Production of L1 protein from different types of HPV in Pichia pastoris using an integrative vector

Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene. However, the use of an episomal vector is not appropriate for protein production on an industrial scale. In the present study, the vectors were integrated into the Pichia genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. Despite the great potential for expression by the P. pastoris system, our results suggest a low yield of L1 recombinant protein, which, however, does not make this system unworkable. The achievement of stable clones containing the expression cassettes integrated in the genome may permit optimizations that could enable the establishment of a platform for the production of VLP-based vaccines.

Simultaneous presence of bovine papillomavirus and bovine leukemia virus in different bovine tissues: in situ hybridization and cytogenetic analysis

Bovine papillomavirus (BPV) DNA sequences were detected in different tissues, in addition to epithelium. Cytogenetic abnormalities were observed in blood lymphocytes. The presence of more than one virus in a single tissue is a difficult aspect to evaluate, especially when the DNA sequences are detected in tissues that are not specifically targeted by the virus. BPV and bovine leukemia virus (BLV) are clastogenic, causing chromosome aberrations in peripheral blood lymphocytes. In the present study, we investigated the simultaneous presence of DNA sequences of both viruses and the possibility of vertical transmission and compared the types of chromosome aberrations related to viral action. BPV 1, 2, and 4 DNA sequences were found in three females of the herd and in their offspring. BLV DNA sequences were not detected in their progeny. A newborn calf that was negative for BLV infection showed specific chromosome rearrangements possibly related to the effect of infection with BPV.

Induction of apoptosis in cancer cells by tumor necrosis factor and butyrolactone, an inhibitor of cyclin-dependent kinases

Induction of apoptosis by tumor necrosis factor (TNF) is modulated by changes in the expression and activity of several cell cycle regulatory proteins. We examined the effects of TNF (1-100 ng/ml) and butyrolactone I (100 µM), a specific inhibitor of cyclin-dependent kinases (CDK) with high selectivity for CDK-1 and CDK-2, on three different cancer cell lines: WEHI, L929 and HeLa S3. Both compounds blocked cell growth, but only TNF induced the common events of apoptosis, i.e., chromatin condensation and ladder pattern of DNA fragmentation in these cell lines. The TNF-induced apoptosis events were increased in the presence of butyrolactone. In vitro phosphorylation assays for exogenous histone H1 and endogenous retinoblastoma protein (pRb) in the total cell lysates showed that treatment with both TNF and butyrolactone inhibited the histone H1 kinase (WEHI, L929 and HeLa) and pRb kinase (WEHI) activities of CDKs, as compared with the controls. The role of proteases in the TNF and butyrolactone-induced apoptosis was evaluated by comparing the number and expression of polypeptides in the cell lysates by gel electrophoresis. TNF and butyrolactone treatment caused the disappearance of several cellular protein bands in the region between 40-200 kDa, and the 110- 90- and 50-kDa proteins were identified as the major substrates, whose degradation was remarkably increased by the treatments. Interestingly, the loss of several cellular protein bands was associated with the marked accumulation of two proteins apparently of 60 and 70 kDa, which may be cleavage products of one or more proteins. These findings link the decrease of cyclin-dependent kinase activities to the increase of protease activities within the growth arrest and apoptosis pathways induced by TNF.

Preparation of human rabies vaccine in VERO cell culture using a microcarrier system

1. The rabies virus (Pasteur PV strain) was propagated in VERO cells attached to microcarriers in a 3.7-1 bioreactor. Virus titers of about 10(6) LD50/ml were obtained regularly. 2. Ultrafiltration was efficient for concentrating the virus suspensions, and the sucrose gradient reduced the residual VERO cell DNA to acceptable levels (less than 50 pg/dose). The remaining cell DNA content was evaluated by dot-blot hybridization with a probe prepared with VERO cell DNA. 3. The final virus preparations were inactivated by B-propiolactone treatment, showed a potency higher than 2.5 IU/dose and protected mice experimentally infected intracerebrally with rabies virus (CVS-13.2). 4. This methodology for the production of a rabies vaccine for human use should be of interest to countries where high technology facilities are not available

The effect of pH and metal chelators upon the sedimentation velocity of Odontophrynus americanus vitellogenin

In the tetraploid amphibian Odontophrynus americans the selective precipitation of vitellogenin by Mg2+ from plasma treated with ethylene diaminetetraacetic acid (EDTA) or ethylene bis (oxyethylenenitrilo)-tetraacetic acid (EGTA) is a pH-dependent phenomenon. Utilizing sucrose gradient centrifugation of whole plasma we have shown that under standardconditions (pH 7.0) the estimated apparent sedimentation coefficient of vitellogenin is 17s.At pH 8.0 and in the presence of EDTA or EGTA there is a decrease of the vitellogenin sedimentation coefficient.This behavior is restricted to vitellogenin as othewr plasma proteins show no alteration in their sedimentation coefficient after similar treatment. The treatment with EDTA at pH 8.0 also induces changes in the vitellogenin molecule which can be detected by partial proteolysis with chymotripsin A