The Molecular and Structural Characterization of Two Vitellogenins from the Free-Living Nematode Oscheius tipulae

This paper describes the purification of yolk proteins, which are important for the reproduction of egg-laying animals, and the structural characterization of two vitellogenins, VT1 and OTI-VIT-6, of the nematode Oscheius tipulae. O. tipulae is an alternative model organism to its relative, the widely used Caenorhabditis elegans, and is a good model to understand reproduction in insect parasitic nematodes of the genus Heterorhabditis. The native purified O. tipulae vitellogenin is composed of three polypeptides (VT1, VT2 and VT3), whereas in C. elegans, vitellogenin is composed of four polypeptides. The gene (Oti-vit-1) encoding yolk polypeptide VT1 has been recently identified in the genome of O. tipulae. Immunoblotting and N-terminal sequencing confirmed that VT1 is indeed coded by Oti-vit-1. Utilizing the same experimental approaches, we showed that the polypeptides VT2 and VT3 are derived from the proteolytic processing of the C- and N-terminal portions of the precursor OTI-VIT-6, respectively. We also showed that the recombinant polypeptide (P40), corresponding to the N-terminal sequence of OTI-VIT-6, preferentially interacts with a 100-kDa polypeptide found in adult worm extracts, as we have previously shown for the native vitellins of O. tipulae. Using the putative nematode vitellogenin amino acid sequences available in the UniProtKB database, we constructed a phylogenetic tree and showed that the O. tipulae vitellogenins characterized in this study are orthologous to those of the Caenorhabditis spp. Together, these results represent the first structural and functional comparative study of nematode yolk proteins outside the Caenorhabditis genus and provide insight into the evolution of these lipoproteins within the Nematode Phylum.     

Antiplatelet properties of novel N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone derivatives

This paper describes the design, synthesis and pharmacological evaluation of new N-acylhydrazone (NAH) compounds, belonging to the N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone class (2a-p). Classical heteroaromatic ring bioisosterism strategies were applied to the previously reported N-phenylpyrazolyl-4-acylhydrazone derivative 1, elected as lead-compound due to its important anti-aggregating profile on arachidonic acid induced platelet aggregation (IC(50)=24+/-0.5 micro M), from which emerge this new series 2. These new compounds 2a-p were readily synthesized, characterized and tested on platelet aggregation assays induced by collagen (5 micro g/mL), ADP (5 micro M) and arachidonic acid (100 micro M) in rabbit citrated platelet-rich plasma. Compounds 2b, 2d, and 2h were found to be the most potent, exhibiting a significant antiplatelet activity on arachidonic acid- and collagen-induced platelet aggregation. In addition, these new antiplatelet agents are free of gastric ulcerogenic effect and presented discrete anti-inflammatory and analgesic properties. The N-para-chlorophenyltriazolyl-4-acylhydrazone compound 2h produced the highest inhibitory effect on collagen (IC(50)=21.6+/-0.4 micro M) and arachidonic acid-induced platelet aggregation (IC(50)=2.2+/-0.06 micro M), suggesting that the nature of the substituent on the phenyl ring of the N-heteroaromatic system of NAH moiety may be an important structural requirement for the improvement of antiplatelet activity, in comparison with lead-series 1.     

Cloning, characterization, and structural analysis of a C-type lectin from Bothrops insularis (BiL) venom

Lectins are carbohydrate-binding molecules that mediate a variety of biological processes. In this work, we identify and characterize a lectin from Bothrops insularis venom, with respect to its biochemical properties and theoretical structure. Initially, from a venom gland cDNA library, we cloned and sequenced a cDNA encoding a protein with high identity to snake venom lectins. A lectin molecule was purified to homogeneity from the venom by affinity column and gel filtration. This protein named BiL displayed hemagglutinating activity that was inhibited by galactose, lactose, and EDTA. Mass spectrometry analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that BiL is a disulfide-linked dimeric protein consisting of monomers with 16,206 m/z. The amino acid sequence, deduced from its cDNA sequence, was confirmed by Edman sequencing and by peptide mass fingerprint analysis. BiL shows similarity to other C-type lectin family members. Modeling studies provide insights into BiL dimeric structure and its structural determinants for carbohydrate and calcium binding.     

