Biomedical subjects

H Urata

Publications and source records attributed to H Urata.

At least 73 records · Page 4Linked to original sources

PubMed1993

Measurement of angiotensin I converting enzyme inhibition in the heart.

Angiotensin (Ang) I converting enzyme (ACE) inhibitors represent a major advance in the treatment of congestive heart failure, and tissue, rather than circulating ACE, may be their major site of action. However, assessments of tissue ACE inhibition in treated patients has not always supported this contention. In these studies, ACE activity was measured in homogenates of sampled tissue by biochemical methods. In the present study, using a model system, we have examined the validity of these tissue-sampling methods. Functional ACE activity was determined by comparing positive inotropic responses to [Pro10]Ang I in either vehicle-pretreated or ACE inhibitor-pretreated papillary muscles. [Pro10]Ang I elicits a response, which is entirely dependent on ACE-mediated conversion to Ang II. The ACE inhibitors studied were captopril, enalaprilat, lisinopril, and quinaprilat. In a parallel study, papillary muscle ACE activity was also measured in homogenates using [125I]MK-351A (a radiolabeled ACE inhibitor) binding. The studies indicate that the tissue-sampling method significantly underestimated functional ACE inhibition in hamster papillary muscles (p < 0.001). Kinetic studies indicated that the half-time for the dissociation of [3H]enalaprilat and [3H]lisinopril from hamster ventricular ACE was 4.5 and 6.2 minutes, respectively. The dissociation of [3H]quinaprilat was biphasic (half-time, 47 and 90 minutes), indicating that the two active sites of somatic ACE differ in their ability to bind to this inhibitor. The rapid rate of ACE inhibitor dissociation suggests that, during the time taken to assay ACE activity biochemically, the enzyme becomes "disinhibited," leading to an underestimation of functional ACE inhibition. ACE inhibitor dissociation rates were partially predictive of the duration of functional ACE inhibition in papillary muscles; other factors that appeared to contribute were "tissue trapping" of the inhibitor and de novo synthesis of ACE in papillary muscles. Quantification of tissue ACE inhibition and its relation to drug efficacy must, therefore, involve a careful consideration of these factors to avoid artifacts in clinical decision making and in assessments of pathogenic mechanisms involved in congestive heart failure.

Angiotensin I↗

PubMed1993

Cellular localization and regional distribution of an angiotensin II-forming chymase in the heart.

The human heart is a target organ for the octapeptide hormone, angiotensin II (Ang II). Recent studies suggest that the human heart contains a dual pathway of Ang II formation in which the major Ang II-forming enzymes are angiotensin I-converting enzyme (ACE) and chymase. Human heart chymase has recently been purified and its cDNA and gene cloned. This cardiac serine proteinase is the most efficient and specific Ang II-forming enzyme described. To obtain insights into the cardiac sites of chymase-dependent Ang II formation, we examined the cellular localization and regional distribution of chymase in the human heart. Electron microscope immunocytochemistry using an anti-human chymase antibody showed the presence of chymase-like immunoreactivity in the cardiac interstitium and in cytosolic granules of mast cells, endothelial cells, and some mesenchymal interstitial cells. In the cardiac interstitium, chymase-like immunoreactivity is associated with the extracellular matrix. In situ hybridization studies further indicated that chymase mRNA is expressed in endothelial cells and in interstitial cells, including mast cells. Tissue chymase levels were determined by activity assays and by Western blot analyses. Chymase levels were approximately twofold higher in ventricles than in atria. There were no significant differences in chymase levels in ventricular tissues obtained from non-failing donor hearts, failing ischemic hearts, or hearts from patients with ischemic cardiomyopathy. These findings suggest that a major site of chymase-dependent Ang II formation in the heart is the interstitium and that cardiac mast cells, mesenchymal interstitial cells, and endothelial cells are the cellular sites of synthesis and storage of chymase. In the human heart, because ACE levels are highest in the atria and chymase levels are highest in ventricles, it is likely that the relative contribution of ACE and chymase to cardiac Ang II formation varies with the cardiac chamber. Such differences may lead to differential suppression of cardiac Ang II levels during chronic ACE inhibitor therapy in patients with congestive heart failure.

Adolescent↗

PubMed1993

Spectroscopic characterization of heterochiral DNAs.

