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Biomedical subjects

H Sugihara

Publications and source records attributed to H Sugihara.

At least 397 records · Page 22Linked to original sources
PubMed1986

Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues.

Bromodeoxyuridine (BrdUrd) immunohistochemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. In order to detect the BrdUrd incorporated into nuclear DNA in formalin-fixed, paraffin-embedded tissues, we tested several different pretreatment procedures including digestion with proteinase and hydrolysis with HCl, prior to immunoperoxidase staining. In order to determine the optimal conditions for detecting nuclear BrdUrd, mice were given BrdUrd and 3H-thymidine simultaneously, and the autoradiographic and immunohistochemical results obtained in BrdUrd-stained sections were compared. It was found that digestion with 0.05% proteinase at 37 degrees C for 20 min and hydrolysis with 1N HCl at 37 degrees C for 20 min was sufficient to detect BrdUrd immunoreactivity in 3H-thymidine-labelled nuclei, the results being virtually unaffected by the orders in which the two pretreatments were performed. Our method extends the range of application for BrdUrd immunohistochemistry in cell-kinetic studies.

Animals↗
PubMed1986

Characterization of Ac3-proteinase from the venom of Agkistrodon acutus (hundred-pace snake).

Ac3-Proteinase from the venom of Agkistrodon acutus was isolated in a homogeneous form by a previously published method. Ac3-Proteinase possessed lethal, hemorrhagic, caseinolytic, azocaseinolytic, dimethylcaseinolytic and hide powder azure hydrolytic activities. These activities were inhibited when Ac3-Proteinase was incubated with the metal chelators ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), tetraethylenepentamine (TEP), 1,10-phenanthroline, phosphoramidon or beta-mercaptoethanol. The toxin also hydrolyzed the oxidized A and B chains of both insulin and fibrinogen. The cleavage sites in the oxidized B chain of insulin were identified as His(10)-Leu(11), Ala(14)-Leu(15), Tyr(16)-Leu(17) and Phe(24)-Phe(25). The A alpha chain of fibrinogen was digested first followed by hydrolysis of the B beta chain. Toxicological and biochemical properties of Ac3-Proteinase were investigated further and are reported in this paper.

Angiotensin I↗
PubMed1986

Purification and characterization of phosphodiesterase from the venom of Trimeresurus mucrosquamatus.

Phosphodiesterase was isolated from the venom of Trimeresurus mucrosquamatus from Taiwan using gel filtration on a Sephadex G-100 column, followed by anion or cation exchange chromatography. Phosphodiesterase was homogeneous as established by a single band on acrylamide gel electrophoresis and immunodiffusion. Phosphodiesterase activity was inhibited by ethylenediamine tetraacetic acid (EDTA), o-phenanthroline, thioglycolic acid or p-chloromercuribenzoate (PCMB) but not by soybean trypsin inhibitor (SBTI) or benzamidine. The molecular weight of this enzyme was determined to be approximately 140,000 and the isoelectric point was found to be pH 7.4 by isoelectric focusing with carrier ampholyte. The Michaelis constant (Km) of this enzyme for p-nitrophenyl thymidine-5'-phosphate and inhibition constant (Ki) for PCMB were found to be 5.6 X 10(-3) and 7.6 X 10(-4) M, respectively.

Amino Acids↗
PubMed1986

Reevaluation of hemorrhagic toxin, HR-I, from Agkistrodon halys blomhoffii venom: proof of proteolytic enzyme.

HR-I is a hemorrhagic toxin originally isolated from Agkistrodon halys blomhoffii (Mamushi) venom by Oshima et al. (1972). It was reported by the original investigators that it was nonproteolytic when casein was used as the substrate. HR-I was isolated again and proteolytic activity was tested using different substrates and assay methods. It is shown that HR-I is indeed a proteolytic enzyme hydrolyzing a number of peptide bonds. This present investigation suggests that more than one method should be used for proteolytic enzyme assay of hemorrhagic toxins. Toxicological and biochemical properties of HR-I were further investigated and are reported in this paper.

Amino Acids↗
PubMed1986

Comparative study of four arginine ester hydrolases, ME-1, 2, 3 and 4 from the venom of Trimeresurus mucrosquamatus (the Chinese habu snake).

