Oxidonitrergic and antioxidant effects of a low molecular weight peptide fraction from hardened bean (Phaseolus vulgaris) on endothelium.

About 3000 tons of beans are not used in human food due to hardening. Several studies on bean-derived bioactive peptides have shown potential to treat some diseases, including those relying on oxidative dysfunctions. We assessed the effects of peptides extracted from hardened bean Phaseolus vulgaris (PV) on reactive oxygen species (ROS) and nitric oxide (NO) production, cytotoxic and cytoprotective effects in endothelial cells, and oxidonitrergic-dependent vasodilating effects. Extract was composed by peptide fraction <3 kDa (PV3) from hardened common bean residue. PV3 sequences were obtained and analyzed with bioinformatics. Human umbilical vein endothelial cells were treated with 10, 20, 30, and 250 µg/mL PV3. Oxidative stress was provoked by 3% H2O2. Cytotoxicity and cytoprotective effects were evaluated by MTT assay, whereas, ROS and NO were quantified using DHE and DAF-FM fluorescent probes by confocal microscopy. NO- and endothelium-dependent vasodilating effects of PV3 were assessed in isolated aortic rings. We found 35 peptides with an average mass of 1.14 kDa. There were no cell deaths with 10 and 20 µg/mL PV3. PV3 at 30 µg/mL increased cell viability, while cytotoxicity was observed only with 250 µg/mL PV3. PV3 at 10 µg/mL was able to protect cells from oxidative stress. PV3 also increased NO release without causing cell death. It also reduced relative ROS production induced by H2O2. PV3 vasodilating effects relied on endothelium-dependent NO release. PV3 obtained from low-commercial-value bean displays little cytotoxicity and exerts antioxidant effects, whereas it increases endothelial NO release.

Oxidonitrergic and antioxidant effects of a low molecular weight peptide fraction from hardened bean (Phaseolus vulgaris) on endothelium

About 3000 tons of beans are not used in human food due to hardening. Several studies on bean-derived bioactive peptides have shown potential to treat some diseases, including those relying on oxidative dysfunctions. We assessed the effects of peptides extracted from hardened bean Phaseolus vulgaris (PV) on reactive oxygen species (ROS) and nitric oxide (NO) production, cytotoxic and cytoprotective effects in endothelial cells, and oxidonitrergic-dependent vasodilating effects. Extract was composed by peptide fraction <3 kDa (PV3) from hardened common bean residue. PV3 sequences were obtained and analyzed with bioinformatics. Human umbilical vein endothelial cells were treated with 10, 20, 30, and 250 µg/mL PV3. Oxidative stress was provoked by 3% H2O2. Cytotoxicity and cytoprotective effects were evaluated by MTT assay, whereas, ROS and NO were quantified using DHE and DAF-FM fluorescent probes by confocal microscopy. NO- and endothelium-dependent vasodilating effects of PV3 were assessed in isolated aortic rings. We found 35 peptides with an average mass of 1.14 kDa. There were no cell deaths with 10 and 20 μg/mL PV3. PV3 at 30 μg/mL increased cell viability, while cytotoxicity was observed only with 250 μg/mL PV3. PV3 at 10 μg/mL was able to protect cells from oxidative stress. PV3 also increased NO release without causing cell death. It also reduced relative ROS production induced by H2O2. PV3 vasodilating effects relied on endothelium-dependent NO release. PV3 obtained from low-commercial-value bean displays little cytotoxicity and exerts antioxidant effects, whereas it increases endothelial NO release.

Proteomic landscape of seminal plasma associated with dairy bull fertility.

Male fertility is the ability of sperm to fertilize the egg and sustain embryo development. Several factors determine the fertilizing capacity of mammalian sperm, including those intrinsic to sperm and components of the seminal plasma. The present study analyzed the seminal fluid proteome of Bos taurus and potential associations between proteins and fertility scores. Mass spectrometry coupled with nano HPLC allowed the identification of 1,159 proteins in the dairy bull seminal plasma. There were 50 and 29 seminal proteins more abundant in high (HF) low fertility (LF) bulls, respectively. Based on multivariate analysis, C-type natriuretic peptide, TIMP-2, BSP5 and sulfhydryl oxidase indicated relationship with HF bulls. Clusterin, tissue factor pathway inhibitor 2, galectin-3-binding protein and 5'-nucleotidase were associated with LF bulls. Abundance of NAD(P)(+)-arginine ADP-ribosyltransferase, prosaposin and transmembrane protein 2 proteins had the highest positive correlations with fertility ranking. Quantities of vitamin D-binding protein, nucleotide exchange factor SIL1 and galectin-3-binding protein showed the highest negative correlations with fertility ranking. A fertility ranking score was calculated and the relationship with these proteins was significant (Spearman's rho = 0.94). The present findings represent a major and novel contribution to the study of bovine seminal proteins. Indicators of fertility can be used to improve reproductive biotechnologies.

