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Biomedical subjects

F Watanabe

Publications and source records attributed to F Watanabe.

At least 19 recordsLinked to original sources
PubMed1992

NADPH-cytochrome c (P-450) reductase has the activity of NADPH-linked aquacobalamin reductase in rat liver microsomes.

To elucidate the mammalian system for synthesis of cobalamin coenzymes, microsomal NADPH-linked aquacobalamin reductase was purified and characterized. The enzyme was purified about 534-fold over rat liver microsomal fraction in a yield of about 32%. The purified enzyme was homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had a monomeric molecular weight of 79,000. The purified aquacobalamin reductase showed a high specific activity (about 55 mumol/min per mg protein) of NADPH-cytochrome c (P-450) reductase. About 33% of the NADPH-cytochrome c reductase activity found in the microsomal fraction was recovered in the final purified preparation. The activity ratio of NADPH-cytochrome c reductase/NADPH-linked aquacobalamin reductase was about 5.0 through the purification steps, indicating that the rat liver microsomal NADPH-linked aquacobalamin reductase is the NADPH-cytochrome c reductase.

Animals↗
PubMed1992

Enzyme immunoassay for serum dexamethasone using 4-(carboxymethylthio)dexamethasone as a new hapten.

A sensitive and simple enzyme immunoassay for direct quantitation of serum dexamethasone was established. An antiserum with high specificity was produced by the immunization of rabbits with a newly synthesized 4-(carboxymethylthio)dexamethasone-bovine serum albumin conjugate. Alkaline phosphatase was used as a labeling enzyme. The minimum amount of dexamethasone detected was 2 pg per tube on the basis of B/Bo 100 - 2 SD (%) of standard curve. However, taking into account the cross-reaction with steroids such as cortisol in dexamethasone-free serum, the measurable range was from approximately 0.13 to 10 micrograms/dl. Intra- and interassay coefficients of variation were 1.5 - 5.4% and 0.6 - 6.5%, respectively. Serum levels of dexamethasone and cortisol in four normal subjects after an oral administration of 1 mg of dexamethasone are also reported.

Animals↗
PubMed1992

Cytochrome b5/cytochrome b5 reductase complex in rat liver microsomes has NADH-linked aquacobalamin reductase activity.

To study the mammalian system for synthesis of cobalamin coenzymes, rat liver microsomal NADH-linked aquacobalamin reductase was characterized. Microsomal NADH-linked aquacobalamin reductase, which was solubilized with 10 g/L sodium deoxycholate, showed identical elution behavior to NADH-cytochrome c reductase (cytochrome b5/cytochrome b5 reductase complex) on DEAE-Toyopearl 650 column chromatography. By mixing the purified cytochrome b5 with cytochrome b5 reductase, cob(II)alamin was immediately formed from aquacobalamin and NADH. These results provide evidence that the NADH-linked aquacobalamin reductase activity is derived from the cytochrome b5/cytochrome b5 reductase complex in rat liver microsomes. Some properties of the cytochrome b5/cytochrome b5 reductase complex in the form of NADH-linked aquacobalamin reductase were studied. The inhibition studies with cobalamin analogues suggested that hydrophobicity of the corrin ring of cobalamin molecule is involved in binding of cobalamin to the cytochrome b5/cytochrome b5 reductase complex.

Animals↗
PubMed1992

Heat distribution in bone during preparation of implant sites: heat analysis by real-time thermography.

The purpose of this experiment was to observe and measure the distribution of heat to bones and the maximum temperature that developed when cutting bone with drills. Generation of heat that spread in the presence or absence of irrigation when drilling with IMZ, Brånemark, and ITI implant (F type) drills was observed in the pig rib via thermography. Without irrigation, the condition of heat spread in each drill and bur differed according to bur shape and drilling site. Maximum heat temperature without irrigation was higher than that with irrigation for any IMZ, ITI, and Brånemark drill.

Animals↗
PubMed1992

[Improvement of pancytopenia and articular and by splenectomy in a patient with Felty's syndrome].

