933 resultados para MULTILOCUS ENZYME ELECTROPHORESIS
Resumo:
Glyoxysomes are specialized peroxisomes present in various plant organs such as germinating cotyledons or senescing leaves. They are the site of beta-oxidation and of the glyoxylate cycle. These consecutive pathways are essential to the maintenance of gluconeogenesis initiated by the degradation of reserve or structural lipids. In contrast to mitochondrial beta-oxidation, which is prevalent in animal cells, glyoxysomal beta-oxidation and the glyoxylate cycle have no direct access to the mitochondrial respiratory chain because of the impermeability of the glyoxysomal membrane to the reduced cofactors. The necessity of NAD(+) regeneration can conceivably be fulfilled by membrane redox chains and/or by transmembrane shuttles. Experimental evidence based on the active metabolic roles of higher plant glyoxysomes and yeast peroxisomes suggests the coexistence of two mechanisms, namely a reductase/peroxidase membrane redox chain and a malate/aspartate shuttle susceptible to transfer electrons to the mitochondrial ATP generating system. Such a model interconnects beta-oxidation, the glyoxylate cycle, the respiratory chain and gluconeogenesis in such a way that glyoxysomal malate dehydrogenase is an essential and exclusive component of beta-oxidation (NAD(+) regeneration). Consequently, the classical view of the glyoxylate cycle is superseded by a tentative reactional scheme deprived of cyclic character.
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To determine the feasibility of data transfer, an interlaboratory comparison was conducted on colon carcinoma cell line (DLD-1) proteins resolved by two-dimensional polyacrylamide gel electrophoresis either on small (6 x 7 cm) or large (16x18 cm) gels. The gels were silver-stained and scanned by laser densitometry, and the image obtained was analyzed using Melanie software. The number of spots detected was 1337+/-161 vs. 2382+/-176 for small vs. large format gels, respectively. After gel calibration using landmarks determined using pl and Mr markers, large- and small-format gels were matched and 712+/-36 proteins were found on both types of gels. Having performed accurate gel matching it was possible to acquire additional information after accessing a 2-D PAGE reference database (http://www.expasy.ch/ cgibin/map2/def?DLD1_HUMAN). Thus, the difference in gel size is not an obstacle for data transfer. This will facilitate exchanges between laboratories or consultation concerning existing databases.
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Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson's index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa ( http://www.dlst.org ), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases.
Resumo:
In recent years, new analytical tools have allowed researchers to extract historical information contained in molecular data, which has fundamentally transformed our understanding of processes ruling biological invasions. However, the use of these new analytical tools has been largely restricted to studies of terrestrial organisms despite the growing recognition that the sea contains ecosystems that are amongst the most heavily affected by biological invasions, and that marine invasion histories are often remarkably complex. Here, we studied the routes of invasion and colonisation histories of an invasive marine invertebrate Microcosmus squamiger (Ascidiacea) using microsatellite loci, mitochondrial DNA sequence data and 11 worldwide populations. Discriminant analysis of principal components, clustering methods and approximate Bayesian computation (ABC) methods showed that the most likely source of the introduced populations was a single admixture event that involved populations from two genetically differentiated ancestral regions - the western and eastern coasts of Australia. The ABC analyses revealed that colonisation of the introduced range of M. squamiger consisted of a series of non-independent introductions along the coastlines of Africa, North America and Europe. Furthermore, we inferred that the sequence of colonisation across continents was in line with historical taxonomic records - first the Mediterranean Sea and South Africa from an unsampled ancestral population, followed by sequential introductions in California and, more recently, the NE Atlantic Ocean. We revealed the most likely invasion history for world populations of M. squamiger, which is broadly characterized by the presence of multiple ancestral sources and non-independent introductions within the introduced range. The results presented here illustrate the complexity of marine invasion routes and identify a cause-effect relationship between human-mediated transport and the success of widespread marine non-indigenous species, which benefit from stepping-stone invasions and admixture processes involving different sources for the spread and expansion of their range.