Four arginine ester hydrolases, ME-1, 2, 3 and 4 from the venom of Trimeresurus mucrosquamatus had been isolated and characterized by Sugihara et al. (1980, 1981, 1982, 1983). Immunologically, ME-1, 2, 3 and 4 are identical. The four enzymes hydrolyzed Pro-Phe-Arg-MCA and z-Phe-Arg-MCA. Furthermore, ME-2 slightly hydrolyzed Boc-Val-Pro-Arg-MCA, Boc-Phe-Ser-Arg-MCA and Boc-Ile-Glu-Gly-Arg-MCA. ME-1 cleaved almost simultaneously the Arg(22)-Gly(23) and Phe(25)-Tyr(26) bond of oxidized insulin B chain. ME-2 and 3 also hydrolyzed the same bond of insulin B chain, but the activity was not as potent as ME-1. ME-4 did not cleave the substrate. The four enzymes hydrolyzed C-terminal of arginine in the biologically active peptides. Four arginine ester hydrolases showed fibrinogenolytic activity. ME-1 and 2 first cleaved B beta-chain and then A alpha-chain. On the contrary, ME-3 and 4 cleaved A alpha- and B beta-chain simultaneously. The four enzymes also hydrolyzed fibrinogen in plasma cleaving B beta- and gamma-chain and slightly digesting A alpha-chain. The various inhibitors affected TAME (tosyl-arginine-methylester) and the fibrinogen hydrolytic activity of the four enzymes. All four enzymes had fibrinolytic activity.

Amino Acid Sequence↗
PubMed1986

Primary cultures of unilocular fat cells: characteristics of growth in vitro and changes in differentiation properties.

Mature white fat tissue consists primarily of unilocular fat cells. Clearly, the study of the biology of these cells would be most helpful for the elucidation of the mechanism of obesity. We describe a new method termed 'ceiling culture' for culturing in vitro unilocular fat cells obtained from humans and rats. These cells can be maintained in culture for long periods of time and, under such conditions, continue to exhibit specific functions such as lipogenesis and lipolysis. Under the culture conditions described, unilocular fat cells change into multilocular fat cells or cells with a fibroblast-like appearance. These cells then proliferate, form a cell monolayer attached to the substratum, and after becoming confluent, exhibit accumulations of intracytoplasmic lipid droplets. These attached dedifferentiated cells continue to exhibit lipogenesis and lipolysis.

Adipose Tissue↗
PubMed1986

Effects of forskolin on the morphology and function of the rat thyroid cell strain, FRTL-5: comparison with the effects of thyrotrophin.

The effect of diterpene forskolin, a potent stimulator of cyclic AMP (cAMP) in the rat thyroid cell strain FRTL-5, was compared with that of TSH. Forskolin stimulated both the release of cAMP into the culture medium and the accumulation of cAMP in the cytoplasm in a dose-dependent manner, within the range of 0.1-1000 mumol/l. Maximum cAMP concentrations were reached within 15 min of stimulation with forskolin. This is comparable with the effects of TSH. Forskolin also induced morphological changes in cultures of FRTL-5 cells, producing conspicuous cell retraction with arborization and numerous microvilli on the cell surface, specific reorganization of the microfilaments and modulation of the distribution of tubulin and fibronectin. Morphological changes induced by forskolin were always observed 20 to 30 min earlier, and in a higher percentage of cells, than the changes induced by TSH. Cell proliferation, however, was stimulated more effectively by TSH than by forskolin. These observations suggest that TSH might exert its effect on the morphology and growth of FRTL-5 cells, at least in part, through cAMP. The control of morphology and growth might not, however, be regulated solely by the adenylate cyclase and cAMP system.

Animals↗
PubMed1986

Modulation of calmodulin function and of Ca2+-induced smooth muscle contraction by the calmodulin antagonist, HT-74.

