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Biomedical subjects

K Ohki

Publications and source records attributed to K Ohki.

At least 163 records · Page 9Linked to original sources
PubMed1984

Effect of alpha-tocopherol on lipid peroxidation and acyl chain mobility of liver microsomes from vitamin E-difficient rat.

Effects of alpha-tocopherol on lipid peroxidation and membrane fluidity were studied in liver microsomes from vitamin E (alpha-tocopherol)-deficient rats using NADPH as a substrate. Microsomes containing various contents of alpha-tocopherol were prepared by incubation with various concentrations of alpha-tocopherol in ethanol solution. NADPH-dependent lipid peroxidation decreased the content of polyunsaturated fatty acids, arachidonic acid and 4, 7, 10, 13, 16, 19-docosahexaenoic acid. The treatment with alpha-tocopherol before peroxidation reduced the production of lipid peroxides and the change in fatty acid composition even at the lowest content of alpha-tocopherol dealt with in this experiment, 0.2 molar fraction, while addition of alpha-tocopherol after peroxidation resulted in a slight inhibition of peroxide production and small alteration in fatty acid composition. By an ESR measurement using stearate spin probe, the alpha-tocopherol incorporated into microsomes did not alter the acyl chain mobility up to 0.2 molar fraction but reduced the mobility above 0.2 molar fraction. The acyl chain mobility was markedly decreased by lipid peroxidation. The decrease of membrane fluidity was repressed in microsomes treated with alpha-tocopherol before peroxidation, but was not repressed in microsomes treated after peroxidation. The experiment using artificial membranes of egg yolk phosphatidylcholine and rat liver phosphatidylcholine revealed that the effect of alpha-tocopherol on membrane fluidity depends on the fatty acid composition of phospholipid, especially the content of arachidonic acid. On the other hand, the mobility of the fatty acyl chain was not affected by spermine at concentrations which could inhibit lipid peroxidation. These results suggest that the inhibitory effect of alpha-tocopherol on lipid peroxidation is due to antioxidant activity rather than the indirect effect of membrane stabilization.

Acylation↗
PubMed1983

An X-ray diffraction study on phase transition temperatures of various membranes isolated from Tetrahymena pyriformis cells grown at different temperatures.

Mitochondrial, microsomal and pellicular membranes were isolated from Tetrahymena cells grown at 39 degrees C or 15 degrees C, and phospholipids, in turn, were separated from total lipids extracted from these membranes. The effect of growth temperature on their solid-to-fluid phase transition temperature was examined by wide-angle X-ray diffraction. The transition temperatures of phospholipids from mitochondria, microsomes and pellicles were 21, 19 and 26 degrees C for cells grown at 39 degrees C and -8, -3 and 6 degrees C for cells grown at 15 degrees C, respectively. All phospholipids were found in a completely fluid state at these growth temperatures. From a comparison between the phospholipids and total lipids from pellicles of cells grown at 39 degrees C, a triterpenoid alcohol, tetrahymanol, caused the transition temperature to increase. The alignment of tetrahymanol in membranes was examined with pellicle'a total lipid oriented in a sample holder.

Cholesterol↗
PubMed1983

Cell hybrids between SV40-transformed macrophage cell lines and a Chinese hamster cell line: growth responsiveness and induction of colony-stimulating factor.

Three cell lines from resident macrophages of BALB/c mice and four from activated macrophages of the same strain were isolated by infection with simian virus 40 (SV40). A majority of these cells showed dependency on L cell-conditioned medium (LCM), which is necessary for proliferation of normal macrophages in vitro. Somatic cell hybridization was applied in the study of macrophage growth responsiveness. A macrophage cell line (BR15) with strict dependency on LCM for growth was fused to a Chinese hamster cell line (hs222-16); it was found that dependency on LCM was a dominant trait in the hybrids. Following fusion of a macrophage cell line (BAM3) which grew without LCM to hs222-16, a large number of colonies appeared in the selection medium containing LCM. Four hybrids not requiring LCM for growth were selected in an LCM-free culture, and their hybrid properties were examined. Three out of the four hybrids secreted colony-stimulating factor (CSF) constitutively, whereas the fourth secreted no CSF. The level of acid phosphatase activity in the hybrids was higher than in the parent cells. Two peaks of CSF activity were observed after gel filtration chromatography of conditioned medium: One was eluted at molecular weight of 36,000 and the other at 17,000.

Acid Phosphatase↗
PubMed1983

Effect of Intralipid-infusion on erythrocyte membrane in rabbits.

Through Intralipid infusion in rabbits, the phospholipids derived from Intralipid were incorporated into erythrocytes, although Intralipid is mainly composed of triglycerides. This is supported by the increase in oleic acid and the compensatory decrease in linoleic acid of the phospholipids in the erythrocyte membrane, corresponding to the content of linoleic acid in the phospholipids from Intralipid. The excess phospholipid rendered the membrane more fluid, probably by overwhelming the rigidifying effect of the increased cholesterol content. Furthermore, the shape of erythrocytes was changed from biconcave to spur, dose dependently. The morphological alterations in erythrocyte membranes could not be completely elucidated by the changes in lipid. These results suggested that the alteration in lipid metabolism in Intralipid-infused rabbits caused various effects on the erythrocyte membrane, through the elevation of triglyceride, cholesterol, and phospholipid contents in plasma.

