Biomedical subjects

S Fujita

Publications and source records attributed to S Fujita.

At least 19 recordsLinked to original sources

PubMed1992

Enzymatic basis for the non-linearity of hepatic elimination of propranolol in the isolated perfused rat liver.

Propranolol (PL) metabolism was studied in the isolated perfused rat liver under single-pass and steady-state conditions. An attempt was made to predict the data observed in the isolated rat liver perfusion at PL infusion rates of 89-1317 nmol/min using the microsomal kinetic parameters obtained in our previous paper (Ishida et al., Biochem Pharmacol 43: 2489-2492, 1992) and the unbound PL fractions in rat liver microsomes and the perfusion medium. The values of kinetic parameters obtained in rat liver microsomes were corrected for the whole liver. Two groups of cytochrome P450 isozymes having high (Km < 0.5 microM)- and low (Km > 20 microM)-affinities participate in the metabolism of PL and sudan III pretreatment induces the low-affinity enzymes rather than the high-affinity enzymes in control rats. Of high-affinity isozyme(s) PL 4-hydroxylase and 7-hydroxylase made a major contribution to the overall activity, while for low-affinity isozymes PL 4-hydroxylase and N-desisopropylase did. A nonlinear relationship between the PL concentrations entering and leaving the liver was predicted from these corrected kinetic parameters using the venous equilibrium model. The outflow concentrations and the metabolic rates of PL for the predicted curves were over-estimated at higher inflow PL concentrations and under-estimated at higher substrate concentrations, respectively. On the other hand, the prediction for them was successfully carried out for the livers whose intrinsic clearance was altered due to the induction of low-affinity enzymes in PL metabolism by sudan III pretreatment. The outflow rates of 4-hydroxypropranolol showed a downward curvature at lower substrate concentrations, followed a linear rise in the livers from control rats, while the outflow rates of 5- and 7-hydroxypropranolol exhibited their respective limiting values. The outflow rates of 4-hydroxypropranolol and N-desisopropylpropranolol were enhanced markedly with increasing the outflow unbound concentration of PL by sudan III pretreatment. These results indicate that non-linear PL first-pass metabolism is due to the saturation of the reactions for the high-affinity enzymes among enzymes engaging in PL ring hydroxylations.

Animals↗

PubMed1992

Sphingolipids are essential for the growth of Chinese hamster ovary cells. Restoration of the growth of a mutant defective in sphingoid base biosynthesis by exogenous sphingolipids.

We previously isolated a temperature-sensitive Chinese hamster ovary cell mutant (strain SPB-1) with thermolabile serine palmitoyltransferase, which is involved in the first step of sphingolipid synthesis (Hanada, K., Nishijima, M., and Akamatsu, Y. (1990) J. Biol. Chem. 265, 22137-22142). In this study, sphingolipid-deficient culture medium was used to examine the effect of exogenous sphingolipids on the cell growth of SPB-1. When cultivated in the sphingolipid-deficient medium, SPB-1 cells ceased growing at non-permissive temperatures. Under these conditions, de novo sphingolipid synthesis ceased in the SPB-1 cells, resulting in a decrease in levels of sphingomyelin and ganglioside sialyl lactosylceramide (GM3), whereas the parental CHO-K1 cells grew logarithmically with normal sphingolipid synthesis. Exogenous sphingosine restored the contents of both sphingomyelin and GM3 in the SPB-1 cells near to the parental levels through metabolic utilization and allowed the mutant cells to grow even at the non-permissive temperature. Similarly, exogenous sphingomyelin restored the sphingomyelin levels and only partly the GM3 levels and also suppressed the temperature-sensitivity of the SPB-1 cell growth. In contrast, exogenous glucosylceramide, which restored the GM3 levels but not the sphingomyelin levels, failed to suppress the temperature sensitivity of the SPB-1 cell growth. Combination of exogenous sphingomyelin with ceramide, glucosylceramide, GM3, or sphingoid bases did not show any synergistic or additive effect on the SPB-1 cell growth enhancement, compared with sphingomyelin alone. The results indicated that the temperature sensitivity of the SPB-1 cell growth was due to the lack of cellular sphingolipids, possibly that of sphingomyelin.

