RESUMO
Methyl 3-(2,4- dinitrophenylamino ) propionimidate hydrochloride (DNP-N-IE), methyl 3-(2,4-dinitrophenyl-N-methylamino) propionimidate hydrochloride (DNP- NMe -IE), and methyl 3-(2,4- dinitrophenylthio ) propionimidate hydrochloride (DNP-S-IE) have been prepared. DNP-N-IE and DNP- NMe -IE both react efficiently at 0-2 degrees C and pH 7-9.5 with sheep immunoglobulin G. At least 12 DNP groups can be introduced into the antibody protein without causing any significant change in its antigen binding affinity or capacity.
Assuntos
Marcadores de Afinidade/metabolismo , Dinitrobenzenos/metabolismo , Haptenos , Imidoésteres/metabolismo , Imunoglobulina G/metabolismo , Nitrobenzenos/metabolismo , Marcadores de Afinidade/síntese química , Animais , Anticorpos Anti-Idiotípicos , Sítios de Ligação de Anticorpos , Concentração de Íons de Hidrogênio , Imunoglobulina G/imunologia , Cinética , Coelhos , OvinosRESUMO
The synthesis of the 9 alpha- and 9 beta-epimers of 8, 9, 10, 11 alpha-tetrahydro-7 alpha H-7, 11-methano-12, 12-dimethylcycloocta [de] naphthyl-9-amine is described.
Assuntos
Proteína Quinase C/antagonistas & inibidores , Estaurosporina/análogos & derivados , Estrutura Molecular , Estaurosporina/síntese química , Estaurosporina/químicaRESUMO
1. When meropenem (I) is incubated with the soluble fraction of rat kidney or its ultrafiltrate the carbapenem disappears at a rate of 50 nmol/min per g of kidney at pH 8.4 and 37 degrees C. 2. In both cases the kinetics are inconsistent with the reaction being enzymic and the product has been shown to be a thiol. 3. The same reaction occurs in similar preparations from the kidney of several other mammalian species. 4. The reaction of meropenem with thiols has been investigated: only those having a vicinal amino group react rapidly. 5. The product of the reaction with cysteine has been characterized as the amide(III) of the latter with meropenoic acid (II), the carboxylic acid formed by the hydrolysis of the beta-lactam ring of meropenem. 6. It is postulated that the destruction of meropenem in the soluble fraction of rat kidney occurs by the non-enzymic reaction of the carbapenem with endogenous cysteine or related thiols. 7. There is no evidence that this reaction can occur in vivo but it should be considered in the future design of carbapenems.
Assuntos
Carbapenêmicos/metabolismo , Rim/metabolismo , Tienamicinas/metabolismo , Animais , Cisteína/metabolismo , Citosol/metabolismo , Ditiotreitol/farmacologia , Endopeptidases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Espectroscopia de Ressonância Magnética , Meropeném , Ratos , Compostos de Sulfidrila/metabolismoRESUMO
13C NMR was used to study the pattern of label incorporation from [2-13C]acetate into trehalose during sporulation in Saccharomyces cerevisiae. A wild-type strain and a strain homozygous for the zwf1 mutation (which affects glucose-6-phosphate dehydrogenase) were used. In the wild-type it was possible to deduce the cycling of glucose 6-phosphate around the hexose monophosphate pathway whilst in the mutant strain this did not occur. The requirement of the hexose monophosphate pathway for providing NADPH for fatty acid biosynthesis was examined using 13C NMR and GC/MS. The wild-type strain produced a typical profile of fatty acids with palmitoleic acid being the most abundant whereas the mutant contained only one-quarter the amount of total fatty acid. As zwf1 homozygous diploids are able to sporulate this indicates that the large amount of fatty acid biosynthesis observed in sporulation of wild-type strains is not essential to the process.
