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1.
Toxicon ; 184: 180-191, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-32585218

RESUMEN

In Colombia, Lachesis acrochorda causes 2-3% of all snake envenomations. The accidents promote a high mortality rate (90%) due to blood and cardiovascular complications. Here, the effects of the snake venom of L. acrochorda (SVLa) were analyzed on human blood cells and on cardiovascular parameters of rats. SVLa induced blood coagulation, as measured by the prothrombin time test, but did not reduce the cell viability of neutrophils and platelets evaluated by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay and by the lactate dehydrogenase (LDH) enzyme assay. In fact, SVLa increased the absorbance in tests made with platelets subjected to the MTT assay. SVLa induced platelet aggregation whose magnitude was comparable to that of the positive control adenosine diphosphate (ADP), and occurred earlier with increasing SVLa concentration. Acetylsalicylic acid (ASA, a cyclooxygenase inhibitor) or clopidogrel (an ADP receptor blocker) inhibited the aggregating effect of SVLa. Inhibition of SVLa-elicited platelet aggregation also resulted from the treatment with disodium ethylenediaminetetraacetate (Na2-EDTA; metalloproteinase inhibitor) and with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, serine protease inhibitor). In isolated right atrium of rats, SVLa increased slightly, but significantly, the magnitude of the spontaneous contractions and, in isolated rat aorta, SVLa relaxed KCl- or phenylephrine-induced contractions. In vivo, SVLa induced hypotension and bradycardia in rats, with detection of hemorrhage in pulmonary and renal tissues. Altogether, under experimental conditions, SVLa induced blood coagulation, platelet aggregation, hypotension and bradycardia. Part of the effects presented here may be explained by the presence of snake venom metalloproteinases (SVMPs) and snake venom serine proteases (SVSPs), constituents of SVLa.


Asunto(s)
Sistema Cardiovascular/efectos de los fármacos , Venenos de Víboras/toxicidad , Viperidae , Animales , Células Sanguíneas , Coagulación Sanguínea , Plaquetas , Colombia , Fibrinógeno , Hemorragia , Humanos , Hipotensión , Metaloproteasas , Agregación Plaquetaria , Tiempo de Protrombina , Ratas , Serina Endopeptidasas , Serina Proteasas , Inhibidores de Serina Proteinasa , Mordeduras de Serpientes
2.
BMC Cancer ; 19(1): 365, 2019 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-30999875

RESUMEN

BACKGROUND: Worldwide, breast cancer is the main cause of cancer mortality in women. Most cases originate in mammary ductal cells that produce the nipple aspirate fluid (NAF). In cancer patients, this secretome contains proteins associated with the tumor microenvironment. NAF studies are challenging because of inter-individual variability. We introduced a paired-proteomic shotgun strategy that relies on NAF analysis from both breasts of patients with unilateral breast cancer and extended PatternLab for Proteomics software to take advantage of this setup. METHODS: The software is based on a peptide-centric approach and uses the binomial distribution to attribute a probability for each peptide as being linked to the disease; these probabilities are propagated to a final protein p-value according to the Stouffer's Z-score method. RESULTS: A total of 1227 proteins were identified and quantified, of which 87 were differentially abundant, being mainly involved in glycolysis (Warburg effect) and immune system activation (activated stroma). Additionally, in the estrogen receptor-positive subgroup, proteins related to the regulation of insulin-like growth factor transport and platelet degranulation displayed higher abundance, confirming the presence of a proliferative microenvironment. CONCLUSIONS: We debuted a differential bioinformatics workflow for the proteomic analysis of NAF, validating this secretome as a treasure-trove for studying a paired-organ cancer type.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Líquido Aspirado del Pezón/metabolismo , Proteoma/análisis , Proteómica/métodos , Microambiente Tumoral , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Pronóstico , Flujo de Trabajo
3.
Anal Chem ; 90(10): 6043-6050, 2018 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-29565564