Secretome of HepG2 cells infected with dengue virus: implications for pathogenesis

Viral hemorrhagic fever is a clinical syndrome that poses serious global health threat. Among the causative agents, dengue virus (DV) has the highest incidence rate and its infection is the major cause of viral hemorrhagic fever in the world. Although the pathophysiological mechanisms of DV-induced diseases are not yet understood, it is well accepted that liver is a site of viral replication. In this study, we used proteomics to analyze infection of a hepatic cell lineage, HepG2, with DV, focusing on the secreted proteins. 1D-electrophoresis and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) were used, allowing the identification of a total of 107 proteins, among which 35 were found only in control secretome and 24 only in infected cells secretome. To validate these data, we performed 2D-eletrophoresis followed by MALDI-TOF/TOF, resulting in the identification of 20 proteins, 8 of them confirming LC-MS/MS results. We discuss the results obtained taking into account the proteins previously described in the secretome of HepG2 cells, proteins present in human plasma and proteins of interest for dengue pathogenesis. Altogether the data presented here provide clues for the progress in the understanding of the role of liver secretion in the progression of the disease.     

Structural features of a snake venom thrombin-like enzyme: thrombin and trypsin on a single catalytic platform?

The Lachesis muta thrombin-like enzyme (LM-TL) is a single chain serine protease that shares 38% sequence identity with the serine protease domain of thrombin and also displays similar fibrinogen-clotting activity. In addition, the 228 amino acid residue LM-TL is 52% identical to trypsin, and cleaves chromogenic substrates with similar specificity. Herein we report a three-dimensional (3D) model validated experimentally for LM-TL based on these two homologous proteins of known 3D structure. Spatial modeling of LM-TL reveals a serine protease with a chymotrypsin fold presenting a hydrophobic pocket on its surface, involved in substrate recognition, and an important 90's loop, involved in restricting the LM-TL catalytic site cleft. Docking analysis showed that LM-TL would not form a stable complex with basic pancreatic trypsin inhibitor and wild-type ecotin since its 90's loop would restrict the access to the catalytic site. LM-TL formed acceptable interactions with fibrinopeptide A and a variant of ecotin; ecotin-TSRR/R in which both the primary and secondary binding sites are mutated Val81Thr, Thr83Ser, Met84Arg, Met85Arg and Asp70Arg. Furthermore, analysis of the primary structures of LM-TL and of the seven snake venom thrombin-like enzymes (SVTLEs) family reveals a subgroup formed by LM-TL, crotalase, and bilineobin, both closely related to thrombin. Therefore, LM-TL provides an initial point to compare SVTLEs with their counterparts, e.g. the mammalian serine proteases, and a basis for the localization of important residues within the little known SVTLEs family.     

The interaction of Bacteroides fragilis with components of the human fibrinolytic system

Bacteroides fragilis is a minor component of the intestinal microbiota and the most frequently isolated from intra-abdominal infections and bacteremia. Previously, our group has shown that molecules involved in laminin-1 (LMN-1) recognition were present in outer membrane protein extracts of B. fragilis MC2 strain. One of these proteins was identified and showed 98% similarity to a putative B. fragilis plasminogen-binding protein precursor, deposited in the public database. Thus, the objective of this work was to overexpress and further characterize this novel adhesin. The ability of B. fragilis MC2 strain and purified protein to convert plasminogen into plasmin was tested. Our results showed that B. fragilis strain MC2 strain adhered to both LMN-1 and plasminogen and this adhesion was inhibited by either LMN-1 or plasminogen. Regarding the plasminogen activation activity, both the whole bacterial cell and the purified protein converted plasminogen into plasmin similar to streptokinase used as a positive control. Bacterial receptors that recognize plasminogen bind to it and enhance its activation, transforming a nonproteolytic bacterium into a proteolytic one. We present in vitro evidence for a pathogenic function of the plasminogen receptor in promoting adherence to laminin and also the formation of plasmin by B. fragilis .     

Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two‐dimensional gel electrophoresis

Introduction – A variety of sample preparation protocols for plant proteomic analysis using two‐dimensional gel electrophoresis (2‐DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied. Objective – This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2‐DE. Methodology – Four sample preparation methods were tested: (1) phenol extraction and methanol–ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid–acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS–PAGE (1‐DE) and 2‐DE. Fifteen selected protein spots were trypsinised and analysed by matrix‐assisted laser desorption/ionisation time‐of‐flight tandem mass spectrometry (MALDI‐TOF‐MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins. Results – Methods number 3 and 4 resulted in large quantities of protein with good 1‐DE separation and were chosen for 2‐DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation. Conclusion – The described sample preparation method allows the proteomic analysis of papaya leaves by 2‐DE and mass spectrometry (MALDI‐TOF‐MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparative proteomic analysis of whole saliva from chronic periodontitis patients

Chronic periodontal disease is a chronic inflammatory process affecting tooth supporting tissues in the presence of pathogenic bacterial biofilm. There is some evidence for changes in the protein composition of whole saliva from chronic periodontitis patients, but there have been no studies using a proteomic approach. Hence, the aim of this study was to compare the protein profiles of unstimulated whole saliva from patients with periodontitis and healthy subjects by two complementary approaches (2D-gel electrophoresis and liquid chromatography). Protein spots of interest were analyzed by MALDI-TOF-TOF, and the data was complemented by an ESI-Q-TOF experiment. The analyses revealed that subjects with periodontal disease have increased amounts of blood proteins (serum albumin and hemoglobin) and immunoglobulin, and they have a lower abundance of cystatin compared to the control group. A higher number of protein spots were observed in the periodontitis group, of which most were identified as alpha-amylase. This higher number of alpha-amylase variants seems to be caused by hydrolysis by cysteine proteases under such inflammatory conditions. This approach gives novel insights into alterations of salivary protein in presence of periodontal inflammation and may contribute to the improvement of periodontal diagnosis.     

A TonB-dependent outer membrane protein as a Bacteroides fragilis fibronectin-binding molecule

The binding of Bacteroides fragilis to plasmatic fibronectin was investigated using strains isolated from healthy subjects and from patients with bacteremia. They were cultivated in a synthetic media in which variations in cysteine concentrations determined alterations in the oxidation–reduction potential (Eh). All the strains assayed were capable of adhering to plasmatic fibronectin when cultivated under oxidizing and reducing conditions. Bacteroides fragilis 1405 showed the greatest difference when the results under these conditions were compared and it was selected for further investigations. Chemical treatments suggested the involvement of a protein in the interaction between B. fragilis and plasmatic fibronectin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of outer membrane proteins (OMPs) revealed differences between the extracts obtained from cultures grown under the two conditions. Protein bands of c . 102, 100, 77, 73, 50 and 40 kDa were more highly expressed under oxidizing than reducing conditions. Dot blot analysis showed a stronger recognition of plasmatic fibronectin by OMPs obtained from cultures grown under higher Eh, and Western blot assays confirmed a band of c . 102 kDa as fibronectin-binding protein. This protein was sequenced and revealed to be a putative TonB-dependent OMPs. PCR analysis confirmed the presence of this gene in all the studied strains.     

RGD- and MLD-disintegrins, jarastatin and EC3, activate integrin-mediated signaling modulating the human neutrophils chemotaxis, apoptosis and IL-8 gene expression

The effects of jarastatin (JT), a monomeric RGD-disintegrin, were compared with those of the heterodimeric MLD-disintegrin, EC3, on human neutrophil activation and functions. Both disintegrins inhibited neutrophil chemotaxis induced by fMet-Leu-Phe and were also potent chemotactic agents. These effects were accompanied by an increase in actin polymerization, and both were inhibited by genistein, a tyrosine kinase inhibitor. While JT, but not other RGD-disintegrins, inhibited EC3-induced chemotaxis, EC3 was not able to inhibit JT effect. The chemotactic effect of JT was blocked by anti-alpha(M) antibody whereas anti-alpha(9)beta(1) inhibited EC3 effect. Both JT and EC3 induced focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) activation. Accordingly, LY294002, a PI3K inhibitor, impaired their chemotactic effect on neutrophils. JT induced Erk-2 translocation to nucleus and a delay of the spontaneous apoptosis of neutrophils in vitro. In contrast, EC3 inhibited Erk-2 activation and had a proapoptotic effect. These effects were reverted by PD98059, an MEK 1/2 inhibitor and blocked by z-VAD-FMK, a caspase inhibitor. In addition, JT, but not EC3, increased the IL-8 mRNA levels in neutrophils. The data indicate that JT and EC3 directly activate an integrin-coupled signaling and modulate the MAPK pathway in different ways, leading the neutrophils to express different functional response.