Extração de esterase de fígado suíno (PLE)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of the administration of a catalase inhibitor into the fourth cerebral ventricle on cardiovascular responses in spontaneously hypertensive rats exposed to sidestream cigarette smoke

OBJECTIVE: ,,,,,Previous studies have demonstrated a relationship between brain oxidative stress and cardiovascular regulation. We evaluated the effects of central catalase inhibition on cardiovascular responses in spontaneously hypertensive rats exposed to sidestream cigarette smoke. ,,,, ,,,, ,,,,,METHODS: ,,,,,Male Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SH) (16 weeks old) were implanted with a stainless steel guide cannula leading into the fourth cerebral ventricle (4th V). The femoral artery and vein were cannulated for arterial pressure and heart rate measurement and drug infusion, respectively. The rats were exposed to sidestream cigarette smoke for 180 minutes/day, 5 days/week for 3 weeks (CO: 100-300 ppm). The baroreflex was tested using a pressor dose of phenylephrine (8 μg/kg, bolus) and a depressor dose of sodium nitroprusside (50 μg/kg, bolus). Cardiovascular responses were evaluated before and 5, 15, 30 and 60 minutes after injection of a catalase inhibitor (3-amino-1,2,4-triazole, 0.001 g/100 μL) into the 4th V. ,,,, ,,,, ,,,,,RESULTS: ,,,,,Vehicle administration into the 4th V did not affect the cardiovascular response, whereas administration of the central catalase inhibitor increased the basal HR and attenuated the bradycardic peak (p<0.05) to a greater extent in WKY rats exposed to sidestream cigarette smoke than in WKY rats exposed to fresh air. However, in spontaneously hypertensive rats, the effect of the catalase inhibitor treatment was stronger in the fresh air condition (p<0.05). ,,,, ,,,, ,,,,,CONCLUSION: ,,,,,Administration of a catalase inhibitor into the 4th V combined with exposure to sidestream cigarette smoke has a stronger effect in WKY rats than in SH rats.

Late administration of a specific COX-2 inhibitor does not treat and/or prevent progression of gastric tumors in rats submitted to duodenogastric reflux procedure

PURPOSE:To assess whether late introduction of a specific COX-2 inhibitor (Meloxicam) can treat and/or prevent the progression of tumors in the stomach of rats submitted to duodenogastric reflux. METHODS: Seventy five male Wistar rats, weighing 150 grams, were submitted to the induction of duodenogastric reflux through the pylorus. At 36 weeks of follow-up were established three experimental groups: DGR36 sacrificed immediately, DGR54 and DGR54MLX both sacrificed at 54th week of follow-up . The animals of the latter group were fed with a rat chow premixed with Meloxicam (2.0 mg/ kg feed; 0.3 mg / kg bw / day) and the other two with standard rat chow. The lesions found in the pyloric mucosa and gastrojejunal anastomosis were analyzed macroscopically and histologically. For statistical analysis was adjusted a generalized linear model assuming a binomial distribution with LOGIT link function. RESULTS: No significant differences were found when comparing the incidences of benign tumor lesions (Adenomatous Hyperplasia), p=0.4915, or malignant (Mucinous Adenocarcinoma), p=0.2731, among groups. CONCLUSION: Late introduction of specific COX-2 inhibitor (Meloxicam) did not treat and was not able to prevent the progression of tumoral lesions induced by duodenogastric reflux in the rat stomachs.

Esterase patterns and phylogenetic relationships of Drosophila species in the saltans subgroup (saltans group)

The esterase patterns of sixteen strains from four species in the saltans subgroup were analyzed using polyacrylamide gel electrophoresis. Thirty-four esterase bands were detected. By using alpha and beta naphthyl acetates as substrates, they were classified in 18 alpha-esterases (they hydrolyse the alpha-naphtyl substrate), 15 beta-esterases (they hydrolyse the beta-naphtyl substrate) and 1 alpha/beta-esterase (it hydrolyses the alpha and beta-naphtyl substrates). Among the alpha-esterases, three were detected exclusively in males. Malathion, Eserine and pCMB were used as inhibitors in order to characterize biochemically the esterases. The results indicated the presence of cholinesterases, carboxylesterases and acetylesterases. The degree of mobility of the bands in the gels, their specificity to alpha and beta naphthyl acetates and the results of the inhibition tests allowed us to recognize tentatively nine genetic loci. Phylogenetic relationships among species inferred on the basis of the esterase patterns by PAUP 4.0 b8, with neighbor-joining search and a bootstrap analysis showed that, although the four species are closely related, D. septentriosaltans, D. saltans and D. austrosaltans are closer to each other than to D. prosaltans. These results showed to be consistent with phylogenetic relationships previously inferred from inversion polymorphism.

