Study protocol to investigate biomolecular muscle profile as predictors of long-term urinary incontinence in women with gestational diabetes mellitus.

BACKGROUND: Pelvic floor muscles (PFM) and rectus abdominis muscles (RAM) of pregnant diabetic rats exhibit atrophy, co-localization of fast and slow fibers and an increased collagen type I/III ratio. However, the role of similar PFM or RAM hyperglycemic-related myopathy in women with gestational diabetes mellitus (GDM) remains poorly investigated. This study aims to assess the frequency of pelvic floor muscle disorders and pregnancy-specific urinary incontinence (PS-UI) 12 months after the Cesarean (C) section in women with GDM. Specifically, differences in PFM/RAM hyperglycemic myopathy will be evaluated. METHODS: The Diamater is an ongoing cohort study of four groups of 59 pregnant women each from the Perinatal Diabetes Research Centre (PDRC), Botucatu Medical School (FMB)-UNESP (São Paulo State University), Brazil. Diagnosis of GDM and PS-UI will be made at 24-26 weeks, with a follow-up at 34-38 weeks of gestation. Inclusion in the study will occur at the time of C-section, and patients will be followed at 24-48 h, 6 weeks and 6 and 12 months postpartum. Study groups will be classified as (1) GDM plus PS-UI; (2) GDM without PS-UI; (3) Non-GDM plus PS-UI; and (4) Non-GDM without PS-UI. We will analyze relationships between GDM, PS-UI and hyperglycemic myopathy at 12 months after C-section. The mediator variables to be evaluated include digital palpation, vaginal squeeze pressure, 3D pelvic floor ultrasound, and 3D RAM ultrasound. RAM samples obtained during C-section will be analyzed for ex-vivo contractility, morphological, molecular and OMICS profiles to further characterize the hyperglycemic myopathy. Additional variables to be evaluated include maternal age, socioeconomic status, educational level, ethnicity, body mass index, weight gain during pregnancy, quality of glycemic control and insulin therapy. DISCUSSION: To our knowledge, this will be the first study to provide data on the prevalence of PS-UI and RAM and PFM physical and biomolecular muscle profiles after C-section in mothers with GDM. The longitudinal design allows for the assessment of cause-effect relationships between GDM, PS-UI, and PFMs and RAMs myopathy. The findings may reveal previously undetermined consequences of GDM.

Modification of the brain proteome of Africanized honeybees (Apis mellifera) exposed to a sub-lethal doses of the insecticide fipronil.

Fipronil is a phenylpyrazole insecticide that is widely used in Brazilian agriculture for pest control. Although honeybees are not targets of fipronil, studies indicate that this pesticide can be harmful to honeybees. To assess the effects of fipronil in the brain of Africanized Apis mellifera workers, this study focused on the toxico-proteome profiling of the brain of newly emerged and aged honeybee workers that were exposed to a sub-lethal dose (10 pg fipronil per day. i.e. (1)/100 of LD50/bee/day during 5 days) of the insecticide. Proteomic analysis identified 25 proteins that were differentially up-regulated or down-regulated when the fipronil-exposed and non-exposed groups were compared. These proteins are potentially related to pathogen susceptibility, neuronal chemical stress, neuronal protein misfolding, and occurrence of apoptosis, ischemia, visual impairment, damaged synapse formation, brain degeneration, memory and learning impairment. The exposure of honeybees to a very low dose of fipronil, even for a short period of time (5 days), was sufficient to cause a series of important neuroproteomic changes in the brains of honeybees.

Hyperalgesic and edematogenic effects of Secapin-2, a peptide isolated from Africanized honeybee (Apis mellifera) venom.

Honeybee stings are a severe public health problem. Bee venom contains a series of active components, including enzymes, peptides, and biogenic amines. The local reactions observed after envenoming include a typical inflammatory response and pain. Honeybee venom contains some well-known polycationic peptides, such as Melittin, Apamin, MCD peptide, Cardiopep, and Tertiapin. Secapin in honeybee venom was described 38 years ago, yet almost nothing is known about its action. A novel, variant form of this peptide was isolated from the venom of Africanized honeybees (Apis mellifera). This novel peptide, named Secapin-2, is 25 amino acid residues long. Conformational analyses using circular dichroism and molecular dynamics simulations revealed a secondary structure rich in strands and turns, stabilized by an intramolecular disulfide bridge. Biological assays indicated that Secapin-2 did not induce hemolysis, mast cell degranulation or chemotactic activities. However, Secapin-2 caused potent dose-related hyperalgesic and edematogenic responses in experimental animals. To evaluate the roles of prostanoids and lipid mediators in the hyperalgesia and edema induced by this peptide, Indomethacin and Zileuton were used to inhibit the cyclooxygenase and lipoxygenase pathways, respectively. The results showed that Zileuton partially blocked the hyperalgesia induced by Secapin-2 and decreased the edematogenic response. In contrast, Indomethacin did not interfere with these phenomena. Zafirlukast, a leukotriene receptor antagonist, blocked the Secapin-2 induced hyperalgesia and edematogenic response. These results indicate that Secapin-2 induces inflammation and pain through the lipoxygenase pathway in both phenomena.