Acyltransferases↗

PubMed1992

Cadherin dysfunction in a human cancer cell line: possible involvement of loss of alpha-catenin expression in reduced cell-cell adhesiveness.

A human lung cancer cell line, PC 9, was analyzed to elucidate the molecular mechanisms of dysfunction of cadherin-mediated cell-cell adhesion in cancer. Although PC 9 cells strongly expressed E-cadherin at the cell membrane, which was indistinguishable immunochemically from functional E-cadherin, they did not show tight cell-cell adhesion and had reduced E-cadherin-mediated aggregation activity. Immunoprecipitation with E-cadherin and Western blot analysis revealed that PC 9 cells did not express alpha-catenin, a cadherin-associated protein, suggesting that this was the cause of the cadherin dysfunction in the cell line. In addition, Northern and Southern blot analyses disclosed homozygous deletion of part of the alpha-catenin gene, which might have resulted in the loss of alpha-catenin expression in PC 9 cells.

Blotting, Northern↗

PubMed1992

Abundant expression of immunoreactive endothelin 1 in mammary phyllodes tumor: possible paracrine role of endothelin 1 in the growth of stromal cells in phyllodes tumor.

Immunoreactive endothelin 1 (irET-1) concentrations were measured in extracts prepared from 4 phyllodes tumors and 14 fibroadenomas. irET-1 was detectable in all tissue extracts by specific radioimmunoassay, and the mean concentration of irET-1 was 18-fold and 27-fold higher in tissue extracts from phyllodes tumors than in those from intracanalicular fibroadenomas and pericanalicular fibroadenomas, respectively. Reverse-phase high-performance liquid chromatography coupled with radioimmunoassay in the extracts from phyllodes tumors revealed one major irET-1 component corresponding to human standard ET-1. Furthermore, immunocytochemical staining for ET-1 revealed that numerous ET-1-immunoreactive cells were seen in the epithelial cells but not in the stromal cells, suggesting that ET-1 is synthesized by the epithelial component of phyllodes tumors. A possible paracrine role of ET-1 in the growth of this rare tumor which is characterized by its prominent stromal cellularity is discussed.

Adenofibroma↗

PubMed1992

Intranuclear topological distribution of HIV-1 trans-activators.

Subcellular localization of human immunodeficiency virus type I (HIV-1) Tat and Rev was examined using a confocal laser scanning microscope (CLSM). In transfected COS-7 cells, Tat resided exclusively in the perinocleolar region, while Rev infiltrated fully into the nucleoli. The chimeric Tat in which the nucleolar targeting signal was replaced by that of Rev, which retains trans-acting activity of Tat, remained still in the perinucleolar region as wild-type Tat. Perinucleolar distribution of Tat protein suggests the existence of a novel nucleolar architecture that affects transcription.

Amino Acid Sequence↗

PubMed1992

Induction of propranolol metabolism by the azo dye sudan III in rats.

Effects of the azo dye sudan III, an inducer of cytochrome P450 isozymes belonging to the CYP1A subfamily, on propranolol (PL) in vitro and in vivo metabolism were investigated in rats. The kinetic parameters of the activity for each metabolic pathway were determined in liver microsomes from control and sudan III-treated rats. Sudan III pretreatment increased extensively PL 4-hydroxylase, 5-hydroxylase and N-desisopropylase activities at high but not at low PL concentrations. On the other hand, kinetic parameters of 7-hydroxylase activity were not affected by sudan III pretreatment. Sudan III pretreatment decreased blood concentrations of PL after intraportal infusion of PL at high doses (12.5 and 20 mg/kg), but not at a low dose (5 mg/kg). These observations were consistent with data obtained from the in intro studies showing that sudan III pretreatment induced low-affinity but not high-affinity cytochrome P450 isozymes involved in PL metabolism in rat liver microsomes.

Animals↗

PubMed1992

Total synthesis of the modified ganglioside de-N-acetyl-GM3 and some analogs.

