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c-Jun N-terminal kinase (JNK) is activated by cellular stress and plays critical roles in diverse types of cell death. However, role of JNK in beta-cell injury is obscure. We investigated the role for JNK in streptozotocin (STZ)-induced beta-cell death. STZ induced JNK activation in insulinoma or islet cells. JNK inhibitors attenuated insulinoma or islet cell death by STZ. STZ-induced JNK activation was decreased by PARP inhibitors, suggesting that JNK activation is downstream of PARP-1. Phosphatase inhibitors induced activation of JNK and abrogated the suppression of STZ-induced JNK activation by PARP inhibitors, suggesting that the inhibition of phosphatases is involved in the activation of JNK by STZ. STZ induced production of reactive oxygen species (ROS) as potential inhibitors of phosphatases, which was suppressed by PARP inhibitors. PARP-1 siRNA attenuated insulinoma cell death and JNK activation after STZ treatment, which was reversed by MKP (MAP kinase phosphatase)-1 siRNA. These results suggest that JNK is activated by STZ downstream of PARP-1 through inactivation of phosphatases such as MKP, which plays important roles in STZ-induced beta-cell death.

In neurodegenerative diseases, such as Alzheimer's and Parkinson's, microglial cell activation is thought to contribute to CNS injury by producing neurotoxic compounds. Prothrombin and kringle-2 increase levels of NO and the mRNA expression of iNOS, IL-1beta, and TNF-alpha in microglial cells. In contrast, the human prothrombin kringle-2 derived peptide NSA9 inhibits NO release and the production of pro-inflammatory cytokines such as IL-1beta, TNF-alpha, and IL-6 in LPS-activated EOC2 microglia. In this study, we investigated the anti-inflammatory effects of NSA9 in human prothrombin- and kringle-2-stimulated EOC2 microglia. Treatment with 20-100 muM of NSA9 attenuated both prothrombin- and kringle-2-induced microglial activation. NO production induced by MAPKs and NF-kappaB was similarly reduced by inhibitors of ERK (PD98059), p38 (SB203580), NF-kappaB (N-acetylcysteine), and NSA9. These results suggest that NSA9 acts independently as an inhibitor of microglial activation and that its effects in EOC2 microglia are not influenced by the presence of kringle-2.

Microglia, the major immune effector cells in the CNS, become activated when the brain suffers injury. In this study, we observed that prothrombin, a zymogen of thrombin, induced NO release and mRNA expression of inducible NO synthase, IL-1β, and TNF-α in rat brain microglia. The effect of prothrombin was independent of the protease activity of thrombin since hirudin, a specific inhibitor of thrombin, did not inhibit prothrombin-induced NO release. Furthermore, factor Xa enhanced the effect of prothrombin on microglial NO release. Kringle-2, a domain of prothrombin distinct from thrombin, mimicked the effect of prothrombin in inducing NO release and mRNA expression of inducible NO synthase, IL-1β, and TNF-α. Prothrombin and kringle-2 both triggered the same intracellular signaling pathways. They both activated mitogen-activated protein kinases and NF-κB in a similar pattern. NO release stimulated by either was similarly reduced by inhibitors of the extracellular signal-regulated kinase pathway (PD98059), p38 (SB203580), NF-κB (N-acetylcysteine), protein kinase C (Go6976, bisindolylmaleimide, and Ro31-8220), and phospholipase C (D609 and U73122). These results suggest that prothrombin can activate microglia, and that, in addition to thrombin, kringle-2 is a domain of prothrombin independently capable of activating microglia.

Activation of hepatic stellate cells (HSC) has been identified as a critical step in hepatic fibrogenesis and is regulated by several factors including cytokines and oxidative stress. However, the molecular mechanism for HSC inactivation is not well understood. We investigated an N-acetyl-L-cysteine (NAC)-mediated signaling pathway involved in HSC inactivation. NAC, which acting through its reducing activity, induced cell arrest at G1 via the mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK pathway in a Ras-independent manner. The sustained activation of this extracellular signal-regulated kinase induced the expression of p21(Cip1/WAF1), a cell cycle-dependent kinase inhibitor, and mediated cell growth arrest through the Sp1 transcription activator-dependent mechanism. These effects of NAC were all reversed by treatment of HSC with MEK inhibitor PD98059 followed by culturing HSC on type I collagen-coated flasks. The collagen-mediated suppression of NAC-induced arrest may be due to an overriding of the cell cycle arrest through an acceleration of integrin-induced cell growth. NAC action is actually dependent on modulating the redox states of cysteine residues of target proteins such as Raf-1, MEK, and ERK. In conclusion, an understanding of the NAC signaling pathway in HSC should provide the theoretical basis for clinical approaches using antioxidant therapies in liver fibrosis.

