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Biomedical subjects

Y Horiuti

Publications and source records attributed to Y Horiuti.

At least 19 recordsLinked to original sources
PubMed1993

Hormonal regulations of alkaline phosphatase, 5'-nucleotidase and gamma-glutamyltransferase activities in adult rat hepatocytes cultured in serum-free medium on collagen gel.

Primary hepatocytes were cultured on collagen gel in serum-free, alpha-modified Eagle's minimum essential medium containing 0.1 microM insulin, 0.1 microM dexamethasone, 10 mM pyruvate and supplements such as glucagon, epinephrine or growth hormone. The activities of alkaline phosphatase, 5'-nucleotidase and gamma-glutamyltransferase were assayed in cell extracts prepared from the cultures. All three enzyme activities were induced by glucagon, epinephrine or dibutyryl cAMP. The maximally effective concentration of glucagon was 5-10 nM for both alkaline phosphatase and 5'-nucleotidase and 100 nM for gamma-glutamyltransferase. Only alkaline phosphatase activity was suppressed by growth hormone, which caused marked suppression at about 1 microU (0.25 ng)/ml. Taurocholate also induced both alkaline phosphatase and gamma-glutamyltransferase activities at 1 mM.

5'-Nucleotidase↗
PubMed1992

Effect of calcium concentration on survival, proliferation and activities of alkaline phosphatase, 5'-nucleotidase, gamma-glutamyltransferase and lactate dehydrogenase of adult rat hepatocytes cultured in serum-free medium.

Mature rat hepatocytes were cultured on collagen coated dishes in serum-free alpha-modified Eagle's minimum essential medium containing 0.1 microM insulin, 0.1 microM dexamethasone, 10 mM pyruvate and Ca2+ at concentrations of 0-2 mM. Survival of nondivided cells was best in medium containing 2 mM Ca2+. Proliferation during 5-day culture was greatest with 0.4 mM Ca2+, but DNA synthesis was scarcely affected by the concentration of Ca2+. Both the activities of alkaline phosphatase, 5'-nucleotidase, gamma-glutamyltransferase and lactate dehydrogenase and the number of cell nuclei of cultures in 0.1 mM and 2 mM Ca2+ media were assayed over a 5-day period, and their activities were calculated as enzyme activities per unit number of cell nuclei. Alkaline phosphatase activity increased rapidly during the first day of culture in both media, and its activity in 0.1 mM medium was higher than that in 2 mM medium after culture for 3 days. The activity of 5'-nucleotidase became higher in 0.1 mM medium than in 2 mM medium from day 2 and was maximal on day 3 in both media. gamma-Glutamyltransferase activity increased and lactate dehydrogenase activity decreased with time in culture, both activities showing no appreciable difference in the two media.

5'-Nucleotidase↗
PubMed1991

Quantification of cell nuclei isolated from hepatocytes by cell lysis with nonionic detergent in citric acid.

A method was developed for determining the number of nuclei of hepatocytes cultured on collagen gel using a nonionic detergent, Nikkol BO-10TX. The cells were recovered in a test tube after solubilizing the gel by incubating it with the detergent in 0.1 M citric acid and then centrifuging the mixture. Nuclei were isolated from the cells with the same detergent solution and collected by centrifugation. The numbers of nuclei in cultures, scored with a hemocytometer or an electronic particle counter, were proportional to the lactate dehydrogenase activities of the cells. This method was also applicable for scoring the number of nuclei of hepatocytes cultured on collagen-coated plastic.

Animals↗
PubMed1989

[The use of intraoperative auditory evoked brainstem responses in the treatment of congenital ossicular anomalies].

Intraoperative auditory brainstem responses (ABRs) were monitored in 3 patients undergoing middle ear surgery for congenital ossicular anomalies. It was investigated whether this technique was capable of providing useful information to the operating surgeon. After the induction of anaesthesia and intubation, standard disc electrodes were attached to the vertex (active), ipsilateral mastoid (reference), and forehead (ground). These electrodes were draped out of the surgical field. The general anaesthesia was maintained by inhalation of enflurane. Stimulus intensity was decreased from 80 dBnHL in 10 dB steps until no response was obtained. Thus latency-intensity functions for wave V of ABR were obtained. First (A) recording was performed before the incision. The tympanomeatal flap was then elevated and the bony annulus overloading the oval window was drilled out. This enabled to visualize and confirm the anomalies of the ossicles. Before reconstructing the ossicles, the tympanomeatal flap was returned to its normal position and second (B) recording was obtained. During the reconstruction, recordings (C1, C2,...) were repeated as indicated. Then it was confirmed whether the effective reconstruction was obtained for auditory function. The results suggest that latency of wave V was significantly decreased when the effective reconstruction of the ossicular chain was done. Latency-intensity function is shifted to the left of A and B functions. Intraoperative auditory assessment using ABR in the reconstructive surgery for the ossicular channel is promising in providing useful information for auditory function and in predicting postoperative hearing improvement.