Methyl[methyl 4,7,8,9-tetra-O-acetyl-5-(tert-butoxycarbonylamino)-3,5- dideoxy-2-thio-D-glycero-alpha-D-galacto-2-nonulopyranosid]onat e was used for the glycosylation of benzyl O-(2,6-di-O-benzyl-beta-D-galactopyranosyl)- and benzyl O-(2,3-di-O-benzyl-beta-D-galactopyranosyl)-(1----4)-3,6-di-O-benzyl- 2-O-pivaloyl-beta-D-glucopyranoside to give benzyl O-[methyl 4,7,8,9-tetra-O-acetyl-5-(tert-butoxycarbonylamino)- 3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonate]-(2-- --3)-O-(2,6-di-O-benzyl-beta-D-galactopyranosyl)-(21) and benzyl O-[methyl 4,7,8,9-tetra-O-acetyl-5-(tert-butoxycarbonylamino)-3,5- dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonate]-(2----6) -O-(2,3-di- O-benzyl-beta-D-galactopyranosyl)-(1----4)-3,6-di-O-benzyl-2-O-pivaloyl- beta-D-glucopyranoside (18), respectively, accompanied by the beta-linked isomers 22 and 19, respectively. Compounds 18, 21, and 22 were converted into the corresponding glycotriosyl donors which, upon coupling with (2S,3R,4E)-3-O-benzoyl-2-N-tetracosanoylsphingenine, afforded completely protected ganglioside analogs 39, 40, and 41, respectively. Deprotection of 40, 41, and 39 completed the synthesis of the modified ganglioside de-N-acetyl-GM3, a stereoisomer, and a regioisomer. The N-deprotected forms of 40 and 39, on successive treatment with methyl isocyanate and O-deprotection, gave the N-(N-methylcarbamoyl) analogs of GM3 and its regioisomer.

Carbohydrate Sequence↗

PubMed1992

A possible mechanism of the impairment of hepatic microsomal monooxygenase activities after multiple administration of propranolol in rats.

The mechanism of selective inhibition of propranolol hydroxylations after multiple administration of the drug was investigated by metabolic inhibition studies in rat liver microsomes. The time course of irreversible binding of a reactive metabolic intermediate(s) of propranolol to liver microsomal protein, which was proposed as the cause of the impairment of enzymatic activities, had a delayed phase followed by a rapid linear rise, while the unmetabolized propranolol remaining in the reaction mixture showed a rapid linear decrease immediately after the onset of incubation. Thus, it was conceivable that the reactive intermediate(s) was not always formed directly from the parent drug, propranolol. Among four primary metabolites of propranolol, 4-hydroxypropranolol was the most potent inhibitor of propranolol hydroxylase activities, and this inhibition was much enhanced by preincubation of 4-hydroxypropranolol with NADPH. The type of inhibition kinetics of propranolol 5- and 7-hydroxylase activities by 4-hydroxypropranolol was changed from a competitive type to a non-competitive type by the preincubation. These results suggest that a reactive metabolite(s) of propranolol which impaired propranolol hydroxylase activities is a further metabolite(s) of 4-hydroxypropranolol.

Animals↗

PubMed1992

Cytochrome P450 isozymes catalyzing 4-hydroxylation of parkinsonism-related compound 1,2,3,4-tetrahydroisoquinoline in rat liver microsomes.

Microsomal 4-hydroxylase of 1,2,3,4-tetrahydroisoquinoline (TIQ), a possible candidate for causing Parkinson disease, was characterized by using rat hepatic microsomes and purified P450 isozymes. Kinetic analysis revealed that Km and Vmax values (mean +/- SE) for hepatic microsomal TIQ 4-hydroxylase of male Wistar rats were 319.6 +/- 26.8 microM and 12.13 +/- 1.43 pmol.min-1.mg-1 protein, respectively. When TIQ 4-hydroxylase activity was compared in Wistar (an animal model of extensive debrisoquine metabolizers) and Dark Agouti (an animal model of poor debrisoquine metabolizers) rats, significant strain (Wistar greater than Dark Agouti) and sex (male greater than female) differences were observed. The microsomal activity toward TIQ 4-hydroxylation was increased by pretreatment of male Wistar rats with P448 inducers (beta-naphthoflavone and sudan I), but not with phenobarbital. Pretreatment with propranolol, an inhibitor of P450 isozymes belonging to the P450 IID gene subfamily, decreased TIQ 4-hydroxylase activity. P450 BTL, a P450 isozyme belonging to the IID subfamily, showed TIQ 4-hydroxylase activity of 64.1 pmol.min-1.nmol P450(-1), which was 3.2-fold that of microsomes (20.9 pmol.min-1.nmol P450(-1)). Antibody (IgG) against this isozyme suppressed microsomal TIQ 4-hydroxylase activity concentration-dependently. A male-specific P450 ml (P450IIC11) catalyzed this reaction to a much lesser extent (10.0 pmol.min-1.nmol P450(-1)), and its antibody did not affect the microsomal activity. These results suggest that TIQ 4-hydroxylation in hepatic microsomes are catalyzed predominantly by a P450 isozyme (or isozymes) belonging to the IID gene subfamily in non-treated rats and its immunochemically related P450 isozyme (or isozymes), and that a P450 isozyme (or isozymes) belonging to the IA subfamily also participates in TIQ 4-hydroxylation in rats pretreated with P448-inducers.

