Snake population venomics and antivenomics of Bothrops atrox: Paedomorphism along its transamazonian dispersal and implications of geographic venom variability on snakebite management

We describe two geographically differentiated venom phenotypes across the wide distribution range of Bothrops atrox, from the Colombian Magdalena Medio Valley through Puerto Ayacucho and El Paují, in the Venezuelan States of Amazonas and Orinoquia, respectively, and S?o Bento in the Brazilian State of Maranh?o. Colombian and Venezuelan venoms show an ontogenetic toxin profile phenotype whereas Brazilian venoms exhibit paedomorphic phenotypes. Venoms from each of the 16 localities sampled contain both population-specific toxins and proteins shared by neighboring B. atrox populations. Mapping the molecular similarity between conspecific populations onto a physical map of B. atrox range provides clues for tracing dispersal routes that account for the current biogeographic distribution of the species. The proteomic pattern is consistent with a model of southeast and southwest dispersal and allopatric fragmentation northern of the Amazon Basin, and trans-Amazonian expansion through the Andean Corridor and across the Amazon river between Monte Alegre and Santarém. An antivenomic approach applied to assess the efficacy towards B. atrox venoms of two antivenoms raised in Costa Rica and Brazil using Bothrops venoms different than B. atrox in the immunization mixtures showed that both antivenoms immunodepleted very efficiently the major toxins (PIII-SVMPs, serine proteinases, CRISP, LAO) of paedomorphic venoms from Puerto Ayacucho (Venezuelan Amazonia) through S?o Bento, but had impaired reactivity towards PLA(2) and P-I SVMP molecules abundantly present in ontogenetic venoms. The degree of immunodepletion achieved suggests that each of these antivenoms may be effective against envenomations by paedomorphic, and some ontogenetic, B. atrox venoms.     

Simultaneous observation of collagen and elastin in normal and pathological tissues: analysis of Sirius-red-stained sections by fluorescence microscopy

In order to observe collagen and elastic fibers simultaneously, sections of human aorta, skin, lung, liver, and bladder were stained by Sirius red and analyzed by fluorescence microscopy. In all cases, the fibers of collagen presented the characteristic fluorescent red-orange color that results from the interaction of this extracellular protein with the dye, whereas elastic fibers showed strong green fluorescence (intrinsic fluorescence). This method efficiently detects collagen and elastic fibers when these two structures are present and could have valuable applications in processes that involves both fibers.L.F.B. received a doctoral fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.     

Verapamil quantification in human plasma by liquid chromatography coupled to tandem mass spectrometry. An application for bioequivalence study

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection (LC-MS/MS) was developed for the determination of Verapamil in human plasma using Metoprolol as the internal standard. The analyte and internal standard were extracted from the plasma samples by liquid-liquid extraction and chromatographed on a C(8) analytical column. The mobile phase consisted of methanol-water (70:30; v/v)+12 mM formic acid. The method had a chromatographic total run time of 3.5 min and was linear within the range 1.00-500 ng/mL. Detection was carried out on a Micromass Quattro Ultima tandem mass spectrometer by multiple reaction monitoring (MRM). The intra-run imprecision was less than 5.1% calculated from the quality control (QC) samples, and 16.3% from the limit of quantification (LOQ). The accuracy determined from QC samples were between 92.9 and 103.1%, and 95.2 and 115.3% from LOQ. Concerning the inter-batch analysis, the imprecision was less than 5.8% and 17.3% from QC samples and LOQ, respectively. The accuracy varied between 98.2 and 100.8% from QC and it was 103.1% from LOQ. The protocol herein described was employed in a bioequivalence study of two tablet formulations of Verapamil.     

New roles of myosin II during vesicle transport and fusion in chromaffin cells

Modified herpes virus (amplicons) were used to express myosin regulatory light chain (RLC) chimeras with green fluorescent protein (GFP) in cultured bovine chromaffin cells to study myosin II implication in secretion. After infection, RLC-GFP constructs were clearly identified in the cytoplasm and accumulated in the cortical region, forming a complex network that co-localized with cortical F-actin. Cells expressing wild type RLC-GFP maintained normal vesicle mobility, whereas cells expressing an unphosphorylatable form (T18A/S19A RLC-GFP) presented severe restrictions in granule movement as measured by individual tracking in dynamic confocal microscopy studies. Interestingly, the overexpression of this mutant form of RLC also affected the initial secretory burst elicited by either high K(+) or BaCl(2), as well as the secretion induced by fast release of calcium from caged compounds in individual cells. Moreover, T18A/S19A RLC-GFP-infected cells presented slower fusion kinetics of individual granules compared with controls as measured by analysis of amperometric spikes. Taken together, our results demonstrate the implication of myosin II in the transport of vesicles, and, surprisingly, in the final phases of exocytosis involving transitions affecting the activity of docked granules, and therefore uncovering a new role for this cytoskeletal element.     

