An effective, simple and low-cost pretreatment for culture clarification in tetanus toxoid production.

Chemically inactivated tetanus toxin (tetanus toxoid, TT), purified from cultures of a virulent Clostridium tetani strain, is the active pharmaceutical ingredient of anti-tetanus vaccines. Culture clarification for TT production and is usually performed by filtration-based techniques. Final clarification of the culture supernatant is achieved by passage through 0.2 µm pore size filtering membranes. Large particles removal (primary clarification) before final filtration (secondary clarification) reduces costs of the overall clarification process. With this aim, chitosan-induced particle aggregation was assessed as an alternative for primary clarification. Three chitosan variants were tested with similar results. Optimal clarification of culture supernatant was achieved by the addition of 8 mg chitosan per l of culture. Extrapolation analysis of filter sizing results indicate that 100 l of chitosan-treated supernatant can be finally filtered with a 0.6 m2 normal filtration cartridge of 0.45 + 0.2 µm pore size. The clarified material is compatible with current standard downstream processing techniques for TT purification. Thus, chitosan-induced particle aggregation is a suitable operation for primary clarification.

Nongenomic steroid- and ceramide-induced maturation in amphibian oocytes involves functional caveolae-like microdomains associated with a cytoskeletal environment.

Stimulation of full-grown amphibian oocytes with progesterone initiates a nontranscriptional signaling pathway that converges in the activation of Cdc2/cyclin B and reentry into meiosis. We observed that cholesterol depletion mediated by methyl-beta-cyclodextrin (MbetaCD) inhibited meiotic maturation, suggesting involvement of membrane rafts. In the present study, we further characterized caveolae-like membranes from Rhinella arenarum oocytes biochemically and functionally. The identification by mass spectrometry of a nonmuscle myosin heavy-chain associated with caveolar membranes showed evidence of direct involvement of the underlying cytoskeletal environment in the structure of oocyte rafts. Biophysical analysis using the fluorescent probe Laurdan revealed that MbetaCD-mediated cholesterol depletion affected membrane lipid order. In line with this finding, cholesterol removal also affected the localization of the raft marker lipid GM1. Results demonstrated that ceramide is an effective inducer of maturation that alters the distribution of the raft markers caveolin-1, SRC, and GM1, while progesterone seems not to affect membrane microdomain integrity. Cholesterol depletion had a greater effect on ceramide-induced maturation, thus suggesting that ceramide is an inducer more vulnerable to changes in the plasma membrane. MbetaCD treatment delayed tyrosine phosphorylation and MAPK activation in progesterone-induced maturation. Functional studies regarding tyrosine phosphorylation raise the possibility that the hormone receptor is located in the nonraft membrane in the absence of ligand and that it translocates to the caveola when it binds to progesterone. The presence of raft markers and the finding of signaling molecules from MAPK cascade functionally associated to oocyte light membranes suggest that this caveolae-rich fraction efficiently recreates, in part, maturation signaling.

Snake venomics of the South and Central American Bushmasters. Comparison of the toxin composition of Lachesis muta gathered from proteomic versus transcriptomic analysis.

We report the proteomic characterization of the venoms of two closely related pit vipers of the genus Lachesis, L. muta (South American Bushmaster) and L. stenophrys (Central American Bushmaster), and compare the toxin repertoire of the former revealed through a proteomic versus a transcriptomic approach. The protein composition of the venoms of Lachesis muta and L. stenophrys were analyzed by RP-HPLC, N-terminal sequencing, MALDI-TOF peptide mass fingerprinting and CID-MS/MS. Around 30-40 proteins of molecular masses in the range of 13-110 kDa and belonging, respectively, to only 8 and 7 toxin families were identified in L. muta and L. stenophrys venoms. In addition, both venoms contained a large number of bradykinin-potentiating peptides (BPP) and a C-type natriuretic peptide (C-NP). BPPs and C-NP comprised around 15% of the total venom proteins. In both species, the most abundant proteins were Zn(2+)-metalloproteinases (32-38%) and serine proteinases (25-31%), followed by PLA(2)s (9-12%), galactose-specific C-type lectin (4-8%), l-amino acid oxidase (LAO, 3-5%), CRISP (1.8%; found in L. muta but not in L. stenophrys), and NGF (0.6%). On the other hand, only six L. muta venom-secreted proteins matched any of the previously reported 11 partial or full-length venom gland transcripts, and venom proteome and transcriptome depart in their relative abundances of different toxin families. As expected from their close phylogenetic relationship, the venoms of L. muta and L. stenophrys share (or contain highly similar) proteins, in particular BPPs, serine proteinases, a galactose-specific C-type lectin, and LAO. However, they dramatically depart in their respective PLA(2) complement. Intraspecific quantitative and qualitative differences in the expression of PLA(2) molecules were found when the venoms of five L. muta specimens (3 from Bolivia and 2 from Peru) and the venom of the same species purchased from Sigma were compared. These observations indicate that these class of toxins represents a rapidly-evolving gene family, and suggests that functional differences due to structural changes in PLA(2)s molecules among these snakes may have been a hallmark during speciation and adaptation of diverging snake populations to new ecological niches, or competition for resources in existing ones. Our data may contribute to a deeper understanding of the biology and ecology of these snakes, and may also serve as a starting point for studying structure-function correlations of individual toxins.