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American bullfrog (Lithobates catesbeianus) is the only species raised in Brazilian commercial frog farms, and the intensive culture of these animals has gained great popularity in Brazil over the past several years. Stress is one of the major obstacles in frog culture. To minimise this problem, the aim of the present study was to investigate the role of added vitamin C in the diet of American bullfrogs as an antistress factor through the assessment of plasma corticosterone, leucocyte levels and macrophage activation in animals raised in confinement. The experimental design was entirely randomised, with six treatments (supplementation of 0, 250, 500, 750, 1000 and 2000 mg of vitamin C/kg of feed) and four replications. The plasma corticosterone level, leucocyte level and macrophage activation were evaluated. It was concluded that vitamin C had no influence on the evaluated parameters due to the possible adaptation of the animals to life in captivity (domestication). The results of this research indicate that farmers should not increase the levels of vitamin C in commercial feeds because this would only enhance production costs.

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Feces are an important viral agent elimination route for infected carrier animals and in aquatic organisms these pathogenic agents can very rapidly propagate due to the habitation environment. The objective of this work is to track viral particles in the intestinal contents of bullfrogs (Lithobates catesbeianus) from five commercial frog farms in the region of Vale do Paraíba, in the State of São Paulo, Brazil, using negative contrast transmission electron microscopy (TEM). The Coronaviridae, Paramyxoviridae, Parvoviridae and Herpesviridae families were observed and photographed in specimens. This work emphasizes the importance of adopting sanitary measures in commercial farms and confirms that observing feces by TEM is an efficient and rapid diagnostic tool for detecting viral agents...

Sabendo-se que as fezes são uma importante via de eliminação de agentes virais pelos animais portadores e que, por estarem na água, os agentes patogênicos podem se propagar mais rapidamente, objetivou-se a pesquisa de vírus em conteúdo intestinal de rãs-touro (Lithobates catesbeianus) de cinco ranários comerciais na região do Vale do Paraíba, no estado de São Paulo, pela técnica de microscopia eletrônica de transmissão. As famílias Coronaviridae, Paramixoviridae, Parvoviridae e Herpesviridae foram observadas e fotografadas. Este trabalho ressalta a importância da adoção de medidas sanitárias nas criações, além da confirmação de que a observação de fezes pela microscopia eletrônica de transmissão é uma eficiente ferramenta de diagnóstico rápido para agentes virais...

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The P2X7 receptor (P2X7R), an ATP-gated cation channel, is expressed predominantly in leukocytes. Activation of P2X7R has been implicated in the formation of a cytolytic pore (i.e., a large conductance channel) that allows the passage of molecules up to 900 Da in macrophages. At least two hypotheses have been presented to explain the conversion of a nonselective cation channel to a cytolytic pore. One hypothesis suggests that the pore is a separate molecular structure activated by P2X7R, and the second asserts that this is an intrinsic property of P2X7R (pore dilation). Based on connexin knockout and hemichannel antagonist studies, some groups have concluded that connexins and pannexins, the hemichannel-forming proteins in vertebrates, are fundamental components of the large conductance channel associated with P2X7R. Dye uptake and electrophysiology experiments were used to evaluate the efficacy and specificity of some hemichannel antagonists under conditions known to open the large conductance channel associated with P2X7R. Hemichannel antagonists and interference RNA (RNAi) targeting pannexin-1 did not affect P2X7R macroscopic currents [ATP, 1,570±189 pA; ATP+100 µM carbenoxolone (CBX), 1,498±100 pA; ATP+1 mM probenecid (Prob), 1,522±9 pA] or dye uptake in a FACS assay (ATP, 63±5%; ATP+100 µM CBX, 51.51±8.4%; ATP+1 mM Prob, 57.7±4.3%) in mouse macrophages. These findings strongly suggest that the high-permeability pore evident after prolonged P2X7R activation does not occur through connexin or pannexin hemichannels in murine macrophages. Another membrane protein may be involved in P2X7R pore formation.

