Gas-phase dissociation of 1,4-naphthoquinone derivative anions by electrospray ionization tandem mass spectrometry

Gas-phase dissociation pathways of deprotonated 1,4-naphthoquinone (NQ) derivatives have been investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The major decomposition routes have been elucidated on the basis of quantum chemical calculations at the B3LYP/6-31+G(d,p) level. Deprotonation sites have been indicated by analysis of natural charges and gas-phase acidity. NQ anions underwent an interesting reaction under collision-induced dissociation conditions, which resulted in the radical elimination of the lateral chain, in contrast with the even-electron rule. Possible pathways have been suggested, and their mechanisms have been elucidated on the basis of Gibbs energy and enthalpy values for the anions previously described at each pathway. Copyright (C) 2009 John Wiley & Sons, Ltd.

What the reasons for no inbreeding and high genetic diversity of the neotropical fig tree Ficus arpazusa ?

Ficus arpazusa Casaretto is a fig tree native to the Atlantic Rain Forest sensu lato. High levels of genetic diversity and no inbreeding were observed in Ficus arpazusa. This genetic pattern is due to the action of its pollinator, Pegoscapus sp., which disperses pollen an estimated distance of 5.6 km, and of Ficus arpazusa`s mating system which, in the study area, is allogamous. This study highlights the importance of adding both ecological and genetic data into population studies, allowing a better understanding of evolutionary processes and in turn increasing the efficacy of forest management and revegetation projects, as well as species conservation.

Serum from children with polyarticular juvenile idiopathic arthritis (pJIA) inhibits differentiation, mineralization and may increase apoptosis of human osteoblasts ""in vitro""

We examined the effects of polyarticular juvenile idiopathic arthritis (pJIA) serum on proliferation, differentiation, mineralization, and apoptosis of human osteoblast cells (hOb) in culture. The hOb were cultured with 10% serum from active pJIA and healthy controls (CT) and were tested for DNA synthesis, alkaline phosphatase (AP) activity, osteocalcin (OC) secretion, calcium levels, caspase 3 activity, and DNA fragmentation. None of the patients had used glucocorticoids for at least 1 month before the study, or any other drug that can affect bone mineral metabolism. Human inflammatory cytokine levels (IL-6, IL-8, IL-10, IL-1 beta, TNF-alpha, and IL-12p70) were measured in pJIA and CT sera. Low levels of AP activity was observed in pJIA cultures compared with CT cultures (67.16 +/- 53.35 vs 100.11 +/- 50.64 mu mol p-nitrophenol/h(-1) mg(-1) protein, P=0.008). There was also a significant decrease in OC secretion (9.23 +/- 5.63 vs 12.82 +/- 7.02 ng/mg protein, P=0.012) and calcium levels (0.475 +/- 0.197 vs 0.717 +/- 0.366 mmol/l, P=0.05) in pJIA hOb cultures. No difference was observed in cell proliferation (323.56 +/- 108.23 vs 328.91 +/- 88.03 dpm/mg protein, P=0.788). Osteoblasts cultured with JIA sera showed lower levels of DNA and increased fragmentation than osteoblasts cultured with CT sera. pJIA sera showed higher IL-6 values than CT (21.44 +/- 9.31 vs 3.58 +/- 2.38 pg/ml, P<0.001), but no difference was observed related to IL-8, IL-10, IL-1 beta, TNF-alpha, and IL-12p70 between pJIA and controls. This study suggests that serum from children with pJIA inhibits differentiation, mineralization and may increase apoptosis of hOb cultures, and inflammatory cytokines such as IL-6 might be a mechanism in this find. These results may represent an alternative therapeutic target for prevention and treatment of bone loss in JIA.

