RESUMO
BACKGROUND: We performed post hoc analyses to evaluate the effect of humanized monoclonal antibody mepolizumab in patients with severe eosinophilic asthma previously treated with omalizumab. METHODS: Data were collected from two randomized double-blind, placebo-controlled studies: MENSA (NCT01691521: 32-week treatment phase) and SIRIUS (NCT01691508: 24-week treatment phase). Active treatment was 75 mg intravenous mepolizumab (MENSA) or 100 mg subcutaneous mepolizumab (MENSA, SIRIUS). Patients had evidence of eosinophilic inflammation ≥150 cells/µl (at screening) or ≥300 cells/µl (during the previous year). Primary outcomes were the rate of exacerbations (MENSA) and the percentage reduction in oral corticosteroid (OCS) dose (SIRIUS). Other outcomes included lung function (forced expiratory volume in 1 s and morning peak expiratory flow), Asthma Control Questionnaire (ACQ-5), St George's Respiratory Questionnaire (SGRQ) scores, and safety. RESULTS: Overall, 576 patients were included from MENSA and 135 from SIRIUS, with 13% and 33% previously receiving omalizumab, respectively. In MENSA, mepolizumab reduced the rate of exacerbations by 57% (prior omalizumab) and 47% (no prior omalizumab) vs placebo. In SIRIUS, reductions in OCS use were comparable regardless of prior omalizumab use. Despite reducing chronic OCS use, mepolizumab also resulted in similar reductions in exacerbation rate relative to placebo in both subgroups. Asthma control and quality of life improved with mepolizumab vs placebo in both studies independent of prior omalizumab use, as shown by ACQ-5 and SGRQ scores. Adverse events were also comparable irrespective of prior omalizumab use. CONCLUSIONS: These post hoc analyses indicate that patients with severe eosinophilic asthma respond positively to mepolizumab regardless of prior use of omalizumab.
Assuntos
Antiasmáticos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Asma/diagnóstico , Asma/tratamento farmacológico , Eosinofilia/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Omalizumab/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Testes de Função Respiratória , Retratamento , Índice de Gravidade de Doença , Resultado do Tratamento , Adulto JovemRESUMO
Chronic non-healing wounds are a major health problem with resident bacteria strongly implicated in their impaired healing. A rapid-screen to provide detailed knowledge of wound bacterial populations would therefore be of value and help prevent unnecessary and indiscriminate use of antibiotics-a process associated with promoting antibiotic resistance. We analysed chronic wound fluid samples, which had been assessed for microbial content, using 20 different fluorescent labelled peptide substrates to determine whether protease activity correlated with the bacterial load. Eight of the peptide substrates showed significant release of fluorescence after reaction with some of the wound samples. Comparison of wound fluid protease activities with the microbiological data indicated that there was no correlation between bacterial counts and enzyme activity for most of the substrates tested. However, two of the peptide substrates produced a signal corresponding with the microbial data revealing a strong positive correlation with Pseudomonas aeruginosa numbers. This demonstrated that short fluorescent labelled peptides can be used to detect protease activity in chronic wound fluid samples. The finding that two peptides were specific indicators for the presence of P. aeruginosa may be the basis for a diagnostic test to determine wound colonisation by this organism.
Assuntos
Carga Bacteriana , Peptídeo Hidrolases/análise , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Infecção dos Ferimentos/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Doença Crônica , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Infecções por Pseudomonas/patologia , Infecção dos Ferimentos/patologia , Adulto JovemRESUMO
AIMS: To determine the effect of pH, temperature, desiccation, ethylenediaminetetraacetic acid (EDTA) and desferrioxamine B (DFO) on Panton-Valentine leukocidin-positive community acquired methicillin-susceptible Staphylococcus aureus (PVL +ve CA-MSSA) biofilm formation. METHODS AND RESULTS: Biofilms from PVL +ve CA-MSSA (clinical isolate) were subjected to pH, temperature, desiccation, EDTA and DFO. PVL +ve CA-MSSA were more resistant to pH and heat than their planktonic equivalents. Desiccation studies demonstrated that PVL +ve CA-MSSA biofilms were more refractory to the treatment than planktonic cells. Significant inhibition of PVL +ve CA-MSSA biofilm formation was observed in the presence of 1 mmol l(-1) EDTA. Low concentrations (2·5 µmol l(-1) ) of DFO enhanced the growth of PVL +ve CA-MSSA biofilms. At higher concentrations (1 mmol l(-1) ), DFO did inhibit the growth but not as much as EDTA. A combination of EDTA and DFO inhibited PVL +ve CA-MSSA biofilm formation at lower concentrations than either alone. CONCLUSIONS: This study demonstrates that PVL +ve CA-MSSA biofilms are resistant to environmental stress but their growth can inhibited effectively by a mixture of EDTA and DFO. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibition of biofilm formation by PVL +ve CA-MSSA using chelating agents has not been previously reported and provides a practical approach to achieve the disruption of these potentially important biofilms formed by an emerging pathogen.
