João Paulo Matos Santos Lima

Mo-CBP3, an Antifungal Chitin-Binding Protein from Moringa oleifera Seeds, Is a Member of the 2S Albumin Family

Mo-CBP3 is a chitin-binding protein from M. oleifera seeds that inhibits the germination and mycelial growth of phytopathogenic fungi. This protein is highly thermostable and resistant to pH changes, and therefore may be useful in the development of new antifungal drugs. However, the relationship of MoCBP3 with the known families of carbohydrate-binding domains has not been established. In the present study, full-length cDNAs encoding 4 isoforms of Mo-CBP3 (Mo-CBP3-1, Mo-CBP3-2, Mo-CBP3-3 and Mo-CBP3-4) were cloned from developing seeds. The polypeptides encoded by the Mo-CBP3 cDNAs were predicted to contain 160 (Mo-CBP3-3) and 163 amino acid residues (Mo-CBP3-1, Mo-CBP3-2 and Mo-CBP3-4) with a signal peptide of 20-residues at the N-terminal region. A comparative analysis of the deduced amino acid sequences revealed that Mo-CBP3 is a typical member of the 2S albumin family, as shown by the presence of an eight-cysteine motif, which is a characteristic feature of the prolamin superfamily. Furthermore, mass spectrometry analysis demonstrated that Mo-CBP3 is a mixture of isoforms that correspond to different mRNA products. The identification of Mo-CBP3 as a genuine member of the 2S albumin family reinforces the hypothesis that these seed storage proteins are involved in plant defense. Moreover, the chitin-binding ability of Mo-CBP3 reveals a novel functionality for a typical 2S albumin.

Structural characterization of a novel peptide with antimicrobial activity from the venom gland of the scorpion Tityus stigmurus: Stigmurin.

A new antimicrobial peptide, herein named Stigmurin, was selected based on a transcriptomic analysis of the Brazilian yellow scorpion Tityus stigmurus venom gland, an underexplored source for toxic peptides with possible biotechnological applications. Stigmurin was investigated in silico, by circular dichroism (CD) spectroscopy, and in vitro. The CD spectra suggested that this peptide interacts with membranes, changing its conformation in the presence of an amphipathic environment, with predominance of random coil and beta-sheet structures. Stigmurin exhibited antibacterial and antifungal activity, with minimal inhibitory concentrations ranging from 8.7 to 69.5μM. It was also showed that Stigmurin is toxic against SiHa and Vero E6 cell lines. The results suggest that Stigmurin can be considered a potential anti-infective drug.

H-3, a new lectin from the marine sponge Haliclona caerulea: Purification and mass spectrometric characterization

A new lectin from the marine sponge Haliclona caerulea (H-3) was isolated using a combination of hydrophobic interaction chromatography and ion-exchange chromatography. H-3 is a protein with three distinct bands on SDS-PAGE: 9 kDa, 16 kDa and 18 kDa. Nevertheless, on gel filtration and N-PAGE, H-3 showed a symmetrical peak and a unique band, respectively. Hemagglutinating activity of H-3 was stable at neutral pH and temperatures up to 60 °C. N-Acetylgalactosamine and porcine stomach mucin were the most potent inhibitors of H-3. Primary structure of the lectin was determined using tandem mass spectrometry, and it showed no similarity to any members of the animal lectin families. Top down fragmentation revealed some posttranslational modifications in H-3, including glycosylation. The glycan composition of H-3 was determined, and its structure was predicted. Furthermore, H-3 is a blue protein, binding to a chromophore(-597) by weak interactions, and this is the first time that the interaction between one lectin and a natural chromophore has been shown.