Pre- and postnatal exposure to ambient levels of urban particulate matter (PM(2.5)) affects mice spermatogenesis

This work characterizes the effects of ambient levels of urban particulate matter (PM(2.5)) from the city of Sao Paulo on spermatogenesis using mice exposed during the embryo-fetal and/or postnatal phases of development. Parental generations (BALB/c mice) were exposed to air pollution in chambers with or without filtering PM(2.5) for 4 months. Animals were mated, and half of the 1-day-old offspring were moved between chambers, which yielded prenatal and postnatal groups. Remaining offspring comprised the non-exposed and pre+postnatal exposed groups. After 90 days, the animals were sacrificed for testis collection and weighing. Optical microscopy was used for the morphometric analyses of the cell counts, spermatogenic cycle, proliferation, and apoptosis. Prenatally exposed animals presented reduced body and testicular weight with an increased gonadosomatic index (GSI). Testicular volume also decreased, as well as the tubular diameter in testes of the same animals. Proliferation, apoptosis, and spermatogenic cycle analyses showed no significant differences among groups. However, the tubules at stage VII of pre- and postnatal animals presented a reduced number of elongated spermatids. Pre+postnatal group presented higher spermatid head retention at stages VIII-XII. These results show that ambient levels of PM(2.5) from Sao Paulo city affect spermatogenesis by damaging sperm production.

Metabolic consequences of intermittent hypoxia: Relevance to obstructive sleep apnea

Obstructive sleep apnea (OSA) is recurrent obstruction of the upper airway leading to sleep fragmentation and intermittent hypoxia (IH) during sleep. There is growing evidence from animal models of OSA that IH is independently associated with metabolic dysfunction, including dyslipidemia and insulin resistance. The precise mechanisms by which IH induces metabolic disturbances are not fully understood. Over the last decade, several groups of investigators developed a rodent model of IH, which emulates the oxyhemoglobin profile in human USA. In the mouse model, IH induces dyslipidemia, insulin resistance and pancreatic endocrine dysfunction, similar to those observed in human USA. Recent reports provided new insights in possible mechanisms by which IH affects lipid and glucose metabolism. IH may induce dyslipidemia by up-regulating lipid biosynthesis in the liver, increasing adipose tissue lipolysis with subsequent free fatty acid flux to the liver, and inhibiting lipoprotein clearance. IH may affect glucose metabolism by inducing sympathetic activation, increasing systemic inflammation, increasing counter-regulatory hormones and fatty acids, and causing direct pancreatic beta-cell injury. IH models of USA have improved our understanding of the metabolic impact of USA, but further studies are needed before we can translate recent basic research findings to clinical practice. (C) 2010 Elsevier Ltd. All rights reserved.

Effects of exercise on leukocyte death: prevention by hydrolyzed whey protein enriched with glutamine dipeptide

Lymphocyte and neutrophil death induced by exercise and the role of hydrolyzed whey protein enriched with glutamine dipeptide (Gln) supplementation was investigated. Nine triathletes performed two exhaustive exercise trials with a 1-week interval in a randomized, double blind, crossover protocol. Thirty minutes before treadmill exhaustive exercise at variable speeds in an inclination of 1% the subjects ingested 50 g of maltodextrin (placebo) or 50 g of maltodextrin plus 4 tablets of 700 mg of hydrolyzed whey protein enriched with 175 mg of glutamine dipeptide dissolved in 250 mL water. Cell viability, DNA fragmentation, mitochondrial transmembrane potential and production of reactive oxygen species (ROS) were determined in lymphocytes and neutrophils. Exhaustive exercise decreased viable lymphocytes but had no effect on neutrophils. A 2.2-fold increase in the proportion of lymphocytes and neutrophils with depolarized mitochondria was observed after exhaustive exercise. Supplementation of maltodextrin plus Gln (MGln) prevented the loss of lymphocyte membrane integrity and the mitochondrial membrane depolarization induced by exercise. Exercise caused an increase in ROS production by neutrophils, whereas supplementation of MGln had no additional effect. MGln supplementation partially prevented lymphocyte apoptosis induced by exhaustive exercise possibly by a protective effect on mitochondrial function.