Specialized roles of the two pathways for the synthesis of mannosylglycerate in osmoadaptation and thermoadaptation of Rhodothermus marinus

Rhodothermus marinus responds to fluctuations in the growth temperature and/or salinity by accumulating mannosylglycerate (MG). Two alternative pathways for the synthesis of MG have been identified in this bacterium: a single-step pathway and a two-step pathway. In this work, the genetic and biochemical characterization of the two-step pathway was carried out with the goal of understanding the function of the two pathways and their regulatory mechanisms. Mannosyl-3-phosphoglycerate synthase (MPGS) of the two-step pathway was purified from R. marinus. Sequence information led to the isolation of two contiguous genes, mpgs (encoding MPGS) and mpgp (encoding mannosyl-3-phosphoglycerate phosphatase). The recombinant MPGS had a low specific activity compared with other homologous MPGSs and contained approximately 30 additional residues at the C terminus. Truncation of this extension produced a protein with a 10-fold higher specific activity. Moreover, the activity of the complete MPGS was enhanced upon incubation with R. marinus cell extracts, and protease inhibitors abolished activation. Therefore, the C-terminal peptide of MPGS was identified as a regulatory site for short term control of MG synthesis in R. marinus. The control of gene expression by heat and osmotic stress was also studied; the level of mannosylglycerate synthase involved in the single-step pathway was selectively enhanced by heat stress, whereas MPGS was overproduced in response to osmotic stress. The concomitant changes in the level of MG were assessed as well. We conclude that the two alternative pathways for the synthesis of MG are differently regulated at the level of expression to play specific roles in the adaptation of R. marinus to two different types of aggression. This is the only example of pathway multiplicity being rationalized in terms of the need to respond efficiently to distinct environmental stresses.     

CTLA-4 blockage increases resistance to infection with the intracellular protozoan Trypanosoma cruzi

Recent studies have revealed an important role for CTLA-4 as a negative regulator of T cell activation. In the present study, we evaluated the importance of CTLA-4 to the immune response against the intracellular protozoan, Trypanosoma cruzi, the causative agent of Chagas' disease. We observed that the expression of CTLA-4 in spleen cells from naive mice cultured in the presence of live trypomastigote forms of T. cruzi increases over time of exposure. Furthermore, spleen cells harvested from recently infected mice showed a significant increase in the expression of CTLA-4 when compared with spleen cells from noninfected mice. Blockage of CTLA-4 in vitro and/or in vivo did not restore the lymphoproliferative response decreased during the acute phase of infection, but it resulted in a significant increase of NO production in vivo and in vitro. Moreover, the production of IFN-gamma in response to parasite Ags was significantly increased in spleen cells from anti-CTLA-4-treated infected mice when compared with the production found in cells from IgG-treated infected mice. CTLA-4 blockade in vivo also resulted in increased resistance to infection with the Y and Colombian strains of T. cruzi. Taken together these results indicate that CTLA-4 engagement is implicated in the modulation of the immune response against T. cruzi by acting in the mechanisms that control IFN-gamma and NO production during the acute phase of the infection.     

Thermococcus kodakarensis Mutants Deficient in Di-myo-Inositol Phosphate Use Aspartate To Cope with Heat Stress

