Effects of resveratrol-related hydroxystilbenes on the nitric oxide production in macrophage cells: structural requirements and mechanism of action

NF-kappaB that plays an important role in iNOS expression is one of the targets of various potential anti-inflammatory agents including resveratrol. Resveratrol contains a structural similarity with estrogen, and there has been speculation about resveratrol as estrogen agonist. In this study, the mechanism and structural requirements of resveratrol and related hydroxystilbenes for the inhibition of LPS-induced nitric oxide production were studied in macrophage cells (RAW 264.7 and J774) by comparing its effect on LPS-induced NF-kappaB translocation and nitric oxide production, and by considering the possibility of involvement of an estrogen receptor. LPS-induced nitric oxide production was inhibited only when cells were treated with resveratrol prior to stimulation with LPS, suggesting that resveratrol does not affect the enzyme itself. A higher concentration of resveratrol than needed for the inhibition of nitric oxide production was required for the inhibition of NF-kappaB mobilization or iNOS expression. Estrogen and diethylstilbesterol, an estrogen agonist, caused only weak inhibition of nitric oxide production, and the effects of resveratrol were not noticeably blocked by ICI-182780, an estrogen antagonist. Structure-activity analysis of resveratrol and nine hydroxystilbenes suggests that the structural balance between oxygen functional groups on the benzene rings is important for their activity. Our results suggest that resveratrol might act on other cellular targets as well as NF-kappaB at the initial stage of gene expression. Unique structural features of hydroxystilbenes are needed for suppression of nitric oxide production and it is unlikely that estrogen receptor is involved in it.     

Catalytic domains of the LAR and CD45 protein tyrosine phosphatases from Escherichia coli expression systems: purification and characterization for specificity and mechanism.

The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates. A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme. A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high kcat/Km value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of 18O from 18O4 inorganic phosphate into H2(16)O at rates of approximately 1 x 10(-2) s-1. The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate. Brief incubation of all three PTPase fragments with a [32P]phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of 32P-labeled enzymes. Pulse/chase studies on the LAR 32P-enzyme showed turnover of the labeled phosphoryl group.     

Enhancement by Putrescine of Gibberellin-Induced Elongation in Hypocotyls of Lettuce Seedlings

Effects of diamines, polyamines, and other basic amino acidson the growth of lettuce hypocotyls were investigated. Putrescine,cadaverine and agmatine enhanced the hypocotyl growth in thepresence of gibberellin, while spermidine and spermine werenon-effective. Arginine and ornithine, which may be precursorsof putrescine, had similar effect. While the growth inhibitiondue to arcaine (1,4-diguanidinobutane), which is a agmatineiminohydrolase inhibitor, was recovered by agmatine, cadaverine,putrescine, and spermidine, putrescine most effectively recoveredits growth-enhancing effect. (Received August 25, 1982; Accepted December 27, 1982)     

Changes in the reproductive function and developmental phenotypes in mice following intramuscular injection of an activin betaA-expressing plasmid

Background  

The TGF-beta family protein activin has numerous reported activities with some uncertainty in the reproductive axis and development. The precise roles of activin in in vivo system were investigated using a transient gain of function model.     

The role of serine 190 in FOXO nuclear export and cell death induction in Drosophila melanogaster

Proteins in the forkhead box O (FOXO) family contain three Akt phosphorylation sites that are important for export of the protein from the nucleus to the cytosol. In mammalian FOXO1, phosphorylation of serine 256 (S256) is a prerequisite for the phosphorylation of the other two sites. Although Drosophila FOXO (dFOXO) contains three well-conserved Akt phosphorylation sites, their role in the regulation of Drosophila physiology is not well understood. In the present study, we examine the regulation and function of phosphorylation at serine 190 (S190), which corresponds to S256 of mammalian FOXO1. Insulin and Akt were shown to increase S190 phosphorylation of dFOXO. Moreover, dFOXO nuclear export was induced by insulin treatment in both fly tissues and transfected Drosophila and human cells, and a protein containing an alanine substitution at S190 (dFOXO^S190A) was defective in these insulin-dependent responses, suggesting that S190 phosphorylation is required for dFOXO nuclear export. Interestingly, dFOXO^S190A and dFOXO^S190D mutants showed lower target gene expression and a reduced ability to induce cell death compared to wild-type dFOXO. These results suggest that the S190 residue is required for dFOXO translocation and is important for the pro-apoptotic function of dFOXO.     