Understanding the genetic diversity, spatial genetic structure and mating system at the hierarchical levels of fruits and individuals of a continuous Theobroma cacao population from the Brazilian Amazon

Understanding the mating patterns of populations of tree species is a key component of ex situ genetic conservation. In this study, we analysed the genetic diversity, spatial genetic structure (SGS) and mating system at the hierarchical levels of fruits and individuals as well as pollen dispersal patterns in a continuous population of Theobroma cacao in Para State, Brazil. A total of 156 individuals in a 0.56 ha plot were mapped and genotyped for nine microsatellite loci. For the mating system analyses, 50 seeds were collected from nine seed trees by sampling five fruits per tree (10 seeds per fruit). Among the 156 individuals, 127 had unique multilocus genotypes, and the remaining were clones. The population was spatially aggregated; it demonstrated a significant SGS up to 15m that could be attributed primarily to the presence of clones. However, the short seed dispersal distance also contributed to this pattern. Population matings occurred mainly via outcrossing, but selfing was observed in some seed trees, which indicated the presence of individual variation for self-incompatibility. The matings were also correlated, especially within ((r) over cap (p(m)) = 0.607) rather than among the fruits ((r) over cap (p(m)) = 0.099), which suggested that a small number of pollen donors fertilised each fruit. The paternity analysis suggested a high proportion of pollen migration (61.3%), although within the plot, most of the pollen dispersal encompassed short distances (28m). The determination of these novel parameters provides the fundamental information required to establish long-term ex situ conservation strategies for this important tropical species. Heredity (2011) 106, 973-985; doi:10.1038/hdy.2010.145; published online 8 December 2010

Crotamine toxicity and efficacy in mouse models of melanoma

Objectives: Selective anticancer cell activity for both cell-penetrating and cationic antimicrobial peptides has previously been reported. As crotamine possesses activities similar to both of these, this study investigates crotamine`s anticancer toxicity in vitro and in vivo. Research design and methods: In vitro cancer cell viability was evaluated after treatment with 1 and 5 mu g/ml of crotamine. In vivo crotamine cytotoxic effects in C57Bl/6J mice bearing B16-F10 primary cutaneous melanoma were tested, with two groups each containing 35 mice. The crotamine-treated group received 1 mu g/day of crotamine per animal, subcutaneously which was well tolerated; the untreated group received a placebo. Results: Crotamine at 5 mu g/ml was lethal to B16-F10, Mia PaCa-2 and SK-Mel-28 cells and inoffensive to normal cells. In vivo crotamine treatment over 21 days significantly delayed tumor implantation, inhibited tumor growth and prolonged the lifespan of the mice. Mice in the crotamine-treated group survived at significantly higher rates (n = 30/35) than those in the untreated group (n = 7/35) (significance calculated with the Kaplan-Meier estimator). The average tumor weight in the untreated group was 4.60 g but was only about 0.27 g in the crotamine-treated mice, if detectable. Conclusions: These data warrant further exploration of crotamine as a tumor inhibition compound.

Apoptosis induced by Oropouche virus infection in HeLa cells is dependent on virus protein expression

Oropouche (OROV) is a single-stranded RNA arbovirus of the family Bunyaviridae, genus Orthobunyavirus, which has caused over half a million cases of febrile illness in Brazil in the past 30 years. OROV fever has been registered almost exclusively in the Amazon region, but global warming, deforestation and redistribution of vectors and animal reservoirs increases the risk of Oropouche virus emergence in other areas. OROV causes a cytolytical infection in cultured cells with characteristic cytopathic effect 48 h post-infection. We have studied the mechanisms of apoptosis induced by OROV in HeLa cells and found that OROV causes DNA fragmentation detectable by gel electrophoresis and by flow cytometric analysis of the Sub-G1 population at 36 h post-infection. Mitochondrial release of cytochrome C and activation of caspases 9 and 3 were also detected by western blot analysis. Lack of apoptosis induced by UV-inactivated OROV reveals that virus-receptor binding is not sufficient to induce cell death. Results obtained in cells treated with chloroquine and cycloheximide indicated that viral uncoating and replication are required for apoptosis induction by OROV. Furthermore, treatment of the cells with pan-caspase inhibitor prevented OROV-induced apoptosis without affecting virus progeny production. The results show that OROV infection in vitro causes apoptosis by an intracellular pathway involving mitochondria, and activated by a mechanism dependent on viral replication and protein synthesis. (C) 2010 Elsevier B.V. All rights reserved.

