Search PubMed⌕ Search

Biomedical subjects

Y Komatsu

Publications and source records attributed to Y Komatsu.

At least 235 records · Page 13Linked to original sources

Purification and some properties of alpha-galactosidase from Penicillium purpurogenum.

alpha-Galactosidase was purified by ion-exchange chromatographies on DEAE-cellulose and SE-cellulose columns from the culture filtrate of Penicillium purpurogenum No. 618. The final preparation was judged homogeneous by SDS-PAGE and its molecular mass and isoelectric point were estimated to be 67 kDa and 4.1, respectively. The N-terminal amino acid sequence of the enzyme was analyzed and aligned with those of other alpha-galactosidases. In addition, the enzyme acted on the stubbed alpha-galactosyl residue connected to the beta-1,4-manno-oligosaccharide chain, indicating that this specificity was quite different from that of Mortierella vinacea alpha-galactosidase.

Amino Acid Sequence↗

An adult case of neurohypophyseal ectopy presenting ACTH deficiency and partial GH deficiency.

A case of ACTH deficiency and partial GH deficiency associated with neurohypophyseal ectopy is described. A 42-year-old woman of short stature was admitted for hypoglycemic coma. The patient had hypocortisolemia, an increase in urinary 17-OHCS after consecutive injections of ACTH-Z, and a low plasma ACTH level which showed no response to corticotropin-releasing factor. This indicated the presence of ACTH deficiency. The plasma GH level showed a blunted response to insulin-induced hypoglycemia, but its response to GRF was preserved. Other hypothalamo-pituitary axes were intact. T1-weighted magnetic resonance imaging demonstrated ectopic neurohypophyseal tissue and a tiny anterior pituitary remnant. ACTH deficiency and partial GH deficiency might have developed as a consequence of pituitary stalk injury and inadequate regeneration of the anterior lobe.

Adrenocorticotropic Hormone↗

[Studies on coagulation-fibrinolysis during normal pregnancy, labor and puerperium using recently developed molecular markers].

The purpose of this study is to further investigate physiologic changes in plasma coagulation-fibrinogenolysis and fibrinolysis in the utero-placental circulation in normal pregnancy, labor and puerperium, employing newly developed molecular markers for coagulation-fibrinogenolysis and fibrinolysis such as prothrombin fragment 1 + 2 (F1.2), active PAI-1, Fgfr and Fbfr. In 30 non-pregnant women and 20 normal pregnant women, the levels of plasma F1.2, TAT, FPA, Fgfr and active PAI-1 were noticeably increased from the first trimester to the full term. tPA/PAI-1/C and B beta 15-42 were also increased as gestation advanced. These findings suggested that noticeably activated states of coagulation, fibrinogenolysis and fibrinolysis exist in normal pregnancy. In 30 cases of placental circulation in healthy pregnant women and at 20-30 minutes after uteroplacental separation, the levels of plasma F1.2, TAT, FPA, tPA/PAI-1/C, PIC, B beta 15-42, Fgfr and Fbfr were noticeably increased, and the level of active PAI-1, was noticeably decreased in the uterine venous blood as compared with those of the peripheral venous blood in healthy pregnant women. At 20-30 minutes after utero-placental separation in the uterine venous blood and in peripheral venous blood, the changes in the levels of these markers were significantly increased, whereas active PAI-1 and PI were significantly depressed compared with those in healthy pregnant women. These findings suggest that further enhancement of local coagulation-fibrinolysis occurred in utero after separation of the placenta.

Biomarkers↗

Nuclear characterization and G2M ploidy in human brain tumors using semiautomated image analysis.

We used image analysis to study nuclear morphometry and DNA content in relation to time to tumor progression in a series of 88 patients with brain tumors. Clinical follow-up was obtained for 73 patients. The patients with diploid tumors had a longer time to tumor progression than those with triploid, tetraploid, or hypertetraploid tumors. Mean SG2M-DNA indices (DIs) increased significantly with a increase in mean DIs in all tumors. The mean DIs appeared to be dependent on the number of SG2M phase cells. We conclude that tumors with hypertetraploid in G2M ploidy are highly malignant. Those tumor cells have a large nuclear size, much deformity in nuclear shape, and great proliferative potential. The G2M-tetraploid tumors showed a shorter time to tumor progression when the number of SG2M fractions was large. In contrast, the G2M-hypotetraploid tumors showed a longer time to tumor progression in comparison with other tetraploid and hypertetraploid tumors, but the difference was not significant.

