RESUMO
OBJECTIVES: First, to investigate the biological variability of N-terminal pro-B-type natriuretic peptide (NT-proBNP) in healthy Labrador retrievers and compare this with current laboratory recommendations for dilated cardiomyopathy screening. Second, to calculate a breed-specific reference interval and validate it in a retrospective cohort. MATERIALS AND METHODS: Plasma NT-proBNP was measured in 51 clinically healthy Labrador retrievers at 0, 2, 4, 6 and 8 weeks. Coefficient of variation for individual dogs over time, the coefficient of variation for the group at each time point and the index of individuality were calculated. A reference interval was derived and tested on a clinical dataset available from four UK cardiology referral centres. RESULTS: Median NT-proBNP was 865 pmol/L (315 to 2064 pmol/L). Mean individual coefficient of variation was 19% (95% CI: 16 to 21%) and group coefficient of variation was 43% (95% CI: 41 to 46%), with index of individuality at 0.44. The breed-specific reference interval was 275 to 2100 pmol/L. In the validation group, 93% of NT-proBNP measurements from healthy dogs were within the reference interval. NT-proBNP measurements exceeded the reference interval in 82% of dogs with dilated cardiomyopathy. The upper bound of the reference interval (2100 pmol/L) had a positive predictive value of 90% and a negative predictive value of 87% for identification of dilated cardiomyopathy in this population. CLINICAL SIGNIFICANCE: Breed-specific reference intervals might improve the diagnostic accuracy of NT-proBNP measurement. Applying the currently recommended general cut-off value to Labradors is likely to result in frequent false positives and diagnosis would be improved by application of the new breed-specific reference interval calculated here.
Assuntos
Peptídeo Natriurético Encefálico , Fragmentos de Peptídeos , Animais , Biomarcadores , Cães , Valores de Referência , Estudos RetrospectivosRESUMO
Real-world applications will inevitably entail divergence between samples on which chemometric classifiers are trained and the unknowns requiring classification. This has long been recognized, but there is a shortage of empirical studies on which classifiers perform best in 'external validation' (EV), where the unknown samples are subject to sources of variation relative to the population used to train the classifier. Survey of 286 classification studies in analytical chemistry found only 6.6% that stated elements of variance between training and test samples. Instead, most tested classifiers using hold-outs or resampling (usually cross-validation) from the same population used in training. The present study evaluated a wide range of classifiers on NMR and mass spectra of plant and food materials, from four projects with different data properties (e.g., different numbers and prevalence of classes) and classification objectives. Use of cross-validation was found to be optimistic relative to EV on samples of different provenance to the training set (e.g., different genotypes, different growth conditions, different seasons of crop harvest). For classifier evaluations across the diverse tasks, we used ranks-based non-parametric comparisons, and permutation-based significance tests. Although latent variable methods (e.g., PLSDA) were used in 64% of the surveyed papers, they were among the less successful classifiers in EV, and orthogonal signal correction was counterproductive. Instead, the best EV performances were obtained with machine learning schemes that coped with the high dimensionality (914-1898 features). Random forests confirmed their resilience to high dimensionality, as best overall performers on the full data, despite being used in only 4.5% of the surveyed papers. Most other machine learning classifiers were improved by a feature selection filter (ReliefF), but still did not out-perform random forests.