Assuntos
Biofilmes/efeitos dos fármacos , Quelantes/farmacologia , Staphylococcus aureus/crescimento & desenvolvimento , Toxinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Desferroxamina/farmacologia , Dessecação , Ácido Edético/farmacologia , Exotoxinas/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Leucocidinas/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Estresse FisiológicoRESUMO
AIMS: To evaluate a new range of chromogenic substrates for the detection of beta-galactosidase activity in coliforms and to compare their performance in agar media and broths. METHODS AND RESULTS: Sixteen novel galactoside substrates were prepared and incorporated into agar and broth. Their performance was compared using Escherichia coli (five strains), Salmonella (two strains), Enterobacter (two strains), Klebsiella, Pseudomonas, Listeria, Serratia, Shigella, Citrobacter, Proteus and Staphylococcus as well as pathological urine samples. The six substrates out of the initial 16 that showed the greatest sensitivity were VQE-gal, VQM-gal, VLPr-gal, VLE-gal, VLM-gal and VBzTM-gal, whose released chromophores were red, brown or purple. VQE-gal and VLPr-gal were studied in greater detail and were incorporated into agar medium. Coliform colonies appeared red and brown respectively, following incubation at 37 degrees C for 24 h; however, positive results were obtained within a working day. The VQE-gal medium was compared with some commercially available media. CONCLUSIONS: The range of substrates described can be used in broths as well as in agars. The VQE agar allows the detection of coliforms within a working day. VQE-gal medium proved to be more sensitive when compared to other available chromogenic media and allows the unambiguous detection of coliforms.
Assuntos
Compostos Cromogênicos/metabolismo , Enterobacteriaceae/isolamento & purificação , Enterobacteriaceae/fisiologia , Ágar/química , Bactérias/classificação , Bactérias/enzimologia , Bactérias/isolamento & purificação , Compostos Cromogênicos/química , Meios de Cultura/química , Enterobacteriaceae/classificação , Enterobacteriaceae/enzimologia , Escherichia coli/enzimologia , Escherichia coli/isolamento & purificação , Escherichia coli/fisiologia , Isopropiltiogalactosídeo/metabolismo , Lactose/metabolismo , Urina/microbiologia , beta-Galactosidase/metabolismoRESUMO
AIMS: Elucidation of the regulation of ChiB production in Aspergillus nidulans. METHODS AND RESULTS: Mutational inactivation of the A. nidulans chiB gene resulted in a nonautolytic phenotype. To better understand the mechanisms controlling both developmental progression and fungal autolysis, we examined a range of autolysis-associated parameters in A. nidulans developmental and/or autolytic mutants. Investigation of disorganization of mycelial pellets, loss of biomass, extra-/intracellular chitinase activities, ChiB production and chiB mRNA levels in various cultures revealed that, in submerged cultures, initialization of autolysis and stationary phase-induced ChiB production are intimately coupled, and that both processes are controlled by the FluG-BrlA asexual sporulation regulatory pathway. ChiB production does not affect the progression of apoptotic cell death in the aging A. nidulans cultures. CONCLUSIONS: The endochitinase ChiB plays an important role in autolysis of A. nidulans, and its production is initiated by FluG-BrlA signalling. Despite the fact that apoptosis is an inseparable part of fungal autolysis, its regulation is independent to FluG-initiated sporulation signalling. SIGNIFICANCE AND IMPACT OF THE STUDY: Deletion of chiB and fluG homologues in industrial filamentous fungal strains may stabilize the hyphal structures in the autolytic phase of growth and limit the release of autolytic hydrolases into the culture medium.
