Biomedical subjects
Publications and source records attributed to A Hara.
1. PAF and LTs may be mediators of the acute gastrointestinal mucosal damage due to endotoxic shock. 2. LTs may be mediators of the acute gastrointestinal mucosal damage due to PAF administration. 3. LMW heparin and unfractionated heparin prevented the increase of LTs due to endotoxin and PAF administration.
The characterization and change of lipids in aorta with the progression of atherosclerosis were elucidated in Watanabe hereditable hyperlipidemic (WHHL) rabbit, an animal model for human familial hypercholesterolemia as compared with those of normal rabbits. The amount of total phospholipids in the aorta of WHHL rabbit increased about 6-fold that of normal level. Sphingomyelin showed the largest increase and phosphatidylcholine the second in the aorta of WHHL rabbit. The fatty acid composition of sphingomyelin was characteristic of higher content of unsaturated fatty acids. It was suggested that the specific increase in both sphingomyelin and its unsaturated fatty acid content might be ascribed to regulation mechanism to recover the membrane fluidity which was reduced by the marked accumulation of cholesterol in the aorta of WHHL rabbit. Glycerophospholipids in the aorta of WHHL rabbit also contained more unsaturated fatty acids than those in normal ones. It was suggested that the higher amounts of unsaturated fatty acids of glycerophospholipids might be derived from those of serum lipoproteins and that they also might help to restore or recover membrane fluidity in the cholesterol-accumulated aorta of WHHL rabbit. Phosphatidylcholine in the aorta of WHHL rabbit contained a large amount of alkylacyl type-homologue (25-fold normal level) which was a precursor of platelet-activating factor (PAF). The increase of lysophosphatidylcholine, a potent chemotactic factor for monocyte, was also observed in WHHL rabbit. The increase of these chemical mediators indicated severe pathologic state in the atherosclerotic lesions of the aorta of WHHL rabbit.
The enzymatic method for isolation and determination of urinary polyamines was modified to measure the polyamines in tissue. High recovery rates of polyamines in tissue by the enzymatic method were obtained, namely, 104.5 +/- 7.4% for diamine, 104.7 +/- 23.3% for spermidine and 104.8 +/- 7.5% for spermine. The results obtained by this method correlated well with those obtained by high performance liquid chromatography, and a close correlation was demonstrated for all fractions: diamine r = 0.8991, y = 0.9916x + 0.0499; spermidine r = 0.8936, y = 1.2073x + 0.1005; spermine r = 0.8921, y = 1.2200x-0.2194, (n = 13).
Polyamine concentrations of human cancerous and non-cancerous tissues from the kidney, ureter, bladder were measured by a new enzymatic method for isolation and determination of polyamines. In cancerous and non-cancerous tissue of the organs studied, the spermine level was highest followed by the spermidine and diamine levels. The concentrations of diamine, spermidine and spermine in cancerous tissues were significantly higher than those in non-cancerous tissues, but there was no significant difference in the spermidine/spermine ratio between the cancerous and non-cancerous tissues. These data suggest that polyamines are produced above the normal levels in pathological conditions such as renal cell carcinoma, ureteral cancer and bladder cancer.
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Dimeric and monomeric proteins containing dihydrodiol dehydrogenase and aldehyde reductase activities were purified from pig lens. The dimeric enzyme of Mr 65,000 specifically oxidized the trans-dihydrodiols of naphthalene and benzene with NADP+ as a strict cofactor, and reduced alpha-diketones, aromatic aldehydes and glyceraldehyde with NADPH as a cofactor. The monomeric enzyme of Mr 35,000, although identical with aldose reductase, oxidized the trans-dihydrodiol of naphthalene at a pH optimum of 7.6. These results suggest that the two enzymes are involved in the pathogenesis of naphthalene cataract.
