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1.
J Proteomics ; 72(2): 256-69, 2009 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-19457351

RESUMEN

The venom proteome of Daboia russelli siamensis, a snake of medical importance in several Asian countries, was analysed by 2-D electrophoresis, subsequent MS/MS and enzymatic assays. The proteome comprises toxins from six protein families: serine proteinases, metalloproteinases, phospholipases A(2), L-amino acid oxidases, vascular endothelial growth factors and C-type lectin-like proteins. The venom toxin composition correlates with the clinical manifestation of the Russell's viper bite and explains pathological effects of the venom such as coagulopathy, oedema, hypotensive, necrotic and tissue damaging effects. The vast majority of toxins are potentially involved in coagulopathy and neurotoxic effects. The predominant venom components are proteinases capable of activating blood coagulation factors and promoting a rapid clotting of the blood, and neurotoxic phospholipase A(2)s. The analysis of the venom protein composition provides a catalogue of secreted toxins. The proteome of D. r. siamensis exhibits a lower level of toxin diversity than the proteomes of other viperid snakes. In comparison to the venoms of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis, the venom from D. r. siamensis showed quantitative differences in the proteolytic, phospholipase A(2), L-amino acid oxidase and alkaline phosphatase activities.


Asunto(s)
Proteómica/métodos , Venenos de Víboras/análisis , Secuencia de Aminoácidos , Animales , Electroforesis en Gel Bidimensional , Femenino , Lectinas/química , Masculino , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Péptidos/química , Proteínas/análisis , Daboia , Mordeduras de Serpientes/terapia , Espectrometría de Masa por Ionización de Electrospray , Venenos de Víboras/química
2.
Invest Ophthalmol Vis Sci ; 50(3): 1112-21, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18997084

RESUMEN

PURPOSE: To evaluate the presence and role of human cationic amino acid transporters (hCATs) at the ocular surface in healthy and pathologic states and under experimental inflammatory conditions. METHODS: Expression of mRNA for hCATs 1, 2, and 3 (SLC7A1, SLC7A2, and SLC7A3) was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts and in an SV40 immortalized human corneal epithelial (HCE) cell line. Localization of hCAT1 and hCAT2 was determined by immunohistochemistry in healthy tissues and in sections of different corneal abnormalities, including keratoconus, Fuchs dystrophy, and herpetic keratitis. Cultured corneal epithelial cells were stimulated with proinflammatory cytokines and supernatants of Staphylococcus aureus and Pseudomonas aeruginosa and were analyzed by real-time PCR. RESULTS: Expression of hCAT1 and hCAT2 mRNA, but not of hCAT3 mRNA, was detected in healthy conjunctiva, cornea, and nasolacrimal ducts. Human lacrimal gland revealed only hCAT2 mRNA expression. Immunohistochemistry demonstrated the presence of hCAT1 and hCAT2 in epithelial cells of cornea, conjunctiva, and nasolacrimal ducts. Goblet cells revealed no reactivity. Moreover, hCAT2 was visible in acinar cells of lacrimal gland. No changes in staining reactivity were obtained for hCAT1 in different corneal abnormalities. In contrast, hCAT2 showed increased subjective staining intensity in all corneal abnormalities. Cell culture experiments revealed that TNF-alpha and supernatant of S. aureus increased hCAT1 and hCAT2 expression significantly. Supernatant of P. aeruginosa led to an increase in hCAT2-expression only. CONCLUSIONS: These results show for the first time the distribution of hCATs in tissues of the ocular surface and lacrimal apparatus. Both transporters seem to be differently regulated under pathologic conditions of the ocular surface. Their physiological functions in amino acid transport make them potential candidates for therapeutic intervention.


Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos/genética , Transportador de Aminoácidos Catiónicos 1/genética , Córnea/metabolismo , Enfermedades de la Córnea/genética , Regulación de la Expresión Génica/fisiología , Anciano , Anciano de 80 o más Años , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Western Blotting , Transportador de Aminoácidos Catiónicos 1/metabolismo , Línea Celular , Conjuntiva/metabolismo , Enfermedades de la Córnea/metabolismo , Citocinas/farmacología , Epitelio Corneal/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Técnicas para Inmunoenzimas , Aparato Lagrimal/metabolismo , Masculino , Persona de Mediana Edad , Pseudomonas aeruginosa/fisiología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Staphylococcus aureus/fisiología
3.
J Proteome Res ; 7(3): 866-86, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18257516

RESUMEN

The venom proteomics of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis, snakes of public health significance and the most poisonous reptiles in Europe, were analyzed by FPLC, 2-D electrophoresis, sequence analysis, and MS/MS. FPLC analysis showed the presence of l-amino acid oxidase, monomeric and heterodimeric phospholipases A2, C-type lectin protein, and proteinases in the venom of V. a. ammodytes. Representatives of the same protein families were found in the venom of the other subspecies, V. a. meridionalis. N-terminally identical PLA2 neurotoxins were identified in both venoms. Difference in the PLA2 compositions of the venoms was also observed: a monomeric protein with phospholipase A2 activity, identical in the first 20 amino acid residues to the catalitically inactive acidic component of the heterodimeric PLA2 present in both venoms, was found only in that of V. a. meridionalis. Probably, this protein represents an intermediate form of the two components of the heterodimer. 2-D electrophoresis and MS/MS analysis showed that the two venoms shared a number of protein families: monomeric and heterodimeric Group II PLA2s, serine proteinases, Group I, II, and III metalloproteinases, l-amino acid oxidases (LAAOs), cysteine-rich secretory proteins, disintegrins, and growth factors. Totally, 38 venom components of the V. a. ammodytes, belonging to 9 protein families, and 67 components of the V. a. meridionalis venom belonging to 8 protein families were identified. The venom proteome of V. a. ammodytes shows larger diversity of proteins (139) in comparison to that of V. a. meridionalis (104 proteins). Most of the proteins are homologues of known representatives of the respective protein families. The protein compositions explain clinical effects of the V. ammodytes snakebites, such as difficulties in the breathing, paralysis, apoptosis, cloting disorders, hemorrhage, and tissue necrosis. The lists of secreted proteins by the two vipers can be used for further study of structure-function relationships in the toxins and for prediction and treatment of snakebite consequences.


