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2.
Scand J Med Sci Sports ; 22(2): 207-16, 2012 Apr.
Article in English | MEDLINE | ID: mdl-20973830

ABSTRACT

Exercise can alter gene transcriptional and protein translational rates leading to changes in protein abundance toward adaptation to exercise. We investigated the alterations in protein abundance in skeletal muscle after one bout of an exhaustive exercise through proteomic analysis. Gastrocnemius muscles were sampled from non-exercised control rats and from rats exercised on a treadmill with incremental increases in speed until exhaustion (approximately 30 min). Rats were sacrificed 3 and 24 h after exercise cessation. Two-dimensional gel electrophoresis was performed and spots with a significant alteration in relative volume were identified by mass spectrometry. Six spots presented statistically significant altered abundances after exercise. The spots identified as the metabolic related proteins triosephosphate isomerase 1, glyceraldehyde-3-phosphate dehydrogenase, the ß subunit of pyruvate dehydrogenase E(1) and carnitine palmitoyltransferase 2 were all more abundant after exercise. One spot identified as heat shock cognate 70 was also more abundant after exercise. One spot demonstrated a decreased abundance after exercise and was identified as α-actin. These results suggest that a single session of exhaustive incremental exercise in untrained muscle can alter thin filaments synthesis/degradation rate and enhance cytosolic and mitochondrial proteins synthesis. The identified proteins may be important to a general preconditioning of skeletal muscle for subsequent exercise sessions.


Subject(s)
Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Physical Conditioning, Animal/physiology , Proteome , Animals , Electrophoresis, Gel, Two-Dimensional , Male , Mass Spectrometry , Proteomics , Rats , Rats, Wistar
3.
Journal of Proteomics ; 72(2): 241-255, Jan 11,2009.
Article in English | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP, SESSP-IBACERVO | ID: biblio-1064340

ABSTRACT

A joint transcriptomic and proteomic approach employing two-dimensional electrophoresis, liquid chromatography and mass spectrometry was carried out to identify peptides and proteins expressed by the venom gland of the snake Bothrops insularis, an endemic species of Queimada Grande Island, Brazil. Four protein families were mainly represented in processed spots, namely metalloproteinase, serine proteinase, phospholipase A2 and lectin. Other represented families were growth factors, the developmental protein G10, a disintegrin and putative novel bradykinin-potentiating peptides. The enzymes were present in several isoforms. Most of the experimental data agreed with predicted values for isoelectric point and Mr of proteins found in the transcriptome of the venom gland. The results also support the existence of posttranslational modifications and of proteolytic processing of precursor molecules which could lead to diverse multifunctional proteins. This study provides a preliminary reference map for proteins and peptides present in Bothrops insularis whole venom establishing the basis for comparative studies of other venom proteomes which could help the search for new drugs and the improvement of venom therapeutics. Altogether, our data point to the influence of transcriptional and post-translational events on the final venom composition and stress the need for a multivariate approach to snake venomics studies.


Subject(s)
Animals , Proteome/analysis , Snake Venoms , Protein Biosynthesis , Bothrops , Poisons/analysis
4.
Toxicon ; 51(1): 54-65, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17889921

ABSTRACT

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding.


Subject(s)
Bothrops/metabolism , Crotalid Venoms/chemistry , Metalloendopeptidases/pharmacology , Metalloproteases/pharmacology , Amino Acid Sequence , Animals , Benchmarking , Blood Coagulation Factors , Cells, Cultured , Crotalid Venoms/pharmacology , Endothelial Cells/drug effects , Hemorrhage/chemically induced , Humans , Metalloendopeptidases/chemistry , Metalloproteases/chemistry , Mice , Molecular Sequence Data , Bothrops jararaca Venom
5.
Br J Sports Med ; 42(5): 386-8, 2008 May.
Article in English | MEDLINE | ID: mdl-17717063

ABSTRACT

OBJECTIVE: We have shown previously that exercise training enhances endothelium-dependent and endothelium-independent vascular relaxation in rabbit kidney. This study aimed to investigate protein expression changes in the rabbit renal cortex induced by chronic dynamic exercise. DESIGN: Kidneys were obtained from New Zealand rabbits either confined to pens (n = 8) or trained on a treadmill (0% grade) for 5 days/week at a speed of 18 m/min for 60-min periods over 12 weeks (n = 8). Expression of proteins in the renal cortex was determined by colloidal Coomassie blue staining after two-dimensional polyacrylamide gel electrophoresis. Differential protein spots were excised and digested with trypsin, and peptides were sequenced by electrospray ionization-ion trap mass spectrometry. RESULTS: Two pairs of matching differentially stained spots displayed an approximate threefold increase in trained compared with sedentary animals. These four spots presented a molecular mass of 23 kDa but different pI values. Mass spectrometric analyses revealed the pairs of matching spots as being rabbit apolipoprotein A-I. CONCLUSION: Chronic dynamic exercise increases apolipoprotein A-I expression in the rabbit renal cortex. This fact could be involved in the alterations observed in the renal circulation after exercise training.