RESUMEN

Cross-linking/Mass spectrometry (XLMS) is a consolidated technique for structural characterization of proteins and protein complexes. Despite its success, the cross-linking chemistry currently used is mostly based on N-hydroxysuccinimide (NHS) esters, which react primarily with lysine residues. One way to expand the current applicability of XLMS into several new areas is to increase the number of cross-links obtainable for a target protein. We introduce a multiplex chemistry (denoted XPlex) that targets Asp, Glu, Lys, and Ser residues. XPlex can generate significantly more cross-links with reactions occurring at lower temperatures and enables targeting proteins that are not possible with NHS ester-based cross-linkers. We demonstrate the effectiveness of our approach in model proteins as well as a target Lys-poor protein, SalBIII. Identification of XPlex spectra requires a search engine capable of simultaneously considering multiple cross-linkers on the same run; to achieve this, we updated the SIM-XL search algorithm with a search mode tailored toward XPlex. In summary, we present a complete chemistry/computational solution for significantly increasing the number of possible distance constraints by mass spectrometry experiments, and thus, we are convinced that XPlex poses as a real complementary approach for structural proteomics studies.


Asunto(s)
Ácido Aspártico/análisis , Biología Computacional , Reactivos de Enlaces Cruzados/química , Ácido Glutámico/análisis , Lisina/análisis , Serina/análisis , Algoritmos , Ésteres/química , Espectrometría de Masas , Proteínas/química , Succinimidas/química , Temperatura
4.
Toxins (Basel) ; 10(2)2018 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-29415440

RESUMEN

Snake venoms are sources of molecules with proven and potential therapeutic applications. However, most activities assayed in venoms (or their components) are of hemorrhagic, hypotensive, edematogenic, neurotoxic or myotoxic natures. Thus, other relevant activities might remain unknown. Using functional genomics coupled to the connectivity map (C-map) approach, we undertook a wide range indirect search for biological activities within the venom of the South American pit viper Bothrops jararaca. For that effect, venom was incubated with human breast adenocarcinoma cell line (MCF7) followed by RNA extraction and gene expression analysis. A list of 90 differentially expressed genes was submitted to biosimilar drug discovery based on pattern recognition. Among the 100 highest-ranked positively correlated drugs, only the antihypertensive, antimicrobial (both antibiotic and antiparasitic), and antitumor classes had been previously reported for B. jararaca venom. The majority of drug classes identified were related to (1) antimicrobial activity; (2) treatment of neuropsychiatric illnesses (Parkinson's disease, schizophrenia, depression, and epilepsy); (3) treatment of cardiovascular diseases, and (4) anti-inflammatory action. The C-map results also indicated that B. jararaca venom may have components that target G-protein-coupled receptors (muscarinic, serotonergic, histaminergic, dopaminergic, GABA, and adrenergic) and ion channels. Although validation experiments are still necessary, the C-map correlation to drugs with activities previously linked to snake venoms supports the efficacy of this strategy as a broad-spectrum approach for biological activity screening, and rekindles the snake venom-based search for new therapeutic agents.


Asunto(s)
Venenos de Crotálidos/farmacología , Descubrimiento de Drogas , Animales , Bothrops , Venenos de Crotálidos/uso terapéutico , Humanos , Células MCF-7 , Transcriptoma/efectos de los fármacos
5.
PLoS Negl Trop Dis ; 11(7): e0005829, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28759578

RESUMEN

Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures. This antitoxic protein can serve as an excellent starting template for the design of novel therapeutics against snakebite envenomation, particularly venom-induced local tissue damage. Therefore, the aim of this work was to produce a recombinant DM64 (rDM64) in the methylotrophic yeast Pichia pastoris and to compare its biological properties with those of native DM64. Yeast fermentation in the presence of Pefabloc, a serine protease inhibitor, stimulated cell growth (~1.5-fold), increased the rDM64 production yield approximately 10-fold and significantly reduced the susceptibility of rDM64 to proteolytic degradation. P. pastoris fermentation products were identified by mass spectrometry and Western blotting. The heterologous protein was efficiently purified from the culture medium by affinity chromatography (with immobilized PLA2 myotoxin) and/or an ion exchange column. Although both native and recombinant DM64 exhibit different glycosylation patterns, they show very similar electrophoretic mobilities after PNGase F treatment. rDM64 formed a noncovalent complex with myotoxin II (Lys49-PLA2) from Bothrops asper and displayed biological activity that was similar to that of native DM64, inhibiting the cytotoxicity of myotoxin II by 92% at a 1:1 molar ratio.