Assuntos
Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Algoritmos , Arabidopsis/química , Arabidopsis/classificação , Arabidopsis/genética , Arabidopsis/metabolismo , Biomassa , Cacau/química , Cacau/classificação , Cacau/genética , Cacau/metabolismo , Análise Discriminante , Metabolômica , Reprodutibilidade dos Testes , Ácido Salicílico/metabolismoRESUMO
OBJECTIVES: Curcumin (diferuloylmethane) is the principal biochemical component of the spice turmeric and has been shown to possess potent anti-catabolic, anti-inflammatory and antioxidant, properties. This article aims to provide a summary of the actions of curcumin on articular chondrocytes from the available literature with the use of a text-mining tool. We highlight both the potential benefits and drawbacks of using this chemopreventive agent for treating osteoarthritis (OA). We also explore the recent literature on the molecular mechanisms of curcumin mediated alterations in gene expression mediated via activator protein 1 (AP-1)/nuclear factor-kappa B (NF-kappaB) signalling in chondrocytes, osteoblasts and synovial fibroblasts. METHODS: A computer-aided search of the PubMed/Medline database aided by a text-mining tool to interrogate the ResNet Mammalian database 6.0. RESULTS: Recent work has shown that curcumin protects human chondrocytes from the catabolic actions of interleukin-1 beta (IL-1beta) including matrix metalloproteinase (MMP)-3 up-regulation, inhibition of collagen type II and down-regulation of beta1-integrin expression. Curcumin blocks IL-1beta-induced proteoglycan degradation, AP-1/NF-kappaB signalling, chondrocyte apoptosis and activation of caspase-3. CONCLUSIONS: The available data from published in vitro and in vivo studies suggest that curcumin may be a beneficial complementary treatment for OA in humans and companion animals. Nevertheless, before initiating extensive clinical trials, more basic research is required to improve its solubility, absorption and bioavailability and gain additional information about its safety and efficacy in different species. Once these obstacles have been overcome, curcumin and structurally related biochemicals may become safer and more suitable nutraceutical alternatives to the non-steroidal anti-inflammatory drugs that are currently used for the treatment of OA.
Assuntos
Cartilagem Articular/metabolismo , Condrócitos/efeitos dos fármacos , Curcumina/farmacologia , Inibidores Enzimáticos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Antígenos CD18/metabolismo , Cartilagem Articular/citologia , Caspase 3/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Humanos , Inflamação/fisiopatologia , Interleucina-1beta/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , NF-kappa B/fisiologia , Osteoartrite/tratamento farmacológico , Osteoartrite/prevenção & controle , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator de Transcrição AP-1/fisiologiaRESUMO
The matrix metalloproteinases (MMPs) and their endogenous regulators, the tissue inhibitors of MMPs (TIMPs) are responsible for the physiological remodelling of the extracellular matrix (ECM) in healthy connective tissues. MMPs are also involved in the regulation of cell behaviour via the release of growth factors and cytokines from the substrates they cleave, increasing the magnitude of their effects. Excess MMP activity is associated with ECM destruction in various inflammatory conditions, such as osteoarthritis (OA), while MMP under-activity potentially impairs healing by promoting fibrosis and preventing the effective removal of scar tissue. Both direct (TIMPs, small molecule MMP inhibitor drugs, blocking antibodies and anti-sense technologies) and indirect (glucocorticoids and non-steroidal anti-inflammatory drugs, statins, anti-sense technologies and various phytochemicals) strategies for MMP inhibition have been proposed and investigated. The strategy of MMP inhibition for degenerative and neoplastic diseases has been relatively unsuccessful due to undesired sequelae, often caused by non-selectivity of the MMP inhibition method. Therapeutic strategies for MMP-related conditions ideally should regulate MMP activity in order to maintain the optimum balance between MMPs and TIMPs. By avoiding complete inhibition it may be possible to prevent the complications of MMP over- and under-activity. Furthermore, MMP sub-type specificity is critical for minimising detrimental off-target effects that have been observed with broad-spectrum MMP inhibitors. Any potential MMP inhibitor or modulator must be subjected to rigorous pharmacokinetic, toxicity and safety studies and data obtained using in vitro models must be verified in clinically relevant animal models before therapeutic use is considered.
Assuntos
Anti-Inflamatórios/uso terapêutico , Inflamação/tratamento farmacológico , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/uso terapêutico , Animais , Anticorpos Bloqueadores/uso terapêutico , Humanos , Inibidores Teciduais de Metaloproteinases/uso terapêuticoRESUMO
The biological reduction of atmospheric N2 to ammonium (nitrogen fixation) provides about 65% of the biosphere's available nitrogen. Most of this ammonium is contributed by legume-rhizobia symbioses, which are initiated by the infection of legume hosts by bacteria (rhizobia), resulting in formation of root nodules. Within the nodules, rhizobia are found as bacteroids, which perform the nitrogen fixation: to do this, they obtain sources of carbon and energy from the plant, in the form of dicarboxylic acids. It has been thought that, in return, bacteroids simply provide the plant with ammonium. But here we show that a more complex amino-acid cycle is essential for symbiotic nitrogen fixation by Rhizobium in pea nodules. The plant provides amino acids to the bacteroids, enabling them to shut down their ammonium assimilation. In return, bacteroids act like plant organelles to cycle amino acids back to the plant for asparagine synthesis. The mutual dependence of this exchange prevents the symbiosis being dominated by the plant, and provides a selective pressure for the evolution of mutualism.