Evaluation of fitness components in strains of Drosophila mulleri carrying different genotypes for an esterase

Several fitness components in strains of Drosophila mulleri carrying the slow or the fast alleles for the major beta esterase (esterase-4) found in this species, as well as in heterozygous flies in which the slow or fast alleles came from one of the parents, were evaluated. Twelve components were analysed including longevity of both virgins and mated males and females, productivity, viability, including the egg-larva, egg-pupa, egg-imago and pupa-imago periods. These parameters were used to estimate the total fitness for each genotype. The best score was reached by individuals having the Est-4(S)/Est-4(S) genotype (scored at 1.000), followed by a fitness value of 0.892 presented by the Est-4(F)/Est-4(S) genotype (with the fast allele from maternal origin), 0.863 for the Est-4(F)/Est-4(F) and 0.842 for the Est-4(S)/Est-4(F) genotypes (with Est-4(F) maternal origin). These results suggested a higher relative adaptability of the Est-4(S)/Est-4(S) genotype followed by the Est-4(F)/Est-4(S) hybrid that possessed the allele Est-4(S) of maternal origin, which was incompatible with predictions of neutral polymorphism.

Esterase patterns and phylogenetic relationships of species and strains included in the Drosophila buzzatii cluster

Ten strains of two species in the Drosophila buzzatii cluster (D. serido and D. seriema) were examined as to esterase patterns using polyacrylamide gel electrophoresis. The migration rate of esterases, and their substrate specificity to alpha and beta naphthyl acetates, were analysed. Other esterase features such as inhibition behaviour, presence in males and females and location in the head, thorax or abdomen of flies, were also examined. The present data,together with results obtained by others for eight strains of D. koepferae, D. serido, D. seriema and D. buzzatii, show that 69 bands have been detected in the eighteen strains studied. This total number of bands was used for comparison of strains and species by similarity index, analysis of dependence and cluster analysis. The comparisons confirmed the existence of a high degree of similarity among D. seriema strains and among D. koepferae strains, but indicated differentiation among the D. serido strains. Two strains (D69R2 and D69R5) which differed from the others of the latter species, showed closer affinities with D. buzzatii, which indicates the need for further work on those strains classified as D. serido.

Neo-clerodane diterpenoid, a new metalloprotease snake venom inhibitor from Baccharis trimera (Asteraceae): anti-proteolytic and anti-hemorrhagic properties

Many plants are used in traditional medicine as active agents against various effects induced by snakebite. Few attempts have been made however to identify the nature of plain natural products with anti-ophidian properties. Baccharis trimera (Less) DC (Asteraceae), known in Brazil as carqueja. has been popularly used to treat liver diseases. rheumatism. diabetes, as well as digestive, hepatic and renal disorders. The active component was identified as 7alpha-hydroxy-3,13-clerodadiene-16,15:18,19-diolide, C20H28O5, (clerodane diterpenoid, Bt-CD). We report now the anti-proteolytic and anti-hemorrhagic propenies against snake venoms of a Bt-CD inhibitor from B. trimera. Bt-CD exhibited full inhibition of hemorrhage and proteolytic activity caused by Bothrops snake venoms. The inhibitor was able to neutralize the hemorrhagic, fibrinogenolytic and caseinolytic activities of class P-I and III metalloproteases isolated from B. neuwiedi and B. jararacussu venoms. No inhibition of the coagulant activity was observed. Bt-CD also partially inhibited the edema induced by other crude venoms, metallopronteases, basic and acidic phospholipases A(2). To further elucidate the inhibitory specificity of Bt-CD against metalloproteases isolated from snake venoms, a deeper understanding of its Structure and function is necessary. Furthermore, the potential use of these inhibitors to complement anti-venom as an alternative treatment of snakebite envenomations needs to be evaluated in future Studies. (C) 2004 Elsevier B.V.. All rights reserved.

