Effects of chemical modifications of crotoxin B, the phospholipase A(2) subunit of crotoxin from Crotalus durissus terrificus snake venom, on its enzymatic and pharmacological activities.

Crotoxin B, the basic Asp49-PLA(2) subunit from crotoxin, the main component of Crotalus durissus terrificus venom, displays myotoxic, edema-inducing, bactericidal (upon Escherichia coli), liposomal-disrupting and anticoagulant activities. Chemical modifications of His (with 4-bromophenacyl bromide, BPB), Tyr (with 2-nitrobenzenesulphonyl fluoride, NBSF), Trp (with o-nitrophenylsulphenyl chloride, NPSC) and Lys (with acetic anhydride) residues of this protein, in addition to cleavage with cyanogen bromide (CNBr) and inhibition with ethylenediaminetetraacetic acid (EDTA), were carried out in order to study their effects on enzymatic and pharmacological activities. Lethality was reduced after modification of His or Lys residues, as well as after cleavage with CNBr, while enzymatic activity was completely abolished after modification of His or incubation with EDTA. Modification of Lys or Tyr, or cleavage with CNBr, partially reduced enzymatic activity. Anticoagulant activity was modified similarly to enzymatic activity, evidencing the dependency of this pharmacological effect on catalytic activity. Myotoxicity was reduced after modification of His or Lys, as well as after cleavage with CNBr, whereas EDTA reduced this effect to a lesser extent. Bactericidal effect was significantly reduced only after modification of Lys and after cleavage with CNBr. Edema-inducing activity was partially inhibited after treatment with EDTA and strongly reduced after acetylation of Lys residues and cleavage with CNBr, being only partially reduced after His alkylation. On the other hand, liposome disrupting activity was only partially reduced after modification of His and Tyr or after cleavage with CNBr. Modification of Trp residue partially reduced lethality and myotoxicity but did not affect enzymatic or anticoagulant activities. These data indicate that enzymatic activity is relevant for some pharmacological effects induced by crotoxin B (mainly lethal, myotoxic and anticoagulant activities), and also evidence that this subunit of crotoxin displays regions different from the active catalytic site which are involved in some of the toxic and pharmacological effects induced by this phospholipase A(2).     

Inhibitory effect of combinations of digoxin and endogenous cardiotonic steroids on Na+/K+-ATPase activity in human kidney membrane preparation

AimsCardiac glycosides have been extensively used in the treatment of congestive heart failure for more than 200 years. Recently, cardenolides and bufadienolides were isolated from mammalian tissue and are considered as a new class of steroidal hormones. The aim of the present work was to characterize the interaction between the most clinical used cardiac glycoside digoxin and the cardiac glycosides known to exist endogenously, i.e., ouabain, marinobufagin and telocinobufagin, on human kidney Na⁺/K⁺-ATPase.Main methodsInhibition of Na⁺/K⁺-ATPase activity from crude membrane preparations of human kidney was performed using increasing concentrations of the drugs alone or mixtures of ouabain:digoxin, telocinobufagin:digoxin and marinobufagin:digoxin in a fixed ratio 1:4, 2:3 and 3:2, respectively. The colorimetric method of Fiske and Subbarow was used to measure the inorganic phosphate released.Key findingsAnalyses of inhibition curves showed that the experimental curves for all combinations were superimposed on the theoretical additive curves indicating that an additive effect occurs among distinct cardenolides and bufadienolides combinations on the human α1β1 Na⁺/K⁺-ATPase protomer.SignificanceConsidering the extensive use of digoxin in the treatment of heart failure and the recent findings that endogenous cardiac glycosides may have altered levels in many diseases, including heart failure, the demonstration of additive effect between cardiac glycosides can help in the understanding of recent clinical observations, including that lower than usual doses of cardiac glycosides are necessary for decreasing mortality in these patients.     

Glucose transport and utilization are altered in the brain of rats deficient in n-3 polyunsaturated fatty acids

Long-chain polyunsaturated (n-3) fatty acids have been reported to influence the efficiency of membrane receptors, transporters and enzymes. Because the brain is particularly rich in docosahexaenoic acid (DHA, 22:6 n-3), the present study addresses the question of whether the 22:6 n-3 fatty acid deficiency induces disorder in regulation of energy metabolism in the CNS. Three brain regions that share a high rate of energy metabolism were studied: fronto-parietal cortex, hippocampus and suprachiasmatic nucleus. The effect of the diet deficient in n-3 fatty acids resulted in a 30-50% decrease in DHA in membrane phospholipids. Moreover, a 30% decrease in glucose uptake and a 20-40% decrease in cytochrome oxidase activity were observed in the three brain regions. The n-3 deficient diet also altered the immunoreactivity of glucose transporters, namely GLUT1 in endothelial cells and GLUT3 in neurones. In n-3 fatty acid deficient rats, GLUT1-immunoreactivity readily detectable in microvessels became sparse, whereas the number of GLUT3 immunoreactive neurones was increased. However, western blot analysis showed no significant difference in GLUT1 and GLUT3 protein levels between rats deficient in n-3 fatty acids and control rats. The present results suggest that changes in energy metabolism induced by n-3 deficiency could result from functional alteration in glucose transporters.     