Assuntos
Ácidos Graxos/biossíntese , Via de Pentose Fosfato , Saccharomyces cerevisiae/fisiologia , Espectroscopia de Ressonância Magnética , Esporos FúngicosRESUMO
13C nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of [2-13C]acetate in a diploid strain of Saccharomyces cerevisiae homozygous for the spo50 mutation. This mutation results in failure to initiate sporulation and suppresses spd mutations (which cause derepressed sporulation). By analysing the pattern of 13C-labelling in glutamate it was deduced that the glyoxylate cycle is responsible for most of the acetate utilization and that there is very little tricarboxylic acid cycle activity. The labelling of alpha,alpha'-trehalose indicated that gluconeogenesis and the hexose monophosphate pathway operate in a similar way to the wild-type. The mutant strain has higher levels of succinate dehydrogenase than the wild-type. All of the physiological alterations caused by the spo50 mutation can be explained by this difference.
Assuntos
Saccharomyces cerevisiae/genética , Acetatos/metabolismo , Ácido Acético , Ciclo do Ácido Cítrico/genética , Gluconeogênese/genética , Glutamatos/metabolismo , Ácido Glutâmico , Glioxilatos/metabolismo , Espectroscopia de Ressonância Magnética , Mutação , Saccharomyces cerevisiae/fisiologia , Esporos Fúngicos/genética , Esporos Fúngicos/fisiologia , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismoRESUMO
The metabolism of [2-13C]acetate by Pseudomonas M27(Icl-) and Pseudomonas MA(Icl+) was studied in vivo using 13C-NMR spectroscopy. The flux of 13C-label into bicarbonate, glutamate and citrate was observed in both organisms. In addition 13C-labelled alpha, alpha-trehalose was synthesized as a major metabolite by Pseudomonas M27 but not by Pseudomonas MA. The presence of this disaccharide in cell extracts of Pseudomonas AM1(Icl-) grown with [13C]methanol was also observed. The data from analysis of the trehalose multiplet signal observed in the spectra of Pseudomonas M27 cell extracts were consistent with the absence of the glyoxylate cycle in this methylotroph.
Assuntos
Acetatos/metabolismo , Metanol/metabolismo , Pseudomonas/metabolismo , Gluconeogênese , Espectroscopia de Ressonância Magnética , Trealose/biossínteseRESUMO
Saccharomyces cerevisiae mutants defective in the structural gene PGI1 lack phosphoglucose isomerase and hence cannot grow on glucose. Spontaneous mutants were isolated by selecting for the regained ability to grow on YEPD (yeast extract/peptone/glucose). Three complementation groups called spg29-31 (suppressor of pgi1 delta) were identified. The metabolism of [2-13C]glucose was studied by 13C NMR spectroscopy. This led to the conclusion that in a spg29 mutant suppression of the glycolytic defect was achieved by increased carbon flux through the hexose monophosphate pathway. The specific activities of enzymes of the hexose monophosphate pathway (except glucose-6-phosphate dehydrogenase) and NAD- and NADP-dependent glutamate dehydrogenase were increased in the bypass mutant.
Assuntos
Genes Fúngicos/genética , Glucose-6-Fosfato Isomerase/genética , Via de Pentose Fosfato/genética , Saccharomyces cerevisiae/genética , Supressão Genética , Enzimas/análise , Deleção de Genes , Teste de Complementação Genética , Glucose/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Glicólise/fisiologia , Espectroscopia de Ressonância Magnética , Mutação , Saccharomyces cerevisiae/metabolismoRESUMO
The metabolism of valine to isobutyl alcohol in yeast was examined by 13C nuclear magnetic resonance spectroscopy and combined gas chromatography-mass spectrometry. The product of valine transamination, alpha-ketoisovalerate, had four potential routes to isobutyl alcohol. The first, via branched-chain alpha-ketoacid dehydrogenase to isobutyryl-CoA is not required for the synthesis of isobutyl alcohol because abolition of branched-chain alpha-ketoacid dehydrogenase activity in an lpd1 disruption mutant did not prevent the formation of isobutyl alcohol. The second route, via pyruvate decarboxylase, is the one that is used because elimination of pyruvate decarboxylase activity in a pdc1 pdc5 pdc6 triple mutant virtually abolished isobutyl alcohol production. A third potential route involved alpha-ketoisovalerate reductase, but this had no role in the formation of isobutyl alcohol from alpha-hydroxyisovalerate because cell homogenates could not convert alpha-hydroxyisovalerate to isobutyl alcohol. The final possibility, use of the pyruvate decarboxylase-like enzyme encoded by YDL080c, seemed to be irrelevant, because a strain with a disruption in this gene produced wild-type levels of isobutyl alcohol. Thus there are major differences in the catabolism of leucine and valine to their respective "fusel" alcohols. Whereas in the catabolism of leucine to isoamyl alcohol the major route is via the decarboxylase encoded by YDL080c, any single isozyme of pyruvate decarboxylase is sufficient for the formation of isobutyl alcohol from valine. Finally, analysis of the 13C-labeled products revealed that the pathways of valine catabolism and leucine biosynthesis share a common pool of alpha-ketoisovalerate.