Assuntos
Aminoácidos/metabolismo , Fixação de Nitrogênio , Pisum sativum/metabolismo , Pisum sativum/microbiologia , Rhizobium/metabolismo , Simbiose , Aminoácidos/biossíntese , Asparagina/biossíntese , Transporte Biológico , Dados de Sequência Molecular , Mutação , Pisum sativum/genética , Rhizobium/genéticaRESUMO
Amino acid transport by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra). However, mutation of these transporters does not prevent this organism from utilizing alanine for growth. An R. leguminosarum permease (MctP) has been identified which is required for optimal growth on alanine as a sole carbon and nitrogen source. Characterization of MctP confirmed that it transports alanine (K(m) = 0.56 mM) and other monocarboxylates such as lactate and pyruvate (K(m) = 4.4 and 3.8 micro M, respectively). Uptake inhibition studies indicate that propionate, butyrate, alpha-hydroxybutyrate, and acetate are also transported by MctP, with the apparent affinity for solutes demonstrating a preference for C3-monocarboxylates. MctP has significant sequence similarity to members of the sodium/solute symporter family. However, sequence comparisons suggest that it is the first characterized permease of a new subfamily of transporters. While transport via MctP was inhibited by CCCP, it was not apparently affected by the concentration of sodium. In contrast, glutamate uptake in R. leguminosarum by the Escherichia coli GltS system did require sodium, which suggests that MctP may be proton coupled. Uncharacterized members of this new subfamily have been identified in a broad taxonomic range of species, including proteobacteria of the beta-subdivision, gram-positive bacteria, and archaea. A two-component sensor-regulator (MctSR), encoded by genes adjacent to mctP, is required for activation of mctP expression.
Assuntos
Alanina/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Rhizobium leguminosarum/metabolismo , Sistemas de Transporte de Aminoácidos/classificação , Sistemas de Transporte de Aminoácidos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácidos Carboxílicos/metabolismo , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Cinética , Dados de Sequência Molecular , Transportadores de Ácidos Monocarboxílicos/classificação , Transportadores de Ácidos Monocarboxílicos/genética , Mutação , Pisum sativum/microbiologia , Filogenia , Rhizobium leguminosarum/genética , Rhizobium leguminosarum/crescimento & desenvolvimento , Sódio/farmacologia , SimbioseRESUMO
Amino acid uptake by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra(Rl)). Characterization of the solute specificity of Bra(Rl) shows it to be the second general amino acid permease of R. leguminosarum. Although Bra(Rl) has high sequence identity to members of the family of hydrophobic amino acid transporters (HAAT), it transports a broad range of solutes, including acidic and basic polar amino acids (L-glutamate, L-arginine, and L-histidine), in addition to neutral amino acids (L-alanine and L-leucine). While amino and carboxyl groups are required for transport, solutes do not have to be alpha-amino acids. Consistent with this, Bra(Rl) is the first ABC transporter to be shown to transport gamma-aminobutyric acid (GABA). All previously identified bacterial GABA transporters are secondary carriers of the amino acid-polyamine-organocation (APC) superfamily. Also, transport by Bra(Rl) does not appear to be stereospecific as D amino acids cause significant inhibition of uptake of L-glutamate and L-leucine. Unlike all other solutes tested, L-alanine uptake is not dependent on solute binding protein BraC(Rl). Therefore, a second, unidentified solute binding protein may interact with the BraDEFG(Rl) membrane complex during L-alanine uptake. Overall, the data indicate that Bra(Rl) is a general amino acid permease of the HAAT family. Furthermore, Bra(Rl) has the broadest solute specificity of any characterized bacterial amino acid transporter.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Rhizobium leguminosarum/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Aminoácidos de Cadeia Ramificada/metabolismo , Transporte Biológico , Cinética , Dados de Sequência Molecular , Mutação , Filogenia , Rhizobium leguminosarum/genética , Especificidade por Substrato , Ácido gama-Aminobutírico/metabolismoRESUMO
The techniques of differential fluorescence induction (DFI) and optical trapping (OT) have been combined to allow the identification of environmentally induced genes in single bacterial cells. Designated DFI-OT, this technique allows the in situ isolation of genes driving the expression of green fluorescent protein (Gfp) using temporal and spatial criteria. A series of plasmid-based promoter probe vectors (pOT) was developed for the construction of random genomic libraries that are linked to gfpUV or egfp. Bacteria that do not express Gfp on laboratory medium (i.e. non-fluorescent) were inoculated into the environment, and induced genes were detected with a combined fluorescence/optical trapping microscope. Using this selection strategy, rhizosphere-induced genes with homology to thiamine pyrophosphorylase (thiE) and cyclic glucan synthase (ndvB) were isolated. Other genes were expressed late in the stationary phase or as a consequence of surface-dependent growth, including fixND and metX, and a putative ABC transporter of putrescine. This strategy provides a unique ability to combine spatial, temporal and physical information to identify environmental regulation of bacterial gene expression.
