Biomedical subjects

S Mochizuki

Publications and source records attributed to S Mochizuki.

At least 55 records · Page 3Linked to original sources

PubMed1999

Identification of a domain affecting agonist potency of meta-chlorophenylbiguanide in 5-HT3 receptors.

The pharmacological properties of rat and human 5-HT3 receptors expressed in Xenopus oocytes were assessed using a two-electrode voltage clamp technique. Meta-chlorophenylbiguanide (mCPBG), a 5-HT3 receptor-selective agonist, elicited typical current responses in both rat and human 5-HT3 receptor-expressing oocytes. However, the EC50 value for rat 5-HT3 receptors was 13-fold lower than for human 5-HT3 receptors. Using several chimeric human-rat 5-HT3 receptors, we identified a potential domain responsible for this difference in mCPBG-response. The domain is in the N-terminal extracellular region adjacent to the first transmembrane domain of rat 5-HT3 receptors and includes a rat-specific seven amino acid sequence (Phe197, Thr198, Lys199, Gln201, Ile205, Thr207 and Ser210). Replacement of corresponding amino acids in human 5-HT3 receptors by rat receptor residues increased the potency of mCPBG on human receptors indicating these amino acids play an important role in the pharmacological response to mCPBG.

Amino Acid Sequence↗

PubMed1999

Ion permeation properties of a cloned human 5-HT3 receptor transiently expressed in HEK 293 cells.

Human 5-HT3 receptors expressed in HEK 293 cells were studied using patch-clamp techniques. The permeability ratios of cations to Na+ were Li+, 1.16; K+, 1.04; Rb+, 1.11; Cs+, 1.11; NMDG+, 0.04; Ca2+, 0.49, and Mg2+, 0.37. The permeability sequence of the alkali metal cations was Li+ > Rb+ = Cs+ > K+ > Na+. Increased external concentrations of Ca2+ or Mg2- decreased 5-HT-induced currents at all potentials tested in a voltage-independent manner. The single-channel conductance of human 5-HT3 receptors measured by fluctuation analysis of whole-cell currents was 790 +/- 100 fS. Differences in the basic properties of 5-HT3 receptors between species may explain interspecies differences in pharmacological properties.

Cations↗

PubMed1999

Flow dependence and time constant of the change in nitric oxide concentration measured in the vascular media.

It has been considered that the concentration of endothelium-derived nitric oxide (NO) in the arterial vascular wall changes in response to flow-induced shear stress. In the present study, using an NO-sensitive electrode, the aim was to directly evaluate the relationship between perfusion rate and NO concentration in the arterial vascular wall. The NO microelectrode (diameter: 100 microns) was inserted into the vascular media of isolated canine femoral arteries, and the vessel was perfused with a Krebs-Henseleit buffer solution. A flow-related change in NO concentration in the vascular media was then evaluated by changing perfusion rate. NO concentration attained a peak value with a first-order time delay by a stepwise increase in perfusion rate, and the peak-level NO concentration was linearly correlated with perfusion rate in each vessel (10-154 pA at 2.1-72.3 ml min-1; n = 7, r2 = 0.89-0.99, p < 0.03). The average time constant for an increase in NO current with a stepwise increase in perfusion rate was 24 +/- 3 s (n = 5). NO production was increased by perfusing a solution containing 1 mmol l-1 L-arginine and was attenuated by 100 mumol l-1 NG-nitro-L-arginine, indicating the intactness of the endothelium, proper insertion of the NO electrode and selective detection of NO by the electrode. It is concluded that the NO microelectrode is applicable to NO measurement in the vascular media where NO controls vascular tone and that the concentration of NO in the arterial vascular media changes with perfusion rate in a rate-dependent manner as well as with a time constant of about 24 s for a stepwise increase in flow.

Animals↗

PubMed1999

Psychophysiological stress induces heat shock cognate protein 70 messenger RNA in the hippocampus of rats.