The effect of dibutyryladenosine 3',5'-cyclic monophosphate (dibutyryl cAMP) on the expression of glutathione S-transferase placental type (GST-P) was examined in rat glioma cell line using an immunohistochemical technique. Cultured T9 glioma cells were negative for GST-P activity under normal conditions. However, treatment with 1 mM dibutyryl cAMP produced GST-P expression in about 50% of the cells, as well as some morphological changes. The expression of GST-P was increased with addition of dibutyryl cAMP together with 1 microgram/ml allyl isothiocyanate (AITC) or 0.1 microgram/ml benzyl isothiocyanate (BITC). With these combinated treatments, almost all cultured cells showed a strong positive reaction for GST-P, although AITC or BITC alone elicited GST-P in only 5% of the cultured cells. The results of the present study indicate that dibutyryl cAMP causes functional as well as morphological differentiation of T9 glioma cells.
1. Five multiple forms of dihydrodiol dehydrogenase (EC 1.3.1.20) with similar molecular weights of around 35,000 were purified from hamster liver cytosol. 2. All the enzymes oxidized trans-dihydrodiols of benzene and naphthalene and reduced various carbonyl compounds, but showed clear differences in specificities for other alcohols and cofactors, and in inhibitor sensitivity. 3. Two NADP+-dependent enzymes were immunologically identified with aldehyde reductase (EC 1.1.1.2) and 3 alpha-hydroxytsteroid dehydrogenase (EC 1.1.1.50). 4. The other enzymes with dual cofactor specificity oxidized xenobiotic alicyclic alcohols, and one of them was active on 3 alpha- and 17 beta-hydroxysteroids with NAD+ as a preferable cofactor.
A commonly used technique to obtain cochlear perilymph for analysis has been the aspiration of samples through the round window membrane. The present study has investigated the influence of the volume withdrawn on sample composition in the guinea pig. Samples of less than 200 nl in volume taken through the round window showed relatively high glycine content, comparable to the level found in samples taken from scala vestibuli. If larger volumes are withdrawn, lower glycine levels are observed. This is consistent with cerebrospinal fluid (having a low glycine content) being drawn into scala tympani through the cochlear aqueduct and contaminating the sample. The existence of a concentration difference for glycine between scala tympani perilymph and cerebrospinal fluid suggests the physiologic communication across the cochlear aqueduct is relatively small in this species. The observation of considerable exchange between cerebrospinal fluid and perilymph, as reported in some studies, is more likely to be an artifact of the experimental procedures, rather than of physiologic significance. Alternative sampling procedures have been evaluated which allow larger volumes of uncontaminated scala tympani perilymph to be collected.
Indanol dehydrogenase was purified to apparent homogeneity from monkey liver cytosol. The enzyme was a monomer with a molecular weight of 36,000 and pI of 8.7. The amino acid composition was determined. The enzyme oxidized alicyclic alcohols including transdihydrodiols of benzene and naphthalene in the presence of both NADP+ and NAD+, and reduced several xenobiotic carbonyl compounds in the presence of NADPH, the 4-pro-R hydrogen atom of which was transferred to the substrate. The results of fluorometric binding and kinetic studies are consistent with an ordered sequential mechanism with NADP+ binding first. The enzyme was inhibited competitively versus NADP+ and uncompetitively versus 1-indanol not only by chelating agents such as 1,10-phenanthroline and 2,2'-bipyridine but also by a nonchelating isomer, 4,4'-bipyridine, which suggests hydrophobic interaction of the aromatic compounds with the enzyme, which did not contain zinc. The enzyme was also inhibited by Cibacron blue dye, synthetic estrogens, and delta 4-3-ketosteroids. The inhibition by Cibacron blue was competitive versus NADP+ and noncompetitive versus 1-indanol, whereas those by hexestrol, medroxyprogesterone acetate, and progesterone were uncompetitive versus NADP+ and competitive versus 1-indanol, corraborating the ordered addition of the coenzyme prior to 1-indanol.
