Protective effect of resveratrol on beta-amyloid-induced oxidative PC12 cell death

Beta-amyloid peptide is considered to be responsible for the formation of senile plaques that accumulate in the brains of patients with Alzheimer's disease. There has been compelling evidence supporting the idea that beta-amyloid-induced cytotoxicity is mediated through the generation of reactive oxygen intermediates (ROIs). Considerable attention has been focused on identifying phytochemicals that are able to scavenge excess ROIs, thereby protecting against oxidative stress and cell death. Resveratrol (3,5,4'-trihydroxy-trans-stilbene), a phytoalexin found in the skin of grapes, has strong antioxidative properties that have been associated with the protective effects of red wine consumption against coronary heart disease ("the French paradox"). In this study, we have investigated the effects of resveratrol on beta-amyloid-induced oxidative cell death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with beta-amyloid exhibited increased accumulation of intracellular ROI and underwent apoptotic death as determined by characteristic morphological alterations and positive in situ terminal end-labeling (TUNEL staining). Beta-amyloid treatment also led to the decreased mitochondrial membrane potential, the cleavage of poly(ADP-ribose)polymerase, an increase in the Bax/Bcl-X(L) ratio, and activation of c-Jun N-terminal kinase. Resveratrol attenuated beta-amyloid-induced cytotoxicity, apoptotic features, and intracellular ROI accumulation. Beta-amyloid transiently induced activation of NF-kappaB in PC12 cells, which was suppressed by resveratrol pretreatment.     

Production and characterization of thermostable cellulases from Streptomyces transformant T3-1

A total of 26 thermophilic isolates, selected from a compost of agricultural waste, which was mostly composed of vegetable, corncob and rice straw, were cultivated at 50 °C for further studies of thermostable cellulase production. The thermostable cellulase gene from the chromosomal DNA of actinomycetes isolate no. 10 was shotgun-cloned and transformed into Streptomyces sp. IAF 10-164. A transformant, T3-1, was found to be a good strain for the production of thermostable cellulases. Cultivation of T3-1 in modified Mandels–Reese broth containing 1% carboxymethylcellulose (CMC)-sodium salt and the optimal condition for microbial growth were studied. Batch cultivation in a flask revealed that CMCase and Avicelase production reached the maximum between the third to fifth day, whereas maximum -glucosidase production occurred on the ninth day. Microbial biomass increased from the first day to the fifth day and then decreased. The crude enzyme had the highest activity at 50 °C and at pH 6.5. The enzyme was shown to be a thermostable cellulase whose activities were stable at 50 °C for more than 7 days.     

The mitogenic and antiapoptotic actions of ghrelin in 3T3-L1 adipocytes

Ghrelin, a stomach-derived hormone, induces adiposity when administered to rodents. Because ghrelin receptor is abundantly expressed in adipose tissue, we investigated the role of ghrelin in adipocyte biology. We observed ghrelin receptor expression in 3T3-L1 preadipocytes and adipocytes. Treatment of preadipocytes with ghrelin induced cellular proliferation and differentiation to mature adipocytes, as well as basal and insulin-stimulated glucose transport, but it inhibited adipocyte apoptosis induced by serum deprivation. Exposure of 3T3-L1 cells to ghrelin caused a rapid activation of MAPKs, especially ERK1/2. Chemical inhibition of MAPK blocked the mitogenic and antiapoptotic effects of ghrelin. Ghrelin also stimulated the insulin receptor substrate-associated phosphatidylinositol 3-kinase/Akt pathway in 3T3-L1 preadipocytes and adipocytes, whereas inhibition of this pathway blocked the effects of ghrelin on cell proliferation, antiapoptosis and glucose uptake. These findings suggest that the direct effects of ghrelin on proliferation, differentiation, and apoptosis in adipocytes may play a role in regulating fat cell number. These effects may be mediated via activation of the MAPK and phosphatidylinositol 3-kinase/Akt pathways.     

Changes of [<Superscript>3</Superscript>H]Muscimol, [<Superscript>3</Superscript>H]Flunitrazepam and [<Superscript>3</Superscript>H]MK-801 Binding in Rat Brain by Prolonged Ventricular Infusion of 7-Nitroindazole

In the present study, we have investigated the effects of prolonged inhibition of nitric oxide synthase (NOS) by infusion of neuronal NOS (nNOS) inhibitor, 7-nitroindazole (7-NI), to examine modulation of NMDA and GABA_A receptor binding in rat brain. The duration of sleeping time was significantly increased by the pre-treatment with 7-NI (100 mg/kg) 30 min before pentobarbital (40 mg/kg) treatment in rats. However, the duration of pentobarbital-induced sleep was shortened by the prolonged infusion of 7-NI into cerebroventricle for 7 days. We have investigated the effect of NOS inhibitor on NMDA and GABA_A receptor binding characteristics in discrete areas of brain regions by using autoradiographic techniques. The GABA_A receptors were analyzed by quantitative autoradiography using [³H]muscimol and [³H]flunitrazepam binding, and NMDA receptor binding was analyzed by using [³H]MK-801 binding in rat brain slices. Rats were infused with 7-NI (500 pmol/10 l/ h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps. The levels of [³H]muscimol were markedly elevated in cortex, caudate putamen, and thalamus while the levels of [³H]flunitrazepam binding were only elevated in cerebellum by NOS inhibitor. However, there was no change in the level of [³H]MK-801 binding except decreasing in the thalamus. These results show that the prolonged inhibition of NOS by 7-NI-infusion highly elevates [³H]muscimol binding in a region-specific manner and decreases the pentobarbital-induced sleep.     