We have synthesized heterochiral dodecadeoxynucleotides having an unnatural L-nucleotide residue, and have investigated their structures by ultraviolet (UV) absorption, circular dichroism (CD) measurements and nuclear magnetic resonance (NMR) spectroscopy. It was clearly shown that the overall structures of the heterochiral 12-mers are a right-handed B-conformation and the unnatural L-nucleotide residue in the heterochiral 12-mer (L-4) forms stable Watson-Crick type base-pairing with the natural complementary residue. In this double helix (L-4), the unnatural G4 residue has an S-type sugar conformer and a low-anti glycosidic torsion angle. From the properties of an enantiomer, natural nucleotides may possibly form a low-anti left-handed B-form duplex with the aid of certain factors.

Base Sequence↗

PubMed1993

Cardiac angiotensin II formation: the angiotensin-I converting enzyme and human chymase.

Angiotensin-I converting enzyme (ACE) inhibitors have provided a remarkable improvement in the treatment of patients with primary hypertension and congestive heart failure. The cardiac renin-angiotensin system is one of the major targets of ACE inhibitor therapy since recent studies show that the human heart contains high affinity angiotensin II (Ang II) receptors and ACE activity. However, it is not clear why ACE inhibitors are more effective than other vasodilators in the treatment of patients with congestive heart failure. This gap in knowledge led us to study the biochemical mechanism of Ang II formation in the human heart. Such studies have only recently been addressed. So far, two Ang II-forming enzymes (ACE and human chymase) have been identified. Unlike in the rat heart, the minor (10%) component of Ang II-forming activity in the left ventricle is due to ACE, whereas the major (80%) component is due to human chymase. This novel cardiac serine proteinase has been purified from the human left ventricle and characterized, and recently, the cDNA and the gene for this enzyme have been cloned. Biochemical characterization revealed that human chymase is the most efficient and specific Ang II-forming enzyme described thus far, but the cellular and regional distribution of two Ang II-forming enzymes seem to be quite different. ACE is localized mainly in endothelial cells and its expression level is higher in atria than ventricles whereas chymase is localized in the interstitial region of the myocardium and its expression is higher in ventricles than atria.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗

PubMed1993

[Body composition (densitometry-hydrostatics), skinfold thickness, BMI and their relationships in junior high school girls].

A study was conducted to evaluate body composition (Hydrostatics = Under-Water Weighing), skinfold thickness, and BMI in 97 junior high school girls, aged 12 to 15, in Nagasaki City. Body density was measured by under-water weighing, and the two compartments--fat weight (Fat) and lean body mass (LBM, or fat free weight: FFW)--were calculated using the formulas of Brozek et al. The results were as follows; 1. Mean values of body density were 1.0501 in first grade girls, 1.0476 in the second grade, and 1.0466 in the third grade. 2. Mean values of percentage body fat (% Fat) were 21.1% in the first grade, 22.1% in the second and 22.5% in the third. 3. Lean body mass (LBM) and LBM/Height increased significantly with advance from the first to the third grade. 4. The correlation coefficient between percent body fat and the sum of two skinfold thicknesses, the sum of three skinfold thicknesses, or the sum of seven skinfold thicknesses were 0.81, 0.80, and 0.82 respectively and all statistically significant (p < 0.001). 5. The correlation coefficient between BMI and the sum of two skinfold thicknesses, the sum of three skinfold thicknesses (r = 0.841) or the sum of seven skinfold thicknesses were 0.85, 0.84, and 0.84 respectively, and all statistically significant (p < 0.001). 6. Mean values of BMI, WSR, ASR, and WHR in all subjects (n = 97) were 19.6, 0.72, 0.39 and 0.43 respectively.

Adolescent↗

PubMed1993

[DNA flow cytometry in smooth muscle tumors of the gastrointestinal tract].

DNA ploidy of 45 smooth muscle tumors of the G.I. tract was determined by flow cytometry and correlated with clinical features and prognosis. The sites of the tumors were: esophagus (1), stomach (24), small intestine (12), large intestine (6), liver (1) and pancreas (1). The histologic type was leiomyoma in 14, leiomyosarcoma in 29, and leiomyoblastoma in 2. DNA aneuploidy was more frequent in leiomyosarcoma (17/29) than leiomyoma (5/14), but the difference was not statistically significant. One leiomyoblastoma was diploid and the other was aneuploid. No patients with leiomyoma died. In patients with leiomyosarcomas, 5-year survival was significantly poorer in those with aneuploid tumors (38%) than in those with diploid tumors (83%). There was no correlation between DNA ploidy and clinico-pathological features of tumors. The present study disclosed that DNA ploidy is a prognostic variable, independent of other variables.

Adult↗

PubMed1992

Effects of 1,3-chelation induced by cis-diamminedichloroplatinum(II) on the stability of DNA duplexes.

