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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.

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.

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.

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.

Objectives: This study aimed to evaluate the feasibility of laparoscopic metastasectomy (LM) in the treatment of ovarian metastases from gastric cancer and to compare the surgical outcomes with patients who underwent open metastasectomy (OM). Methods: The cases of 73 patients who underwent LM (n = 16) or OM (n = 57) were retrospectively reviewed. All patients were diagnosed with gastric cancer and, subsequently, underwent a metastasectomy at Yonsei University Health System between December 2002 and March 2011. Results: Sixteen operations were completed laparoscopically with no conversion to laparotomy. Complete cytoreduction surgery was achievable in 13 patients (81.3%). Operating time, complete cytoreduction, and occurrence of perioperative complications were comparable between the 2 groups. The LM group had less blood loss (25 vs 400 mL, P G 0.0001), earlier return to a general diet (3 vs 4 days, P = 0.005), shorter postoperative hospital stay (4.5 vs 7 days, P G 0.0001), and lower postoperative pain scores after 6, 24, and 48 hours than those in the OM group. There were no operative complications in the LM group. Conclusions: As a surgical treatment for ovarian metastases from gastric cancer, LM is feasible and provides benefits to patients without detrimental effects on the clinical outcomes for selected patients.