Pharmacokinetic profile of tert-butylaminoethanethiol

A preliminary study of the pharmacokinetic parameters of t-Butylaminoethanethiol (TBAESH) was performed after administration of a single dose (35 mg/kg) either orally or intravenously. Plasma or blood samples were treated with dithiothreitol, perchloric acid and, after filtration, submitted to further purification with anionic resin. In the final step the drug was retained on a cationic resin column, eluted with NaCl lM and detected according to the method of Ellman (1958). The results suggested a pharmacokinetic behavior related to a one open compartment model with the following values for the total drug: area under the intravenous curve (AUC i.v.): 443(+ ou -) 24.0; AUC oral: 85.5(+ ou -) 14.5 ug min.ml(elevado a -1); elimination rate constant: 0.069(+ ou -) 0.0055 min(elevado a -1), biological half-life: 10.0(+ ou -) 0.80 min; distribution volume 1.15(+ ou -) 0.15 ml/g; biodisponibility: 0.19(+ ou -) 0.02. From a pharmacokinetic standpoint, TBAESH seems to have no advantage over the analogous disulfide compound.

Toxoplasma gondii: a rapid method for the isolation of pure tachyzoites: preliminary characterization of its genome

A rapid and simple technique for the purification of Toxoplasma gondii tachyzoites was developed. Highly purified parasites were obtained from the peritoneal exudates of infected mice by means of two consecutive discontinous sucrose gradients run at low speed (10,000xg, 30 min). Parasites obtained by this method conserved its biological activity. Hybridizations tudies with DNA from healthy mice and from purified tachyzoites preparations demonstrated that Toxoplasma gondii tachyzoites DNA could be obtained with better than 90 per cents purity. Preliminary studies with DNA endonucleases showed the presence in the tachyzoites genome of highly repetitives sequences.

Analgesic activity of a triterpene isolated from Scoparia dulcis L. (vassourinha)

Analgesic and anti-inflammatory activities of water (WE) and ethanolic (EE) extracts of Scoparia dulcis L. were investigated in rats and mice, and compared to the effects induced by Glutinol, a triterpene isolated by purification of EE. Oral adminsitration (p.o.) of either WE or EE (up to 2 g/Kg) did not alter the normal spontaneous activity of mice and rats. The sleeping time induced by sodium pentobarbital (50 mg/Kg, i.p.) was prolonged by 2 fold in mice pretreated with 0.5 g/Kg EE, p.o. Neither extract altered the tail flick response of mice in immersion test, but previous administration of EE (0.5 g/Kg, p.o.) reduced writhings induced by 0.8% acetic acid (0.1 ml/10 g, i.p.) in mice by 47% EE (0.5 and 1 g/Kg, p.o.) inhibited the paw edema induced by carrageenan in rats by respectively 46% and 58% after 2 h, being ineffective on the paw edema induced by dextran. No significant analgesic or anti-edema effects were detected in animals pretreated with WE (1 g/Kg, p.o.). Administration of Glutinol (30 mg/Kg, p.o.) reduced writhing induced by acetic acid in mice by 40% and the carrageenan induced paw edema in rats by 73%. The results indicate that the analgesic activity of S dulcis L. may be explained by explained by an anti-inflammatory activity probably related to the triterpene Glutinol.

Histopathological alterations induced by non-viable cells and biochemical fractions from Paracoccidioides brasiliensis in mice

Non-viable cells and biochemical fractions from Paracoccidioides brasiliens were obtained for experimental inoculation in mice and posterior histopatological analysis. Dead total fungus, total fungus disrupted by sonorous waves, lipids of the fungus, supernatant of the lipid purification, integral and disrupted fungus free of lipids were obtained. The six preparations arised from masses of lyophilized yeasts of a recent isolate of P. brasiliensis (strain JT-1) and from a "Pool" equitably constituted by four strains maintained in laboratory for a long time (SN, 2, 18 and 192). Different doses of the 12 preparations were intraperitonially inoculated and histopathological analysis were done 30 days later. This analysis showed that all the inoculated preparations gave origin to inflamatory foci, except the one designated "supernatant of lipid purification". The alterations were detected exclusively in the liver of the animals and occurred from the smallest dose tested (1 mg), with exception of the lipids of the fungus, where the foci appeared only from a 3 mg dose onwards. No difference in the capacity of inducing histopathological alterations was found between the preparations obtained from the recent isolate (JT-1) and from the older ones ("Pool"). On the other hand, an increase of the number of inflammatory foci in function of the inoculated dose was observed.

