RESUMO
This study examined whether the neighborhood built environment moderated gestational weight gain (GWG) in LIFE-Moms clinical trials. Participants were 790 pregnant women (13.9 weeks' gestation) with overweight or obesity randomized within four clinical centers to standard care or lifestyle intervention to reduce GWG. Geographic information system (GIS) was used to map the neighborhood built environment. The intervention relative to standard care significantly reduced GWG (coefficient = 0.05; p = 0.005) and this effect remained significant (p < 0.03) after adjusting for built environment variables. An interaction was observed for presence of fast food restaurants (coefficient = -0.007; p = 0.003). Post hoc tests based on a median split showed that the intervention relative to standard care reduced GWG in participants living in neighborhoods with lower fast food density 0.08 [95% CI, 0.03,0.12] kg/week (p = 0.001) but not in those living in areas with higher fast food density (0.02 [-0.04, 0.08] kg/week; p = 0.55). Interaction effects suggested less intervention efficacy among women living in neighborhoods with more grocery/convenience stores (coefficient = -0.005; p = 0.0001), more walkability (coefficient -0.012; p = 0.007) and less crime (coefficient = 0.001; p = 0.007), but post-hoc tests were not significant. No intervention x environment interaction effects were observed for total number of eating establishments or tree canopy. Lifestyle interventions during pregnancy were effective across diverse physical environments. Living in environments with easy access to fast food restaurants may limit efficacy of prenatal lifestyle interventions, but future research is needed to replicate these findings.
Assuntos
Ambiente Construído/estatística & dados numéricos , Ganho de Peso na Gestação/fisiologia , Estilo de Vida , Complicações na Gravidez/epidemiologia , Adulto , Feminino , Humanos , Obesidade/epidemiologia , Sobrepeso/epidemiologia , Gravidez , Características de Residência , Caminhada/estatística & dados numéricosRESUMO
Sugar alcohols (polyols) exist widely in nature. While some specific sugar alcohol phosphatases are known, there is no known phosphatase for some important sugar alcohols (e.g., sorbitol-6-phosphate). Using liquid chromatography-mass spectrometry-based metabolomics, we screened yeast strains with putative phosphatases of unknown function deleted. We show that the yeast gene YNL010W, which has close homologues in all fungi species and some plants, encodes a sugar alcohol phosphatase. We term this enzyme, which hydrolyzes sorbitol-6-phosphate, ribitol-5-phosphate, and (d)-glycerol-3-phosphate, polyol phosphatase 1 or PYP1. Polyol phosphates are structural analogs of the enediol intermediate of phosphoglucose isomerase (Pgi). We find that sorbitol-6-phosphate and ribitol-5-phosphate inhibit Pgi and that Pyp1 activity is important for yeast to maintain Pgi activity in the presence of environmental sugar alcohols. Pyp1 expression is strongly positively correlated with yeast growth rate, presumably because faster growth requires greater glycolytic and accordingly Pgi flux. Thus, yeast express the previously uncharacterized enzyme Pyp1 to prevent inhibition of glycolysis by sugar alcohol phosphates. Pyp1 may be useful for engineering sugar alcohol production.
Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fosfatos Açúcares/metabolismo , Deleção de Genes , Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Hidrólise , Monoéster Fosfórico Hidrolases/genética , Proteínas de Saccharomyces cerevisiae/genética , Fosfatos Açúcares/químicaRESUMO
Carboxylic acid reductases (CARs) selectively reduce carboxylic acids to aldehydes using ATP and NADPH as cofactors under mild conditions. Although CARs attracts significant interest, only a few enzymes have been characterized to date, whereas the vast majority of CARs have yet to be examined. Herein the authors report that 12 bacterial CARs reduces a broad range of bifunctional carboxylic acids containing oxo-, hydroxy-, amino-, or second carboxyl groups with several enzymes showing activity toward 4-hydroxybutanoic (4-HB) and adipic acids. These CARs exhibits significant reductase activity against substrates whose second functional group is separated from the carboxylate by at least three carbons with both carboxylate groups being reduced in dicarboxylic acids. Purified CARs supplemented with cofactor regenerating systems (for ATP and NADPH), an inorganic pyrophosphatase, and an aldo-keto reductase catalyzes a high conversion (50-76%) of 4-HB to 1,4-butanediol (1,4-BDO) and adipic acid to 1,6-hexanediol (1,6-HDO). Likewise, Escherichia coli strains expressing eight different CARs efficiently reduces 4-HB to 1,4-BDO with 50-95% conversion, whereas adipic acid is reduced to a mixture of 6-hydroxyhexanoic acid (6-HHA) and 1,6-HDO. Thus, our results illustrate the broad biochemical diversity of bacterial CARs and their compatibility with other enzymes for applications in biocatalysis.