Asunto(s)
Proteínas Sanguíneas/química , Inhibidores de Fosfolipasa A2/química , Fosfolipasas A2/química , Proteínas de Reptiles/química , Venenos de Serpiente/química , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/biosíntesis , Bothrops , Brasil , Línea Celular , Espectrometría de Masas , Ratones , Zarigüeyas , Pichia , Proteínas Recombinantes/biosíntesis
6.
J Proteomics ; 117: 86-94, 2015 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-25638022

RESUMEN

UNLABELLED: NAF is a breast fluid that is closely related to the tumor microenvironment and a valuable sample for studying breast cancer. To perform an in-depth proteomic analysis of this sample, aliquots of a single NAF digest were analyzed by the following peptide-centric fractionation strategies: a) 30-cm reversed-phase (RP) column on-line with an LTQ-Orbitrap XL; b) off-line strong cation-exchange (SCX) column; and c) pI-based OFFGEL fractionation. All fractions from approaches (b) and (c) were further analyzed on a 10-cm RP column hyphenated to the same mass spectrometer. The RP-30cm, SCX/RP-10cm, and OFFGEL/RP-10cm approaches identified 1676, 2930, and 3240 peptides, which corresponded to 193, 390 and 528 proteins, respectively. In our cumulative dataset, 4466 distinct NAF peptides corresponded to a total of 557 proteins, of which only 34% were identified by all three approaches. No exclusive protein identification was associated to the RP-30cm approach, while SCX/RP-10cm and OFFGEL/RP-10cm contributed to 28 and 166 exclusive identifications, respectively. Each approach provided additional information related to energy metabolism (fermentation process/carbohydrate biosynthesis). In conclusion, the pre-fractionation platforms used were complementary for the comprehensive characterization of NAF and our work provides methodological information for future quantitative cancer-related NAF sample studies. BIOLOGICAL SIGNIFICANCE: High-resolution peptide separation is a sine qua non condition for achieving extensive proteome coverage. Various techniques have been employed to improve peptide fractionation prior to LC-MS/MS, thus allowing a comprehensive characterization of complex biological samples. Although fractionation efficiency is very sample-dependent, this issue is commonly overlooked, and a "cookbook" approach is routinely used during this critical step. The present study provides a systematic comparison of analytical information needed for the successful large-scale differential proteomic analysis of NAF samples from breast cancer patients, a precious and volume-limited biological sample. It reinforces the importance of optimizing sample-specific fractionation protocols for information retrieval from mass spectrometric analysis.


Asunto(s)
Fibroadenoma/metabolismo , Proteínas de Neoplasias/metabolismo , Líquido Aspirado del Pezón/metabolismo , Péptidos/metabolismo , Proteómica , Microambiente Tumoral , Adulto , Femenino , Humanos , Espectrometría de Masas
7.
Curr Top Med Chem ; 14(3): 359-68, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24304313

RESUMEN

Breast cancer is the leading cause of cancer related deaths in women. Most breast cancers stem from mammary ductal cells that secrete nipple aspirate fluid (NAF), a biological sample that contains proteins associated with the tumor microenvironment. In this study, NAF samples from both breasts of 7 Brazilian patients with unilateral breast cancer were analyzed. These samples were systematically compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional fluorescence difference gel electrophoresis (2D-DIGE); substantial qualitative individual differences were observed. In general, when NAF samples were compared from both breasts within the same patient their electrophoretic patterns were very similar, regardless of their cancer status. A comparison of all patients identified 2 main NAF protein profiles. The HomEP, homogeneous expression profile, was characterized by typical SDS-PAGE and 2D-DIGE protein patterns that were observed in patients with a good breast cancer prognosis and were similar to previous Type I NAF classifications that used one-dimensional electrophoresis. The HetEP, heterogeneous expression profile, was characterized by distinct protein patterns that have not been reported in previous studies and have been primarily observed in breast cancer patients with a poor prognosis. The NAF samples were rich in metal-dependent proteolytic enzymes, as visualized by SDS-PAGE zymography. They varied qualitatively with respect to their gelatinolytic band distribution. However, there were no correlations between these characteristics and the pathologic features of these tumors. A comparative analysis of NAF samples taken from each breast in a single patient showed conserved zymographic patterns. In conclusion, the present study highlights important distinctions in the protein content of individual NAF samples and provides insight into the composition of the tumor microenvironment. These data reinforce breast cancer as a heterogeneous disease with a diverse natural history, which is becoming increasingly evident through other recent studies.


Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Electroforesis en Gel de Poliacrilamida , Proteínas de Neoplasias/biosíntesis , Líquido Aspirado del Pezón/metabolismo , Proteómica/métodos , Electroforesis en Gel Bidimensional , Femenino , Humanos , Proteínas de Neoplasias/análisis
8.
Mem Inst Oswaldo Cruz ; 107(6): 752-9, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22990964

RESUMEN

Angiostrongylus costaricensis is a nematode that causes abdominal angiostrongyliasis, a widespread human parasitism in Latin America. This study aimed to characterize the protease profiles of different developmental stages of this helminth. First-stage larvae (L1) were obtained from the faeces of infected Sigmodon hispidus rodents and third-stage larvae (L3) were collected from mollusks Biomphalaria glabrata previously infected with L1. Adult worms were recovered from rodent mesenteric arteries. Protein extraction was performed after repeated freeze-thaw cycles followed by maceration of the nematodes in 40 mM Tris base. Proteolysis of gelatin was observed by zymography and found only in the larval stages. In L3, the gelatinolytic activity was effectively inhibited by orthophenanthroline, indicating the involvement of metalloproteases. The mechanistic class of the gelatinases from L1 could not be precisely determined using traditional class-specific inhibitors. Adult worm extracts were able to hydrolyze haemoglobin in solution, although no activity was observed by zymography. This haemoglobinolytic activity was ascribed to aspartic proteases following its effective inhibition by pepstatin, which also inhibited the haemoglobinolytic activity of L1 and L3 extracts. The characterization of protease expression throughout the A. costaricensis life cycle may reveal key factors influencing the process of parasitic infection and thus foster our understanding of the disease pathogenesis.


Asunto(s)
Angiostrongylus/enzimología , Proteolisis , Angiostrongylus/clasificación , Animales , Heces/parasitología , Femenino , Larva/enzimología , Masculino , Sigmodontinae
9.
Mem. Inst. Oswaldo Cruz ; 107(6): 752-759, set. 2012. ilus, tab
Artículo en Inglés | LILACS | ID: lil-649490

RESUMEN

Angiostrongylus costaricensis is a nematode that causes abdominal angiostrongyliasis, a widespread human parasitism in Latin America. This study aimed to characterize the protease profiles of different developmental stages of this helminth. First-stage larvae (L1) were obtained from the faeces of infected Sigmodon hispidus rodents and third-stage larvae (L3) were collected from mollusks Biomphalaria glabrata previously infected with L1. Adult worms were recovered from rodent mesenteric arteries. Protein extraction was performed after repeated freeze-thaw cycles followed by maceration of the nematodes in 40 mM Tris base. Proteolysis of gelatin was observed by zymography and found only in the larval stages. In L3, the gelatinolytic activity was effectively inhibited by orthophenanthroline, indicating the involvement of metalloproteases. The mechanistic class of the gelatinases from L1 could not be precisely determined using traditional class-specific inhibitors. Adult worm extracts were able to hydrolyze haemoglobin in solution, although no activity was observed by zymography. This haemoglobinolytic activity was ascribed to aspartic proteases following its effective inhibition by pepstatin, which also inhibited the haemoglobinolytic activity of L1 and L3 extracts. The characterization of protease expression throughout the A. costaricensis life cycle may reveal key factors influencing the process of parasitic infection and thus foster our understanding of the disease pathogenesis.