Psychophysiological stress has been shown to increase 70,000 mol. wt heat shock protein messenger RNAs with northern blotting in rats. However, its localization is unknown. With in situ hybridization, we tested our hypothesis that restraint water-immersion stress may induce heat shock cognate protein 70 messenger RNA expression simultaneously with some morphological changes selectively in the hippocampus of rats. Stress for 6 h significantly increased heat shock cognate protein 70 messenger RNAs in the hippocampus, with maximal intensity in the CA3 subfield of the Ammon's horn and to a lesser extent in CA2. Stress for 12 h significantly increased heat shock cognate protein 70 messenger RNAs in the whole hemisphere including the cerebral cortex, the thalamus, the hypothalamus, and the hippocampus with the highest density in CA3. Heat shock cognate protein 70 messenger RNA in rats with stress for 6 h followed by recovery for 6 h significantly increased at CA3 and CA2 compared with the controls or rats stressed for 6 h without recovery. No overt histological changes were detected in neuronal or glial cells in the slides of hematoxylin-eosin or Cresyl Violet staining. These results show that psychophysiological stress induces heat shock cognate protein 70 messenger RNA in the most stress-vulnerable brain structure, hippocampal CA3, probably for cytoprotection.

Animals↗

PubMed1999

Implantation of the undulation pump total artificial heart in the goat.

The undulation pump total artificial heart (UPTAH) was developed by using small-size continuous-flow displacement-type blood pumps (undulation pump). To clarify and improve the problems accompanied by the implantation in the chest, 14 animal experiments were performed on goats weighing 41.3-79.2 kg. The UPTAH could be implanted in the chest of all goats and was driven with a modulation pulsatile mode. The first problem was the atrial suction effect. This problem could be prevented to some extent by developing the soft disk and by improving atrial cuffs. An automatic detection and releasing of the atrial suction effect was also tried. The next problem was acute lung edema accompanied by the postural change of the animal. Development of the automatic control of left atrial pressure could prevent this problem. Small blood leakage from a pinpoint hole in the seal membrane was the next problem. Improvement of the manufacturing procedure of the membrane prevented this. With these improvements, a 10 day survival could be obtained with this unique implantable total artificial heart.

Acute Disease↗

PubMed1999

Present status of the total artificial heart at the University of Tokyo.

At the University of Tokyo, various types of total artificial heart (TAH) systems have been studied since 1959. At the present time, 2 types of implantable TAH have been developed. One is an undulation pump TAH (UPTAH) and the other is a flow transformed pulsatile TAH (FTPTAH). Using the UPTAH, 14 cases of implantation were performed in goats and 10 days' survival obtained. The new type of FTPTAH is under a prototype study. To prevent ring thrombus, a polyurethane membrane valve, a jellyfish valve, has been developed. The longest in vivo experiences with this valve in the TAH blood pump have been 312 days in the left side blood pump and 414 days in the right side blood pump. Conductance and arterial pressure based control (1/R control) can realize the physiological control of the TAH. Using 1/R control, 532 days of survival could be obtained in a goat with a paracorporeal TAH. The technique required to apply this control method to a implantable TAH is under development. We have proposed a new 5 year research project of the implantable TAH entitled "Comprehensive Basic Research on the Development of a Japanese Original Implantable Total Artificial Heart" to The Ministry of Welfare.

Animals↗

PubMed1999

Crystal structure of human serum albumin at 2.5 A resolution.

A new triclinic crystal form of human serum albumin (HSA), derived either from pool plasma (pHSA) or from a Pichia pastoris expression system (rHSA), was obtained from polyethylene glycol 4000 solution. Three-dimensional structures of pHSA and rHSA were determined at 2.5 A resolution from the new triclinic crystal form by molecular replacement, using atomic coordinates derived from a multiple isomorphous replacement work with a known tetragonal crystal form. The structures of pHSA and rHSA are virtually identical, with an r.m. s. deviation of 0.24 A for all Calpha atoms. The two HSA molecules involved in the asymmetric unit are related by a strict local twofold symmetry such that the Calpha atoms of the two molecules can be superimposed with an r.m.s. deviation of 0.28 A in pHSA. Cys34 is the only cysteine with a free sulfhydryl group which does not participate in a disulfide linkage with any external ligand. Domains II and III both have a pocket formed mostly of hydrophobic and positively charged residues and in which a very wide range of compounds may be accommodated. Three tentative binding sites for long-chain fatty acids, each with different surroundings, are located at the surface of each domain.

