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Biomedical subjects

M Watanuki

Publications and source records attributed to M Watanuki.

At least 37 records · Page 2Linked to original sources
PubMed1996

Effects of calcium gluconate on the utilization of magnesium and the nephrocalcinosis in rats fed excess dietary phosphorus and calcium.

The effects of calcium gluconate on the utilization of magnesium and nephrocalcinosis in male Wistar rats made magnesium-deficient by adding excess dietary phosphorus (1.195 g of phosphorus/100 g of diet) and calcium (1.04 g of calcium/100 g of diet) were compared with the effects of calcium carbonate. The effects of dietary magnesium concentration on the magnesium status and nephrocalcinosis were also examined. Adding excess dietary phosphorus and calcium decreased the apparent magnesium absorption ratios and the concentrations of magnesium in the serum and femur and increased the deposition of calcium in the kidney, and the low magnesium condition (0.024 g of magnesium/100 g of diet) aggravated the deposition of calcium and the low magnesium status. The apparent magnesium absorption ratios and femur magnesium concentration in the rats fed a calcium gluconate diet (an equimolar mixture of calcium gluconate and calcium carbonate was used as a source of calcium) were significantly higher than in the rats fed a calcium carbonate diet (only calcium carbonate was used as a source of calcium), irrespective of dietary magnesium concentration. Dietary calcium gluconate lessened the accumulation of calcium in the kidney and increased the serum magnesium concentration compared with dietary calcium carbonate, when the rats were fed the normal magnesium diet (0.049 g of magnesium/100 g of diet) but not the low magnesium diet. We speculate that the increased utilization of magnesium by feeding the calcium gluconate diet to a limited extent prevented the low magnesium status and the severity of nephrocalcinosis caused by adding excess dietary phosphorus and calcium.

Absorption↗
PubMed1996

The effect of 6'-galactooligosaccharides on bone mineralization of rats adapted to different levels of dietary calcium.

6'-galactooligosaccharides (6'-GOS), a mixture of galactosyl oligosaccharides formed from lactose by the transgalactosyl reaction with beta-D-galactosidase derived from Aspergillus oryzae and Streptococcus thermophillus, are unhydrolyzed in the small intestine and are fermented by the intestinal bacteria. The effects of 6'-GOS on calcium (Ca) absorption and bone mineralization were examined in male Wistar rats adapted to different levels of dietary Ca for 30 days. Dietary 6'-GOS (5 g/100 g of diet) were more potent than control in stimulating Ca absorption in rats fed the Normal-Ca diet (0.5 g of Ca/100 g of diet) after 8-10 days and 18-20 days, and the bone (femur and tibia) Ca content of rats fed the Normal-Ca diet with 6'-GOS were significantly higher than those of the control animals. However, in rats fed the Low-Ca diet (0.05 g of Ca/100 g of diet), 6'-GOS feeding did not affect both the absorption of Ca and the bone mineralization. Ca content in the liquid phase of the cecal digesta was significantly elevated by 6'-GOS feeding in the rats fed the Normal-Ca diet, however, this was unchanged in the rats fed the Low-Ca diet. We conclude that the effect of 6'-GOS on the bone mineralization is affected by dietary Ca concentration used in the experiment, and the stimulatory effect of 6'-GOS on Ca absorption may be partly associated with increased solubility of Ca in the intestinal digesta.

Absorption↗
PubMed1995

Effects of some diethylstilbestrol metabolites and analogs on cytotoxicity and aneuploidy induction in Chinese hamster V79 cells.