Many of the marine microorganisms which are adapted to grow at temperatures above 80°C accumulate di-myo-inositol phosphate (DIP) in response to heat stress. This led to the hypothesis that the solute plays a role in thermoprotection, but there is a lack of definitive experimental evidence. Mutant strains of Thermococcus kodakarensis (formerly Thermococcus kodakaraensis), manipulated in their ability to synthesize DIP, were constructed and used to investigate the involvement of DIP in thermoadaptation of this archaeon. The solute pool of the parental strain comprised DIP, aspartate, and α-glutamate. Under heat stress the level of DIP increased 20-fold compared to optimal conditions, whereas the pool of aspartate increased 4.3-fold in response to osmotic stress. Deleting the gene encoding the key enzyme in DIP synthesis, CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase, abolished DIP synthesis. Conversely, overexpression of the same gene resulted in a mutant with restored ability to synthesize DIP. Despite the absence of DIP in the deletion mutant, this strain exhibited growth parameters similar to those of the parental strain, both at optimal (85°C) and supraoptimal (93.7°C) temperatures for growth. Analysis of the respective solute pools showed that DIP was replaced by aspartate. We conclude that DIP is part of the strategy used by T. kodakarensis to cope with heat stress, and aspartate can be used as an alternative solute of similar efficacy. This is the first study using mutants to demonstrate the involvement of compatible solutes in the thermoadaptation of (hyper)thermophilic organisms.Hyperthermophilic bacteria and archaea isolated from saline environments accumulate unusual organic solutes in response to osmotic as well as heat stress. Mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, mannosyl-di-myo-inositol phosphate (DIP), diglycerol phosphate, and glycero-phospho-myo-inositol are examples of compatible solutes highly restricted to thermophiles and hyperthermophiles (27, 31). Our team has, over several years, examined the compatible solute composition in a large number of hyperthermophiles and their accumulation under stressful conditions. The data reveal a trend toward specialization of roles in thermoadaptation and osmoadaptation. Indeed, mannosylglycerate and diglycerol phosphate typically accumulate in response to increased NaCl concentration in the growth medium, whereas the levels of DIP and derivatives consistently increase at supraoptimal growth temperatures (11, 16, 17, 27, 31).DIP is widespread among extreme archaeal hyperthermophiles, such as Methanotorris igneus, Aeropyrum pernix, Stetteria hydrogenophila, Pyrodictium occultum, Pyrolobus fumarii, Archaeoglobus spp., and all the members of the Thermococcales examined thus far, except Palaeococcus ferrophilus (5, 7, 11, 13, 16, 18, 31). This organic solute has also been found in representatives of the two hyperthermophilic bacterial genera, Aquifex and Thermotoga (14, 17, 22).The specific chemical nature of solutes encountered in hyperthermophiles, together with their accumulation in response to elevated temperatures, led to the hypothesis that they play a role in thermoprotection of cellular components in vivo. However, there is a lack of convincing experimental evidence, such as that obtained with suitable mutants. Progress toward understanding the physiological functions of these solutes critically depends on two conditions: the availability of genetic tools to manipulate hyperthermophilic organisms and knowledge about the genes and enzymes implicated in the synthesis of these unusual solutes.Thermococcus kodakarensis (formerly Thermococcus kodakaraensis) is a member of the order Thermococcales with an optimal growth temperature of 85°C and is able to grow at temperatures up to 94°C in batch cultures. The NaCl concentration for optimal growth matches that of seawater (1). T. kodakarensis is the only marine hyperthermophile for which a number of genetic tools have been developed, including Escherichia coli-T. kodakarensis shuttle vectors and a reliable gene disruption system (19, 29, 32, 34). The genome of T. kodakarensis possesses a gene encoding CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase (IPCT/DIPPS), a key enzyme in DIP synthesis (2, 25, 26). This enzyme catalyzes the synthesis of CDP-inositol from CTP and inositol-1-phosphate as well as the transfer of the inositol group from CDP-inositol to a second molecule of inositol-1-phosphate to yield a phosphorylated form of DIP (2). Therefore, we set out to investigate whether DIP was involved in thermoadaptation of T. kodakarensis. A DIP-deficient mutant was constructed by deleting the IPCT/DIPPS gene; subsequently, this strain was complemented in this activity by inserting the gene under the control of a constitutive promoter, resulting in a construct with restored ability to synthesize DIP. The effects of heat and osmotic stress on the pattern of solute accumulation and on the growth profiles of the two mutants provided evidence for the involvement of DIP in thermoprotection.     

Peptide gomesin triggers cell death through L-type channel calcium influx,MAPK/ERK,PKC and PI3K signaling and generation of reactive oxygen species

Gomesin is an antimicrobial peptide isolated from hemocytes of a common Brazilian tarantula spider named Acanthoscurria gomesiana. This peptide exerts antitumor activity in vitro and in vivo by an unknown mechanism. In this study, the cytotoxic mechanism of gomesin in human neuroblastoma SH-SY5Y and rat pheochromocytoma PC12 cells was investigated. Gomesin induced necrotic cell death and was cytotoxic to SH-SY5Y and PC12 cells. The peptide evoked a rapid and transient elevation of intracellular calcium levels in Fluo-4-AM loaded PC12 cells, which was inhibited by nimodipine, an L-type calcium channel blocker. Preincubation with nimodipine also inhibited cell death induced by gomesin in SH-SY5Y and PC12 cells. Gomesin-induced cell death was prevented by the pretreatment with MAPK/ERK, PKC or PI3K inhibitors, but not with PKA inhibitor. In addition, gomesin generated reactive oxygen species (ROS) in SH-SY5Y cells, which were blocked with nimodipine and MAPK/ERK, PKC or PI3K inhibitors. Taken together, these results suggest that gomesin could be a useful anticancer agent, which mechanism of cytotoxicity implicates calcium entry through L-type calcium channels, activation of MAPK/ERK, PKC and PI3K signaling as well as the generation of reactive oxygen species.