Binding Sites↗

PubMed1999

Alterations in sarcoplasmic reticulum function and gene expression in ischemic-reperfused rat heart.

In view of the critical role of sarcoplasmic reticular (SR) Ca(2+) release and the Ca(2+) pump in cardiac contraction-relaxation, this study was undertaken to assess the status of SR function, protein content, and gene expression in isolated rat hearts subjected to global ischemia for 30 min followed by 60 min of reperfusion (I/R). Attenuated recovery of contractile function in the I/R hearts was associated with reduced SR Ca(2+) uptake, Ca(2+) release, and ryanodine-binding activities. mRNA levels and protein contents for SR Ca(2+) pump ATPase and Ca(2+) release channels were markedly depressed in the I/R hearts. Perfusion of hearts with superoxide dismutase plus catalase, well-known scavengers of oxyradicals, prevented the I/R-induced alterations in cardiac function and partially prevented SR Ca(2+) transport activities and mRNA abundance. In hearts perfused with xanthine plus xanthine oxidase or H(2)O(2), changes similar to those in the I/R hearts were observed. These results indicate that oxyradicals may participate in depressing the SR Ca(2+) handling and gene expression in the I/R heart. It is suggested that treatment of hearts with antioxidants may improve the recovery of cardiac function by preserving the SR function and partially protecting the SR gene expression.

Animals↗

PubMed1999

Mechanisms of Hydrogen Peroxide-Induced Increase in Intracellular Calcium in Cardiomyocytes.

BACKGROUND: Hydrogen peroxide (H(2)O(2)) in high concentrations has been implicated in heart dysfunction attributable to ischemia-reperfusion. Although H(2)O(2) is also known to increase the intracellular concentration of Ca(2+) ([Ca(2+)](i)) in cardiomyocytes, the mechanisms for such a change are not clear. In this study, the sources and mechanisms of increase in [Ca(2+)](i) caused by high concentrations of H(2)O(2) in cardiomyocytes were explored. METHODS AND RESULTS: Cardiomyocytes were isolated from adult male Sprague-Dawley rats. Cell viability was examined by trypan blue exclusion test. [Ca(2+)](i) was measured by employing cell suspension at room temperature and Fura-2 fluorescence technique. Incubation of cells with 0.25-l mmol/L H(2)O(2) increased [Ca(2+)](i) in a time- and concentration-dependent manner. Catalase attenuated the H(2)O(2)-induced increase in [Ca(2+)](i) significantly, whereas mannitol showed no effect. Neither the presence of verapamil, a sarcolemmal Ca(2+) channel blocker, nor the removal of Ca(2+) from the medium produced any significant reduction in the H(2)O(2)-induced increase in [Ca(2+)](i). Conversely, treatment of cardiomyoctes with staurosporin, a protein kinase C inhibitor, thapsigargin, a sarcoplasmic reticulum Ca(2+)-pump adenosine triphosphatase inhibitor, as well as ryanodine, a sarcoplasmic reticulum Ca(2+)-release channel blocker, markedly prevented the 0.5-mmol/L H(2)O(2)-induced increase in [Ca(2+)](i). The responses of cardiomyoctes to H(2)O(2) and other Ca(2+)-mobilizing agents, such as KCl or adenosine triphosphate, were additive. No changes in cardiomyocyte viability were seen on incubation with 0.5 and 1 mmol/L H(2)O(2). Perfusion of the isolated heart with H(2)O(2) (0.1-0.5 mmol/L) depressed the left ventricular developed pressure, rate of contraction, and rate of relaxation, whereas the left ventricular end-diastolic pressure was increased. CONCLUSIONS: These results indicate that formation of H(2)O(2) under pathophysiological conditions such as ischemic heart disease may induce changes in Ca(2+) homeostasis in cardiomyocytes and may induce contractile dysfunction. Furthermore, the sarcoplasmic reticulum involving a protein kinase C-mediated mechanism appears to be the main site of action of H(2)O(2) in cardiomyocytes.

Journal Article↗

PubMed1999

Coexpression of mineralocorticoid receptors and 11beta-hydroxysteroid dehydrogenase 2 in human gastric mucosa.