Assuntos
Proteínas de Bactérias , Engenharia Metabólica/métodos , Oxirredutases , Trifosfato de Adenosina/metabolismo , Adipatos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácidos Carboxílicos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroxibutiratos , NADP/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismoRESUMO
OBJECTIVES: To assess the effect of iron chelation therapy with deferasirox on cardiac iron and function in patients with transfusion-dependent thalassemia major, sickle cell disease (SCD), and myelodysplastic syndromes (MDS). METHODS: This phase IV, single-arm, open-label study over 53 wk evaluated the change in cardiac and liver iron load with deferasirox (up to 40 mg/kg/d), measured by magnetic resonance imaging (MRI). RESULTS: Cardiac iron load (myocardial T2*) significantly improved (P = 0.002) overall (n = 46; n = 36 thalassemia major, n = 4 SCD, n = 6 MDS). Results were significant for patients with normal and moderate baseline cardiac iron (P = 0.017 and P = 0.015, respectively), but not in the five patients with severe cardiac iron load. Liver iron concentration (LIC) significantly decreased overall [mean LIC 10.4 to 8.2 mg Fe/g dry tissue (dw); P = 0.024], particularly in those with baseline LIC >7 mg Fe/g dw (19.9 to 15.6 mg Fe/g dw; P = 0.002). Furthermore, myocardial T2* significantly increased in patients with LIC <7 mg Fe/g dw, but not in those with a higher LIC. Safety was consistent with previous reports. CONCLUSIONS: Once-daily deferasirox over 1 yr significantly increased myocardial T2* and reduced LIC. This confirms that single-agent deferasirox is effective in the management of cardiac iron, especially for patients with myocardial T2* >10 ms (Clinicaltrials.gov identifier: NCT00673608).
Assuntos
Benzoatos/uso terapêutico , Transfusão de Sangue , Terapia por Quelação , Quelantes de Ferro/uso terapêutico , Sobrecarga de Ferro/tratamento farmacológico , Ferro/metabolismo , Miocárdio/metabolismo , Triazóis/uso terapêutico , Adolescente , Adulto , Idoso , Benzoatos/farmacologia , Deferasirox , Feminino , Ferritinas/sangue , Testes de Função Cardíaca , Hemoglobinopatias/complicações , Hemoglobinopatias/terapia , Humanos , Quelantes de Ferro/farmacologia , Sobrecarga de Ferro/diagnóstico , Sobrecarga de Ferro/etiologia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Reação Transfusional , Resultado do Tratamento , Triazóis/farmacologia , Adulto JovemRESUMO
S-adenosylmethionine (SAM)-dependent methyltransferases regulate a wide range of biological processes through the modification of proteins, nucleic acids, polysaccharides, as well as various metabolites. TYW3/Taw3 is a SAM-dependent methyltransferase responsible for the formation of a tRNA modification known as wybutosine and its derivatives that are required for accurate decoding in protein synthesis. Here, we report the crystal structure of Taw3, a homolog of TYW3 from Sulfolobus solfataricus, which revealed a novel α/ß fold. The sequence motif (S/T)xSSCxGR and invariant aspartate and histidine, conserved in TYW3/Taw3, cluster to form the catalytic center. These structural and sequence features indicate that TYW3/Taw3 proteins constitute a distinct class of SAM-dependent methyltransferases. Using site-directed mutagenesis along with in vivo complementation assays combined with mass spectrometry as well as ligand docking and cofactor binding assays, we have identified the active site of TYW3 and residues essential for cofactor binding and methyltransferase activity.