Avaliação proteica do humor aquoso de equinos / Evaluation of equine aqueous humor protein

A avaliação proteica do humor aquoso (HA) pode ser utilizada como método diagnóstico nas uveítes. Entretanto, estudos sobre as proteínas nesse fluido, em equinos hígidos, são escassos e apresentam variações conforme a metodologia empregada. Dessa forma, objetivou-se realizar a análise proteica e citológica do HA nessa espécie, bem como verificar sua correlação com as proteínas plasmáticas. Foram avaliados 13 equinos adultos (26 olhos), sem raça definida, machos ou fêmeas. Mediante aqueocentese, foi coletado 0,5 mL de humor aquoso de cada olho. Cada amostra foi encaminhada para quantificação proteica pelo método de Bradford modificado e pela eletroforese em gel de poliacrilamida - dodecil sulfato de sódio (SDS-PAGE), bem como para avaliação citológica. Por meio de venopunção, coletou-se sangue para determinação da concentração de proteínas séricas. Treze olhos (50% das amostras) apresentaram valor proteico médio de 40,3 mg/dL±6,45 e a eletroforese demonstrou presença de proteínas de massas mais elevadas que 43 KDa. Houve ausência de células em 96,15% das amostras (25 olhos). Equinos hígidos apresentaram baixa concentração de proteínas no HA. Já a correlação entre proteína no humor aquoso/proteína plasmática total foi de 0,56%.(AU)

Evaluation of equine aqueous humor (AH) proteins can help the diagnosis of uveitis. However, studies on proteins in this fluid in healthy horses are scarce and present variations according to the methodology employed. This study aimed to perform protein analysis and cytology of equine aqueous humor of healthy horses and verify its correlation with plasmatic proteins. Thirteen adult horses (26 eyes), mixed breed, male or female were evaluated. A volume of 0.5 mL of aqueous humor was collected through aqueocentesis from both eyes. The samples were submitted to protein quantification by modified Bradford method and to sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE), and to cytological evaluation. Blood was collected for determination of plasmatic protein concentration. Thirteen eyes (50% of the samples) had values larger than zero by the Bradford method, with an average of 40.3 mg/dl±6.45. Electrophoresis showed presence of higher masses of proteins (43 KDa). There were no cells in 96.15% of the samples (25 eyes). Healthy equines presented low protein concentration in the HA. The ratio between protein concentration in the aqueous humor / total plasma protein of 0.56%.(AU)

Neutrophil proteomic analysis reveals the participation of antioxidant enzymes, motility and ribosomal proteins in the prevention of ischemic effects by preconditioning.

Intestinal ischemia and reperfusion injury are widely used models, which result into tissue injury and multiple organ failure also observed after trauma and surgery. Ischemic preconditioning (IPC) preceding ischemia and reperfusion (IR) was shown to attenuate this injury and has a potential therapeutic application; however the exact underlying mechanism is not clear. Neutrophils play an important role in the mechanism of injuries caused by ischemia and reperfusion while IPC led to a decrease in neutrophil stimulation and activation. The effect of preconditioning on the neutrophil proteome is unclear. Proteomic analysis has been ratified as an appropriate tool for studying complex systems. In order to evaluate the effect of IPC preceding 45min of ischemia on the proteome of neutrophils we used Wistar rats divided in four experimental groups: Control, sham laparotomy, intestinal ischemia reperfusion and ischemic preconditioning. After neutrophil separation, proteins were extracted, trypsin digested and the resulting peptides were iTRAQ labeled followed by HILIC fractionation and nLC-MS/MS analysis. After database searches, normalization and statistical analysis our proteomic analysis resulted in the identification of 2437 protein groups that were assigned to five different clusters based on the relative abundance profiles among the experimental groups. The clustering followed by statistical analysis led to the identification of significantly up and downregulated proteins in IR and IPC. Cluster based KEGG pathways analysis revealed up- regulation of actin cytoskeleton, metabolism, Fc gamma R mediated phagocytosis, chemokine signaling, focal adhesion and leukocyte transendothelial migration whereas downregulation in ribosome, spliceosome, RNA transport, protein processing in endoplasmic reticulum and proteasome, after intestinal ischemic preconditioning. Furthermore, enzyme prediction analysis revealed the regulation of some important antioxidant enzymes and having their role in reactive oxygen species production. To our knowledge, this work describes the most comprehensive and detailed quantitative proteomic study of the neutrophil showing the beneficial role of ischemic preconditioning and its effects on the neutrophil proteome. This data will be helpful to understand the effect of underlying protective mechanisms modulating the role of PMNs after IPC and provide a trustworthy basis for future studies. BIOLOGICAL SIGNIFICANCE: Preconditioning is a relevant strategy to overcome clinical implications from ischemia and reperfusion. Such implications have the neutrophil as a major player. Although many publications describe specific biochemical and physiological roles of the neutrophil in such conditions, there is no report of a proteomic study providing a broader view of this scenario. Here we describe a group of proteins significantly regulated by ischemia and reperfusion being such regulation prevented by preconditioning. Such finding may provide relevant information for a deeper understanding of the mechanisms involved, as well as serve as basis for future biomarker or drug target assays.