Enhanced tolerance against freezing stress in<Emphasis Type="Italic">Escherichia coli</Emphasis> cells expressing an algal cyclophilin gene

Members of the cyclophilin (Cyp) family are known to function as co-chaperones, interacting with chaperones such as heat shock protein 90, and perform important roles in protein folding under high temperature stress. In addition, they have been isolated from a wide range of organisms. However, there have been no reports on the functions of algal Cyps under other stress conditions. To study the functions of the cDNAGjCyp-1 isolated from the red alga (Griffithsia japonica), a recombinant GjCyp-1 containing a hexahistidine tag at the amino-terminus was constructed and expressed inEscherichia coli. Most of the gene product expressed inE. coli was organized as aggregate insoluble particles known as inclusion bodies. Thus, the optimal time, temperature, and concentration ofl(+)-arabinose for expressing the soluble and nonaggregated form of GjCyp-1 inE. coli were examined. The results indicate that the induction of Cyp, at 0.2%l(+)-arabinose for 2 h at 25°C, had a marked effect on the yield of the soluble and active form of the co-chaperone as PPlase. An expressed fusion protein, H₆GjCyp-1, maintained the stability ofE. coli proteins up to-75°C. In a functional bioassay of the recombinant H₆GjCyp-1, the viability ofE. coli cells overexpressing H₆GjCyp-1 was compared to that of cells not expressing H₆GjCyp-1 at −75°C. For all the cycles of a freeze/thaw treatment, a significant increase in viability was observed in theE. coli cells overexpressing H₆GjCyp-1. The results of the GjCyp-1 bioassays, as well asin vitro studies, strongly suggest that the algal Cyp confers freeze tolerance toE. coli.     

Fungal utilization of a known and safe macroalga for pigment production using solid-state fermentation

Saccharina (Laminaria) japonica, a safe, cheap, and readily available macroalga can be used as a substrate for various microbial fermentations. This work investigated the feasibility of S. japonica as a substrate for production of pigments by the fungus Talaromyces amestolkiae GT11 in solid-state fermentation without additional salt and/or nitrogen sources. Under optimized conditions, the pigment exhibited maximum absorption spectrum at 410 (yellow) and 510 nm (red), and the pigment yield of 1,153.5 (yellow) and 506.2 (red) OD units g^?1 of dry fermented substrate were achieved with a particle size of 1.0 mm and pH 7, although visually the pigment was reddish in color. The optimum incubation period, pH, moisture, inoculum size, and temperature were observed to be at 192 h, pH 7.0, 80 % (w/w) moisture, 1.8?×?10⁶ spores mL^?1 of inoculum g^-1 of dry substrate and 28 °C. Hence, this study indicates the suitability of utilization of S. japonica as a substrate for natural pigment production by T. amestolkiae GT11 which can be used in food, cosmetics and pharmaceutical industries for various applications.     

Reconstituted fusion pore

Fusion pores or porosomes are basket-like structures at the cell plasma membrane, at the base of which, membrane-bound secretory vesicles dock and fuse to release vesicular contents. Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. ImmunoAFM studies demonstrated the release of vesicular contents through the pores. Transmission electron microscopy (TEM) further confirmed the presence of fusion pores, and immunoAFM, and immunochemical studies demonstrated t-SNAREs to localize at the base of the fusion pore. In the present study, the morphology, function, and composition of the immunoisolated fusion pore was investigated. TEM studies reveal in further detail the structure of the fusion pore. Immunoblot analysis of the immunoisolated fusion pore reveals the presence of several isoforms of the proteins, identified earlier in addition to the association of chloride channels. TEM and AFM micrographs of the immunoisolated fusion pore complex were superimposable, revealing its detail structure. Fusion pore reconstituted into liposomes and examined by TEM, revealed a cup-shaped basket-like morphology, and were functional, as demonstrated by their ability to fuse with isolated secretory vesicles.     

Alterations in mRNA expression of ribosomal protein S9 in hydrogen peroxide-treated neurotumor cells and in rat hippocampus after transient ischemia

This study was designed to isolate new genes related to apoptosis in rat pheochromocytoma (PC12) cells treated with hydrogen peroxide (H₂O₂), and to characterize the roles of the genes using both in vitro and in vivo models of oxidative injury. cDNA libraries were prepared from H₂O₂-treated and -untreated PC12 cells, and a ribosomal protein S9 (RPS9) clone was isolated by a differential screening method. Increase of RPS9 expression in both H₂O₂-treated PC12 and neuroblastoma (Neuro-2A) cells was shown by Northern blot analysis. Viability of the antisense-transfected Neuro-2A (RPS9-AS) cells following H₂O₂ treatment was significantly reduced in a dose-dependent manner. In an in vivo model of transient forebrain ischemia, an increase in RPS9 expression was prominent by 1 day postischemia in the granule cell layer neurons of the dentate gyrus. Both activation of caspase-3 and significant recovery of viability following pretreatment with cycloheximide were shown in RPS9-AS cells treated with H₂O₂. These data suggest that RPS9 plays a protective role in oxidative injury of neuronal cells.