Increased susceptibility to streptozotocin-induced beta-cell apoptosis and delayed autoimmune diabetes in alkylpurine-DNA-N-glycosylase-deficient mice

Type I diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta -cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta -cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in A-PNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta -cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(-/-) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.

Application of nox-restriction fragment length polymorphism for the differentiation of Brachyspira intestinal spirochetes isolated from pigs and poultry in Australia

Sixty-nine intestinal spirochetes isolated from pigs and poultry in eastern Australia were selected to evaluate the effectiveness of a species-specific PCR-based restriction fragment length polymorphism (RFLP) analysis of the Brachyspira nox gene. For comparative purposes, all isolates were subjected to species-specific PCRs for the pathogenic species Brachyspira hyodysenteriae and Brachyspira pilosicoli, and selected isolates were examined further by sequence analysis of the nox and 16S ribosomal RNA genes. Modifications to the original nox-RFLP method included direct inoculation of bacterial cells into the amplification mixture and purification of the PCR product, which further optimized the nox-RFLP for use in a veterinary diagnostic laboratory, producing sufficient product for both species identification and future comparisons. Although some novel profiles that prevented definitive identification were observed, the nox-RFLP method successfully classified 45 of 51 (88%) porcine and 15 of 18 (83%) avian isolates into 5 of the 6 recognized species of Brachyspira. This protocol represents a significant improvement over conventional methods currently used in veterinary diagnostic laboratories for rapid specific identification of Brachyspira spp. isolated from both pigs and poultry.