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.

Proposed modifications to metabolic model for glycogen-accumulating organisms under anaerobic conditions

Filipe et al. (2001) proposed an anaerobic metabolic model for glycogen-accumulating organisms (GAO) in which the succinate-propionate pathway was used to describe the production of propionyl-CoA. However, propionyl-CoA is only an intermediate product in the above pathway. Stopping at propionyl-CoA instead of propionate (the end product of the pathway) results in the consumption of one ATP from succinate to succinyl-CoA, which was not accounted for in the model of Filipe et al. (2001). This resulted in significant errors in the stoichiometric coefficients in the final metabolic model. A modified model is presented in this communication and is shown to fit the experimental data significantly better than the original model. (C) 2002 Wiley Periodicals, Inc.

Virulence of Streptococcus pneumoniae: PsaA mutants are hypersensitive to oxidative stress

psaA encodes a 37-kDa pneumococcal lipoprotein which is part of an ABC Mn(II) transport complex. Streptococcus pneumoniae D39 psaA mutants have previously been shown to be significantly less virulent than wild-type D39, but the mechanism underlying the attenuation has not been resolved. In this study, we have shown that psaA and psaD mutants are highly sensitive to oxidative stress, i.e., to superoxide and hydrogen peroxide, which might explain why they are less virulent than the wild-type strain. Our investigations revealed altered expression of the key oxidative-stress response enzymes superoxide dismutase and NADH oxidase in psaA and psaD mutants, suggesting that PsaA and PsaD may play important roles in the regulation of expression of oxidative-stress response enzymes and intracellular redox homeostasis.

Factors affecting the rate of phosphocreatine resynthesis following intense exercise

Within the skeletal muscle cell at the onset of muscular contraction, phosphocreatine (PCr) represents the most immediate reserve for the rephosphorylation of adenosine triphosphate (ATP). As a result, its concentration can be reduced to less than 30% of resting levels during intense exercise. As a fall in the level of PCr appears to adversely affect muscle contraction, and therefore power output in a subsequent bout, maximising the rate of PCr resynthesis during a brief recovery period will be of benefit to an athlete involved in activities which demand intermittent exercise. Although this resynthesis process simply involves the rephosphorylation of creatine by aerobically produced ATP (with the release of protons), it has both a fast and slow component, each proceeding at a rate that is controlled by different components of the creatine kinase equilibrium. The initial fast phase appears to proceed at a rate independent of muscle pH. Instead, its rate appears to be controlled by adenosine diphosphate (ADP) levels; either directly through its free cytosolic concentration, or indirectly, through its effect on the free energy of ATP hydrolysis. Once this fast phase of recovery is complete, there is a secondary slower phase that appears almost certainly rate-dependant on the return of the muscle cell to homeostatic intracellular pH. Given the importance of oxidative phosphorylation in this resynthesis process, those individuals with an elevated aerobic power should be able to resynthesise PCr at a more rapid rate than their sedentary counterparts. However, results from studies that have used phosphorus nuclear magnetic resonance (P-31-NMR) spectroscopy, have been somewhat inconsistent with respect to the relationship between aerobic power and PCr recovery following intense exercise. Because of the methodological constraints that appear to have limited a number of these studies, further research in this area is warranted.

Bracken (Pteridium aquilinum)-induced DNA adducts in mouse tissues are different from the adduct induced by the activated form of the bracken carcinogen ptaquiloside

Following treatment with bracken fern (Pteridium aquilinum) extract and bracken spores a number of DNA adducts were detected by P-32-postlabeling. Three of these adducts have been described previously (Povey et al., Br. J. Cancer (1996) 74, 1342-1348) and in this study, using a slightly different protocol, four new adducts, with higher chromatographic mobility, were detected at levels ranging from 50 to 230% of those previously described, When DNA was treated in vitro with activated ptaquiloside (APT) and analysed by butanol extraction or nuclease P1 treatment, only one adduct was detected by P-32-postlabeling, This adduct was not present in the DNA from mice treated with bracken fern or spores, suggesting either that bracken contains genotoxins other than ptaquiloside or that the metabolism of ptaquiloside produces genotoxins not reflected by activated ptaquiloside. However, as the ATP-derived adduct has been detected previously in ileal DNA of bracken-fed calves, species-specific differences in the metabolism of bracken genotoxins may exist, thereby leading to differences in their biological outcomes. (C) 2001 Academic Press.