A forty seven year-old woman with a 18 years history of rheumatoid arthritis presented with recurrent infection and pancytopenia. A diagnosis of Felty's syndrome was made hand on clinical and laboratory findings. In spite of drug therapy using nonsteroidal anti-inflammatory drugs, D-penicillamine and corticosteroid, the hematological abnormalities and active joint symptoms continued. Recurrences of severe bacterial infection and the rupture of esophageal varices necessitated splenectomy. Soon after the operation, dramatic improvement of hematological abnormalities were observed. Furthermore, after 3 months postoperatively, marked decrease in rheumatoid activities and disappearance of subcutaneous nodules were noticed. Titer of rheumatoid factor also showed a significant reduction and became negative 6 months after the operation. Although the efficacy of splenectomy on hematological abnormalities in Felty's syndrome has been well documented, its effect on rheumatic disease has not been clarified. The case presented here might suggested that suppression of reticuloendothelial system may have therapeutic effects in rheumatoid arthritis. The possible mechanisms involved in these findings have been discussed.

Felty Syndrome↗
PubMed1991

Vitamin B12 metabolism in a photosynthesizing green alga, Chlamydomonas reinhardtii.

To elucidate vitamin B12 (cobalamin) metabolism in green algae, Chlamydomonas reinhardtii was labeled with radioactive cobalamin for 4 days. The Chlamydomonas cells did not require cobalamin for growth, but nevertheless took up and accumulated exogenous cobalamin. The level of the accumulated cobalamin reached a maximum (about 20 fmol/10(6) cells) at day 1 of the cell growth. The cobalamin accumulated by the 4-day cells was partly converted to the coenzymes (5'-deoxyadenosylcobalamin, 20.7% and methylcobalamin, 4.8%). The cells contained NADH- and NADPH-linked aquacobalamin reductases (35.9 and 31.5 nmol/min per mg protein, respectively) involved in the synthesis of the coenzymes. Most of the accumulated cobalamin was associated with two heat-labile macromolecules; the coenzymes synthesized were recovered in the macromolecular fraction with a molecular weight of 230,000, while non-coenzyme forms (cyanocobalamin and hydroxocobalamin) predominated in that with a molecular weight of 40,000. Evidence for de novo biosynthesis of cobalamin was also obtained in the alga, indicating that it has both ability to synthesize cobalamin and take up the exogenous vitamin.

Chlamydomonas↗
PubMed1991

Vitamin B-12 deficiency increases the specific activities of rat liver NADH- and NADPH-linked aquacobalamin reductase isozymes involved in coenzyme synthesis.

Rat liver contains both NADH- and NADPH-linked aquacobalamin reductases, which are involved in the synthesis of the vitamin B-12 coenzymes and are distributed in both the mitochondrial and microsomal membranes. To clarify the physiological roles of these hepatic enzymes, vitamin B-12-deficient rats were used to study the effect of the deficiency on the enzyme activities. Male rats fed a vitamin B-12-deficient diet for 11 wk developed a severe vitamin B-12 deficiency with a high urinary methylmalonate excretion (214.3 +/- 115.2 mumol/d) and approximately 96% lower hepatic vitamin B-12 content. Tissues of the vitamin B-12-deficient rats were assayed for NADH- and NADPH-linked aquacobalamin reductase activities. The specific activities of both enzymes in homogenates of liver, kidney or upper intestine were shown to be three- to 20-fold greater in the vitamin-deficient rats than in the control rats. In liver, the vitamin deficiency specifically elevated the specific activities of the mitochondrial NADH-linked and microsomal NADPH-linked enzymes. These are likely the isozymes involved in vitamin B-12 coenzyme synthesis.

Animals↗
PubMed1991

Studies on Phe-228 and Leu-307 recombinant mutants of porcine kidney D-amino acid oxidase: expression, purification, and characterization.

Two recombinant mutants of porcine kidney D-amino acid oxidase [EC 1.4.3.3, DAO], in which Tyr(228) and His(307) are replaced with Phe and Leu, respectively, have been expressed in Escherichia coli and purified to apparent homogeneity. The molecular size and amino-terminal sequence of the two mutants were the same as those of the native DAO. Kinetic analysis revealed that the Michaelis constants of the Phe-228 and Leu-307 mutants for D-alanine were 71- and 10-fold and the inhibition constants for benzoate, a potent competitive inhibitor, were 1,189- and 18-fold greater than those of the native DAO, respectively. The maximum velocities of the Phe-228 and Leu-307 mutants were 66 and 58% that of the native DAO. The kinetically estimated dissociation constant of the Leu-307 mutant for FAD was 28-fold greater than that of the native DAO, whereas the value of the Phe-228 mutant was comparable to that of the native DAO. The Leu-307 mutant and the recombinant wild-type DAO were inactivated by D-propargylglycine (D-PG), a suicide substrate. However, the Phe-228 mutant was resistant to the inactivation. Absorption peaks of the Phe-228 mutant were blue-shifted about 10 nm from the corresponding peaks of the wild-type DAO, and the oxidized form was fully reduced by D-alanine without appearance of the purple intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Synthesis of metabolites of S-1452, an orally active thromboxane A2 receptor antagonist.

