Shigetada Teshima

Space shuttle flight (STS-90) enhances degradation of rat myosin heavy chain in association with activation of ubiquitin–proteasome pathway

To elucidate the mechanisms of microgravity‐induced muscle atrophy, we focused on fast‐type myosin heavy chain (MHC) degradation and expression of proteases in atrophied gastrocnemius muscles of neonatal rats exposed to 16‐d spaceflight (STS‐90). The spaceflight stimulated ubiquitination of proteins, including a MHC molecule, and accumulation of MHC degradation fragments in the muscles. Semiquantitative reverse transcriptase‐polymerase chain reaction revealed that the spaceflight significantly increased mRNA levels of cathepsin L, proteasome components (RC2 and RC9), polyubiquitin, and ubiquitin‐conjugating enzyme in the muscles, compared with those of ground control rats. The levels of μ‐calpain, m‐calpain, cathepsin B, and cathepsin H mRNAs were not changed by the spaceflight. We also found that tail‐suspension of rats for 10 d or longer caused the ubiquitination and degradation of MHC in gastrocnemius muscle, as was observed in the spaceflight rats. In the muscle of suspended rats, these changes were closely associated with activation of proteasome and up‐regulation of expression of mRNA for the proteasome components and polyubiquitin. Administration of a cysteine protease inhibitor, E‐64, to the suspended rats did not prevent the MHC degradation. Our results suggest that spaceflight induces the degradation of muscle contractile proteins, including MHC, possibly through a ubiquitin‐dependent proteolytic pathway.

Type I Helicobacter pylori Lipopolysaccharide Stimulates Toll-Like Receptor 4 and Activates Mitogen Oxidase 1 in Gastric Pit Cells

ABSTRACT Guinea pig gastric pit cells express an isozyme of gp91-phox, mitogen oxidase 1 (Mox1), and essential components for the phagocyte NADPH oxidase (p67-, p47-, p40-, and p22-phox). Helicobacter pylori lipopolysaccharide (LPS) andEscherichia coli LPS have been shown to function as potent activators for the Mox1 oxidase. These cells spontaneously secreted about 10 nmol of superoxide anion (O2−)/mg of protein/h under LPS-free conditions. They expressed the mRNA and protein of Toll-like receptor 4 (TLR4) but not those of TLR2. LPS from type I H. pylori at 2.1 endotoxin units/ml or higher stimulated TLR4-mediated phosphorylations of transforming growth factor β-activated kinase 1 and its binding protein 1 induced TLR4 and p67-phox and up-regulated O2−production 10-fold. In contrast, none of these events occurred withH. pylori LPS from complete or partial deletion mutants of the cag pathogenicity island. Lipid A was confirmed to be a bioactive component for the priming effects, while removal of bisphosphates from lipid A completely eliminated the effects, suggesting the importance of the phosphorylation pattern besides the acylation pattern for the bioactivity. H. pylori LPS is generally accepted as having low toxicity; however, our results suggest that type I H. pylori lipid A may be a potent stimulator for innate immune responses of gastric mucosa by stimulating the TLR4 cascade and Mox1 oxidase in pit cells.

Glutathione depletion impairs transcriptional activation of heat shock genes in primary cultures of guinea pig gastric mucosal cells.

When primary cultures of guinea pig gastric mucosal cells were exposed to heat (43 degree C), ethanol, hydrogen peroxide (H2O2), or diamide, heat shock proteins (HSP90, HSP70, HSP60, and HSC73) were rapidly synthesized. The extent of each HSP induction varied with the type of stress. Ethanol, H2O2, and diamide increased the syntheses of several other undefined proteins besides the HSPs. However, none of these proteins were induced by exposure to heat or the reagents, when intracellular glutathione was depleted to <10% of the control level by pretreatment with DL-buthionine-[S,R]-sulfoximine. Gel mobility shift assay using a synthetic oligonucleotide coding HSP70 heat shock element showed that glutathione depletion inhibited the heat- and the reagent-initiated activation of the heat shock factor 1 (HSF1) and did not promote the expression of HSP70 mRNA. Immunoblot analysis with antiserum against HSF1 demonstrated that the steady-state level of HSF1 was not changed in glutathione-depleted cells, but glutathione depletion inhibited the nuclear translocation of HSF1 after exposure to heat stress. These results suggest that intracellular glutathione may support early and important biochemical events in the acquisition by gastric mucosal cells of an adaptive response to irritants.