Assuntos
Aspergillus nidulans/enzimologia , Aspergillus nidulans/genética , Autólise , Quitinases/metabolismo , Proteínas Fúngicas/metabolismo , Anticorpos Antifúngicos/imunologia , Aspergillus nidulans/crescimento & desenvolvimento , Aspergillus nidulans/imunologia , Autólise/genética , Autólise/metabolismo , Biomassa , Quitina/metabolismo , Quitinases/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica , Fenótipo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismoRESUMO
Mutations in the PKD1 gene are responsible for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). This gene encodes a large membrane associated glycoprotein, polycystin-1, which is predicted to contain a number of extracellular protein motifs, including a C-type lectin domain between amino acids 403--532. We have cloned and expressed the PKD1 C-type lectin domain, and have demonstrated that it binds carbohydrate matrices in vitro, and that Ca(2+) is required for this interaction. This domain also binds to collagens type I, II and IV in vitro. This binding is greatly enhanced in the presence of Ca(2+) and can be inhibited by soluble carbohydrates such as 2-deoxyglucose and dextran. These results suggest that polycystin-1 may be involved in protein-carbohydrate interactions in vivo. The data presented indicate that there may a direct interaction between the PKD1 gene product and an ubiquitous extracellular matrix (ECM) protein.
Assuntos
Cálcio/metabolismo , Metabolismo dos Carboidratos , Proteínas da Matriz Extracelular/metabolismo , Lectinas/química , Proteínas/metabolismo , Sequência de Aminoácidos , Cálcio/química , Sequência de Carboidratos , Carboidratos/química , Cátions Bivalentes , Linhagem Celular , Colágeno/química , Colágeno/metabolismo , Proteínas da Matriz Extracelular/química , Humanos , Lectinas Tipo C , Dados de Sequência Molecular , Proteínas/química , Proteínas Recombinantes de Fusão/química , Alinhamento de Sequência , Transdução de Sinais , Canais de Cátion TRPPRESUMO
Cyst fluid samples obtained from eight patients with autosomal dominant polycystic kidney disease (ADPKD) were analysed for the presence of the basement membrane component laminin and its breakdown products, using ELISA and immunoblotting techniques. Whole laminin was not detected, whereas laminin fragments of 270, 155, 87, 56, and 14 kDa were detected at a mean total value of approximately 0.5 microgram/ml. The laminin fragments were assessed for their effect on cultured normal and ADPKD epithelial cells. Both cell types showed accelerated growth under these conditions. These findings suggest that basement membrane degradative fragments present in cyst fluid may contribute to cystic epithelial cell proliferation and may therefore be important in the pathogenesis of ADPKD.
Assuntos
Laminina/análise , Rim Policístico Autossômico Dominante/química , Adulto , Divisão Celular/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Exsudatos e Transudatos/química , Feminino , Humanos , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Laminina/química , Laminina/fisiologia , Masculino , Pessoa de Meia-Idade , Peso Molecular , Dodecilsulfato de SódioRESUMO
The kinetic characteristics of beta-galactosidases from bovine liver and testes, Escherichia coli, Aspergillus niger and Jack bean were studied using five newly-developed colorimetric substrates. All the chromophores released by enzyme hydrolysis had high extinction coefficients in the visible region of the spectrum. Varying amounts of substrate inhibition were found with each of these substrates (VBzTM-Gal, VLM-Gal, VLPr-Gal, VQM-Gal and VQPr-Gal), but this was not a significant problem if the correct assay conditions were used. The substrates attached particularly tightly to the active centre of E. coli beta-D-galactosidase resulting in low Km values. The data suggest that the chemical properties of the heterocyclic portion of the aglycone distant from the glycosidic oxygen do not affect the substrate specificity and the substrate inhibition can be attributed to interactions not involving the catalytic site. When the product of the maximum observed velocity (Vm) and the molar absorption coefficient is calculated for each substrate, the relative merits of the substrates for the assay of each enzyme can be assessed. The beta-D-galactosidases from fungal and bacterial sources hydrolysed the substrates most efficiently, indicating that they may be of particular value in areas of molecular biology and biotechnology.