Aneuploidy↗

The correlative evidence suggesting that trehalose stabilizes membrane structure in the yeast Saccharomyces cerevisiae.

The contribution of trehalose and hsp104 to membrane fluidity and the mobility of non-freezing cell water were examined on the basis of whole cell NMR analysis of the yeast Saccharomyces cerevisiae. Membrane fluidity was dependent on the accumulation of trehalose not hsp104 and non-freezing cell water was dependent on the accumulation of hsp104 not trehalose. Thus, the correlative evidence suggesting that trehalose protects yeast cells from temperature extremes by stabilizing the membrane structure was observed in vivo.

Cell Membrane↗

Design and anti-HIV-1 activity of ribozymes that cleave HIV-1 LTR.

A hairpin ribozyme (HR112) and two hammerhead ribozymes (RZ115 and RZ119) containing a 5'C(UUCG)G3' loop were designed to cleave the long terminal repeat (LTR) of HIV-1. When the ribozyme catalyzed RNA cleavage reaction for a chemically synthesized 19 mer (LTR 19) was measured, the t 1/2 value of LTR 19 mediated by RZ115 was smaller than that of the RZ119 case. Moreover, the transformed CEM cells harboring the gene encoding these ribozymes were challenged with a HIV-1IIIB strain, two ribozymes, HR112 and RZ119 possessed strong anti-HIV-1 activity. However, the anti-HIV-1 activity displayed by RZ115 was weak. On the basis of secondary structure predictions of the RNA transcribed with the gene encoding ribozymes, the secondary structure of the transcribed RNA with RZ115 sequences was observed to be different from those with the other ribozymes. It has been demonstrated that the secondary structures of transcribed RNAs can possibly influence the anti-HIV-1 activity.

Antiviral Agents↗

Construction of new hairpin ribozymes with replaced domains.

We have constructed a new type of hairpin ribozyme by cleaving and reverse-joining one of the two catalytic domains to conserve an essential bending structure. The two domains of the new ribozymes were tethered by different lengths of cytidylate linkers. These ribozymes retained the cleavage activity and the cleavage activities depended on the linker lengths. Although these rejoined ribozymes showed weak turnover abilities, those with 18 cytidylates showed a larger kcat/K(m) value than the natural hairpin ribozymes. These modifications in the primary structure of the hairpin ribozyme confirm the bent conformation for the catalytic reaction of this ribozyme, and provide a new approach for the design of highly efficient ribozymes.

Base Sequence↗

Identification of genetic events involved in early steps of immortalization of mouse fibroblasts.

The spontaneously immortalized early passaged fibroblasts from three different strains of mouse are observed to represent two distinct stages of immortalization. The cells at stage I are characterized by slow growth rate, contact inhibition and requisition of serum factors for their growth and proliferation. Stage II cells are marked by fast, multilayer growth that is independent of serum supplementation in growth medium and by the elevated levels of the two marker proteins, i.e., p53 and p81. The change from cytosolic distribution of mortalin, a senescence inducing protein (J. Biol. Chem. (1993) 268, 6615-6621; 22239-22242) to the perinuclear locale is detected as an early event during cellular immortalization. Furthermore, the distinct stages could be characterized by thermal analysis of intact cells, that to the best of our knowledge is employed for the first time for the analysis of cellular mortal and immortal phenotypes. The study characterizes at least two distinct end points in rodent transformation suggesting that there are multiple routes to immortalization.

Animals↗

Natriuretic peptide clearance receptor is transcriptionally down-regulated by beta 2-adrenergic stimulation in vascular smooth muscle cells.

