Technical improvement to prevent DNA degradation of Leptospira spp. in pulsed field gel electrophoresis

Leptospirosis is a public health problem. Infection with pathogenic Leptospira occurs by exposure to many environments and is traditionally associated with occupational risk activities. Pulsed-field gel electrophoresis was used to investigate the epidemiological relatedness among Leptospira isolates. However, analysis by PFGE yielded inconclusive data as a result of extensive DNA degradation. This degradation can be significantly reduced by the inclusion of thiourea in the electrophoresis buffer, improving the analysis of DNA banding patterns.     

Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase‐D (dermonecrotic toxin) from brown spider venom

Brown spiders have world‐wide distribution and are the cause of health problems known as loxoscelism. Necrotic cutaneous lesions surrounding the bites and less intense systemic signs like renal failure, DIC, and hemolysis were observed. We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase‐D , causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose‐dependent and time‐dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP‐phospholipase‐D protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin‐treated erythrocytes reacted with annexin‐V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase‐D , which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine‐protease inhibitor) that had no effect on hemolysis. By site‐directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxin's ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that L. intermedia venom phospholipase‐D triggers direct human blood cell hemolysis in a catalytic‐dependent manner. J. Cell. Biochem. 107: 655–666, 2009. © 2009 Wiley‐Liss, Inc.     

Nitrated plasma albumin as a marker of nitrative stress and neonatal encephalopathy in perinatal asphyxia

Reactive nitrogen species (RNS) have been shown to play a major role in the pathophysiology of hypoxic–ischemic cerebral injury. Using a novel sensitive ELISA allowing the quantification of nitrated albumin (nitroalbumin) in plasma, we tested the hypothesis that perinatal asphyxia increases nitrating RNS generation by verifying whether the concentration of one of its target proteins is correlated with the clinical outcome. We assayed nitroalbumin in 114 plasma samples collected during the first hour, at day 1, and at day 4 of life from 48 term newborns suffering from perinatal asphyxia and correlated this marker with neurological and systemic neonatal outcomes. Nitroalbumin levels at day 1, but not at days 0 and 4, were significantly increased in patients who developed moderate or severe encephalopathy compared to those who had a normal neurological evolution or developed mild encephalopathy (median: 14.4 ng/ml versus 7.3 ng/ml, respectively). In contrast, nitroalbumin concentration at day 1 was not associated with systemic complications. First-hour and fourth-day nitroalbumin concentrations did not differ with respect to the neonatal neurological course. At day 0, nitroalbumin levels also correlated with circulating leukocytes. We conclude that plasma nitroalbumin seems to be a specific marker of neurological injury after perinatal asphyxia and may serve as a secondary end-point in neuroprotective clinical trials.     

Pollen morphology of the shrub and arboreal flora of mangroves of Northeastern Brazil

The Brazilian coast, especially in the Northeastern one, possesses a vast area of mangrove which acts as the interface that links terrestrial ecosystems and estuaries. After an inventory of the shrub and tree species present in the mangroves of Northeastern Brazil, polliniferous material was collected for morphological study of the pollen grains, which were acetolysed, measured and analyzed using light and scanning electron microscopy. Fourteen species belonging to seven families were grouped as restricted and peripheral, according to their position into the mangrove vegetation. The restricted species are six: Avicennia germinans and A. schaueriana (Avicenniaceae), Rhizophora harrisonii, R. mangle and R. racemosa (Rhizophoraceae), Laguncularia racemosa (Combretaceae). The peripheral species are eight: Annona glabra (Annonaceae), Conocarpus erectus (Combretaceae), Dalbergia ecastophyllum, Inga subnuda, Sophora tomentosa (Leguminosae), Hibiscus pernambucensis, H. tiliaceus (Malvaceae) and Suriana maritima (Surianaceae). All species had their pollen grains described and illustrated in that first atlas of the pollen flora of the Brazilian mangroves.     

A TonB-dependent outer membrane protein as a Bacteroides fragilis fibronectin-binding molecule

The binding of Bacteroides fragilis to plasmatic fibronectin was investigated using strains isolated from healthy subjects and from patients with bacteremia. They were cultivated in a synthetic media in which variations in cysteine concentrations determined alterations in the oxidation–reduction potential (Eh). All the strains assayed were capable of adhering to plasmatic fibronectin when cultivated under oxidizing and reducing conditions. Bacteroides fragilis 1405 showed the greatest difference when the results under these conditions were compared and it was selected for further investigations. Chemical treatments suggested the involvement of a protein in the interaction between B. fragilis and plasmatic fibronectin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of outer membrane proteins (OMPs) revealed differences between the extracts obtained from cultures grown under the two conditions. Protein bands of c . 102, 100, 77, 73, 50 and 40 kDa were more highly expressed under oxidizing than reducing conditions. Dot blot analysis showed a stronger recognition of plasmatic fibronectin by OMPs obtained from cultures grown under higher Eh, and Western blot assays confirmed a band of c . 102 kDa as fibronectin-binding protein. This protein was sequenced and revealed to be a putative TonB-dependent OMPs. PCR analysis confirmed the presence of this gene in all the studied strains.     

A new method for measurement of annual growth rings in cork by means of autofluorescence

A new method for measuring annual growth rings in cork has been developed. It is based on the fact that excitation of Quercus suber L. cork cross-sectional planes with UV light and also with blue light results in enhanced autofluorescence at the growth ring borders. This distinct autofluorescence band is located in the cork produced at the end of the vegetation period, with its smaller cells and thicker cell walls. The enhanced autofluorescence of polyphenolics mainly results from a very thick suberin layer in the walls of the small late cork cells. The gradient in autofluorescence from late cork to first early cork is steep. The best visibility of cork annual rings was found under the epifluorescence microscope in 60 μm thick microtome cork cross sections. For fast screening of high sample numbers scanning the blue-excited (473 nm) fluorescence of water wetted polished cross-sectional surfaces of cork pieces with a laser-equipped fluorescence image analyzer was most suitable. Evaluation of visibility of cork rings showed a clear improvement in comparison with reflected light image analysis; thus data obtained with this new autofluorescence scanning method are excellent for modeling the yearly growth increment of cork.     

Omega-3 consumption and sudden cardiac death in schizophrenia

People with schizophrenia show a two- to three-fold increased risk to die prematurely. Mortality is accounted for by a combination of factors (patients’ life style, suicide, premature cardiovascular disease, metabolic syndromes and, not so often mentioned, sudden death). The cause of sudden death in schizophrenia is unknown, but cardiac arrhythmia plays a potential role. Patients with schizophrenia are at high risk for cardiovascular disease, and some antipsychotics may be associated with cardiovascular adverse events (e.g., electrocardiograph QT interval prolongation), suggesting that this could lead to sudden cardiac death. Animal and clinical studies have shown that omega-3 fatty acids could be useful in the prevention and treatment of schizophrenia. As omega-3 fatty acids have been considered a cardioprotector agent, reducing cardiac arrhythmias and hence sudden cardiac deaths and given their relative safety and general health benefits, our update article summarizes the knowledge by the possible positive effects of omega-3 supplementation and fish consumption against sudden cardiac death in patients with schizophrenia. However, fish species should be selected with caution due to contamination with toxic methylmercury.