Assuntos
Compostos Cromogênicos/metabolismo , Galactosídeos/metabolismo , beta-Galactosidase/metabolismo , Animais , Aspergillus niger/enzimologia , Sítios de Ligação , Sequência de Carboidratos , Bovinos , Escherichia coli/enzimologia , Galactosídeos/química , Cinética , Fígado/enzimologia , Masculino , Dados de Sequência Molecular , Estrutura Molecular , Especificidade por Substrato , Testículo/enzimologiaRESUMO
High field 1H-NMR spectra of fluid collected from the cysts of six renal transplant recipients with autosomal dominant polycystic kidney disease (ADPKD) have been measured and the major metabolite signals assigned. Quantitative NMR measurements have revealed a combination of unusual biochemical features of the cystic fluids that shows them to be distinct from both blood plasma and urine. Isoleucine, lysine, threonine and valine were present at mM concentrations, in cyst fluid and in some cases levels up to 2 orders of magnitude higher than normal plasma or urine were recorded. Mean glucose concentrations in the cyst fluids ranged from 3.4-9.6 mM and a number of organic acids and bases, including acetate, lactate, succinate, creatinine and dimethylamine were also present at high concentration and in different ratios to those found in either plasma or urine. The majority of cyst fluids examined also contained significant quantities of glycoproteins with characteristic 1H-NMR signals from N-acetyl groups of amino-sugar and sialic acid side chains which had a high degree of molecular mobility (as indicated by their relatively long T2 relaxation times, greater than 120 ms). High levels of ethanol (0.5-12.6 mM/l) were found in all fluid samples from the six transplanted patients (confirmed by conventional analysis). In general there was little variation in the 1H-NMR spectral patterns of either the intra- or interpatient cyst fluids, although the contribution of the protein macromolecules to individual spectra was lower in a few cysts. This constancy of biochemical composition probably reflects the chronic nature of the accumulation of cyst fluid and a long turnover of the cystic fluid components which has the effect of averaging composition. These findings suggest that the dynamic composition of cyst fluid from ADPKD patients is unique among the other body fluids and that the unusual composition may be related to epithelial polarity reversal of the cystic epithelium which could also contribute to the growth of the cysts.
Assuntos
Rim Policístico Autossômico Dominante/química , Aminoácidos/análise , Transporte Biológico , Humanos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Concentração OsmolarRESUMO
Laminin isolated from human placenta was subjected to prolonged pepsin digestion. Seven peptide fragments (designated N1 to N7) were separated by ion-exchange chromatography and gel filtration and characterised by SDS-polyacrylamide gel electrophoresis and immunoblotting. The molecular size of the laminin fragments varied from approx. 900,000 (N1) to 28,000 (N7). Epithelial cells obtained from normal kidneys and patients with autosomal dominant polycystic kidney disease (ADPKD) were cultured. The incorporation of [3H]thymidine was measured over 96 h to determine the effect of the addition of the different fragments and whole laminin from EHS tumour to the cells. The rate of growth of both normal and polycystic cells was increased in the presence of the laminin fragments but this effect was more pronounced in the ADPKD cells.
Assuntos
Rim/efeitos dos fármacos , Laminina/farmacologia , Fragmentos de Peptídeos/farmacologia , Placenta/metabolismo , Rim Policístico Autossômico Dominante/patologia , Divisão Celular , Células Cultivadas , Epitélio/efeitos dos fármacos , Humanos , Rim/patologia , Laminina/isolamento & purificação , Pepsina A , Fragmentos de Peptídeos/isolamento & purificação , Rim Policístico Autossômico Dominante/metabolismoRESUMO
The labelling of pepsin-digested human glomerular basement membrane (pHGBM) with a newly developed fluorescent iodine acceptor 7-hydroxy-coumarin-3-acetic acid N-hydroxysucciniimydyl ester (CASE) is described. The binding of a monoclonal antibody to pHGBM was assessed by radiobinding assays, and when directly iodinated pHGBM was used there was no apparent binding. When CASE was conjugated to pHGBM prior to iodination 11% binding was achieved. CASE acting as an iodine acceptor may be useful for proteins containing few or inaccessible tyrosine residues or which are destroyed by introduction of 125I. Since CASE is fluorescent, small amounts of material can be detected during isolation prior to iodination.