Assuntos
Proteínas de Bactérias/genética , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica , Rhizobium leguminosarum/genética , Rhizobium leguminosarum/fisiologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sondas de DNA , Vetores Genéticos , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Dados de Sequência Molecular , Pisum sativum/microbiologia , Raízes de Plantas/microbiologia , Rhizobium leguminosarum/citologia , Microbiologia do SoloRESUMO
The ATP-binding cassette (ABC) transporter superfamily is one of the most widespread of all gene families and currently has in excess of 1100 members in organisms ranging from the Archaea to manQ1. The movement of the diverse solutes of ABC transporters has been accepted as being strictly unidirectional, with recent models indicating that they are irreversible. However, contrary to this paradigm, we show that three solute-binding protein-dependent (SBP) ABC transporters of amino acids, i.e. the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra) of Rhizobium leguminosarum and the histidine permease (His) of Salmonella typhimurium, are bidirectional, being responsible for efflux in addition to the uptake of solutes. The net solute movement measured for an ABC transporter depends on the rates of uptake and efflux, which are independent; a plateau is reached when both are saturated. SBP ABC transporters promote active uptake because, although the Vmax values for uptake and efflux are not significantly different, there is a 103-104 higher affinity for uptake of solute compared with efflux. Therefore, the SBP ABC transporters are able to support a substantial concentration gradient and provide a net uptake of solutes into bacterial cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Sistemas de Transporte de Aminoácidos Básicos , Aminoácidos/metabolismo , Proteínas de Bactérias , Proteínas de Saccharomyces cerevisiae , Transportadores de Cassetes de Ligação de ATP/genética , Sistemas de Transporte de Aminoácidos , Ácidos Aminoisobutíricos/metabolismo , Transporte Biológico , Proteínas Fúngicas , Cinética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Periplasma/metabolismo , Rhizobium leguminosarum/genética , Rhizobium leguminosarum/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismoRESUMO
One of the paradigms of symbiotic nitrogen fixation has been that bacteroids reduce N2 to ammonium and secrete it without assimilation into amino acids. This has recently been challenged by work with soybeans showing that only alanine is excreted in 15N2 labelling experiments. Work with peas shows that the bacteroid nitrogen secretion products during in vitro experiments depend on the experimental conditions. There is a mixed secretion of both ammonium and alanine depending critically on the concentration of bacteroids and ammonium concentration. The pathway of alanine synthesis has been shown to be via alanine dehydrogenase, and mutation of this enzyme indicates that in planta there is likely to be mixed secretion of ammonium and alanine. Alanine synthesis directly links carbon catabolism and nitrogen assimilation in the bacteroid. There is now overwhelming evidence that the principal carbon sources of bacteroids are the C4-dicarboxylic acids. This is based on labelling and bacteroid respiration data, and mutation of both the dicarboxylic acid transport system (dct) and malic enzyme. L-malate is at a key bifurcation point in bacteroid metabolism, being oxidized to oxaloacetate and oxidatively decarboxylated to pyruvate. Pyruvate can be aminated to alanine or converted to acetyl-CoA where it either enters the TCA cycle by condensation with oxaloacetate or forms polyhydroxybutyrate (PHB). Thus regulation of carbon and nitrogen metabolism are strongly connected. Efficient catabolism of C4-dicarboxylates requires the balanced input and removal of intermediates from the TCA cycle. The TCA cycle in bacteroids may be limited by the redox state of NADH/NAD+ at the 2-ketoglutarate dehydrogenase complex, and a number of pathways may be involved in bypassing this block. These pathways include PHB synthesis, glutamate synthesis, glycogen synthesis, GABA shunt and glutamine cycling. Their operation may be critical in maintaining the optimum redox poise and carbon balance of the TCA cycle. They can also be considered to be overflow pathways since they act to remove or add electrons and carbon into the TCA cycle. Optimum operation of the TCA cycle has a major impact on nitrogen fixation.
