Biomedical subjects

H Okuda

Publications and source records attributed to H Okuda.

At least 181 records · Page 10Linked to original sources

PubMed1995

Glycosylation and secretion of lipoprotein lipase by 3T3-L1 adipocytes: effects of brefeldin A.

Time courses of synthesis and secretion of lipoprotein lipase (LPL) were examined in 3T3-L1 adipocytes. LPL was glycosylated in the endoplasmic reticulum (ER) within 10 min after synthesis, and was transported after 20-30 min to the trans Golgi where it was converted to the mature form with M(r) = 55,000-58,000, which was resistant to endoglycosidase H (endo H). LPL subunits with M(r) = 55,000-58,000 appeared in the medium within 30 min after synthesis. The effects of brefeldin A (BFA), which inhibits transport of glycoproteins in various types of cells, on secretion and glycosylation of LPL were also examined. BFA completely blocked release of LPL activity into the medium, causing accumulation of the activity in cells. The suppressive effect of BFA on release of LPL activity was reversible. BFA-treated cells synthesized LPL with M(r) = 53,000-55,000 consisting of 2 types of subunits, the main type being totally endo H-sensitive and the other partially endo H-sensitive. No LPL were secreted into the medium by BFA-treated cells.

Adipocytes↗

PubMed1995

[A clinico-pathological study of renal cell carcinoma with a diameter of less than 5 cm].

To examine the indication of nephron sparing operations, we searched 31 nephrectomized kidneys with renal cell carcinoma for small associated lesions in the normal-appearing portion of these kidneys. The diameter of the predominant tumors was between 15 and 50 mm. The kidneys were serially sectioned at 5-mm intervals, and all sections were examined microscopically. Two of the 31 kidneys had a small renal cell carcinoma, and four had a small adenoma in the normal-appearing portion. We recommend that the nephron-sparing operation should be is performed limitedly and that radical nephrectomy is yet a standard operation in the presence of a normal contralateral kidney.

Adenoma↗

PubMed1994

The synthesis of fatty acid ethyl ester by carboxylester lipase.

Carboxylester lipase obtained from pig pancreas is associated with fatty acid ethyl ester synthase as judged by their elution in the same fraction from a heparin-Sepharose column, coprecipitations by antibody against purified carboxylester lipase and identical profiles of inhibition by diisopropyl fluorophosphate. Only one polypeptide of molecular mass 74-kDa in purified carboxylester lipase was labeled by immunostaining and affinity labeling with [3H]diisopropyl fluorosphate. Bovine serum albumin decreased the fatty-acid-ethyl-ester-synthesizing activity in a concentration-dependent manner. On incubation of purified carboxylester lipase with trioleylglycerol in an ethanol/water mixture, fatty acid ethyl ester was formed in the presence of a high concentration of bovine serum albumin. The acyltransfer activities from trioleylglycerol to ethanol (ethanolysis) were approximately 25-30 times higher than the acyltransfer activities to water (hydrolysis). When cholesterol was used as an acceptor, acyltransfer activity from trioleylglycerol to cholesterol (cholesterolysis) was also observed. We propose the following mechanism of fatty acid ethyl ester formation from triacyl glycerol. The enzyme attacks triacyl glycerol forming an acyl-enzyme intermediate, and during the deacylation process, alcohol binds to fatty acid as an acceptor. These results suggest that during lipid (triacyl glycerol) degradation, carboxylester lipase contributes to non-oxidative ethanol metabolism in the intestinal lumen.

Acyltransferases↗

PubMed1994

Effects of alpha- and beta-adrenergic antagonist on epinephrine-induced aggregation and intracellular free calcium concentration in human platelets.

Recently, it has been reported that an alpha-adrenergic antagonist phentolamine inhibits epinephrine-induced platelet aggregation but a beta-adrenergic antagonist propranolol did not. In this study, we examined the effects of alpha- and beta-adrenergic antagonist on cytosolic free calcium movements induced by epinephrine. An alpha-adrenergic antagonist phentolamine was found to inhibit the elevation of cytosolic free calcium concentration induced by epinephrine, while a beta-adrenergic antagonist propranolol had no effect on cytosolic free calcium concentration induced by epinephrine.

Adrenergic alpha-Antagonists↗

PubMed1994

Novel theta class glutathione S-transferases Yrs-Yrs' and Yrs'-Yrs' in rat liver cytosol: their potent activity toward 5-sulfoxymethylchrysene, a reactive metabolite of the carcinogen 5-hydroxymethylchrysene.