Three natriuretic peptide receptors (the ANP-A, ANP-B, and clearance (C) receptors) have been reported. The regulation of these receptors by catecholamines was examined in cultured rat vascular smooth muscle cells. Treatment with norepinephrine decreased the maximum 125I-ANP binding. The competitive binding assay with des[Gln18,Ser19,Gly20,Leu21, Gly22]ANP-(4-23)-NH2 (C-ANF-4-23), a specific ligand for the C receptor, revealed that the decrease in the 125I-ANP binding by norepinephrine was caused by the down-regulation of the C receptor. Isoproterenol also down-regulated the C receptor in a time- and dose-dependent manner. The catecholamine-induced down-regulation of the C receptor was antagonized by a beta 2-selective adrenergic antagonist, ICI 118,551 but not by an alpha 1-, alpha 2-, or beta 1-adrenergic antagonist. Forskolin, NaF and, 8-bromo-cyclic AMP also decreased the C receptor density. The isoproterenol-induced decrease in the C receptor level was further confirmed by affinity cross-linking and Western blot analysis. Northern blot analysis revealed that isoproterenol and 8-bromo-cyclic AMP decreased the steady-state level of C receptor mRNA. By contrast, neither the ANP-A receptor nor the ANP-B receptor mRNA level was affected by 8-bromo-cyclic AMP. The nuclear run-on assay showed that the transcriptional rate of the C receptor gene was decreased by isoproterenol, whereas those of the ANP-A and ANP-B receptor genes were unchanged. Isoproterenol attenuated the clearance of exogenously added ANP and augmented the ANP-stimulated intracellular cyclic GMP production to the same extent as the selective occupancy of the C receptor with C-ANF-(4-23), suggesting that the isoproterenol-induced enhancement of responsiveness to ANP could result not from the sensitization of the ANP-A or ANP-B receptor but from the down-regulation of the C receptor, which leads to the attenuated clearance of ANP. These findings suggest that the beta 2-adrenergic receptor stimulation down-regulates the C receptor through the decrease in the transcriptional rate of the C receptor gene and that the activation of the sympathetic nervous system augments the biological responsiveness to natriuretic peptides by attenuating their metabolic clearance in vascular walls.

8-Bromo Cyclic Adenosine Monophosphate↗

Modulation of KC/gro protein (interleukin-8 related protein in rodents) release from hepatocytes by biologically active mediators.

Modulation of the release of KC/gro protein (a chemoattractant for neutrophils; IL-8 related protein in rodents) from isolated hepatocytes after stimulation with biologically active mediators was investigated. The release of KC/gro protein from hepatocytes of control rats was enhanced by stimulation with lipopolysaccharide, IL-1 beta or TNF-alpha in a dose-dependent manner, but was not enhanced by IL-6. In contrast, although spontaneous release of KC/gro protein from the hepatocytes of chronically ethanol-fed rats was markedly enhanced as compared with control rats, the relative increase by stimulation with lipopolysaccharide, IL-1 beta or TNF-alpha was significantly smaller than in controls. These findings suggest that the regulation of hepatocyte KC/gro protein production might be disturbed in chronically ethanol-fed rats.

Alcoholism↗

Expression of biologically active receptors for natriuretic peptides in the human uterus during pregnancy.

Human amnion cells secrete a large amount of brain natriuretic peptide (BNP). In the present study, to elucidate the possible roles of amniotic BNP during the course of human pregnancy, the tissue distributions of biologically active receptors of natriuretic peptides, atrial natriuretic peptide (ANP)-A receptor and ANP-B receptor in the human uterus during pregnancy were investigated by Northern blot analysis and an in vitro guanosine 3',5'-cyclic phosphate (cGMP) generation assay, the second messenger of natriuretic peptides, using crude membrane preparations. Both ANP-A and ANP-B receptor mRNAs were detected in the tissues of decidua vera, chorion laeve, myometrium and placenta. The in vitro cGMP generation assay revealed that the expression of ANP-A receptor was more prominent than that of ANP-B receptor in these tissues. On the other hand, in amniotic tissue only ANP-B receptor mRNA was weakly expressed but ANP-A receptor mRNA was not detected.

Atrial Natriuretic Factor↗

Enzymic O-sulfation of tyrosine residues in hirudins by sulfotransferase from Eubacterium A-44.