Agelaia MP-I: a peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets.

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K(+) (K(ATP)) channels, increasing intracellular Ca(2+) concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 µM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 µM diazoxide, a K(ATP) channel opener and 10 µM nifedipine, a Ca(2+) channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K(ATP) and L-type Ca(2+) channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling.

A single nucleotide deletion at the C1 inhibitor gene as the cause of hereditary angioedema: insights from a Brazilian family.

BACKGROUND: Hereditary angioedema is an autosomal dominant disease characterized by episodes of subcutaneous and submucosal edema. It is caused by deficiency of the C1 inhibitor protein, leading to elevated levels of bradykinin. More than 200 mutations in C1 inhibitor gene have been reported. The aim of this study was to analyze clinical features of a large family with an index case of hereditary angioedema and to determine the disease-causing mutation in this family. METHODS: Family pedigree was constructed with 275 individuals distributed in five generations. One hundred and sixty-five subjects were interviewed and investigated for mutation at the C1 inhibitor gene. Subjects reporting a history of recurrent episodes of angioedema and/or abdominal pain attacks underwent evaluation for hereditary angioedema. RESULTS: We have identified a novel mutation at the C1 inhibitor gene, c.351delC, which is a single-nucleotide deletion of a cytosine on exon 3, resulting in frameshift with premature stop codon. Sequencing analysis of the hypothetical truncated C1 inhibitor protein allowed us to conclude that, if transcription occurs, this protein has no biological activity. Twenty-eight members of the family fulfilled diagnostic criteria for hereditary angioedema and all of them presented the c.351delC mutation. Variation in clinical presentation and severity of disease was observed among these patients. One hundred and thirty-seven subjects without hereditary angioedema did not have the c.351delC mutation. CONCLUSION: The present study provides definitive evidence to link a novel genetic mutation to the development of hereditary angioedema in patients from a Brazilian family.

Understanding the structure, activity and inhibition of chorismate synthase from Mycobacterium tuberculosis.

Tuberculosis is considered a worldwide health problem mainly due to co-infection with HIV and proliferation of multi-drug-resistant strains. The enzymes of the shikimate pathway are potential targets for the development of new therapies because they are essential for bacteria, but absent from mammals. The last step in this pathway is performed by chorismate synthase (CS), which catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate (EPSP) to chorismate. The aim of this article is to review the available information on chorismate synthase from Mycobacterium tuberculosis.

Shikimate kinase (EC 2.7.1.71) from Mycobacterium tuberculosis: kinetics and structural dynamics of a potential molecular target for drug development.

The enzymes of the shikimate pathway represent potential molecular targets for the development of non-toxic antimicrobial agents and anti-parasite drugs. One of the most promising of these enzymes is shikimate kinase (EC 2.7.1.71), which is responsible for the fifth step in the shikimate pathway. This enzyme phosphorylates shikimic acid to yield shikimate-3-phosphate, using ATP as a substrate. In this work, the conformational dynamics of the shikimate kinase from Mycobacterium tuberculosis was investigated in its apostate in solution. For this study, the enzyme was subjected to a gradient of temperatures from 15°C to 45°C in the presence or absence of deuterium oxide, and the amide H/D exchange was monitored using ESI-mass spectrometry. We observed: i) the phosphate binding domain in the apo-enzyme is fairly rigid and largely protected from solvent access, even at relatively high temperatures; ii) the shikimate binding domain is highly flexible, as indicated by the tendency of the apo-enzyme to exhibit large conformational changes to permit LID closure after the shikimate binding; iii) the nucleotide binding domain is initially conformationally rigid, which seems to favour the initial orientation of ADP/ATP, but becomes highly flexible at temperatures above 30°C, which may permit domain rotation; iv) part of the LID domain, including the phosphate binding site, is partially rigid, while another part is highly flexible and accessible to the solvent.

Analysis of agonist and antagonist effects on thyroid hormone receptor conformation by hydrogen/deuterium exchange.

Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T(3)) or antagonist (NH3). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, C-terminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T(3), but not NH3, increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T(3) but not NH3. We present data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes.