Assuntos
Proteínas Arqueais/química , Metiltransferases/química , Nucleosídeos/química , S-Adenosilmetionina/química , Sulfolobus solfataricus/química , Motivos de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Metiltransferases/genética , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Nucleosídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Sulfolobus solfataricus/enzimologiaRESUMO
OBJECTIVE: To compare the efficacy and adverse effects of using bronchial blockers (BBs) and double-lumen endobronchial tubes (DLTs). DESIGN: Systematic review and meta-analysis of randomized controlled trials (RCTs) comparing BBs and DLTs. SETTING: Hospital units undertaking thoracic surgery PARTICIPANTS: Patients undergoing thoracic surgery requiring lung isolation. INTERVENTIONS: BBs and DLTs. MEASUREMENTS AND MAIN RESULTS: A systematic literature search was conducted for RCTs comparing BBs and DLTs using Google Scholar, Ovid Medline, and Cochrane library databases up to October 2013. Inclusion criteria were RCTs comparing BBs and DLTs, intubation carried out by qualified anesthesiologists or trainee specialists, outcome measures relating to either efficacy or adverse effects. Studies that were inaccessible in English were excluded. Mantel-Haenszel fixed-effect meta-analysis of recurring outcome measures was performed using RevMan 5 software. The search produced 39 RCTs published between 1996 and 2013. DLTs were quicker to place (mean difference: 51 seconds, 95% confidence intervals [CI] 8-94 seconds; p = 0.02) and less likely to be incorrectly positioned (odds ratio [OR] 2.70; 95% CI 1.18-6.18, p = 0.02) than BBs. BBs were associated with fewer patients having a postoperative sore throat (OR 0.39, 95% CI: 0.23-0.68, p = 0.0009), less hoarseness (OR: 0.43,95%, CI 0.24-0.75, p = 0.003), and fewer airway injuries (OR 0.40, 95% CI 0.21-0.75, p = 0.005) than DLTs. CONCLUSION: While BBs are associated with a lower incidence of airway injury and a lower severity of injury, DLTs can be placed quicker and more reliably.
Assuntos
Intubação Intratraqueal/instrumentação , Intubação Intratraqueal/métodos , Ensaios Clínicos Controlados Aleatórios como Assunto/métodos , Procedimentos Cirúrgicos Torácicos/instrumentação , Procedimentos Cirúrgicos Torácicos/métodos , Rouquidão/diagnóstico , Rouquidão/etiologia , Humanos , Intubação Intratraqueal/efeitos adversos , Faringite/diagnóstico , Faringite/etiologia , Procedimentos Cirúrgicos Torácicos/efeitos adversos , Resultado do TratamentoRESUMO
Inhibition of enzyme activity by high concentrations of substrate and/or cofactor is a general phenomenon demonstrated in many enzymes, including aldehyde dehydrogenases. Here we show that the uncharacterized protein BetB (SA2613) from Staphylococcus aureus is a highly specific betaine aldehyde dehydrogenase, which exhibits substrate inhibition at concentrations of betaine aldehyde as low as 0.15 mM. In contrast, the aldehyde dehydrogenase YdcW from Escherichia coli, which is also active against betaine aldehyde, shows no inhibition by this substrate. Using the crystal structures of BetB and YdcW, we performed a structure-based mutational analysis of BetB and introduced the YdcW residues into the BetB active site. From a total of 32 mutations, those in five residues located in the substrate binding pocket (Val288, Ser290, His448, Tyr450, and Trp456) greatly reduced the substrate inhibition of BetB, whereas the double mutant protein H448F/Y450L demonstrated a complete loss of substrate inhibition. Substrate inhibition was also reduced by mutations of the semiconserved Gly234 (to Ser, Thr, or Ala) located in the BetB NAD(+) binding site, suggesting some cooperativity between the cofactor and substrate binding sites. Substrate docking analysis of the BetB and YdcW active sites revealed that the wild-type BetB can bind betaine aldehyde in both productive and nonproductive conformations, whereas only the productive binding mode can be modeled in the active sites of YdcW and the BetB mutant proteins with reduced substrate inhibition. Thus, our results suggest that the molecular mechanism of substrate inhibition of BetB is associated with the nonproductive binding of betaine aldehyde.