We have previously reported the inhibitory effects of diethylstilbestrol (1) and optically active indenestrol derivatives on microtubule polymerization in vitro and their disruptive effect on cytoplasmic microtubules and cytotoxicity in cultured Chinese hamster V79 cells. In the present study, the cytotoxicities of (+-)-diethylstilbestrol oxide (2), (+)-, (-)- and (+-)-monomethyl ethers (4) of 2, (+-)-dimethyl ether (5) of 2, diethylstilbestrol pinacolone (3), E,E-dienestrol (6), Z,Z-dienestrol (7), meso-hexestrol (8), a mixture of (1R,1'S)4-hydroxyhexestrol and (1R,1'S)4'-hydroxyhexestrol (9), and the 4-hydroxy derivative (10) of diethylstilbestrol dimethyl ether were investigated in Chinese hamster V79 cells. The results indicated that the cytotoxic activity of 10 was the strongest of the compounds tested, although its activity was the almost same as that of 1. Moreover, as the activity of (-)-4 was greater than those of 2 and 1 monomethyl ethers, the effect of 4 on cytotoxic activities was elucidated. In conclusion, the present results indicate that the cytotoxic activities of hydroxylated metabolites are greater than those of each mother compound, although epoxidation of 1 leads to a product which can be broken down more readily than the parent compound.

Aneuploidy↗
PubMed1995

Effect of galactooligosaccharides on calcium absorption and preventing bone loss in ovariectomized rats.

The effects of galactooligosaccharides (GOS), a mixture of galactosyl oligosaccharides formed from lactose by the transgalactosyl reaction of beta-D-galactosidase derived from Bacillus circulans, on calcium absorption and prevention of bone loss were examined in ovariectomized (OVX) Wistar rats. Rats fed on a diet containing GOS absorbed calcium more efficiently than those on the control diet after 8-10 days and 18-20 days, and the bone (femur and tibia) ash weight and tibia calcium content of OVX rats fed on the GOS diet were significantly higher than those of the control animals. Although the serum total cholesterol of the ovariectomized rats was significantly elevated, GOS produced a significant hypocholesterolemic effect in the OVX rats. GOS, which is fermented by bacteria in the lower part of the intestine, enhanced volatile fatty acid production, and thus prevented bone loss and lower serum total cholesterol concentration in the ovariectomized rats.

Absorption↗
PubMed1995

Effect of galactooligosaccharides on calcium absorption in rats.

The effect of transgalactosylated oligosaccharides (TOS), which are oligosaccharides that are unhydrolyzed in the small intestine and are fermented by the intestinal bacteria, on calcium absorption was examined in male Wistar rats for 10 days. The apparent calcium absorption ratios and the apparent retention ratios were significantly higher in the rats fed TOS-containing diets (5 or 10 g/100 g of diet). In the second experiment, the cecum was ligated in situ and calcium absorption from the cecum was observed after injecting TOS into the cecal lumen. Four hours after the injection, the calcium concentration in the cecal vein of the rats given TOS was significantly higher than that of the control. The calcium content in the liquid phase of the cecal lumen and the liquid phase weight were also increased by the injection of TOS into the cecum. Although the extent of calcium absorption from the cecum of rats fed TOS is due to overall calcium absorption is not known, under the experimental conditions used in the present study the stimulatory effect of TOS on calcium absorption may be partly associated with increased solubility of calcium and the fluid content in the intestinal lumen.

Animals↗
PubMed1990

Thrombolytic properties of staphylokinase.

We evaluated the properties of recombinant staphylokinase in comparison with those of tissue-type plasminogen activator (t-PA) and streptokinase (SK). The presence of fibrin(ogen) fragment FCB-2 in the reaction mixture increased plasminogen activation by staphylokinase more than 20-fold. Such characteristics are similar to those of t-PA. On the other hand, SK was not affected by the presence of FCB-2. The thrombolytic properties of staphylokinase were studied in a system consisting of a radioactive human plasma clot (125I-fibrinogen-labeled) suspended in the circulating citrated plasma. Significant thrombolysis (50% in 3 hours) was obtained with 2 micrograms/mL of staphylokinase and 4.45 micrograms/mL t-PA, as compared with 12 micrograms/mL for SK. The relative molar potency of staphylokinase, calculated from the molecular weight, was about two times more effective than that of SK, but about half of that of t-PA. Systemic fibrinolytic activation and fibrinogen breakdown was not observed with staphylokinase or t-PA, but was observed with SK. The thrombolytic efficiency of staphylokinase, which was calculated as the ratio of the degree of thrombolysis/the degree of fibrinogenolysis, was about five times greater than that of SK, and about half of that of t-PA. These findings suggest that staphylokinase has higher specific thrombolytic properties and lesser fibrinogenolytic properties than those of SK.