Resumo:
BACKGROUND: Genome-wide association studies have linked CYP17A1 coding for the steroid hormone synthesizing enzyme 17α-hydroxylase (CYP17A1) to blood pressure (BP). We hypothesized that the genetic signal may translate into a correlation of ambulatory BP (ABP) with apparent CYP17A1 activity in a family-based population study and estimated the heritability of CYP17A1 activity. METHODS: In the Swiss Kidney Project on Genes in Hypertension, day and night urinary excretions of steroid hormone metabolites were measured in 518 participants (220 men, 298 women), randomly selected from the general population. CYP17A1 activity was assessed by 2 ratios of urinary steroid metabolites: one estimating the combined 17α-hydroxylase/17,20-lyase activity (ratio 1) and the other predominantly 17α-hydroxylase activity (ratio 2). A mixed linear model was used to investigate the association of ABP with log-transformed CYP17A1 activities exploring effect modification by urinary sodium excretion. RESULTS: Daytime ABP was positively associated with ratio 1 under conditions of high, but not low urinary sodium excretion (P interaction <0.05). Ratio 2 was not associated with ABP. Heritability estimates (SE) for day and night CYP17A1 activities were 0.39 (0.10) and 0.40 (0.09) for ratio 1, and 0.71 (0.09) and 0.55 (0.09) for ratio 2 (P values <0.001). CYP17A1 activities, assessed with ratio 1, were lower in older participants. CONCLUSIONS: Low apparent CYP17A1 activity (assessed with ratio 1) is associated with elevated daytime ABP when salt intake is high. CYP17A1 activity is heritable and diminished in the elderly. These observations highlight the modifying effect of salt intake on the association of CYP17A1 with BP.
Resumo:
Cytochrome P450 (CYP) enzymes play a pivotal role in the metabolism of many drugs. Inhibition of CYP enzymes usually increases the plasma concentrations of their substrate drugs and can thus alter the safety and efficacy of these drugs. The metabolism of many widely used nonsteroidal antiinflammatory drugs (NSAIDs) as well as the metabolism of the antidepressant venlafaxine is nown to be catalyzed by CYP enzymes. In the present studies, the effect of CYP inhibition on the armacokinetics and pharmacodynamics of NSAIDs and venlafaxine was studied in clinical trials with healthy volunteers and with a crossover design, by using different antifungal agents as CYP inhibitors. The results of these studies demonstrate that the inhibition of CYP enzymes leads to increased concentrations of NSAIDs. In most cases, the exposure to ibuprofen, diclofenac, etoricoxib, and meloxicam was increased 1.5to 2 fold when they were used concomitantly with antifungal agents. CYP2D6 inhibitor, terbinafine, substantially increased the concentration of parent venlafaxine, whereas the concentration of active moiety of venlafaxine (parent drug plus active metabolite) was only slightly increased. Voriconazole, an inhibitor of the minor metabolic pathway of venlafaxine, produced only minor changes in the pharmacokinetics of venlafaxine. These studies show that an evident increase in the concentrations of NSAIDs may be expected, if they are used concomitantly with CYP inhibitors. However, as NSAIDs are generally well tolerated, use of single doses of NSAIDs concomitantly with CYP inhibitors is not likely to adversely affect patient safety, whereas clinical relevance of longterm concomitant use of NSAIDs with CYP inhibitors needs further investigation. CYP2D6 inhibitors considerably affect the pharmacokinetics of venlafaxine, but the clinical significance of this interaction remains unclear.
Resumo:
This report outlines the discovery, the design and development of new compounds, and, structure-activity relationships for this drug category. Updated approaches to planned syntheses of new worthy ACE-inhibitors are also exploited.
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The effects of diet composition and ration size on the activities of key enzymes involved in intermediary metabolism were studied in the liver of gilthead sea bream (Sparus aurata). Highcarbohydrate, low-protein diets stimulated 6-phosphofructo 1-kinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) enzyme activities, while they decreased alanine aminotransferase (EC 2.6.1.2) activity. A high degree of correlation was found between food ration size and the activity of the enzymes 6-phosphofructo 1-kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (positive correlations) and fructose-1,6-bisphosphatase (EC 3.1.3.11) (negative correlation). These correlations matched well with the high correlation also found between ration size and growth rate in starved fish refed for 22 d. Limited feeding (5 g/kg body weight) for 22 d decreased the activities of the key enzymes for glycolysis and lipogenesis, and alanine aminotransferase activity. The findings presented here indicate a high level of metabolic adaptation to both diet type and ration size. In particular, adaptation of enzyme activities to the consumption of a diet with a high carbohydrate level suggests that a carnivorous fish like Sparus aurata can tolerate partial replacement of protein by carbohydrate in the commercial diets supplied in culture. The relationship between enzyme activities, ration size and fish growth indicates that the enzymes quickly respond to dietary manipulations of cultured fish.