Two novel theta class glutathione S-transferases (GSTs), designated Yrs-Yrs' and Yrs'-Yrs', were isolated from rat liver cytosol and purified to homogeneity. Polyclonal antibody raised against the previously reported theta class GST Yrs-Yrs (Hiratsuka, A. et al., J. Biol. Chem. 265, 11973 (1990)) cross-reacted with GSTs Yrs-Yrs' and Yrs'-Yrs'. These three theta class GSTs had different pI values and were separated by chromatofocusing. The enzyme subunit Yrs' was separated from Yrs by reverse partition HPLC, but identical to Yrs in the first 37 N-terminal amino acid sequence. Like GST Yrs-Yrs, both GSTs Yrs-Yrs' and Yrs'-Yrs' were not retained on an S-hexyl-GSH affinity column and had little activity toward 1-chloro-2,4-dinitrobenzene. However, they showed potent activities toward the reactive sulfate ester of the carcinogen, 5-hydroxymethylchrysene. GSTs Yrs-Yrs and Yrs'-Yrs' showed much higher GSH peroxidase activities toward arachidonate hydroperoxide than did rat liver alpha class GST Ya-Ya.

Amino Acid Sequence↗

PubMed1994

Role of processing of the oligosaccharide chains in the affinity of lipoprotein lipase for heparin.

The role of processing of the oligosaccharide chains in the affinity of lipoprotein lipase (LPL) for heparin was examined in 3T3-L1 adipocytes. 43% of 35S-labeled LPL subunits in tunicamycin (TUN)-treated cells did not bind to a heparin-Sepharose column and 46% was eluted with 0.6 M NaCl. 11% of LPL subunits in castanospermine (CSTP)-treated cells did not bind to the column and 38% was eluted with 0.6 M NaCl. In contrast, as in untreated cells, LPL subunits in 1-deoxymannojirimycin (dMM)-treated and swainsonine (SW)-treated cells almost all bound to the column and over 93% of the subunits bound were eluted with 1.5 M NaCl. Thus, core glycosylation and subsequent removal of the distal glucose residue from oligosaccharide chains of LPL in the endoplasmic reticulum (ER) is required for acquisition of a higher affinity for heparin.

1-Deoxynojirimycin↗

PubMed1994

Fatty acid ethyl ester-synthesizing activity of lipoprotein lipase from rat postheparin plasma.

Lipoprotein lipase (LPL) was obtained from rat postheparin plasma by chromatographies on heparin-Sepharose and hydroxyapatite. The enzyme was associated with fatty acid ethyl ester synthase (FAEE synthase) as judged by their co-elution profiles and identical profiles of inhibition by diisopropyl fluorophosphate. Only one polypeptide of molecular weight 57,000 in purified LPL fraction was labeled by affinity labeling with [3H]-diisopropyl fluorophosphate. The FAEE synthase activity of LPL was not affected by addition of apolipoprotein C-II. Digestion of the enzyme with trypsin resulted in almost complete loss of the triolein-hydrolyzing activity without change in FAEE synthase activity. The tributyrin-hydrolyzing activity of LPL was also not affected by addition of apolipoprotein C-II or trypsin digestion. On addition at progressively higher concentrations, bovine serum albumin increased FAEE synthesis to a maximum at 2 mg/ml and at higher concentrations inhibited its activity. On incubation of purified LPL with chylomicrons in an ethanol/water mixture, FAEE was formed in the presence of a high concentration of bovine serum albumin. The specific activity of FAEE synthesis from chylomicrons was about 65 times that from oleic acid. Triolein/gum arabic emulsion was used for identification of reaction products. We propose the following mechanism of FAEE formation from chylomicrons by LPL. The enzyme attacks chylomicrons forming an acyl-enzyme intermediate, and during the deacylation process, ethanol binds to fatty acids as an acceptor. These results suggest that LPL contributes to nonoxidative ethanol metabolism (FAEE formation) through degradation of triglyceride-rich lipoproteins such as chylomicrons.

Acyltransferases↗

PubMed1994

Lead exposure in vivo alters the fertility potential of sperm in vitro.