Assuntos
Glomérulos Renais/ultraestrutura , Pepsina A/metabolismo , Umbeliferonas/metabolismo , Membrana Basal/metabolismo , Fluorescência , Humanos , Ensaio RadioliganteRESUMO
Polycystin-1 is a putative 460 kDa membrane protein with a unique structure and is possibly representative of a new family of proteins. Its structure suggests an involvement in cell signalling and cell-matrix interactions. The amino acid sequence of polycystin-1 has to date been predicted from its gene sequence. This, to our knowledge, is the first report of the isolation and analysis of polycystin-1 at the protein level using mass spectrometry to confirm its predicted structure. The availability of purified polycystin-1 will allow a new approach to unravelling the complexity of the cell-cell and cell-matrix interactions of this large molecule in normal cells and its perturbation in disease.
Assuntos
Espectrometria de Massas/métodos , Proteínas/química , Proteínas/isolamento & purificação , Western Blotting , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Concentração de Íons de Hidrogênio , Ligação Proteica , Conformação Proteica , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Canais de Cátion TRPP , Tripsina/metabolismoRESUMO
An improved method is described for the detection of Escherichia coli by an enzyme linked immunofilter assay (ELIFA) using nitrocellulose membrane sandwiched between two 96-well plates. The incorporation of a pumping system permits a continuous flow of reagents and/or wash fluids through the membrane and provides an assay procedure capable of detecting 10(3) bacteria per well within 40 min. Quantitative bacterial detection was based on precipitated chromogen determined by scanning densitometry. The procedure represents a significant improvement in assay time and/or sensitivity over previously described ELIFA and ELISA methods for whole bacterial cells.
Assuntos
Escherichia coli/isolamento & purificação , Técnicas Imunoenzimáticas , Escherichia coli/imunologia , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/imunologia , Filtração/métodos , Técnicas Imunoenzimáticas/instrumentação , Sensibilidade e EspecificidadeRESUMO
Damage to the kidneys is one of the primary toxic actions of metals. Nephrotoxic substances not only cause renal disease directly, but they can also destroy renal reserve capacity, potentially placing those people with additional risk factors, such as diabetes, hypertension, cardiovascular disease, and genetic predispositions, at greater risk. To detect nephrotoxicity in people at a stage where intervention can be effective, sensitive methods are needed. One of the major advantages of using sensitive biomarkers of renal damage is that people who may be particularly susceptible to renal damage can be identified early, at a reversible stage of damage, and the progression to end-stage renal disease may be halted or delayed. Various categories of tests can be used to detect effects of nephrotoxic substances on the kidney. Through the use of biomarkers of damage to various parts of the nephron, U.S. and European studies have both shown a similar pattern of damage among men occupationally exposed to cadmium. These studies indicate various thresholds of renal effects, which researchers suggest represent a cascade of progressively severe damage to the kidney. Research into new biomarkers of damage caused by exposure to nephrotoxic substances centers around mechanisms of cell death, including necrosis and apoptosis; mechanisms of cell growth, regeneration, and proliferation, including factors that control cell cycle, influence gene expression, and modulate nucleic acid synthesis; and genetic factors that increase susceptibility to renal disease. Examples of types of candidate biomarkers include cytokines, lipid mediators, growth factors, transcription factors and protooncogenes, extracellular matrix components (collagen, glycoproteins, and proteoglycans), and cell adhesion molecules. Research into new categories of biomarkers may provide additional insights into the mechanisms of damage caused by nephrotoxins.
