of transcriptional activation. TF moonlighting, which is not present in the other enzyme variant (PAPS-red B) encoded by the T. melanosporum genome, relies on a transplantable C-terminal polypeptide containing an alternating hydrophobic/hydrophilic amino acid motif. A similar moonlighting activity was demonstrated for six additional proteins, suggesting that multitasking is a relatively frequent event. PAPS-red A is sulfur-state-responsive and highly expressed, especially in fruitbodies, and likely acts as a recruiter of transcription components involved in S-metabolism gene network activation. PAPS-red B, instead, is expressed at low levels and localizes to a highly methylated and silenced region of the genome, hinting at an evolutionary mechanism based on gene duplication, followed by epigenetic silencing of this non-moonlighting gene variant. PMID:27121330

  13. Cloning, sequencing, and expression of the gene encoding the Clostridium stercorarium alpha-galactosidase Aga36A in Escherichia coli.

    PubMed

    Suryani; Kimura, Tetsuya; Sakka, Kazuo; Ohmiya, Kunio

    2003-10-01

    The alpha-galactosidase gene aga36A of Clostridium stercorarium F-9 was cloned, sequenced, and expressed in Escherichia coli. The aga36A gene consists of 2,208 nucleotides encoding a protein of 736 amino acids with a predicted molecular weight of 84,786. Aga36A is an enzyme classified in family 36 of the glycoside hydrolases and showed sequence similarity with some enzymes of family 36 such as Geobacillus (formerly Bacillus) stearothermophilus GalA (57%) and AgaN (52%). The enzyme purified from a recombinant E. coli is optimally active at 70 degrees C and pH 6.0. The enzyme hydrolyzed raffinose and guar gum with specific activities of 3.0 U/mg and 0.46 U/mg for the respective substrates.

  14. Architecture for VLSI design of Reed-Solomon encoders

    NASA Technical Reports Server (NTRS)

    Liu, K. Y.

    1981-01-01

    The logic structure of a universal VLSI chip called the symbol-slice Reed-Solomon (RS) encoder chip is discussed. An RS encoder can be constructed by cascading and properly interconnecting a group of such VLSI chips. As a design example, it is shown that a (255,223) RD encoder requiring around 40 discrete CMOS ICs may be replaced by an RS encoder consisting of four identical interconnected VLSI RS encoder chips. Besides the size advantage, the VLSI RS encoder also has the potential advantages of requiring less power and having a higher reliability.

  15. Schizosaccharomyces pombe encodes a mutated AP endonuclease 1.

    PubMed

    Laerdahl, Jon K; Korvald, Hanne; Nilsen, Line; Dahl-Michelsen, Kristin; Rognes, Torbjørn; Bjørås, Magnar; Alseth, Ingrun

    2011-03-01

    Mutagenic and cytotoxic apurinic/apyrimidinic (AP) sites are among the most frequent lesions in DNA. Repair of AP sites is initiated by AP endonucleases and most organisms possess two or more of these enzymes. Saccharomyces cerevisiae has AP endonuclease 1 (Apn1) as the major enzymatic activity with AP endonuclease 2 (Apn2) being an important backup. Schizosaccharomyces pombe also encodes two potential AP endonucleases, and Apn2 has been found to be the main repair activity, while Apn1 has no, or only a limited role in AP site repair. Here we have identified a new 5' exon (exon 1) in the apn1 gene and show that the inactivity of S. pombe Apn1 is due to a nonsense mutation in the fifth codon of this new exon. Reversion of this mutation restored the AP endonuclease activity of S. pombe Apn1. Interestingly, the apn1 nonsense mutation was only found in laboratory strains derived from L972 h(-) and not in unrelated isolates of S. pombe. Since all S. pombe laboratory strains originate from L972 h(-), it appears that all experiments involving S. pombe have been conducted in an apn1(-) mutant strain with a corresponding DNA repair deficiency. These observations have implications both for future research in S. pombe and for the interpretation of previously conducted epistatis analysis.

  16. Biochemical characterization of exoribonuclease encoded by SARS coronavirus.

    PubMed

    Chen, Ping; Jiang, Miao; Hu, Tao; Liu, Qingzhen; Chen, Xiaojiang S; Guo, Deyin

    2007-09-30

    The nsp14 protein is an exoribonuclease that is encoded by severe acute respiratory syndrome coronavirus (SARS-CoV). We have cloned and expressed the nsp14 protein in Escherichia coli, and characterized the nature and the role(s) of the metal ions in the reaction chemistry. The purified recombinant nsp14 protein digested a 5'-labeled RNA molecule, but failed to digest the RNA substrate that is modified with fluorescein group at the 3'-hydroxyl group, suggesting a 3'-to-5' exoribonuclease activity. The exoribonuclease activity requires Mg2+ as a cofactor. Isothermal titration calorimetry (ITC) analysis indicated a two-metal binding mode for divalent cations by nsp14. Endogenous tryptophan fluorescence and circular dichroism (CD) spectra measurements showed that there was a structural change of nsp14 when binding with metal ions. We propose that the conformational change induced by metal ions may be a prerequisite for catalytic activity by correctly positioning the side chains of the residues located in the active site of the enzyme.

  17. Recombinant Exon-Encoded Resilins for Elastomeric Biomaterials

    PubMed Central

    Qin, Guokui; Rivkin, Amit; Lapidot, Shaul; Hu, Xiao; Arinus, Shira B.; Dgany, Or; Shoseyov, Oded; Kaplan, David L.

    2011-01-01

    Resilin is an elastomeric protein found in specialized regions of the cuticle of most insects, providing outstanding material properties including high resilience and fatigue lifetime for insect flight and jumping needs. Two exons (1 and 3) from the resilin gene in Drosophila melanogaster were cloned and the encoded proteins expressed as soluble products in Escherichia coli. A heat and salt precipitation method was used for efficient purification of the recombinant proteins. The proteins were solution cast from water and formed into rubber-like biomaterials via horseradish peroxidase-mediated cross-linking. Comparative studies of the two proteins expressed from the two different exons were investigated by Fourier Transform Infrared Spectroscopy (FTIR) and Circular Dichrosim (CD) for structural features. Little structural organization was found, suggesting structural order was not induced by the enzyme-mediateed dityrosine cross-links. Atomic Force Microscopy (AFM) was used to study the elastomeric properties of the uncross-linked and cross-linked proteins. The protein from exon 1 exhibited 90% resilience in comparison to 63% for the protein from exon 3, and therefore may be the more critical domain for functional materials to mimic native resilin. Further, the cross-linking of the recombinant exon 1 via the citrate-modified photo-Fenton reaction was explored as an alternative dityrosine mediated polymerization method and resulted in both highly elastic and adhesive materials. The citrate-modified photo-Fenton system may be suitable for in-vivo applications of resilin biomaterials. PMID:21963157

  18. Chlamydia pneumoniae encodes a functional aromatic amino acid hydroxylase.

    PubMed

    Abromaitis, Stephanie; Hefty, P Scott; Stephens, Richard S

    2009-03-01

    Chlamydia pneumoniae is a community-acquired respiratory pathogen that has been associated with the development of atherosclerosis. Analysis of the C. pneumoniae genome identified a gene (Cpn1046) homologous to eukaryotic aromatic amino acid hydroxylases (AroAA-Hs). AroAA-Hs hydroxylate phenylalanine, tyrosine, and tryptophan into tyrosine, dihydroxyphenylalanine, and 5-hydroxytryptophan, respectively. Sequence analysis of Cpn1046 demonstrated that residues essential for AroAA-H enzymatic function are conserved and that a subset of Chlamydia species contain an AroAA-H homolog. The chlamydial AroAA-Hs are transcriptionally linked to a putative bacterial membrane transport protein. We determined that recombinant Cpn1046 is able to hydroxylate phenylalanine, tyrosine, and tryptophan with roughly equivalent activity for all three substrates. Cpn1046 is expressed within 24 h of infection, allowing C. pneumoniae to hydroxylate host stores of aromatic amino acids during the period of logarithmic bacterial growth. From these results we can conclude that C. pneumoniae, as well as a subset of other Chlamydia species, encode an AroAA-H that is able to use all three aromatic amino acids as substrates. The maintenance of this gene within a number of Chlamydia suggests that the enzyme may have an important role in shaping the metabolism or overall pathogenesis of these bacteria. PMID:19141112

  19. Enzyme therapeutics for systemic detoxification.

    PubMed

    Liu, Yang; Li, Jie; Lu, Yunfeng

    2015-08-01

    Life relies on numerous biochemical processes working synergistically and correctly. Certain substances disrupt these processes, inducing living organism into an abnormal state termed intoxication. Managing intoxication usually requires interventions, which is referred as detoxification. Decades of development on detoxification reveals the potential of enzymes as ideal therapeutics and antidotes, because their high substrate specificity and catalytic efficiency are essential for clearing intoxicating substances without adverse effects. However, intrinsic shortcomings of enzymes including low stability and high immunogenicity are major hurdles, which could be overcome by delivering enzymes with specially designed nanocarriers. Extensive investigations on protein delivery indicate three types of enzyme-nanocarrier architectures that show more promise than others for systemic detoxification, including liposome-wrapped enzymes, polymer-enzyme conjugates, and polymer-encapsulated enzymes. This review highlights recent advances in these nano-architectures and discusses their applications in systemic detoxifications. Therapeutic potential of various enzymes as well as associated challenges in achieving effective delivery of therapeutic enzymes will also be discussed.

