Antiretroviral resistance mutations in human immunodeficiency virus type 1 infected patients enrolled in genotype testing at the Central Public Health Laboratory, São Paulo, Brazil: preliminary results

Antiretroviral resistance mutations (ARM) are one of the major obstacles for pharmacological human immunodeficiency virus (HIV) suppression. Plasma HIV-1 RNA from 306 patients on antiretroviral therapy with virological failure was analyzed, most of them (60%) exposed to three or more regimens, and 28% of them have started therapy before 1997. The most common regimens in use at the time of genotype testing were AZT/3TC/nelfinavir, 3TC/D4T/nelfinavir and AZT/3TC/efavirenz. The majority of ARM occurred at protease (PR) gene at residue L90 (41%) and V82 (25%); at reverse transcriptase (RT) gene, mutations at residue M184 (V/I) were observed in 64%. One or more thymidine analogue mutations were detected in 73%. The number of ARM at PR gene increased from a mean of four mutations per patient who showed virological failure at the first ARV regimens to six mutations per patient exposed to six or more regimens; similar trend in RT was also observed. No differences in ARM at principal codon to the three drug classes for HIV-1 clades B or F were observed, but some polymorphisms in secondary codons showed significant differences. Strategies to improve the cost effectiveness of drug therapy and to optimize the sequencing and the rescue therapy are the major health priorities.

Estudo do valor nutritivo do farelo de guandu [Cajanus cajan (L.) Miilps] var. fava larga na alimentação de frangos de corte. 1. Avaliação do tratamento térmico

The experiment was conducted to evaluate pigeon pea (Cajanus cajan) protein in broiler chicks (up to 28 days of age) feeding, as compared to soybean (Glycine mar) protein, In the experiment the effects of temperature and autoclaving on antinutritional factors on pigeon pea meal were studied. Temperatures of 100, 110 e 120 degrees C and times of autoclaving of 10, 20 and 30 minutes were applied using a two-way factorial design, replicated in four times. Underheating and overheating during the process of inactivation of proteases inhibiting were evaluated by ureatic activity and protein solubility analysis, after the grains were dried and ground. After 28 days, the best time of autoclaving was 20 minutes (p<,05) irrespectively of temperature, which lead to a lower feed consuption. There were no histopathological alterations in kidney, liver, pancreas, heart and intestines of the broilers.

Chondroitin sulfate proteoglycans are structural renewable constituents of the rabbit vitreous body

Purpose: To characterize the vitreous intrinsic proteoglycans, investigate their dynamics, and examine their role in the supramolecular organization of the vitreous. Methods: Vitreous from normal rabbits was collected and processed for observation with the transmission electron microscope after treatment with glycosidases. Also, rabbits were injected intravitreally with [S-35]-sodium sulfate and sacrificed at several time intervals after the injection. Proteoglycans (PGs) were assayed in the vitreous supernatant or in whole samples extracted with guanidine hydrochloride by polyacrylamide or agarose gel electrophoresis, followed respectively by fluorography or autoradiography, and ion-exchange chromatography and gel-filtration chromatography, combined with glycolytic treatment of the samples. The sulfated glycosaminoglycans (GAGs) were characterized by agarose gel electrophoresis after treating vitreous samples with protease and specific glycosidases. Results: the electron microscopic study revealed a network with hyaluronic acid ( HA) as thin threads coating and connecting collagen fibrils. The elimination of the HA coat showed chondroitin sulfate granules (8-25 nm) arranged at regular intervals on the fibril surface. The chondroitinase ABC digestion, besides removing the granules, also caused the formation of thicker bundles of the collagen fibrils. The PG and GAG analysis indicated that there are three renewable PGs in the vitreous ( e. g., one heparan-and two chondroitin-sulfate ones). Conclusions: At least one of the chondroitin sulfate PGs is involved in the interactions that occur in the vitreous structure, mainly by providing adequate spacing between the collagen fibrils, a condition that is probably required for the transparency of the vitreous.

Effect of early feed restriction and enzyme supplementation on digestive enzyme activities in broilers

The effect of feed restriction and enzymatic supplementation on intestinal and pancreatic enzyme activities and weight gain was studied in broiler chickens. Quantitative feed restriction was applied to chickens from 7 to 14 d of age. An enzyme complex mainly consisting of protease and amylase was added to the chicken ration from hatching to the end of the experiment. Birds subjected to feed restriction whose diet was not supplemented showed an increase in sucrase, amylase, and lipase activities immediately after the restriction period. Amylase, lipase, and chymotrypsin activities were higher in chickens subjected to feed restriction and fed a supplemented diet than in those only subjected to feed restriction. Trypsin activity increased after feed restriction and after supplementation, but there was no interaction between these effects. Early feed restriction had no effect on enzyme activity in 42-d-old chickens. Chickens subjected to early restriction and fed the supplemented diet presented higher sucrase, maltase, and lipase activities than nonsupplemented ones (P < 0.05). There was no effect of early feed restriction or diet supplementation on weight gain to 42 d. Percentage weight gain from 14 to 42 d of age was equivalent in feed-restricted and ad libitum fed birds. Feed-restricted broilers fed a supplemented diet showed a higher percentage weight gain than nonsupplemented birds. We conclude that enzymatic supplementation potentiates the effect of feed restriction on digestive enzyme activity and on weight gain.

Production of extracellular alkaline proteases by Aspergillus clavatus

The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37degreesC. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25degreesC for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH4NO3 showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40degreesC and the half-lives at 40, 45 and 50degreesC were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0.

Purification and characterization of jararassin-I, a thrombin-like enzyme from Bothrops jararaca snake venom

A thrombin-like serine protease, jararassin-I, was isolated from the venom of Bothrops jararaca. The protein was obtained in high yield and purity by a single chromatographic step using the affinity resin Benzamidine-Sepharose CL-6B. SDS-PAGE and dynamic light scattering analyses indicated that the molecular mass of the enzyme was about 30 kD. The enzyme possessed fibrinogenolytic and coagulant activities. The jararassin-I degraded the Bbeta chain of fibrinogen while the Aalpha chain and gammachain were unchanged. Proteases inhibitors, PMSF and benzamidine inhibited the coagulant activity. These results showed jararassin-I is a serine protease similar to coagulating thrombin-like snake venom proteases, but it specifically cleaves Bbeta chain of bovine fibrinogen. Single crystals of enzyme were obtained (0.2 mmx0.2 mmx0.2 mm) and used for X-ray diffraction experiments.