Transferência de imunidade passiva (TIP) e dinâmica de anticorpos específicos em bezerros naturalmente expostos para as viroses respiratórias / Passive immune transfer (PIT) and dynamics of specific antibodies in calves naturally exposed to the respiratory viruses

Esta pesquisa avaliou a TIP e a dinâmica de anticorpos (ACs) específicos em bezerros naturalmente expostos aos agentes causadores da doença respiratória bovina (DRB). Foram selecionados 19 bezerros Holandeses alimentados com colostro proveniente de doadoras vacinadas para DRB. Amostras de soro foram obtidas antes e após a ingestão do colostro (48h) para a soroneutralização (SN). Os valores médios (log2) detectados após colostragem foram de 11,5±1,6 (BVDV), 8,8±1,3 (BoHV-1), 5,5±1,6 (BRSV) e 8,4±1,5 (BPIV-3). Cinco bezerros foram criados do nascimento aos 240 dias de vida, observando-se decréscimo nos títulos de ACs para BVDV, BoHV-1 e BPIV-3 ao longo do tempo (P≤0,001). As taxas de infecções detectadas entre o D14 e o D240 foram de 40% (2/5), 20% (1/5), 80% (4/5), e 60% (3/5), respectivamente, para BVDV, BoHV-1, BRSV e BPIV-3. A maioria dos bezerros manifestou broncopneumonia após as infecções virais. Os bezerros apresentaram ACs para todas as viroses às 48 horas de vida, porém os títulos adquiridos para o BRSV foram baixos. A susceptibilidade para as infecções variou de acordo com os níveis e a duração dos títulos de ACs maternos.(AU)

This research evaluated the PIT and the dynamics of specific antibody (Ab) for calves naturally exposed to the viral agents involved in Bovine Respiratory Disease (BRD). Nineteen Holstein calves fed colostrum from vaccinated donors for DRB. Serum samples were obtained before and after colostrum intake (48h) for serum neutralization (SN). Mean values (log2) detected after colostrum feeding were 11.5±1.6 (BVDV), 8.8 ±1.3 (BoHV-1) 5.5±1.6 (BRSV) and 8.4±1.5 (BPIV-3). Five calves were raised from birth to 240 days of life and presented a decrease in Ab titers for BVDV, BoHV-1 and BPIV-3 over time (P≤ 0.001). Infection rates from D14 to D240 were of 40% (2/5), 20% (1/5), 80% (4/5) and 60% (3/5), respectively for BVDV, BoHV-1, BRSV and BPIV-3. Most of the calves presented bronchopneumonia after seroconversion to the virus. Calves presented Ab for all viruses at 48 hours of life, however BRSV Ab titer were low. Levels and persistence of maternal antibody titers determined the susceptibility to viral infections.(AU)

The Brazilian investment in science and technology

An analysis of Brazilian federal expenditures in science and technology is presented is this study. The 1990-1999 data were compiled from records provided by two federal agencies (MCT and CNPq) responsible for managing most of the national budget related to these activities. The results indicate that the federal investments in Brazilian science and technology stagnated during the last decade (US$ 2.32 billion in 1990, US$ 2.39 billion in 1996, and US$ 2.36 billion in 1999). In contrast, a great increase in private investments in research was acknowledged both by industry and by the government during the same period, from US$ 2.12 to US$ 4.64 billion. However, this investment did not result in an increase in invention patents granted to residents (492 in 1990 and only 232 in 1997) or in a reduction of patent costs. Despite this unfavorable scenario, the number of graduate programs in the country has increased two-fold in the last decade and the contribution of Brazilians to the database of the Institute for Scientific Information has increased 4.7-fold from 1990 (2,725 scientific publications) to 2000 (12,686 scientific publications). Unstable federal resources for science, together with the poor returns of private resources in terms of developing new technologies, may jeopardize the future of Brazilian technological development

Hydrogen peroxide detoxification in the midgut of the blood-sucking insect, Rhodnius prolixus.

