Excessive activation of poly(ADP-ribose) polymerase 1 (PARP1) leads to NAD ؉ depletion and cell death during ischemia and other conditions that generate extensive DNA damage. When activated by DNA strand breaks, PARP1 uses NAD ؉ as substrate to form ADP-ribose polymers on specific acceptor proteins. These polymers are in turn rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG), a ubiquitously expressed exo-and endoglycohydrolase. In this study, we examined the role of PARG in the PARP1-mediated cell death pathway. Mouse neuron and astrocyte cultures were exposed to hydrogen peroxide, N-methyl-D-aspartate (NMDA), or the DNA alkylating agent, N-methyl-N-nitro-N-nitrosoguanidine (MNNG). Cell death in each condition was markedly reduced by the PARP1 inhibitor benzamide and equally reduced by the PARG inhibitors gallotannin and nobotanin B. The PARP1 inhibitor benzamide and the PARG inhibitor gallotannin both prevented the NAD ؉ depletion that otherwise results from PARP1 activation by MNNG or H2O2. However, these agents had opposite effects on protein poly(ADP-ribosyl)ation. Immunostaining for poly(ADP-ribose) on Western blots and neuron cultures showed benzamide to decrease and gallotannin to increase poly(ADP-ribose) accumulation during MNNG exposure. These results suggest that PARG inhibitors do not inhibit PARP1 directly, but instead prevent PARP1-mediated cell death by slowing the turnover of poly(ADP-ribose) and thus slowing NAD ؉ consumption. PARG appears to be a necessary component of the PARP-mediated cell death pathway, and PARG inhibitors may have promise as neuroprotective agents.oly(ADP-ribose) polymerase (PARP) can contribute to both DNA repair and cell death (1, 2). Poly(ADP-ribose) polymerase 1 (PARP1) is the most abundant and best characterized member of the PARP family (3, 4). PARP1 appears to function in the detection and intracellular signaling of DNA damage, because PARP1 is activated by DNA strand breaks or kinks (1). PARP1 transfers ADP-ribose moieties from NAD ϩ to specific acceptor proteins to form complex, branched chains with lengths of up to 200 residues. Known acceptor proteins include many proteins that function in DNA repair and cell cycle regulation, such as histones, DNA polymerases, DNA ligases, p53, and Fos (1, 5, 6). PARP1 itself is also an acceptor protein, and PARP1 is strongly inhibited when extensively poly(ADP-ribosyl)ated (1, 7).Despite its function in DNA repair, overactivation of PARP has long been recognized to induce cell death under some conditions (8). PARP inhibitors and PARP1 gene disruption can reduce cell death resulting from oxidative stress (9), radiation (10), nitric oxide, peroxynitrite (11-13), and other agents that damage DNA (13,14). Oxidative stress contributes to cell death in cerebral ischemia (15, 16), and genetic or pharmacological inhibition of PARP1 reduces ischemic cell death (13, 17). PARP1 inactivation can also prevent neuronal death induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (18) and other oxidants (2, 13).The mechanism ...

During the last half-century, incidences of breast cancer have increased globally. Various factors—genetic and environmental— have been implicated in the initiation and progression of this disease. One potential environmental risk factor that has not received a lot of attention is the exposure to heavy metals. While several mechanisms have been put forth describing how high concentrations of heavy metals play a role in carcinogenesis, it is unclear whether chronic, low-level exposure to certain heavy metals (i.e. cadmium and nickel), can directly result in the development and progression of cancer. Cadmium and nickel have been hypothesized to play a role in breast cancer development by acting as metalloestrogens— metals that bind to estrogen receptors and mimic the actions of estrogen. Since the lifetime exposure to estrogen is a well-established risk factor for breast cancer, anything that mimics its activity will likely contribute to the etiology of the disease. However, heavy metals, depending on their concentration, are capable of binding to a variety of proteins and may exert their toxicities by disrupting multiple cellular functions, complicating the analysis of whether heavy metal-induced carcinogenesis is mediated by the estrogen receptor. The purpose of this review is to discuss the various epidemiological, in vivo, and in vitro studies that show a link between the heavy metals, cadmium and nickel, and breast cancer development. We will particularly focus on the studies that test whether or not these two metals act as metalloestrogens in order to assess the strength of the data supporting this hypothesis.

The α2,8-polysialyltransferases (polySTs) from embryonic chick brain catalyze the α2,8-specific polysialylation of endogenous neural cell adhesion molecules (N-CAMs). This posttranslation glycosylation decreases N-CAM-dependent cell adhesion and migration. The enzymatic properties of the membrane-bound form of the polyST activity was investigated in vitro. Our results show that the polyST activity was developmentally expressed with maximum specific activity appearing about 12 days after fertilization. This time shortly precedes maximal expression of the cognate polysialylated N-CAMs. Kinetic studies showed the K M and V max for CMPNeu5Ac were 133 µM and 0.13 µM/h, respectively, at pH 6.1, 33_C. CMP-Neu5Gc was not a donor substrate. PolyST activity was increased 5-to 6-fold in the presence of 10 mM MnCl 2, the preferred divalent cation, and 1 mM dithiothreitol (DTT). Heparin (3 kDa) was a noncompetitive inhibitor of polysialylation with a K i of 9 µM. Based on the affinity of the enzyme for heparin, the polyST activity was partially purified (∼30-fold) by heparin-Sepharose affinity chromatography, after differential solubilization with the zwitterionic detergent, CHAPS. DTT and chemical modification studies using the thiol-directed alkylating reagents, N-ethylmaleimide (NEM) and iodoacetamide (IAA), were used to show that at least one cysteinyl residue in the polyST was of critical importance for polysialylation, but of lesser importance for monosialylation, catalyzed by the α2,3-, α2,6-, and α2,8-monosialyltransferases (monoSTs). A sulfhydryl residue is implicated in chain initiation. Two important structural differences between the mono-and polySTs were revealed by sequence analyses. First, the polySTs contain heparin-like, positively charged amino acid clusters upstream of both sialylmotif L and S. Second, the polySTs contain a uniquely extended basic amino acid region (pI 11.6-12.0) of 31 residues immediately upstream of sialylmotif S. This extended, positively charged region may function in the processive mechanism of polymerization by allowing nascent polySia chains to remain bound to the polyST during the repetitive addition of each new Sia residue to the nonreducing termini of the growing chain. The importance of these studies is that they provide new information on the enzymatic basis of polysialylation. They also reveal that sulfhydryl residues and extended basic amino acid domains are two structural features unique to polysialylation, in contrast to monosialylation. Both may be important distinguishing features between the classes of distributive (monoSTs) and processive polysialyltransferases, which have not been previously described.

Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in the prostanoid biosynthesis pathway, converting arachidonic acid into prostaglandin H 2 . COX-2 exists as 72 and 74 kDa glycoforms, the latter resulting from an additional oligosaccharide chain at residue Asn 580 . In this study, Asn 580 was mutated to determine the biological significance of this variable glycosylation. COS-1 cells transfected with the mutant gene were unable to express the 74 kDa glycoform and were found to accumulate more COX-2 protein and have five times greater COX-2 activity than cells expressing both glycoforms. Thus, COX-2 turnover appears to depend upon glycosylation of the 72 kDa glycoform. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

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