Assuntos
Butanóis/metabolismo , Saccharomyces cerevisiae/metabolismo , Valina/metabolismo , Isótopos de Carbono , Divisão Celular/genética , Proteínas Fúngicas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Hemiterpenos , Cetoácidos/metabolismo , Leucina/biossíntese , Espectroscopia de Ressonância Magnética , Piruvato Descarboxilase/metabolismoRESUMO
Two different mechanisms have been proposed previously for initiating the biodegradation of monomethyl sulfate (MeSO4) in bacteria. For a Hyphomicrobium species, a sulfatase enzyme has been proposed to hydrolyse MeSO4 to methanol and inorganic sulfate. For an Agrobacterium sp., an alternative proposal involves monooxygenation of MeSO4 (hydroxylation) to produce methanediol monosulfate, which decomposes spontaneously to formaldehyde and inorganic sulfate. In the present study, 13C-NMR was used to monitor metabolic intermediates of [13C]MeSO4 in real time in each species in order to resolve the issue of mechanism of biodegradation. Agrobacterium sp. M3C grew on MeSO4 but not on methanol. MeSO4-grown cells catabolised [13C]MeSO4 but not [13C]methanol, and [13C]methanol did not accumulate from MeSO4 in the presence of a known inhibitor of methanol dehydrogenase (cyclopropanol). Hyphomicrobium MS223 grew on MeSO4 and, in contrast with the Agrobacterium sp., also on methanol. The normally rapid metabolism of [13C]methanol by methanol-grown cells was arrested by cyclopropanol, but metabolism of [13C]MeSO4 by MeSO4-grown cells was unaffected. Moreover there was no accumulation of [13C]methanol from [13C]MeSO4 under conditions in which methanol dehydrogenase was shown to be inactive. The results provided strong evidence against the intermediacy of methanol in the biodegradation of MeSO4 in either species, and thereby render untenable mechanisms involving sulfatase-mediated hydrolysis of MeSO4. The data are consistent with the hydroxylation of MeSO4 via a monooxygenation mechanism and subsequent spontaneous hydrolysis of the methanediol monosulfate intermediate.
Assuntos
Ésteres do Ácido Sulfúrico/metabolismo , Oxirredutases do Álcool/antagonistas & inibidores , Bactérias/metabolismo , Inibidores Enzimáticos/farmacologia , Éteres Cíclicos/farmacologia , Formaldeído/metabolismo , Espectroscopia de Ressonância Magnética , Rhizobium/metabolismo , Tetraciclina/farmacologiaRESUMO
The biosynthesis of alginate by a mucoid strain of Pseudomonas aeruginosa, isolated from a cystic-fibrosis patient, was monitored by using 13C-n.m.r. spectroscopy of bacterial cultures incubated with 1-13C- or 2-13C-enriched fructose. When 1-13C- or 2-13C-enriched fructose was used as the precursor of alginate, enrichment with 13C in the constituent uronic acid monomers of the polysaccharide could only be detected in C-1 or C-2 respectively, indicating that alginate is synthesized in Ps. aeruginosa directly from fructose, with the hexose molecule being retained intact; this rules out the involvement of C3 intermediates, which occurs when glucose is the alginate precursor. The absence of detectable poly-L-gluluronate block sequences from the alginate of Ps. aeruginosa was confirmed, and it was shown that there is no modification of the arrangement of the constituent uronic acids between polymerization to form alginate and the appearance of the mature alginate in the extracellular medium. The 13C-n.m.r. data also provided independent evidence for acetylation on D-mannuronate residues and for the ratio of D-mannuronate to L-guluronate residues in newly synthesized alginate, which had previously been determined only for material secreted from bacteria into the extracellular medium.