Amino Acid Sequence↗

PubMed1992

Loss of heterozygosity on the long arm of chromosome 22 in pheochromocytoma.

To identify the putative common deleted region on the long arm of chromosome 22 in pheochromocytoma, restriction fragment length polymorphism analysis was performed in 17 pheochromocytomas. All cases were heterozygous for at least one of the eight marker loci on 22q. Loss of heterozygosity (LOH) was observed in nine pheochromocytomas, of which eight were hereditary and one nonhereditary. Three pheochromocytomas had interstitial deletions that enabled us to localize the commonly deleted region as distal to D22S10 and proximal to D22S22. Hereditary pheochromocytoma frequently occurs in association with medullary thyroid carcinoma (MTC). Therefore, we also studied allelic loss on 22q in 23 hereditary MTCs. Only one of the MTCs showed LOH on 22q. Recent studies have mapped tumor suppressor loci associated with meningioma and neurofibromatosis type 2 (NF2) to 22q. The commonly deleted region in pheochromocytoma found by us encompasses the regions to which tumor suppressor genes associated with NF2 and meningioma have been mapped. The exact role of the pheochromocytoma tumor suppressor gene on 22q and its relationship to the suppressor genes involved in NF2 and meningioma remain unknown.

Adrenal Gland Neoplasms↗

PubMed1992

3-D observation of actin filaments during cardiac myofibrinogenesis in chick embryo using a confocal laser scanning microscope.

Using a confocal laser scanning microscope (CLSM), we observed subcellular three-dimensional (3-D) arrangements of actin filaments stained with fluorescein-labeled phalloidin during myofibrinogenesis of chick embryonic heart (7- to 13-somite stages). Serial optical tomograms were obtained from whole-mounted heart tubes and reconstructed into stereoscopic images. Development of myofibrils in myocardial differentiation considerably differed in inner and outer myocardial cell layers. In the outer layer, initial myofibrils appeared along cell membranes at the 8-somite stage. They increased rapidly and constituted network structures with spatial extension over cell-cell junctions. In the inner layer, myofibrils appeared at the bottom, facing the cardiac jelly, at the 10-somite stage, and, when the straight heart tube began to bend, they were already aligned circumferentially in the direction of the heart tube. Double staining of fluorescein-phalloidin and DiI [1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate; DiI-C18-(3)] of the looped heart revealed that while myocytes in the outer layer were round, those of the inner layer were spindle-shaped, and their long axes coincided with the circumferential direction. These results suggest that the circumferentially arranged myofibrils at the bottom of the inner layer may play an important role in the looping of the heart tube.

Actins↗

PubMed1992

Histochemical study on the maturation of human megakaryocytes using microfluorometry.

A microcytofluorometrical DNA measurement was basically studied and was applied to single megakaryocytes previously identified on a Wright-Giemsa stained smear. The smear was first photographed and the location of each megakaryocyte was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained with 4',6-diamidino-2-phenylindole (DAPI) reagent (pH 7.4) at 4 degrees C. Nuclear blue fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA with the coefficient of variation (CV) of 3.6% when stained for 30 min. After 30 min DAPI staining, the DNA measurement was microcytofluorometrically performed in single megakaryocytes which had been morphologically classified into 4 groups on the basis of cytoplasmic maturation, Bessis' classification, assessed on Wright-Giemsa-stained bone-marrow smears from normal human beings. The histograms of the cells did not show any difference in DNA ploidy distribution among the classes: that is, the DNA histograms disclosed ploidy distribution from 4 N to 64 N with the largest population of 16 N. These findings suggest that nuclear DNA synthesis is completed before platelet production starts. This method is useful for comparing the morphological features and DNA content of single megakaryocytes.