Assuntos
Betaína-Aldeído Desidrogenase/genética , Betaína-Aldeído Desidrogenase/metabolismo , Betaína/análogos & derivados , Inibidores Enzimáticos/metabolismo , Staphylococcus aureus/enzimologia , Substituição de Aminoácidos , Betaína/metabolismo , Betaína-Aldeído Desidrogenase/antagonistas & inibidores , Betaína-Aldeído Desidrogenase/química , Sítios de Ligação , Análise Mutacional de DNA , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação ProteicaAssuntos
Comportamento do Adolescente , Comportamentos Relacionados com a Saúde , Conhecimentos, Atitudes e Prática em Saúde , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/administração & dosagem , Neoplasias do Colo do Útero/prevenção & controle , Vacinação/estatística & dados numéricos , Feminino , HumanosRESUMO
Maf (for multicopy associated filamentation) proteins represent a large family of conserved proteins implicated in cell division arrest but whose biochemical activity remains unknown. Here, we show that the prokaryotic and eukaryotic Maf proteins exhibit nucleotide pyrophosphatase activity against 5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, and 7-methyl-GTP, which represent the most abundant modified bases in all organisms, as well as against canonical nucleotides dTTP, UTP, and CTP. Overexpression of the Maf protein YhdE in E. coli cells increased intracellular levels of dTMP and UMP, confirming that dTTP and UTP are the in vivo substrates of this protein. Crystal structures and site-directed mutagenesis of Maf proteins revealed the determinants of their activity and substrate specificity. Thus, pyrophosphatase activity of Maf proteins toward canonical and modified nucleotides might provide the molecular mechanism for a dual role of these proteins in cell division arrest and house cleaning.
Assuntos
Proteínas de Escherichia coli/metabolismo , Fatores de Transcrição Maf/química , Fatores de Transcrição Maf/metabolismo , Nucleotídeos/química , Nucleotídeos/metabolismo , Pirofosfatases/metabolismo , Bacillus subtilis/enzimologia , Sequência Conservada , Cristalografia por Raios X , Escherichia coli/enzimologia , Humanos , Fatores de Transcrição Maf/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Saccharomyces cerevisiae/enzimologia , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética , Especificidade por SubstratoAssuntos
Artroscopia/métodos , Cartilagem Articular/cirurgia , Medicina Baseada em Evidências/métodos , Ortopedia/normas , Osteoartrite do Joelho/cirurgia , Guias de Prática Clínica como Assunto , Sociedades Médicas , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/diagnóstico , Estados UnidosRESUMO
Nucleotide degradation is a universal metabolic capability. Here we combine metabolomics, genetics and biochemistry to characterize the yeast pathway. Nutrient starvation, via PKA, AMPK/SNF1, and TOR, triggers autophagic breakdown of ribosomes into nucleotides. A protein not previously associated with nucleotide degradation, Phm8, converts nucleotide monophosphates into nucleosides. Downstream steps, which involve the purine nucleoside phosphorylase, Pnp1, and pyrimidine nucleoside hydrolase, Urh1, funnel ribose into the nonoxidative pentose phosphate pathway. During carbon starvation, the ribose-derived carbon accumulates as sedoheptulose-7-phosphate, whose consumption by transaldolase is impaired due to depletion of transaldolase's other substrate, glyceraldehyde-3-phosphate. Oxidative stress increases glyceraldehyde-3-phosphate, resulting in rapid consumption of sedoheptulose-7-phosphate to make NADPH for antioxidant defense. Ablation of Phm8 or double deletion of Pnp1 and Urh1 prevent effective nucleotide salvage, resulting in metabolite depletion and impaired survival of starving yeast. Thus, ribose salvage provides means of surviving nutrient starvation and oxidative stress.