Assuntos
Alginatos/metabolismo , Pseudomonas aeruginosa/metabolismo , Isótopos de Carbono , Frutose/metabolismo , Espectroscopia de Ressonância MagnéticaRESUMO
The enantiomers of the aromatase inhibitors 3-(4-aminophenyl)-pyrrolidine-2,5-dione (WSP-3, II), its N-pentyl derivative (III), and the antifungal econazole (IV), all possessing a benzylic proton at the chiral centre, are rapidly racemised in vitro in phosphate buffer (0.01 M) at pH 7.4 and 23 degrees C with t 1/2 values of 7, 6, and 5 h respectively. In vivo studies in rats show that (+)-econazole is racemised after intraperitoneal injection with t 1/2 = 1.24h. The enantiomers of the antifungal 1-[(benzofuran-2-yl)-4-chlorophenylmethyl] imidazole (V) were stable at pH 7.4, attributable to steric hindrance to carbanion formation in the racemisation step.
Assuntos
Compostos de Anilina/química , Econazol/química , Pirrolidinonas/química , Succinimidas/química , Compostos de Anilina/isolamento & purificação , Compostos de Anilina/farmacologia , Animais , Inibidores da Aromatase , Soluções Tampão , Econazol/isolamento & purificação , Econazol/farmacologia , Meia-Vida , Concentração de Íons de Hidrogênio , Cinética , Fosfatos , Prótons , Pirrolidinonas/isolamento & purificação , Pirrolidinonas/farmacologia , Ratos , Estereoisomerismo , Succinimidas/isolamento & purificação , Succinimidas/farmacologiaRESUMO
The metabolism of isoleucine to active amyl alcohol (2-methylbutanol) in yeast was examined by the use of (13)C nuclear magnetic resonance spectroscopy, combined gas chromatography-mass spectrometry, and a variety of mutants. From the identified metabolites a number of routes between isoleucine and active amyl alcohol seemed possible. All involved the initial decarboxylation of isoleucine to alpha-keto-beta-methylvalerate. The first, via branched chain alpha-ketoacid dehydrogenase to alpha-methylbutyryl-CoA, was eliminated because abolition of branched-chain alpha-ketoacid dehydrogenase in an lpd1 disruption mutant did not prevent the formation of active amyl alcohol. However, the lpd1 mutant still produced large amounts of alpha-methylbutyrate which initially seemed contradictory because it had been assumed that alpha-methylbutyrate was derived from alpha-methylbutyryl-CoA via acyl-CoA hydrolase. Subsequently it was observed that alpha-methylbutyrate arises from the non-enzymic oxidation of alpha-methylbutyraldehyde (the immediate decarboxylation product of alpha-keto-beta-methylvalerate). Mutant studies showed that one of the decarboxylases encoded by PDC1, PDC5, PDC6, YDL080c, or YDR380w must be present to allow yeast to utilize alpha-keto-beta-methylvalerate. Apparently, any one of this family of decarboxylases is sufficient to allow the catabolism of isoleucine to active amyl alcohol. This is the first demonstration of a role for the gene product of YDR380w, and it also shows that the decarboxylation steps for each alpha-keto acid in the catabolic pathways of leucine, valine, and isoleucine are accomplished in subtly different ways. In leucine catabolism, the enzyme encoded by YDL080c is solely responsible for the decarboxylation of alpha-ketoisocaproate, whereas in valine catabolism any one of the isozymes of pyruvate decarboxylase will decarboxylate alpha-ketoisovalerate.