Molecular approaches for structural characterization of a new potassium channel blocker from Tityus stigmurus venom: cDNA cloning, homology modeling, dynamic simulations and docking

Potassium channels are involved in the maintenance of resting membrane potential, control of cardiac and neuronal excitability, neurotransmitters release, muscle contractility and hormone secretion. The Tityus stigmurus scorpion is widely distributed in Northeastern Brazil and known to cause severe human envenomations, inducing pain, hypoesthesia, edema, erythema, paresthesia, headaches and vomiting. Most potassium channel blocking peptides that have been purified from scorpion venoms contain 30-40 amino acids with three or four disulfide bridges. These peptides belong to α-KTx subfamily. On the other hand, the β-KTx subfamily is poorly characterized, though it is very representative in some scorpion venoms. A transcriptomic approach of T.stigmurus scorpions developed by our group revealed the repertoire of possible molecules present in the venom, including many toxins of the β-KTx subfamily. One of the ESTs found, named TSTI0003C has a cDNA sequence of 538 bp codifying a mature protein with 47 amino acid residues, corresponding to 5299 Da. This β-KTx peptide is a new member of the BmTXKβ-related toxins, and was here named TstKMK. The three-dimensional structure of this potassium channel toxin of the T. stigmurus scorpion was obtained by computational modeling and refined by molecular dynamic simulations. Furthermore, we have made docking simulations using a Shaker kV-1.2 potassium channel from rats as receptor model and proposed which amino acid residues and interactions could be involved in its blockade.

Phylogenetic relationships within Chamaecrista sect. Xerocalyx (Leguminosae, Caesalpinioideae) inferred from the cpDNA trnE-trnT intergenic spacer and nrDNA ITS sequences.

Chamaecrista belongs to subtribe Cassiinae (Caesalpinioideae), and it comprises over 330 species, divided into six sections. The section Xerocalyx has been subjected to a profound taxonomic shuffling over the years. Therefore, we conducted a phylogenetic analysis using a cpDNA trnE-trnT intergenic spacer and nrDNA ITS/5.8S sequences from Cassiinae taxa, in an attempt to elucidate the relationships within this section from Chamaecrista. The tree topology was congruent between the two data sets studied in which the monophyly of the genus Chamaecrista was strongly supported. Our analyses reinforce that new sectional boundaries must be defined in the Chamaecrista genus, especially the inclusion of sections Caliciopsis and Xerocalyx in sect. Chamaecrista, considered here paraphyletic. The section Xerocalyx was strongly supported as monophyletic; however, the current data did not show C. ramosa (microphyllous) and C. desvauxii (macrophyllous) and their respective varieties in distinct clades, suggesting that speciation events are still ongoing in these specimens.

In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae

BackgroundThe PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new in silico melting curve approach. This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively.ResultsA phylogenetic tree based on RdRp amino acid sequences was constructed, and eight monophyletic groups were defined. Alignments of RdRp RNA sequences from each group were screened to identify RNA regions with conserved secondary structure. One region in the second half of ORF2 was identified and individually modeled using the RNAfold tool. Afterwards, each DNA or RNA sequence was denatured in silico using the softwares MELTSIM and RNAheat that generate melting curves considering the denaturation of a double stranded DNA and single stranded RNA, respectively. The same groups identified in the RdRp phylogenetic tree were retrieved by a clustering analysis of the melting curves data obtained from RNAheat. Moreover, the same approach was used to successfully discriminate different variants of Trichomonas vaginalis virus, which was not possible by the visual comparison of the double stranded melting curves generated by MELTSIM.ConclusionIn silico analysis indicate that ssRNA melting curves are more informative than dsDNA melting curves. Furthermore, conserved RNA structures may be determined from analysis of individuals that are phylogenetically related, and these regions may be used to support the reconstitution of their phylogenetic groups. These findings are a robust basis for the development of in vitro systems to ssRNA melting curves detection.