Lead is a male reproductive toxicant. We previously reported that in vivo lead exposure results in a suppression of the hypothalamic-pituitary-testicular axis in male rats. This study was designed to assess if lead exposure in vivo alters (1) the ability of sperm to fertilize ova in vitro, (2) the morphology of the spermatozoa, and (3) the relationship of cell types in the testes as evaluated by DNA flow cytometry. Male Sprague-Dawley rats were given access to either lead-free (0.0%) or 0.3% lead acetate-containing water for 14, 30, or 60 days starting at 100 days of age. On the day of termination, sperm harvested from the caudae epididymis were incubated with eggs harvested from superovulated non-lead-treated females and were scored for stages of penetration. Sperm were also studied by electron microscopy. The testes of animals treated for 60 days were processed for DNA flow cytometry. The overall distribution of the stages of fertilization was significantly different between control and lead-treated animals. The lead-treated groups fertilized significantly fewer eggs than did sperm from the control group. Increased duration of exposure to lead acetate did not result in a more significant percentage of eggs not fertilized. No ultrastructural changes were noted in the spermatozoa of animals treated with lead compared to control animals. There were no differences in the histogram patterns of testicular cells collected from lead-treated animals and control animals. We conclude that lead alters sperm function by altering the hormonal control of spermatogenesis rather than by direct toxic action on spermatozoa.

Administration, Oral↗

PubMed1994

Immunocytochemical localization of antigens in adult worms of Trichinella spiralis recognized by Fischer rats.

We demonstrate the tissue localization, in adult Trichinella spiralis, of antigens recognized by Fischer rat sera at 32 weeks postinfection. Immunodominant antigens were located in a wide variety of tissues, including type 1 stichocyte granules, stichocyte cytoplasm, the canalicular tree, hemolymph, hypodermis, hypodermal glands, cord cytoplasm, intestinal-gland cell granules, membranous structures in the midgut epithelium, midgut-occupying substance, brush border, hindgut epithelial cytoplasm, hindgut cuticle, vaginal cuticle, epithelial cytoplasm of the female genital tract, microvilli and discrete areas of the ovum, embryo sheath, intersperm space, discrete areas in immature sperm, small granules and cup-shaped membrane structures of sperm, and exocrine granules in the seminal vesicle and ejaculatory duct. A small amount of antigen was located in the inner layers of the genital portion of the body cuticle. The precise localization of antigens in adult worms should form a basis for better analysis of T. spiralis-related immunology.

Animals↗

PubMed1994

Molecular cloning and functions of rat liver hydroxysteroid sulfotransferases catalysing covalent binding of carcinogenic polycyclic arylmethanols to DNA.

Three sulfotransferases (STs) catalysing the metabolic activation of potent carcinogenic polycyclic arylmethanols were purified from female Sprague-Dawley (SD) rat liver cytosol without loss of their enzyme activities in the presence of Tween 20 used for preventing the enzymes from aggregation during purification and identified as hydroxysteroid sulfotransferases (HSTs). All the purified HSTs, STa, STb, and STc, with different electric charges had an apparently equal size of subunit (30.5 kDa) and cross-reacted with polyclonal antibody raised against STa. Our study on molecular cloning of cDNA libraries from two female SD rat livers indicated that both contained cDNA inserts coding for 5 different HST subunits, consisting of 284-285 amino acid residues (M(r), 33,084-33,535) and sharing strong amino acid sequence identity (> 83%). Of the 5 HST subunits, two had an identical amino acid sequence except for only one amino acid residue, and the other two contained only 6 amino acid substitutions in their sequences.

Amino Acid Sequence↗

PubMed1994

Major hydroxysteroid sulfotransferase STa in rat liver cytosol may consist of two microheterogeneous subunits.

The possible existence of two microheterogeneous subunits, designated ST-40P and ST-41P, of hydroxysteroid sulfotransferases in female Sprague-Dawley rat liver cytosol was demonstrated by cloning and sequencing of cDNAs, both isolated from two rat liver cDNA libraries. These subunits consisted of an equal number of amino acid residues with only one amino acid substitution. ST-40P and ST-41P expressed as homodimers from the ST-40 and ST-41 cDNAs in Escherichia coli had enzyme activities toward all of the examined 20 hydroxysteroids, 13 bile acids, and the carcinogen 5-hydroxymethylchrysene (5-HCR), with formation of the reactive metabolite 5-HCR sulfate, at rates very similar to those by STa, the major hydroxysteroid sulfotransferase in rat liver cytosol. This strongly suggested that they are essential components of STa. The present study carried out by using the recombinant enzymes provides the first direct evidence for the identity of sulfotransferases catalysing the sulfation of hydroxysteroids and bile acids and proposes that the current nomenclature system used for distinguishing hydroxysteroid sulfotransferases from bile acid sulfotransferases should be improved.