Several 1,3-intra-strand cross-linked decadeoxynucleotide duplexes, modified with cis-diamminedichloroplatinum(II) (cis-DDP), and their base substitution analogues at the complementary site to the intervening base of the coordination sites, were synthesized and measured for UV-melting profiles to determine melting temperature (Tm) values. The results indicated the thermal stability of the oligonucleotide duplexes containing Pt-induced 1,3-intra-strand cross-linking did not depend on the kind of intervening base of the coordination site but rather on its complementary base. These results may explain the mutagenicity of cis-DDP from a chemical aspect.

Base Sequence↗

PubMed1992

Synthesis and properties of mirror-image DNA.

We have investigated the conformations of the hexadeoxyribonucleotide, L-d(CGCGCG) composed of L-deoxyribose, the mirror image molecule of natural D-deoxyribose. In this paper, we report the synthesis of four L-deoxynucleosides and the L-oligonucleotide-ethidium bromide interactions. The L-deoxyribose synthon 9 was synthesized from L-arabinose with an over all yield of 28.5% via the Barton-McCombie reaction. The L-deoxynucleosides were obtained by a glycosylation of appropriate nucleobase derivatives with the 1-chloro sugar 9. After derivatization to nucleoside phosphoramidites, L-deoxycytidine and L-deoxyguanosine were incorporated into a hexadeoxynucleotide, L-d(CGCGCG) by a solid-phase beta-cyanoethylphosphoramidite method. This L-hexanucleotide was resistant to digestion with nuclease P1. The conformations of L-d(CGCGCG) were an exact mirror image of that of the corresponding natural one as described previously, and the conformations of the L-d(CGCGCG)-ethidium bromide complex were also the mirror images of those of the D-d(CGCGCG)-ethidium bromide complex under both low and high salt conditions. These results suggest that ethidium bromide prefers not a right-handed helical sense, but the base-base stacking geometry of the B-form rather than that of the Z-form. Thus, L-DNA would be a useful tool for studying DNA-drug interactions.

Chromatography, High Pressure Liquid↗

PubMed1992

Application of L-DNA to the study of the specific DNA recognition mechanism of bleomycin.

The hexadeoxynucleotide analog, L-d(CGCGCG) composed of L-deoxyribose was synthesized and clearly shown to have the same conformation and dynamic properties with natural D-d(CGCGCG) except for chirality with CD spectra. This unnatural hexanucleotide was not cleaved by bleomycin, an antitumor DNA cleaving drug, but was able to bind to the DNA binding domain of bleomycin to a similar extent with the natural one. These results strongly suggest the importance of the other moiety than the DNA binding domain for the specific DNA recognition of bleomycin. Thus, L-oligonucleotides are useful for the study of DNA-drug interactions.

Binding Sites↗

PubMed1991

Multiple determinants for the high substrate specificity of an angiotensin II-forming chymase from the human heart.

Human heart chymase, a chymotrypsin-like serine proteinase that hydrolyzes the Phe8-His9 bond in angiotensin I (Ang I) to yield the octapeptide hormone angiotensin II (Ang II) and His-Leu, is the most specific, efficient Ang II-forming enzyme described. Other mammalian chymases display a much broader substrate specificity. To better define its substrate specificity, we have mapped the extended substrate-binding site of human heart chymase using Ang I analogs. The enzyme has a preference for aromatic amino acids phenylalanine, tyrosine, and tryptophan at the P1 site. At the S2 subsite there is a significant preference for proline over hydrophobic or hydrophilic amino acids. There is no clear preference for hydrophobic or hydrophilic amino acids at the S'1 and S'2 subsites, but an Ang I analog containing a P'1 proline is not hydrolyzed and one with a P'2 proline is hydrolyzed poorly. An increasing reduction in reactivity occurs when the P position amino acids in Ang I are deleted sequentially from the N terminus. An increase or decrease in the length of the His-Leu leaving group also produces a marked decrease in reactivity. No single determinant in Ang I is preeminently required for efficient catalysis, but several factors acting synergistically appear to be important. Thus, we propose that ideal substrates for human heart chymase should contain the structure nXaa-Pro-[Phe, Tyr, or Trp]-Yaa-Yaa, where n greater than or equal to 6; Xaa = any amino acid; Yaa = any amino acid except proline. This structure exists in Ang I and neurotensin, both of which are good substrates for human heart chymase. These findings indicate that the selection of the scissile bond by the extended substrate-binding site of human heart chymase is more restricted than that in other chymases.

Amino Acid Sequence↗

PubMed1991

Cloning of the gene and cDNA for human heart chymase.

