Physiology of vertebrates under micro-gravity with special reference to the Ca metabolism]

On April 12, 1961, Major Yurii A. Gagarin of the former-U.S.S.R. Air Force circled the Earth in a spacecraft named "Vostok", a word which means "east". He spent 1 hour and 48 minutes in space. Since then, the U.S.S.R. and the U.S.A. have sent many astronauts into space. In one case, the stay in space exceeded a year in length, reaching 438 days. Through these experiences, it became clear that micro-gravity caused various problems in human physiology. One of the most serious problems was the loss of Ca from bones, as a result of the negative expenditure of Ca. Under 1G on the ground, bone absorption and bone formation proceed in accordance. Under micro-gravity, however, this balance is broken. Although this phenomenon has been widely analyzed from the viewpoint of molecular biology as well, studies to clarify the mechanism that causes the disorder of Ca metabolism in bones have just started. At present, no perfect treatment to prevent the loss of Ca from bones is available.     

Increased expression of cardiotrophin-1 during ventricular remodeling in hypertensive rats

Cardiotrophin-1 (CT-1) stimulates longitudinal myocardial cell hypertrophy. We examined the expression of CT-1, leukemia inhibitory factor (LIF), and gp130 by competitive RT-PCR and Western blotting in Dahl salt-sensitive (DS) rats with a high-salt diet, which showed a distinct transition from left ventricular hypertrophy (LVH) to congestive heart failure (CHF). The expression levels of CT-1 mRNA and protein were significantly increased at the CHF stage compared with the LVH stage and age-matched Dahl salt-resistant (DR) rats (n = 6 for each group). mRNA expression of LIF was not changed in the left ventricle at any stage by RT-PCR. gp130 mRNA and protein levels of DS rats at 11 and 17 wk were significantly increased compared with age-matched DR rats. The isolated myocyte length of DS rats at 17 wk was the longest among the four groups of rats. The LV end-diastolic dimension (LVDd) of DS rats, determined by echocardiography, was significantly increased at the CHF stage. There was a significant correlation between the CT-1 protein level and LVDd. CT-1 may play a role in ventricular remodeling during transition from LVH to CHF in the rat hypertensive model.     

A comparative study of body wall structures of a pogonophore and an annelid from a phylogenetic viewpoint

Pogonophores are tube worms that live in reducing deep-sea waters where sunlight does not penetrate. They are highly adapted for their special habitat in lacking guts and possessing endosymbiotic chemosynthetic bacteria. Because of these peculiar characteristics, it is not yet clear whether they should be classified as annelids or not. Electron-microscopic observations of sections of a Japanese pogonophore (Oligobrachia mashikoi) show that the body wall has circular and longitudinal muscular systems. These muscular systems, however, differ from the annelid (Branchiura sowerbyi) in these ways: (1) The outer circular muscle of the pogonophore was constructed of smooth muscle cells. In contrast, that of the annelid was composed of obliquely-striated muscle cells, even though the cells were small and bore undeveloped characteristics. (2) The inner longitudinal muscle of the pogonophore was constructed of undeveloped obliquely-striated muscle cells, whereas that of the annelid was composed of well-developed ones. These observations suggest that this pogonophore can not be classified as an annelid, although many previous studies have placed pogonophores in that phylum.     

<Emphasis Type="Italic">N</Emphasis>-acetylneuraminic acid coupled human recombinant TNFα exhibits enhanced anti-tumor activity against Meth-A fibrosarcoma and reduced toxicity

In order to study the effect of glycosylation on its biological activities and to develop tumor necrosis factor α (TNFα) with less deleterious effects, N-acetylneuraminic acid (NeuAc) with a C9 spacer was chemically coupled to human recombinant TNFα. NeuAc-coupled TNFα (NeuAc-TNFα) exhibited reduced activities in vitro by about threefold compared to native TNFα. In this study, we examined a variety of TNFα activities in vivo. NeuAc-TNFα reduced activities in the up-regulation of serum levels of IL-6 and NOx, but comparable activity as native TNFα in the down-regulation of the serum level of glucose. However, NeuAc-TNFα was more potent than TNFα in the up-regulation of the serum level of serum amyloid A (SAA). NeuAc-TNFα was less toxic to mice. In addition, NeuAc-TNFα exhibited an augmented anti-tumor activity against Meth-A fibrosarcoma without hemorrhagic necrosis. These results indicate that coupling with NeuAc enabled us to develop neoglycoTNFα with selective activities in vivo, including enhanced anti-tumor activity but reduced toxicity.     