The enzymic O-sulfation of Tyr residues in a recombinant hirudin variant-1 (rHV-1) and its analog in which Glu61 and Glu62 were replaced by Tyr, [E61Y, E62Y]rHV-1, was carried out by use of sulfotransferase isolated from an anaerobic bacterium from the human intestine, Eubacterium A-44. Although rHV-1 was not sulfated by this enzyme, the sulfation of [E61Y, E62Y]rHV-1 was observed, and three kinds of sulfated analog, whose C-terminal six amino acid residues were -PYY(SO3H)YLQ, -PYYY(SO3H)LQ, and -PYY(SO3H)Y(SO3H)LQ, were obtained. Among the sulfated hirudin analogs tested here, the Tyr62 and Tyr63 bisulfated [E61Y, E62Y]rHV-1 showed the strongest thrombin inhibition with the inhibition constant (Ki) of 0.0430 pM, followed by the Tyr63 monosulfated analog (Ki = 0.0593 pM) and the Tyr62 monosulfated one (Ki = 0.158 pM). The Tyr63 monosulfated analog and Tyr62 and Tyr63 bisulfated one were more potent inhibitors of thrombin than unsulfated rHV-1. The increase in affinity caused by sulfation was predominantly due to an increase in the association-rate constant.

Amino Acid Sequence↗

Detection of C-type natriuretic peptide in human circulation and marked increase of plasma CNP level in septic shock patients.

We have previously reported that C-type natriuretic peptide (CNP), the third member of natriuretic family, was produced in vascular endothelial cells and hypothesized that CNP might be a local regulator of vascular tone and/or growth from endothelial cells. In order to clarify the pathophysiological significance of CNP in humans, we examined the presence of CNP in human circulation and determined plasma levels of CNP in patients with various cardiovascular disorders. The plasma level of CNP in healthy persons was 1.4 +/- 0.6 fmol/ml (n = 13). The plasma level of CNP was markedly increased in patients with septic shock (13.2 +/- 10.1 fmol/ml, n = 11), while there was no alteration in patients with congestive heart failure or hypertension. There was two-fold increase of the plasma CNP level in patients with chronic renal failure. These results indicate that CNP, which can be considered as an endothelium-derived relaxing peptide, is detectable in human circulation and suggest the pathophysiological significance of endothelial CNP in humans.

Aged↗

Production of hematopoietic stem cell-chemotactic factor by bone marrow stromal cells.

Chemotactic factors produced by stromal cells in the bone marrow are characterized. Two kinds of factors produced by stromal cell lines are identified using blind-well Boyden's Chambers; one is a neutrophil-chemotactic factor and the other a hematopoietic stem cell (HSC)-chemotactic factor. The latter attracts blastic cells in a low-density fraction, which are Thy1lo, wheat germ agglutinin (WGA)hi, H-2Khi, Ly-1-, Ly-2-, L3T4-, Ly5-, and slg-. The molecular weight of this HSC-chemotactic factor is estimated to be more than 200 kD. Putative cytokines and growth factors, such as granulocyte colony-stimulating factor (CSF), macrophage CSF, granulocyte-macrophage CSF, stem cell factor (SCF), interleukin (IL)-6, and IL-3, do not possess HSC-chemotactic activity. These findings strongly suggest that bone marrow stromal cells produce a new factor that attracts HSCs.

Animals↗

Sequence-specific cleavage of RNA by a hybrid ribonuclease H.

Site-specific cleavage of the 22-, 132- and 534-base RNAs by the DNA/protein hybrid RNase H were examined. The 22-base RNA was chemically synthesized, and 132- and 534-base RNAs were prepared by run-off transcription. The hybrid enzyme cleaves these RNAs, which contain a single target sequence, primarily at the unique phosphodiester bond within the target sequence. The hybrid enzyme performs multiple turnovers, and at a substrate/enzyme ratio of 10:1 the RNAs are almost completely cleaved by the hybrid enzyme at 37 degrees C within 1 h. We propose that hybrid RNase H molecules with various oligodeoxyribonucleotides function as RNA restriction enzymes and are useful for structural and functional studies of RNA.