Dihydrodiol dehydrogenase activity was detected in the cytosol of various mouse tissues, among which kidney exhibited high specific activity comparable to the value for liver. The enzyme activity in the kidney cytosol was resolved into one major and three minor peaks by Q-Sepharose chromatography: one minor form cross-reacted immunologically with hepatic 3 alpha-hydroxysteroid dehydrogenase and another with aldehyde reductase. The other minor form was partially purified and the major form was purified to homogeneity. These two forms, although different in their charges, were monomeric proteins with the same molecular weight of 39,000 and had similar catalytic properties. They oxidized cis-benzene dihydrodiol and alicyclic alcohols as well as trans-dihydrodiols of benzene and naphthalene in the presence of NADP+ or NAD+, and reduced several xenobiotic aldehydes and ketones with NAD(P)H as a cofactor. The enzymes also catalyzed the oxidation of 3 alpha-hydroxysteroids and epitestosterone, and the reduction of 3- and 17-ketosteroids, showing much lower Km values (10(-7)-10(-6) M) for the steroids than for the xenobiotic alcohols. The results of mixed substrate experiments, heat stability, and activity staining on polyacrylamide gel electrophoresis suggested that, in the two enzymes, both dihydrodiol dehydrogenase and 3(17)alpha-hydroxysteroid dehydrogenase activities reside on a single enzyme protein. Thus, dihydrodiol dehydrogenase existed in four forms in mouse kidney cytosol, and the two forms distinct from the hepatic enzymes may be identical to 3(17)alpha-hydroxysteroid dehydrogenases.
Homogeneous indanol dehydrogenase from monkey liver catalyzed the reversible conversion of 3 alpha- or 20 alpha-hydroxy groups of several bile acids and 5 beta-pregnanes to the corresponding 3- or 20-ketosteroids. The kcat values for the steroids determined at pH 7.4 were low, but the kcat/Km values for the 3-ketosteroids were comparable to or exceeded those for 1-indanol and xenobiotic carbonyl substrates. The enzyme transferred the 4-pro-R-hydrogen atom of NADPH to the 3 beta- or 20 beta-face of the ketosteroid substrate. Competitive inhibition of the hydroxysteroid dehydrogenase activity of the enzyme by medroxyprogesterone acetate, hexestrol, and 1,10-phenanthroline suggests that both 1-indanol and hydroxysteroid are oxidized at the same active site on the enzyme. The specific inhibitor of the enzyme, 1,10-phenanthroline, suppressed the 3 alpha-hydroxysteroid dehydrogenase activity in the crude extract of monkey liver by 50%. The results strongly suggest that indanol dehydrogenase acts as a 3(20)alpha-hydroxysteroid dehydrogenase in the metabolism of certain steroid hormones and bile acids.
Chemical cross-linking and NADPH binding studies suggested that the native dihydrodiol dehydrogenase from monkey kidney is a basic dimer having a molecular weight of 78,000 and one active site per the subunit. The enzyme oxidized specifically trans-dihydrodiols of benzene and naphthalene, whereas it catalyzed the reduction of dihydroxyacetone and dihydroxyacetone phosphate at a physiological pH, 7.4. The Km and kcat values for dihydroxyacetone phosphate were 5.0 mM and 4.3 s-1, respectively. The enzyme transferred the 4-pro-R hydrogen atom of NADPH to the carbonyl substrate. Immunochemical experiments using an antibody against the dimeric enzyme revealed the specific distribution of the enzyme in the kidney of this animal. By immunohistochemical staining with the specific antibody, the immunoreactivity was found in proximal and distal tubules of the cortex, and in the loop of Henle of the medulla.
The modifying effect of ethanol (EtOH) on aflatoxin B1 (AFB1)-induced hepatocarcinogenesis was examined in male ACI/N rats by chronic treatment at the post-initiation phase. Rats received an ip injection of AFB1 (1.5 mg/kg) twice a week for 10 weeks (a total of 20 doses). Following a week of acclimation, they were given 10% EtOH as drinking water for 56 weeks. The effect of EtOH on the hepatocarcinogenesis was evaluated in terms of the incidence of altered hepatocellular foci and neoplasms at the end of the experiment. Exposure to AFB1 alone induced a substantial number of altered foci (6.98 iron-excluding foci/cm2) in rats. The number of altered liver cell foci in rats receiving AFB1 followed by EtOH was significantly increased (26.39 iron-excluding foci/cm2). In the rats given EtOH after AFB1, the total area and mean diameter of both iron-excluding foci and altered foci identified in hematoxylin and eosin-stained sections were significantly higher than in the rats exposed to AFB1 alone. The incidence of liver cell tumors of the group given AFB1 and EtOH (3/15, 20%) was higher than that of the group treated with AFB1 alone (0/14, 0%). Treatment with EtOH alone for 56 weeks did not induce either. These results indicate an enhancing effect of EtOH on AFB1-induced hepatocarcinogenesis when it is given in the promotion phase.