  20. Enzyme Mimics: Advances and Applications.

    PubMed

    Kuah, Evelyn; Toh, Seraphina; Yee, Jessica; Ma, Qian; Gao, Zhiqiang

    2016-06-13

    Enzyme mimics or artificial enzymes are a class of catalysts that have been actively pursued for decades and have heralded much interest as potentially viable alternatives to natural enzymes. Aside from having catalytic activities similar to their natural counterparts, enzyme mimics have the desired advantages of tunable structures and catalytic efficiencies, excellent tolerance to experimental conditions, lower cost, and purely synthetic routes to their preparation. Although still in the midst of development, impressive advances have already been made. Enzyme mimics have shown immense potential in the catalysis of a wide range of chemical and biological reactions, the development of chemical and biological sensing and anti-biofouling systems, and the production of pharmaceuticals and clean fuels. This Review concerns the development of various types of enzyme mimics, namely polymeric and dendrimeric, supramolecular, nanoparticulate and proteinic enzyme mimics, with an emphasis on their synthesis, catalytic properties and technical applications. It provides an introduction to enzyme mimics and a comprehensive summary of the advances and current standings of their applications, and seeks to inspire researchers to perfect the design and synthesis of enzyme mimics and to tailor their functionality for a much wider range of applications. PMID:27062126

  1. Enzyme actuated bioresponsive hydrogels

    NASA Astrophysics Data System (ADS)

    Wilson, Andrew Nolan

    Bioresponsive hydrogels are emerging with technological significance in targeted drug delivery, biosensors and regenerative medicine. Conferred with the ability to respond to specific biologically derived stimuli, the design challenge is in effectively linking the conferred biospecificity with an engineered response tailored to the needs of a particular application. Moreover, the fundamental phenomena governing the response must support an appropriate dynamic range and limit of detection. The design of these systems is inherently complicated due to the high interdependency of the governing phenomena that guide the sensing, transduction, and the actuation response of hydrogels. To investigate the dynamics of these materials, model systems may be used which seek to interrogate the system dynamics by uni-variable experimentation and limit confounding phenomena such as: polymer-solute interactions, polymer swelling dynamics and biomolecular reaction-diffusion concerns. To this end, a model system, alpha-chymotrypsin (Cht) (a protease) and a cleavable peptide-chromogen (pro-drug) covalently incorporated into a hydrogel, was investigated to understand the mechanisms of covalent loading and release by enzymatic cleavage in bio-responsive delivery systems. Using EDC and Sulfo-NHS, terminal carboxyl groups of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, a cleavable chromogen, were conjugated to primary amines of a hydrated poly(HEMA)-based hydrogel. Hydrogel discs were incubated in buffered Cht causing enzyme-mediated cleavage of the peptide and concomitant release of the chromophore for monitoring. To investigate substrate loading and the effects of hydrogel morphology on the system, the concentration of the amino groups (5, 10, 20, and 30 mol%) and the cross-linked density (1, 5, 7, 9 and 12 mol%) were independently varied. Loading-Release Efficiency of the chromogen was shown to exhibit a positive relation to increasing amino groups (AEMA). The release rates demonstrated a

  2. The Arabidopsis thaliana ortholog of a purported maize cholinesterase gene encodes a GDSL-lipase.

    PubMed

    Muralidharan, Mrinalini; Buss, Kristina; Larrimore, Katherine E; Segerson, Nicholas A; Kannan, Latha; Mor, Tsafrir S

    2013-04-01

    Acetylcholinesterase is an enzyme that is intimately associated with regulation of synaptic transmission in the cholinergic nervous system and in neuromuscular junctions of animals. However the presence of cholinesterase activity has been described also in non-metazoan organisms such as slime molds, fungi and plants. More recently, a gene purportedly encoding for acetylcholinesterase was cloned from maize. We have cloned the Arabidopsis thaliana homolog of the Zea mays gene, At3g26430, and studied its biochemical properties. Our results indicate that the protein encoded by the gene exhibited lipase activity with preference to long chain substrates but did not hydrolyze choline esters. The At3g26430 protein belongs to the SGNH clan of serine hydrolases, and more specifically to the GDS(L) lipase family. PMID:23430565

  3. Length-encoded multiplex binding site determination: application to zinc finger proteins.

    PubMed Central

    Desjarlais, J R; Berg, J M

    1994-01-01

    The screening of combinatorial libraries is becoming a powerful method for identifying or refining the structures of ligands for binding proteins, enzymes, and other receptors. We describe an oligonucleotide library search procedure in which the identity of each member is encoded in the length of oligonucleotides. This encoding scheme allows binding-site preferences to be evaluated via DNA length determination by denaturing gel electrophoresis. We have applied this method to determine the binding-site preferences for 18 Cys2His2 zinc finger domains as the central domain within a fixed context of flanking zinc fingers. An advantage of the method is that the relative affinities of all members of the library can be estimated in addition to simply determining the sequence of the optimal or consensus ligand. The zinc finger domain specificities determined will be useful for modular zinc finger protein design. Images PMID:7972017

  4. Human TOP3: a single-copy gene encoding DNA topoisomerase III.

    PubMed Central

    Hanai, R; Caron, P R; Wang, J C

    1996-01-01

    A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12. Images Fig. 2 PMID:8622991

  5. Design, synthesis and selection of DNA-encoded small-molecule libraries.

    PubMed

    Clark, Matthew A; Acharya, Raksha A; Arico-Muendel, Christopher C; Belyanskaya, Svetlana L; Benjamin, Dennis R; Carlson, Neil R; Centrella, Paolo A; Chiu, Cynthia H; Creaser, Steffen P; Cuozzo, John W; Davie, Christopher P; Ding, Yun; Franklin, G Joseph; Franzen, Kurt D; Gefter, Malcolm L; Hale, Steven P; Hansen, Nils J V; Israel, David I; Jiang, Jinwei; Kavarana, Malcolm J; Kelley, Michael S; Kollmann, Christopher S; Li, Fan; Lind, Kenneth; Mataruse, Sibongile; Medeiros, Patricia F; Messer, Jeffrey A; Myers, Paul; O'Keefe, Heather; Oliff, Matthew C; Rise, Cecil E; Satz, Alexander L; Skinner, Steven R; Svendsen, Jennifer L; Tang, Lujia; van Vloten, Kurt; Wagner, Richard W; Yao, Gang; Zhao, Baoguang; Morgan, Barry A

    2009-09-01

    Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase. PMID:19648931

  6. Cellobiohydrolase I enzymes

    DOEpatents

    Adney, William S; Himmel, Michael E; Decker, Stephen R; Knoshaug, Eric P; Nimlos, Mark R; Crowley, Michael F; Jeoh, Tina

    2014-01-28

    Provided herein is an isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide, wherein the mutations reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. Also provided herein is an isolated Cel7A polypeptide comprising increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The increased O-linked glycosylation is a result of the addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide. In some embodiments, the isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide further comprises increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The mutations in the catalytic domain reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. The addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide increases O-linked glycosylation of the isolated polypeptide. Further provided are compositions comprising such polypeptides and nucleic acids encoding such polypeptides. Still further provided are methods for making such polypeptides.