Amino Acid Sequence↗
PubMed1989

Mechanism of fibrin-specific fibrinolysis by staphylokinase: participation of alpha 2-plasmin inhibitor.

When the extent of plasminogen activation by staphylokinase (SAK) or streptokinase (SK) was measured in human plasma, SAK barely induced plasminogen activation, whereas SK activated plasminogen significantly. When the plasma was clotted with thrombin, the plasminogen activation by SAK was markedly enhanced, but that of SK was little enhanced. Similarly, in a purified system composed of plasminogen, fibrinogen and alpha 2-plasmin inhibitor (alpha 2-PI, alpha 2-antiplasmin), such a fibrin clot increased the activity of SAK significantly. However, when alpha 2-PI was removed from the reaction system, enhancement of the SAK reaction was not observed. In addition, SAK as distinct from SK, showed very little interference with the action of alpha 2-PI. Plasminogen activation by SAK is thus essentially inhibited by alpha 2-PI, but this reaction is not inhibited in fibrin clots. These results suggest that SAK forms a complex with plasminogen, which binds to fibrin and induces fibrinolysis.

Blood Coagulation↗
PubMed1983

Effect of soybean protein on coprostanol production and cholesterol metabolism in cholesterol-fed rats.

The effect of soybean protein on coprostanol production and cholesterol metabolism was studied in cholesterol-fed rats. Plasma cholesterol was decreased in the soybean protein diet group compared to the casein diet group. Although coprostanol was produced more in rats fed soybean protein than in those fed casein, no difference was observed in the levels of total neutral steroids at any part of the intestine. The activity of microbial conversion from cholesterol to coprostanol was evidently high in rats fed soybean protein. The total amount of neutral steroids excreted in feces had a tendency to increase. These data seem to indicate that the increase of the unabsorbed soybean protein causes the increase of intestinal coprostanol production.

Animals↗
PubMed1980

Selective inhibition by benzaldehyde of the uptake of nucleosides and sugar into simian virus 40-transformed cells.

The effects of benzaldehyde, which has been found in figs as a carcinostatic element, were studied on the uptake of nucleosides, 2-deoxy-D-glucose, and amino acids into simian virus 40-transformed rat fibroblast cells (SV40-transformed cells) and into the parent normal cells (normal cells). Benzaldehyde, at the concentrations of 25 to 100 microgram/ml at which the selective growth inhibition against SV40-transformed cells was revealed, markedly inhibited the uptake of thymidine, other nucleosides, and 2-deoxy-D-glucose into SV40-transformed cells without any significant inhibition of the uptake of these compounds into normal cells. The uptake of amino acids into both transformed and normal cells was not inhibited by benzaldehyde. Selectively cytotoxic benzaldehyde-related compounds such as 4-nitrobenzaldehyde, 4-acetaminobenzaldehyde, thiophene-3-carboxaldehyde, etc., showed a similar inhibitory effect on thymidine uptake. The deprivation of glucose from the incubation medium strikingly diminished the inhibitory effect of benzaldehyde on the uptake of thymidine and 2-deoxy-D-glucose into SV40-transformed cells. The intracellular adenosine 5'-triphosphate level of SV40-transformed cells was reduced to less than one-half by treatment with benzaldehyde (50 microgram/ml) in glucose-containing medium. This effect was not observed in glucose-free medium. Treatment with benzaldehyde caused no change of the intracellular adenosine 5'-triphosphate level of normal cells. Based on the above results, the selective cytotoxicity of benzaldehyde was attributed to the reduction of intracellular adenosine 5'-triphosphate level of transformed cells, accompanied by the poor uptake of thymidine, glucose, etc., into SV40-transformed cells.

Adenosine Triphosphate↗
PubMed1979

Adrenocortical cytochrome P-450 side chain cleavage. Preparation of membrane-bound side chain cleavage system from purified components.