Assuntos
Intoxicação por Cádmio/diagnóstico , Nefropatias/induzido quimicamente , Nefropatias/diagnóstico , Nefrologia/métodos , Biomarcadores/análise , Limiar Diferencial , Humanos , Nefrologia/tendênciasRESUMO
A number of chemicals may adversely affect one or more of the anatomical structures of the kidney, such as the glomerulus, the tubular apparatus, the medullary, or interstitial cells. To recognize subclinical renal dysfunction, a battery of new, non-invasive tests was applied in comparison to established ones. The study on cadmium exposed subjects, performed within the framework of a collaborative European research project, exemplifies the concept of target selectivity within a nephron. One hundred seventy-two subjects were classified according to urinary cadmium excretion as controls (< 1.5 micrograms/g creatinine), or subjects with moderate or high cadmium body burden (1.5 to 5 micrograms/g creatinine, > 5 micrograms/g creatinine). Twenty-six urinary analytes (such as serum derived proteins, tubular enzymes, eicosanoids) and four plasma markers, related to the function or integrity of specific nephron segments, were investigated in a cross-sectional study. The group with the moderate cadmium body burden showed alterations of proximal tubular integrity, that is, increased excretion of tubular brush-border antigens. The group with higher cadmium body burden revealed an involvement of the whole nephron. The most prominent quantitative changes were found for the glomerular markers high molecular weight proteins, and thromboxane B2 and for the proximal tubular markers retinol binding protein, alpha 1-microglobulin, N-acetyl-beta-D-glucosaminidase, and the intestinal alkaline phosphatase. A diagnostic approach to screen for nephrotoxicity due to environmental hazards like cadmium should include proximal tubular markers (alpha 1-microglobulin and tubular enzymes, that is, intestinal alkaline phosphatase) but the measurement of glomerular markers is also advisable.
Assuntos
Biomarcadores/urina , Cádmio/toxicidade , Néfrons/efeitos dos fármacos , Adulto , Biomarcadores/sangue , Carga Corporal (Radioterapia) , Análise Discriminante , Feminino , Humanos , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/fisiopatologia , Túbulos Renais Distais/efeitos dos fármacos , Túbulos Renais Distais/fisiopatologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/fisiopatologia , Alça do Néfron/efeitos dos fármacos , Alça do Néfron/fisiopatologia , Masculino , Pessoa de Meia-Idade , Peso Molecular , Néfrons/fisiopatologia , Exposição Ocupacional , Proteínas/química , Proteinúria/urina , Tromboxano B2/urinaRESUMO
The use of a new substrate 2-(2-(4-(beta-D-galactopyranosyloxy)-3- methoxyphenyl)-vinyl)-3-methylbenzothiazolium toluene-4-sulphonate (VB-zTM-gal) is described for the detection of beta-galactosidase activity in colonies of wild type and mutant strains of Escherichia coli. On enzymic hydrolysis this substrate, which is soluble in water, released a chromophore which is red at pH 7 and bound to cellulose and nitrocellulose. The best procedure for the detection of activity was to grow colonies on standard nitrocellulose membranes (pore size 0.45 microns) laid onto an agar plate and to float the membranes over a solution of the substrate. Coloured colonies developed within 3 min, which were stable at 4 degrees C for several days, and this identified the expression of beta-galactosidase activity. This was found to be more specific than methods using triphenyltetrazolium or Eosin Methylene Blue media, and more economical than methods using X-gal (5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside). VBzTM-gal should have applications in gene cloning technology and in the detection of coliform organisms in polluted water.
Assuntos
Compostos Cromogênicos , Escherichia coli/enzimologia , Galactosídeos , Compostos de Vinila , beta-Galactosidase/análise , Galactosídeos/análise , Indóis/análiseRESUMO
The propionate (Pro), decanoate (Dec) and laurate (Lau) esters of 5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothiazoline++ +-3-acetic acid were assessed as substrates for lipase and esterase. On hydrolysis these substrates yield an intensely red coloured phenol which could be assayed at 505 nm. The Pro ester was an effective substrate for porcine esterase and was hydrolysed at a rate 20 times greater than the Lau and Dec esters. Conversely, Pseudomonas lipase had a high activity towards the Lau and Dec esters, especially in the presence of bovine serum albumin, but little activity towards the Pro ester. The Dec and Lau were used to detect lipolytic activity in Pseudomonas strains associated with milk spoilage. For this purpose, the substrates were absorbed onto filter paper disks, which were placed over bacterial colonies growing on agar plates; activity was indicated by bright red colouration of discs within 2 h. Escherichia coli colonies hydrolysed the Pro but not the Lau or Dec esters.