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Immune-mediated inflammation plays a major role in atherosclerosis and atherothrombosis, two essential features for cardiovascular disease (CVD) development, currently considered as the leading cause of death in the western world. There is accumulating evidence showing that humoral autoimmunity might play an important role in CVD and that some autoantibodies could represent emerging cardiovascular risk factors. Recent studies demonstrate that IgG autoantibodies against apolipoprotein A-1 (apoA-1) are raised in many diseases associated with a high cardiovascular risk, such as systemic lupus erythematosus, acute coronary syndrome, rheumatoid arthritis, severe carotid stenosis, and end-stage renal disease. In this work, we aimed at reviewing current data in the literature pointing to anti-apolipoprotein A-1 antibodies (anti-apoA-1 IgG) as a possible prognostic and diagnostic biomarker of cardiovascular risk and appraising their potential role as active mediators of atherogenesis.

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Chronic Chagas' disease cardiomyopathy (CCC) is an often fatal outcome of Trypanosoma cruzi infection, with a poorer prognosis than other cardiomyopathies. CCC is refractory to heart failure treatments, and is the major indication of heart transplantation in Latin America. A diffuse myocarditis, plus intense myocardial hypertrophy, damage and fibrosis, in the presence of very few T. cruzi forms, are the histopathological hallmarks of CCC. To gain a better understanding of the pathophysiology of CCC, we analyzed the protein profile in the affected CCC myocardium. Homogenates from left ventricular myocardial samples of end-stage CCC hearts explanted during heart transplantation were subjected to two-dimensional electrophoresis with Coomassie blue staining; protein identification was performed by MALDI-ToF mass spectrometry and peptide mass fingerprinting. The identification of selected proteins was confirmed by immunoblotting. We demonstrated that 246 proteins matched in gels from two CCC patients. They corresponded to 112 distinct proteins. Along with structural/contractile and metabolism proteins, we also identified proteins involved in apoptosis (caspase 8, caspase 2), immune system (T cell receptor ss chain, granzyme A, HLA class I) and stress processes (heat shock proteins, superoxide dismutases, and other oxidative stress proteins). Proteins involved in cell signaling and transcriptional factors were also identified. The identification of caspases and oxidative stress proteins suggests the occurrence of active apoptosis and significant oxidative stress in CCC myocardium. These results generated an inventory of myocardial proteins in CCC that should contribute to the generation of hypothesis-driven experiments designed on the basis of the classes of proteins identified here.

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Chronic Chagas' disease cardiomyopathy (CCC) is an often fatal outcome of Trypanosoma cruzi infection, with a poorer prognosis than other cardiomyopathies. CCC is refractory to heart failure treatments, and is the major indication of heart transplantation in Latin America. A diffuse myocarditis, plus intense myocardial hypertrophy, damage and fibrosis, in the presence of very few T. cruzi forms, are the histopathological hallmarks of CCC. To gain a better understanding of the pathophysiology of CCC, we analyzed the protein profile in the affected CCC myocardium. Homogenates from left ventricular myocardial samples of end-stage CCC hearts explanted during heart transplantation were subjected to two-dimensional electrophoresis with Coomassie blue staining; protein identification was performed by MALDI-ToF mass spectrometry and peptide mass fingerprinting. The identification of selected proteins was confirmed by immunoblotting. We demonstrated that 246 proteins matched in gels from two CCC patients. They corresponded to 112 distinct proteins. Along with structural/contractile and metabolism proteins, we also identified proteins involved in apoptosis (caspase 8, caspase 2), immune system (T cell receptor ß chain, granzyme A, HLA class I) and stress processes (heat shock proteins, superoxide dismutases, and other oxidative stress proteins). Proteins involved in cell signaling and transcriptional factors were also identified. The identification of caspases and oxidative stress proteins suggests the occurrence of active apoptosis and significant oxidative stress in CCC myocardium. These results generated an inventory of myocardial proteins in CCC that should contribute to the generation of hypothesis-driven experiments designed on the basis of the classes of proteins identified here.

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Epifluorescence microscope methods, namely BacLight, direct epifluorescence filter technique and Rhodamine 123, consistently underestimated plate bacterial counts in a 4-chlorophenol degrading consortium. Cells capable of passing through 0.2 microm filters, referred as 'ultramicrocells', were found. Although cell counts were higher when traditional methods were used, BacLight and direct epifluorescence filter technique were convenient techniques for the systematic monitoring of bacteria involved in biodegradation processes, as results were consistent and available within a short time.