Cm38: A New Antimicrobial Peptide Active Against Klebsiella pneumoniae is Homologous to Cn11

Antimicrobial peptides (AMPs) represent a large and ubiquitous group of peptides. The current crisis in antibiotic therapy has led to an intensified search for new antimicrobial agents. In this regard, scorpion venom constitutes a rich source of biologically active peptides including AMPs. In the present study, the purification of a novel peptide with antimicrobial activity against the Gram-negative bacteria Klebsiella pneumoniae is described. This antimicrobial peptide, named Cm38, was purified from Centruroides margaritatus scorpion venom using a two-step chromatographic strategy using C-8 and C-18 columns. This toxin inhibits the proliferation of the Gram-negative bacteria Klebsiella pneumoniae with a Minimal Inhibitory Concentration (MIC) of 64 mu M. An analysis of the N-terminal sequence of Cm38 revealed a close structural relationship to Cn11, a Na+-channel modulator toxin previously isolated from Centruroides noxius scorpion venom. Therefore, to test Cm38 for effects on ion channels, we measured its effects on action potential firing in cultured dorsal root ganglion neurons. Cm38 depolarized and increased action potential firing in a subset of neurons tested. The present work reports a new peptide related to Cn11 with antimicrobial properties that is also active in neurons.

Anticancer peptides and proteins: a panoramic view.

Despite progress in understanding carcinogenesis, cancer is still a major cause of morbidity and mortality worldwide. Treatment is complicated by the resistance to chemotherapeutics acquired by tumors, ability to repair damaged DNA, alteration of the interaction between drug and its target, and defects in the machinery that leads to apoptosis. The development of novel therapies based on biologically active peptides, especially antimicrobial peptides (AMPs), and proteins has emerged as a new strategy to defeat cancer.

Neutrophil proteome: lessons from different standpoints.

The present review brings a timeline of some of the major steps given throughout the years towards the development of our knowledge regarding the biology of the neutrophil. The contribution of early articles and their elementary biochemical approach is highlighted. The importance of the development of proteomic techniques is paralleled to the shift in neutrophil research towards high throughput molecular methods. As a last change of standpoint, the study of the neutrophil is presented integrated with other life- sciences technologies such as lipidomics, genomics and systems biology. The paper also brings a perspective/tendency overview at the same time that it discusses some of the difficulties encountered in the research of the neutrophil.

Proteome analysis of Nelore bull (Bos taurus indicus) seminal plasma.

The Nelore bull (Bos taurus indicus) seminal plasma proteome was analyzed by MALDI-TOF MS and two-dimensional gel electrophoresis. A total of 260 spots were visualized in the 2-DE gel (pI range 3-10) and 13 spots could be identified by peptide mass fingerprinting corresponding to 11 different polypeptides. The results allowed the creation of the first proteomic map of Bos taurus indicus seminal plasma. The roles of the identified proteins in the bull seminal plasma are discussed.

Purification and characterization of a new xylanase from Acrophialophora nainiana.

A new xylanase activity (XynII) was isolated from liquid state cultures of Acrophialophora nainiana containing birchwood xylan as carbon source. XynII was purified to apparent homogeneity by gel filtration and ion exchange chromatographies. The enzyme was optimally active at 55 degrees C and pH 7.0. XynII had molecular mass of 22630+/-3.0 and 22165 Da, as determined by mass spectrometry and SDS-PAGE, respectively. The purified enzyme was able to act only on xylan as substrate. The apparent K(m) values on soluble and insoluble birchwood xylans were 40.9 and 16.1 mg ml(-1), respectively. The enzyme showed good thermal stability with half lives of 44 h at 55 degrees C and ca. 1 h at 60 degrees C The N-terminal sequence of XynII showed homology with a xylanase grouped in family G/11. The enzyme did not show amino acid composition similarity with xylanases from some fungi and Bacillus amyloliquefaciens.

Improving the recovery of lysine in automated protein sequencing.

Automated protein sequencing is an important tool in protein characterization. Most instruments use tetrahydrofuran (THF) as the HPLC eluent for separation of the derivatized amino acids residues. THF is highly perishable when exposed to air, generating peroxides which can degrade amino acids, mainly lysine, leading to uncertainty in chromatogram interpretations. Modifications of the existing HPLC equipment were introduced to create a permanent inert atmosphere inside the bottle of THF solution. This was carried out by changing the argon plumbing and some electrical connections and by reprogramming the software of the protein sequencer. The positive results of this procedure were demonstrated by comparing the decreasing lysine peak area during 28 days before and after the modifications. In the modified instrument, lysine recovery was much better as a function of the age of the THF eluent. Since these modifications improved the instrument performance, they have been adopted for routine use in our laboratory.