Assuntos
Carbono/metabolismo , Fixação de Nitrogênio , Rhizobium/metabolismo , Alanina/metabolismo , Ciclo do Ácido Cítrico , Ácidos Dicarboxílicos/metabolismo , Gluconeogênese , Ácido Glutâmico/metabolismo , Compostos de Amônio Quaternário/metabolismoRESUMO
N2-fixation by Rhizobium-legume symbionts is of major ecological and agricultural importance, responsible for producing a substantial fraction of the biosphere's nitrogen. On the basis of 15N-labelling studies, it had been generally accepted that ammonium is the sole secretion product of N2-fixation by the bacteroid and that the plant is responsible for assimilating it into amino acids. However, this paradigm has been challenged in a recent 15N-labelling study showing that soybean bacteroids only secrete alanine. Hitherto, nitrogen secretion has only been assessed from in vitro 15N-labelling studies of isolated bacteroids. We show that both ammonium and alanine are secreted by pea bacteroids. The in vitro partitioning between them will depend on whether the system is open or closed, as well as the ammonium concentration and bacteroid density. To overcome these limitations we identified and mutated the gene for alanine dehydrogenase (aldA) and demonstrate that AldA is the primary route for alanine synthesis in isolated bacteroids. Bacteroids of the aldA mutant fix nitrogen but only secrete ammonium at a significant rate, resulting in lower total nitrogen secretion. Peas inoculated with the aldA mutant are green and healthy, demonstrating that ammonium secretion by bacteroids can provide sufficient nitrogen for plant growth. However, plants inoculated with the mutant are reduced in biomass compared with those inoculated with the wild type. The labelling and plant growth studies suggest that alanine synthesis and secretion contributes to the efficiency of N2-fixation and therefore biomass accumulation.
Assuntos
Alanina/metabolismo , Aminoácido Oxirredutases/metabolismo , Pisum sativum/microbiologia , Compostos de Amônio Quaternário/metabolismo , Rhizobium/enzimologia , Alanina Desidrogenase , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dados de Sequência Molecular , Mutação , Nitrogênio/metabolismo , Fixação de Nitrogênio , Rhizobium/genética , Rhizobium/crescimento & desenvolvimento , Rhizobium/metabolismo , SimbioseRESUMO
A gene, cpaA, with similarity to calcium proton antiporters has been identified adjacent to lpcAB in Rhizobium leguminosarum. LpcA is a galactosyl transferase while LpcB is a 2-keto-3-deoxyoctonate transferase, both of which are required to form the lipopolysaccharide (LPS) core in R. leguminosarum. Mutations in lpcAB result in a rough LPS phenotype with a requirement for elevated calcium concentrations to allow growth, suggesting that truncation of the LPS core exposes a highly negatively charged molecule. This is consistent with the LPS core being one of the main sites for binding calcium in the Gram-negative outer membrane. Strain RU1109 (cpaA::Tn5-lacZ) has a normal LPS layer, as measured by silver staining and Western blotting. This indicates that cpaA mutants are not grossly affected in their LPS layer. LacZ fusion analysis indicates that cpaA is constitutively expressed and is not directly regulated by the calcium concentration. Over-expression of cpaA increased the concentration of calcium required for growth, consistent with CpaA mediating calcium export from the cytosol. The location of lpcA, lpcB and cpaA as well as the phenotype of lpcB mutants suggests that CpaA might provide a specific export pathway for calcium to the LPS core.