  7. Computational enzyme design: transitioning from catalytic proteins to enzymes.

    PubMed

    Mak, Wai Shun; Siegel, Justin B

    2014-08-01

    The widespread interest in enzymes stem from their ability to catalyze chemical reactions under mild and ecologically friendly conditions with unparalleled catalytic proficiencies. While thousands of naturally occurring enzymes have been identified and characterized, there are still numerous important applications for which there are no biological catalysts capable of performing the desired chemical transformation. In order to engineer enzymes for which there is no natural starting point, efforts using a combination of quantum chemistry and force-field based protein molecular modeling have led to the design of novel proteins capable of catalyzing chemical reactions not catalyzed by naturally occurring enzymes. Here we discuss the current status and potential avenues to pursue as the field of computational enzyme design moves forward.

  8. The Large Binocular Telescope azimuth and elevation encoder system

    NASA Astrophysics Data System (ADS)

    Ashby, David S.; Sargent, Tom; Cox, Dan; Rosato, Jerry; Brynnel, Joar G.

    2008-08-01

    A typical high-resolution encoder interpolator relies on careful mechanical alignment of the encoder read-heads and tight electrical tolerances of the signal processing electronics to ensure linearity. As the interpolation factor increases, maintaining these tight mechanical and electrical tolerances becomes impractical. The Large Binocular Telescope (LBT) is designed to utilize strip-type encoders on the main axes. Because of the very large scale of the telescope, the accumulative length of the azimuth and elevation encoder strips exceeds 80 meters, making optical tape prohibitively expensive. Consequently, the designers of the LBT incorporated the far less expensive Farrand Controls Inductosyn® linear strip encoder to encode the positions of the main axes and the instrument rotators. Since the cycle pitch of these encoders is very large compared to that of optical strip encoders, the interpolation factor must also be large in order to achieve the 0.005 arcsecond encoder resolution as specified. The authors present a description of the innovative DSP-based hardware / software solution that adaptively characterizes and removes common systematic cycle-to-cycle encoder interpolation errors. These errors can be caused by mechanical misalignment, encoder manufacturing flaws, variations in electrical gain, signal offset or cross-coupling of the encoder signals. Simulation data are presented to illustrate the performance of the interpolation algorithm, and telemetry data are presented to demonstrate the actual performance of the LBT main-axis encoder system.

  9. Co-expression of a Saccharomyces diastaticus glucoamylase-encoding gene and a Bacillus amyloliquefaciens alpha-amylase-encoding gene in Saccharomyces cerevisiae.

    PubMed

    Steyn, A J; Pretorius, I S

    1991-04-01

    A glucoamylase-encoding gene (STA2) from Saccharomyces diastaticus and an alpha-amylase-encoding gene (AMY) from Bacillus amyloliquefaciens were cloned separately into a yeast-integrating shuttle vector (YIp5), generating recombinant plasmids pSP1 and pSP2, respectively. The STA2 and AMY genes were jointly cloned into YIp5, generating plasmid pSP3. Subsequently, the dominant selectable marker APH1, encoding resistance to Geneticin G418 (GtR), was cloned into pSP3, resulting in pSP4. For enhanced expression of GtR, the APH1 gene was fused to the GAL10 promoter and terminated by the URA3 terminator, resulting in pSP5. Plasmid pSP5 was converted to a circular minichromosome (pSP6) by the addition of the ARS1 and CEN4 sequences. Laboratory strains of Saccharomyces cerevisiae transformed with plasmids pSP1 through pSP6, stably produced and secreted glucoamylase and/or alpha-amylase. Brewers' and distillers' yeast transformed with pSP6 were also capable of secreting amylolytic enzymes. Yeast transformants containing pSP1, pSP2 and pSP3 assimilated soluble starch with an efficiency of 69%, 84% and 93%, respectively. The major starch hydrolysis products produced by crude amylolytic enzymes found in the culture broths of the pSP1-, pSP2- and pSP3-containing transformants, were glucose, glucose and maltose (1:1), and glucose and maltose (3:1), respectively. These results confirmed that co-expression of the STA2 and AMY genes synergistically enhanced starch degradation.

  10. Isolation of α-glucuronidase enzyme from a rumen metagenomic library.

    PubMed

    Lee, Charles C; Kibblewhite, Rena E; Wagschal, Kurt; Li, Ruiping; Orts, William J

    2012-03-01

    α-Glucuronidase enzymes play an essential role in the full enzymatic hydrolysis of hemicellulose. Up to this point, all genes encoding α-glucuronidase enzymes have been cloned from individual, pure culture strains. Using a high-throughput screening strategy, we have isolated the first α-glucuronidase gene (rum630-AG) from a mixed population of microorganisms. The gene was subcloned into a prokaryotic vector, and the enzyme was overexpressed and biochemically characterized. The RUM630-AG enzyme had optimum activity at pH 6.5 and 40 °C. When birchwood xylan was used as substrate, the RUM630-AG functioned synergistically with an endoxylanase enzyme to hydrolyze the substrate.

  11. ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia

    PubMed Central

    Landt, Stephen G.; Marinov, Georgi K.; Kundaje, Anshul; Kheradpour, Pouya; Pauli, Florencia; Batzoglou, Serafim; Bernstein, Bradley E.; Bickel, Peter; Brown, James B.; Cayting, Philip; Chen, Yiwen; DeSalvo, Gilberto; Epstein, Charles; Fisher-Aylor, Katherine I.; Euskirchen, Ghia; Gerstein, Mark; Gertz, Jason; Hartemink, Alexander J.; Hoffman, Michael M.; Iyer, Vishwanath R.; Jung, Youngsook L.; Karmakar, Subhradip; Kellis, Manolis; Kharchenko, Peter V.; Li, Qunhua; Liu, Tao; Liu, X. Shirley; Ma, Lijia; Milosavljevic, Aleksandar; Myers, Richard M.; Park, Peter J.; Pazin, Michael J.; Perry, Marc D.; Raha, Debasish; Reddy, Timothy E.; Rozowsky, Joel; Shoresh, Noam; Sidow, Arend; Slattery, Matthew; Stamatoyannopoulos, John A.; Tolstorukov, Michael Y.; White, Kevin P.; Xi, Simon; Farnham, Peggy J.; Lieb, Jason D.; Wold, Barbara J.; Snyder, Michael

    2012-01-01

    Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode.org/) portals. PMID:22955991

  12. The Collaborative Encoding Deficit is Attenuated with Specific Warnings

    PubMed Central

    Barber, Sarah J.; Rajaram, Suparna; Paneerselvam, Bavani

    2012-01-01

    Individuals learning together do so less effectively than individuals learning alone, an effect known as the collaborative encoding deficit (Barber, Rajaram, & Aron, 2010). In the present studies we examined whether providing participants with a warning about the collaborative encoding deficit would increase their encoding task performance, and reduce subsequent memory deficits. Across two experiments, specific warnings were beneficial for memory. Collaborating participants who were told about the collaborative encoding deficit, and who received suggestions for how to complete the encoding task, had superior memory than participants who received no warning. This benefit was not due to qualitative changes in encoding task performance, was unrelated to the type of collaboration utilized, was absent when a more general warning was utilized, and was unrelated to self-reported task motivation. Rather, specific warnings appear to protect against the collaborative encoding deficit by increasing time spent on, and attention directed to, the encoding task. PMID:23296389

  13. Porcine Hypothalamic Aromatase Cytochrome P450: Isoform Characterization, Sex-Dependent Activity, Regional Expression, and Regulation by Enzyme Inhibition in Neonatal Boars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta and pre-implantation blastocyst. All catalyze estrogen synthesis, but the “gonadal” type enzyme is unique in also synthesizing a nonaromat...

  14. Making the Rate: Enzyme Dynamics

    ERIC Educational Resources Information Center

    Ragsdale, Frances R.

    2004-01-01

    An enzyme exercise to address the problem of students inability to visualize chemical reaction at the molecular level is described. This exercise is designed as a dry lab exercise but can be modified into a classroom activity then can be augmented by a wet lab procedure, thereby providing students with a practical exposure to enzyme function.

  15. 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.

  16. Molecular cloning, nucleotide sequence and expression of a Sulfolobus solfataricus gene encoding a class II fumarase.

    PubMed

    Colombo, S; Grisa, M; Tortora, P; Vanoni, M

    1994-01-01

    Fumarase catalyzes the interconversion of L-malate and fumarate. A Sulfolobus solfataricus fumarase gene (fumC) was cloned and sequenced. Typical archaebacterial regulatory sites were identified in the region flanking the fumC open reading frame. The fumC gene encodes a protein of 438 amino acids (47,899 Da) which shows several significant similarities with class II fumarases from both eubacterial and eukariotic sources as well as with aspartases. S. solfataricus fumarase expressed in Escherichia coli retains enzymatic activity and its thermostability is comparable to that of S. solfataricus purified enzyme despite a 11 amino acid C-terminal deletion.