Adult↗
PubMed1989

[High-resolution computed tomography of posterior tympanum].

A complex set of sinuses, eminences and ridges lies in the posterior border of the tympanic cavity (posterior tympanum). The facial nerve canal is located between the facial sinus and the sinus tympani. The posterior limit is the posterior semicircular canal. The posterior tympanum is often the site of residual collection of granulation tissue or cholesteatoma, and is not directly visualized by the usual surgical approaches. Thus preoperative knowledge of cholesteatoma in these area is of obvious importance. It has been accepted that high-resolution computed tomography (HRCT) has the significant advantage to provide specific information of the middle ear. Contribution of the HRCT for diagnosis of posterior tympanum was examined by three temporal bone specimens and one hundred subjects with normal middle ear. In the experiments using the temporal bone specimens, each structure in the posterior tympanum was labeled by a fine needle. HRCTs were subsequently obtained to confirm the labeled structure. In one hundred subjects, it was examined whether each structure was also confirmed by the routine HRCT. Results are as follows: (1) Axial HRCT sections provided essential informations for the preoperative evaluation of the posterior tympanum. (2) Such bony structures as the pyramidal eminence and the pyramidal ridge were the most prominent structures on the posterior wall. (3) Laterally close to the pyramidal eminence lies one sinus which was marked by the needle placed in the facial sinus proper or in the lateral tympanic sinus; It is the facial sinus in a wide sense.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1982

Role of serum in maintenance of functional hepatocytes in primary culture.

Serum at 5 to 10% is required for maintenance of functional adult rat hepatocytes in primary culture. One effect of the serum is to induce attachment and spreading of hepatocytes on plates as monolayers. Another role is to maintain cell viability for over 2 days. For the first effect, serum could be replaced completely by fibronectin (Fn). The effects of Fn on attachment and spreading of cells were dose-dependent and maximum at 10 micrograms/ml. Cells in serum-free medium on Fn-coated dishes showed similar activities of glycogenolysis and glycogenesis to cells cultured in medium containing 5% calf serum on untreated dishes in response to glucagon, dibutyryl cyclic AMP (bt2c AMP), isoproterenol and insulin. The increase in alkaline phosphatase [EC 3.1.3.1] activity and induction of tyrosine aminotransferase [EC 2.6.1.5] by dexamethasone (Dex) in cells under the two conditions were also similar. However, the inductions of tryptophan oxygenase [EC 1.13.11.11] by Dex, glucagon, and bt2cAMP were 4-7 times higher in cells cultured in serum-free medium. The inductions by Dex plus glucagon in the two types of cultures were inhibited similarly by insulin. In serum-free medium containing Dex and insulin in Fn-coated dishes, the cells survived as monolayers for about 50 h without detachment from the dishes, but for longer survival it was necessary to add 5% serum to the medium. A fraction with a molecular weight of over 50,000 from serum was separated by ultrafiltration and this fraction showed a similar effect to serum in increasing survival. A similar factor, but with about 70 times higher specific activity, was found in an extract of bovine pituitary gland.

Animals↗
PubMed1980

Purification of phospholipase B from Penicillium notatum by hydrophobic chromatography on palmitoyl cellulose.

Phospholipase B (lysolecithin acyl-hydrolase, EC 3.1.1.5) from the mycelia of Penicillium notatum (Institute for Fermentation, Osaka, Japan; No.4640) was adsorbed from a crude solution to palmitoyl cellulose. Adsorption was efficient at pH 4 at low ionic strength (10 mM buffer), and at pH 4-9 at higher ionic strength (1-2M NaCl in 10 mM buffer). The adsorbed enzyme was eluted from the cellulose with a suitable detergent, such as Adekatol SO-120, Triton X-100, or deoxycholate. As an application of this procedure, the enzyme was purified from an extract of the mycelia by column chromatography on a palmitoylated textile (palmitoylated gauze) with an overall recovery of 59% and a 38-fold increase in specific activity. By subsequent column chromatographies on Amberlite XAD-2, Sephadex G-100 and G-150, and DEAE-Sephadex A-50, the enzyme was purified about 4,000-fold to a nearly homogeneous state from a mycelium extract with an overall recovery of 37%.

Chromatography, Affinity↗
PubMed1979

Purification of Streptomyces chromofuscus phospholipase D by hydrophobic affinity chromatography on palmitoyl cellulose.