Here we investigated H2O2 production and detoxification in the hematophagous hemiptera, Rhodnius prolixus. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide radical (O2-). This reaction produces hydrogen peroxide, which is scavenged by antioxidant enzymes such as catalase (CAT). SOD and CAT activities were found in all tissues studied, being highest in the midgut. CAT was dose-dependently inhibited in vivo by injections of 3-amino-1,2,4-triazole (AT). Insects treated with AT showed a twofold increase in H2O2 levels. Injection of DL-buthionine-[S, R]-sulfoximine (BSO), an inhibitor of glutathione synthesis, also resulted in a fourfold increase in H2O2, together with stimulation of CAT activity. Simultaneous administration of both AT and BSO had a synergistic effect on midgut H2O2 content. Taken all together, our results suggest that CAT and glutathione-dependent mechanisms cooperate to control H2O2 concentration in the midgut cell and prevent hydroxyl radical generation by Fenton reaction in this tissue.

Ochratoxin A in Brazilian roasted and instant coffees.

Thirty-four samples of roast and ground coffee, 14 samples of instant coffee and two samples of decaffeinated instant coffee were collected in markets and supermarkets in the city of Campinas, Brazil, and analysed for ochratoxin A using immunoaffinity columns for clean-up and HPLC with fluorescence detection for quantification. The limit of detection was 0.2 ng/g ochratoxin A. Twenty-three samples of ground and roast coffee were found to be contaminated with the toxin at levels ranging between 0.3 and 6.5 ng/g. The average concentration in all 34 samples was 0.9 ng/g. All samples of instant coffee contained ochratoxin A at levels ranging from 0.5 to 5.1 ng/g, with an average figure of 2.2 ng/g. Roast and ground coffee is the type of coffee most used by Brazilians for the preparation of the beverage. Considering that an average Brazilian adult takes five cups of coffee per day, which corresponds to 30 g of roast and ground coffee, the probable daily intake of ochratoxin A by a 70 kg adult would be 0.4 ng/kg bw, which is far below the current Provisional Tolerable Daily Intake of 14 ng/kg bw for ochratoxin A as set by the Codex Alimentarius. To study the transfer of ochratoxin A into coffee brew, the beverage was prepared by two methods: (a) the drip method and (b) the Brazilian country style method. No significant difference was observed between the two methods in terms of extraction of the toxin using five contaminated samples containing between 0.8 and 6.5 ng/g ochratoxin A. The drip method extracted 86 +/- 15% and the Brazilian country style 74 +/- 20% of the ochratoxin A initially present in the roast and ground coffee.

HeLp, a heme lipoprotein from the hemolymph of the cattle tick, Boophilus microplus.

The main protein of the hemolymph of the cattle tick Boophilus microplus has been isolated and shown to be a heme lipoprotein (HeLp). HeLp has an apparent molecular mass of 354,000 and contains two apoproteins (103 and 92 kDa) found in equal amounts. HeLp presents a pI of 5.8 and a density of 1.28 g/ml and contains 33% lipids, containing both neutral lipids and phospholipids, and 3% of sugars. A remarkable feature of HeLp is the abundance of cholesterol ester (35% of total lipids), a lipid not previously reported in invertebrate lipoproteins. Western blot analysis showed HeLp in hemolymph from adult females and males, but not in eggs. Although HeLp contains 2 heme molecules, it is capable of binding 6 additional molecules of heme. Boophilus feeds large amount of blood, and we recently showed that this tick is unable to perform de novo synthesis of heme (Braz, G. R. C., Coelho, H. S. L., Masuda, H., and Oliveira, P. L. (1999) Curr. Biol. 9, 703-706). Injection of tick females with (55)Fe-labeled heme-HeLp indicated that this protein transports heme from hemolymph to tissues. HeLp is suggested to be an essential adaptation to the loss of the heme synthesis pathway.

Urate synthesis in the blood-sucking insect rhodnius prolixus. Stimulation by hemin is mediated by protein kinase C.