Subject(s)
Apolipoprotein A-I/metabolism , Kidney Cortex/physiology , Physical Conditioning, Animal/physiology , Animals , Electrophoresis, Gel, Two-Dimensional , Proteomics , Rabbits , Random Allocation
6.
Biochimie ; 89(11): 1332-42, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17544198

ABSTRACT

Pacu (Piaractus mesopotamicus Holmberg, 1887, Characiformes) dwells in waters of Pantanal, in which it has adapted for alternate concentrations of dissolved oxygen. Intracellular antioxidant protection should be vital for such an adaptation. Accordingly, we found that cytosol from liver of pacu has the highest antioxidant glutathione peroxidase activity so far reported for fish and murine species. To clarify whether this activity was due to a selenium independent glutathione S-transferase or to a glutathione peroxidase, we purified it and studied its kinetics. The substrates cumene hydroperoxide and hydrogen peroxide were promptly reduced by the enzyme, but peroxidized phosphatidylcholine had to undergo previous fatty acid removal with phospholipase A(2). Augmenting concentrations (from 2 to 6 mM) of reduced glutathione activated the pure enzyme. Curves of velocity versus different micromolar concentrations of hydrogen peroxide in the presence of 2, 4 or 8 mM reduced glutathione indicated that at least 2.5 mM reduced glutathione should be available in vivo for an efficient continuous destruction of micromolar concentrations of hydrogen peroxide by this peroxidase. Molecular exclusion HPLC and SDS-polyacrylamide gel electrophoresis indicated that the purified peroxidase is a homotetramer. Data from internal sequences showed selenocysteine in its primary structure and that the enzyme was a homologue of the type-1 glutathione peroxidase found in rat, bull, trout, flounder and zebra fish. Altogether, our data establish that in liver cells of pacu, a hypoxia-tolerant fish from South America, there are high levels of a cytosolic GPX-1 capable of quenching hydrogen peroxide and fatty acid peroxides, providing an effective antioxidant action.


Subject(s)
Cytosol/enzymology , Fishes/metabolism , Glutathione Peroxidase/isolation & purification , Glutathione Peroxidase/metabolism , Hypoxia/metabolism , Liver/cytology , Liver/enzymology , Amino Acid Sequence , Ammonium Sulfate , Animals , Antioxidants/metabolism , Benzene Derivatives/metabolism , Chemical Fractionation , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Glutathione/metabolism , Glutathione Peroxidase/chemistry , Hydrogen Peroxide/metabolism , Isoelectric Focusing , Molecular Sequence Data , Oxidation-Reduction , Phosphatidylcholines/metabolism , South America , Wetlands , Glutathione Peroxidase GPX1
7.
Biochimie ; 87(8): 687-99, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16054523

ABSTRACT

A novel family of proteins with kininogenase activity and unique primary structure was characterized using combined pharmacological, proteomic and transcriptomic approaches of Thalassophryne nattereri fish venom. The major venom components were isolated and submitted to bioassays corresponding to its main effects: nociception and edema. These activities were mostly located in one fraction (MS3), which was further fractionated. The isolated protein, named natterin, was able to induce edema, nociception and cleave human kininogen and kininogen-derived synthetic peptides, releasing kallidin (Lys-bradykinin). The enzymatic digestion was inhibited by kallikrein inhibitors as Trasylol and TKI. Natterin N-terminal peptide showed no similarity with already known proteins present in databanks. Primary structure of natterin was obtained by a transcriptomic approach using a representative cDNA library constructed from T. nattereri venom glands. Several expressed sequence tags (ESTs) were obtained and processed by bioinformatics revealing a major group (18%) of related sequences unknown to gene or protein sequence databases. This group included sequences showing the N-terminus of isolated natterin and was named Natterin family. Analysis of this family allowed us to identify five related sequences, which we called natterin 1-4 and P. Natterin 1 and 2 sequences include the N-terminus of the isolated natterin. Furthermore, internal peptides of natterin 1-3 were found in major spots of whole venom submitted to mass spectrometry/2DGE. Similarly to the ESTs, the complete sequences of natterins did not show any significant similarity with already described tissue kallikreins, kininogenases or any proteinase, all being entirely new. These data present a new task for the knowledge of the action of kininogenases and may help in understanding the mechanisms of T. nattereri fish envenoming, which is an important medical problem in North and Northeast of Brazil.