Soluble cytochrome P-450 from bovine adrenocortical mitochrondria, capable of side chain cleavage, can be incorporated into membranes prepared by dispersion of phospholipids in aqueous buffer when cholate is added to the membrane suspension. In addition, the complete protein side chain cleavae system (i.e. including the ancillary proteins adrenodoxin and adrenodoxin reductase and the substrate cholesterol) can be incorporated into such membranes so that on addition of TPNH, pregnenolone is formed. These components remain in the membrane through gel filtration (which removes almost all the cholate) and sedimentation through sucrose density gradients which separate vesicles without protein and soluble enzyme from the membrane-bound P-450 remains associated with the membrane during and following lysis of vesicles. The vesicles which do not leak [14C]glucose were seen on electron microscopy to show a mean diameter of 350 to 450 A. A number of phospholipids are capable of accomodating P-450 in this manner: mitochondrial lipid extracts, synthetic dipalmitoyl phosphatidylcholine, synthetic dipalmitoyl phosphatidylserine, and egg lecithin, separately or in various combinations. Cholesterol is not necessary for incorporation of the side chain cleavage system. Membrane-bound P-450 shows a Vmax of 28.1 nmol of pregnenolone/min/mg of protein, more than 10 times that of soluble P-450. The spectral properties of the soluble P-450 are altered to become predominantly low spin in the membrane and the enzyme is more stable at 4 degrees C than is soluble p-450.

Adrenal Cortex↗
PubMed1979

Composition of lipids bound to pure cytochrome P-450 of cholesterol side-chain cleavage enzyme from bovine adrenocortical mitochondria.

Phospholipids bound to highly purified cytochrome P-450 from bovine adrenocortical mitochondria, part of the enzyme complex responsible for catalyzing the conversion of cholesterol to pregnenolone, have been examined for comparison with the bulk phospholipids of the mitochondria from the same tissue. In both cases, the major phospholipids are phosphatidylcholine (PC) (37%) and phosphatidylethanolamine (PE) (56%), as well as smaller amounts of sphingomyelin and diphosphatidylglycerol. The fatty acid compositions of the four classes of phospholipids and of the neutral lipids bound to the pure enzyme are indistinguishable from those of the respective mitochondrial lipids. They are also similar to those of mitochondria from other organs except for high levels of arachidonate and low levels of diphosphatidylglycerol.

Adrenal Cortex↗
PubMed1978

Cytochrome P-450 for 11beta- and 18-hydroxylase activities of bovine adrenocortical mitochondria: one enzyme or two?

The 11beta- and 18-hydroxylase activities of a highly purified cytochrome P-450 from bovine adrenocortical mitochondria have been examined in detail with a view to determining whether the two activities are shown by a single protein or by two distinct proteins. The purified enzyme shows a single N-terminal residue (glutamic acid) and its amino acid composition is reported. Both enzyme activities decay considerably during storage at 4 degrees C for 11 days and the rates of decay are similar for the two activities. Metyrapone inhibits both activities competitively (Ki = 1.50 and 1.43 micrometer for 11beta- and 18-hydroxylase, respectively). Carbon monoxide inhibits both activities and the ratio CO:O2 for 50% inhibition is similar for the two activities (K = 1.69 and 1.53). A variety of nonspecific inhibitors produce approximately the same inhibition of both activities. Finally, antiserum produced by rabbits to the purified enzyme on double diffusion in agarose gels gives a single band with the purified enzyme. Increasing concentrations of antiserum added to the assay system produce increasing and proportionate inhibition of both activities. The evidence strongly supports earlier suggestions that the two hydroxylase activities occur in a single protein.

Adrenal Cortex↗
PubMed1978

On the role of protein synthesis in the response of adrenal tumor cells to ACTH.

