Nitric oxide: a signaling molecule against mitochondrial permeability transition- and pH-dependent cell death after reperfusion

Reperfusion of ischemic tissue can precipitate cell death. Much of this cell killing is related to the return of physiological pH after the tissue acidosis of ischemia. The mitochondrial permeability transition (MPT) is a key mechanism contributing to this pH-dependent reperfusion injury in hepatocytes, myocytes, and other cell types. When ATP depletion occurs after the MPT, necrotic cell death ensues. If ATP levels are maintained, at least in part, the MPT initiates apoptosis caused by mitochondrial swelling and release of cytochrome c and other proapoptotic factors. Cyclosporin A and acidotic pH inhibit opening of permeability transition pores and protect cells against oxidative stress and ischemia/reperfusion injury, whereas Ca²⁺, mitochondrial reactive oxygen species, and pH above 7 promote mitochondrial inner membrane permeabilization. Reperfusion with nitric oxide (NO) donors also blocks the MPT via a guanylyl cyclase and protein kinase G-dependent signaling pathway, which in turn prevents reperfusion-induced cell killing. In isolated mitochondria, a combination of cGMP, cytosolic extract, and ATP blocks the Ca²⁺-induced MPT, an effect that is reversed by protein kinase G inhibition. Thus, NO prevents pH-dependent cell killing after ischemia/reperfusion by a guanylyl cyclase/cGMP/protein kinase G signaling cascade that blocks the MPT.     

Epigallocatechin gallate markedly enhances formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the reaction of 2'-deoxyguanosine with hypochlorous acid

A tea polyphenol, (-)-epigallocatechin gallate (EGCG), which can scavenge a variety of reactive oxygen species, enhances the yield of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) up to 20-fold in the reaction of 2'-deoxyguanosine with hypochlorous acid (HOCl), compared with the reaction without EGCG. Certain concentrations of EGCG inhibited HOCl-mediated oxidation of 2'-deoxyguanosine to 8-oxo-dG to a limited extent, but efficiently inhibited further oxidation of 8-oxo-dG to spiroiminodihydantoin nucleoside, resulting in the accumulation of 8-oxo-dG in the reaction mixture. Conversely, EGCG inhibited dose-dependently an increase in 8-oxo-dG levels in calf thymus DNA incubated with HOCl. However, addition of HOCl to the DNA preoxidized with an oxidant-generating system (CuCl2, ascorbate, H2O2), led to the extensive loss of 8-oxo-dG due to its further oxidation. EGCG effectively inhibited this HOCl-mediated loss of 8-oxo-dG in the oxidized DNA, resulting in an apparent increase in 8-oxo-dG levels in the oxidized DNA, compared with the levels found without EGCG. The conversion of 8-oxo-dG into other oxidized lesions will inevitably affect recognition by DNA repair enzymes as well as the rates of mutations and DNA synthesis. Thus, our results suggest that as a biomarker of oxidative DNA damage, not only 8-oxo-dG but also the products of its further oxidation should be analyzed.     

Orally active factor Xa inhibitors: 4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine derivatives

In our investigation of factor Xa inhibitors, a series of 1-(6-chloronaphthalen-2-yl)sulfonyl-4-(4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine-2-carbonyl)piperazines 3a-i were synthesized. In vitro inhibitory activities of the compounds against factor Xa and coagulation are summarized. Among the compounds, 3c and 3d, possessing a carbamoyl or N-methylcarbamoyl moiety, showed potent inhibitory activities when administered orally to rats.     

Seasonal variation of phosphatidylcholine hydroperoxides in blood of sweet smelt Plecoglossus altivelis

Sweet smelt was reared at two fishery experimental stations for 5 months from June to October. Every 2 weeks blood was collected from the caudal vessels and, subsequently, the phosphatidylcholine hydroperoxide contents and the fatty acid compositions in the blood were determined by high-performance liquid chromatography and gas chromatography, respectively. The seasonal variation of the contents of accumulated hydroperoxides and fatty acids in the sweet smelt blood were observed in both experimental stations. Sweet smelt started performance of cucumber-like or watermelon-like aroma in the middle of July and the aroma was enhanced in August. The content of phosphatidylcholine hydroperoxides and the amount of total fatty acid in the fish blood, in terms of possible precursors of volatile compounds, were also extremely high in the same period. According to lipid peroxidation mechanisms, the strong characteristic aroma of sweet smelt during July to August might be due to the high contents of accumulated lipid hydroperoxides and polyunsaturated fatty acids in their tissues.     

Propagation of rat parvovirus in thymic lymphoma cell line C58(NT)d and subsequent appearance of a resistant cell clone after lytic infection

Rat parvovirus (RPV) is nonpathogenic in rats but causes persistent lymphocytotropic infection. We found that RPV was propagated in rat thymic lymphoma cell line C58(NT)D and induced apoptosis. Interestingly, a resistant subclone, C58(NT)D/R, from surviving cells after lytic infection had differentiated phenotypic modifications, such as increased cell adherence, resistance to apoptosis, and suppressed tumorigenicity.     