We have recently identified and characterized a chymotrypsin-like serine proteinase in human heart (human heart chymase) that is the most catalytically efficient enzyme described, thus far, for the cleavage of angiotensin I to yield angiotensin II and the dipeptide His-Leu. Compared to other chymases, this enzyme also has an unusually high degree of specificity for the substrate angiotensin I. We report here the molecular cloning and nucleotide sequence of the gene and cDNA encoding human heart chymase, and determination of its entire deduced amino acid sequence. These data indicate that human heart chymase is highly homologous to other members of the chymase subfamily of chymotrypsin-like proteinases and, most likely, all evolved from a common ancestral gene. Potential regulatory elements found in the 5'-untranslated region of other chymases are also found in the human heart chymase gene. However, this gene lacks mast cell-specific sequences found in the 5'- and 3'-untranslated regions of the rat chymase II gene. In addition, human heart chymase contains clusters of unique amino acid sequences located at key positions likely involved in substrate binding, which may contribute to its high substrate specificity. These contrasting features of the human heart chymase gene and cDNA, and the potential determinants of its primary structure that underlie its unique functional characteristics are considered.

Amino Acid Sequence↗

PubMed1991

Photo-induced formation of the 2-deoxyribonolactone-containing nucleotide for d(ApCpA); effects of neighboring bases and modification of deoxycytidine.

Previously, we reported the formation of a 2-deoxyribonolactone-containing nucleotide by UV irradiation of d(ApCpA). In this paper, we report some mechanical feature of this novel photoreaction. From the results of photolysis of d(GpCpG), this reaction is independent of neighboring bases. On photolysis of deoxycytidine at acidic pH, 2-deoxyribonolactone was produced with 74.2% isolated yield. And effects of some modifications of deoxycytidine were investigated. The results suggest that hydrates are possible intermediates for this reaction and the protonation may be essential not for deoxycytidine, but for the intermediates.

Adenine↗

PubMed1991

Interstrand cross-linking of the hexadeoxynucleotide d(TACGTA) upon reaction with trans-diamminedichloroplatinum(II).

The reaction of trans-diamminedichloroplatinum(II), an inactive isomer of cisplatin, with d(TACGTA) was performed in aqueous solution, and the interstrand cross-linked adduct was isolated on a preparative scale. This adduct was characterized by 1 M thiourea treatment, atomic absorption spectroscopy, enzymatic digestion and 1H NMR as platinum coordinated to two molecules of d(TACGTA) at the N7 atom of the guanine residue. This interstrand cross-linked adduct did not form a duplex structure in the conditions where the free hexanucleotide adopts it.

Chromatography, High Pressure Liquid↗

PubMed1990

Identification of a highly specific chymase as the major angiotensin II-forming enzyme in the human heart.

Although angiotensin II (Ang II)-forming enzymatic activity in the human left cardiac ventricle is minimally inhibited by angiotensin I (Ang I) converting enzyme inhibitors, over 75% of this activity is inhibited by serine proteinase inhibitors (Urata, H., Healy, B., Stewart, R. W., Bumpus, F. M., and Husain, A. (1990) Circ. Res. 66, 883-890). We now report the identification and characterization of the major Ang II-forming, neutral serine proteinase, from left ventricular tissues of the human heart. A 115,150-fold purification from human cardiac membranes yielded a purified protein with an Mr of 30,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based upon its amino-terminal sequence, the major human cardiac Ang II-forming proteinase appears to be a novel member of the chymase subfamily of chymotrypsin-like serine proteinases. Human heart chymase was completely inhibited by the serine proteinase inhibitors, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, and chymostatin. It was partially inhibited by p-tosyl-L-phenylalanine chloromethyl ketone, but was not inhibited by p-tosyl-L-lysine chloromethyl ketone, and aprotinin. Also, human heart chymase was not inhibited by inhibitors of the other three classes of proteinases. Human heart chymase has a high specificity for the conversion of Ang I to Ang II and the Ang I-carboxyl-terminal dipeptide His-Leu (Km = 60 microM; Kcat = 11,900 min-1; Kcat/Km = 198 min-1 microM-1). Human heart chymase did not degrade several peptide hormones, including Ang II, bradykinin, and vasoactive intestinal peptide, nor did it form Ang II from angiotensinogen. The high substrate specificity of human heart chymase for Ang I distinguishes it from other Ang II-forming enzymes including Ang I converting enzyme, tonin, kallikrein, cathepsin G, and other known chymases.

Amino Acid Sequence↗

PubMed1990

Angiotensin II-forming pathways in normal and failing human hearts.