  17. Multigene families of Cellulomonas flavigena encoding endo-beta-1,4-glucanases (CM-cellulases).

    PubMed

    Akhtar, M W; Duffy, M; Dowds, B C; Sheehan, M C; McConnell, D J

    1988-12-30

    Multiple genes coding for endo-beta-1,4-glucanases (CM-cellulases) have been isolated from a newly discovered highly cellulolytic strain of Cellulomonas flavigena. Clones of C. flavigena DNA were isolated in Escherichia coli and screened for gene expression on CM-cellulose plates staining with congo red. Six clones produced CM-cellulase activity as detected in liquid assays, and on activity gels. They fell into three groups within which the sequences cross-hybridised. There were small differences in the pH and temperature optima of the enzymes encoded by representatives of the three groups of clones.

  18. Evaluating standard terminologies for encoding allergy information

    PubMed Central

    Goss, Foster R; Zhou, Li; Plasek, Joseph M; Broverman, Carol; Robinson, George; Middleton, Blackford; Rocha, Roberto A

    2013-01-01

    Objective Allergy documentation and exchange are vital to ensuring patient safety. This study aims to analyze and compare various existing standard terminologies for representing allergy information. Methods Five terminologies were identified, including the Systemized Nomenclature of Medical Clinical Terms (SNOMED CT), National Drug File–Reference Terminology (NDF-RT), Medication Dictionary for Regulatory Activities (MedDRA), Unique Ingredient Identifier (UNII), and RxNorm. A qualitative analysis was conducted to compare desirable characteristics of each terminology, including content coverage, concept orientation, formal definitions, multiple granularities, vocabulary structure, subset capability, and maintainability. A quantitative analysis was also performed to compare the content coverage of each terminology for (1) common food, drug, and environmental allergens and (2) descriptive concepts for common drug allergies, adverse reactions (AR), and no known allergies. Results Our qualitative results show that SNOMED CT fulfilled the greatest number of desirable characteristics, followed by NDF-RT, RxNorm, UNII, and MedDRA. Our quantitative results demonstrate that RxNorm had the highest concept coverage for representing drug allergens, followed by UNII, SNOMED CT, NDF-RT, and MedDRA. For food and environmental allergens, UNII demonstrated the highest concept coverage, followed by SNOMED CT. For representing descriptive allergy concepts and adverse reactions, SNOMED CT and NDF-RT showed the highest coverage. Only SNOMED CT was capable of representing unique concepts for encoding no known allergies. Conclusions The proper terminology for encoding a patient's allergy is complex, as multiple elements need to be captured to form a fully structured clinical finding. Our results suggest that while gaps still exist, a combination of SNOMED CT and RxNorm can satisfy most criteria for encoding common allergies and provide sufficient content coverage. PMID:23396542

  19. Novel encoding methods for DNA-templated chemical libraries.

    PubMed

    Li, Gang; Zheng, Wenlu; Liu, Ying; Li, Xiaoyu

    2015-06-01

    Among various types of DNA-encoded chemical libraries, DNA-templated library takes advantage of the sequence-specificity of DNA hybridization, enabling not only highly effective DNA-templated chemical reactions, but also high fidelity in library encoding. This brief review summarizes recent advances that have been made on the encoding strategies for DNA-templated libraries, and it also highlights their respective advantages and limitations for the preparation of DNA-encoded libraries.

  20. Positron emitter labeled enzyme inhibitors

    SciTech Connect

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-04-03

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  1. Moonlighting enzymes in parasitic protozoa.

    PubMed

    Collingridge, Peter W; Brown, Robert W B; Ginger, Michael L

    2010-08-01

    Enzymes moonlight in a non-enzymatic capacity in a diverse variety of cellular processes. The discovery of these non-enzymatic functions is generally unexpected, and moonlighting enzymes are known in both prokaryotes and eukaryotes. Importantly, this unexpected multi-functionality indicates that caution might be needed on some occasions in interpreting phenotypes that result from the deletion or gene-silencing of some enzymes, including some of the best known enzymes from classic intermediary metabolism. Here, we provide an overview of enzyme moonlighting in parasitic protists. Unequivocal and putative examples of moonlighting are discussed, together with the possibility that the unusual biological characteristics of some parasites either limit opportunities for moonlighting to arise or perhaps contribute to the evolution of novel proteins with clear metabolic ancestry.

  2. Engineering Cellulase Enzymes for Bioenergy

    NASA Astrophysics Data System (ADS)

    Atreya, Meera Elizabeth

    Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current

  3. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    1987-05-22

    This invention involved a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide in activators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

  4. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, Joanna S.; MacGregor, Robert R.; Wolf, Alfred P.; Langstrom, Bengt

    1990-01-01

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  5. Doppler imaging using spectrally-encoded endoscopy

    PubMed Central

    Yelin, Dvir; Bouma, B. E.; Rosowsky, J. J.; Tearney, G. J.

    2009-01-01

    The capability to image tissue motion such as blood flow through an endoscope could have many applications in medicine. Spectrally encoded endoscopy (SEE) is a recently introduced technique that utilizes a single optical fiber and miniature diffractive optics to obtain endoscopic images through small diameter probes. Using spectral-domain interferometry, SEE is furthermore capable of three-dimensional volume imaging at video rates. Here we show that by measuring relative spectral phases, this technology can additionally measure Doppler shifts. Doppler SEE is demonstrated in flowing Intralipid phantoms and vibrating middle ear ossicles. PMID:18795020

  6. Error correction for encoded quantum annealing

    NASA Astrophysics Data System (ADS)

    Pastawski, Fernando; Preskill, John

    2016-05-01

    Recently, W. Lechner, P. Hauke, and P. Zoller [Sci. Adv. 1, e1500838 (2015), 10.1126/sciadv.1500838] have proposed a quantum annealing architecture, in which a classical spin glass with all-to-all pairwise connectivity is simulated by a spin glass with geometrically local interactions. We interpret this architecture as a classical error-correcting code, which is highly robust against weakly correlated bit-flip noise, and we analyze the code's performance using a belief-propagation decoding algorithm. Our observations may also apply to more general encoding schemes and noise models.

  7. Polynucleotides encoding TRF1 binding proteins

    DOEpatents

    Campisi, Judith; Kim, Sahn-Ho

    2002-01-01

    The present invention provides a novel telomere associated protein (Trf1-interacting nuclear protein 2 "Tin2") that hinders the binding of Trf1 to its specific telomere repeat sequence and mediates the formation of a Tin2-Trf1-telomeric DNA complex that limits telomerase access to the telomere. Also included are the corresponding nucleic acids that encode the Tin2 of the present invention, as well as mutants of Tin2. Methods of making, purifying and using Tin2 of the present invention are described. In addition, drug screening assays to identify drugs that mimic and/or complement the effect of Tin2 are presented.

  8. Encoding of Memory in Sheared Amorphous Solids

    NASA Astrophysics Data System (ADS)

    Fiocco, Davide; Foffi, Giuseppe; Sastry, Srikanth

    2014-01-01

    We show that memory can be encoded in a model amorphous solid subjected to athermal oscillatory shear deformations, and in an analogous spin model with disordered interactions, sharing the feature of a deformable energy landscape. When these systems are subjected to oscillatory shear deformation, they retain memory of the deformation amplitude imposed in the training phase, when the amplitude is below a "localization" threshold. Remarkably, multiple persistent memories can be stored using such an athermal, noise-free, protocol. The possibility of such memory is shown to be linked to the presence of plastic deformations and associated limit cycles traversed by the system, which exhibit avalanche statistics also seen in related contexts.

  9. The ENCODE (ENCyclopedia Of DNA Elements) Project.

    PubMed

    2004-10-22

    The ENCyclopedia Of DNA Elements (ENCODE) Project aims to identify all functional elements in the human genome sequence. The pilot phase of the Project is focused on a specified 30 megabases (approximately 1%) of the human genome sequence and is organized as an international consortium of computational and laboratory-based scientists working to develop and apply high-throughput approaches for detecting all sequence elements that confer biological function. The results of this pilot phase will guide future efforts to analyze the entire human genome.

  10. Gene encoding herbicide safener binding protein

    DOEpatents

    Walton, Jonathan D.; Scott-Craig, John S.