Amino Acid Sequence↗

PubMed1994

Effects of toxic levels of lead on gene regulation in the male axis: increase in messenger ribonucleic acids and intracellular stores of gonadotrophs within the central nervous system.

Lead is a male reproductive toxicant. Lead exposure results in a general suppression of the hypothalamic-pituitary-testicular (HPT) axis in male rats. The mechanism(s) for this disruption by lead is unknown. Toxic lead levels seem to disrupt central nervous system (CNS) control of the HPT system, resulting in a decrease in serum testosterone levels and sperm concentrations. A study designed to elucidate the mechanisms accounting for the disruption of the normal function of the male axis by toxic lead levels at the molecular level demonstrated a 2-3-fold enhancement of mRNA levels of GnRH and the tropic hormone LH. A 3-fold increase of intracellular stores of LH was also found. Because mRNA levels of LH and GnRH and pituitary levels of stored LH are proportional to blood levels of lead, we hypothesize that lead interferes with the normal release of tropic hormones and disrupts hormonal feedback mechanisms. The observed pleiotropic effects of lead upon the male axis and other systems may be explained by simple and unique competition by lead with normal metal ion binding sites that govern genetic control of specific genes.

Animals↗

PubMed1994

Existence of lipoprotein lipase in human sarcomas and carcinomas.

Aqueous extracts of acetone/ether powders of surgically obtained specimens of human tumors hydrolyzed 3H-labeled triolein in a dose-dependent manner. The lipolytic activity in these extracts was inhibited by anti-lipoprotein lipase (LPL) IgG dose-dependently, 25 micrograms of anti-LPL IgG causing 95% inhibition of the activity. Thus, LPL accounts for most of the lipolytic activity in extracts of acetone/ether powders of the tumors. All sarcomas and carcinomas examined contained LPL activity. Western blotting showed that they gave a band corresponding to that of human adipose tissue LPL (M(r) = 57,000). Immunocytochemical studies showed that LPL was present in cultured human osteosarcoma cells and distributed throughout the cells. We determined the proliferating cell nuclear antigen (PCNA)-labeling index as an indicator of the proliferative activity of tumor cells and measured LPL activity in extracts of tumors in areas corresponding to those used for determining the PCNA-labeling index. In malignant fibrous histiocytomas, the PCNA-labeling index in area a, which corresponds to the subcapsular region, was higher than that in area b, which corresponds to the central region. The LPL activity in area a was 10 times that in area b. In rectal cancer, the index in area c, which corresponds to the subserosal region, was higher than that in area d, which corresponds to the submucosal region. The LPL activity in area c was 1.9 times that in area d. These findings indicate heterogeneity in the distributions of LPL activity within tumors and higher levels of LPL activity in tumors that are proliferating actively.

Antigens, Neoplasm↗

PubMed1994

Effects of aging on acute toxicity of nicotine in rats.

Acute toxicity of nicotine was examined in old (24 months) and young (6 weeks) Wistar rats. There were no significant age differences in the mortality and convulsive responses induced by an intraperitoneal injection of nicotine (24.5 mg/kg). The lethal nicotine levels in blood and cortex and the latent period to death in old rats were larger than those in young rats. Cortical and blood nicotine levels 15 min. after the nicotine injection in old rats were also higher than those in young rats. Nicotine significantly increased dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid levels in striatum and hippocampus in young rats, but not those in old rats. Moreover, nicotine-induced elevations in blood levels of corticosterone in old rats were also less than those in young rats. On the other hand, the cytochrome P-450 content, the nicotine oxidase activity and the flavin-containing monooxygenase activity in liver showed age-related decreases in the young, the middle-aged (12 months) and the old rats. These results indicate that the acute toxicity of nicotine in old rats reflects the decreases in hepatic nicotine metabolism and in brain sensitivity to nicotine.

Aging↗

PubMed1994

[Molecular diversity and properties of brain nicotinic acetylcholine receptor].

To date, cDNA studies and purification of nicotinic acetylcholine receptors (nAChRs) have shown that there are many different subtypes of the receptors in the central and peripheral nervous systems. This review focuses on the animal and avian brain nAChRs, their diversity, pharmacological properties, distribution and regulation. nAChR subtypes are classified as alpha beta types and alpha-bungarotoxin binding proteins. Each subtype contains two or more subunits. Each subunit combination leads to a distinct pattern of sensitivity to nicotinic agonists in reconstituted systems of Xenopus oocytes. Nicotine has been shown to increase the levels of intracellular free Ca through both nAChR types. The mRNAs for each subunit are uniquely expressed in animal and avian brains, although beta 2 is expressed in most regions of the brains. This shows somewhat overlapping patterns of gene expression. Monoclonal antibodies against each subunit make it possible to investigate the distribution of the subunits in brains and neurons. nAChRs have been shown to be regulated by nicotine administration and phosphorylation of the receptors. The regulatory mechanisms have been extensively studied, but still remain obscure. The structural and functional diversity of neuronal nAChRs is probably important in nicotine addiction, tolerance and reverse tolerance.

