Inhibition of poly(ADP-ribose) polymerase attenuates inflammation in a model of chronic colitis.

Crohn's disease is a chronic disease characterized by oxidant-induced tissue injury and increased intestinal permeability. A consequence of oxidative damage is the accumulation of DNA strand breaks and activation of poly(ADP-ribose) polymerase (PARP), which subsequently catalyzes ADP-ribosylation of target proteins. In this study, we assessed the role of PARP in the colitis seen in interleukin (IL)-10 gene-deficient mice. IL-10 gene-deficient mice demonstrated significant alterations in colonic cellular energy status in conjunction with increased permeability, proinflammatory cytokine release, and nitrosative stress. After 14 days of treatment with the PARP inhibitor 3-aminobenzamide, IL-10 gene-deficient mice demonstrated normalized colonic permeability; reduced tumor necrosis factor-alpha and interferon-gamma secretion, inducible nitric oxide synthase expression, and nitrotyrosine levels; and significantly attenuated inflammation. Time course studies demonstrated that 3-aminobenzamide rapidly altered cellular metabolic activity and decreased cellular lactate levels. This was associated with normalization of colonic permeability and followed by a downregulation of proinflammatory cytokine release. Our data demonstrate that inhibition of PARP activity results in a marked improvement of colonic inflammatory disease and a normalization of cellular metabolic function and intestinal permeability.

Detection of primary DNA damage in Chlamydomonas reinhardtii by means of modified microgel electrophoresis.

The assessment of genotoxic potential in surface water requires test methods, among which are those that detect initial DNA damage in organisms of aquatic biocenosis. The microgel electrophoresis (MGE) "comet assay" was applied to a ubiquitous unicellular green alga (Chlamydomonas reinhardtii) to detect DNA damage caused by genotoxins. For this, the test protocol described by Singh NP et al. [Exp Cell Res 175: 184-191, 1988] was modified. Major modifications were the use of alkaline lysis buffer with ionic detergents and the reduction of preincubation and electrophoresis times. Short-time exposure of Chlamydomonas to the well-known genotoxicants 4-nitroquinoline-1-oxide (4-NQO), N-nitrosodimethylamine, and hydrogen peroxide led to dose-dependent DNA damage. Chlamydomonas responded very sensitively to treatment with increasing doses of 4-NQO. At a concentration of 25 nM, significant DNA damage was observed. At higher 4-NQO doses (> 100 nM), DNA damage was visible as complete DNA fragmentation into fine granules. N-Nitrosodimethylamine caused genotoxic effects at a concentration range from 0.014 to 0.14 mM without producing complete DNA fragmentation at the concentrations tested (highest dose, 140 mM). To evaluate the influence of illumination conditions during exposure, cells were incubated with increasing doses of H2O2 (0.25-1.0 mM) in darkness and in light. Our results indicate that incubation in light enables Chlamydomonas to cope with oxidative stress more efficiently than under dark conditions. To a certain extent, cytotoxic as well as genotoxic effects of H2O2 depend on the illumination condition or repair and anti-oxidative protection mechanisms activated by light, respectively.

Polymorphism in an HLA linked proteasome gene influences phenotypic expression of disease in HLA-B27 positive individuals.

OBJECTIVE: To investigate the potential influence of the HLA-linked LMP (low molecular weight polypeptide) genes on disease susceptibility in HLA-B27 individuals with ankylosing spondylitis (AS). METHODS: A polymorphic CfoI restriction enzyme site in the coding region of one proteasome gene was evaluated in 125 genomic DNA samples from B27 individuals with well documented AS, 55 of whom had had acute iritis, and 42 samples from normal, ethnically matched B27 blood donors where AS was excluded. RESULTS: Analysis of individuals with B27 AS with iritis revealed significant differences in allelic distribution of this biallelic locus compared to patients with B27 AS without iritis. Furthermore, homozygosity for the disease associated allele was significantly more prevalent in patients with AS with iritis (72.7%) than in patients without iritis (38.6%) (p(uncorrected) = 0.0003) or B27 controls (45.2%) (p(uncorrected) = 0.01). CONCLUSION: Our findings support the involvement of additional HLA linked genes in the phenotypic expression of disease in B27 individuals and suggest a role for the non-B27 HLA haplotype in susceptibility to iritis.

Insect lipid transfer particle catalyzes bidirectional vectorial transfer of diacylglycerol from lipophorin to human low density lipoprotein.

Insect plasma lipid transfer particle (LTP) catalyzes vectorial net transfer of diacylglycerol (DAG) from Manduca sexta larval high density lipophorin (HDLp-L) to human low density lipoprotein (LDL) producing an LDL of lower density and lipophorin subspecies of higher density. At equilibrium, a stable DAG-depleted very high density lipophorin species (density = 1.25 g/ml) is formed. Electrophoretic analysis of the substrate and product lipoproteins showed that apoprotein exchange or transfer between human LDL and lipophorin did not occur during the lipid transfer reaction. Facilitated net transfer of cholesteryl ester, free cholesterol, and phospholipid occurred to a much lower extent than DAG net transfer, indicating that under these conditions, LDL serves as a sink for lipophorin-associated DAG. This reaction, therefore, provides a method whereby the mass of lipid associated with human LDL can be modified in vitro without alteration of its apoprotein component. The DAG content of LDL increased in a linear manner with respect to LTP concentration and time during the initial phase of the reaction, demonstrating the utility of this system as a quantitative assay method for LTP-mediated net DAG transfer. When [3H]DAG-labeled LDL was prepared and employed in transfer experiments with unlabeled lipophorin, labeled DAG was recovered in the HDLp-L fraction. The amount of labeled DAG recovered in the HDLp-L fraction was dependent on the ratio of LDL to HDLp-L in the reaction. Thus, in this system, LTP-mediated DAG redistribution is bidirectional, suggesting that the final equilibrium distribution of lipid may be dictated by the properties of potential donor/acceptor lipoproteins rather than by an inherent particle substrate specificity of LTP.

Synthesis of 4R- and 4S-tritium labeled NADPH for the determination of the coenzyme stereospecificity of NADPH: protochlorophyllide oxidoreductase.

A rapid and easy method for the production of both the 4R and 4S tritium labeled isomers of either NADH or NADPH has been developed. The method requires the use of only a single labeled compound (D-[1(-3)H] glucose), and two enzymes (glucose dehydrogenase from Bacillus sp. and alcohol dehydrogenase from Thermoanaerobium brockii) which are specific for the pro S and pro R hydrogens, respectively, of either NADH or NADPH. The 4R and 4S tritium labeled isomers of NADPH have been used to determine that NADPH:protochlorophyllide oxidoreductase from etiolated wheat was specific for the pro S hydrogen of NADPH.