Determination of the amino acid sequence of the plant cytolysin enterolobin.

The cytolytic seed protein enterolobin from seeds of Enterolobium contortisiliquum was purified by using FPLC on a Mono Q column giving a single peak in capillary electrophoresis. The complete amino acid sequence of the plant cytolysin was determined by an automated method, yielding a molecular mass of 54,806 Da. Databank searches and sequence alignment demonstrated a high degree of sequence identity and similarity between enterolobin and bacterial aerolysins from Aeromonas hydrophila and A. sobria. Several key residues involved in oligomerization of A. hydrophila aerolysin are conserved in enterolobin. Circular dichroism measurements and structural predictions revealed that enterolobin is very rich in beta sheet, like aerolysin. Light-scattering studies revealed that enterolobin oligomerizes as a hexamer at pH levels below 7.0. NaCl concentrations above 50 mM caused dimerization of enterolobin. Dithiothreitol did not cause oligomerization.

Homology between the seed cytolysin enterolobin and bacterial aerolysins.

Enterolobin, a 55-kDa cytolytic, inflammatory, and insecticidal protein isolated from seeds of the Brazilian tree Enterolobium contortisiliquum (Leguminosae-Mimosoideae) has been further purified and partially sequenced by using both manual and automated methods. A computational search of enterolobin partial amino acid sequence against the PIR database revealed possible sequence similarities with aerolysins, cytolytic proteins from Aeromonas species. An alignment of enterolobin partial sequence to the amino acid sequences of A. hydrophila and A. sobria aerolysins showed several similar regions with many residue identities. The seed protein enterolobin and the bacterial aerolysins may be homologous proteins despite the distant phylogenetic relationship.

Percutaneous catheterization of the subclavian vein in hemophiliac patients: report of 47 cases.

Hemophiliac patients frequently require venous access, but usually their peripheral veins are collapsed and not puncturable. Several procedures employed to overcome this difficulty present a high complication rate. The authors report 47 cases of percutaneous subclavian vein catheterization (PSC) in hemophiliacs with an overall complication rate of 23%. Six patients (13%) had complications attributed to the coagulation disorder. The complications attributed to the coagulopathy and presenting major clinical importance occurred early in the series. After the improvement of the coagulation control with a routine AHG administration policy, the authors observed that among the last 22 patients there were 3 complications related to the coagulopathy, all of them without major clinical importance. The authors conclude that, in the lack of a safer access, PSC seems to be an acceptable procedure in hemophiliac patients, if employed with strict indication, accurate technique and rigorous control of the coagulopathhy.

[Syphilitic aneurysm communicating with an aortic sinus of Valsalva. A case report]. / Aneurisma sifilítico comunicando-se com um seio de Valsalva aórtico. Relato de caso.

The authors present the case of a 27-year old woman with an aneurysm, possibly originating from an ectopic coronary sprout and communicating with the right anterior sinus of Valsalva. Secondary syphilitic lesions were observed. By virtue of its great size and localization, this aneurysm produced obstruction of the outlet of the right ventricle and dislocation of the left coronary artery. The authors conclude that this aneurysm was a congenital anomaly because of its great volume, configuration, the way it opened in the aortic right anterior sinus of Valsalva, the normal aortic wall and valve, and normal sinuses of Valsalva, observed at surgery. The follow-up was uneventful.

The efficacy of loop colostomy for complete fecal diversion.

In an attempt to evaluate the real efficacy of loop colostomy for fecal diversion, the authors studied 62 patients previously colostomized under emergency conditions. Radiologic series of the abdomen were taken after 200 gm of barium meal. The results showed that the colostomy provided complete diversion of the radiologic contrast in 53 patients (85 percent) and incomplete diversion in nine patients (15 percent). Analysis of the results revealed that incomplete fecal diversion was: 1) observed as of the 86th postoperative day, with a significantly higher frequency following the 10th postoperative month, and 2) significantly correlated with either colostomy retraction or prolapse. The authors present a diagram showing a possible interaction of factors responsible for incomplete fecal diversion in loop colostomy and conclude that: 1) retraction is probably the basic contributing factor for colostomy failure; 2) the prolapse, once reduced, propitiates sinking of the stoma, facilitating colostomy failure; 3) the common assumption that loop colostomy eventually fails to provide complete fecal diversion is further supported; 4) loop colostomy assures, over its usual duration, a satisfactory defunctionalization of the colon; and 5) use of improved techniques of colostomy construction may prolong complete fecal diversion.