Animals↗

PubMed1994

Immunohistochemical analysis of P-glycoprotein expression in diverse histological types of epithelial ovarian tumors.

P-glycoprotein is a transmembrane protein which acts as an energy-dependent drug efflux pump for a variety of anti-cancer drugs. The mdr-1 gene which encodes P-glycoprotein was successfully cloned in 1986. To investigate P-glycoprotein expression in diverse ovarian tumors, including benign, low malignant potential and malignant, immunohistochemical study was done using a monoclonal antibody (C 219). Overall, 8 out of the 59 epithelial ovarian tumors (13.6%) expressed P-glycoprotein. It was noted that 5 of the 12 mucinous tumors were found to express P-glycoprotein, while none of the 31 serous tumors were immunohistochemically positive. In 10 malignant ovarian tumors, P-glycoprotein immunostaining was examined both prior to and after chemotherapy. Nine of them did not express any P-glycoprotein before or after chemotherapy. However, one tumor expressed P-glycoprotein after six courses of multidrug resistance-related drug administration. These findings indicate that P-glycoprotein expression is not so common in ovarian tumors, regardless of their malignant potential. Nevertheless, the results suggest a strong association between P-glycoprotein expression and certain histological cell types in epithelial ovarian tumors. It is also possible that P-glycoprotein appears as a result of chemotherapy, but such a phenomenon can not occur unless chemotherapy is administered at high doses for a long period of time.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Existence of 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ols in lipoproteins from diabetic patients and normal subjects.

We report evidence for the presence of 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ols (cholesterol 7-hydroperoxides, Ch 7 alpha-OOH and Ch 7 beta-OOH, respectively) in human plasma lipoproteins in vivo, which had been reported to be markers of aging in rat skin. A comparative study was carried out focusing on the detection of Ch 7-OOHs in plasma lipoproteins from diabetic patients whose plasma has been suggested to be under high oxidative stress. Blood samples were collected from healthy volunteers (control) and diabetics with and without hypercholesterolemia. Ch 7-OOHs were isolated from low and high density lipoproteins (LDL and HDL, respectively) in the plasma of these subjects, identified, and determined by high-performance liquid chromatography with a chemiluminescence detector. The percent detection of Ch 7-OOHs in LDL from diabetics without hypercholesterolemia was similar to that in the control group. However, it was significantly higher in diabetics with hypercholesterolemia than in those without hypercholesterolemia. The percent detection of Ch 7-OOHs in HDL from diabetics without hypercholesterolemia was higher than both that in LDL from the same group and that in HDL from the control group.

Adult↗

PubMed1994

[Flow cytometric DNA analysis on renal cell carcinoma--a study of 116 cases on fresh surgical specimens].

DNA ploidy was investigated using flow cytometry on fresh surgical specimens from 116 cases with renal cell carcinoma. DNA diploid tumor was observed in 39 cases (33.6%) and aneuploid in 77 cases (66.4%). DNA aneuploid group was further classified into 3 subgroups by DNA index (D.I.); near-diploid group (D.I.: 0.8-1.2, 21.6%), near-tetraploid group (D.I.: 1.8-2.2, 12.9%) and other aneuploid group (31.9%). DNA ploidy pattern correlated with clinical stages and histological grading, indicating significantly a higher incidence of DNA diploid in cases in stage I and grade 1. DNA clonal heterogeneity was observed in 48.1%, and homogeneously diploid was in 28.6% of 77 cases who were analyzed more than 2 specimens. Incidences of DNA heterogeneity and homogeneous diploid correlated with the number of analyzed specimens and our results showed that more than 4 specimens were necessary to diagnose the DNA ploidy patterns. The near-tetraploid group was shown to have an extremely poor prognosis compared with the diploid group and the other aneuploid group. There were no significant difference between the diploid group and the aneuploid group in terms of the early prognosis, however, the incidence of disease recurrence was significantly higher in the aneuploid group. Our results demonstrated that DNA ploidy was an useful prognostic factor in the evaluation of patients with renal cell carcinoma.

Adult↗