Phospholipase D [phosphatidylcholine cholinehydrolase, EC 3.1.4.4] excreted from Streptomyces chromofuscus was purified from the culture supernatant by precipitation with acetone and column chromatographies on palmitoylated gauze (Pal-G), DEAE-cellulose, and Sephadex G-150 with an overall recovery of 46% and 1000-fold increase in specific activity. The purified enzyme preparation showed a single band on sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis. The enzyme had a molecular weight of about 50,000 by gel filtration on Sephadex G-150 or about 57,000 by SDS-polyacrylamide disc gel electrophoresis and an isoelectric point (pI) of pH 5.1 on isoelectric focusing. The enzyme hydrolyses lecithin, lysolecithin, sphingomyelin, and cephalin; the relative reaction velocities and Km's for choline-phospholipids were 87% and 1.43 mM for lecithin, 100% and 1.67 mM for lysolecithin, and 22% and 0.56 mM for sphingomyelin. The enzymatic reaction was optimal at pH 8, and its velocity was appreciably increased by either detergent (Triton X-100, deoxycholate), Ca2+ or both detergent and Ca2+. Diethyl ether stimulated the enzymatic activity by 30%; SDS and EDTA inhibited the activity. Bovine serum albumin, Triton X-100, and lipids (lecithin, lysolecithin, phosphatidic acid, lysophosphatidic acid, palmitic acid, and oleic acid) inhibited adsorption of the purified enzyme onto palmitoyl cellulose (Pal-C) and affected both the enzyme activity and stability: albumin and Triton X-100 increased the activity and enhanced the heat-stability; lysophospholipids decreased the activity but other lipids increased the activity; all the lipids lowered the heat-stability. The enzyme adsorbed on Pal-C was active, although its activity was about one-ninth of that of free enzyme, and was protected from heat-inactivation. Thus this enzyme appears to possess a hydrophobic site distinct from its catalytic site and to be adsorbed onto Pal-C through the hydrophobic site. Albumin, Triton X-100, and lipids seem to bind to the hydrophobic site and to have an appreciable effect on the enzyme activity and stability.

Cations, Divalent↗
PubMed1979

Purification of phospholipase C from Bacillus cereus by hydrophobic chromatography on palmitoyl cellulose.

Phospholipase C (phosphatidylcholine choline-phosphohydrolase, EC 3.1.4.E) from Bacillus cereus (IAM-1208) was adsorbed to palmitoyl cellulose from a crude enzyme solution at pH 5--9. The adsorption was not influenced by ionic strength up to 2 M NaCl. The adsorbed enzyme was eluted almost completely by washing the cellulose with a suitable detergent, such as Triton X-100, Adekatol SO-120, Cation DT-205, or sodium deoxycholate. The enzyme was then purified by column chromatography on a palmitoylated textile (palmitoylated gauze) with an overall recovery of 91% and a 467-fold increase in specific activity over that of enzyme in the crude culture supernatant. Subsequent fractionation with acetone and chromatography on a Sephadex G-75 column separated two nearly homogeneous phospholipase C's. The enzyme adsorbed on palmitoyl cellulose was active, although its activity was about one-fourth that of free phospholipase C. Therefore, the enzyme appeared to be adsorbed to the cellulose through a hydrophobic site that was distinct from the catalytic site on the enzyme molecule.

Bacillus cereus↗
PubMed1978

Enzymatic determination of phospholipase D activity with choline oxidase.

A new enzymatic method was developed for the assay of phospholipase D [phosphatidylcholine phosphatidohydrolase EC 3.1.4.4] from cabbage leaves using choline oxidase from Arthrobacter globiformis cells. The method was based on the estimation of choline by the following series of enzymatic reactions after ending the phospholipase D reaction: Choline + 202 + h2o Choline oxidase Betaine + 2H2O2 2H2O2 + Phenol + 4-Aminoantipyrine Peroxidase Quinoneimine dye + 4H2O The amount of choline was proportional to the amount of resulting quinoneimine dye with an absorbance maximum at 500 nm. The phospholipase D reaction (choline liberation) was carried out at pH 5.5 in the presence of Ca2+ ions and ended by adding EDTA in conc. Tris-HCl buffer, pH 8, to give a final pH of around 8. The initial rate of the phospholipase D reaction was proportional to the enzyme concentration over the absorbance change range of 0 to 0.25 (equivalent to 0-21 micron of choline) under the optimal reaction conditions.

Alcohol Oxidoreductases↗
PubMed1978

Stimulation of Chromobacterium lipase activity and prevention of its adsorption to palmitoyl cellulose by hydrophobic binding of fatty acids.