Assuntos
Proteínas de Bactérias , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Lipopolissacarídeos/metabolismo , Rhizobium leguminosarum/genética , Rhizobium leguminosarum/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Meios de Cultura , Elementos de DNA Transponíveis , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Concentração de Íons de Hidrogênio , Magnésio/metabolismo , Dados de Sequência Molecular , Plasmídeos/genética , Rhizobium leguminosarum/crescimento & desenvolvimento , Alinhamento de Sequência , Análise de Sequência de DNA , Açúcares Ácidos/metabolismo , Transferases/genética , Transferases/metabolismoRESUMO
The lipopolysaccharide (LPS) core of the Gram-negative bacterium Rhizobium leguminosarum is more amenable to enzymatic study than that of Escherichia coli because much of it is synthesized from readily available sugar nucleotides. The inner portion of the R. leguminosarum core contains mannose, galactose, and three 3-deoxy-D-manno-octulosonate (Kdo) residues, arranged in the order: lipid A-(Kdo)2-Man-Gal-Kdo-[O antigen]. A mannosyltransferase that uses GDP-mannose and the conserved precursor Kdo2-[4'-32P]lipid IVA (Kadrmas, J. L., Brozek, K. A., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 32119-32125) is proposed to represent a key early enzyme in R. leguminosarum core assembly. Conditions for demonstrating efficient galactosyl- and distal Kdo-transferase activities are now described using a coupled assay system that starts with GDP-mannose and Kdo2-[4'-32P]lipid IVA. As predicted, mannose incorporation precedes galactose addition, which in turn precedes distal Kdo transfer. LPS core mutants with Tn5 insertions in the genes encoding the putative galactosyltransferase (lpcA) and the distal Kdo-transferase (lpcB) are shown to be defective in the corresponding in vitro glycosylation of Kdo2-[4'-32P]lipid IVA. We have also discovered the new gene (lpcC) that encodes the mannosyltransferase. The gene is separated by several kilobase pairs from the lpcAB cluster. All three glycosyltransferases are carried on cosmid pIJ1848, which contains at least 20 kilobase pairs of R. leguminosarum DNA. Transfer of pIJ1848 into R. meliloti 1021 results in heterologous expression of all three enzymes, which are not normally present in strain 1021. Expression of the lpc genes individually behind the T7 promoter results in the production of each R. leguminosarum glycosyltransferase in E. coli membranes in a catalytically active form, demonstrating that lpcA, lpcB, and lpcC are structural genes.
Assuntos
Glicosiltransferases/genética , Lipopolissacarídeos/biossíntese , Rhizobium leguminosarum/enzimologia , Bacteriófago T7/genética , Sequência de Bases , Sequência de Carboidratos , Clonagem Molecular , Cosmídeos , Primers do DNA , Glicosilação , Lipopolissacarídeos/química , Dados de Sequência Molecular , Mutagênese InsercionalRESUMO
A 2-kb region that complements the Tn5-derived lipopolysaccharide (LPS) rough mutant Rhizobium leguminosarum RU301 was sequenced. Two open reading frames (ORFs) were identified. The first ORF (lpcA) is homologous to a family of bacterial sugar transferases involved in LPS core tetrasaccharide biosynthesis. ORF2 (lpcB), in which Tn5 transposed, has no significant homology to any DNA in the GenBank-EMBL databases. Chemical characterization of LPS produced by strain RU301 demonstrated that the 3-deoxy-D-manno-2-octulosonic acid (Kdo) residue which normally attaches the core tetrasaccharide to the O chain was missing, suggesting that IpcB may encode a CMP-Kdo:LPS Kdo transferase.