Relationship between first polar body morphology before intracytoplasmic sperm injection and fertilization rate, cleavage rate, and embryo quality

Objectives: To evaluate the influence of the morphology of the first polar body (PB) on intracytoplasmic sperm injection (ICSI) outcomes. Methods: The morphology of the first PB was assessed in 3177 metaphase 11 oocytes and classified as: intact and normal size, fragmented, or enlarged size. The rates of fertilization, cleavage, and embryo quality were evaluated on day 2. Results: The rates of fertilization, cleavage, and formation of good quality embryos resulting from the insemination of oocytes with an enlarged first PB (20.7%, 18.7%, and 5.0%, respectively) were significantly lower than those for oocytes with an intact first PB of normal size (70.8%, 62.5%, and 19%, respectively) or a fragmented first PB (69.7%, 60.5%, and 17.1%, respectively). Rates did not differ significantly between oocytes with an intact first PB of normal size and oocytes with a fragmented first PB (P>0.05). Conclusions: The presence of an enlarged PB is related to poorer rates of fertilization, cleavage, and top quality embryos. However, identification of first PB fragmentation does not seem to interfere with ICSI outcomes. (C) 2008 International Federation ofGynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

INCREASED SARCOLEMMAL PERMEABILITY AS AN EARLY EVENT IN EXPERIMENTAL SEPTIC CARDIOMYOPATHY: A POTENTIAL ROLE FOR OXIDATIVE DAMAGE TO LIPIDS AND PROTEINS

This study describes increased sarcolemmal permeability and myofilamentar damage that occur together with lipid peroxidation and protein nitration in the myocardium in severe sepsis induced by cecal ligation and puncture. Male C57BL/6 mice were submitted to moderate and severe septic injury and sham operation. Using light and laser confocal microscopy, diffuse foci of myocytolysis associated with focal disruption of the actin/myosin contractile apparatus could be seen in hearts with severe septic injury. The myocardial expressions of the sarcomeric proteins myosin and actin were downregulated by both severe and moderate injuries. The detection of albumin staining in the cytoplasm of myocytes to evaluate sarcolemmal permeability provided evidence of severe and mild injury of the plasma membrane in hearts with severe and moderate septic injury, respectively. The administration of a superoxide scavenger caused marked reduction of sarcolemmal permeability, indicating the involvement of free radicals in its genesis. On electron microscopy, these changes were seen to correspond to spread blocks of a few myocytes with fragmentation and dissolution of myofibrils, intracellular edema, and, occasionally, rupture of the sarcolemma. In addition, oxidative damage to lipids, using anti-4-hydroxynonenal, an indicator of oxidative stress and disruption of plasma membrane lipids, and to proteins, using antinitrotyrosine, a stable biomarker of peroxynitrite-mediated protein nitration, was demonstrated. These findings make plausible the hypothesis that increased sarcolemmal permeability might be a primary event in myocardial injury in severe sepsis possibly due to oxidative damage to lipids and proteins that could precede phenotypic changes that characterize a septic cardiomyopathy.

Microsatellite diversity and effective population size in a germplasm bank of Hymenaea courbaril var. stilbocarpa (Leguminosae), an endangered tropical tree: recommendations for conservation

Deforestation in southeast Brazil has led to the extinction of Hymenaea courbaril var. stilbocarpa and ex situ conservation has been established. In this study, the levels of genetic diversity and the effective population size of H. courbaril in a germplasm bank were investigated using six nuclear microsatellite loci. A total of 79 and 91 alleles were found in 65 seed-trees and their 176 offspring, respectively. Offspring have a higher average number of alleles per locus (A = 15.2) than seed-trees (A = 13.2), but lower observed heterozygosity (offspring: H (o) = 0.566; seed-trees: H (o) = 0.607). The estimate of outcrossing rate shows that the study population is perfectly outcrossed (t (m) = 0.978, P > 0.05). Significant deviations from random mating were detected through mating among relatives and correlated matings. The average variance in effective population size for each family was 2.63, with a total effective population size retained in the bank of 170.1. These results confirm that the preserved population of H. courbaril retains substantial genetic variability.