The relationship between the functions of calmodulin (CaM) and Ca2+-induced smooth muscle contraction was investigated using a newly synthesized CaM antagonist, 3-(2-benzothiazolyl)-4,5-dimethoxy-N-[3-(4- -phenylpiperidinyl)propyl]benzenesulfonamide (HT-74). We noted a selectivity of HT-74 for CaM, compared to other calcium-binding proteins and target enzymes of CaM. As HT-74 had no significant effect on the intensity of 8-anilino-1-naphthalene-sulfonic acid (ANS) fluorescence in the presence of the Ca2+-CaM complex, the HT-74-binding sites may differ from those of naphthalenesulfonamides and phenothiazines which decrease ANS fluorescence. The Ca2+ binding to CaM was inhibited significantly by 1.0 microM HT-74, in sharp contrast to phenothiazines and naphthalenesulfonamides which increase the extent of the Ca2+ binding to CaM. Increasing CaM concentrations reversed the HT-74-induced inhibition of CaM-dependent enzymes such as myosin light chain kinase and Ca2+-dependent cyclic nucleotide phosphodiesterase, with Ki values of 0.5 microM and 0.4 microM, respectively. In the presence of 0.3 microM HT-74, potassium-depolarized rabbit aortic strips pre-contracted with 0.3 mM CaCl2 relaxed, and this relaxation was completely reversed by the addition of an excess amount of CaCl2 (10 mM). This compound shifted the dose-response curve for CaCl2 to the right, in a competitive manner. However, HT-74 inhibited the phenylephrine-induced contraction elicited in Ca2+-free solution and the calcium ionophore A23187-induced contraction in the presence of calcium ion. Therefore, this agent affects intracellular actions of Ca2+ rather than membrane receptors or the influx of Ca2+. HT-74 is a CaM antagonist which binds to CaM in a manner different from that heretofore reported. It inhibits Ca2+ binding to CaM and produces a competitive inhibition of Ca2+-induced contractions of depolarized vascular smooth muscle.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1986

DNA ploidy patterns of minute carcinomas in the stomach.

DNA ploidy patterns of minute carcinomas in the stomach were determined by cytofluorometric measurement, using paraffin sections which had been prepared for histological examination. The examples studied were 19 minute carcinomas less than 5 mm in diameter, of which 12 were adenocarcinomas, and 7 were signet ring cell carcinomas. By measuring the fluorescence intensity of more than 30 mitotic nuclei, the DNA ploidy pattern of each tumor was determined. The control diploid DNA content was obtained by measuring the fluorescence intensity of non-cancerous mitoses in the gastric mucosa. In the present study, heteroploidy was seen in 5 carcinomas; 4 adenocarcinomas and one signet ring cell carcinoma. The remaining 14 carcinomas were composed of a diploid stem cell line. In 3 adenocarcinomas, polyploid cells were seen. The occurrence of heteroploidy in the minute cancers was similar to that found in advanced cancers, whereas polyploid cells appeared to occur less frequently in the minute cancers than in the advanced cancers.

Aged↗
PubMed1986

Cell differentiation and cell cycle effects on human promyelocytic leukemia cells induced by 12-O-tetradecanoylphorbol-13-acetate.

As has been reported, doses of 12-O-tetradecanoylphorbol-13-acetate (TPA) as small as 1 to 100 ng/ml induced human promyelocytic leukemia HL-60 cells to differentiate terminally into macrophage-like cells rather than toward cells of the granulocytic series. This differentiation was accompanied by the appearance of monocyte/macrophage markers and by the disappearance of myeloid markers from the view point of enzyme cytochemistry. Contrasted to untreated HL-60 cells, TPA-treated cells increased in cell size and showed increased phagocytotic activities against opsonized sheep blood red cells and activated yeast. Nitroblue tetrazolium-positive cells increased rapidly after TPA exposure. The alterations of the cell cycle traverse of HL-60 cells by TPA were analyzed by [3H]thymidine autoradiography, flow microfluorimetry, and mitotic cell counting. TPA sequentially caused (a) inhibition of cells to move from G1 to S near G1/S boundary in G1; (b) temporary inhibition in G2; (c) growth arrest of most cells in G1 within 2 to 3 days after TPA exposure.

Cell Cycle↗
PubMed1986

[A case of extraskeletal osteosarcoma of the retroperitoneum].

A case of extraskeletal osteosarcoma of the retroperitoneum in a 57-year-old woman is reported. A rapidly growing intra-abdominal tumor was noticed eight months before her death. The resected tumor, weighing 1.9 kg, consisted of a large amount of solid mucinous tissue with significant bony materials. Histologically, the tumor showed fibrosarcomatous, chondrosarcomatous and osteosarcomatous growth patterns. A diagnosis of osteosarcoma was made. The CT scan and autopsy findings confirmed that the tumor did not originate from the preexisting bones, but from the retroperitoneum. Differences distinguishing retroperitoneal osteosarcoma from other soft tissue sarcomas were also described from the histological point of view.

Female↗