Effect of P(2)' site tryptophan and P(20)' site deletion of Momordica charantia trypsin inhibitor II on inhibition of proteinases

Momordica charantia trypsin inhibitor II (MCTI-II) inhibits the amidolytic activity of factor Xa with a K(i) value 10-100-fold smaller than those of other squash family inhibitors. It also inhibits factor X activation mediated by factor VIIa-tissue factor complex or factor IXa. Comparison of other squash family inhibitors reveal Trp at position 7 (P(2)') and a deletion at position 25 (P(20)') are characteristics of MCTI-II. In order to elucidate the effect of these positions on the inhibitory activity, we chemically synthesized three inhibitors: S-MCTI-II whose amino acid sequence is identical to natural MCTI-II, S-MCTI-II(7L) whose P(2)'(Trp) is substituted with Leu, and S-MCTI-II(25N) whose P(20)'(deletion) is filled with Asn. The dissociation constants of the complexes of human factor Xa with S-MCTI-II, S-MCTI-II(7L), and S-MCTI-II(25N) were 1.3x10(-6) M, 2.8x10(-5) M, and 7.3x10(-6) M, respectively. They inhibited factor X activation mediated by factor VIIa with the same degree. As in the case of natural MCTI-II, S-MCTI-II suppressed factor X activation mediated by factor IXa, while S-MCTI-II(7L) and S-MCTI-II(25N) did not. Both the Trp at the P(2)' position and deletion at the P(20)' position are thus likely required for the inhibition of factor Xa, trypsin, and factor IXa, while these two positions do not affect factor X activation initiated by the factor VIIa-tissue factor complex.     

Immunohistochemical study on the expression of c-Fos and c-Jun in human skin wounds

An immunohistochemical study on the temporal expression of c-Fos and c-Jun, both of which designate proto-oncogene products, was performed on 60 human skin wounds with different post-infliction intervals. In unwounded skin, c-Fos or c-Jun was immunolocalized at the nuclei of the epidermal cells in the basal layer, hair follicle cells and sweat gland cells. During the early inflammatory phase of wound healing, the nuclei of polymorphonuclear cells (probably neutrophils), mainly infiltrating at the wound site, were labeled with anti-c-Fos or -c-Jun antibody. As the wound age increased, the neutrophils had disappeared at the wound site, and both mononuclear cells (probably macrophages) and spindle-shaped fibroblastic cells, which expressed a c-Fos or c-Jun positive reaction in the nuclei, were mainly observed. Morphometrically, the distribution of the c-Fos-positive ratio was very similar to that of the c-Jun-positive ratio; the positive ratio was considerably increased in wound specimens with a post-infliction interval of 1 day, thus indicating the late inflammatory or proliferative phase. This study showed that c-Fos and c-Jun were closely involved in the inflammatory phase as well as the proliferative phase of the wound healing process.     

Immunohistochemical study on Fas and Fas ligand in skin wound healing

An immunohistochemical study on the expression of Fas and Fas ligand (Fas L) was performed in order to examine the role of apoptosis through Fas–Fas L in mouse skin wound healing. After a 1-cm-long incision in the central dorsum skin, mice were sacrificed at intervals ranging from 0.5 to 240h, followed by the sampling of wound margin. The expression of Fas and Fas L in the wound margins and in uninjured skin controls was studied using frozen sections. In uninjured skin controls, a very weak expression of Fas and Fas L was detected immunohistochemically in hair follicles, sebaceous glands and epidermal cells. In wounded specimens, polymorphonuclear cells and inflammatory mononuclear ones (round-shaped and spindle-shaped types) were evident. A single immunostaining showed that Fas or Fas L was detectable in inflammatory mononuclear cells involved in the skin wound healing process. Double immunostaining for Fas and Fas L revealed that inflammatory mononuclear cells co-expressed both antigens. In situ TUNEL combined with immunostaining showed that the inflammatory mononuclear cells expressing Fas or Fas L and the polymorphonuclear cells were TUNEL-stained, although neither Fas nor Fas L was detected in the polymorphonuclear cells. The number of TUNEL-positive, inflammatory mononuclear cells expressing Fas or Fas L per 0.01×0.01cm² was counted. The average number of 10 randomly selected microscope fields reached a peak at the fibro-proliferative phase of wound healing. These results indicate that apoptosis through Fas and Fas L may play an important role for reducing the cellularity during skin wound healing in mice.     

Nitric oxide prevents gamma-radiation-induced cell cycle arrest by impairing p53 function in MCF-7 cells

We previously reported that nitric oxide (NO) released from S-nitrosoglutathione induces conformational change of the p53 tumor-suppressor protein that impairs its DNA-binding activity in vitro. We now demonstrate that MCF-7 cells preincubated in the presence of 0.5-1 mM S-nitrosoglutathione for 4 h before gamma-irradiation failed to arrest in the G1 phase of the cell cycle, whereas those gamma-irradiated without S-nitrosoglutathione exhibited a normal cell cycle arrest. The S-nitrosoglutathione-treated cells did not express the p53 target gene p21(waf-1) after gamma-irradiation, although p21(waf-1) was strongly expressed in cells irradiated in the absence of S-nitrosoglutathione. These results strongly suggest that NO impairs the function of p53 possibly via conformational change and/or amino acid modifications. On the other hand, cells incubated for 16 h in the presence of 1 mM S-nitrosoglutathione underwent apoptosis with accumulation of the pro-apoptotic protein Bax. This Bax accumulation, however, was shown to occur via a p53-independent pathway.