Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 μg/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox's lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.     

Free-energy profiles for catalysis by dual-specificity phosphatases

PTPs (protein tyrosine phosphatases) are fundamental enzymes for cell signalling and have been linked to the pathogenesis of several diseases, including cancer. Hence, PTPs are potential drug targets and inhibitors have been designed as possible therapeutic agents for Type II diabetes and obesity. However, a complete understanding of the detailed catalytic mechanism in PTPs is still lacking. Free-energy profiles, obtained by computer simulations of catalysis by a dual-specificity PTP, are shown in the present study and are used to shed light on the catalytic mechanism. A highly accurate hybrid potential of quantum mechanics/molecular mechanics calibrated specifically for PTP reactions was used. Reactions of alkyl and aryl substrates, with different protonation states and PTP active-site mutations, were simulated. Calculated reaction barriers agree well with experimental rate measurements. Results show the PTP substrate reacts as a bi-anion, with an ionized nucleophile. This protonation state has been a matter of debate in the literature. The inactivity of Cys-->Ser active-site mutants is also not fully understood. It is shown that mutants are inactive because the serine nucleophile is protonated. Results also clarify the interpretation of experimental data, particularly kinetic isotope effects. The simulated mechanisms presented here are better examples of the catalysis carried out by PTPs.     

Enteric Neuronal Damage,Intramuscular Denervation and Smooth Muscle Phenotype Changes as Mechanisms of Chagasic Megacolon: Evidence from a Long-Term Murine Model of Tripanosoma cruzi Infection

We developed a novel murine model of long-term infection with Trypanosoma cruzi with the aim to elucidate the pathogenesis of megacolon and the associated adaptive and neuromuscular intestinal disorders. Our intent was to produce a chronic stage of the disease since the early treatment should avoid 100% mortality of untreated animals at acute phase. Treatment allowed animals to be kept infected and alive in order to develop the chronic phase of infection with low parasitism as in human disease. A group of Swiss mice was infected with the Y strain of T. cruzi. At the 11^th day after infection, a sub-group was euthanized (acute-phase group) and another sub-group was treated with benznidazole and euthanized 15 months after infection (chronic-phase group). Whole colon samples were harvested and used for studying the histopathology of the intestinal smooth muscle and the plasticity of the enteric nerves. In the acute phase, all animals presented inflammatory lesions associated with intense and diffuse parasitism of the muscular and submucosa layers, which were enlarged when compared with the controls. The occurrence of intense degenerative inflammatory changes and increased reticular fibers suggests inflammatory-induced necrosis of muscle cells. In the chronic phase, parasitism was insignificant; however, the architecture of Aüerbach plexuses was focally affected in the inflamed areas, and a significant decrease in the number of neurons and in the density of intramuscular nerve bundles was detected. Other changes observed included increased thickness of the colon wall, diffuse muscle cell hypertrophy, and increased collagen deposition, indicating early fibrosis in the damaged areas. Mast cell count significantly increased in the muscular layers. We propose a model for studying the long-term (15 months) pathogenesis of Chagasic megacolon in mice that mimics the human disease, which persists for several years and has not been fully elucidated. We hypothesize that the long-term inflammatory process mediates neuronal damage and intramuscular and intramural denervation, leading to phenotypic changes in smooth muscle cells associated with fibrosis. These long-term structural changes may represent the basic mechanism for the formation of the Chagasic megacolon.     

Construction of cloning vectors for Bacillus thuringiensis.

The replication region of the Bacillus thuringiensis plasmid, pHT1030, was treated with hydroxylamine. Various copy-number mutants were selected and subsequently used to construct shuttle vectors with multiple cloning sites. These recombinant plasmids are very stable and allowed the cloning of a delta-endotoxin-encoding gene in B. thuringiensis. Comparison between gene expression level and vector copy-number indicated that a plateau in delta-endotoxin production is reached with a copy-number of about fifteen per equivalent chromosome.     

Genetic relationships between sympatric populations of Bacillus cereus and Bacillus thuringiensis, as revealed by rep-PCR genomic fingerprinting

The bacterial strain Bacillus cereus is closely related to Bacillus thuringiensis, although any genetic relationship between the two strains is still in debate. Using rep-PCR genomic fingerprinting, we established the genetic relationships between Brazilian sympatric populations of B. cereus and B. thuringiensis simultaneously collected from two geographically separate sites. We observed the formation of both B. thuringiensis and B. cereus clusters, as well as strains of B. cereus that are more closely related to B. thuringiensis than to other B. cereus strains. In addition, lower genetic variability was observed among B. thuringiensis clusters compared to B. cereus clusters, indicating that either the two species should be categorized as separate or that B. thuringiensis may represent a clone from a B. cereus background.     

Assessment of Total and Organic Mercury Levels in Blue Sharks (Prionace glauca) from the South and Southeastern Brazilian Coast

Mercury occurrence was evaluated in samples of edible muscle tissue of 27 blue sharks (Prionace glauca) caught in the Atlantic Ocean, adjacent to the south and southeastern Brazilian coast, indicating a slight increase in comparison with previous data obtained for the same studied area and being higher than those fish caught at different sites of the Atlantic Ocean. Total Hg concentrations ranged from 0.46 to 2.40 mg kg^?1 with the organic Hg fraction ranging between 0.44 and 2.37 mg kg^?1. A negative correlation between total Hg concentration in muscle tissue and blue shark size was obtained, and 40 % of samples analyzed had Hg concentrations higher than 1.0 mg kg^?1 Hg, the maximum concentration permitted in Brazilian predator fish. Data obtained showed that total Hg can be used as a reliable predictor of organic Hg in blue shark muscle because 95 to 98 % of the total Hg measured was found to be organic mercury. The wide range of Hg concentrations obtained for our set of samples can be explained by the heterogeneity of sampled population and the large size of the studied area. Given the adverse toxicological effects of Hg on animals and humans, a regular monitoring program of Hg contamination in Brazilian marine ecosystem can be recommended.     