Animals↗
PubMed1983

Electron microscopic study on the structures formed by mixtures containing synthetic glyceroglycolipids.

Structures of aqueous dispersions prepared from monoglycosyldiglycerides and from mixtures of monoglycosyldiglyceride and phosphatidylcholine were studied by freeze-fracture electron microscopy. Aqueous dispersions of glucosyl or galactosyl dipalmitylglycerol formed lamellar structures when quenched both from below and above the gel-liquid crystalline phase transition temperature. With mixtures of monoglycosyl dipalmitylglycerol and dipalmitoylphosphatidylcholine, tubular structures could be observed in the extended bilayer structures. Diglucosyl dipalmitylglycerols showed only a smooth fracture surface, however, when mixed with dipalmitoylphosphatidylcholine. In the fractured face of dispersions composed of egg yolk phosphatidylcholine and glucosyl dipalmitylglycerol, particles having a diameter of 3-6 nm, instead of tubular structures, were observed, suggesting that the occurrence of tubular structure may depend on the fatty acid compositions of the mixtures. A mixture of dipalmitoylphosphatidylcholine and galactosylceramide did not show any distinct tubular or globular structures on the fractured face. These findings indicate that structure in the polar group of molecules as well as structure in the non-polar group may be important for the formation of a non-bilayer, tubular structure.

Chemical Phenomena↗
PubMed1983

Purification and properties of inorganic pyrophosphatase from porcine brain.

Inorganic pyrophosphatase [EC 3.6.1.1] was purified from porcine brain to an electrophoretically homogeneous state. The molecular weight of the enzyme was estimated to be 62,000 by gel filtration and that of the subunit to be 33,000 by gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the enzyme consists of two identical subunits. The stability of the purified enzyme was dependent on its protein concentration. The enzyme was stable above 50 micrograms/ml at 20 degrees C, but it was gradually inactivated below this concentration, even at 0 degree C unless other proteins such as bovine serum albumin, calmodulin, etc. were present. Those added proteins not only protected the enzyme from inactivation, but also completely reactivated the enzyme after it had been once inactivated. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate but not that of other phosphate esters. Only Mg2+ was required as an activating cation, and other divalent cations inhibited the activity to some degree. The addition of sulfhydryl reagents prevented the inhibition of activity by divalent cations.

Animals↗
PubMed1983

Stimulation of tubulin-dependent ATPase activity in microtubule proteins from porcine brain by taxol.

Taxol, an antimitotic agent that induces microtubule assembly, stimulated tubulin-dependent Mg2+-ATPase activity of microtubule-associated proteins (MAPs). A concentration-dependent increase in the rate of ATP hydrolysis was observed. Taxol acted through its binding to the tubulin molecule on MAP ATPase, and maximal stimulation, which was found at approximately equal concentrations of taxol and tubulin, reached about 140% of the original level in the absence of taxol. Taxol enhanced ATP hydrolysis by a mixture of MAPs and tubulin, and this continued at a steady linear rate even when the polymerization had approached a plateau. In the presence of taxol, a large portion of ATPase activity and protein was recovered in the pellet after centrifugation at 70,000 g for 60 min at 25 degrees C. Both colchicine and podophyllotoxin inhibited taxol-stimulated ATPase activity via the same mechanism by which they inhibited taxol-induced microtubule polymerization. The stimulation by taxol was not found in the presence of Ca2+ alone but required Mg2+. We conclude that tubulin effectively stimulates Mg2+-ATPase activity of MAPs under conditions that induce tubulin polymerization.

Adenosine Triphosphatases↗
PubMed1983

Targeting of asialofetuin sugar chain-bearing liposomes to liver lysosomes.

Specific direction of liposomes bearing an asialofetuin sugar chain (AFSC) to liver parenchymal cells was examined both in vivo and in vitro. The AFSC-bearing liposomes were preferentially recovered in the liver within several minutes after an intravenous injection into mice and were found to be predominantly localized in mitochondrial-lysosomal fraction. The massive distribution of the AFSC-liposomes in this fraction was also confirmed by using a lysosomal protease inhibitor, E-64-d. In isolated rat hepatocytes, the uptake of AFSC-liposomes was increased 2-3-fold as compared with the control liposomes without AFSC. Thus liposomes bearing AFSC would be useful to target enzymes to liver lysosomes.

Animals↗
PubMed1982

Evidence for a correlation between swimming velocity and membrane fluidity of Tetrahymena cells.

