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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.

Summary A short distance migrating cationic peroxidase from Korean radish seeds (Raphanus sativus) was detected. Cationic peroxidase C s was purified to apparent homogeneity and characterized. The molecular mass of the purified cationic peroxidase C s was estimated to be about 44 kDa on SDS-PAGE. After reconstitution of apoperoxidase C s with protohemin, the absorption spectra revealed a new peak in the Soret region around 400 nm, which is typical in a classical type III peroxidase family. The optimum pH of peroxidase activity for o-dianisidine oxidation was observed at pH 7.0. Kinetic studies revealed that the reconstituted cationic peroxidase C s has K m values of 1.18 mM and of 1.27 mM for o-dianisidine and H2O2, respectively. The cationic peroxidase C s showed the peroxidase activities for native substrates, such as coumaric acid, ferulic acid, and scopoletin. This result suggested that cationic peroxidase Cs plays an important role in plant cell wall formation during seed germination.

Regulation of the Korean radish cationic peroxidase (KRCP) promoter by auxins–cytokinins are described in this study. Various fragments of the KRCP promoter have been cloned and transcriptionally fused to the GUS gene to transform tobacco BY-2 cells, arabidopsis, and tobacco plants. Cytokinins decreased the dose- and time-dependent GUS expression of the pBK12 construct in the BY-2 cells. In contrast, the GUS expression of the pBK12 construct was significantly induced by a low ratio of cytokinin to auxin, although the GUS expression was not observed in any organ of the transgenic arabidopsis and tobacco plants at any stage of normal development under no hormone treatment. The GUS activities of the pBK12 construct driven by a low ratio of cytokinin to auxin were over 400- and 58-fold higher in the leaves and stems, respectively, than in those of the untreated arabidopsis plants. The induced GUS staining was mainly localized in the leaves and stems treated with the low ratio of cytokinin to auxin. These results suggested that the promoter activity of the KRCP gene be up-regulated by the low ratio of cytokinin to auxin. The KRCP promoter exhibited a higher degree of specificity to a low ratio of cytokinin to auxin rather than to either auxin or cytokinin alone in tobacco and arabidopsis plants. To date this study provides the first evidence that the combination of cytokinin and auxin takes part more in the regulation of the activity of KRCP promoter than in each hormone alone.

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.

The Korean radish cationic peroxidase promoter (KRPP) and the β -glucuronidase (GUS) gene were fused on the genome of tobacco BY-2 cells via Agrobacterium -mediated transformation method. A fragment of the KRPP comprising nucleotides −471 to +704 relative to the transcriptional initiation site confers constituent expression of GUS in transgenic tobacco BY-2 cells (BYK12). The expressed GUS activity was increased by as much as 40% at 577 nM gibberellic acid (GA 3 ). This increase was time and dose dependent. The abscisic acid (ABA) from 10 to 100 μM slowly decreased the GUS activity and at 100 μM reduced completely the inductive effect of GA 3 on GUS expression. Thus, GA 3 and ABA have antagonistic effects on the GUS expression mediated by the Korean radish cationic peroxidase promoter in transgenic BYK12 cells. Exogenous GA 3 activated the activity of the Korean radish isoperoxidases and also increased the intensity of two bands of Korean radish cationic isoperoxidases, when hypocotyl portions of the Korean radish seedlings grown in a green house for 10 days were examined using starch gel electrophoresis. However, ABA decreased the intensity of the Korean radish cationic isoperoxidase bands and also reduced the inductive effect of GA 3 in seedlings.

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.

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.

Angiogenesis, a multi-step process which involves endothelial cell proliferation, adhesion, migration, and basement membrane (BM) degradation, is essential for tumor metastasis. Here we show that recombinant human prothrombin kringle-2 (rk-2) inhibited bovine capillary endothelial cell migration with an IC(50) (concentration for half maximal inhibition) of 38 nM and inhibited adhesion to extracellular matrix (ECM) proteins. Because tumor metastasis requires angiogenesis, we examined whether rk-2 could inhibit metastases induced by injection of B16F10 melanoma cells into mice. The results revealed that the metastatic tumors in mouse lung were markedly decreased in a dose-dependent manner and acute lung injury induced by B16F10 melanoma metastasis was diminished by systemic rk-2 treatment. In immunohistochemical analysis, rk-2 reduced expression of vascular endothelial growth factor, which is a potent angiogenic activator and neovascularization in the mouse lung. Also, rk-2 diminished the expression of matrix metalloproteinase-2 and -9 in the mouse lung which induces tumor metastasis and angiogenesis. These data suggest that inhibition of B16F10 melanoma metastasis by rk-2 was caused by inhibition of neovascularization and reduction of matrix metalloproteinase expression.

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.

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.