Partial isolation and some properties of enterotoxin produced by Bacillus cereus strains

Extracellular proteins produced by Bacillus cereus AL-42 and AL-15 were fractioned by chromatography on QAE-Sephadex and Sephadex G75. This last chromatographic process resulted in three peaks. The major peak showed vascular permeability activity to rabbits, lethality to mice, and cytotoxicity to Vero and Hela cells. The analysis by SDS-PAGE after ultrafiltration confirm recent findings that the enterotoxin is a compound with molecular mass > 30.000.

The imidazoline receptors and the central regulation of the arterial blood pressure: a minireview

Recently, we proposed the hypothesis according to wich the central hypotensive effect of clonidine and related substances could be related to an action upon specific receptors, requiring the imidazoline or imidazoline-like structures, rather than alpha2-adrenoceptors. Since then, direct evidences have been accumulated to confirm the existence of a population of imidazoline specific binding sites in the brainstem of animals and man, more precisely in the Nucleus Reticularis Lateralis (NRL) region of the ventrolateral medulla (VLM), site of the antihypertensive action of clonidine. The purification of the putative endogenous ligand of the imidazoline receptors - named endazoline - is currently being attempted from human brain extracts. This new concept might at last lead to the expected dissociation of the pharmacological mechanisms involved, on the one hand, in the therapeutic antihypertensive effect, and on the other, in their major side-effect, which is sedation. In fact, it has been recently confirmed that hypotension is mediated by the activation of imidazoline preferring receptors (IPR) within the NRL region, while sedation is attributed to the inhibition of alpha2-adrenergic mechanisms in the locus coeruleus, which is involved in the control of the sleep-waking cycle. The IPRmay constitute on interesting target for new drugs in the treatment of arterial hypertension. Finally, dysfunctions of this modulatory system which could be involved in the pathophysiologyof some forms of the hypertensive disease are under investigation.

Biological characterization of clones derived from the edmonston strain of measles virus in comparison with schwarz and CAM-70 vaccine strains

Four virus clones were derived from the Edmonston strain of measles virus by repeated plaque purification. These clones were compared with the vaccine strains Schwarz and CAM-70 in terms of biological activities including plaque formation, hemagglutination, hemolysis and replication in Vero cells and chick embryo fibroblasts (CEF). Two clones of intermediate plaque yielded mixed plaque populations on subcultivation whereas the other two, showing small and large plaque sizes, showed stable plaque phenotypes. The vaccine strains showed consistent homogeneous plaque populations. All the Edmonston clones showed agglutination of monkey erythrocytes in isotonic solution while both vaccine strains hemagglutinated only in the presence of high salt concentrations. Variation in the hemolytic activity was observed among the four clones but no hemolytic activity was detected for the vaccine virus strains. Vaccine strains replicated efficiently both in Vero cells and CEF. All four clones showed efficient replication in Vero cells but different replication profiles in CEF. Two of them replicated efficiently, one was of intermediate efficiency and the other showed no replication in CEF. Two of the clones showed characteristics similar to vaccine strains. One in terms of size and homogeneity of plaques, the other for a low hemolytic activity and both for the efficiency of propagation in CEF.

Immunodiagnosis of Trypanosoma cruzi (Chagas' Disease) Infection in Naturally Infected Dogs

This study reports on the standardization of an enzyme-linked immunosorbent assay (ELISA) for detecting specific antibodies anti-Trypanosoma cruzi in naturally infected dogs. Sera from 182 mongrel dogs of all ages residing in four rural villages in Santiago del Estero, Argentina, were collected in November 1994 and preserved in buffered neutral glycerin. All sera were tested by indirect hemagglutination test (IHAT), indirect immunofluorescence test (IFAT), and ELISA using the flagellar fraction of T. cruzi as antigen. Dog sera from an area without vectorial transmission were used to calculate ELISA specificity and cut-off value. Eighty-six percent of sera had concordant results for all tests. All sera reactive for IHAT and IFAT were also reactive for ELISA, except in one case. Sera tested by ELISA when diluted 1:200 allowed a clearer division between non-reactive and reactive sera than when 1:100 with greater agreement among serologic techniques. The specificity of ELISA was 96.2%. Among 34 adult dogs with a positive xenodiagnosis, sensitivity was 94% both for ELISA and IFAT. ELISA is the first choice for screening purposes and one of the pair of techniques recommended for diagnostic studies in dog populations

Protective immunity induced in mice by F8.1 and F8.2 antigens purified from Schistosoma mansoni eggs

