Alexsandro Sobreira Galdino

Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae

An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa) by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH(4) (+) and inhibited by Cu(+2) and Hg(+2). Significant biochemical and structural discrepancies between wild-type and recombinant α-amylase with respect to K(m) values, enzyme specificity, and secondary structure content were found. Far-UV CD spectra analysis at pH 7.0 revealed the high thermal stability of both proteins and the difference in folding pattern of Amy1 compared with wild-type amylase from C. flavus, which reflected in decrease (10-fold) of enzymatic activity of recombinant protein. Despite the differences, the highest activity of Amy1 towards soluble starch, amylopectin, and amylase, in contrast with the lowest activity of Amy1(w), points to this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels.

Cloning, purification, and partial characterization of Bacillus subtilis urate oxidase expressed in Escherichia coli

Urate oxidase (EC 1.7.3.3) is an enzyme involved in purine metabolism which is used in the treatment of gout and as diagnostic reagent for detection of uric acid. In order to produce this enzyme in large quantities for biotechnological purposes, the gene coding for the Bacillus subtilis urate oxidase was cloned and heterologously expressed in Escherichia coli. Time course induction in E. coli showed an induced protein with an apparent molecular mass of ∼60 kDa. Soluble recombinant enzyme was purified in a single-step procedure using Ni-NTA column. The enzyme was purified 2.1-fold with a yield of 56% compared to the crude extract. MALDI-TOF analysis revealed an ion with a mass of 58675 Da which is in agreement with the expected mass of the recombinant protein. The purified enzyme showed an optimal pH and temperature of 8.0 and 37°C, respectively, and retained 90% of its activity after 72 hours of incubation at −20°C and 4°C.

Genetic variability of brycon orbignyanus (valenciennes, 1850) (characiformes: characidae) in cultivated and natural populations of the upper paraná river, and implications for the conservation of the species

This study aimed to evaluate the genetic variability of B. orbignyanus in cultivated and natural populations of the Upper Parana River, using molecular RAPD markers and mtDNA control region. Specimens were collected in the Parana River and in the Piracema fish farm in Maringa, State of Parana, Brazil. RAPD primers produced 82 loci with consistent expression. The population from the Parana River showed 28 polymorphic loci, whereas the population from the fish farm presented only 12. Data revealed genetic differentiation between the two populations, although not very pronounced. These results were corroborated by the principal coordinate analysis and by neighbor-joining clustering. The alignment of the D-loop sequences of B. orbignyanus indicated the existence of polymorphism only in the natural population. These data could be helpful for the formulation of management strategies and conservation of the genetic diversity of the species.

A Recombinant Multiepitope Protein for Hepatitis B Diagnosis

Hepatitis B is a liver inflammation caused by hepatitis B virus (HBV) and can be diagnosed in clinical stage by hepatitis B core antibody from IgM class (anti-HBcIgM). Hepatitis B core antibody from IgG class (Anti-HBcIgG) appears quickly after IgM, reaching high titers in chronic hepatitis, and remains even after cure. Since hepatitis B core antibody (anti-HBc) is the first antibody identified and sometimes the only marker detected during the course of infection, it can be used both to indicate HBV acute infection (anti-HBc-IgM) and to identify individuals who have come into contact with the virus (anti-HBc-IgG). In this work we propose a recombinant hepatitis B core multiepitope antigen (rMEHB) to be used for diagnosis of hepatitis B. For this purpose, a synthetic gene coding for rMEHB was designed and cloned into vector pET21a with a 6xHis tag at the C-terminal. Time course induction in E. coli showed an induced protein with an apparent molecular mass of ~21 kDa. Protein purification was performed by a single step with affinity chromatography Ni-NTA. Circular dichroism spectroscopy indicated rMEHB as a thermal stable protein at pH 7.0 and 8.0. In these conditions rMEHB was successfully used to perform an enzyme linked immuno sorbent assay (ELISA) with positive and negative sera.