Assuntos
Regulação Fúngica da Expressão Gênica , N-Glicosil Hidrolases/genética , Nucleotídeos/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Ribose/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Gliceraldeído 3-Fosfato/metabolismo , N-Glicosil Hidrolases/deficiência , NADP/metabolismo , Via de Pentose Fosfato/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Purina-Núcleosídeo Fosforilase/deficiência , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Estresse Fisiológico/genética , Fosfatos Açúcares , Transaldolase/genética , Transaldolase/metabolismoRESUMO
The uncharacterized α/ß-hydrolase protein OLEI01171 from the psychrophilic marine bacterium Oleispira antarctica belongs to the PF00756 family of putative esterases, which also includes human esterase D. In the present paper we show that purified recombinant OLEI01171 exhibits high esterase activity against the model esterase substrate α-naphthyl acetate at 5-30°C with maximal activity at 15-20°C. The esterase activity of OLEI01171 was stimulated 3-8-fold by the addition of chloride or several other anions (0.1-1.0 M). Compared with mesophilic PF00756 esterases, OLEI01171 exhibited a lower overall protein thermostability. Two crystal structures of OLEI01171 were solved at 1.75 and 2.1 Å resolution and revealed a classical serine hydrolase catalytic triad and the presence of a chloride or bromide ion bound in the active site close to the catalytic Ser148. Both anions were found to co-ordinate a potential catalytic water molecule located in the vicinity of the catalytic triad His257. The results of the present study suggest that the bound anion perhaps contributes to the polarization of the catalytic water molecule and increases the rate of the hydrolysis of an acyl-enzyme intermediate. Alanine replacement mutagenesis of OLEI01171 identified ten amino acid residues important for esterase activity. The replacement of Asn225 by lysine had no significant effect on the activity or thermostability of OLEI01171, but resulted in a detectable increase of activity at 35-45°C. The present study has provided insight into the molecular mechanisms of activity of a cold-active and anion-activated carboxyl esterase.
Assuntos
Ânions/metabolismo , Carboxilesterase/química , Carboxilesterase/metabolismo , Oceanospirillaceae/enzimologia , Óleos/metabolismo , Sequência de Aminoácidos , Regiões Antárticas , Carboxilesterase/genética , Catálise , Domínio Catalítico , Cristalografia por Raios X , Hidrólise , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Conformação Proteica , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , TemperaturaRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas proteins represent an adaptive microbial immunity system against viruses and plasmids. Cas3 proteins have been proposed to play a key role in the CRISPR mechanism through the direct cleavage of invasive DNA. Here, we show that the Cas3 HD domain protein MJ0384 from Methanocaldococcus jannaschii cleaves endonucleolytically and exonucleolytically (3'-5') single-stranded DNAs and RNAs, as well as 3'-flaps, splayed arms, and R-loops. The degradation of branched DNA substrates by MJ0384 is stimulated by the Cas3 helicase MJ0383 and ATP. The crystal structure of MJ0384 revealed the active site with two bound metal cations and together with site-directed mutagenesis suggested a catalytic mechanism. Our studies suggest that the Cas3 HD nucleases working together with the Cas3 helicases can completely degrade invasive DNAs through the combination of endo- and exonuclease activities.
Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , DNA Helicases/metabolismo , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo , Sequências Repetidas Invertidas , Methanococcales/enzimologia , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/genética , Bacteriófagos , Domínio Catalítico , Cristalografia por Raios X , DNA Viral/metabolismo , Desoxirribonucleases/genética , Methanococcales/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Plasmídeos , Conformação ProteicaRESUMO
Concentrations of endocrine disrupting chemicals and endocrine disruption in fish were determined in 11 lakes across Minnesota that represent a range of trophic conditions and land uses (urban, agricultural, residential, and forested) and in which wastewater treatment plant discharges were absent. Water, sediment, and passive polar organic integrative samplers (POCIS) were analyzed for steroidal hormones, alkylphenols, bisphenol A, and other organic and inorganic molecular tracers to evaluate potential non-point source inputs into the lakes. Resident fish from the lakes were collected, and caged male fathead minnows were deployed to evaluate endocrine disruption, as indicated by the biological endpoints of plasma vitellogenin and gonadal histology. Endocrine disrupting chemicals, including bisphenol A, 17ß-estradiol, estrone, and 4-nonylphenol were detected in 90% of the lakes at part per trillion concentrations. Endocrine disruption was observed in caged fathead minnows and resident fish in 90% of the lakes. The widespread but variable occurrence of anthropogenic chemicals in the lakes and endocrine disruption in fish indicates that potential sources are diverse, not limited to wastewater treatment plant discharges, and not entirely predictable based on trophic status and land use.
Assuntos
Disruptores Endócrinos/análise , Sistema Endócrino/efeitos dos fármacos , Monitoramento Ambiental , Água Doce/química , Poluentes Químicos da Água/análise , Animais , Compostos Benzidrílicos , Disruptores Endócrinos/toxicidade , Estradiol/análise , Estradiol/toxicidade , Estrona/análise , Estrona/toxicidade , Peixes/metabolismo , Sedimentos Geológicos/química , Minnesota , Fenóis/análise , Fenóis/toxicidade , Esteroides/análise , Esteroides/toxicidade , Vitelogeninas/metabolismo , Poluentes Químicos da Água/toxicidadeRESUMO
The use of aspirin for venous thromboembolism (VTE) prophylaxis after major orthopaedic surgery is controversial. The hypothesis of the present study is that aspirin will decrease the rate of operative site bleeding without increasing thromboembolic events when aspirin is used for VTE prophylaxis after major orthopaedic surgery. A pooled analysis of 14 randomized controlled trials (RCTs) cited by the American College of Chest Physicians (ACCP) guidelines was performed to determine pooled rates of symptomatic deep vein thromboses, pulmonary emboli (PE), fatal PE, and operative site bleeding rates. The VTE rates with aspirin were not significantly different than the rates for vitamin K antagonists (VKA), low molecular weight heparins (LMWH), and pentasaccharides. The operative site bleeding relative risks of VKA, LMWH, and pentasaccharides versus aspirin, are 4.9, 6.4, and 4.2, respectively. A pooled analysis of RCTs supports the use of aspirin for VTE prophylaxis after major orthopaedic surgery.
Assuntos
Anticoagulantes/uso terapêutico , Procedimentos Ortopédicos/métodos , Tromboembolia Venosa/prevenção & controle , Aspirina/uso terapêutico , Heparina de Baixo Peso Molecular/uso terapêutico , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto , Vitamina K/antagonistas & inibidoresRESUMO
Inorganic polyphosphate (polyP) is a linear polymer of tens or hundreds of phosphate residues linked by high-energy bonds. It is found in all organisms and has been proposed to serve as an energy source in a pre-ATP world. This ubiquitous and abundant biopolymer plays numerous and vital roles in metabolism and regulation in prokaryotes and eukaryotes, but the underlying molecular mechanisms for most activities of polyP remain unknown. In prokaryotes, the synthesis and utilization of polyP are catalyzed by 2 families of polyP kinases, PPK1 and PPK2, and polyphosphatases. Here, we present structural and functional characterization of the PPK2 family. Proteins with a single PPK2 domain catalyze polyP-dependent phosphorylation of ADP to ATP, whereas proteins containing 2 fused PPK2 domains phosphorylate AMP to ADP. Crystal structures of 2 representative proteins, SMc02148 from Sinorhizobium meliloti and PA3455 from Pseudomonas aeruginosa, revealed a 3-layer alpha/beta/alpha sandwich fold with an alpha-helical lid similar to the structures of microbial thymidylate kinases, suggesting that these proteins share a common evolutionary origin and catalytic mechanism. Alanine replacement mutagenesis identified 9 conserved residues, which are required for activity and include the residues from both Walker A and B motifs and the lid. Thus, the PPK2s represent a molecular mechanism, which potentially allow bacteria to use polyP as an intracellular energy reserve for the generation of ATP and survival.
