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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.

Collagen is critical for skin strength and elasticity, and its degradation leads to wrinkles that accompany aging. Based emphasis on the aesthetics, we tried to make a new compound that can highly stimulate collagen biosynthesis and synthesized ascorbyl conjugated peptide that is a complex form connected by succinoyl linker. We conducted several in vitro and in vivo experiments to identify if the compound has a potent activity, comparing to the ascorbic acid only for collagen biosynthesis. Our in vitro and in vivo result identified that ascorbyl conjugated peptide can stimulate collagen biosynthesis in human dermis and is assumably stable in the rat skin extracts. In conclusion, we strongly suggest that ascorbyl conjugated peptide can be used as a main ingredient for cosmetic products as well as wound healing agents.

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.

Prothrombin, a protein involved in blood coagulation, is a plasma glycoprotein composed of the Gla domain, two adjacent kringle domains, and a serine protease domain. Kringles are triple-disulfide-loop folding domains, which are found in several other blood proteins. In this study, we showed that recombinant human prothrombin kringle-1, -2. and -1-2 (rk-1, -2, -1-2) all have potent anti-angiogenic activities, which inhibit Lewis lung carcinoma (LLC) tumor growth and metastases. Recombinant human prothrombin kringles were expressed by an E. coli expression system and purified to apparent homogeneity from crude E. coli extracts. Purified rk-1, -2, -1-2 migrated with a molecular mass of 14, 19, and 31 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. rk-1, -2, -1-2 exhibited potent inhibitory effects on bFGF-stimulated bovine capillary endothelial cell growth with half-maximal concentrations (ED50) of approximately 41, 55, and 156 nM, respectively. All of the recombinant human prothrombin kringles also inhibited angiogenesis in the chorioallantoic membrane (CAM) of chick embryos at a dose of 20 microg. Systemic administration of rk-1, -2, -1-2 at a dose of 0.5 mg/kg/day suppressed the growth of primary LLC and at dose of 0.5 and 1.0 mg/kg/day inhibited LLC metastases in C57BL6/J mice lungs through their anti-angiogenic effects.

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.

An inhibitor of the proliferation of hepatic stellate cells (HSC) was purified from rat liver by a combination of gel filtration and ion exchange chromatography. The molecular mass of this non-arginase growth inhibitory factor (NAGIF) was determined to be 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The proliferation of HSC was inhibited by NAGIF with a 50% inhibitory dose of 5 nmol/liter. The inhibitory activity of NAGIF was not limited to HSC but also affected the growth of bovine endothelial cells and 3T6 fibroblasts. However, the growth of B16 mouse melanoma was not inhibited by NAGIF. The NH(2)-terminal sequence of NAGIF, AEPVEPWS, is identical to an internal sequence of rat Zn-alpha(2)-glycoprotein. Although the action mode of this inhibitor remains to be investigated, it seems very likely that NAGIF is involved in the negative control mechanism of HSC growth.

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.

The carbohydrate moieties of two anionic peroxidases, termed A1 and A2, and one cationic peroxidase, named C3, from Korean radish (Raphanus sativus) were studied. For profiling of N-glycans, each peroxidase was treated with peptidyl N-glycosidase F and hydrazine. These peroxidases were more susceptible to hydrazine than to peptidyl N-glycosidase F. When these three peroxidases were subjected to trifluoroacetic acid treatment, mannose, fucose and N-acetylglucosamine were released. Two major N-glycans of peroxidase C3 were isolated and treated with several glycohydrolases. Analysis of digested products of the two major N-glycans on polyacrylamide gel suggested that core-fucosylated trimannosylchitobiose may contain a different linkage from the typical α-1,6 of native N-linked oligosaccharide.

The valine sensitive acetolactate synthase (ALS) isozyme from Serratia marcescens ATCC 25419 was purified to homogeneity. Analysis of the native molecular weight of the purified enzyme by the native pore gradient polyacrylamide gel electrophoresis indicated the molecular weight of about 178,000 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the enzyme to be composed of two different types of subunits with molecular weights of 62,000 and 35,000. The molar ratio of the two polypeptides was estimated to be 1, suggesting that native enzyme is composed of two large subunits and two small subunits. The enzyme exhibits homotropic allosterism with pyruvate unlike other enteric ALS isozymes. The specificity ratio R (V[acetohydroxybutyrate]/V[acetolactate] = R.[alpha-ketobutyrate]/pyruvate]), of the enzyme was found to be 0 suggesting that the Serratia ALS has very high specificity for pyruvate. The pH optimum was around 7.5, and the enzyme was stable at 50 degrees C for 30 min. The pI value for the purified enzyme was 5.2. The concentration of branched chain amino acids for 50% inhibition of the enzyme was 0.1 mM for valine, and 1 mM for leucine and isoleucine, respectively.