Antigenicity, immunogenicity and protective efficacy of a conserved Leishmania hypothetical protein against visceral leishmaniasis

In this study, a Leishmania hypothetical protein, LiHyS, was evaluated regarding its antigenicity, immunogenicity and protective efficacy against visceral leishmaniasis (VL). Regarding antigenicity, immunoblottings and an enzyme-linked immunosorbent assay using human and canine sera showed high sensitivity and specificity values for the recombinant protein (rLiHyS) in the diagnosis of VL. When evaluating the immunogenicity of LiHyS, which is possibly located in the parasites flagellar pocket, proliferative assays using peripheral blood mononuclear cells from healthy subjects or VL patients showed a high proliferative index in both individuals, when compared to the results obtained using rA2 or unstimulated cultures. Later, rLiHyS/saponin was inoculated in BALB/c mice, which were then challenged with Leishmania infantum promastigotes. The vaccine induced an interferon-γ, interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor production, which was maintained after infection and which was associated with high nitrite and IgG2a antibody levels, as well as low IL-4 and IL-10 production. Significant reductions in the parasite load in liver, spleen, bone marrow and draining lymph nodes were found in these animals. In this context, the present study shows that the rLiHyS has the capacity to be evaluated as a diagnostic marker or vaccine candidate against VL.

A Novel Structurally Stable Multiepitope Protein for Detection of HCV

Hepatitis C virus (HCV) has emerged as the major pathogen of liver diseases in recent years leading to worldwide blood-transmitted chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Accurate diagnosis for differentiation of hepatitis C from other viruses is thus of pivotal importance for proper treatment. In this work we developed a recombinant multiepitope protein (rMEHCV) for hepatitis C diagnostic purposes based on conserved and immunodominant epitopes from core, NS3, NS4A, NS4B, and NS5 regions of the virus polyprotein of genotypes 1a, 1b, and 3a, the most prevalent genotypes in South America (especially in Brazil). A synthetic gene was designed to encode eight epitopes in tandem separated by a flexible linker and bearing a his-tag at the C-terminal end. The recombinant protein was produced in Escherichia coli and purified in a single affinity chromatographic step with >95% purity. Purified rMEHCV was used to perform an ELISA which showed that the recombinant protein was recognized by IgG and IgM from human serum samples. The structural data obtained by circular dichroism (CD) spectroscopy showed that rMEHCV is a highly thermal stable protein at neutral and alkaline conditions. Together, these results show that rMEHCV should be considered an alternative antigen for hepatitis C diagnosis.

SEED PROTEIN VARIATION AMONG PEPPER (CAPSICUM SP.) GENOTYPES REVEALED BY MALDI- TOF ANALYSIS

A method for seed proteome analysis using MALDI-TOF mass spectrometry is described. The data were used to estimate the genetic diversity degree among twelve genotypes of pepper (Capsicum). The resulting spectra were converted into a binary matrix consisting of 23 protein data sets, and genetic similarity values were calculated with the FreeTree software and Jaccards coefficient of similarity. We have also been able to identify the presence of certain proteins in the extracts, by checking their masses on on-line databases.

Recombinant small glutamine-rich tetratricopeptide repeat-containing protein of Leishmania infantum: Potential vaccine and diagnostic application against visceral leishmaniasis.

Graphical abstract Figure. No caption available. HighlightsUse of the Leishmania SGT protein against visceral leishmaniasis.Serological marker to identify VL patients, but without presents cross‐reactivity.Partial protection induced in BALB/c mice against L. infantum infection.Immunogenicity in PBMCs from recovered and treated VL patients with IFN‐&ggr; production.A new candidate to studies as vaccine or serological marker against human VL. &NA; Different Leishmania proteins have been evaluated in order to find a potential vaccine candidate or diagnostic marker capable of providing long lasting protection against infection or helping to identify infected mammalian hosts, respectively. However, just few molecules have fulfilled all the requirements to be evaluated. In the current study, we evaluated the prophylactic and diagnostic value against visceral leishmaniasis (VL) of a small glutamine‐rich tetratricopeptide repeat‐containing (SGT) protein from Leishmania infantum species. In a first step, the immune response elicited by the immunization using the recombinant protein (rSGT) plus saponin was evaluated in BALB/c mice. Immunized animals had a low parasitism in all evaluated organs. They developed a specific Th1 immune response, which was based on protein‐specific production of IFN‐&ggr;, IL‐12 and GM‐CSF, and a humoral response dominated by antibodies of the IgG2a isotype. Both CD4+ and CD8+ T cells contributed to the IFN‐&ggr; production, showing that both T cell subtypes contribute to the resistance against infection. Regarding its value as a diagnostic marker, rSGT showed maximum sensitivity and specificity to serologically identify L. infantum‐infected dog and human sera. No cross‐reactivity with sera from humans or dogs that had other diseases was found. Although further studies are necessary to validate these findings, data showed here suggest immunogenicity of rSGT and its protective effect against murine VL, as well as its potential for the serodiagnosis of human and canine VL.

