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A phosphodiesterase was purified from the venom of the snake Bothrops alternatus by a combination of gel filtration and ion exchange chromatographies. In SDS-PAGE, the enzyme gave a single band with a molecular mass of 105 kDa, which was unaltered in the presence of beta-mercaptoethanol, indicating that the protein contained no subunits. A single protein band was also observed in native PAGE. There were no contaminating 5'-nucleotidase, alkaline phosphatase and protease activities. The enzyme was recognized by commercial bothropic antiserum and gave a single band in immunoblotting. The enzyme had a pH optimum in the range of 7.5-9.5 and the optimum temperature was 60 degrees C, with activity being rapidly lost within 1 min at > or = 70 degrees C. The Km of the enzyme was 2.69 mM. PDE activity was potentiated by cobalt and, to a lesser extent, by calcium, whereas copper, manganese, zinc, EDTA, and beta-mercaptoethanol were inhibitory. These properties show that this enzyme is very similar to that isolated from other snake venoms.

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OBJECTIVE:To determine the incidence of Mycobacterium tuberculosis complex and non-tuberculous mycobacterial isolates in the routine setting of a large general hospital using an "in-house" multiplex polymerase chain reaction method and to establish a paradigm for the definitive identification of mycobacteria isolated using semi-automated equipment.METHODS:Established tests, including polymerase chain reaction restriction enzyme analysis, PNB, and NAP inhibition tests as the gold standard, showed 100% agreement with an IS6110/hsp65 multiplex polymerase chain reaction when used to identify stock strains (n = 117).RESULTS:In a subsequent study, 8,790 clinical specimens producing 476 isolates were evaluated with multiplex PCR and also showed 100% agreement in identification using PRA-polymerase chain reaction as the gold standard. The application of this technique to routine analysis was demonstrated in this study. A method was established with the initial application of multiplex PCR for all positive liquid cultures and the subsequent identification of non-tuberculous mycobacteria by polymerase chain reaction restriction enzyme analysis. In total, 77% of isolates belonged to the Mycobacterium tuberculosis complex, and 23% were non-tuberculous mycobacteria.CONCLUSIONS:Several non-tuberculous mycobacterial species were identified, primarily M. avium, but other potentially pathogenic species were also frequently observed, including M. fortuitum, M. abscessus, and M. kansasii. The expeditious communication of these data to the clinical staff was fundamental for the diagnosis of clinical cases. Even in settings where tuberculosis is of major importance, the incidence of non-tuberculous mycobacteria infection is substantial.

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Severe cases of human envenoming by caterpillars of the saturniid moth Lonomia obliqua in Brazil can result in renal damage, leading to renal failure, and intracerebral hemorrhaging. In this work, we used immunohistochemical staining with rabbit antiserum raised against L. obliqua venom to examine venom distribution in selected tissues of the brain (cerebellum and hippocampus), kidneys, and liver of male Wistar rats injected with a single dose of venom (200 microg/kg, i.v.) and sacrificed 6, 18, 24, and 72 hours later. The immunolabeling of GFAP was also examined to assess the venom effects on perivascular astrocytic end-feet in the microvasculature of the hippocampus and cerebellum. Venom was detected in the kidneys (6 and 18 hours) and in the liver (6 hours) but not in the brain at any of the time intervals examined. In contrast, immunolabeling for GFAP revealed astrogliosis in the cerebellum and enhanced expression of this protein in the glial processes of the cerebellum and hippocampus, with a maximum response from 24 hours onwards. The high immunoreactivity seen in the kidneys agreed with the renal damage and dysfunction reported for some patients. The lack of venom detection in the brain, despite the altered expression of GFAP in astrocytes, suggested either that the venom does not enter this organ or that its entrance is transient and fast. Alternatively, the circulating venom may induce the release of mediators that could serve as second messengers to provoke the late astrocytic reactivity and astrogliosis. It is possible that both of these mechanisms may contribute to the effects observed.

Magnetic nanoparticles (MNps) have become powerful tools for multiple biomedical applications such as hyperthermia drivers, magnetic resonance imaging (MRI) vectors, as well as drug-delivery systems. However, their toxic effects on human health have not yet been fully elucidated, especially in view of their great diversity of surface modifications and functionalizations. Citrate-coating of MNps often results in increased hydrophilicity, which may positively impact their performance as drug-delivery systems. Nonetheless, the consequences on the intrinsic toxicity of such MNps are unpredictable. Herein, novel magnetite (Fe3O4) nanoparticles covered with citrate were synthesized and their potential intrinsic acute toxic effects were investigated using in vitro and in vivo models. The proposed synthetic pathway turned out to be simple, quick, inexpensive, and reproducible. Concerning toxicity risk assessment, these citrate-coated iron oxide nanoparticles (IONps) did not affect the in vitro viability of different cell lines (HaCaT and HepG2). Moreover, the in vivo acute dose assay (OECD test guideline #425) showed no alterations in clinical parameters, relevant biochemical variables, or morphological aspects of vital organs (such as brain, liver, lung and kidney). Iron concentrations were slightly increased in the liver, as shown by Graphite Furnace Atomic Absorption Spectrometry and Perls Prussian Blue Staining assays, but this finding was considered non-adverse, given the absence of accompanying functional/clinical repercussions. In conclusion, this study reports on the development of a simple, fast and reproducible method to obtain citrate-coated IONps with promising safety features, which may be used as a drug nanodelivery system in the short run. (263 words)