The synthesis of 16 metabolites of S-1452, an orally active thromboxane A2 (TXA2) receptor antagonist, is described. Regioselective hydroxylation at C-5 or C-6 of the bicyclo[2.2.1]heptane skeleton of the optically active intermediate 16 was attempted by using 9-borabicyclo[3.3.1]nonane followed by H2O2 or m-chloroperbenzoic acid (m-CPBA) and then LiA1H4, to obtain the hydroxylated product 17a or 17b, respectively. Modification of the C-2 substituent of 17a and 17b afforded eight metabolites of S-1452. Eight non-hydroxylated metabolites were synthesized by using a similar reaction sequence.

Animals↗
PubMed1991

Occurrence and subcellular location of NADH- and NADPH-linked aquacobalamin reductases in human liver.

1. Both activities of NADH- and NADPH-linked aquacobalamin reductases were found in some human tissues, liver, kidney pancreas, heart, spleen, lung, cerebrum, cerebellum, adrenal glands, stomach, duodenum, jejunum, ileum, colon and bone marrow. 2. Human liver contained both enzymes with higher specific activities than any other tissues. 3. The liver NADH-linked enzyme was distributed in both mitochondrial (approx. 60%) and microsomal (40%) fractions; similar to the distribution of the NADPH-linked enzyme, but of which 40% activity was found in the mitochondria and the remaining activity was recovered in the microsomes. 4. The results suggest that the synthetic systems of the cobalamin coenzymes occur in both mitochondria and microsomes of human liver.

Cell Fractionation↗
PubMed1990

The transition of the higher-order structure of rat liver chromatin takes place at about 85% neutralization of DNA charges.

The salt dependence of the stoichiometry in the binding of linker histone to rat liver chromatin was analysed. It was found that the transition of the higher-order structure of chromatin (between folded and unfolded) takes place at the point of approximately 85% neutralization of DNA charges in chromatin, as in a related process in the B-Z transition of double-helical DNA.

Animals↗
PubMed1990

Mutagenicity of isoquinoline alkaloids, especially of the aporphine type.

The mutagenicity of 44 isoquinoline alkaloids was tested in Salmonella typhimurium TA100 and TA98 in the presence or absence of S9 mix. The alkaloids tested included compounds from the isoquinoline, benzylisoquinoline, bisbenzylisoquinoline, monoterpene isoquinoline, berberine, morphinane, hasubanan, benzo[c]phenanthridine and aporphine groups. Among the alkaloids tested, liriodenine was the most potent mutagen for TA100 and roemerine was the most potent for TA98. A clear structure-mutagenicity relationship was observed in a series of aporphine alkaloids (aporphine, dehydroaporphine, 7-oxoaporphine and 4,5-dioxoaporphine), and 10,11-non-substituted aporphines were suggested to exert their mutagenicity through metabolic activation of the 10,11 positions, possibly as the 10,11-epoxides.

Alkaloids↗
PubMed1990

[Heat distribution within the bone tissue by rotary cutting instrument for IMZ implant. Heat analysis by a real-time thermography].

A highly skilled surgical technic is an important factor for the success of implant. But the heat generation during bone preparation will greatly affect the healing process of bone and prognosis of implant. In this study, the heat occurrence and distribution during bone preparation which performed on an in vitro mandible model (ribs of pig) were considered with the aid of a real-time thermography. IMZ implant drills and bur, designed for cylindrical type implant were used for the bone preparation. The conclusion are as follows: 1. Any drill or bur generates higher heat without water irrigation. 2. Heat distribution produced different shape of range according to different type of drills. 3. On a relationship of preparating time to heat generation, spiral drill takes the longest time to complete drilling but heat rising ratio is slow. Round bur and Canon drill take short time to complete drilling but it is accompanied with rapid heat rising.

Animals↗