Preimplantational embryo development and incidence of blastomere apoptosis in bovine somatic cell nuclear transfer embryos reconstructed with long-term cultured donor cells

This study was performed to investigate whether types and/or age of donor cells affect preimplantational embryo development and the incidence of apoptosis in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine fetal or adult ear fibroblasts were isolated, cultured in vitro and categorized into fresh or long-term cultured cells in terms of population doublings (PD): in fetal fibroblasts, <16 being considered fresh and >50 being long-term cultured; in adult ear fibroblasts, <16 being considered fresh and >30 being long-term cultured. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199, enucleated and reconstructed by SCNT. The reconstructed oocytes were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) at 39 degrees C in a humidified atmosphere of 5% CO(2) air for 7 days. The early development of SCNT embryos was monitored under a microscope and the quality of blastocysts was assessed by differential counting of inner cell mass (ICM) and trophectoderm (TE) cells and by apoptosis detection in blastomeres using a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay. As results, types and/or age of donor cells did not affect the rate of blastocyst formation and the number of ICM and TE cells. However, a significant increase in apoptotic blastomeres was observed in SCNT embryos reconstructed with long-term cultured fetal or adult ear fibroblasts compared to those in SCNT embryos derived from fresh fetal or adult ear fibroblasts. In conclusion, these results indicated that the long-term culture of donor cells caused increased the incidence of apoptosis in bovine SCNT embryos but did not affect the developmental competence and the cell number of blastocysts.     

Design of novel analogues with potent antibiotic activity based on the antimicrobial peptide, HP(2-9)-ME(1-12)

To develop novel antibiotic peptides useful as therapeutic drugs, a number of analogues were designed to increase the hydrophobic helix region either by Trp-substitution or net positive charge increase by Lys-substitution, from HP(2-9)-ME(1-12). The antibiotic activities of these peptides were evaluated using bacterial (Salmonella tryphimurium, Proteus vulgaris, Bacillus subtilis and Staphylococcus aureus), fungi (Saccharomyces cerevisiae, Trichosporon beigelii and Candida albicans), tumor and human erythrocyte cells. The substitution of Lys for Thr at position 18 and 19 of HP(2-9)-ME(1-12) (HM5) increased activity against Proteus vulgaris and fungal strains without hemolysis. In contrast, substitution of Trp for Lys and Thr at positions 2, 15 and 19 of HP(2-9)-ME(1-12), respectively (HM3 and HM4), decreased activity but increased hemolysis against human erythrocytes. This suggests that an increase in positive charge increases antimicrobial activity whereas an increase in hydrophobicity by introducing Trp residues at C-terminus of HP(2-9)-ME(1-12) causes a hemolytic effect. Circular dichroism spectra suggested that the alpha-helical structure of these peptides plays an important role in their antibiotic effect but that the alpha-helical property is not connected with the enhanced antibiotic activity.     

Degradation of phytochrome interacting factor 3 in phytochrome-mediated light signaling

Plant photoreceptors regulate various developmental processes. Among the photoreceptors, phytochromes, red and far-red light receptors, regulate light responses through many signaling components, including phytochrome-interacting proteins. The functional relationships among phytochromes and their interacting proteins, however, have not been clearly established. Here, we sought to identify a functional relationship between phytochromes and phytochrome interacting factor 3 (PIF3). We demonstrate that PIF3 is polyubiquitinated rapidly and subsequently degraded in PHYA and PHYB-mediated light signaling. We also show that the degradation of PIF3 is mediated by the 26S proteasome. Our data indicate that light-stimulated phytochromes cause the degradation of their interacting protein, PIF3, by the 26S proteasome.     

Altered cAMP signaling induced by lysophosphatidic acid in senescent human diploid fibroblasts

Lysophosphatidic acid (LPA) is a lipid mitogen that acts through G-protein-coupled receptors. LPA responsiveness has been reported to be dependent on the senescent state of the cells. To solve the mechanism underlying, we observed LPA-dependent cAMP status and found its age-dependent contrasting profile such as high level of cAMP in the senescent cells vs its low level in the young cells. In order to clarify the molecular mechanism of the ageing effect, we examined various molecular species involved in the cAMP signaling pathway by semi-quantitative RT-PCR. EDG-1 and EDG-4 were unchanged, but EDG-2 and EDG-7 were reduced with age. Senescent cells showed a partial reduction of Gi1, Gi2, and Gi3, but no change in the level of Gq. Decreased Gis and Gi-coupled LPA receptors may reduce the inhibitory effect of Gi alpha on adenylyl cyclases (ACs), resulting in cAMP accumulation via activation of adenylyl cyclase in senescent fibroblasts. We also observed an age-dependent increase in some of AC isoforms: II, IV, and VI. In conclusion, multiple changes in the cAMP signaling pathway of the senescent cells might explain the altered responsiveness to the mitogenic stimuli.