Y-1 adrenal tumor cells were incubated with aminoglutethimide with and without ACTH. Greater production of pregnenolone from endogenous cholesterol was observed (after washing to remove aminoglutethimide) in mitochondria from cells incubated with aminoglutethimide and ACTH than in those from cells incubated with aminoglutethimide alone. This response was inhibited by cycloheximide and puromycin but not by chloramphenicol or actinomycin D. ACTH increased the incorporation of [3H]tyrosine into protein associated with mitochondria but not into total cell protein or protein of postmitochondrial supernatant. This response did not require aminoglutethemide block and was inhibited by cycloheximide and puromycin but not by chloramphenicol or actinomycin D. Dibutyryl cyclic AMP produced both of these responses (increased production of pregnenolone and synthesis of protein associated with mitochondria). The concentration of cycloheximide required to cause 50% inhibition of the responses to ACTH and dibutyryl cyclic AMP was approximately the same for steroidogenesis by whole cells, for production of pregnenolone by isolated mitochondria, for incorporation of [3H]tyrosine into Y-1 cell protein and for the increase in synthesis of protein associated with mitochondria produced by ACTH (0.08--0.2 microgram/ml). Disc gel electrophoresis revealed that the increased incorporation of [3H]tyrosine involved two proteins corresponding to molecular weight of approximately 27,000 and 13,000 respectively. These observations suggest that ACTH promotes synthesis of protein(s) by cytoplasmic ribosomes on stable messenger RNA, that the protein(s) becomes associated with mitochondria and that the protein(s) includes one or more which are associated with the increase in production of pregnenolone produced in mitochondria by the addition of ACTH to adrenal cells.

Adrenal Gland Neoplasms↗
PubMed1977

Side-chain cleavage P-450 from bovine adrenocortical mitochondria. Reconstitution of enzyme activity.

The subunit structure of the cytochrome P-450 from bovine adrenocortical mitochondria responsible for the conversion of cholesterol to pregnenolone (side-chain cleavage) has been studied. Isoelectric focusing in 6 M urea reveals two fractions of identical amino acid composition which differ in apparent isoelectric points and in phospholipid content: fraction SI shows 0.6-1.8 nmol phospholipid per 53 000 daltons and pI approx. 4.0; SII shows 6.6-8.9 nmol phospholipid per 53 000 daltons and pI approx. 7.0. SII can be made to behave on isoelectric focusing like SI by removal of phospholipid and SI like SII when the extracted phospholipid is added to the protein (SI). Enzymatic activity can be restored to SII by addition of heme and to SI by addition of heme together with the phospholipid extracted from P-450 from the fractions SI and SII. This phospholipid contains at least four classes of phospholipid of which two have been tentatively identified as phosphatidylcholine and phosphatidylethanolamine. A variety of phospholipids from commercial sources do not permit reconstitution of enzyme activity. Evidence is presented to show that minor contaminants seen on polyacrylamide SDS gels are not essential for enzyme activity nor do they appear greatly to influence enzymatic activity. The possible role of phospholipid in reconstituting cytochrome P-450 activity is considered.

Adrenal Cortex↗
PubMed1977

Preparation and properties of side-chain cleavage cytochrome P-450 from bovine adrenal cortex by affinity chromatography with pregnenolone as ligand.

A method is described for preparing cytochrome P-450 (side-chain cleavage) from bovine adrenocortical mitochondria, by affinity chromatography on pregnenolong-Sepharose beads. The cytochrome P-450 appears in two fractions, a large form of heterogeneous molecular weight (large P-450) and a form of molecular weight 850 000 composed of 16 apparently identical subunits (molecular weight 52 000-53 000); this form is referred to as protein 16. Electrophoresis on polyacrylamide gel yields one main band and two minor bands; the appearance of the gels is identical whether the starting material is large P-450 or protein 16 or protein 16 prepared by an entirely different method. Yields of protein 16 can be increased by rechromatography on pregnenolone-Sepharose of large P-450 made 0.1 mM in NADPH. Large P-450 shows greater than 10 heme groups per 16 subunits and is less active enzymatically than protein 16. Chromatography on Sepharose and analytical ultracentrifugation show that large P-450 is heterogeneous with respect to molecular weight. Protein 16 shows a heme content of 8 nmol/mg protein and for both large P-450 and protein 16 heme content by CO-difference spectroscopy is in agreement with values by pyridine hemochromogen. This method of preparing P-450 is convenient and both large P-450 and protein 16 are highly purified.

Adrenal Cortex↗