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5-Aminolevulinic acid (ALA) is a heme precursor that accumulates in lead poisoning and inborn porphyrias. It has been shown to produce reactive oxygen species upon metal-catalyzed aerobic oxidation and to cause oxidative damage to proteins, liposomes, DNA, and subcellular structures. Studies have also shown that ALA may condense to yield the cyclic product 3,6-dihydropyrazine-2,5-dipropanoic acid (DHPY). Here we propose that DHPY could be involved in DNA damage in the presence of high concentrations of ALA. Exposure of plasmid pUC19 DNA to low concentrations of DHPY (2-10 microM) in the presence of 0.1 mM Cu2+ ions causes DNA strand breaks, as demonstrated by agarose gel electrophoresis. It was also shown that in the presence of Cu2+ ions DHPY is able to increase the oxidation of monomeric 2'-deoxyguanosine to form 8-oxo-7,8-dihydro-2'-deoxyguanosine as inferred from high performance liquid chromatography measurements using electrochemical detection. Addition of a metal chelator (bathocuproine, 0.5 mM), the DNA compacting polyamines spermidine (1 mM) and spermine (1 mM) or antioxidant enzymes such as superoxide dismutase (10 microg/ml) and catalase (20 pg/ml) protect the DNA against these damages. The data presented here are discussed with respect to the increased frequency of liver cancer in patients with acute intermittent porphyria.

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Interactions between visible or infrared (IR) and ultraviolet (UV, 254 nm) radiation have been studied in E. coli. Pre-illumination with non-coherent monochromatic 446, 466, 570 and 685 nm radiation, as well as with polychromatic red and IR radiation at room temperature, leads to increased cell survival after a subsequent irradiation with UV light. In the thermic range of the spectrum (red and IR), IR but not red light pre-treatment is able to increase cell survival to a subsequent lethal heat (51 degrees C) challenge, suggesting that increased UV survival may be due to IR-induced heat-shock response. On the other hand, visible-light-induced resistance may be due to a different mechanism, possibly involved with unknown bacterial light receptors.

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Cellular accumulation of 5-aminolevulinic acid (ALA), the first specific intermediate of heme biosynthesis, is correlated in liver biopsy samples of acute intermittent porphyria affected patients with an increase in the occurrence of hepatic cancers and the formation of ferritin deposits in hepatocytes. 5-Aminolevulinic acid is able to undergo enolization and to be subsequently oxidized in a reaction catalyzed by iron complexes yielding 4,5-dioxovaleric acid (DOVA). The released superoxide radical (O(*-)(2)) is involved in the formation of reactive hydroxyl radical ((*)OH) or related species arising from a Fenton-type reaction mediated by Fe(II) and Cu(I). This leads to DNA oxidation. The metal catalyzed oxidation of ALA may be exalted by the O(*-)(2) and enoyl radical-mediated release of Fe(II) ions from ferritin. We report here the potentiating effect of ferritin on the ALA-mediated cleavage of plasmid DNA and the enhancement of the formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodGuo). Plasmid pBR322 was incubated with ALA and varying amounts of purified ferritin. DNA damage was assessed by gel electrophoresis analysis of the open and the linear forms of the plasmid from the native supercoiled structure. Addition of either the DNA compacting polyamine spermidine or the metal chelator ethylenediaminetetraacetic acid (EDTA) inhibited the damage. It was also shown that ALA in the presence of ferritin is able to increase the oxidation of the guanine moiety of monomeric 2'-deoxyguanosine (dGuo) and calf thymus DNA (CTDNA) to form 8-oxodGuo as inferred from high performance liquid chromatography (HPLC) measurements using electrochemical detection. The formation of the adduct dGuo-DOVA was detected in CTDNA upon incubation with ALA and ferritin. In a subsequent investigation, the aldehyde DOVA was also able to induces strand breaks in pBR322 DNA.

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Survival of Lactobacillus bulgaricus during spray drying was studied at various outlet air temperatures. During spray drying the logarithmic survival ratio decreased with increased outlet air temperature with first-order kinetics; the pseudo-z value for Lactobacillus bulgaricus was 17.3°C. Plots of the death-rate constant for Lactobacillus bulgaricus versus reciprocal outlet temperature during spray drying in skim milk show a curve with two different activation energies (Ea). The calculated Ea values were 33.47 kJ/mol above 70°C and 85.77 kJ/mol below 70°C. Thermodynamic quantities for spray drying of Lactobacillus bulgaricus are also presented. Results show that the relationship between the entropy of activation and the enthalpy of activation for both spray drying and heating in liquid medium is linear, with all the data for drying falling in the range of a negative entropy.

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