Resumo:
The effects of diet composition and ration size on the activities of key enzymes involved in intermediary metabolism were studied in the liver of gilthead sea bream (Sparus aurata). Highcarbohydrate, low-protein diets stimulated 6-phosphofructo 1-kinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) enzyme activities, while they decreased alanine aminotransferase (EC 2.6.1.2) activity. A high degree of correlation was found between food ration size and the activity of the enzymes 6-phosphofructo 1-kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (positive correlations) and fructose-1,6-bisphosphatase (EC 3.1.3.11) (negative correlation). These correlations matched well with the high correlation also found between ration size and growth rate in starved fish refed for 22 d. Limited feeding (5 g/kg body weight) for 22 d decreased the activities of the key enzymes for glycolysis and lipogenesis, and alanine aminotransferase activity. The findings presented here indicate a high level of metabolic adaptation to both diet type and ration size. In particular, adaptation of enzyme activities to the consumption of a diet with a high carbohydrate level suggests that a carnivorous fish like Sparus aurata can tolerate partial replacement of protein by carbohydrate in the commercial diets supplied in culture. The relationship between enzyme activities, ration size and fish growth indicates that the enzymes quickly respond to dietary manipulations of cultured fish.
Resumo:
ELISAs have been applied to pesticide residue analysis due to their high sensitivity and selectivity. However, some ELISAs performance may be affected by matrix components. In this work, ELISA for carbaryl in water samples was checked for interference by naturally occurring fulvic acids. The results suggested that the high fulvic acid concentration (ž³30 mg L-1) and acidic pH conditions (pH 4.0) interfere with the signal detection decreasing the method sensitivity. A dilution of the samples and adjust to pH 8.0 are appropriate to minimize the matrix interferences in the ELISA method. Good correlation between ELISA and HPLC-DAD results was observed.
Resumo:
Fungi and bacteria are key agents in plant litter decomposition in freshwater ecosystems. However, the specific roles of these two groups and their interactions during the decomposition process are unclear. We compared the growth and patterns of degradativeenzymes expressed by communities of bacteria and fungi grown separately and in coexistence on Phragmites leaves. The two groups displayed both synergistic and antagonistic interactions. Bacteria grew better together with fungi than alone. In addition, there was a negative effect of bacteria on fungi, which appeared to be caused by suppression of fungal growth and biomass accrual rather than specifically affecting enzyme activity. Fungi growing alone had a high capacity for the decomposition of plant polymers such as lignin, cellulose, and hemicellulose. In contrast, enzyme activities were in general low when bacteria grew alone, and the activity of key enzymes in the degradation of lignin and cellulose (phenol oxidase and cellobiohydrolase) was undetectable in the bacteria-only treatment. Still, biomass-specific activities of most enzymes were higher in bacteria than in fungi. The low total activity and growth of bacteria in the absence of fungi in spite of apparent high enzymatic efficiency during the degradation of many substrates suggest that fungi provide the bacteria with resources that the bacteria were not able to acquire on their own, most probably intermediate decomposition products released by fungi that could be used by bacteria
Resumo:
Fumonisins are mycotoxins occurring worldwide, mainly in maize and maize-based food products, which could affect animal and human health. This paper reviews analytical methodologies for the determination of these fungal toxins in foods. It includes extraction, cleanup, derivatization procedures, detection, quantification, and confirmation procedures. Initial attempts at gas chromatographic methods and thin layer chromatography were supplanted by liquid chromatographic methods, mainly performed with fluorometric detection, or mass spectrometry detection, enabling the analysis of polar and thermolabile chemicals without chemical derivatization, which results in lower limits of detection. Alternative methods, such as enzyme linked immunosorbent assay or zone capillary zone electrophoresis, are also described.
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The inhibition of the enzyme acetylcholinesterase is considered as a strategy for the treatment of Alzheimer's disease, senile dementia, ataxia, and myasthenia gravis. Three lanostane- and two cycloartane-type triterpenes, together with two mulinane-type diterpenes were isolated from petroleum ether extract of the whole shrub of Azorella trifurcata (Gaertn.) Pers. Their effect on the enzyme acetylcholinesterase was assessed as well. In addition, this is the first report of these triterpenes in the genus Azorella.