    1999-01-01

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is set forth in FIG. 5 and SEQ ID No. 1. The deduced amino acid sequence is provided in FIG. 5 and SEQ ID No. 2. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with said vectors and seeds from said plants.

  11. Human gamma-glutamyl hydrolase: cloning and characterization of the enzyme expressed in vitro.

    PubMed Central

    Yao, R; Schneider, E; Ryan, T J; Galivan, J

    1996-01-01

    A cDNA encoding human gamma-glutamyl hydrolase has been identified by searching an expressed sequence tag data base and using rat gamma-glutamyl hydrolase cDNA as the query sequence. The cDNA encodes a 318-amino acid protein of Mr 35,960. The deduced amino acid sequence of human gamma-glutamyl hydrolase shows 67% identity to that of rat gamma-glutamyl hydrolase. In both rat and human the 24 amino acids preceding the N terminus constitute a structural motif that is analogous to a leader or signal sequence. There are four consensus asparagine glycosylation sites in the human sequence, with three of them conserved in the rat enzyme. Expression of both the human and rat cDNA in Escherichia coli produced antigenically related proteins with enzyme activities characteristic of the native human and rat enzymes, respectively, when methotrexate di- or pentaglutamate were used as substrates. With the latter substrate the rat enzyme cleaved the innermost gamma-glutamyl linkage resulting in the sole production of methotrexate as the pteroyl containing product. The human enzyme differed in that it produced methotrexate tetraglutamate initially, followed by the triglutamate, and then the diglutamate and methotrexate. Hence the rat enzyme is an endopeptidase with methotrexate pentaglutamate as substrate, whereas the human enzyme exhibits exopeptidase activity. Another difference is that the expressed rat enzyme is equally active on methotrexate di- and pentaglutamate whereas the human enzyme has severalfold greater activity on methotrexate pentaglutamate compared with the diglutamate. These properties are consistent with the enzymes derived from human and rat sources. Images Fig. 3 PMID:8816764

  12. Visual Encoding Mechanisms and Their Relationship to Text Presentation Preference

    ERIC Educational Resources Information Center

    Pammer, Kristen; Lavis, Ruth; Cornelissen, Piers

    2004-01-01

    This study was designed to investigate the importance of spatial encoding in reading, with particular emphasis on visuo-spatial encoding mechanisms. Thirty one school children participated in the first study in which they were measured on their ability to solve a centrally presented spatial encoding task, as well as their sensitivity to the…

  13. Optical Pseudocolor Encoding Of Gray-Scale Image

    NASA Technical Reports Server (NTRS)

    Chao, Tien-Hsin; Liu, Hua-Kuang

    1990-01-01

    Optical encoding much faster than digital electronic encoding. In optical pseudocolor-encoding apparatus brightness modulation in image from television camera transformed into polarization modulation in LCTV, and then into pseudocolor modulation in image on projection screen. Advantageous for such purposes as thermography, inspection of circuit boards, mammography, and mapping.

  14. Cloning, expression and characterization of a new agarase-encoding gene from marine Pseudoalteromonas sp.

    PubMed

    Lu, Xinzhi; Chu, Yan; Wu, Qianqian; Gu, Yuchao; Han, Feng; Yu, Wengong

    2009-10-01

    The beta-agarase gene agaA, cloned from a marine bacterium, Pseudoalteromonas sp. CY24, consists of 1,359 nucleotides encoding 453 amino acids in a sequence corresponding to a catalytic domain of glycosyl hydrolase family 16 (GH16) and a carbohydrate-binding module type 13 (CBM13). The recombinant enzyme is an endo-type agarase that hydrolyzes beta-1,4-linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the predominant products. In two cleavage patterns, AgaA digested the smallest substrate, neoagarooctaose, into neoagarobiose, neoagarotetraose and neoagarohexaose. Site directed mutation was performed to investigate the differences between AgaA and AgaD of Vibrio sp. PO-303, identifying residues V(109)VTS(112) as playing a key role in the enzyme reaction. PMID:19504047

  15. Arabidopsis TCH4, regulated by hormones and the environment, encodes a xyloglucan endotransglycosylase

    NASA Technical Reports Server (NTRS)

    Xu, W.; Purugganan, M. M.; Polisensky, D. H.; Antosiewicz, D. M.; Fry, S. C.; Braam, J.

    1995-01-01

    Adaptation of plants to environmental conditions requires that sensing of external stimuli be linked to mechanisms of morphogenesis. The Arabidopsis TCH (for touch) genes are rapidly upregulated in expression in response to environmental stimuli, but a connection between this molecular response and developmental alterations has not been established. We identified TCH4 as a xyloglucan endotransglycosylase by sequence similarity and enzyme activity. Xyloglucan endotransglycosylases most likely modify cell walls, a fundamental determinant of plant form. We determined that TCH4 expression is regulated by auxin and brassinosteroids, by environmental stimuli, and during development, by a 1-kb region. Expression was restricted to expanding tissues and organs that undergo cell wall modification. Regulation of genes encoding cell wall-modifying enzymes, such as TCH4, may underlie plant morphogenetic responses to the environment.

  16. [Lignocellulose degrading bacteria and their genes encoding cellulase/hemicellulase in rumen--a review].

    PubMed

    Chen, Furong; Zhu, Yaxin; Dong, Xiuzhu; Liu, Lihua; Huang, Li; Dai, Xin

    2010-08-01

    Rumen of ruminant animals is known as a natural reactor involved in highly efficient lignocelluloses degradation. Rumen fibrolytic microbes have attracted an increasing attention for their potential value in biofuel research. Studies on rumen microbes have traditionally entailed the isolation of fibrolytic bacteria and subsequent analysis of fibrolytic enzymes. Developments in genomic and metagenomic approaches have made it possible to isolate directly genes and gene clusters encoding fibrolytic activities from rumen samples, permitting a global analysis of mechanisms of degradation of lignocellulose in rumen. Research in this field shows that lignocellulose degradation in rumen is a complex process involving a number of different microbes and is effected by a huge array of hydrolytic enzymes in a concerted fashion. This review briefly summarizes results from recent studies, especially metagenomic studies, on lignocellulose degradation in rumen.

  17. Encoded Library Synthesis Using Chemical Ligation and the Discovery of sEH Inhibitors from a 334-Million Member Library

    PubMed Central

    Litovchick, Alexander; Dumelin, Christoph E.; Habeshian, Sevan; Gikunju, Diana; Guié, Marie-Aude; Centrella, Paolo; Zhang, Ying; Sigel, Eric A.; Cuozzo, John W.; Keefe, Anthony D.; Clark, Matthew A.

    2015-01-01

    A chemical ligation method for construction of DNA-encoded small-molecule libraries has been developed. Taking advantage of the ability of the Klenow fragment of DNA polymerase to accept templates with triazole linkages in place of phosphodiesters, we have designed a strategy for chemically ligating oligonucleotide tags using cycloaddition chemistry. We have utilized this strategy in the construction and selection of a small molecule library, and successfully identified inhibitors of the enzyme soluble epoxide hydrolase. PMID:26061191

  18. Encoded Library Synthesis Using Chemical Ligation and the Discovery of sEH Inhibitors from a 334-Million Member Library.

    PubMed

    Litovchick, Alexander; Dumelin, Christoph E; Habeshian, Sevan; Gikunju, Diana; Guié, Marie-Aude; Centrella, Paolo; Zhang, Ying; Sigel, Eric A; Cuozzo, John W; Keefe, Anthony D; Clark, Matthew A

    2015-01-01

    A chemical ligation method for construction of DNA-encoded small-molecule libraries has been developed. Taking advantage of the ability of the Klenow fragment of DNA polymerase to accept templates with triazole linkages in place of phosphodiesters, we have designed a strategy for chemically ligating oligonucleotide tags using cycloaddition chemistry. We have utilized this strategy in the construction and selection of a small molecule library, and successfully identified inhibitors of the enzyme soluble epoxide hydrolase. PMID:26061191

  19. Optical Security System with Fourier Plane encoding.

    PubMed

    Javidi, B; Ahouzi, E

    1998-09-10

    We propose a new technique for security verification of personal documents and other forms of personal identifications such as ID cards, passports, or credit cards. In this technique a primary pattern that might be a phase-encoded image is convolved by a random code. The information is phase encoded on the personal document. Therefore the information cannot be reproduced by an intensity detector such as a CCD camera. An optical processor based on the nonlinear joint transform correlator is used to perform the verification and the validation of documents with this technique. By verification of the biometrics information and the random code simultaneously, the proposed optical system determines whether a card is authentic or is being used by an authorized person. We tested the performance of the optical system for security and validation in the presence of input noise and in the presence of distortion of the information on the card. The performance of the proposed method is evaluated by use of a number of metrics. Statistical analysis of the system is performed to investigate the noise tolerance and the discrimination against false inputs for security verification. PMID:18286124

  20. V123 BEAM SYNCHRONOUS ENCODER MODULE.

    SciTech Connect

    KERNER,T.; CONKLING,C.R.; OERTER,B.