A new scenario of bioprospecting of Hymenoptera venoms through proteomic approach

Venoms represent a huge and essentially unexplored reservoir of bioactive components that may cure diseases that do not respond to currently available therapies. This review select advances reported in the literature from 2000 to the present about the new scenario of Hymenoptera venom composition. On account of new technologies in the proteomic approach, which presents high resolution and sensitivity, the combination of developments in new instruments, fragmentation methods, strategic analysis, and mass spectrometry have become indispensable tools for interrogation of protein expression, molecule interaction, and post- translational modifications. Thus, the biochemical characterization of Hymenoptera venom has become a major subject of research in the area of allergy and immunology, in which proteomics has been an excellent alternative to assist the development of more specific extracts for diagnosis and treatment of hypersensitive patients to Hymenoptera venoms.

Proteomic analysis reveals suppression of bark chitinases and proteinase inhibitors in citrus plants affected by the citrus sudden death disease.

Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges grafted on Rangpur lime rootstock, the most important rootstock in Brazilian citriculture. We performed a proteomic analysis to generate information related to this plant pathogen interaction. Protein profiles from healthy, CSD-affected and CSD-tolerant stem barks, were generated using two-dimensional gel electrophoresis. The protein spots were well distributed over a pI range of 3.26 to 9.97 and a molecular weight (MW) range from 7.1 to 120 kDa. The patterns of expressed proteins on 2-DE gels made it possible to distinguish healthy barks from CSD-affected barks. Protein spots with MW around 30 kDa and pI values ranging from 4.5 to 5.2 were down-regulated in the CSD-affected root-stock bark. This set of protein spots was identified as chitinases. Another set of proteins, ranging in pI from 6.1 to 9.6 with an MW of about 20 kDa, were also suppressed in CSD-affected rootstock bark; these were identified as miraculin-like proteins, potential trypsin inhibitors. Down-regulation of chitinases and proteinase inhibitors in CSD-affected plants is relevant since chitinases are well-known pathogenesis-related protein, and their activity against plant pathogens is largely accepted.

Changes in amounts of total salivary gland proteins of Lutzomyia longipallpis (Diptera: Psychodidae) according to age and diet.

Saliva plays important roles in facilitation of a bloodmeal, lubrication of mouthparts, and parasite transmission for some vector insects. Salivary composition changes during the lifetime of an insect, and differences in the salivary profile may influence its functions. In this report, the amount and profile of salivary gland protein of the American visceral leishmaniasis vector Lutzomyia longipalpis (Lutz & Neiva, 1912) were analyzed at different times of insect development and diet. Protein content from unfed female sand flies increased significantly with age, and a significant difference was observed in sugar-fed females during the first 10 d of adult life. Salivary protein content sharply decreased 1 d after blood feeding, with gradual increase in concentration the following days. SDS-polyacrylamide gel electrophoresis analysis revealed that most polypeptides present in the saliva of sugar-fed also were present in the saliva of blood-fed females. Understanding changes in sand fly's saliva contents at distinct days after emergence and the influence of a bloodmeal in this aspect may reveal the role played by saliva during leishmaniasis transmission.

Conformation and lytic activity of eumenine mastoparan: a new antimicrobial peptide from wasp venom.

Eumenine mastoparan-AF (EMP-AF) is a novel membrane active tetradecapeptide recently isolated from the venom of solitary wasp, Anterhynchium flavomarginatum micado. It was reported previously that EMP-AF peptide presented low cytolytic activities in human erythrocytes and in RBL-2H3 mast cells. In the present work, we observed that this peptide is able to permeate anionic liposomes, and in less extension also the neutral ones. We present evidences showing that the permeation ability is well correlated with the amount of helical conformation assumed by the peptides in these environments. This peptide also showed a broad-spectrum inhibitory activity against Gram-positive and Gram-negative bacteria. The permeability of liposomes and the antibiotic effect showed a significant reduction when C-terminus was deamidated (in acidic form). The removal of the three first amino acid residues from the N-terminus rendered the peptide inactive both in liposomes and in bacteria. The results suggest that the mechanism of action involves a threshold in the accumulation of the peptide at level of cell membrane.

Insects as biological models to assay spider and scorpion venom toxicity

This study was undertaken to develop an experimental protocol using insects as biological models to assay venom toxicity of the following spiders Loxosceles gaucho, Phoneutria nigriventer, Nephilengys cruentata and Tityus serrulatus scorpion. Three different insect species were bioassayed: Apis mellifera (Hymenoptera), Grillus assimilis (Orthoptera), and Diatraea saccharalis (Lepidoptera). Venoms were injected into the hemocele of insects with a microsyringe at concentrations that caused dose/weight-dependent effects; doses causing either paralysis (ED50) or death (LD50) were recorded for each venom and insect test-species. T. serrulatus and L. gaucho venoms were lethal to all tested species, while P. nigriventer venom caused paralysis and death, and N. cruentata venom caused only paralysis at the doses assayed. A comparison between the insect test species described above revealed that A. mellifera was highly sensitive to all venoms tested; even a tiny amount of N. cruentata non-lethal venom caused a change in the walking pattern leading to transient paralysis. D. saccharalis larvae were very resistant to all four venoms.

Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis.

A novel antimicrobial peptide, anoplin, was purified from the venom of the solitary wasp Anoplius samariensis. The sequence was mostly analyzed by mass spectrometry, which was corroborated by solid-phase synthesis. Anoplin, composed of 10 amino acid residues, Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2, has a high homology to crabrolin and mastoparan-X, the mast cell degranulating peptides from social wasp venoms, and, therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the circular dichroism (CD) spectra of anoplin in the presence of trifluoroethanol or sodium dodecyl sulfate showed a high content, up to 55%, of the alpha-helical conformation. A modeling study of anoplin based on its homology to mastoparan-X supported the CD results. Biological evaluation using the synthetic peptide revealed that this peptide exhibited potent activity in stimulating degranulation from rat peritoneal mast cells and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, this is the first antimicrobial component to be found in the solitary wasp venom and it may play a key role in preventing potential infection by microorganisms during prey consumption by their larvae. Moreover, this peptide is the smallest among the linear alpha-helical antimicrobial peptides hitherto found in nature, which is advantageous for chemical manipulation and medical application.

ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized ß-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the ß-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA

ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity.

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized beta-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the beta-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA.

Preliminary cryocrystallography analysis of an eumenine mastoparan toxin isolated from the venom of the wasp Anterhynchium flavomarginatum micado.

Mastoparans are tetradecapeptides found to be the major component of vespid venoms. These peptides present a wide spectrum of biological activities, such as mast cell degranulation, hemolytic activity and also reveals antimicrobial activity. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized. At room temperature these crystals diffracted to 2.8 A resolution. However, upon cooling to cryogenic temperature around 85 K, the original resolution limit could be improved to 2.0 A. Crystals were determined to belong to the space group P3(1) (P3(2)). This is the first mastoparan to be crystallized and it will provide further insights in the conformational significance of mastoparan toxins, with respect to their potency and activity in G protein regulation.

Expression and processing of recombinant sarafotoxins precursor in Pichia pastoris.

Sarafotoxins are peptides isolated from the Atractaspis snake venom, with strong constrictor effect on cardiac and smooth muscle. They are structurally and functionally related to endothelins. The sarafotoxins precursor cDNA predicts an unusual structure 'rosary-type', with 12 successive similar stretches of sarafotoxin (SRTX) and spacer. In the present work, the recombinant precursor of SRTXs was sub-cloned and expressed in the yeast Pichia pastoris, and secreted to the culture medium. Characterization by SDS-PAGE, immunoblot, mass spectrometry and biological activity, suggests that intact precursor was expressed but processing into mature toxins also occurred. Furthermore, our results indicate that the correct proportion of sarafotoxin types as contained in the precursor, is obtained in the yeast culture medium. Contractile effects of the expressed toxins, on rat and Bothrops jararaca isolated aorta, were equivalent to 5x10(-10)M and 5x10(-11)M of sarafotoxin b, respectively. The enzymes responsible for the complete maturation of sarafotoxins precursor are still unknown. Our results strongly suggest that the yeast Pichia pastoris is able to perform such a maturation process. Thus, the yeast Pichia pastoris may offer an alternative to snake venom gland to tentatively identify the molecular process responsible for SRTXs release.

Isolation and sequence determination of peptides in the venom of the spider wasp (Cyphononyx dorsalis) guided by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry.

Micro-scale (sub-pmol) isolation and sequence determination of three peptides from the venom of the solitary spider wasp Cyphononyx dorsalis is described. We isolated two novel peptides Cd-125 and Cd-146 and a known peptide Thr(6)-bradykinin from only two venom sacs of solitary spider wasp Cyphononyx dorsalis without bioassay-guided fractionation, but instead guided by MALDI-TOF MS. The MALDI-TOF MS analysis of each fraction showed the purity and molecular weight of the components, which led to the isolation of the peptides virtually without loss of sample amount. The sequences of the novel peptides Cd-125 (Asp-Thr-Ala-Arg-Leu-Lys-Trp-His) and Cd-146 (Ser-Glu-Thr-Gly-Asn-Thr-Val-Thr-Val-Lys-Gly-Phe-Ser-Pro-Leu-Arg) were determined by Edman degradation together with mass spectrometry, and finally corroborated by solid-phase synthesis. The known peptide Thr(6)-bradykinin (Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg) was identified by comparison with the synthetic authentic specimen. This is the first example for any kinins to be found in Pompilidae wasp venoms. The procedure reported here can be applicable to studies on many other components of solitary wasp venoms with limited sample availability.