Base Sequence↗

Structure and chromosomal localization of the aminomethyltransferase gene (AMT)

The gene for human aminomethyltransferase (AMT), also known as the T-protein of the glycine cleavage system, was isolated from a human placental cosmid library and examined by restriction mapping, polymerase chain reaction analysis, and DNA sequencing. The gene is about 6 kb in length and consists of nine exons. The 5'-flanking region of the gene lacks typical TATAA sequence but has a single defined transcription initiation site detected by the primer extension method. Two putative glucocorticoid-responsive elements and a putative thyroid hormone-responsive element are present. The AMT gene was assigned to subband 3p21.2-p21.1 by fluorescence in situ hybridization.

Amino Acid Metabolism, Inborn Errors↗

Detection of alpha-fetoprotein mRNA, an indicator of hematogenous spreading hepatocellular carcinoma, in the circulation: a possible predictor of metastatic hepatocellular carcinoma.

We attempted to detect circulating hepatocellular carcinoma by demonstrating hepatocyte-associated mRNA in the nuclear cell component of peripheral blood using nested reverse transcription-polymerase chain reaction because of the extremely small number of tumor cells in the circulation. Albumin mRNA was demonstrated not only in the liver tissue (hepatocytes) and HepG2 cells but also in nuclear cells of the blood from normal healthy volunteers (neutrophils and lymphocytes) by reverse transcription-polymerase chain reaction. In contrast, alpha-fetoprotein mRNA was demonstrated in the liver tissue, as well as in HepG2 cells, but not in peripheral blood of normal healthy volunteers, indicating the possibility of using alpha-fetoprotein mRNA for detection of benign and malignant hepatocytes among the population of neutrophils and lymphocytes. alpha-Fetoprotein mRNA in peripheral blood was detected in 17 of 33 cases of hepatocellular carcinoma (52%), 2 of 13 cases of cirrhosis (15%) and 2 of 17 cases of chronic hepatitis (12%). alpha-Fetoprotein mRNA was not demonstrated in 26 cases of normal healthy volunteers (0%). Among the patients with hepatocellular carcinoma, total volume of tumor tissue, maximum size of tumor and serum alpha-fetoprotein level were markedly increased in the patients with alpha-fetoprotein mRNA in blood. In addition, alpha-fetoprotein mRNA was detected in the blood of all 6 patients showing metastasis at extrahepatic organs (100%), in contrast to 11 of 27 cases without metastasis (41%). From these results, we conclude that the presence of alpha-fetoprotein mRNA in peripheral blood may be an indicator of circulating malignant or benign hepatocytes, which might predict hematogenous spreading metastasis of tumor cells in patients with hepatocellular carcinoma.

Adult↗

Role of polymorphonuclear leukocytes in galactosamine hepatitis: mechanism of adherence to hepatic endothelial cells.

To investigate the role of polymorphonuclear leukocytes in galactosamine-induced hepatic injury, we injected rats intraperitoneally with antiserum against rat polymorphonuclear leukocytes to deplete circulating neutrophils, then administered galactosamine plus lipopolysaccharide. Polymorphonuclear leukocytes in the hepatic sinusoids were increased after administration of galactosamine plus lipopolysaccharide, whereas pretreatment with the antiserum decreased the number of circulating leukocytes and reduced the mortality and the severity of hepatic injury. Serum collected 1 hr after galactosamine/lipopolysaccharide treatment enhanced in vitro polymorphonuclear leukocyte adherence to hepatic endothelial cells and induced leukocyte superoxide production. Intercellular adhesion molecule-1 expression on hepatic endothelial cells was also enhanced after stimulation with the serum. Polymorphonuclear leukocyte adhesion was partially inhibited by an antibody against tumor necrosis factor-alpha but not by superoxide dismutase. These results suggest that polymorphonuclear leukocytes play an important role in galactosamine-induced hepatic injury and that the accumulation and activation of leukocytes, as well as the enhanced expression of adhesion molecules on hepatic endothelial cells, can be induced by biologically active mediators such as tumor necrosis factor-alpha. In addition, prostaglandins E1 and E2 lessened the enhanced adherence of polymorphonuclear leukocytes and thus contributed to protection against hepatic injury.

Alprostadil↗