Schistosoma mansoni soluble egg antigens (SEA) were fractionated by isoelectric focusing, resulting in 20 components, characterized by pH, absorbance and protein concentration. The higher absorbance fractions were submitted to electrophoresis, and fraction 8 (F8) presented a specific pattern of bands on its isoelectric point. Protein 3 was observed only on F8, and so, it was utilized to rabbit immunization, in order to evaluate its capacity of inducing protective immunity. IgG antibodies from rabbit anti-F8 serum were coupled to Sepharose, and used to obtain the specific antigen by affinity chromatography. This antigen, submitted to electrophoresis, presented two proteic bands (F8.1 and F8.2), which were transferred to nitrocellulose membrane (PVDF) and sequenciated. The homology of F8.2 to known proteins was determined using the Basic Local Alignment Search Tool program (BLASTp). Significant homologies were obtained for the rabbit cytosolic Ca2+ uptake inhibitor, and for the bird a1-proteinase inhibitor. Immunization of mice with F8.1 and F8.2, in the presence of Corynebacterium parvum and Al(OH)3 as adjuvant, induced a significant protection degree against challenge infection, as observed by the decrease on worm burden recovered from portal system.

r-Sm14 - pRSETA efficacy in experimental animals

Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system.

Evaluation by ELISA of Anisakis simplex Larval Antigen Purified by Affinity Chromatography

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of 1/400 and 1 µg/ml of antigenic concentration.

Predation Potential of the Water Bugs Sphaerodema rusticum on the Sewage Snails Physa acuta

The sewage snail Physa acuta is a serious threat to certain economic plants and to the purification plant of sewage works by rendering the biofilters ineffective. Various attempts are being made to control it. The efficacy of the predacious water bugs Sphaerodema rusticum was judged experimentally, in the laboratory in the potential control of P. acuta. It is revealed that, when supplied separately, the first, second and third instar and the adult S. rusticum did not attack P. acuta belonging to 3.1-8 mm, 5.1-8 mm, 7.1-8 mm and <= 3 mm size classes respectively. In the remaining trials predation rate varied from zero to eight (average 2.3) individuals per predator per day. In experiments with P. acuta belonging to all the size classes supplied together, none, except the first instar S. rusticum, attacked the prey individuals belonging to the lowest (<= 3 mm) size class. The first and second instar S. rusticum, in both trials did not attack P. acuta larger than 4 mm and 5 mm in shell length respectively. The water bugs belonging to the third, fourth, fifth instar and adult stages though preyed upon P. acuta with 3.1-8 mm shell length. The average rate of predation by a single S. rusticum varied from 0.14-3.08 individuals per day depending upon the size of P. acuta and the stage of S. rusticum. A single S. rusticum, irrespective of instar and adult stages, destroyed on average 4.16 P. acuta daily irrespective of sizes. It is estimated that one S. rusticum could destroy 1,360 P. acuta in its life time. The results clearly indicate that the water bug S. rusticum may be used to control the snails P. acuta.

In vitro antibacterial activity of a 7-O-beta-D-glucopyranosyl-nutanocoumarin from Chaptalia nutans (Asteraceae)

Ethanolic crude extracts from the roots of Chaptalia nutans, traditionally used in Brazilian folk medicine, were screened against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa by using the disk diffusion test technique. S. aureus with 14 mm inhibition zone was considered susceptible. E. coli and P. aeruginosa without such a zone were considered resistant. As a result of this finding, the ethanolic crude extract was fractionated on silica gel column chromatography into five fractions. The ethyl acetate fraction was active against S. aureus and Bacillus subtilis. Further column chromatography separation of the ethyl acetate fraction afforded 30 fractions, which were assayed against S. aureus. Fractions 16 and 17 showed inhibition zones with S. aureus, indicating the presence of active compounds, and were subjected to purification by repeated preparative thin layer chromatography. The pure compound 7-O-beta-D-glucopyranosyl-nutanocoumarin inhibited B. subtilis and S. aureus at concentrations of 62.5 µg/ml and 125 µg/ml, respectively. The antibacterial property of C. nutans appears to have justified its use for the treatment of wounds, which are contaminated through bacterial infections.

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity.

Trypanosoma (Duttonella) vivax: its biology, epidemiology, pathogenesis, and introduction in the New World - a review

The biology, epidemiology, pathogenesis, diagnostic techniques, and history of the introduction of Trypanosoma (Duttonella) vivax in the New World are reviewed. The two main immunological responses of trypanosome-infected animals - antibody production and immunodepression - are discussed in the context of how these responses play a role in disease tolerance or susceptibility. Isolation and purification of T. vivax are briefly discussed. The recent reports of bovine trypanosomiasis diagnosed in cattle on farms located in the Pantanal region of the states of Mato Grosso do Sul and Mato Grosso, Brazil, are also discussed.