Hemin is a catalyst of the formation of reactive oxygen species. We proposed that hematophagous insects are exposed to intense oxidative stress because of hemoglobin hydrolysis in their midgut (Petretsky, M. D., Ribeiro, J. M. C., Atella, G. C., Masuda, H., and Oliveira, P. L. (1995) J. Biol. Chem. 270, 10893-10896). We have shown that hemin stimulates urate synthesis in the blood-sucking insect Rhodnius prolixus (Graça-Souza, A. V., Petretsky, J. H., Demasi, M., Bechara, E. J. H., and Oliveira, P. L. (1997) Free Radical Biol. Med. 22, 209-214). Once released by fat body cells, urate accumulates in the hemolymph, where this radical scavenger constitutes an important defense against blood-feeding derived oxidative stress. Incubation of Rhodnius fat bodies with okadaic acid raises the level of urate synthesis, suggesting that urate production can be controlled by protein phosphorylation/dephosphorylation. Urate synthesis is stimulated by dibutyryl cAMP and inhibited by N(2((p-bromocinnamil)amino)ethyl)-5-isoquinolinesulfonamide (H-89), an inhibitor of protein kinase A, as well as activated by the protein kinase C activator phorbol 12-myristate 13-acetate. In the presence of hemin, however, inhibition of urate synthesis by H-89 does not occur, suggesting that the hemin stimulatory effect is not mediated by protein kinase A. Calphostin C completely inhibits the hemin-induced urate production, suggesting that the triggering of urate antioxidant response depends on protein kinase C activation. This conclusion is reinforced by the observation that in fat bodies exposed to hemin, both protein kinase C activity and phosphorylation of specific endogenous polypeptides are significantly increased.

Isolation of an aspartic proteinase precursor from the egg of a hard tick, Boophilus microplus.

An aspartic proteinase precursor, herein named BYC (Boophilus Yolk pro-Cathepsin) was isolated from eggs of the hard tick, Boophilus microplus. As judged by electrophoresis on sodium dodecyl sulfate polyacrylamide slab gel (SDS-PAGE), purified BYC presented 2 bands of 54 and 49 kDa, bearing the same NH2-terminal amino acid sequence. By Western blot analysis, BYC was also found in the haemolymph, indicating an extraovarian site of synthesis. Several organs were incubated in culture medium with [35S]methionine, and only the gut and fat body showed synthesis of BYC polypeptides. Protein sequencing of both the NH2-terminal and an internal sequence obtained after cyanogen bromide (CNBr) cleavage of BYC revealed homology with several aspartic proteinase precursors. Incubation at pH 3.5 resulted in autoproteolysis of BYC, which produced the mature form of the enzyme, that displayed pepstatin-sensitive hydrolytic activity against haemoglobin. Western blot analysis using anti-BYC monoclonal antibodies showed proteolytic processing of BYC during embryogenesis and suggested activation of the enzyme during development. A role of BYC in degradation of vitellin, the major yolk protein of tick eggs, is discussed.

A heme-binding protein from hemolymph and oocytes of the blood-sucking insect, Rhodnius prolixus. Isolation and characterization.

A heme-binding protein has been isolated and characterized from both the hemolymph and oocytes of the blood-sucking insect, Rhodnius prolixus. The protein from both sources is identical in most aspects studied. The Rhodnius heme-binding protein (RHBP) is composed of a single 15-kDa polypeptide chain coiled in a highly alpha-helical structure which binds non-covalently one heme/polypeptide chain. This RHBP is not produced by limited degradation of hemoglobin from the vertebrate host, since specific polyclonal antibodies against it do not cross-react with rabbit hemoglobin, and since it differs from hemoglobin in having a distinct amino-acid composition and NH2-terminal sequence. The spectrum of the dithionite-reduced protein has peaks at 426, 530, and 559 nm and resembles that of a b-type cytochrome. RHBP from hemolymph is not saturated with heme and promptly binds heme added to the solution. The oocyte protein, on the other hand, is fully saturated and is not capable of binding additional heme.