Caracterização molecular de acessos de Cratylia argentea e sua relação filogenética com outras leguminosas

The objective of this work was to molecularly characterize 11 Cratylia argentea accessions, based on the ITS (ITS1/5.8S/ITS2) region sequencing, as well to establish its phylogenetic relationship with other legume species. The phylogenetic relationship of this species with other 15 legume ones was established using a gene sequence that codes the subunit 18S of the rRNA (rDNA 18S). DNA amplification of the ITS/5.8S region of these 11 accessions revealed an amplicon with around 650 bp. ITS/5.8S sequences were obtained from all accessions analysed, and then aligned with the region ITS/5.8S of Galactia striata legume. The size of ITS/5.8S region ranged from 565 to 615 bp. Average G + C contents in the ITS1 and ITS2 regions ranged between 46 and 47%. The multiple sequence alignment between the ITS sequences from C. argentea accessions and Galactia striata revealed the presence of deletions and insertions. C. argentea accessions formed a unique politomic clade. Cratylia argentea phylogenetic analysis demonstrated that this species is placed into the true Diocleinae Clade, and that Calopogonium and Pachyrhizus are not included in subtribe Diocleinae.

Vaccination with a CD4+ and CD8+ T-cell epitopes-based recombinant chimeric protein derived from Leishmania infantum proteins confers protective immunity against visceral leishmaniasis

&NA; Vaccination seems to be the best approach to control visceral leishmaniasis (VL). Resistance against infection is based on the development of a Th1 immune response characterized by the production of interferons‐&ggr; (IFN‐&ggr;), interleukin‐12 (IL‐12), granulocyte‐macrophage‐colony‐stimulating factor (GM‐CSF), and tumor necrosis factor‐&agr; (TNF‐&agr;), among others. A number of antigens have been tested as potential targets against the disease; few of them are able to stimulate human immune cells. In the present study, 1 prediction of MHC class I and II molecules‐specific epitopes in the amino acid sequences of 3 Leishmania proteins: 1 hypothetical, prohibitin, and small glutamine‐rich tetratricopeptide repeat‐containing proteins, was performed using bioinformatics tools, and a T‐cell epitopes‐based recombinant chimeric protein was constructed, synthetized and purified to be evaluated in invitro and in vivo experiments. The purified protein was tested regarding its immunogenicity in peripheral blood mononuclear cells (PBMCs) from healthy subjects and VL patients, as well as to its immunogenicity and protective efficacy in a murine model against Leishmania infantum infection. Results showed a Th1 response based on high IFN‐&ggr; and low IL‐10 levels derived from in chimera‐stimulated PBMCs in both healthy subjects and VL patients. In addition, chimera and/or saponin‐immunized mice presented significantly lower parasite burden in distinct evaluated organs, when compared to the controls, besides higher levels of IFN‐&ggr;, IL‐2, IL‐12, and GM‐CSF, and an IgG2a isotype‐based humoral response. In addition, the CD4+ and CD8+ T‐cell subtypes contributed to IFN‐&ggr; production in the protected animals. The results showed the immunogenicity in human cells and the protective efficacy against L. infantum in a murine model, and well indicate that this recombinant chimera can be considered as a promising strategy to be used against human disease.