Role of xanthine oxidase inhibitor as free radical scavenger: a novel mechanism of action of allopurinol and oxypurinol in myocardial salvage

Xanthine oxidase (XO) has been hypothesized to be a potential source of oxygen-derived free radicals during reperfusion of ischemic myocardium based on the fact that allopurinol, a XO-inhibitor, can reduce reperfusion injury. In this communication we report that both allopurinol and oxypurinol, the principle metabolite of allopurinol, prevent the reperfusion injury in isolated pig heart. However, we found that neither pig heart nor pig blood contain any XO activity. Our study showed a direct free radical scavenging action of these XO-inhibitors during ischemia and reperfusion, as judged by the reduction of free radical signals when compared using an Electron Paramagnetic Resonance Spectrometer. Using a Luminometer, we also confirmed that both allopurinol and oxypurinol can scavenge ClO2, HOCl, and significantly inhibit free radical signals generated by activated neutrophils. These XO-inhibitors, however, failed to scavenge O2. and OH. radicals. Our results suggest that these XO-inhibitors salvaged the ischemic-reperfused myocardium by scavenging free radicals, and not by inhibiting XO in the pig heart.     

Comparison by 1H-nmr spectroscopy of the conformation of the 2600 dalton antifreeze glycopeptide of polar cod with that of the high molecular weight antifreeze glycoprotein

The antifreeze glycopeptide (AFGP-8) from polar cod, B. saida, is a 14-amino acid polypeptide having alternating glycotripeptide sequences of Ala-[Gal(β1 → 3)GalNAc(β1 → O)]-Thr-Pro and Ala-[Gal(β1 → 3)GalNAc(β1 → O)]-Thr-Ala, with alanyl residues at amino and carboxy terminals. Conformational studies of AFGP-8 have been carried out by ¹H-nmr and empirical energy calculations to investigate the difference in its antifreeze behavior from that of the more active high-molecular weight AFGP 1-4 of P. borchgrevinki. The ¹H-nmr spectra, including the resonances of the exchangeable amide protons, were assigned by two-dimensional correlated spectroscopy (COSY), one-dimensional difference decoupling, and nuclear Overhauser effect (NOE) measurements. For the four threonyl residues, the amide proton coupling constants and the small coupling constants between H^α and H^β indicate similar conformations, despite significant chemical shift differences. The strong NOE between the α protons and the amide protons of the residue following together with large temperature coefficients of chemical shifts, indicate an extended conformation not consisting of α-helix, turns or bends. Energy computations indicate several low-energy conformations consistent with the observed coupling constants for ?. Among these, a left-handed helical conformation with three repeating residues per turn has been proposed, which is in accordance with the observed NOE between the methyl group of the α-GalNAc and Ala H^βs. While the observed Overhauser effects in the threonyl side chain suggest a certain amount of conformational averaging, the effect involving the acetmido methyl of α-GalNAc and H^βs of Ala indicate that it as is a major conformer. In view of the close similarity between the conformations of AFGP-8 and the more active antifreeze polymer, AFGP 1-4, we propose that the difference in their activities is due to the length of the regular repeating structure with glycosylation at every third amino acid residue, and not due to any fundamental difference in their conformations.     

Prostaglandins can modify gamma-radiation and chemical induced cytotoxicity and genetic damage in vitro and in vivo

The effect of prostaglandin E1, E2, and F2 alpha on gamma-radiation, benzo(a)pyrene and diphenylhydantoin-induced cytotoxicity in vivo and genotoxicity in vitro was investigated. Prostaglandin E1 prevented both cytotoxic and genotoxic actions of all the three agents, where as both PGE2 and PGF2 alpha were ineffective. In fact, it was seen that both PGE2 and PGF2 alpha are genotoxic by themselves. Gamma-linolenic acid and dihomogamma-linolenic acid, the precursor of PGE1 were also as protective as that of PGE1, where as arachidonic acid, the precursor of 2 series PGs, has genotoxic actions to human lymphocytes in vitro. These results suggest that prostaglandins and their precursors can determine the susceptibility of cells to cytotoxic and genotoxic actions of chemicals and radiation. This study is particularly interesting since, it is known that some tumor cells contain excess of PGE2 and PGF2 alpha and many carcinogens can augment the synthesis of 2 series of PGs.     

Protective role of fructose 1,6-bisphosphate during CCl4 hepatotoxicity in rats.

Rats were injected intraperitoneally with CCl4 (2.5 ml/kg body wt.) and the hepatotoxicity was compared with that of rats receiving the same dose of CCl4 and an intraperitoneal injection of fructose 1,6-bisphosphate (2 g/kg body wt.). A 50-70% decrease in plasma aspartate aminotransferase and alanine aminotransferase activities was observed in the latter treatment, indicating a protective role of the sugar bisphosphate in CCl4 hepatotoxicity. The protection was accompanied by elevated hepatic activities of ornithine decarboxylase at 2, 6 and 24 h, S-adenosylmethionine decarboxylase at 6 h, and spermidine N1-acetyltransferase at 2 h. The increase in the enzymes involved in polyamine metabolism was shown in our previous work [Rao, Young & Mehendale (1989) J. Biochem. Toxicol. 4, 55-63] to correlate with increased polyamine synthesis or interconversion, which was related to the extent of hepatocellular regeneration. The hepatic contents of fructose 1,6-bisphosphate and ATP significantly decreased after CCl4 treatment, and administration of the sugar bisphosphate increased hepatic ATP. Fructose 1,6-bisphosphate, an intermediary metabolite of the glycolytic pathway, may decrease CCl4 toxicity by increasing the ATP in the hepatocytes. The ATP generated is useful for hepatocellular regeneration and tissue repair, events which enable the liver to overcome CCl4 injury.     