We have shown that prothrombin kringle-2 (pKr-2), a domain of human prothrombin distinct from thrombin could activate cultured rat brain microglia in vitro. However, little is known whether pKr-2-induced microglial activation could cause neurotoxicity on dopaminergic (DA) neurons in vivo. To address this question, pKr-2 was injected into the rat substantia nigra (SN). Tyrosine hydroxylase (TH) immunohistochemistry experiments demonstrate significant loss of DA neurons seven days after injection of pKr-2. In parallel, pKr-2-activated microglia were detected in the SN with OX-42 and OX-6 immunohistochemistry. Reverse transcription PCR and double-label immunohistochemistry revealed that activated microglia in vivo exhibit early and transient expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and several proinflammatory cytokines. The pKr-2-induced loss of SN DA neurons was partially inhibited by the NOS inhibitor N(G)-nitro-L-arginine methyl ester hydrochloride, and the COX-2 inhibitor DuP-697. Extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were activated in the SN as early as 1 hr after pKr-2 injection, and localized within microglia. Inhibition of these kinases led to attenuation of mRNA expression of iNOS, COX-2 and several proinflammatory cytokines, and rescue of DA neurons in the SN. Intriguingly, following treatment with pKr-2 in vitro, neurotoxicity was detected exclusively in co-cultures of mesencephalic neurons and microglia, but not microglia-free neuron-enriched mesencephalic cultures, indicating that microglia are required for pKr-2 neurotoxicity. Our results strongly suggest that microglia activated by endogenous compound(s), such as pKr-2, are implicated in the DA neuronal cell death in the SN.

Pro-inflammatory mediators, such as nitric oxide (NO), prostaglandin E{sub 2} (PGE{sub 2}), and several cytokines (tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-1{beta}, and IL-6) are responsible for central nervous system (CNS) injuries that include ischemia, Alzheimer's disease, and neural death. Inhibition of these pro-inflammatory mediators would be an effective therapy to reduce the progression of neurodegenerative diseases. In this study, we examined the anti-inflammatory effects of a human prothrombin fragment-2-derived peptide, NSA9 (NSAVQLVEN), on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-activated brain microglia. NSA9 significantly inhibited the release of NO, PGE{sub 2}, and pro-inflammatory cytokines in a dose-dependent manner. Furthermore, NSA9 reduced the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 mRNA and protein, which control the production of NO and PGE{sub 2}, respectively. Moreover, NSA9 suppressed the LPS-induced nuclear translocation and activation of nuclear factor-{kappa}B (NF-{kappa}B). These results suggest that NSA9 strongly inhibits the pro-inflammatory responses of microglia through the modulation of NF-{kappa}B activity.

Prothrombin is a plasma glycoprotein involved in blood coagulation and, as we have previously reported, prothrombin kringles inhibit BCE (bovine capillary endothelial) cell proliferation. To reveal the mechanism, we investigated the influence of rk-2 (recombinant human prothrombin kringle-2) on the BCE cell cycle progression and ROS (reactive oxygen species) generation using FACS (fluorescence-activated cell sorter) analysis. Cell cycle analysis showed a decrease of G(1) phase cells in cells treated with bFGF (basic fibroblast growth factor) and an increase in cells treated with rk-2, as compared with the control cells. But, the portion of the S phase was reversed. In Western blot analysis, bFGF induced cytoplasmic translocation of p21(Waf1/Cip1) and p27(Kip1) and phosphorylation of p27(Kip1) but rk-2 treatment inhibited translocation of p21(Waf1/Cip1) and p27(Kip1) from nucleus to cytoplasm and phosphorylation of p27(Kip1). Also, rk-2 induced up-regulation of p53 and nuclear p21(Waf1/Cip1) and inhibited the cyclin D1/CDK4 (cyclin-dependent kinase 4) complex. The ROS level of rk-2-treated BCE cells was increased 2-fold when compared with the control, but treatment with NAC (N-Acetyl-L: -cysteine), an anti-oxidant, decreased ROS generation about 55% as compared with the rk-2 treatment. NAC treatment also restored cell cycle progression inhibited by rk-2 and down-regulated p53 and nuclear p21(Waf1/Cip1) expression induced by rk-2.These data suggest that rk-2 induces the BCE cell cycle arrest at G(0)-G(1) phase through inhibition of the cyclin D1/CDK4 complex caused by increase of ROS generation and nuclear cyclin-dependent kinase inhibitors.

