Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

Transient transfection of a synthetic hammerhead ribozyme targeted against human MGMT gene to cells in culture potentiates the genotoxicity of the alkylation damage induced by mitozolomide.

Unmodified and chemically modified forms of a synthetic hammerhead ribozyme with the mRNA of methylguanine-DNA methyltransferase (MGMT) gene as substrate were characterized for their in vitro and in vivo activities. The unmodified ribozyme efficiently cleaved in vitro a short synthetic substrate, and it was rapidly degraded in fetal bovine serum (FBS). The introduction of phosphorothioates and the substitution of uridine with thymidine at probable nuclease-sensitive sites slightly increased the nuclease resistance of the ribozyme. Conversely, pyrimidine nucleoside substitution with 2'NH2 and 2'F nucleosides strongly enhanced nuclease resistance. The in vivo activity was determined by measuring the genotoxicity induced by the alkylating drug mitozolomide, the damage of which is repaired by MGMT enzyme. CHO/47 cells, temporarily depleted of the MGMT protein, were first transfected with the various synthetic ribozymes and subsequently treated with mitozolomide. At equivalent concentration of the drug, the induction of sister chromatid exchanges was higher in ribozyme-transfected than in untransfected cells, indicating that the synthetic ribozymes potentiated the genotoxicity of mitozolomide. Moreover, the concomitant occurrence of messenger RNA reduction in ribozyme-transfected cells indicated that the inhibition of MGMT resynthesis was the basis of the enhanced genotoxicity.

Absence of mutations in the highest mutability region of the p53 gene in tumour-derived CHEF18 Chinese hamster cells.

A series of independent tumour-derived Chinese hamster CHEF18 cell lines was analyzed for the presence of mutations in the cDNA region (exons 5-9) of the p53 gene, where the great majority of p53 mutations of human tumours accumulate. Since the gene is highly conserved among species, we used two primers designed on the basis of the human cDNA sequence to isolate the cDNA region from total RNA of Chinese hamster cells. The amplified fragments of 614 bp were digested with cleavase I endonuclease and fragment length polymorphism analysis showed that the restriction pattern of the p53 exons 5-9 region of tumour-derived cell lines was identical to that of diploid Chinese hamster CHL fibroblasts. Sequencing of the amplified fragments showed 100% homology between sequences, which demonstrated the absence of p53 mutations in the exons 5-9 cDNA region expected to have the highest mutability. Nevertheless, the antibody DO-7 recognized the presence of a stabilized p53 protein only in tumour-derived cell lines, which indicated that p53 expression correlated with transformed status.

The sensitization of cells treated with O6-methylguanine to alkylation damage is affected by the number of O6-methylguanine-DNA methyltransferase molecules escaped from inactivation.

O6-Methylguanine (MeG) can bind to the active site of O6-methylguanine-DNA methyltransferase (MGMT) as a free base. The subsequent methyl transfer reaction inactivates the repair protein. Hence, MeG is used to deplete the active MGMT pools in Chinese hamster cell lines (CHO) transfected to express varying amounts of human MGMT. After treatment with the free base, a residual population of active protein molecules remains localized mostly in the cytoplasm. Depleted cells are then challenged with the alkylating drug mitozolomide. Genotoxicity of this agent varied among the cell lines, and the compound sensitivity seemed to be regulated by a steady state equilibrium of residual MGMT molecules between nucleus and cytoplasm.

Absence of UV-induced non-homologous recombination in repair-deficient CHO cell lines transfected with ERCC genes.

The nucleotide excision repair pathway removes a broad spectrum of DNA lesions, including UV-induced damage. To ascertain whether the repair of the latter has a causative role in the enhancement of non-homologous recombination, Chinese hamster CHO cell lines proficient and deficient in the ability to repair UV-induced damage were transfected with a plasmid containing the bacterial neoR gene. Following UV-treatment an enhancement of non-homologous recombination above the spontaneous level was observed in repair-proficient cells, whereas no increase was observed in repair-deficient cell lines. Hence, the latter were transfected with the corresponding excision repair cross complementing human genes and the resulting repair-proficient transfectants were tested for UV-induced non-homologous recombination. In both untreated and UV-treated transfectants, the frequencies of the event were not significantly different. Cumulatively, the results suggest that non-homologous recombination induced by UV-irradiation is not restored by the correction of the excision repair defect.