Prebiotic processes in planetary atmospheres

Within 40 years of experimental studies in prebiotic chemistry, most of the building blocks of the living systems have been synthesized in plausible conditions of the primitive Earth. The starting ingredients correspond to two complementary classes: volatile organics, and their non volatile oligomers. They may have been formed in the atmosphere on the primitive Earth and/or imported by extra-terrestrial sources. Organic chemistry is involved in meteorites, comets, in the giant planets and several of their satellites. Again this chemistry presents the two complementary aspects. In particular, with a dense reduced atmosphere rich in organic compounds in gas and aerosol phases, Titan appears as a natural laboratory for studying prebiotic chemistry at a planetary scale.     

Improving Quality Control of Contagious Caprine Pleuropneumonia Vaccine with Tandem Mass Spectrometry

Vaccines to protect livestock against contagious caprine pleuropneumonia (CCPP) consist of inactivated, adjuvanted antigens. Quality control of these vaccines is challenging as total protein quantification provides no indication of protein identity or purity, and culture is not an option. Here, a tandem mass spectrometry approach is used to identify the mycoplasma antigen contained in reference samples and in commercial CCPP vaccines. By the same approach, the relative amounts of mycoplasma antigen and residual proteins originating from the production medium are determined. Mass spectrometry allows easy and rapid identification of the peptides present in the vaccine samples. Alongside the most probable mycoplasma species effectively present in the vaccines, a very high proportion of peptides from medium constituents are detected in the commercial vaccines tested.     

X-ray diffraction characterization of the dense phases formed by nucleosome core particles

Multiple dense phases of nucleosome core particles (NCPs) were formed in controlled ionic conditions (15-160 mM monovalent salt, no divalent ions), under osmotic pressures ranging from 4.7 x 10(5) to 2.35 x 10(6) Pa. We present here the x-ray diffraction analysis of these phases. In the lamello-columnar phase obtained at low salt concentration (<25 mM), NCPs stack into columns that align to form bilayers, kept separated from one another by a layer of solvent. NCPs form a monoclinic lattice in the plane of the bilayer. For high salt concentration (>50 mM), NCPs order into either a two-dimensional columnar hexagonal phase or into three-dimensional orthorhombic (quasi-hexagonal) crystals. The lamellar and hexagonal (or quasi-hexagonal) organizations coexist in the intermediate salt range; their demixing requires a long time. For an applied pressure P = 4.7 10(5) Pa, the calculated NCPs concentration ranges from approximately 280 to 320 mg/ml in the lamello-columnar phase to 495 to 585 mg/ml in the three-dimensional orthorhombic phase. These concentrations cover the concentration of the living cell.     

Prevalence and seasonal variations of six bee viruses in Apis mellifera L. and Varroa destructor mite populations in France

A survey of six bee viruses on a large geographic scale was undertaken by using seemingly healthy bee colonies and the PCR technique. Samples of adult bees and pupae were collected from 36 apiaries in the spring, summer, and autumn during 2002. Varroa destructor samples were collected at the end of summer following acaricide treatment. In adult bees, during the year deformed wing virus (DWV) was found at least once in 97% of the apiaries, sacbrood virus (SBV) was found in 86% of the apiaries, chronic bee paralysis virus (CBPV) was found in 28% of the apiaries, acute bee paralysis virus (ABPV) was found in 58% of the apiaries, black queen cell virus (BQCV) was found in 86% of the apiaries, and Kashmir bee virus (KBV) was found in 17% of the apiaries. For pupae, the following frequencies were obtained: DWV, 94% of the apiaries; SBV, 80% of the apiaries; CBPV, none of the apiaries; ABPV, 23% of the apiaries; BQCV, 23% of the apiaries; and KBV, 6% of the apiaries. In Varroa samples, the following four viruses were identified: DWV (100% of the apiaries), SBV (45% of the apiaries), ABPV (36% of the apiaries), and KBV (5% of the apiaries). The latter findings support the putative role of mites in transmitting these viruses. Taken together, these data indicate that bee virus infections occur persistently in bee populations despite the lack of clinical signs, suggesting that colony disease outbreaks might result from environmental factors that lead to activation of viral replication in bees.     

Trans-Arctic dispersals and the evolution of a circumpolar marine fish species complex, the capelin (Mallotus villosus)

Trans-Arctic dispersals and population and range expansions during the Pleistocene enhanced opportunities for evolutionary diversification and contributed to the process of speciation within the capelin, a northern marine-fish complex exhibiting a circumpolar distribution. Capelin is composed of four highly divergent and geographically discrete mitochondrial DNA (mtDNA) clades (609 bp; cytochrome b). Two clades occur in the North Atlantic, one associated with Canadian Atlantic waters, including Hudson Bay, and the second distributed from West Greenland to the Barents Sea. Two additional clades occur in the Arctic and northeast Pacific Oceans, representing the most recent divergence within the capelin phylogenetic tree. Judged from mtDNA diversity, capelin populations comprising all clades experienced at least one demographic and spatial reduction-expansion episode during recent Pleistocene glaciations that imprinted their molecular architecture. The large contemporary populations in the northeast Pacific and Arctic Oceans exhibited significant genetic structure whereas no such structure was detected in the equally extensive North Atlantic clades. All clades are characterized by one or two prevalent mtDNA haplotypes distributed over the entire range of the clade. Assuming a Pacific ancestor for capelin, we infer that capelin dispersed on two separate occasions to the North Atlantic. A more recent event resulted in the isolation of eastern Pacific and Arctic clades, with the Arctic clade positioned for a potential third Atlantic invasion, as revealed by the presence of this clade in the Labrador Sea. The Labrador Sea is a potential contact zone for three of the four capelin clades.