The role of mineralocorticoids in human gastrointestinal tract is well established. In the stomach, aldosterone is thought to regulate electrolyte transport associated with gastric acid secretion. In mineralocorticoid target organs, the action of the glucocorticoid inactivating enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) facilitates aldosterone binding to a nonselective mineralocorticoid receptor (MR) in the face of high levels of circulating glucocorticoids. In the present study, we examined 25 specimens of human stomach for the presence of MR and 11beta-HSD2 using a [3H]aldosterone binding assay, Northern blot analysis, RT-PCR, and immunohistochemistry. Specific [3H]aldosterone binding sites were detected in gastric fundic mucosa, but not in the antrum. In fundic mucosa the Kd was 0.72+/-0.05 nmol/L (mean +/- SE), and Bmax was 6.0+/-1.4 fmol per milligram of protein. Northern blot analysis demonstrated a faint band for MR mRNA at 6.0 kb, although message for 11beta-HSD2 was undetectable. However, RT-PCR demonstrated specific PCR products for both MR and 11beta-HSD2. Immunohistochemistry demonstrated the colocalization of MR and 11beta-HSD2 only in parietal cells. MR-positive cells were further characterized by electron microscopy, confirming the identity of parietal cells. This study shows that parietal cells contain both MR and 11beta-HSD2, suggesting that the human stomach is a novel target organ for mineralocorticoids. Aldosterone may, therefore, regulate biological functions of parietal cells including gastric acid secretion.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1999

Homozygous deletion of the p16/MTS-1/CDKN2 gene in malignant gliomas is infrequent among Japanese patients.

We have analyzed the status of the p16/MST-1/CDKN2 gene in 63 brain tumors from Japanese patients. With quantitative multiplex polymerase chain reaction (PCR) assay using the exon 2 primers of the p16 gene and control chromosome 9qSTS primers, we found homozygous deletion of the p16 gene in 7 cases; in 1 out of 10 cases of anaplastic astrocytomas (WHO grade III), 6 out of 35 cases of glioblastoma multiformes (grade IV) but in none of the tumors of grade I or II. We also found mobility-shifted PCR products in 8 cases using the single-strand conformation polymorphism technique. DNA sequencing of the aberrantly migrated products revealed that 5 cases of glioblastoma multiforme had mutations which caused amino acid substitutions. We found one case with silent mutations and two cases with nucleotide changes in the non-coding region. The frequency of the alteration of the p16 gene, either homozygous deletion or mutation accompanied with amino acid substitutions, increased in malignant brain tumors (grade III and IV) compared with that in low grade tumors (grade I and II) (p=0.0275), suggesting possible role(s) of the gene in the progression of brain tumors. In addition, the low frequency of homozygous deletions shown in this study is quite different from previous reports that demonstrated frequently deleted p16 gene in malignant gliomas from Caucasian patients. We have also shown the presence of heterogeneous cell populations within the glioblastoma masses based on the variety of the mutated p16 sequences. The present study, therefore, suggests a possible racial difference in the mechanism of the tumorigenesis and a heterogeneity of malignant gliomas developed during the tumor progression.

Adolescent↗

PubMed1999

[Estimation of left internal thoracic artery flow reserve after coronary artery bypass grafting using Doppler flow wire].

The usefulness of the left internal thoracic artery (LITA) for aortocoronary bypass grafting is now established. Reports of variation in LITA graft function are rare. Graft flow was measured using a Doppler flow wire to estimate graft function in 27 patients (24 males, 3 females) who underwent LITA grafting to the left anterior descending artery. Patients were classified into the following 3 groups: Group A consisted of 9 patients with normal left ventricular function and no native flow; Group B consisted of 11 patients with normal ventricular function and good native flow; Group C consisted of 7 patients with abnormal left ventricular function and no native flow. LITA graft function was also estimated 1 year after operation in 12 of these 27 patients (4 in each group). Diastolic/systolic velocity ratio (DSVR) and flow reserve were determined in the proximal, middle, and distal portions of the LITA graft and native left anterior descending artery before and after papaverine administration (8-10 mg). DSVR was significantly higher in the distal portion than in the proximal portion (p < 0.01), but this value did not change after papaverine administration. After 1 year, DSVR in the proximal portion was significantly higher in Group C than in Groups A or B (p < 0.05 for both). Four weeks after operation, the flow reserve of the distal portion was significantly higher in Group A than in Group C (p < 0.001). After 1 year, this value was significantly higher in Group A than in either Groups B or C (p < 0.01, p < 0.001, respectively). The rate of increase in flow reserve in the distal portion was significantly greater in Group A (23.4%) than in groups B (2.53%) or C (1.94%; p < 0.05 for both). The distal portion of the LITA was the best measurement site, since the flow pattern in the LITA graft varied throughout all portions. Diastolic flow velocity in the LITA graft was dominant in patients with myocardial damage. The results indicate that flow reserve of the LITA graft depends on antegrade native coronary flow and distal myocardial damage.