Asunto(s)
Animales , Femenino , Masculino , Angiostrongylus/enzimología , Proteolisis , Angiostrongylus/clasificación , Heces/parasitología , Larva/enzimología , Sigmodontinae
10.
Proteomics ; 12(17): 2753-65, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22744933

RESUMEN

The resistance of the opossum Didelphis aurita to Bothrops snake venoms is attributed to the opossum's antihemorrhagic (DM43) and antimyotoxic (DM64) acidic serum glycoproteins. The aim of this study was to characterize the N-glycosylation sites of these antiophidic proteins and to determine whether their glycans influence the biological activity measured by in vitro assays. Our experimental pipeline included the sequential enzymatic digestion of the inhibitors with two different proteinases (trypsin and endoproteinase Asp-N) and eventually with trypsin, peptide-N-glycosidase F (PNGase F) and endoproteinase Asp-N, used in that order. All of the peptide and protein samples were analyzed by MALDI-TOF/TOF MS. The results experimentally confirmed the putative N-glycosylation sites of DM43 (Asn23, Asn156, Asn160, and Asn175) and DM64 (Asn46, Asn179, Asn183, and Asn379). Following treatments with specific glycosidases, complex-type oligosaccharides containing galactose and sialic acid could be assigned to both proteins. The removal of these monosaccharide units by exoglycosidase digestion did not measurably affect the inhibitory activity. In contrast, partially deglycosylated DM43 treated with PNGase F under nondenaturing conditions was half as effective as native DM43. In conclusion, we have demonstrated that the contribution of the carbohydrate portion of these potentially therapeutic molecules, for their mechanism of action, should not be overlooked.


Asunto(s)
Bothrops/metabolismo , Venenos de Crotálidos/antagonistas & inhibidores , Didelphis/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Polisacáridos/análisis , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Venenos de Crotálidos/metabolismo , Glicosilación , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/metabolismo , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Polisacáridos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tripsina/metabolismo
11.
J Exp Ther Oncol ; 7(4): 285-90, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-19227008

RESUMEN

Perillyl alcohol (POH) is a naturally occurring monoterpene with antiangiogenic and anti-tumoral properties. This chemotherapeutic agent has proven effectiveness in several clinical trials, including an ongoing phase I, comprising patients with recurrent glioblastoma multiform (GBM) under treatment with POH by intranasal administration. Proteomics offers tools to distinguish states of biological systems according to protein expression differences and therefore, can be used to gain pathological insights and to search for disease follow-up biomarkers. In this work, a differential gel electrophoresis (DIGE) proteomic approach was used to search for plasma proteins that correlated with the disease progression in 10 of these patients. Our results pointed antithrombin (down) and fibrinogen (up) regulated after a four months treatment deserving to be further verified as prognostic markers for this treatment. Possible links between tumor progression and anti-thrombin expression level are also discussed.


Asunto(s)
Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Neoplasias del Sistema Nervioso Central/sangre , Fibrina/biosíntesis , Regulación Neoplásica de la Expresión Génica , Glioblastoma/sangre , Monoterpenos/farmacología , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Progresión de la Enfermedad , Electroforesis en Gel de Poliacrilamida , Fibrinógeno/biosíntesis , Glioblastoma/tratamiento farmacológico , Humanos , Espectrometría de Masas , Proteómica/métodos , Recurrencia
12.
Proteomics ; 6(5): 1495-511, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16447160

RESUMEN

A proteomic analysis of a wild-type and of a phoB mutant showed that Vibrio cholerae expresses genes of two major regulons in response to phosphate starvation. The Pho regulon, expressed by the wild-type, allowed the cells to adapt to the new environment. Induction of the general stress regulon was mainly observed in the phoB mutant as a strategy to resist stress and survive. Some functions of the adaptative and survival responses play roles in the pathogenicity of the bacteria. Among the members of the Pho regulon, we found a porin described as an important factor for the intestinal colonisation. Other functions not obviously related to phosphate metabolism, expressed preferentially by the wild-type cells, have also been implicated in virulence. These findings might explain the lack of virulence of the phoB mutant. The Pho regulon picture of V. cholerae, however, will not be complete until minor members and membrane proteins are identified. Among the phosphate-starvation induced genes we have found 13 hypothetical ones and for some of them functions have been assigned. The majority of the genes identified here have not been described before, thus they could be used to expand the proteomic reference map of V. cholerae El Tor.


Asunto(s)
Adaptación Fisiológica , Proteínas Bacterianas/metabolismo , Fosfatos/metabolismo , Proteoma/análisis , Regulón , Vibrio cholerae O1/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Electroforesis en Gel Bidimensional , Datos de Secuencia Molecular , Operón , Vibrio cholerae O1/genética , Vibrio cholerae O1/patogenicidad
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