Geranylgeranylacetone stimulates mucin synthesis in cultured guinea pig gastric pit cells by inducing a neuronal nitric oxide synthase

Abstract: Nitric oxide (NO) has been considered to play an important role in the regulation of blood flow, mucosal integrity, and mucus production in the stomach. We investigated the stimulatory actions of epidermal growth factor (EGF) and a cytoprotective compound, geranylgeranylacetone (GGA), on mucin synthesis in guinea pig gastric pre-pit cells, maintained in a serum-free culture system. GGA increased [3H]glucosamine uptake and the accumulation of mucus granules positive for galactose oxidase-Schiff reaction in the cells. This stimulatory action of GGA was equivalent to that of EGF, but GGA did not stimulate the cell growth. Both EGF and GGA increased the release of NO degeneration products, NO2− and NO3−. The [3H]glucosamine uptake was completely inhibited by the non-selective NO synthase (NOS) inhibitors, NG-nitro-l-arginine and NG-monomethyl-l-arginine, and it was only partially inhibited by a more selective inhibitor for inducible NOS isoform (iNOS), aminoguanidine. Northern blotting with a cDNA probe for rat iNOS, and Western blotting with a polyclonal antibody against iNOS, demonstrated that GGA did not up-regulate the iNOS mRNA expression nor induce its protein. In contrast, GGA and EGF induced neuronal NOS, but not endothelial NOS, which was confirmed by immunoblot analyses with antibodies against these constitutive NOS isoforms. Thus, the present experiments suggests that GGA, as well as EGF, stimulates mucin synthesis at least in part through an NO-dependent pathway, leading to an increase in the integrity of the gastric mucosa.

Helicobacter pylori lipopolysaccharide enhances the expression of NADPH oxidase components in cultured guinea pig gastric mucosal cells

Recently, we showed that cultured guinea pig gastric pit cells possess a phagocyte NADPH oxidase‐like activity, which was up‐regulated by Helicobacter pylori lipopolysaccharide. We demonstrate here that these cells express all of the phagocyte NADPH oxidase components (gp91‐, p22‐, p67‐, p47‐, and p40‐phoxes). Treatment with lipopolysaccharide increased the expression of gp91‐, p22‐, and p67‐phoxes, but not that of p47‐ and p40‐phoxes. Intriguingly, the p67‐phox expression consistently correlated with up‐regulation of superoxide anion‐producing ability. Thus, the gastric pit cell NADPH oxidase may play an important role in regulation of the inflammatory response associated with H. pylori infection.

Effects of a Soy Protein Diet on Exercise-Induced Muscle Protein Catabolism in Rats

OBJECTIVE We examined effects of dietary soy protein isolate on muscle calpain activity and myosin heavy chain (MHC) degradation in rats performing an acute running exercise. METHODS In rats fed a 20% casein diet, the treadmill running exercise, fixed at 80 kg/m, transiently increased calpain activity in gastrocnemius muscles in parallel with the release of creatine kinase into plasma. The fixed running also caused an accumulation of immunoreactive degradation fragments of MHC in the muscle. Feeding a 20% soy protein isolate diet as opposed to the control casein diet to rats significantly suppressed the running-induced activation of mu- and m-calpains, fragmentation of MHC, and release of creatine kinase into plasma (P < 0.05). RESULTS Rats fed the soy protein isolate diet had significantly higher calpastatin activity in gastrocnemius muscle than did rats fed the casein diet (P < 0.05), indicating that this increase inhibits the exercise-induced autoactivation of calpain. Activities of proteasome, cathepsin B + L, and antioxidant enzymes and the levels of glutathione and thiobarbituric acid-reactive substances in the muscle did not differ between the diet groups at the end of the exercise. CONCLUSIONS Our results suggest that diets containing soy protein prevent exercise-induced protein degradation in skeletal muscle, possibly through inhibiting the calpain-mediated proteolysis.