Virulence in murine model shows the existence of two distinct populations of Brazilian Vaccinia virus strains

Brazilian Vaccinia virus had been isolated from sentinel mice, rodents and recently from humans, cows and calves during outbreaks on dairy farms in several rural areas in Brazil, leading to high economic and social impact. Some phylogenetic studies have demonstrated the existence of two different populations of Brazilian Vaccinia virus strains circulating in nature, but little is known about their biological characteristics. Therefore, our goal was to study the virulence pattern of seven Brazilian Vaccinia virus strains. Infected BALB/c mice were monitored for morbidity, mortality and viral replication in organs as trachea, lungs, heart, kidneys, liver, brain and spleen. Based on the virulence potential, the Brazilian Vaccinia virus strains were grouped into two groups. One group contained GP1V, VBH, SAV and BAV which caused disease and death in infected mice and the second one included ARAV, GP2V and PSTV which did not cause any clinical signals or death in infected BALB/c mice. The subdivision of Brazilian Vaccinia virus strains into two groups is in agreement with previous genetic studies. Those data reinforce the existence of different populations circulating in Brazil regarding the genetic and virulence characteristics.     

Shorter leukocyte telomere length is associated with severity of COVID-19 infection.

The infection by COVID-19 is a serious global public health problem. An efficient way to improve this disease's clinical management would be to characterize patients at higher risk of progressing to critically severe infection using prognostic biomarkers. The telomere length could be used for this purpose. Telomeres are responsible for controlling the number of maximum cell divisions. The telomere length is a biomarker of aging and several diseases. We aimed to compare leukocyte telomere length (LTL) between patients without COVID-19 and patients with different clinical severity of the infection. Were included 53 patients who underwent SARS-CoV-2 PCR divided in four groups. The first group was composed by patients with a negative diagnosis for COVID-19 (n = 12). The other three groups consisted of patients with a confirmed diagnosis of COVID-19 divided according to the severity of the disease: mild (n = 15), moderate (n = 17) and severe (n = 9). The LTL was determined by Q-PCR. The severe group had the shortest LTL, followed by the moderate group. The negative and mild groups showed no differences. There is an increase of patients with hypertension (p = 0.0099) and diabetes (p = 0.0067) in moderate and severe groups. Severe group was composed by older patients in comparison with the other three groups (p = 0.0083). Regarding sex, there was no significant difference between groups (p = 0.6279). In an ordinal regression model, only LTL and diabetes were significantly associated with disease severity. Shorter telomere length was significantly associated with the severity of COVID-19 infection, which can be useful as a biomarker or to better understand the SARS-CoV-2 pathophysiology.     

A low-protein diet during pregnancy prevents modifications in intercellular communication proteins in rat islets

Background

Gap junctions between β-cells participate in the precise regulation of insulin secretion. Adherens junctions and their associated proteins are required for the formation, function and structural maintenance of gap junctions. Increases in the number of the gap junctions between β-cells and enhanced glucose-stimulated insulin secretion are observed during pregnancy. In contrast, protein restriction produces structural and functional alterations that result in poor insulin secretion in response to glucose. We investigated whether protein restriction during pregnancy affects the expression of mRNA and proteins involved in gap and adherens junctions in pancreatic islets. An isoenergetic low-protein diet (6% protein) was fed to non-pregnant or pregnant rats from day 1–15 of pregnancy, and rats fed an isocaloric normal-protein diet (17% protein) were used as controls.

Results

The low-protein diet reduced the levels of connexin 36 and β-catenin protein in pancreatic islets. In rats fed the control diet, pregnancy increased the levels of phospho-[Ser^279/282]-connexin 43, and it decreased the levels of connexin 36, β-catenin and beta-actin mRNA as well as the levels of connexin 36 and β-catenin protein in islets. The low-protein diet during pregnancy did not alter these mRNA and protein levels, but avoided the increase of levels of phospho-[Ser^279/282]-connexin 43 in islets. Insulin secretion in response to 8.3 mmol/L glucose was higher in pregnant rats than in non-pregnant rats, independently of the nutritional status.

Conclusion

Short-term protein restriction during pregnancy prevented the Cx43 phosphorylation, but this event did not interfer in the insulin secretion.     

Conformational flexibility of the complete catalytic domain of Cdc25B phosphatases

Cdc25B phosphatases are involved in cell cycle checkpoints and have become a possible target for developing new anticancer drugs. A more rational design of Cdc25B ligands would benefit from detailed knowledge of its tertiary structure. The conformational flexibility of the C‐terminal region of the Cdc25B catalytic domain has been debated recently and suggested to play an important structural role. Here, a combination of experimental NMR measurements and molecular dynamics simulations for the complete catalytic domain of the Cdc25B phosphatase is presented. The stability of the C‐terminal α‐helix is confirmed, but the last 20 residues in the complete catalytic domain are very flexible, partially occlude the active site and may establish transient contacts with the protein core. This flexibility in the C‐terminal tail may modulate the molecular recognition of natural substrates and competitive inhibitors by Cdc25B. Proteins 2016; 84:1567–1575. © 2016 Wiley Periodicals, Inc.