Snake venomics of the Siamese Russell's viper (Daboia russelli siamensis) -- relation to pharmacological activities.

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.

Detection and regulation of cationic amino acid transporters in healthy and diseased ocular surface.

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.

Comparative analysis of the venom proteomes of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis.

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.

Regulation of aryl hydrocarbon receptor activity in porcine cumulus-oocyte complexes in physiological and toxicological conditions: the role of follicular fluid.

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.

Apoptosis in bovine cumulus-oocyte complexes after exposure to polychlorinated biphenyl mixtures during in vitro maturation.

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.