Glutathione depletion inhibits oxidant-induced activation of nuclear factor-kappa B, AP-1, and c-Jun/ATF-2 in cultured guinea-pig gastric epithelial cells

Abstract: The aim of this study was to reveal the role of intracellular glutathione in the oxidative stress responses of gastric epithelial cells. Metabolic radiolabeling with L-[35S]methionine and analysis of synthesized proteins by gel electrophoresis and fluorography showed that upon exposure to hydrogen peroxide (H2O2) or diamide, primary cultures of guinea-pig gastric epithelial cells rapidly induced several undefined proteins, as well as heat shock proteins. When intracellular glutathione was depleted to less than 10% of the control value by treatment with buthionine-[S,R]-sulfoximine, these inductions were completely inhibited. Gel mobility shift assay demonstrated that H2O2 and diamide rapidly activated nuclear factor-kappa B (NF-κB), and diamide activated activator protein (AP)-1, and c-Jun/activating transcription factor (ATF)-2, suggesting that the response may be coupled to these reduction-oxidation (redox)-sensitive transcription factors, as well as heat shock transcription factor 1. The activations of NF-κB, AP-1, and c-Jun/ATF-2 by the oxidants did not occur in glutathione-depleted cells. Northern blot analysis showed that glutathione depletion markedly or completely suppressed the diamide-induced expression of c-fos and c-jun mRNAs. These results suggest that intracellular glutathione redox may participate in the initiation of oxidative stress responses; thereby, it plays an important role in gastric mucosal defense.

Oxidant-induced activation of nuclear factor-kappa B in cultured guinea pig gastric epithelial cells.

The aim of this study was to revealoxidant-sensitive components in gastric epithelialcells, which may regulate inflammatory processes ingastric mucosa. Gel mobility shift assay showed thattreatment of cultured guinea pig gastric epithelial cellswith hydrogen peroxide or diamide produced a κBoligonucleotide-protein complex within 5 min. Thebinding proteins consisted of a p50/p65 heterodimer, which was identified by immunosupershift, UVcrosslinking, and immunoprecipitation analyses.Immunocytochemical study demonstrated that surfaceepithelial cells and parietal cells expressed p50 andp65 mainly in the cytosol, and the oxidants rapidlyinitiated the nuclear translocation of the components.The oxidants caused the up-regulation of p105 (a p50precursor) synthesis and the expression of inducible nitric oxide synthase mRNA. These resultssuggest that the oxidant-sensitive p50/p65 heterodimerin gastric epithelial cells may play an important rolein transcriptional activation of genes involved in inflammatory responses of thestomach.

Vitamin D3 elicits calcium response and activates blood monocyte-derived macrophages from patients with vitamin D dependent rickets type II

We studied the effects of vitamin D3 metabolites on intracellular free Ca2+ concentration ([Ca2+]i) and the respiratory burst of monocyte‐derived macrophages (MDM) from patients with vitamin D dependent rickets type II. Treatment of MDM from the patients and healthy donors with 1 nM 1,25(OH)2D3 produced a rapid elevation of [Ca2+]i and similarly primed both types of cells for enhanced capacity for O2 − release with phorbol diester. These results suggest that macrophages may have distinct non‐genomic pathways of vitamin D3, which partly explain the absence of immunodeficiency and the disappearance of rickets after treatment with vitamin D3 in the patients.

Regulation of Mucin Synthesis by Neuronal Nitric Oxide Synthase in Gastric Pit Cells

We investigated the stimulatory actions of epidermal growth factor (EGF) and a cytoprotective compound, geranylgeranylacetone (GGA), on mucin synthesis of guinea pig gastric pre-pit cells, maintained in a serum-free culture system. GGA increased [3H]glucosamine uptake and accumulation of mucous granules positive for galactose oxidase-Schiff reaction in the cells. This stimulatory action of GGA was equivalent to that of EGF, whereas GGA did not stimulate cell growth. EGF and GGA increased the release of nitric oxide (NO) degeneration products, NO2- and NO3-. [3H]Glucosamine uptake by the cells was completely inhibited by the non-selective NO synthase (NOS) inhibitors, N G-nitro-L-arginine, and N G-monomethyl-L-arginine; and it was only partially inhibited by a more selective inhibitor for inducible NOS isoform, aminoguanidine. Immunoblot analysis with antibodies against three isozymes of NOS showed that GGA and EGF induced neuronal NOS but not endothelial NOS or inducible NOS. Our results suggest that GGA and EGF stimulate mucin synthesis at least in part through the NO-dependent pathway, leading to increased integrity of the gastric mucosa.