Characterization of poly(ADP-ribosyl)ated domains of rat pachytene chromatin.

Poly(ADP-ribosyl)ation of nuclear proteins was several-fold higher in the pachytene spermatocytes than in the premeiotic germ cells of the rat. Among the histones of the pachytene nucleus, histone subtypes H2A, H1 and H3 were poly(ADP-ribosyl)ated. Based on the immunoaffinity fractionation procedure of Malik, Miwa, Sugimara & Smulson [(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2554-2558] we have fractionated DNAase-II-solubilized chromatin into poly(ADP-ribosyl)ated chromatin (PAC) and non-poly(ADP-ribosyl)ated chromatin (non-PAC) domains on an anti-[poly(ADP-ribose)] IgG affinity matrix. Approx. 2.5% of the pachytene chromatin represented the PAC domains. A significant amount of [alpha-32P]dATP-labelled pachytene chromatin (labelled in vitro) was bound to the affinity matrix. The DNA of pachytene PAC domains had internal strand breaks, significant length of gaps and ligatable ends, namely 5'-phosphoryl and 3'-hydroxyl termini. On the other hand, the PAC domains from 18 h regenerating liver had very few gaps, if any. The presence of gaps in the pachytene PAC DNA was also evident from thermal denaturation studies. Although many of the polypeptides were common to the PAC domains of both pachytene and regenerating liver, the DNA sequences associated with these domains were quite different. A 20 kDa protein and the testis-specific histone H1t were selectively enriched in the pachytene PAC domains. The pachytene PAC domains also contained approx. 10% of the messenger coding sequences present in the DNAase-II-solubilized chromatin. The pachytene PAC domains, therefore, may represent highly enriched DNA-repair domains of the pachytene nucleus.     

Hydrogen peroxide permeation across liposomal membranes: a novel method to assess structural flaws in liposomes

Hydrogen peroxide permeation across large multilamellar vesicles of defined and complex lipid composition was shown to obey precise kinetic relationships for the activity of the occluded catalase. Careful assay conditions precluded simultaneous peroxidative damage to the lipids. The kinetic data was consistent with a barrier role for the bilayer for hydrogen peroxide permeation. More interestingly, hydrogen peroxide permeation across liposomes of complex lipid mixtures exhibited osmotic inhibition of permeation of hydrogen peroxide. On the other hand, purified egg lecithin vesicles did not exhibit any effect of external osmolality on hydrogen peroxide permeation in an experimentally defined non-lytic zone of external osmolarity. These results argue in favour of a heterogeneous, heteroporous structure of bilayers with complex lipid composition.     

Developmental and Dimensional Changes in the Cambial Fusiform Initials ofHoloptelea integrifolia (Roxb.) Planch.

Divisional activity, intrusive growth of the cell wall and loss of fusiform initials have been studied in Holoptelea integrifolia. The dimensional changes in relation to mean length, length frequency, mean width and length variation in relation to fibre length have also been analysed.     

Effects of nucleotide bromination on the stabilities of Z-RNA and Z-DNA: a molecular mechanics/thermodynamic perturbation study

The structures of ZI- and ZII-form RNA and DNA oligonucleotides were energy minimized in vacuum using the AMBER molecular mechanics force field. Alternating C-G sequences were studied containing either unmodified nucleotides, 8-bromoguanosine in place of all guanosine residues, 5-bromocytidine in place of all cytidine residues, or all modified residues. Some molecules were also energy minimized in the presence of H2O and cations. Free energy perturbation calculations were done in which G8 and C5 hydrogen atoms in one or two residues of Z-form RNAs and DNAs were replaced in a stepwise manner by bromines. Bromination had little effect on the structures of the energy-minimized molecules. Both the minimized molecular energies and the results of the perturbation calculations indicate that bromination of guanosine at C8 will stabilize the Z forms of RNA and DNA relative to the nonbrominated Z form, while bromination of cytidine at C5 stabilizes Z-DNA and destabilizes Z-RNA. These results are in agreement with experimental data. The destabilizing effect of br5C in Z-RNAs is apparently due to an unfavorable interaction between the negatively charged C5 bromine atom and the guanosine hydroxyl group. The vacuum-minimized energies of the ZII-form oligonucleotides are lower than those of the corresponding ZI-form molecules for both RNA and DNA. Previous x-ray diffraction, nmr, and molecular mechanics studies indicate that hydration effects may favor the ZI conformation over the ZII form in DNA. Molecular mechanics calculations show that the ZII-ZI energy differences for the RNAs are greater than three times those obtained for the DNAs. This is due to structurally reinforcing hydrogen-bonding interactions involving the hydroxyl groups in the ZII form, especially between the guanosine hydroxyl hydrogen atom and the 3'-adjacent phosphate oxygen. In addition, the cytidine hydroxyl oxygen forms a hydrogen bond with the 5'-adjacent guanosine amino group in the ZII-form molecule. Both of these interactions are less likely in the ZI-form molecule: the former due to the orientation of the GpC phosphate away from the guanosine ribose in the ZI form, and the latter apparently due to competitive hydrogen bonding of the cytidine 2'-hydroxyl hydrogen with the cytosine carbonyl oxygen in the ZI form. The hydrogen-bonding interaction between the cytidine hydroxyl oxygen and the 5'-adjacent guanosine amino group in Z-RNA twists the amino group out of the plane of the base. This may be responsible for differences in the CD and Raman spectra of Z-RNA and Z-DNA.