Reduced preload and afterload to the heart are important effects of angiotensin converting enzyme (ACE) inhibitors in the treatment of congestive heart failure. However, since angiotensin II (Ang II) directly increases the strength of myocardial contraction, suppression of Ang II formation by ACE inhibitors could potentially reduce the beneficial effects of Ang II on the failing heart. To study how ACE inhibition suppresses cardiac Ang II formation in man, we characterized ACE-dependent and ACE-independent Ang II-forming pathways in eight normal and 24 failing human hearts obtained at cardiac transplantation. Ang II-forming activity in left ventricular (LV) membrane preparations was assessed by measuring the conversion of [125I]angiotensin I (Ang I) to [125I]Ang II. LV [125I]Ang II-forming activity in normal hearts (35.5 +/- 2.7 fmol/min/mg, n = 8) was not different from that in hearts from patients with ischemic cardiomyopathy (25.5 +/- 2.9 fmol/min/mg, n = 9) and was 48% lower (p less than 0.001) in hearts from patients with idiopathic cardiomyopathy (18.5 +/- 1.9 fmol/min/mg, n = 15).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1990

Angiotensins and the failing heart. Enhanced positive inotropic response to angiotensin I in cardiomyopathic hamster heart in the presence of captopril.

We examined the hypothesis that the positive inotropic effect of angiotensin I (Ang I) may be retained in the presence of angiotensin converting enzyme inhibitors so that it may have a direct beneficial effect on the heart. Accordingly, isolated perfused hearts (Langendorff preparation) of 300-day-old cardiomyopathic hamsters (a model of spontaneous cardiomyopathy) and age-matched normal hamsters (controls) were infused with Ang I in the presence of captopril; propranolol was added to the perfusing medium to block catecholamine-mediated effects of angiotensins on the heart. Left ventricular developed pressure and the rate of increase in left ventricular developed pressure increased significantly (p less than 0.001) in both the cardiomyopathic and the normal hamster heart despite concomitant reduction in myocardial flow rate favoring a direct inotropic effect of Ang I in both normal and myopathic hearts; these changes were significantly higher by almost threefold in the cardiomyopathic than in the normal hamsters (p less than 0.01) and were blocked by the angiotensin II (Ang II) antagonist [Sar1,Thr8]Ang II. Comparing dose-left ventricular contractility response curves for Ang I and Ang II, ED50 for responses was identical in both normal and myopathic hearts, whereas peak responses to Ang II were double those to Ang I in normal hearts but were almost identical in the myopathic hearts. Binding of [125I]Ang II in six cardiomyopathic and four normal hamster hearts was of high affinity, but there was no evidence for Ang I-saturable high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin I↗

PubMed1989

A 2-deoxyribonolactone-containing nucleotide: isolation and characterization of the alkali-sensitive photoproduct of the trideoxyribonucleotide d(ApCpA).

It has been reported that ACA sequences in DNA are mutagenic hot spots in UV-induced mutagenesis and are sites of an alkali-sensitive lesion produced by UV irradiation. In order to characterize the UV-induced lesion of an ACA site, chemically synthesized trideoxyribonucleotide d(ApCpA) was irradiated with UV light and the alkali-sensitive photoproduct was isolated. The structure of this photoproduct was characterized as the trinucleotide containing 2-deoxyribonolactone at the internal residue by 2D DQF-COSY, FT-IR, FAB-MS, and chemical properties. It is known that this lesion is also produced by gamma-irradiation, neocarzinostatin, the 1,10-phenanthroline-copper complex, and hydrogen peroxide and is highly mutagenic because of its resistance to cleavage by certain apurinic/apyrimidinic (AP) endonucleases. Thus, 2-deoxyribonolactone may be one of the lethal DNA lesions induced by UV irradiation to organisms and one of the intermediates of UV-induced DNA strand breaks because the DNA strand is cleaved at this site with beta- and subsequent delta-elimination mechanisms.

Base Composition↗

PubMed1989

Mechanism of the reversal reaction of platinated DNA with thiourea studied by platinated 5'-GMP.

The reversal reactions of cis-[Pt(NH3)2(5'-GMP)2] 2-(1) and trans-[Pt(NH3)2(5'-GMP)2] 2-(2) with thiourea were examined by reversed phase HPLC and monothioureido intermediate cis-[Pt(NH3)2(5'-GMP) (tu)] (4) was detected. This result suggested that Pt-[5'-GMP-N(7)] bond was more labile than Pt-NH3 bond and the release of ammonia from cis-Pt(II)-DNA base complexes is a result of trans-labilizing effect of sulfur containing molecule displaced with DNA base.

Ammonia↗