Syndromic and non-syndromic aneurysms of the human ascending aorta share activation of the Smad2 pathway

Common features such as elastic fibre destruction, mucoid accumulation, and smooth muscle cell apoptosis are co-localized in aneurysms of the ascending aorta of various aetiologies. Recent experimental studies reported an activation of TGF-beta in aneurysms related to Marfan (and Loeys-Dietz) syndrome. Here we investigate TGF-beta signalling in normal and pathological human ascending aortic wall in syndromic and non-syndromic aneurysmal disease. Aneurysmal ascending aortic specimens, classified according to aetiology: syndromic MFS (n = 15, including two mutations in TGFBR2), associated with BAV (n = 15) or degenerative forms (n = 19), were examined. We show that the amounts of TGF-beta 1 protein retained within and released by aneurysmal tissue were greater than for control aortic tissue, whatever the aetiology, contrasting with an unchanged TGF-beta 1 mRNA level. The increase in stored TGF-beta 1 was associated with enhanced LTBP-I protein and mRNA levels. These dysiregulations of the extracellular ligand are associated with higher phosphorylated Smad2 and Smad2 mRNA levels in the ascending aortic wall from all types of aneurysm. This activation correlated with the degree of elastic fibre fragmentation. Surprisingly, there was no consistent association between the nuclear location of pSmad2 and extracellular TGF-beta 1 and LTBP-I staining and between their respective mRNA expressions. In parallel, decorin. was focally increased in aneurysmal media, whereas biglycan was globally decreased in aneurysmal aortas. In conclusion, this study highlights independent dysregulations of TGF-beta retention and Smad2 signalling in syndromic and non-syndromic aneurysms of the ascending aorta. Copyright (C) 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Protease activity and the ultrastructure of broiler chicken PSE (pale, soft, exudative) meat

The biological cause of broiler PSE meat seems to be an excessive release of Ca(2+), promoted by a genetic mutation of ryanodine receptors located in the sarcoplasmic reticulum of skeletal muscle cells. Excessive Ca(2+), associated with protein denaturation in meat, enhances protease activity and influences the functional properties of PSE meat. Twenty-four-hour post-mortem Pectoralis major m. samples exhibited lower values for pH, water-holding capacity, and shear force than did control samples, in contrast to colour (L*) and cooking loss values. Protease activity, measured as myofibril fragmentation index, presented higher values in PSE meat than in control samples. Ultrastructural examination revealed shrinking and depolymerisation of myofilaments and Z-lines disorganisation within the sarcomere in PSE meat. Intense calpain activity was also observed, indicating that the process may initiate at the filaments, because of protein denaturation, and spread through Z-lines, resulting in the collapse of the sarcomere structure. (C) 2009 Elsevier Ltd. All rights reserved.

Wild canids, domestic dogs and their pathogens in Southeast Brazil: disease threats for canid conservation

Wild canids are under many pressures, including habitat loss, fragmentation and disease. The current lack of information on the status of wildlife health may hamper conservation efforts in Brazil. In this paper, we examined the prevalence of canine pathogens in 21 free-ranging wild canids, comprising 12 Cerdocyon thous (crab-eating fox), 7 Chrysocyon brachyurus (maned wolf), 2 Lycalopex vetulus (hoary fox), and 70 non-vaccinated domestic dogs from the Serra do Cip National Park area, Southeast Brazil. For wild canids, seroprevalence of antibodies to canine parvovirus, canine adenovirus, canine coronavirus and Toxoplasma gondii was 100 (21/21), 33 (7/21), 5 (1/19) and 68 (13/19) percent, respectively. Antibodies against canine distemper virus, Neospora caninum or Babesia spp. were not found. We tested domestic dogs for antibodies to canine parvovirus, canine distemper virus and Babesia spp., and seroprevalences were 59 (41/70), 66 (46/70), and 42 (40/70) percent, respectively, with significantly higher prevalence in domestic dogs for CDV (P < 0.001) and Babesia spp. (P = 0.002), and in wild canids for CPV (P < 0.001). We report for the first time evidence of exposure to canine coronavirus in wild hoary foxes, and Platynossomun sp. infection in wild maned wolves. Maned wolves are more exposed to helminths than crab-eating foxes, with a higher prevalence of Trichuridae and Ancylostomidae in the area. The most common ectoparasites were Amblyomma cajennense, A. tigrinum, and Pulex irritans. Such data is useful information on infectious diseases of Brazilian wild canids, revealing pathogens as a threat to wild canids in the area. Control measures are discussed.