    1999-03-29

    The V123 Synchronous Encoder Module transmits events to distributed trigger modules and embedded decoders around the RHIC rings where they are used to provide beam instrumentation triggers [1,2,3]. The RHIC beam synchronous event link hardware is mainly comprised of three VMEbus board designs, the central input modules (V201), and encoder modules (V123), and the distributed trigger modules (V124). Two beam synchronous links, one for each ring, are distributed via fiber optics and fanned out via twisted wire pair cables. The V123 synchronizes with the RF system clock derived from the beam bucket frequency and a revolution fiducial pulse. The RF system clock is used to create the beam synchronous event link carrier and events are synchronized with the rotation fiducial. A low jitter RF clock is later recovered from this carrier by phase lock loops in the trigger modules. Prioritized hardware and software triggers fill up to 15 beam event code transmission slots per revolution while tracking the ramping RF acceleration frequency and storage frequency. The revolution fiducial event is always the first event transmitted which is used to synchronize the firing of the abort kicker and to locate the first bucket for decoders distributed about the ring.

  1. Intensity encoding in unsupervised neural nets.

    PubMed

    Parkinson, Alan M.; Parpia, Dawood Y.

    1998-06-01

    The requirement of input vector normalisation in unsupervised neural nets results in a loss of information about the intensity of the signal contained in the input datastream. We show through a simple algebraic analysis that the introduction of an additional input channel encoding the root-mean-square intensity in the signals cannot restore this information if the input vectors have to be, nevertheless, all of the same length. We suggest an alternative method of encoding the input vectors where each of the input channels is split into two components in such a way that the resultant input vector is then of fixed length and retains information of the intensity in the signals. We further demonstrate, by using synthetic data, that a Kohonen Net is capable of forming topological maps of signals of different intensity, where an adjacency relationship is maintained both among the signals of the same frequency composition at different intensities and between signals of different frequency compositions at the same intensity. A second experiment reported here shows the same behaviour for less artificial inputs (based on a cochlear model) and additionally demonstrates that the trained network can respond appropriately to signals not previously encountered.

  2. Directed forgetting benefits motor sequence encoding.

    PubMed

    Tempel, Tobias; Frings, Christian

    2016-04-01

    Two experiments investigated directed forgetting of newly learned motor sequences. Concurrently with the list method of directed forgetting, participants successively learned two lists of motor sequences. Each sequence consisted of four consecutive finger movements. After a short distractor task, a recall test was given. Both experiments compared a forget group that was instructed to forget list-1 items with a remember group not receiving a forget instruction. We found that the instruction to forget list 1 enhanced recall of subsequently learned motor sequences. This benefit of directed forgetting occurred independently of costs for list 1. A mediation analysis showed that the encoding accuracy of list 2 was a mediator of the recall benefit, that is, the more accurate execution of motor sequences of list 2 after receiving a forget instruction for list 1 accounted for better recall of list 2. Thus, the adaptation of the list method to motor action provided more direct evidence on the effect of directed forgetting on subsequent learning. The results corroborate the assumption of a reset of encoding as a consequence of directed forgetting. PMID:26471189

  3. SpyRings Declassified: A Blueprint for Using Isopeptide-Mediated Cyclization to Enhance Enzyme Thermal Resilience.

    PubMed

    Schoene, C; Bennett, S P; Howarth, M

    2016-01-01

    Enzymes often have marginal stability, with unfolding typically leading to irreversible denaturation. This sensitivity is a major barrier, both for de novo enzyme development and for expanding enzyme impact beyond the laboratory. Seeking an approach to enhance resilience to denaturation that could be applied to a range of different enzymes, we developed SpyRing cyclization. SpyRings contain genetically encoded SpyTag (13 amino acids) on the N-terminus and SpyCatcher (12kDa) on the C-terminus of the enzyme, so that the Spy partners spontaneously react together through an irreversible isopeptide bond. SpyRing cyclization gave major increases in thermal resilience, including on a model for enzyme evolution, β-lactamase, and an industrially important enzyme in agriculture and nutrition, phytase. We outline the SpyRing rationale, including comparison of SpyRing cyclization to other cyclization strategies. The cloning strategy is presented for the simple insertion of enzyme genes for recombinant expression. We discuss structure-based approaches to select suitable enzyme cyclization targets. Approaches to evaluate the cyclization reaction and its effect on enzyme resilience are described. We also highlight the use of differential scanning calorimetry to understand how SpyRing cyclization promotes enzyme refolding. Efficiently searching sequence space will continue to be important for enzyme improvement, but the SpyRing platform may be a valuable rational adjunct for conferring resilience. PMID:27586332

  4. Myxoma Virus Encodes an α2,3-Sialyltransferase That Enhances Virulence

    PubMed Central

    Jackson, Ronald J.; Hall, Diana F.; Kerr, Peter J.

    1999-01-01

    A 4.7-kb region of DNA sequence contained at the right end of the myxoma virus EcoRI-G2 fragment located 24 kb from the right end of the 163-kb genome has been determined. This region of the myxoma virus genome encodes homologs of the vaccinia virus genes A51R, A52R, A55R, A56R, and B1R; the myxoma virus gene equivalents have been given the prefix M. The MA55 gene encodes a protein belonging to the kelch family of actin-binding proteins, while the MA56 gene encodes a member of the immunoglobulin superfamily related to a variety of cellular receptors and adhesion molecules. A novel myxoma virus early gene, MST3N, is a member of the eukaryotic sialyltransferase gene family located between genes MA51 and MA52. Detergent lysates prepared from myxoma virus-infected cell cultures contained a virally encoded sialyltransferase activity that catalyzed the transfer of sialic acid (Sia) from CMP-Sia to an asialofetuin glycoprotein acceptor. Analysis of the in vitro-sialylated glycoprotein acceptor by digestion with N-glycosidase F and by lectin binding suggested that the MST3N gene encodes an enzyme with Galβ1,3(4)GlcNAc α2,3-sialyltransferase specificity for the N-linked oligosaccharide of glycoprotein. Lectin binding assays demonstrated that α2,3-sialyltransferase activity is expressed by several known leporipoxviruses that naturally infect Sylvilagus rabbits. The sialyltransferase is nonessential for myxoma virus replication in cell culture; however, disruption of the MST3N gene caused attenuation in vivo. The possible implications of the myxoma virus-expressed sialyltransferase in terms of the host’s defenses against infection are discussed. PMID:9971821

  5. R-plasmid-encoded adhesive factor in Klebsiella pneumoniae strains responsible for human nosocomial infections.

    PubMed Central

    Darfeuille-Michaud, A; Jallat, C; Aubel, D; Sirot, D; Rich, C; Sirot, J; Joly, B

    1992-01-01

    Klebsiella pneumoniae strains involved in hospital outbreaks of nosocomial infections, such as suppurative lesions, bacteremia, and septicemia, were resistant to multiple antibiotics including broad-spectrum cephalosporins. Epidemiologic investigations revealed that the reservoir for these K. pneumoniae strains was the gastrointestinal tracts of the patients. The study of the adherence ability of the strains reported here showed that these bacteria adhered to the microvilli of the Caco-2 cell line. This adhesion was mediated by a nonfimbrial protein with a molecular mass of 29,000 Da designated CF29K. Pretreatment of bacteria with antibodies raised against CF29K or Caco-2 cells with purified CF29K prevented the adhesion of K. pneumoniae strains to Caco-2 cells. CF29K immunologically cross-reacted with the CS31A surface protein of Escherichia coli strains involved in septicemia in calves. Genes encoding CF29K were located on a high-molecular-weight conjugative R plasmid, which transferred to E. coli K-12. Transconjugants expressed a large amount of CF29K protein and adhered to the brush border of Caco-2 cells. These findings show that K. pneumoniae strains were able to colonize the human intestinal tract through a plasmid-encoded 29,000-Da surface protein. Hybridization experiments indicated that the gene encoding resistance to broad-spectrum cephalosporins by the production of CAZ-1 enzyme and the gene encoding the adhesive property to intestinal cells were both located on a 20- to 22-kb EcoRI restriction DNA fragment. Genes encoding aerobactin and the ferric aerobactin receptor were also found on this R plasmid. Images PMID:1345909

  6. Conservation of the prohormone convertase gene family in metazoa: analysis of cDNAs encoding a PC3-like protein from hydra.