Assuntos
Proteínas de Bactérias/genética , Hexosiltransferases/genética , Lipopolissacarídeos/biossíntese , Rhizobium leguminosarum/enzimologia , Transferases/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano , Dados de Sequência Molecular , Mutação , Rhizobium leguminosarum/genética , Homologia de Sequência de AminoácidosRESUMO
Effects of the two-component sensor-regulator pairs DctBD and NtrBC upon the expression of a dctA::phoA fusion from Rhizobium meliloti were determined under excess and limiting nitrogen concentrations in Escherichia coli. Results indicated that NtrBC affected transcription from the dctA promoter on a number of regulatory levels and under different physiological conditions in the heterologous host. However, NtrBC-dependent cross-talk was not observed in free-living R. meliloti under the conditions tested. Comparisons of the predicted amino acid sequences of DctD and NtrC from various sources indicated a specific region of the NtrC from rhizobia, which may have diverged from a consensus NtrC/DctD sequence to minimise interference between the two component systems, NtrBC and DctBD.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Transporte/genética , Proteínas de Ligação a DNA/biossíntese , Transportadores de Ácidos Dicarboxílicos , Regiões Promotoras Genéticas/genética , Sinorhizobium meliloti/genética , Transativadores , Sequência de Aminoácidos , Evolução Biológica , Ácidos Dicarboxílicos/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli , Dados de Sequência Molecular , Proteínas PII Reguladoras de Nitrogênio , Proteínas Recombinantes/biossíntese , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Sinorhizobium meliloti/metabolismo , Especificidade da Espécie , Simbiose/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The effect of 1-octen-3-ol (octenol) on catches of estuarine biting midges in encephalitis vector surveillance (EVS) traps was examined in southeastern Queensland. Octenol by itself was not attractive but appeared to act as a synergist with CO2 to increase catches of most species. For four of six species tested, a medium (about 6 mg/h) release rate of octenol captured the most individuals. Addition of light also increased the catch size of most species. Octenol in combination with CO2 could be used to enhance biting midge catch size, improving the sensitivity of surveillance for midges and the pathogens they vector.
Assuntos
Ceratopogonidae/efeitos dos fármacos , Ceratopogonidae/efeitos da radiação , Animais , Austrália , Dióxido de Carbono/farmacologia , Insetos Vetores , Luz , Octanóis/farmacologia , Vigilância da PopulaçãoRESUMO
The neurotoxin gene from Clostridium barati ATCC43756 was cloned as a series of overlapping polymerase chain reaction (PCR) generated fragments using primers designed to conserve toxin sequences previously published. The toxin gene has an open reading frame (ORF) of 1268 amino acids giving a calculated molecular mass of 141,049 Da. The sequence identity between the C. barati ATCC43756 and non-proteolytic C. botulinum 202F neurotoxins is 64.2% for the light chain and 73.6% for the heavy chain. This is much lower than reported identities for the type E neurotoxins from C. botulinum and C. butyricum (96% identity between light chains and 98.8% between the heavy chains). Previously identified conserved regions in other botulinal neurotoxins were also conserved in that of C. barati. An ORF upstream of the toxin coding region was revealed. This shows strong homology to the 3' end of the gene coding for the nontoxic-nonhemagglutinin (NTNH) component of the progenitor toxin from C. botulinum type C neurotoxin.
Assuntos
Toxinas Bacterianas/genética , Clostridium/genética , Genes Bacterianos/genética , Neurotoxinas/genética , Toxina Tetânica/genética , Sequência de Aminoácidos , Proteínas de Bactérias , Sequência de Bases , Clonagem Molecular , Sequência Conservada , Variação Genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Toxina Tetânica/classificaçãoRESUMO
Primers designed to conserved regions of botulinum and tetanus clostridial toxins were used to amplify DNA fragments from non-proteolytic Clostridium botulinum type F (202F) DNA using polymerase chain reaction technology. The fragments were cloned and the complete nucleotide sequence of the gene encoding type F toxin determined. Analysis of the nucleotide sequence demonstrated the presence of an open frame encoding a protein of 1274 amino acids, similar to other botulinum neurotoxins. Upstream of the toxin gene is the end of an open reading frame which encodes the C-terminus of a protein with homology to non-toxic-non-hemagglutinin component of type C progenitor toxin.
Assuntos
Toxinas Botulínicas/genética , Clostridium botulinum/genética , Genes Bacterianos , Neurotoxinas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Homologia de Sequência de AminoácidosRESUMO
Amplification of the gene encoding 23S rRNA of Plesiomonas shigelloides by polymerase chain reaction (PCR), with primers complementary to conserved regions of 16S and the 3' end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in a further two clones. In one the sequence of a single tRNA(Glu) was found which was absent from the other two. This variation in sequence suggests that the different clones may be derived from different ribosomal RNA operons.