Assuntos
Difosfato de Adenosina/biossíntese , Trifosfato de Adenosina/biossíntese , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Polifosfatos/metabolismo , Pseudomonas aeruginosa/enzimologia , Sinorhizobium meliloti/enzimologia , Alanina/genética , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Cristalografia por Raios X , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Fosfotransferases (Aceptor do Grupo Fosfato)/isolamento & purificação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
We have identified a novel family of proteins, in which the N-terminal cystathionine beta-synthase (CBS) domain is fused to the C-terminal Zn ribbon domain. Four proteins were overexpressed in Escherichia coli and purified: TA0289 from Thermoplasma acidophilum, TV1335 from Thermoplasma volcanium, PF1953 from Pyrococcus furiosus, and PH0267 from Pyrococcus horikoshii. The purified proteins had a red/purple color in solution and an absorption spectrum typical of rubredoxins (Rds). Metal analysis of purified proteins revealed the presence of several metals, with iron and zinc being the most abundant metals (2-67% of iron and 12-74% of zinc). Crystal structures of both mercury- and iron-bound TA0289 (1.5-2.0 A resolution) revealed a dimeric protein whose intersubunit contacts are formed exclusively by the alpha-helices of two cystathionine beta-synthase subdomains, whereas the C-terminal domain has a classical Zn ribbon planar architecture. All proteins were reversibly reduced by chemical reductants (ascorbate or dithionite) or by the general Rd reductase NorW from E. coli in the presence of NADH. Reduced TA0289 was found to be capable of transferring electrons to cytochrome C from horse heart. Likewise, the purified Zn ribbon protein KTI11 from Saccharomyces cerevisiae had a purple color in solution and an Rd-like absorption spectrum, contained both iron and zinc, and was reduced by the Rd reductase NorW from E. coli. Thus, recombinant Zn ribbon domains from archaea and yeast demonstrate an Rd-like electron carrier activity in vitro. We suggest that, in vivo, some Zn ribbon domains might also bind iron and therefore possess an electron carrier activity, adding another physiological role to this large family of important proteins.
Assuntos
Cistationina beta-Sintase/química , Cistationina beta-Sintase/metabolismo , Zinco/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Ácido Ascórbico/farmacologia , Cálcio/análise , Cálcio/química , Sequência Conservada , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/isolamento & purificação , Cisteína/química , Citocromos c/metabolismo , Dimerização , Ditionita/farmacologia , Escherichia coli/genética , Cavalos , Ferro/análise , Ferro/química , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Miocárdio/enzimologia , NAD/metabolismo , Oxirredução , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pyrococcus furiosus/química , Pyrococcus furiosus/isolamento & purificação , Pyrococcus furiosus/metabolismo , Pyrococcus horikoshii/química , Pyrococcus horikoshii/isolamento & purificação , Pyrococcus horikoshii/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rubredoxinas/análise , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Thermoplasma/química , Thermoplasma/isolamento & purificação , Thermoplasma/metabolismo , Zinco/análiseRESUMO
Based on the lead compound BX-517, a series of C-4' substituted indolinones have been synthesized and evaluated for PDK1 inhibition. Modification at C-4' of the pyrrole afforded potent compounds (7b and 7d) with improved solubility and ADME properties. In this letter, we describe the synthesis, selectivity profile, and pharmacokinetic data of selected compounds.