    PubMed Central

    Chan, S J; Oliva, A A; LaMendola, J; Grens, A; Bode, H; Steiner, D F

    1992-01-01

    A subclass of proteolytic enzymes that correctly cleave precursor proteins at paired basic residues and are structurally related to the bacterial subtilisins has recently been identified. In yeast, a single membrane-bound proteolytic processing enzyme encoded by the kex2 gene has been found, whereas in higher vertebrates cDNAs encoding four distinct enzymes (PC2, PC3, furin, and PACE 4) have been identified. Like kex2, furin (also known as PACE) contains a hydrophobic transmembrane domain, but PC2, PC3, and PACE 4 lack this feature. All five enzymes exhibit striking similarities in their catalytic domains, and this suggests that they have arisen from a common ancestral subtilisin-like gene. We report here the identification of cDNAs encoding a protein that is similar in structure to PC3 from a simple metazoan, Hydra vulgaris (formerly Hydra attenuata). cDNAs encoding two isoforms of this PC3-like enzyme were obtained that differ only in their carboxyl-terminal sequences, probably due to alternative splicing of a common pre-mRNA. Neither form contains a transmembrane domain. Predicted amino acid sequence comparisons revealed that the hydra PC3-like enzyme is 55.4% and 56.7% identical in the catalytic domain to mouse PC3 and human furin, respectively. RNA blot analyses revealed that the PC3-like RNA is expressed predominantly in the hydra body column and not in the head region, although the hydra head contains a high density of nerve cells, which synthesize a variety of neuropeptides. For this reason, we suspect that another proprotein cleavage enzyme isoform may be expressed in head nerve cells. The isolation of a PC3-like cDNA from hydra is consistent with the presence of neuroendocrine cells and indicates that the PC/furin gene family has been well conserved in all metazoa. A simplified nomenclature for the group of mammalian processing proteases is proposed. Images PMID:1495957

  7. The rnhB gene encoding RNase HII of Streptococcus pneumoniae and evidence of conserved motifs in eucaryotic genes.

    PubMed Central

    Zhang, Y B; Ayalew, S; Lacks, S A

    1997-01-01

    A single RNase H enzyme was detected in extracts of Streptococcus pneumoniae. The gene encoding this enzyme was cloned and expressed in Escherichia coli, as demonstrated by its ability to complement a double-mutant rnhA recC strain. Sequence analysis of the cloned DNA revealed an open reading frame of 290 codons that encodes a polypeptide of 31.9 kDa. The predicted protein exhibits a low level of homology (19% identity of amino acid residues) to RNase HII encoded by rnhB of E. coli. Identification of the S. pneumoniae RNase HII translation start site by amino-terminal sequencing of the protein and of mRNA start sites by primer extension with reverse transcriptase showed that the major transcript encoding rnhB begins at the protein start site. Comparison of the S. pneumoniae and E. coli RNase HII sequences and sequences of other, putative bacterial rnhB gene products surmised from sequencing data revealed three conserved motifs. Use of these motifs to search for homologous genes in eucaryotes demonstrated the presence of rnhB genes in a yeast and a roundworm. Partial rnhB gene sequences were detected among expressed sequences of mouse and human cells. From these data, it appears that RNase HII is universally present in living cells. PMID:9190796

  8. The genome of the mustard leaf beetle encodes two active xylanases originally acquired from bacteria through horizontal gene transfer

    PubMed Central

    Pauchet, Yannick; Heckel, David G.

    2013-01-01

    The primary plant cell wall comprises the most abundant polysaccharides on the Earth and represents a rich source of energy for organisms which have evolved the ability to digest them. Enzymes able to degrade plant cell wall polysaccharides are widely distributed in micro-organisms but are generally absent in animals, although their presence in insects, especially phytophagous beetles from the superfamilies Chrysomeloidea and Curculionoidea, has recently begun to be appreciated. The observed patchy distribution of endogenous genes encoding these enzymes in animals has raised questions about their evolutionary origins. Recent evidence suggests that endogenous plant cell wall degrading enzymes-encoding genes have been acquired by animals through a mechanism known as horizontal gene transfer (HGT). HGT describes how genetic material is moved by means other than vertical inheritance from a parent to an offspring. Here, we provide evidence that the mustard leaf beetle, Phaedon cochleariae, possesses in its genome genes encoding active xylanases from the glycoside hydrolase family 11 (GH11). We also provide evidence that these genes were originally acquired by P. cochleariae from a species of gammaproteobacteria through HGT. This represents the first example of the presence of genes from the GH11 family in animals. PMID:23698014

  9. Targeting enzymes for cancer therapy: old enzymes in new roles.

    PubMed Central

    Deonarain, M. P.; Epenetos, A. A.

    1994-01-01

    Enzymes which traditionally have played no role in cell-directed cytotoxicity are finding their way into schemes for prodrug activation and immunotoxins owing to such useful enzymatic activity. Alkaline phosphatase, carboxypeptidases, beta-glucosidases and beta-lactamases among many others are being utilised to regenerate potent anti-cancer drugs or toxic small molecules from precursors in a bid to enhance their activity in tumours. These prodrug activation systems require the pretargeting of the enzyme to the surface of a tumour cell, usually by an antibody or its immunoreactive fragment. A recent novel approach proposes the intracellular delivery of appropriate enzymes, such as phosphodiesterases, to particular cellular compartments. There, enzyme activity can cause substantive damage resulting in cell death. Cell targeting of mammalian phosphodiesterase promises to improve upon conventional immunotoxins because of their increased cytotoxicity when targeted to the appropriate compartment and their expected lack of, or lower, immunogenicity in clinical use. PMID:7947082

  10. Architecture for VLSI design of Reed-Solomon encoders

    NASA Technical Reports Server (NTRS)

    Liu, K. Y.

    1982-01-01

    A description is given of the logic structure of the universal VLSI symbol-slice Reed-Solomon (RS) encoder chip, from a group of which an RS encoder may be constructed through cascading and proper interconnection. As a design example, it is shown that an RS encoder presently requiring approximately 40 discrete CMOS ICs may be replaced by an RS encoder consisting of four identical, interconnected VLSI RS encoder chips, offering in addition to greater compactness both a lower power requirement and greater reliability.

  11. Negative base encoding in optical linear algebra processors

    NASA Technical Reports Server (NTRS)

    Perlee, C.; Casasent, D.

    1986-01-01

    In the digital multiplication by analog convolution algorithm, the bits of two encoded numbers are convolved to form the product of the two numbers in mixed binary representation; this output can be easily converted to binary. Attention is presently given to negative base encoding, treating base -2 initially, and then showing that the negative base system can be readily extended to any radix. In general, negative base encoding in optical linear algebra processors represents a more efficient technique than either sign magnitude or 2's complement encoding, when the additions of digitally encoded products are performed in parallel.

  12. Characterization of cDNAs encoding serine proteases and their transcriptional responses to Cry1Ab protoxin in the gut of Ostrinia nubilalis larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serine proteases, such as trypsin and chymotrypsin, are the primary digestive enzymes in lepidopteran larvae, and are also involved in Bacillus thuringiensis (Bt) protoxin activation and protoxin/toxin degradation. We isolated and sequenced 34 cDNAs putatively encoding trypsins, chymotrypsins and th...