Synthesis of glycosylated human tumor necrosis factor α coupled with <Emphasis Type="Italic">N</Emphasis>-acetylneuraminic acid

In order to study the effect of glycosylation on its biological activities, and to develop TNFα with less deleterious effects, recombinant human TNFα was chemically coupled with N-acetylneuraminic acid (NeuAc). NeuAc with C9 spacer was coupled to TNFα by acyl azide method. Two glycosylated TNFαs, designated L NeuAc-TNFα and H NeuAc-TNFα, were purified by anion-exchange chromatography. NeuAc coupling to TNFα was confirmed by lectin blotting. Average number of carbohydrate molecules introduced per molecule of L NeuAc-TNFα and H NeuAc-TNFα were estimated to be 1.0 and 1.5, respectively. We examined a variety of TNFα activities in vitro, including antiproliferative or cytotoxic activities to tumor cells, proliferative effect on fibroblast cells, stimulatory effects on IL-6 production by melanoma cells and NF-κB activation in hepatoma cells. L NeuAc-TNFα and H NeuAc-TNFα exhibited reduced activities about 1/3 and 1/10 as compared to native TNFα in all the activities performed in vitro.     

Crystallization and preliminary X-ray crystallographic analysis of extracellular giant hemoglobin from pogonophoran Oligobrachia mashikoi

An extracellular giant hemoglobin of Oligobrachia mashikoi, composed of 24 globins with the molecular mass of approximately 400 kDa was crystallized in its intact form. Two crystal forms were obtained by the vapor-diffusion method. Form I crystals obtained using sodium acetate as a precipitant belong to the space group P6(1)22 or P6(5)22, with unit-cell parameters a=112.41, c=621.25 A, and diffracted X-rays beyond 3.0 A resolution. Form II crystals obtained using PEG 10000 as a precipitant belong to the space group R32, with unit-cell parameters a=111.50, c=276.84 A, and diffracted X-rays beyond 2.9 A resolution. The crystals are suitable for X-ray crystallography to determine the supramacromolecular assembly of this giant hemoglobin.     

Amlodipine inhibits the production of cytokines induced by ouabain

Recent studies have suggested that cytokines are capable of modifying cardiovascular function and that drugs used in the treatment of heart failure have various modulating properties on the production of cytokines. More recently, we have found that ouabain induces the production of cytokines. This study was performed to examine the effects of calcium channel blockers on the production of cytokines induced by a cardiac glycoside. Human peripheral blood mononuclear cells (PBMC) were obtained from healthy volunteers. PBMC were cultured in 0.1, 1, 10, and 30 micromol/l amlodipine, diltiazem, and nifedipine in presence of 1 micromol/l ouabain. After 24 h of incubation, IL-1alpha, IL-1beta, IL-6, and TNF-alpha were measured in the culture supernatants by enzyme-linked immunosorbent assay. Ouabain induced the production of IL-1alpha, IL-1beta and IL-6, but not of TNF-alpha. Induction of IL-1beta was most prominent. The production of IL-1alpha, and IL-6 was inhibited by amlodipine in a concentration-dependent manner and was significantly decreased at a concentration of 10 micromol/l. IL-1beta production was also inhibited by 30 micromol/l amlodipine. In contrast, neither diltiazem nor nifedipine inhibited the production of these cytokines. The unique property of amlodipine to inhibit the production of IL-1alpha, IL-1beta and IL-6 may contribute to its beneficial effects in heart failure patients.     

Basic fibroblast growth factor protects cardiac myocytes from iNOS-mediated apoptosis.

Basic fibroblast growth factor (bFGF) is an important angiogenic factor produced by hearts subjected to ischemia. However, the direct effects of bFGF on myocardial cells are unknown. Primary cultured cardiac myocytes from neonatal rats were stimulated with lipopolysaccharide (LPS), a potent inducer of inducible nitric oxide synthase (iNOS), in the presence or the absence of bFGF. LPS induced the expression of iNOS in cardiac myocytes, demonstrated at both mRNA and protein levels. We showed that LPS activated the apoptotic pathway, evidenced by TUNEL staining, DNA ladder formation, and morphologic features. LPS-induced apoptosis was blocked by the administration of L-NAME, an inhibitor of NOS. This indicates that LPS induces apoptosis via an iNOS-dependent pathway. Administration of bFGF completely inhibited myocardial cell apoptosis induced by hydrogen peroxide or acidic medium as well as LPS. To determine signaling pathways for this inhibitory effect, we utilized PD098059, an MEK-1-specific inhibitor. PD098059 blocked bFGF-induced activation of ERK (extracellularly responsive kinase)-1/2 and neutralized the apoptotic inhibitory effect of bFGF. These findings demonstrate that LPS induces myocardial cell apoptosis in an iNOS-dependent manner. The results also suggest that bFGF is a protective factor against myocardial cell apoptosis and that this protection requires the MEK-1-ERK pathway.