Subject(s)
Batrachoidiformes/metabolism , Fish Venoms/isolation & purification , Kallikreins/isolation & purification , Amino Acid Sequence , Animals , Base Sequence , Chromatography, Gel , Electrophoresis, Gel, Two-Dimensional , Fish Venoms/chemistry , Fishes, Poisonous , Gene Library , Kallikreins/chemistry , Molecular Sequence Data , Sequence Alignment
8.
Biochimie ; 87(8): 687-699, aug.2005.
Article in English | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP, SESSP-IBACERVO | ID: biblio-1060829

ABSTRACT

A novel family of proteins with kininogenase activity and unique primary structure was characterized using combined pharmacological, proteomic and transcriptomic approaches of Thalassophryne nattereri fish venom. The major venom components were isolated and submitted to bioassays corresponding to its main effects: nociception and edema. These activities were mostly located in one fraction (MS3), which was further fractionated. The isolated protein, named natterin, was able to induce edema, nociception and cleave human kininogen and kininogen-derived synthetic peptides, releasing kallidin (Lys-bradykinin). The enzymatic digestion was inhibited by kallikrein inhibitors as Trasylol and TKI. Natterin N-terminal peptide showed no similarity with already known proteins present in databanks. Primary structure of natterin was obtained by a transcriptomic approach using a representative cDNA library constructed from T. nattereri venom glands. Several expressed sequence tags (ESTs) were obtained and processed by bioinformatics revealing a major group (18%) of related sequences unknown to gene or protein sequence databases. This group included sequences showing the N-terminus of isolated natterin and was named Natterin family. Analysis of this family allowed us to identify five related sequences, which we called natterin 1-4 and P. Natterin 1 and 2 sequences include the N-terminus of the isolated natterin. Furthermore, internal peptides of natterin 1-3 were found in major spots of whole venom submitted to mass spectrometry/2DGE. Similarly to the ESTs, the complete sequences of natterins did not show any significant similarity with already described tissue kallikreins, kininogenases or any proteinase, all being entirely new. These data present a new task for the knowledge of the action of kininogenases and may help in understanding the mechanisms of T. nattereri fish envenoming, which is an important medical problem in North and Northeast of Brazil.


Subject(s)
Animals , Batrachoidiformes/metabolism , Kallikreins/isolation & purification , Kallikreins/chemistry , Fishes, Poisonous/classification , Fish Venoms/isolation & purification , Fish Venoms/chemistry , Gene Library , Brazil , Chromatography, Gel , Molecular Sequence Data , Electrophoresis, Gel, Two-Dimensional , Proteins , Amino Acid Sequence
9.
Eur J Biochem ; 268(10): 3042-52, 2001 May.
Article in English | MEDLINE | ID: mdl-11358523

ABSTRACT

Fractionation of the serum of the venomous snake Bothrops jararaca with (NH4)2SO4, followed by phenyl-Sepharose and C4-reversed phase chromatographies, resulted in the isolation of the anti-hemorrhagic factor BJ46a. BJ46a is a potent inhibitor of the SVMPs atrolysin C (class P-I) and jararhagin (P-III) proteolytic activities and B. jararaca venom hemorrhagic activity. The single-chain, acidic (pI 4.55) glycoprotein has a molecular mass of 46 101 atomic mass units determined by MALDI-TOF MS and 79 kDa by gel filtration and dynamic laser light scattering, suggesting a homodimeric structure. mRNA was isolated from the liver of one specimen and transcribed into cDNA. The cDNA pool was amplified by PCR, cloned into a specific vector and used to transform competent cells. Clones containing the complete coding sequence for BJ46a were isolated. The deduced protein sequence was in complete agreement with peptide sequences obtained by Edman degradation. BJ46a is a 322-amino-acid protein containing four putative N-glycosylation sites. It is homologous to the proteinase inhibitor HSF (member of the fetuin family, cystatin superfamily) isolated from the serum of the snake Trimeresurus flavoviridis, having 85% sequence identity. This is the first report of a complete cDNA sequence for an endogenous inhibitor of snake venom metalloproteinases (SVMPs). The sequence reveals that the only proteolytic processing required to obtain the mature protein is the cleavage of the signal peptide. Gel filtration analyses of the inhibitory complexes indicate that inhibition occurs by formation of a noncovalent complex between BJ46a and the proteinases at their metalloproteinase domains. Furthermore, the data shows that the stoichiometry involved in this interaction is of one inhibitor monomer to two enzyme molecules, suggesting an interesting mechanism of metalloproteinase inhibition.


Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Metalloendopeptidases/antagonists & inhibitors , Snake Venoms/enzymology , Viper Venoms/chemistry , Viper Venoms/pharmacology , Amino Acid Sequence , Animals , Base Sequence , Bothrops/blood , Crotalid Venoms/pharmacology , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Endopeptidases/chemistry , Indicators and Reagents/pharmacology , Iodoacetamide/analogs & derivatives , Iodoacetamide/pharmacology , Isoelectric Focusing , Light , Liver/metabolism , Metalloendopeptidases/chemistry , Metalloendopeptidases/pharmacology , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , RNA, Messenger/metabolism , Scattering, Radiation , Sequence Analysis, DNA , Sequence Analysis, Protein , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Time Factors , Bothrops jararaca Venom
10.
Toxicon ; 37(10): 1417-29, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10414866

ABSTRACT

The antibothropic factor (ABF) from D. marsupialis was collected from perforated hollow plastic golf balls which were surgically implanted subcutaneously in anesthetized opossums, a technique originally described for the production of polyclonal antibodies. Two months after the implantation of the balls, approximately 15 ml of seromatous fluid from D. marsupialis (SFDm-50 mg total protein/ml) could be recovered monthly. Opossum serum as well as SFDm showed similar SDS-PAGE profiles and antihemorrhagic potencies against Bothrops jararaca snake venom (Bjv). The presence of ABF in SFDm was confirmed by immunoblotting, using rabbit polyclonal antibodies raised against ABF isolated from opossum serum. ABF isolated from SFDm or from serum by ion-exchange chromatography showed identical chromatographic and electrophoretic profiles. ABF fromboth sources displayed very similar antihemorrhagic and anticaseinolytic activities against Bjv. In the case of B. jararaca, polyethylene perforated tubes were inserted in the abdominal cavity and two months after implantation, approximately 4 ml of seromatous fluid from B. jararaca (SFBj-23 mg total protein/ml) were recovered. B.jararaca serum and SFBj showed the same native and SDS-PAGE band pattern. Both serum and SFBj inhibited Bjv hemorrhagic activity. We conclude that this new methodology is very suitable for continuously obtaining opossum ABF and SFBj, in large scale and in an easier way, avoiding animal suffering and eventual sacrifice.


Subject(s)
Antivenins/isolation & purification , Bothrops , Crotalid Venoms/antagonists & inhibitors , Opossums , Animals , Antibody Formation , Blotting, Western , Chromatography, Ion Exchange , Crotalid Venoms/immunology , Electrophoresis, Polyacrylamide Gel , Hemorrhage/prevention & control , Methods , Rabbits
11.
Toxicon ; 36(6): 901-13, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9663696

ABSTRACT

The non-covalent interaction between two molecular entities namely, phospholipase A2 and crotapotin, results in the main toxin, crotoxin, present in the venom of the South American rattlesnake Crotalus durissus terrificus. High performance liquid chromatography has enabled us the isolation of three phospholipase A2 isoforms (F1, F2 and F3), characterized through denaturing and non-denaturing polyacrylamide gel electrophoresis and also through the N-terminal amino acid sequence analysis. The effect of each purified phospholipase A2 isoform on isolated rat liver mitochondria was determined through mitochondrial swelling and O2 consumption during respiratory state 4. F1 showed a dose-dependent stimulation of O2 consumption while F2 and F3 caused stimulation only at low doses and inhibition at high amounts. These effects were completely suppressed by the presence of 0.1% bovine serum albumin or 0.5 mM EGTA in the incubation medium. Taking the mitochondrial swelling as an activity parameter, all of them presented the same behaviour at different intensities, leading to permeabilization of the mitochondrial membrane. In this case, addition of EGTA prevented it whereas bovine serum albumin was ineffective, indicating that the lipid microenvironment was affected. These results suggest that free fatty acids are directly responsible for the observed effects induced by phospholipase A2 isoforms on oxygen consumption experiments. The protection conferred by cyclosporin-A on swelling induced by the isoforms, when present in low concentrations, may suggest that cyclosporin-A binds to a mitochondrial membrane site protecting the membrane against the phospholipase A2 attack.


Subject(s)
Crotalid Venoms/enzymology , Crotalus , Mitochondria, Liver/drug effects , Phospholipases A/toxicity , Amino Acid Sequence , Animals , Egtazic Acid/pharmacology , Female , Isoenzymes/isolation & purification , Mitochondria, Liver/metabolism , Mitochondria, Liver/pathology , Mitochondrial Swelling , Molecular Sequence Data , Oxygen Consumption/drug effects , Phospholipases A/isolation & purification , Phospholipases A2 , Rats , Rats, Wistar , Sequence Homology, Amino Acid , Serum Albumin, Bovine/pharmacology
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