  13. Metabolic regulation via enzyme filamentation

    PubMed Central

    Aughey, Gabriel N.; Liu, Ji-Long

    2016-01-01

    Abstract Determining the mechanisms of enzymatic regulation is central to the study of cellular metabolism. Regulation of enzyme activity via polymerization-mediated strategies has been shown to be widespread, and plays a vital role in mediating cellular homeostasis. In this review, we begin with an overview of the filamentation of CTP synthase, which forms filamentous structures termed cytoophidia. We then highlight other important examples of the phenomenon. Moreover, we discuss recent data relating to the regulation of enzyme activity by compartmentalization into cytoophidia. Finally, we hypothesize potential roles for enzyme filament formation in the regulation of metabolism, development and disease. PMID:27098510

  14. Odor recognition memory: two encoding trials are better than one.

    PubMed

    Nguyen, Lauren A; Ober, Beth A; Shenaut, Gregory K

    2012-10-01

    The primary purpose of this study was to determine the effect of one versus two encoding trials in the classical yes/no recognition memory paradigm using olfactory stimuli. A group of 24 young adults rated 18 standard microencapsulated odorant targets for familiarity (first encoding block) or pleasantness (second encoding block). Once-encoded targets were in only one block and twice-encoded targets were in both, with items counterbalanced across participants. Participants performed a 20-min nonverbal distractor task followed by a yes/no recognition test incorporating 18 additional odors as foils. Memory performance for twice-encoded targets was superior to that for once-encoded targets. For once-encoded targets, performance did not differ between those rated for familiarity versus those rated for pleasantness. Less pleasant odors produced overall better recognition, with a tendency for less familiar odors to produce overall better recognition. There was a tendency for the second encoding trial to have a larger effect for less pleasant or familiar odors than for more pleasant or familiar odors. The main conclusion is that recognition memory for odors is better for items encoded two times than for items encoded only once. Implications of these findings and suggestions for future research are discussed. PMID:22843762

  15. Adenylating Enzymes in Mycobacterium tuberculosis as Drug Targets

    PubMed Central

    Duckworth, Benjamin P.; Nelson, Kathryn M.; Aldrich, Courtney C.

    2013-01-01

    Adenylation or adenylate-forming enzymes (AEs) are widely found in nature and are responsible for the activation of carboxylic acids to intermediate acyladenylates, which are mixed anhydrides of AMP. In a second reaction, AEs catalyze the transfer of the acyl group of the acyladenylate onto a nucleophilic amino, alcohol, or thiol group of an acceptor molecule leading to amide, ester, and thioester products, respectively. Mycobacterium tuberculosis encodes for more than 60 adenylating enzymes, many of which represent potential drug targets due to their confirmed essentiality or requirement for virulence. Several strategies have been used to develop potent and selective AE inhibitors including high-throughput screening, fragment-based screening, and the rationale design of bisubstrate inhibitors that mimic the acyladenylate. In this review, a comprehensive analysis of the mycobacterial adenylating enzymes will be presented with a focus on the identification of small molecule inhibitors. Specifically, this review will cover the aminoacyl tRNA-synthetases (aaRSs), MenE required for menaquinone synthesis, the FadD family of enzymes including the fatty acyl-AMP ligases (FAAL) and the fatty acyl-CoA ligases (FACLs) involved in lipid metabolism, and the nonribosomal peptide synthetase adenylation enzyme MbtA that is necessary for mycobactin synthesis. Additionally, the enzymes NadE, GuaA, PanC, and MshC involved in the respective synthesis of NAD, guanine, pantothenate, and mycothiol will be discussed as well as BirA that is responsible for biotinylation of the acyl CoA-carboxylases. PMID:22283817

  16. When two is too many: Collaborative encoding impairs memory.

    PubMed

    Barber, Sarah J; Rajaram, Suparna; Aron, Arthur

    2010-04-01

    Humans routinely encode and retrieve experiences in interactive, collaborative contexts. Yet much of what we know about human memory comes from research on individuals working in isolation. Some recent research has examined collaboration during retrieval, but not much is known about how collaboration during encoding affects memory. We examined this issue. Participants created episodes by elaborating on study materials alone or collaboratively, and they later performed a cued-recall task alone, with the study partner, or with a different partner (Experiment 1). Collaborative encoding impaired recall. This counterintuitive outcome was found for both individual and group recall, even when the same partners collaborated across encoding and retrieval. This impairment was significantly reduced, but persisted, when the encoding instructions encouraged free-flowing collaboration (Experiment 2). Thus, the collaborative-encoding deficit is robust in nature and likely occurs because collaborative encoding produces less effective cues for later retrieval. PMID:20234016

  17. A Multi-Encoding Approach for LTL Symbolic Satisfiability Checking

    NASA Technical Reports Server (NTRS)

    Rozier, Kristin Y.; Vardi, Moshe Y.

    2011-01-01

    Formal behavioral specifications written early in the system-design process and communicated across all design phases have been shown to increase the efficiency, consistency, and quality of the system under development. To prevent introducing design or verification errors, it is crucial to test specifications for satisfiability. Our focus here is on specifications expressed in linear temporal logic (LTL). We introduce a novel encoding of symbolic transition-based Buchi automata and a novel, "sloppy," transition encoding, both of which result in improved scalability. We also define novel BDD variable orders based on tree decomposition of formula parse trees. We describe and extensively test a new multi-encoding approach utilizing these novel encoding techniques to create 30 encoding variations. We show that our novel encodings translate to significant, sometimes exponential, improvement over the current standard encoding for symbolic LTL satisfiability checking.

  18. Molecular duplexes with encoded sequences and stabilities.

    PubMed

    Gong, Bing

    2012-12-18

    Through specific molecular shapes and repeating polymeric sequences, biomacromolecules encode information about both structure and function. Inspired by DNA molecules, we have conceived a strategy to encode linear molecular strands with sequences that specify intermolecular association, and we and our collaborators have supported this idea through our experimental work. This Account summarizes the design and development of a class of molecular duplexes with programmable hydrogen-bonding sequences and adjustable stabilities. The specific system involves oligoamide strands synthesized from readily available monomeric modules based on standard amide (peptide) chemistry. By covalently linking three types of basic building blocks in different orders, we create oligoamide strands with various arrangements of amide O and H atoms that provide arrays of hydrogen bonding sequences. Because one of the two edges of these molecules presents the sequences of hydrogen-bond donors and acceptors, these oligoamide strands associate via their hydrogen-bonding edges into double-stranded pairs or duplexes. Systematic studies have demonstrated the strict sequence specificity and tunable stability of this system. These structurally simple duplexes exhibit many features associated with DNA sequences such as programmable sequence specificity, shape and hydrogen-bonding complementarity, and cooperativity of multipoint interactions. Capable of specifying intermolecular associations, these duplexes have formed supramolecular structures such as β-sheets and non-covalent block copolymers and have templated chemical reactions. The incorporation of dynamic covalent interactions into these H-bonded duplexes has created association units that undergo sequence-specific association and covalent ligation in both nonpolar solvents and polar media including water. These new association units may facilitate the development of new dynamic covalent structures, and new properties are emerging from these

  19. Isolation and Characterization of Anaerobic Ethylbenzene Dehydrogenase, a Novel Mo-Fe-S Enzyme

    PubMed Central

    Johnson, Hope A.; Pelletier, Dale A.; Spormann, Alfred M.

    2001-01-01

    The first step in anaerobic ethylbenzene mineralization in denitrifying Azoarcus sp. strain EB1 is the oxidation of ethylbenzene to (S)-(−)-1-phenylethanol. Ethylbenzene dehydrogenase, which catalyzes this reaction, is a unique enzyme in that it mediates the stereoselective hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen. We purified ethylbenzene dehydrogenase to apparent homogeneity and showed that the enzyme is a heterotrimer (αβγ) with subunit masses of 100 kDa (α), 35 kDa (β), and 25 kDa (γ). Purified ethylbenzene dehydrogenase contains approximately 0.5 mol of molybdenum, 16 mol of iron, and 15 mol of acid-labile sulfur per mol of holoenzyme, as well as a molydopterin cofactor. In addition to ethylbenzene, purified ethylbenzene dehydrogenase was found to oxidize 4-fluoro-ethylbenzene and the nonaromatic hydrocarbons 3-methyl-2-pentene and ethylidenecyclohexane. Sequencing of the encoding genes revealed that ebdA encodes the α subunit, a 974-amino-acid polypeptide containing a molybdopterin-binding domain. The ebdB gene encodes the β subunit, a 352-amino-acid polypeptide with several 4Fe-4S binding domains. The ebdC gene encodes the γ subunit, a 214-amino-acid polypeptide that is a potential membrane anchor subunit. Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes. PMID:11443088

  20. Molybdenum enzymes in higher organisms

    PubMed Central

    Hille, Russ; Nishino, Takeshi; Bittner, Florian

    2010-01-01

    Recent progress in our understanding of the structural and catalytic properties of molybdenum-containing enzymes in eukaryotes is reviewed, along with aspects of the biosynthesis of the cofactor and its insertion into apoprotein. PMID:21516203