Fatty acids prevented adsorption of purified Chromobacterium lipase [triacylglycerol acylhydrolase, EC 3.1.1.3] onto palmitoyl cellulose (Pal-C) and also increased the activity of the purified lipase. These effects increased with increase in the concentration and chainlength (up to 16 carbon atoms) of the fatty acids, and long-chain unsaturated fatty acids, such as oleic acid, linoleic acid and erucic acid, were most effective. When the lipase was adsorbed (immobilized) on Pal-C, its activity was elevated to 20 times that of the free lipase in detergent-free reaction mixture (olive oil-buffer system). Thus lipase was adsorbed to Pal-C through a hydrophobic site distinct from its catalytic site and the binding of fatty acids to the hydrophobic site seems to result in stimulation of the lipase activity.

Adsorption↗
PubMed1977

Purification of lipase from Chromobacterium viscosum by chromatography on palmitoyl cellulose.

Palmitoyl cellulose was used to adsorb the extracellular lipase [triacylglycerol acyl-hydrolase EC 3.1.1.3] of Chromobacterium viscosum from crude enzyme solution, and the adsorbed enzyme was eluted with a suitable detergent, such as Adekatol 45-S-8 or Triton X-100. The enzyme was then purified by chromatography on a palmitoylated gauze column with an overall recovery of 71% and an increase in the specific activity of 11-fold from the supernatant fluid of bacterial cultures. Further purification procedures included fractionation with acetone, and chromatography on Sephadex G-150 and G-75 columns. Two isoenzymes were obtained, each in a homogeneous state on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: one had a molecular weight of 120,000 and pI of 3.7 and the other a molecular weight of 30,000 with a pI of 7.3.

Cellulose↗
PubMed1977

Oxidative pathway of choline to betaine in the soluble fraction prepared from Arthrobacter globiformis.

One strain of bacteria which showed high H2O2-generating activity was isolated from soil and characterized as Arthrobacter globiformis based on its morphological, nutritional, and physiological characteristics. The activities of H2O2 generation, NAD reduction and oxygen consumption in the bacterial cells were examined using choline, betaine aldehyde or betaine as substrate. Choline was oxidized to betaine aldehyde under aerobic conditions in a reaction coupled with H2O2 generation and oxygen consumption. On the other hand, betaine aldehyde seemed to be oxidized to betaine through two distinct oxidative reactions, H2O2 generation (oxygen consumption) under aerobic conditions and NAD reduction under either aerobic or anaerobic conditions. These enzyme activities were found in the supernatant fraction of the sonicated cell preparation.

Aldehydes↗
PubMed1977

Purification and characterization of choline oxidase from Arthrobacter globiformis.

Choline oxidase was purified from the cells of Arthrobacter globiformis by fractionations with acetone and ammonium sulfate, and column chromatographies on DEAE-cellulose and on Sephadex G-200. The purified enzyme preparation appeared homogeneous on disc gel electrophoresis. The enzyme was a flavoprotein having a molecular weight of approx. 83,000 (gel filtration) or approx. 71,000 (sodium dodecyl sulfate--polyacrylamide disc gel electrophoresis) and an isoelectric point (pI) around pH 4.5. Identification of the reaction products showed that the enzyme catalyzed the following reactions: choline + O2 leads to betaine aldehyde + H2O2, betaine aldehyde + O2 + H2O leads to betaine + H2O2. The enzyme was highly specific for choline and betaine aldehyde (relative reaction velocities: choline, 100%; betaine aldehyde, 46%; N,N-dimethylaminoethanol, 5.2%; triethanolamine, 2.6%; diethanolamine, 0.8%; monoethanolamine, N-methylaminoethanol, methanol, ethanol, propanol, formaldehyde, acetaldehyde, and propionaldehyde, 0%). Its Km values were 1.2 mM for choline and 8.7 mM for betaine aldehyde. The optimum pH for the enzymic reaction was around pH 7.5.

Alcohol Oxidoreductases↗
PubMed1976

Effective method for activity assay of lipase from Chromobacterium viscosum.

A method was devised for activity assay of the lipase [triacylglycerol acyl-hydrolase, EC 3.1.1.3] excreted from Chromobacterium viscosum into the culture medium; olive oil emulsified with the aid of Adekatol 45-S-8 (a non-ionic detergent, the ethoxylate of linear sec-alcohols having chain lengths of 10--16 carbon atoms) was used as the substrate. This method was specifically effective for Chromobacterium lipase acitvity assay, and was approximately twice as sensitive as the conventional method, in which polyvinyl alcohol is used for the emulsification of the substrate.

Chromobacterium↗