Coronary Artery Bypass↗

PubMed1998

Osteoclastogenesis inhibitory factor suppresses osteoclast survival by interfering in the interaction of stromal cells with osteoclast.

Osteoclastogenesis inhibitory factor (OCIF) was originally identified as a factor inhibiting osteoclast (OC) formation. The number of OC in rats treated with OCIF decreased, suggesting that OCIF inhibits OC formation in vivo; however, it is also possible that OCIF affects the number of OC by promoting apoptosis of OC. To address this issue, the effects of OCIF on the survival of OC were examined using well established mouse culture systems. OCIF dose-dependently inhibited OC formation in mouse marrow cultures. Addition of OCIF during day 0-3 did not alter the peak levels of OC formation on day 7 and 8. However, the addition of OCIF during day 5 and thereafter resulted in the rapid decrease of the number of OC. OCIF inhibited the survival of OC formed in mouse marrow cultures in dose- and time-dependent manners. The involvement of stromal cells in OC survival was examined using crude and stromal cell-depleted OC populations. OCIF dramatically inhibited the survival of crude OC populations rich with stromal cells. However, in stromal cell-depleted OC populations, OC spontaneously decreased in the absence of OCIF and OCIF did not enhance the decrease further at least up to 48 h. Apoptotic OC were detected in detached cell populations treated with OCIF in mouse marrow cultures and a specific inhibitor for caspase-3 rescued the death of OC. OCIF mutant lacking the death domain homologous regions inhibited OC survival, though the potency was much less than native OCIF. Taken together, OCIF inhibited not only OC recruitment but also OC survival. OCIF inhibited OC survival by interfering the interaction of stromal cells with OC.

Animals↗

PubMed1998

Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin.

Osteoclasts are multinucleated cells that resorb bone. Osteoclastogenesis inhibitory factor (OCIF), also called osteoprotegerin (OPG), acts as a naturally occurring decoy receptor for osteoclast differentiation factor, which mediates an essential signal to osteoclast progenitors for their differentiation into osteoclasts. Here we show that the OCIF/OPG knockout mice exhibited severe osteoporosis due to enhanced osteoclastogenesis when they grew to be adults. These mice were viable and fertile. They exhibited marked bone loss accompanied by destruction of growth plate and lack of trabecular bone in their femurs. The strength of their bones dramatically decreased. These results demonstrate that OCIF/OPG is a key factor acting as a negative regulator against osteoclastogenesis. The OCIF/OPG knockout mice provide the first animal model for osteoporosis without other obvious abnormalities.

Animals↗

PubMed1998

Osteoclast differentiation factor mediates an essential signal for bone resorption induced by 1 alpha,25-dihydroxyvitamin D3, prostaglandin E2, or parathyroid hormone in the microenvironment of bone.

Osteoclast differentiation factor (ODF), a ligand for osteoprotegerin (OPG)/osteoclastogenesis-inhibitory factor (OCIF), induces osteoclast-like cell formation in vitro. To elucidate the role of ODF in the microenvironment of bone, we examined effects of ODF, OPG/OCIF, and anti-ODF polyclonal antibody on bone resorption using a fetal mouse long bone culture system. A genetically engineered soluble-form ODF (sODF) elicited bone resorption in a concentration-dependent manner and OPG/OCIF blocked the bone resorption. Anti-ODF polyclonal antibody, which neutralizes ODF activity, negated bone resorption induced by 1 alpha,25-dihydroxyvitamin D3, parathyroid hormone, or prostaglandin E2. OPG/OCIF also abolished bone-resorbing activity elicited by these bone-resorbing agents. Interleukin 1 alpha-stimulated bone resorption was also significantly suppressed by anti-ODF polyclonal antibody and OPG/OCIF. Thus, we conclude that ODF plays a critical role in bone resorption in the microenvironment of bone.