Assuntos
Indóis/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ureia/análogos & derivados , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Linhagem Celular Tumoral , Humanos , Indóis/farmacologia , Inibidores de Proteínas Quinases/química , Ureia/química , Ureia/farmacologiaRESUMO
The Escherichia coli genes frmB (yaiM) and yeiG encode two uncharacterized proteins that share 54% sequence identity and contain a serine esterase motif. We demonstrated that purified FrmB and YeiG have high carboxylesterase activity against the model substrates, p-nitrophenyl esters of fatty acids (C2-C6) and alpha-naphthyl acetate. However, both proteins had the highest hydrolytic activity toward S-formylglutathione, an intermediate of the glutathione-dependent pathway of formaldehyde detoxification. With this substrate, both proteins had similar affinity (Km = 0.41-0.43 mM), but FrmB was almost 5 times more active. Alanine replacement mutagenesis of YeiG demonstrated that Ser145, Asp233, and His256 are absolutely required for activity, indicating that these residues represent a serine hydrolase catalytic triad in this protein and in other S-formylglutathione hydrolases. This was confirmed by inspecting the crystal structure of the Saccharomyces cerevisiae S-formylglutathione hydrolase YJG8 (Protein Data Bank code 1pv1), which has 45% sequence identity to YeiG. The structure revealed a canonical alpha/beta-hydrolase fold and a classical serine hydrolase catalytic triad (Ser161, His276, Asp241). In E. coli cells, the expression of frmB was stimulated 45-75 times by the addition of formaldehyde to the growth medium, whereas YeiG was found to be a constitutive enzyme. The simultaneous deletion of both frmB and yeiG genes was required to increase the sensitivity of the growth of E. coli cells to formaldehyde, suggesting that both FrmB and YeiG contribute to the detoxification of formaldehyde. Thus, FrmB and YeiG are S-formylglutathione hydrolases with a Ser-His-Asp catalytic triad involved in the detoxification of formaldehyde in E. coli.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Formaldeído/metabolismo , Formaldeído/toxicidade , Tioléster Hidrolases/química , Sequência de Aminoácidos , Cristalografia por Raios X , Hidrólise , Cinética , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de AminoácidosRESUMO
To find proteins with nucleotidase activity in Escherichia coli, purified unknown proteins were screened for the presence of phosphatase activity using the general phosphatase substrate p-nitrophenyl phosphate. Proteins exhibiting catalytic activity were then assayed for nucleotidase activity against various nucleotides. These screens identified the presence of nucleotidase activity in three uncharacterized E. coli proteins, SurE, YfbR, and YjjG, that belong to different enzyme superfamilies: SurE-like family, HD domain family (YfbR), and haloacid dehalogenase (HAD)-like superfamily (YjjG). The phosphatase activity of these proteins had a neutral pH optimum (pH 7.0-8.0) and was strictly dependent on the presence of divalent metal cations (SurE: Mn(2+) > Co(2+) > Ni(2+) > Mg(2+); YfbR: Co(2+) > Mn(2+) > Cu(2+); YjjG: Mg(2+) > Mn(2+) > Co(2+)). Further biochemical characterization of SurE revealed that it has a broad substrate specificity and can dephosphorylate various ribo- and deoxyribonucleoside 5'-monophosphates and ribonucleoside 3'-monophosphates with highest affinity to 3'-AMP. SurE also hydrolyzed polyphosphate (exopolyphosphatase activity) with the preference for short-chain-length substrates (P(20-25)). YfbR was strictly specific to deoxyribonucleoside 5'-monophosphates, whereas YjjG showed narrow specificity to 5'-dTMP, 5'-dUMP, and 5'-UMP. The three enzymes also exhibited different sensitivities to inhibition by various nucleoside di- and triphosphates: YfbR was equally sensitive to both di- and triphosphates, SurE was inhibited only by triphosphates, and YjjG was insensitive to these effectors. The differences in their sensitivities to nucleotides and their varied substrate specificities suggest that these enzymes play unique functions in the intracellular nucleotide metabolism in E. coli.