Structure and in vitro activities of a Copper II-chelating anionic peptide from the venom of the scorpion Tityus stigmurus

HIGHLIGHTSTanP is anionic peptide rich in aspartic acid.TanP presented chelation potential of copper metal.TanP structure is sensitive to pH variation of the medium.TanP induced macrophage proliferation, though decreasing the release of NO. ABSTRACT Anionic Peptides are molecules rich in aspartic acid (Asp) and/or glutamic acid (Glu) residues in the primary structure. This work presents, for the first time, structural characterization and biological activity assays of an anionic peptide from the venom of the scorpion Tityus stigmurus, named TanP. The three‐dimensional structure of TanP was obtained by computational modeling and refined by molecular dynamic (MD) simulations. Furthermore, we have performed circular dichroism (CD) analysis to predict TanP secondary structure, and UV–vis spectroscopy to evaluate its chelating activity. CD indicated predominance of random coil conformation in aqueous medium, as well as changes in structure depending on pH and temperature. TanP has chelating activity on copper ions, which modified the peptides secondary structure. These results were corroborated by MD data. The molar ratio of binding (TanP:copper) depends on the concentration of peptide: at lower TanP concentration, the molar ratio was 1:5 (TanP:Cu2+), whereas in concentrated TanP solution, the molar ratio was 1:3 (TanP:Cu2+). TanP was not cytotoxic to non‐neoplastic or cancer cell lines, and showed an ability to inhibit the in vitro release of nitric oxide by LPS‐stimulated macrophages. Altogether, the results suggest TanP is a promising peptide for therapeutic application as a chelating agent.

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.

Direct sequencing of the white spot syndrome virus from Brazil: Genome assembly and new insights on phylogeny

White spot syndrome virus (WSSV) has been the cause of great economic losses in world shrimp farming. In this work the genome of a Brazilian WSSV isolate was determined from direct sequencing of total DNA extracted from an infected whiteleg shrimp, and assembled based on a chimera template approach. Comparisons between WSSV-BR and other isolates revealed that the Brazilian virus has a relatively small genome, and is very similar to isolates from Thailand and Mexico. A phylogenetic relationship using different approaches has demonstrated that these isolates share a common evolutionary history. An analysis of conflicting phylogenetic signals also considering genomes of other isolates revealed that the evolutionary history of WSSV may be related to recombination events. We observed that these events can also be traced at some level by analyzing the homologous regions in the WSSV genome. The existence of recombination events introduces a new point of view that must be considered in the evolutionary history of WSSV.

Exercise training ameliorates matrix metalloproteinases 2 and 9 messenger RNA expression and mitigates adverse left ventricular remodeling in streptozotocin-induced diabetic rats☆

BACKGROUND The aim was to investigate whether exercise training (ExT) would ameliorate expression of key genes for myocardial morphostructure and mitigate adverse left ventricular (LV) remodeling in experimental type 1 diabetes (T1D). METHODS AND RESULTS Male Wistar rats were divided into four groups: sedentary control (SC, n=9), trained control (TC, n=13), sedentary diabetic (SD, n=20), and trained diabetic (TD, n=17). T1D was induced by 40 mg/kg streptozotocin (single dose, i.v.). Training program consisted of 4-week treadmill running (60 min/day, 5 days/wk). Structure of the LV was evaluated using histomorphometric techniques. Gene expression changes of LV collagens I and III, metalloproteinases (MMPs) 2 and 9, and transforming growth factor-β1 were detected by reverse transcriptase quantitative polymerase chain reaction. Compared with SC, SD rats presented LV eccentric remodeling, myocyte hypertrophy, and fibrosis, whereas TD animals showed normal LV geometry and collagen content but thinner myocytes. Expression of collagens and type I/III collagen messenger RNA (mRNA) ratio were diminished in diabetic hearts compared with SC. MMP-2 gene was down-regulated in SD, whereas TD group showed decreased MMP-9 mRNA levels and MMP-2 expression comparable to that of SC rats. CONCLUSIONS Attenuation of MMP-2 down-regulation and reduction in MMP-9 mRNA expression may constitute an underlying mechanism by which ExT counteracts progression of adverse LV remodeling in T1D.