A phosphoprotein phosphatase (PPase: EC 3.1.3.2) was recently purified from rat epidermis. The enzyme dephosphorylates phosphoprotein, and its properties, such as pH optimum, inhibitor spectrum, and Fe2+ activation, differ from those of other soluble phosphatases. We investigated in 2-day-old rat skin the distribution of immunologically detectable PPase and intracellular localization of PPase activity. The reaction of rabbit monospecific anti-PPase IgG was identified in granular and cornified cells by the avidin-biotin complex method. For activity staining, basic principles of the Gomori lead-salt method and azo dye technique with the substrates p-nitrophenylphosphate (p-NPP) and alpha-naphthyl phosphate (NP), respectively, were modified according to the biochemical properties of PPase activity which is resistant to formalin, Na tartrate, and NaF. Activity was detectable in granular cells including keratohyalin granules and the lower strata of cornified cells. The activity was inhibited by 1 mM CuSO4 and enhanced by a mixture of 0.5 mM FeSO4 and 1 mM ascorbic acid. We consider that PPase may be involved in dephosphorylation of histidine-rich proteins in granular and cornified cells and may play a key role in intracellular catabolism associated with epidermal cell differentiation.
Tannic acid was found to be a highly potent inhibitor of human placenta aldose reductase. The most potent inhibitory component of the tannic acid was isolated and identified as penta-O-galloyl-beta-D-glucose, which showed an IC50 value of 70 nM. The inhibition by the gallotannin was reversible and of mixed type with respect to DL-glyceraldehyde as the varied substrate.
Plasminogen activator (PA) and PA inhibitor were partially purified from 2-d-old rat epidermis and characterized in order to elucidate the enzyme-inhibitor interaction in epidermis. PA extracted with buffer containing KSCN was first purified by Blue-Sepharose affinity chromatography. Separation of two PAs, with relative molecular mass (Mr) of 66000 and 44000, was accomplished by Con A-Sepharose column chromatography. The Mr 66000 enzyme had the properties of tissue-type PA (t-PA), while the Mr 44000 enzyme showed those of urokinase-type PA (u-PA) as determined by immunological and fibrin-binding studies. PA inhibitor was extracted in 1,4-piperazinediethanesulphonic acid buffer and purified by (NH4)2SO4 precipitation, gel filtration followed by a Mono Q column in an FPLC system. This inhibitor showed Mr 60000 and inhibited human u-PA activity in a dose- and time-dependent manner, but did not inhibit the activity of t-PA from human and murine melanoma cells or plasmin. It inhibited epidermal PA, Mr 44000, more effectively than it did the Mr 66000 epidermal PA. It was stable at 60 degrees C for 60 min or between pH 5 and 11. This study indicates that both u-PA and t-PA function in normal rat epidermis. On the other hand, an inhibitor which preferentially acts against u-PA exists, but inhibitor to t-PA does not appear to operate under normal epidermal functions.
Two major and two minor forms of dihydrodiol dehydrogenase with similar molecular weights of around 36000 were purified from monkey liver cytosol. All the forms oxidized trans-dihydrodiols of benzene and naphthalene and reduced aromatic aldehydes, but showed differences in charge, specificity for other substrates and inhibitor sensitivity. One major (pI 8.7) and one minor (pI 7.9) form of the enzyme exhibited high activity for alicyclic alcohols and sensitivity to o-phenanthroline. The other major form (pI 6.2) oxidized 3 alpha-hydroxysteroids and was inhibited by dexamethasone and indomethacin, whereas the other minor form (pI 5.8) showed high reductase activity for aldehydes including D-glucuronate and sensitivity to barbital and sorbinil, and cross-reacted with human aldehyde reductase. The results indicate that the multiple forms of monkey liver dihydrodiol dehydrogenase are indanol dehydrogenases, 3 alpha-hydroxysteroid dehydrogenase and aldehyde reductase.