Aldolase A (AldB) gene is one of the liver-specific genes, which is activated in the fetal stage. As a first step to investigate the functional relationship between transcription and DNA replication, we intended to determine the initiation zone of replication nearest to the AldB gene region. BrdU-labeled nascent DNA was obtained from G1/S arrested hepatoma cells at various times after entering S phase. Hybridization of the newly synthesized, BrdU-labeled DNA with probes corresponding to regions spanning about 26 Kb, revealed that replication zone locates within the AldB gene region. This result, together with the result of hybridization of nascent DNA obtained by alkaline sucrose density-gradient centrifugation, suggested that the initiation zone is located within a more defined region (about 1.0 Kb) containing AldB promoter. In the predicted initiation zone, a purine-rich element which shows high homology to known mammalian origin sequences and other replication components are found. Further, autonomously replicating activity of this initiation zone was examined by DNA transfection. The results showed that the predicted initiation zone confers replication initiation in Cos-1 cells.

There is currently an urgent need to develop safe and effective adjuvants for enhancing vaccine-induced antigen-specific immune responses. We demonstrate here that intranasal immunization with clinically used polypeptide antibiotics, polymyxin B (PMB) and colistin (CL), along with ovalbumin (OVA), increases OVA-specific humoral immune responses in a dose-dependently manner at both mucosal and systemic compartments. Enhanced immunity by boosting was found to persist during 8 months of observation. Moreover, mice intranasally immunized with OVA plus various doses of PMB or CL showed neither inflammatory responses in the nasal cavity and olfactory bulbs nor renal damages, compared to those given OVA alone. These data suggest that polymyxins may serve as novel and safe mucosal adjuvants to induce humoral immune responses. The polymyxin adjuvanticity was found to be independent of endotoxins liberated by its bactericidal activity, as indicated by similar enhancing effects of PMB in lipopolysaccharide (LPS)-hyporesponsive and LPS-susceptible mice. However, despite the presence of preexisting anti-PMB antibodies, we observed no reduction in the adjuvant function of polymyxins when they were given intranasally. Furthermore, the titers of OVA-specific Abs in mice intranasally immunized with OVA plus PMB or CL were significantly higher than those in mice administered with polymyxin analogues, such as polymyxin B nonapeptide and colistin methanesulfonate. The levels of released β-hexosaminidase and histamine in mast cell culture supernatants stimulated by PMB or CL were also significantly higher than those stimulated by their analogues. These results suggest that both the hydrophobic carbon chain and hydrophilic cationic cyclic peptide contribute to the mucosal adjuvanticity of PMB and CL.

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We describe a novel pharmacological activity of the gentian root, an ingredient of Chinese medicines. Root extract from Gentiana triflora triggered cell death of human Daudi cells in culture. In addition, daily administration of the extract to mice inhibited growth of implanted solid tumors. Extract treatment of cultured cells resulted in the appearance of shranken, fragmented, or condensed cell and nuclear morphologies, and in chromosomal DNA degradation. But, the extract-treated cells did not show DNA fragmentation, which exhibits a nucleosome ladder, suggesting that extract-triggered cell death is not mediated through a typical apoptotic pathway.