Studies of the regulation of the activity of the Korean radish cationic peroxidase ( KRCP ) promoter during dedifferentiation and redifferentiation are reported here. Histochemical staining with 5-bromo-4-chloro-indolyl glucuronide (X-gluc) showed that only dedifferentiated marginal cells of leaf discs of the transgenic plants, but not of the interior region, were stained blue, as leaf discs were incubated on dedifferentiation-inducing medium from 5 days after callus induction (DACI). The levels of cationic peroxidase activity and of KRCP transcripts in Korean radish seedlings ( Raphanus sativus L. F1 Handsome Fall) were also upregulated by a low ratio of cytokinin to auxin, but not by high concentrations of cytokinin. To identify important cis -regulatory regions controlling callus-specific expression, a series of 5′ promoter deletions was carried out with KRCP::GUS gene fusion systems. The data suggest that at least two positively regulatory regions are involved in the KRCP::GUS expression during dedifferentiation induced by a low ratio of cytokinin to auxin: one from –471 to –242 and another from –241 to +196. GUS expression, however, was quickly decreased to a basal level during regeneration of root and shoot. Thus, the downstream region between +197 and +698 seems to be enough to suppress GUS expression of all constructs during regeneration. We further show that the 142-bp fragment (–471 to –328) has at least one cis -element to bind to the nuclear proteins from Korean radish seedlings induced by dedifferentiation.

We constructed the synthetic peptide library representing human prothrombin fragment 2 (F2) sequence and explored the inhibitory sequence for prothrombinase, which was reconstituted in vitro by adding factor Xa, factor Va, and calcium into phospholipids. The nonapeptide NSAVLQVEN (NSA9) suppressed prothrombinase reconstituted not only on phospholipid vesicles but also on the bovine capillary endothelial (BCE) cell surface. Kinetic analyses demonstrated that NSA9 is a mixed-type inhibitor of Xa. Furthermore, the nonapeptide inhibited the proliferation of BCE cells and also suppressed angiogenesis in chicken embryos. The inhibitory activities of NSA9 were abrogated by pre-incubation with anti-F2 monoclonal antibody, 4E7. These data demonstrate that anti-angiogenic activity of F2 may be related to its ability to inhibit prothrombinase.

Previously, we reported that the rabbit prothrombin fragment 2 (kringle 2 domain) has an anti-endothelial cell proliferative effect (Lee et al., J. Biol. Chem., in press). In this report, we show that not only rabbit prothrombin fragment 2 but also human prothrombin fragment 1 and 2 have an inhibitory effect on bFGF-stimulated BCE cell growth. Human prothrombin fragment 1 and 2 obtained as proteolytic fragments of human prothrombin display potent inhibitory effects on bovine capillary endothelial cells with a half-maximal concentration (ED50) of approximately 100 nM and 120 nM, respectively. As rabbit prothrombin fragment 2, the human prothrombin fragment 1 and 2 also inhibit angiogenesis in the chorioallantoic membrane (CAM) of chick embryos.

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.

A TNF-like factor was purified from lipopolysaccharide (LPS) induced New Zealand white rabbit serum. The TNF-like factor was purified by DEAE-Sephacel, Sephacryl S-200, Mono-Q, CM-affi gel Blue, Superose 12 H/R preparative columns to the specific activity of 4 x 10(6) U/mg protein. The purified protein was 45 kDa in its oligomeric form and 22 kDa in its monomeric form. Rabbit TNF-like factor had a pI value of 5.0 and was resistant to trypsin digestion. The TNF-like factor reacted with polyclonal-Ab against human TNFalpha on immunoblot and immunoprecipitation analysis and interacted with human TNF receptors. Taken together, rabbit TNF-like factor might be a high molecular weight form of rabbit TNFalpha.

Two cationic isoperoxidases (designated C1 and C3) and four anionic isoperoxidases (designated A1, A2, A3n and A3) from Korean radish (Raphanus sativus L.) root have been purified to apparent homogeneity, and some of their enzymatic properties were characterized. All six isoperoxidases are glycoproteins composed of a single polypeptide chain. The Mrs Of C1, C3, A1 and A2 were ca 44 000, while anionic isoperoxidase A3n and A3 have Mrs of 31 000 and 50 000, respectively. Deglycosylated A2 and C3 by trifluoromethanesulphonic acid treatment showed Mrs of 37 000 and 40 000, respectively, suggesting that the carbohydrate contents for these isoenzymes are 14 and 9%, respectively. Relative amino acid compositions of four anionic isoperoxidases (designated A1, A2, A3n and A3) and one cationic isoperoxidase C3 were determined. N-Terminal amino acid sequences were determined for A1, A3n and C3, while A2 was found to have a blocked amino terminal residue. Kinetic studies with respect to various synthetic and naturally occurring substrates were also investigated.

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.