The influence of the physical state of the membrane on the swimming behaviour of Tetrahymena pyriformis was studied in cells with lipid-modified membranes. When the growth temperature of Tetrahymena cells was increased from 15 degrees C to 34 degrees C or decreased from 39 degrees C to 15 degrees C, their swimming velocity changed gradually in a similar to the adaptive change in membrane lipid composition. Therefore, such adaptive changes in swimming velocity were not observed during short exposures to a different environment. Tetrahymena cells adapted to 34 degrees C swam at 570 microns/s. On incubation at 15 degrees C these cells swam at 100 microns/s. When the temperature was increased to 34 degrees C after a 90-min incubation at 15 degrees C, the initial velocity was immediately recovered. On replacement of tetrahymanol with ergosterol, the swimming velocity of 34 degrees C-grown cells decreased to 210 microns/s, and the cells ceased to move when the temperature was decreased to 15 degrees C. To investigate the influence of the physical state of the membrane on the swimming velocity, total phospholipids were prepared from Tetrahymena cells grown under these different conditions. The fluidities of liposomes of these phospholipid were measured using stearate spin probe. The membrane fluidity of the cells cooled to 15 degrees C increased gradually during incubation at 15 degrees C. On the other hand, the fluidity of the heated cell decreased during incubation at 34 degrees C. Replacement of tetrahymanol with ergosterol decreased the membrane fluidity markedly. Consequently, a good correlation was observed between swimming velocity and membrane fluidity; as the membrane fluidity increased, the swimming velocity increased linearly up to 600 microns/s. These results provide evidence for the regulation of the swimming behaviour by physical properties of the membrane.

Animals↗
PubMed1982

Effect of phosphatidylinositol replacement by diacylglycerol on various physical properties of artificial membranes with respect to the role of phosphatidylinositol response.

In an attempt to gain insight into the physiological role of phosphatidylinositol turnover enhanced by extracellular stimuli, the physical properties of artificial membranes (egg yolk phosphatidylcholine/bovine brain phosphatidylserine) containing phosphatidylinositol or diacylglycerol were studied by ESR using spin probes and freeze-fracture electron microscopy. Diacylglycerol lost both the ability to form lipid bilayer structures and its susceptibility to calcium ions. Yeast phosphatidylinositol included in dipalmitoylphosphatidylcholine liposomes lowered the phase transition temperature of dipalmitoylphosphatidylcholine and expanded the temperature range of phase transition. However, diacylglycerol at the same concentration did not undergo the effects caused by phosphatidylinositol but the phase transition temperature was slightly raised. Phase separation of phosphatidylserine induced by calcium ions was enhanced when the phosphatidylinositol was replaced by diacylglycerol in phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) mixtures. The mobility of phosphatidylcholine spin probe was decreased in phosphatidylcholine/phosphatidylserine/diacylglycerol (3:5:2, by molar ratio) liposomes compared with phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) liposomes. An additional component from protonated stearic acid spin probes was observed in phosphatidylcholine/phosphatidylinositol (8:2, by molar ratio) liposomes at 40 degrees C, whereas the component was not seen in phosphatidylcholine/diacylglycerol (8:2, by molar ratio) liposomes. This may indicate the alteration of surface charge induced by the replacement of phosphatidylinositol by diacylglycerol. Indeed, in the presence of 1 mM Ca2+, the additional component was removed by an electrostatic interaction between Ca2+ and phosphatidylinositol molecules in phosphatidylcholine/phosphatidylinositol liposomes at 40 degrees C. These results support the hypothesis that the enhanced turnover of phosphatidylinositol may play a triggering role for various cellular responses to exogenous stimuli by altering membrane physical states.

Diglycerides↗
PubMed1982

Stimulation of tubulin-dependent ATPase activity in microtubule proteins from porcine brain by vinblas tine.

Vinblastine, a plant alkaloid which inhibits tubulin polymerization, stimulated an ATPase activity in microtubules. When microtubule proteins were separated into microtubule-associated proteins (MAPs) and tubulin by phosphocellulose column chromatography, vinblastine did not stimulate an ATPase activity recovered in the MAPs fraction unless tubulin was present. Therefore, vinblastine is considered to act through its binding to the tubulin molecule on MAPs ATPase. Divalent cations that activate tubulin-dependent MAPs ATPase activity were also required for the stimulation by vinblastine. In the presence of Ca2+ and vinblastine the ATPase activity was most active and the extent of stimulation reached about 200% of the original level in the absence of vinblastine. Half-maximal stimulation was attained when the molar ratio of vinblastine to tubulin was 0.5. The concentration of tubulin for half-maximal stimulation was increased in the presence of vinblastine, while divalent cation requirements were decreased. Several factors such as KCl (100 mM), alkaline pH (pH 7.5), and low temperature (10 degrees C) were not responsible for the disappearance of the stimulation. Vincristine stimulated tubulin-dependent MAPs ATPases activity as vinblastine did, whereas the activity was scarcely affected by colchicine, podophyllotoxin, strychnine, and chlorpromazine. Actin had no effect on MAPs ATPase activity in the absence and presence of vinblastine when it was used in place of tubulin.

Actins↗