Cytotoxicity and morphological analysis of cell death induced by Bothrops venoms from the northeast of Argentina

Bothrops snake venoms have been proved toxic to a variety of cell types, in both in vivo and in vitro models. Studies on the pharmacological actions of Bothrops venoms from Argentina are relatively secarce and the direct action of the crude venoms has not been assessed using cell culture models. In this work, we investigated the cytotoxicity of crude venoms from B. alternatus and B. diporus in a skeletal muscle (C2C12) cell line, which is commonly used as a model for studying the myotoxic action of snake venom. Both venoms (1.25-50 miug/mL) induced an early and significant decrease in cell viability. The cytotoxic concentration 50 (CC50), determined three hours after exposure, revealed that B. diporus venom was significantly more cytotoxic (CC50: 2 miug/mL) than B. aftematus (CC50: 5.8 miug/mL). To investigate the cell death mechanism involved, myoblast cells were examined by phase contrast microscopy and after acridine orange and ethidium bromide fluorescence staining, respectively. Our data clearly demonstrated that an apoptotic mediated this cell line destruction. The current study aimed to provide new information on the citotoxicity meohanisms of Argentine Bothrops snake venoms on a skeletal muscle cell line

Lysyl oxidase: an oxidative enzyme and effector of cell function.

Lysyl oxidase (LOX) oxidizes the side chain of peptidyl lysine converting specific lysine residues to residues of alpha-aminoadipic-delta-semialdehyde. This posttranslational chemical change permits the covalent crosslinking of the component chains of collagen and those of elastin, thus stabilizing the fibrous deposits of these proteins in the extracellular matrix. Four LOX-like (LOXL) proteins with varying degrees of similarity to LOX have been described, constituting a family of related proteins. LOX is synthesized as a preproprotein which emerges from the cell as proLOX and then is processed to the active enzyme by proteolysis. In addition to elastin and collagen, LOX can oxidize lysine within a variety of cationic proteins, suggesting that its functions extend beyond its role in the stabilization of the extracellular matrix. Indeed, recent findings reveal that LOX and LOXL proteins markedly influence cell behavior including chemotactic responses, proliferation, and shifts between the normal and malignant phenotypes.

Cloning and expression of a yeast gene encoding a protein with ATPase activity and high identity to the subunit 4 of the human 26 S protease.

The cloning, expression, and biochemical characterization of an essential gene of Saccharomyces cerevisiae that encodes for a new member of the TBP1-like subfamily of putative ATPases are described. The protein is 72% identical at the amino acid level to subunit four (S4) of the human 26 S protease and 73% identical to Schizosaccharomyces pombe MTS2 gene product. The purified, recombinant protein, designated Yhs4p, has an estimated molecular mass of 49 kDa and exhibits a Mg(2+)-dependent ATPase activity with nucleotide specificity and Km for ATP similar to those exhibited by the human 26 S protease. The observed ATPase activity was reduced by 73% upon the introduction of point mutation K229Q in the "P-loop" domain of the ATP-binding site relative to the nonmutated form of the protein. This is the first direct biochemical evidence supporting the putative ATPase activity of a member of the TBP1-like subfamily. Furthermore, the experimental results demonstrate a regulatory function for the amino-terminal region of the molecule. The amino-terminal truncated form of Yhs4p lacking two clusters of positively charged amino acids exhibits a greater ATPase activity. The ATPase activity of both the truncated and complete forms of Yhs4p is stimulated by polyanions. Polylysine partially inhibits the ATPase activity of the amino-terminal truncated form having no observable effect on the complete protein. N-Ethylmaleimide inhibits the ATPase activity of both forms of Yhs4p. We propose that Yhs4p ATPase may play an essential role in the regulatory function of the proteolytic activity of the yeast 26 S protease.

Stromal serine protein kinase activity in spinach chloroplasts.

At least twelve 32P-labeled stromal proteins were detected by electrophoresis under denaturing conditions when intact chloroplasts were incubated with 32Pi, in the light but only three were detected in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or in the dark. Incubation of isolated stroma with [gamma-32P]ATP resulted in the preferential phosphorylation of one of them, a 70-kDa polypeptide, in serine residues. Thylakoid membranes in the dark promoted the phosphorylation of two additional stromal polypeptides of 55 and 40 kDa. Illumination during the phosphorylation of stroma in the presence of thylakoids stimulated severalfold the labeling of the 40-kDa polypeptide but not when DCMU was added. The protein kinase activity present in isolated stroma phosphorylated exogenous substrates like histone III, phosvitin, histone II, and casein with specific activities of 3, 1.8, 0.7, and 0.2 pmol X mg-1 X min-1. Histone III polypeptides were phosphorylated differently by stroma and by thylakoids in the dark. Moreover, histone III phosphorylated by thylakoids in the dark yielded a pattern of phosphopeptides after V8 protease treatment that was different from the pattern obtained when histone III was phosphorylated by stroma.