Genetic variation along time in a brazilian population of Aedes aegypti (Diptera : Culicidae), detected by changes in the esterase patterns

Aedes aegypti from the Brazilian cities of Sao Jose do Rio Preto (SJ) and Goiania (GO) were analyzed as to their esterase patterns and the results were compared with data obtained about 5 years before for SJ population. Esterase bands not detected in the previous study were now observed in mosquitoes from both SJ and GO populations, being the last considered a population resistant to insecticides. Other similarities between SJ and GO populations in this study, and some differences in comparison with the previous data on SJ were observed, involving, in addition to changes in band type, changes in frequency of mosquitoes expressing them and differential gene activation during development. As it is generally true for genetic features, changes in the esterase patterns are expected to be the result of factors such as selection by environmental conditions and genetic drift. In the present case, continuous use of insecticides aiming mosquito population size control in SJ by sanitary authorities could be involved in the observed changes. Changed esterases were classified as carboxylesterases and cholinesterases, which are enzymes already shown to take part in the development of resistance in several organisms. In addition, data obtained in the elapsed time by authorities responsible for the mosquito control has shown increasing insecticide resistance of SJ population mosquitoes parallel to increase in the total amount of esterases, reinforcing the mentioned possibility.

Variability of esterase patterns in adult flies of the saltans species group of Drosophila (subgenus Sophophora)

Esterases are known for their involvement in several physiological processes and high degree of polymorphism, in many organisms. Such polymorphism has been used to characterize species and species groups and to study genetic changes occurred in their evolutionary history. In the present study, the esterase patterns of 19 strains from 10 species representative of the five subgroups of the saltans species group were analyzed using polyacrylamide gel electrophoresis and alpha- and beta- naphthyl acetates as substrates. Fifty-one esterase bands were detected and classified as 31 alpha-esterases, 18 beta-esterases and two alpha/beta-esterases. on the basis of the inhibition patterns using Malathion and eserine sulfate, 34 bands were classified as carboxylesterases, 14 as acethylesterases and three as cholinesterases. Ten gene loci were tentatively established on the basis of data on band position in the gel, substrate preference and inhibition pattern. Twenty bands were species-specific, the remaining being shared by species from the same or different subgroups. Bands detected exclusively in males and bands with a different frequency or degree of expression between sexes were also detected. In the gels prepared for analysis of gene expression in the body parts (head, thorax and abdomen), the degree of expression of the beta-esterases was higher in the thorax, while the alpha-esterases were expressed predominantly in the abdomen and thorax. A global view of the data available at present on the esterases of the species from the saltans group and their degree of polymorphism are presented, as well as the possibility of using some beta-esterases, because of their characteristics in the gels, as markers for species identification.

Effects of maternal exposure to an aromatase inhibitor on sexual behaviour and neurochemical and endocrine aspects of adult male rat

The present study examined the effects of letrozole exposure during brain sexual differentiation on endocrine, behavioural and neurochemical parameters in male rat descendants. Pregnant female rats received 1 mg kg(-1) day(-1) letrozole or vehicle by oral gavage on gestational Days 21 and 22. Exposure to letrozole reduced anogenital distance in males on postnatal Day (PND) 22. At adulthood (PND 75), plasma testosterone levels and hypothalamic dopaminergic activity were increased, but sexual competence was impaired, because fewer successful sexual behaviours (mount, intromission and principally ejaculation) were observed. The impairment of reproductive function by prenatal exposure to an aromatase inhibitor reinforces the importance of adequate oestrogenic activity during perinatal sexual differentiation for complete masculinisation of the hypothalamus.

Esterase patterns in species of the triatome bug Rhodnius

The aim of the present study was to analyse esterase patterns in three triatomine species of Rhodnius genus. Four loci, Est 1, Est 2, Est 3 and Est 4, were found. The corresponding enzymes were characterized as carboxylesterases (E.C. 3.1.1.1) or cholinesterases (E.C. 3.1.1.8) based on inhibitory experiments, using eserine sulphate, malathion, mercury chloride, p-chloromercuribenzoate (pCMB) and iodoacetamide. Low genetic variability was observed: Est 1, Est 2 and Est 3 were monomorphic in Rhodnius domesticus, Rhodnius robustus and Rhodnius neivai, whereas locus Est 4 was polymorphic in the first two species. The UPGMA analysis based on esterase genotypic frequencies indicated greater similarity between R. domesticus and R. robustus when compared with R. neivai. The present study expands our knowledge about genetic variability among triatomines and accords with the hypothesis that R. domesticus is a species derived from R. robustus.