Assuntos
Isoleucina/metabolismo , Pentanóis/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Dados de Sequência Molecular , Piruvato Descarboxilase/metabolismoRESUMO
OBJECTIVE: To evaluate a new rapid diagnostic test for bacterial vaginosis. DESIGN: Comparison of a new biochemical diamine test with low technology tests and microbiological culture. SETTING: General practice, family planning clinic. MAIN OUTCOME MEASURES: Comparison of the new diamine test with microbiological culture for Gardnerella vaginalis, with clue cells, and with the amine test. RESULTS: Two hundred and twenty-nine vaginal swabs were assayed quantitatively by the new diamine test. When compared with microbiological culture of Gardnerella vaginalis, the sensitivities and specificities were 86% and 81%, respectively. When compared with clue cell findings, the sensitivities and specificities were 97% and 83%, respectively. In the third comparison with the amine test the sensitivity was 94% and the specificity was 84%. Since microbiological diagnosis of organisms related to bacterial vaginosis is difficult, the new test and existing sideroom tests may be more sensitive to the condition and the true frequency of false positives may be less than the specificity in this study suggests. CONCLUSIONS: The new diamine test is accurate, sensitive and specific, and provides the basis for the rapid diagnosis of bacterial vaginosis. Such a test is needed if bacterial vaginosis is to be diagnosed and managed effectively in both general and specialist practice.
Assuntos
Diaminas/análise , Vaginose Bacteriana/diagnóstico , Adolescente , Adulto , Idoso , Feminino , Gardnerella vaginalis , Humanos , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Vaginose Bacteriana/microbiologiaRESUMO
A model is described for the binding of non-steroidal inhibitors of aromatase to the enzyme with interaction of a ligand with the Fe3+-haem of the cytochrome. The model suggests that the inhibitors utilise a common binding site with ring A of the steroidal substrates.
Assuntos
Androstenodiona/análogos & derivados , Androstenodiona/farmacologia , Inibidores da Aromatase , Androstenodiona/síntese química , Sítios de Ligação , Enzima de Clivagem da Cadeia Lateral do Colesterol/antagonistas & inibidores , Simulação por Computador , Feminino , Humanos , Cinética , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Placenta/enzimologia , Gravidez , Relação Estrutura-AtividadeRESUMO
The metabolism of leucine to isoamyl alcohol in yeast was examined by 13C nuclear magnetic resonance spectroscopy. The product of leucine transamination, alpha-ketoisocaproate had four potential routes to isoamyl alcohol. The first, via branched-chain alpha-keto acid dehydrogenase to isovaleryl-CoA with subsequent conversion to isovalerate by acyl-CoA hydrolase operates in wild-type cells where isovalerate appears to be an end product. This pathway is not required for the synthesis of isoamyl alcohol because abolition of branched-chain alpha-keto acid dehydrogenase activity in an lpd1 disruption mutant did not prevent the formation of isoamyl alcohol. A second possible route was via pyruvate decarboxylase; however, elimination of pyruvate decarboxylase activity in a pdc1 pdc5 pdc6 triple mutant did not decrease the levels of isoamyl alcohol produced. A third route utilizes alpha-ketoisocaproate reductase (a novel activity in Saccharomyces cerevisiae) but with no role in the formation of isoamyl alcohol from alpha-hydroxyisocaproate because cell homogenates could not convert alpha-hydroxyisocaproate to isoamyl alcohol. The final possibility was that a pyruvate decarboxylase-like enzyme encoded by YDL080c appears to be the major route of decarboxylation of alpha-ketoisocaproate to isoamyl alcohol although disruption of this gene reveals that at least one other unidentified decarboxylase can substitute to a minor extent.