Asunto(s)
Proteoma , Venenos de Víboras/química , Secuencia de Aminoácidos , Animales , Electroforesis en Gel Bidimensional , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Ionización de Electrospray , Viperidae
4.
Reproduction ; 133(5): 887-97, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17616719

RESUMEN

The arylhydrocarbon receptor (AhR) mediates the adverse effects of dioxin-like compounds. However, it has also been reported that the AhR may exert a role in ovarian physiology. In the present study, porcine cumulus-oocyte complexes (COCs) were matured in vitro in the presence of 10% follicular fluid. Expression of AhR and its partner, AhR nuclear translocator occurs in immature COCs. After in vitro maturation (IVM), an up-regulation of AhR and cytochrome P450 1A1 (CYP1A1; the main AhR-target gene) was observed. To explore the role of the AhR during IVM, we exposed the COCs to 50 microM beta-napthoflavone (betaNF). The treatment induced a marked up-regulation of CYP1A1 mRNA, indicating both constitutive and inducible AhR activity. However, in contrast to what was observed in other cell types, no sign of toxicity was observed in COCs. To investigate if components of porcine follicular fluid may exert a protective role against AhR ligands, we exposed porcine COCs to betaNF, in the absence of follicular fluid. In these conditions, a marked decrease in the percentage of matured oocytes, concomitant with an increase in oocyte degeneration, was observed. Furthermore, betaNF increased apoptosis in cumulus cells in the absence of follicular fluid, whereas betaNF has no effects when COCs were treated in the presence of porcine follicular fluid (pFF). In conclusion, these results suggest the presence of unknown endogenous AhR-ligand(s) during porcine IVM and that a dysregulation of this mechanism may result in ovotoxicity by inducing apoptosis in cumulus cells. However, this phenomenon is interrupted by the presence of follicular fluid, indicating a putative protective role for follicular fluid components against exogenous insults.


Asunto(s)
Líquido Folicular/metabolismo , Oocitos/metabolismo , Folículo Ovárico/fisiología , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Apoptosis/efectos de los fármacos , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Western Blotting/métodos , Citocromo P-450 CYP1A1/metabolismo , Femenino , Fertilización In Vitro , Etiquetado Corte-Fin in Situ , Meiosis/efectos de los fármacos , Oogénesis/efectos de los fármacos , ARN Mensajero/análisis , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , beta-naftoflavona/farmacología
5.
Reproduction ; 130(6): 857-68, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16322545

RESUMEN

Aroclor-1254 (A-1254) is a commercial mixture of coplanar (dioxin-like) and non-coplanar (non dioxin-like) polychlorinated biphenyls (PCBs) affecting bovine oocyte in vitro maturation (IVM) and developmental competence. In the present study, the role of cumulus cell apoptosis in mediating the toxic effects of PCBs during in vitro maturation has been investigated. Results indicate that exposure of cumulus-oocyte complexes (COCs) to A-1254 significantly induced apoptosis of cumulus cells. Furthermore, A-1254 significantly increased the expression of the pro-apoptotic gene, Bax, concomitantly reducing the level of the anti-apoptotic gene, Bcl-2, in the cumulus cell compartment. The effects of pure mixtures of coplanar (PCB 77, 126 and 169) or non-coplanar (PCB 52, 101 and 153) PCBs were examined. Exposure of COCs to coplanar PCBs affected maturation at doses as low as 100.6 pg/ml. Furthermore, a significant increase in apoptosis and in Bax mRNA expression was observed. No variations in maturation or apoptosis were observed in the non-coplanar PCB group. To further analyze the role of cumulus cells, COCs and denuded oocytes (DOs) have been exposed to A-1254 or coplanar PCBs during IVM. Exposure of COCs significantly reduced the percentage of matured oocytes after 24 h of culture in both treatments. In contrast, exposure of DOs significantly decreased the maturation rate only at the highest dose investigated (100-fold greater than that affecting COCs). Taken together, the results indicate a direct role of cumulus cell apoptosis in mediating PCB toxicity on bovine oocytes, and a direct relationship between congener planarity and toxicity in bovine oocytes is suggested.


Asunto(s)
/toxicidad , Oocitos/citología , Oogénesis/efectos de los fármacos , Teratógenos/toxicidad , Animales , Apoptosis , Bovinos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Femenino , Expresión Génica/efectos de los fármacos , Genes bcl-2 , Etiquetado Corte-Fin in Situ , Oocitos/efectos de los fármacos , Bifenilos Policlorados/farmacología , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , Teratógenos/química , Proteína X Asociada a bcl-2/genética
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