Assuntos
Benzoatos/metabolismo , Compostos Cromogênicos/metabolismo , Escherichia coli/enzimologia , Esterases/metabolismo , Lipase/metabolismo , Pseudomonas/enzimologia , Tiazóis/metabolismo , Ésteres/metabolismo , Hidrólise , Pseudomonas fluorescens/enzimologiaRESUMO
Immunochemical methods were developed for monitoring cyclodextrin (CD) glycosyltransferase (CGTase) production and growth of an industrial CD-producing Bacillus macerans strain. Extracellular concentrations of CGTase released into a non-transparent culture medium during a 44 h long fermentation were detected by an indirect antigen inhibition enzyme-linked immunosorbent assay (ELISA). The ELISA was sensitive (minimal detection level 6 ng ml-1) and highly reproducible (coefficients of variation < or = 1.2 and 5.9%, within-runs and between-runs, respectively) compared to assays of CGTase activity (coefficients of variation < or = 4.2 and 7.0%, respectively). The ELISA, in combination with enzyme activity measurements, was useful to detect the decrease in the specific CGTase activities after 36 h of incubation, which was clearly indicative of the proteolytic degradation of CGTase. B. macerans cell numbers were estimated using an enzyme-linked immunofilter assay (ELIFA). The assay took less than 1 h and the coefficients of variation within and between-runs (2.9-6.4%) were considerably less than for viable counting (10.6-15.4%). In the exponential phase of growth, ELIFA results correlated more closely with the cell counting based on total protein than with viable counts. Nevertheless, in the phase of cell lysis, the bacterial cell number was systematically underestimated by ELIFA in comparison to both viable cell number and total protein determinations. Thus cell antigens detected with immunological procedures might be lost during the transition from vegetative cells to spores. On the other hand, the ELIFA procedure was specific for B. macerans cells and was a better indicator of the onset of the different growth phases than the cell numbers calculated from the protein assay.
Assuntos
Bacillus/enzimologia , Bacillus/crescimento & desenvolvimento , Ensaio de Imunoadsorção Enzimática/métodos , Glucosiltransferases/biossíntese , Filtração , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A large number of compounds, which are in common usage in industry and medicine, are potentially nephrotoxic. Renal damage and disease resulting from toxic exposure is progressive and will, if unarrested, culminate in irreversible renal disease. There is, therefore, a need to develop a battery of tests with which to monitor and characterise the nephrotoxic cascade. A European-wide study compared biomarker profiles of adult male workers who were exposed to heavy metals or solvents. It became apparent that the urinary profiles varied with the nature of the toxin, reflecting the functional region of the kidney affected and also the severity of the damage. Children are a particularly vulnerable group and the investigation of range of biomarkers indicated that they were indeed susceptible to nephrotoxic pollutants in their environment. It is proposed that a small cohort of tests should be used to monitor the early (pre-clinical stages) of renal damage or dysfunction; these can be supplemented if necessary by additional specific tests. In the future better information on at-risk populations and genetic information will enable the determination of individual susceptibility to be assessed more precisely.
Assuntos
Biomarcadores/urina , Poluentes Ambientais/toxicidade , Rim/efeitos dos fármacos , Adulto , Humanos , Masculino , Exposição Ocupacional , UrináliseRESUMO
Renal transplant recipients were divided into five categories according to their clinical course from transplantation to their discharge from hospital. Total N-acetyl-beta-D-glucosaminidase (NAG) activity in urine was determined using a chromogenic substrate 2-methoxy-4-(2'-nitrovinyl)-phenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside. The isoenzyme composition of the urine of each patient was determined by semi-automated DEAE-cellulose chromatography. Although raised NAG activity was found in stable transplant patients compared to controls, the level of activity was constant and no change in the isoenzyme profile was found. In reversible rejection there was a marked increase in the intermediate forms, particularly I2 and a concomitant fall in the relative amount of the A-form present but the profile became normal when the patient stabilised. Much more complex patterns were observed in patients who did not respond to treatment. Both the B and I forms were elevated with a fall in the A-form and in one case excretion of the serum As form was observed. The intermediate forms were always increased in rejection.