Neural control of the heart: developmental changes in ionic conductances in mammalian intrinsic cardiac neurons

The expression and properties of ionic channels were investigated in dissociated neurons from neonatal and adult rat intracardiac ganglia. Changes in the hyperpolarization-activated and ATP-sensitive K+ conductances during postnatal development and their role in neuronal excitability were examined. The hyperpolarization-activated nonselective cation current, I-h, was observed in all neurons studied and displayed slow time-dependent rectification. An inwardly rectifying K+ current, I-K(I), was present in a population of neurons from adult but not neonatal rats and was sensitive to block by extracellular Ba2+. Using the perforated-patch recording configuration, an ATP-sensitive K+ (K-ATP) conductance was identified in greater than or equal to 50% of intracardiac neurons from adult rats. Levcromakalim evoked membrane hyperpolarization, which was inhibited by the sulphonylurea drugs. glibenclamide and tolbutamide. Exposure to hypoxic conditions also activated a membrane current similar to that induced by levcromakalim and was inhibited by glibenclamide. Changes in the complement of ion channels during postnatal development may underlie observed differences in the function of intracardiac ganglion neurons during maturation. Furthermore, activation of hyperpolarization-activated and KATP channels in mammalian intracardiac neurons may play a role in neural regulation of the mature heart and cardiac function during ischaemia-reperfusion. (C) 2002 Elsevier Science B.V All rights reserved.

Mechanisms for the inhibition of amiloride-sensitive Na+ absorption by extracellular nucleotides in mouse trachea

Purinergic stimulation of airway epithelial cells induces Cl- secretion and modulates Na+ absorption by an unknown mechanism. To gain insight into this mechanism, we used a perfused micro-Ussing chamber to assess transepithelial voltage (V-te) and amiloride-sensitive short-circuit current (Isc-Amil) in mouse trachea. Exposure to apical ATP or UTP (each 100 mumol/l) caused a large initial increase in lumen negative V-te and I-sc corresponding to a transient Cl- secretion, while basolateral application of ATP/UTP induced only a small secretory response. Luminal, but not basolateral, application of nucleotides was followed by a sustained and reversible inhibition of Isc-Amil that was independent of extracellular Ca2+ or activation of protein kinase C and was not induced by carbachol (100 mumol/l) or the Ca2+ ionophore ionomycin (1 mumol/l). Removal of extracellular Cl- or exposure to 200 muM DIDS reduced UTP-mediated inhibition of Isc-Amil Substantially. The phospholipase inhibitor U73122 (10 mumol/l) and pertussis toxin (PTX 200 ng/ml) both attenuated UTP-induced Cl- secretion and inhibition of Isc-Amil. Taken together, these data imply a contribution of Cl- conductance and PTX-sensitive G proteins to nucleotide-dependent inhibition of the amiloride-sensitive Na+ current in the mouse trachea.

Vps20p and Vta1p interact with Vps4p and function in multivesicular body sorting and endosomal transport in Saccharomyces cerevisiae

Vps4p (End13p) is an AAA-family ATPase that functions in membrane transport through endosomes, sorting of soluble vacuolar proteins to the vacuole, and multivesicular body (MVB) sorting of membrane proteins to the vacuole lumen. In a yeast two-hybrid screen with Vps4p as bait we isolated VPS20 (YMR077c) and the novel open reading frame YLA181c, for which the name VTA1 has recently been assigned (Saccharomyces Genome Database). Vps4p directly binds Vps20p and Vta1p in vitro and binding is not dependent on ATP-conversely, Vps4p binding to Vps20p is partially sensitive to ATP hydrolysis. Both ATP binding [Vps4p-(K179A)] and ATP hydrolysis [Vps4p-(E233Q)] mutant proteins exhibit enhanced binding to Vps20p and Vta1p in vitro. The Vps4p-Vps20p interaction involves the coiled-coil domain of each protein, whereas the Vps4p-Vta1p interaction involves the (non-coiled-coil) C-terminus of each protein. Deletion of either VPS20 (vps20Delta) or VTA1 (vta1Delta) leads to similar class E Vps(-) phenotypes resembling those of vps4Delta, including carboxypeptidase Y (CPY) secretion, a block in ubiquitin-dependent MVB sorting, and a delay in both post-internalisation endocytic transport and biosynthetic transport to the vacuole. The vacuole resident membrane protein Sna3p (whose MVB sorting is ubiquitin-independent) does not appear to exit the class E compartment or reach the vacuole in cells lacking Vps20p, Vta1p or Vps4p, in contrast to other proteins whose delivery to the vacuole is only delayed. We propose that Vps20p and Vta1p regulate Vps4p function in vivo.