To investigate the protection mechanism of Bcl-2 against tumour necrosis factor (TNF)-mediated cell death, the bcl2 gene was transfected into the L929 cells and stably expressed. Two clones having different sensitivity among bcl2 -transfected L929 clones had been isolated, and termed clone R1 and R2. It was observed that activation of manganese superoxide dismutase (MnSOD) and suppression of Jun kinase of clone R1 and R2 were correlated with protection from TNF cytotoxicity. Upon treatment with TNF, clone R1 and R2 were more resistant than control L929 cells against TNF cytotoxicity and the protective effect of clone R1 was stronger than clone R2. However, in case of TNF plus actinomycin D treatment, clone R1 was still resistant against TNF cytotoxicity, whereas clone R2 became more sensitive than control L929 cells. The JNK activities of clone R1 and R2 were suppressed upon TNF treatment and in case of TNF plus actinomycin D treatment, clone R2 showed a marked increase in JNK activities and had higher activity than control L929 cells. The specific activities of MnSOD of clone R1 and R2 upon TNF treatment were 70 U/ml and 33 U/ml, respectively, while the MnSOD activity was not detectable in control L929 cells. When TNF and actinomycin D were treated simultaneously, MnSOD activity was not detectable in control L929 cells and bcl2 -transfected L929 cells (clone R1, R2). Consistent with these results, both clone R1 and R2 showed higher levels of MnSOD mRNA expression than control L929 cells after TNF treatment. These data suggest that suppression of Jun kinase and increase of MnSOD may be involved in inhibitory action of Bcl-2 against TNF, and the balance between MnSOD and JNK signalling pathway may be an important factor for the protection of bcl2 -transfected L929 cells from TNF cytotoxicity.

To understand the effects of bcl-2 on glucose metabolism and tumor necrosis factor-alpha (TNF-alpha) mediated cytotoxicity, the activities of glycolytic enzymes (hexokinase, 6-phosphofructo-1-kinase, and pyruvate kinase), lactate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase were examined with or without TNF-alpha treatment in TNF-alpha sensitive L929 cells and TNF-alpha resistant bcl-2 transfected L929 cells. In TNF-alpha-treated L929 cells, the activities of the glycolytic enzymes and lactate dehydrogenase greatly increased, but there was no detectable change in phosphoenolpyruvate carboxykinase. Pyruvate carboxylase activity decreased by about 25% between 6 and 12 h after TNF-alpha treatment. The activities of the glycolytic enzymes and lactate dehydrogenase in bcl-2 transfected L929 cells were lower than in L929 cells upon TNF-alpha treatment. On the other hand, the activity of pyruvate carboxylase was 20-100% greater after 6 h of TNF-alpha treatment than in the L929 cells. The activity of phosphoenolpyruvate carboxykinase of bcl-2 trasfected L929 cells was lower by up to 25% than in L929 cells after 12 h. The increase of pyruvate carboxylase activity and decrease of phosphoenolpyruvate carboxykinase activity in bcl-2 transfected L929 cells may contribute to the protective effects of bcl-2 against TNF-alpha mediated cytotoxicity.

Recently, O’Reilly et al.(O’Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994) Cell 79, 315–328; O’Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R., and Folkman, J. (1997) Cell 88, 277–285) developed a simple in vitro angiogenesis assay system using bovine capillary endothelial cell proliferation and purified potent angiogenic inhibitors, including angiostatin and endostatin. Using a simplein vitro assay for angiogenesis, we purified a protein molecule that showed anti-endothelial cell proliferative activity from the serum of New Zealand White rabbits, which was stimulated by lipopolysaccharide. The purified protein showed only bovine capillary endothelial cell growth inhibition and not any cytotoxicity. This molecule was identified as a prothrombin kringle-2 domain (fragment-2) using Edman degradation and the amino acid sequence deduced from the cloned cDNA. Both the prothrombin kringle-2 domain released from prothrombin by factor Xa cleavage and the angiogenic inhibitor purified from rabbit sera exhibited anti-endothelial cell proliferative activity. The recombinant rabbit prothrombin kringle-2 domain showed potent inhibitory activity with half-maximal concentrations (ED50) of 2 μg/ml media. As in angiostatin, the recombinant rabbit prothrombin kringle-2 domain also inhibited angiogenesis in the chorioallantoic membrane of chick embryos.

Some chemical properties of the purified biodegredative threonine dehydratases ( l -threonine hydro-lyase (deaminating), EC 4.2.1.16) from Escherichia coli and Salmonella typhimurium are described. The overall amino acid compositions of the two enzymes appear similar with some variations in several amino acid residues. Tryptic peptide maps show that in S. typhimurium four peptides of E. coli origin are missing, whereas six peptides unique to Salmonella protein are present. Carboxymethylation reaction with iodo[14C]acetate to detect half-cystine residues indicates that peptides 21 and S5 in S. typhimurium, but not in E. coli enzyme, are labeled, and the reverse is true for peptide 22; four other peptides of S. typhimurium have more half-cystine residues than their counterparts in E. coli. In addition, the Salmonella enzyme appears to have several disulfide bonds. Despite these differences, the amino acid sequence of the amino termini of the two proteins reveals a highly conserved structure, with only three out of 25 residues being different. Reduction with tritium-labeled borohydride followed by tryptic fingerprinting of the two proteins shows that one peptide contains active-site pyridoxal phosphate. Modifier binding studies with the S. typhimurium enzyme indicate that pyruvate and glyoxylate occupy separate sites on the enzyme molecules. Further, there are two distinct sites for glyoxylate binding: in the monoglyoxylated form of the enzyme, only peptide 22 becomes labeled, whereas both peptides 22 and 21 of the tetraglyoxylated form of the dehydratase contain bound glyoxylate. These results support the earlier findings that these two metabolites regulate enzyme activity by two separate, mutually exclusive, mechanisms.