Animals↗

PubMed1998

Characterization of monomeric and homodimeric forms of osteoclastogenesis inhibitory factor.

Osteoclastogenesis inhibitory factor (OCIF) is present naturally as two molecular forms, a monomer and a homodimer. The two forms of recombinant human OCIF (rOCIF) produced by Chinese hamster ovary (CHO) cells were purified to homogeneity. Determination of the C-terminal amino-acid sequences of the two forms of rOCIF revealed that the monomeric rOCIF lacked several amino acids including Cys379, which is involved in the intermolecular disulfide bond, in its C-terminal region. The two forms of rOCIF were indistinguishable in stability, sialic acid content, and specific activity in inhibition of osteoclastogenesis. In contrast, the homodimeric rOCIF was stronger in heparin-binding ability than the monomeric rOCIF. The homodimeric rOCIF was significantly shorter in initial half-life and smaller in AUC value in rats than the monomeric rOCIF, but exerted more potent biological activity in reducing the calcium concentration in serum of rats than did the monomeric rOCIF.

Amino Acid Sequence↗

PubMed1998

Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL.

Osteoclasts, the multinucleated cells that resorb bone, develop from hematopoietic cells of monocyte/macrophage lineage. Osteoclast-like cells (OCLs) are formed by coculturing spleen cells with osteoblasts or bone marrow stromal cells in the presence of bone-resorbing factors. The cell-to-cell interaction between osteoblasts/stromal cells and osteoclast progenitors is essential for OCL formation. Recently, we purified and molecularly cloned osteoclastogenesis-inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF is a secreted member of the tumor necrosis factor receptor family and inhibits osteoclastogenesis by interrupting the cell-to-cell interaction. Here we report the expression cloning of a ligand for OPG/OCIF from a complementary DNA library of mouse stromal cells. The protein was found to be a member of the membrane-associated tumor necrosis factor ligand family and induced OCL formation from osteoclast progenitors. A genetically engineered soluble form containing the extracellular domain of the protein induced OCL formation from spleen cells in the absence of osteoblasts/stromal cells. OPG/OCIF abolished the OCL formation induced by the protein. Expression of its gene in osteoblasts/stromal cells was up-regulated by bone-resorbing factors. We conclude that the membrane-bound protein is osteoclast differentiation factor (ODF), a long-sought ligand mediating an essential signal to osteoclast progenitors for their differentiation into osteoclasts. ODF was found to be identical to TRANCE/RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only osteoclastogenesis but also immune system.

Animals↗

PubMed1998

Sister chromatid exchange analysis of human cells carrying retroviral DNA.

Chromosomal abnormalities have been seen in human cells following infection with human T lymphotropic virus type I (HTLV-I). Effects on sister chromatid exchange (SCE) in cells carrying HTLV-I related DNA were examined by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Peripheral blood specimens were obtained from 16 donors. Cells were processed for SCE analysis and sera were tested for HTLV-I antibodies by Western blot analysis. DNA from fresh lymphoid cells was analyzed by PCR-SSCP for HTLV-I genes. Of 16 donors, eight were HTLV-I seropositive and eight seronegative. Five HTLV-I infected lymphocyte cultures were also analyzed for SCE. Cells were stained immunocytochemically with anti-p19 for the viral expression. By MNNG treatment, SCE was significantly enhanced in cells derived from two seropositive donors with HTLV-I genes. In cells of six seropositive and eight seronegative donors with or without HTLV-I related DNA, SCE frequency was influenced insignificantly. Cells from all persons were negative for the p19 expression. SCE was greatly enhanced by MNNG treatment in cells from three productively infected cultures when compared with SCE in two nonproductively infected cultures.

Adult↗