Bone Marrow Cells↗

PubMed1992

Management of giant serpentine aneurysms of the middle cerebral artery--review of literature and report of a case successfully treated by STA-MCA anastomosis only.

Thirty-eight cases of giant serpentine aneurysms (GSA), including 17 GSA of the middle cerebral artery (MCA), were reviewed in the literature. The treatment possibilities of GSA of the MCA are discussed together with our own case who was a 39-year-old male with a GSA of the right MCA and was treated only by STA-MCA anastomosis. The pathogenetic mechanism of progressive enlargement of the aneurysm is also discussed.

Adult↗

PubMed1992

Quality of life after total proctocolectomy and ileal J-pouch-anal anastomosis.

We investigated the daily life and opinions of patients after total proctocolectomy and ileal J-pouch-anal anastomosis. Eighty patients answered a questionnaire, completed Cattell's anxiety scale, and recorded bowel function for one month. Both colitis and polyposis patients defecated five times daily, had 1.6 episodes of nocturnal motions weekly, and soiled twice weekly. However, colitis patients were more satisfied with their bowel function and surgical outcome than were polyposis patients. This difference had a close relationship to the personality factors of ego strength and frustration. All patients with either disease who defecated less than three times daily, had no nocturnal motions, and had no soiling showed normal ego strength and frustration and were completely satisfied with their bowel status and operation. In contrast, patients defecating more than seven times daily or more than once per week nocturnally and soiling more than four times weekly had high frustration and were dissatisfied. Thus, satisfaction with surgery correlated not only with the objective outcome but also with personality and lifestyle.

Adenomatous Polyposis Coli↗

PubMed1992

Steroid complications in patients with ulcerative colitis.

Physicians treating patients with ulcerative colitis are confronted with the difficult task of deciding whether medical or surgical treatment is best for their patients. There are no definitive criteria to indicate when medical therapy should be exchanged for definitive surgery. Even in patients who respond well to glucocorticoid treatment, the side effects of these drugs may necessitate surgery. We reviewed the steroid complications of our operative cases retrospectively. Although ulcerative colitis was usually in remission, severe steroid complications were no longer tolerable and definitive surgery was required. We also reviewed the literature regarding the adverse effects of steroid. Because of advances in sphincter-preserving surgery, re-evaluation of the treatment of ulcerative colitis is necessary. Although conservative treatment remains the first choice, tolerance of irreversible side effects (especially in children) no longer seems to be justified. In such patients, early definitive surgery may offer more than it appears to sacrifice.

Adult↗

PubMed1992

Tumoricidal activity of Kupffer cells augmented by anticancer drugs.

The effect of Mitomycin-C (MMC) and Adriamycin (ADM) on the antitumor-associated function of Kupffer cells was examined. MMC and ADM enhanced the production of superoxide by Kupffer cells in cultures at low concentrations likely to occur in clinical use. The expression of interleukin-2 receptor, Ia antigen and asialoGM1 antigen, measured by flowcytometry, was increased by contact with MMC. Growth inhibition of AH130, rat ascites hepatoma, and P815, murine mastocytoma, by Kupffer cells treated with anticancer drugs was greater than that by Kupffer cells alone or anticancer agent alone. These results show that MMC and ADM activate Kupffer cells, leading to synergistic antitumor activity. The results suggest that some anticancer agents act through immunological mechanisms as well as through direct antineoplastic activity.

Animals↗

PubMed1992

Effect of PGE2 on interleukin-1 and superoxide release from primary-cultured human hepatic macrophages.

In order to learn more about how human hepatic macrophages function, we analyzed the effect of exogenous PGE2 on the amounts of interleukin-1 (IL-1) and superoxide (O2-) released from primary-cultured human hepatic macrophages (HHM phi). When endogenous PGE2 production was blocked by indomethacin, exogenous PGE2 reduced IL-1 release from HHM phi in a dose-dependent manner, whereas it tended to increase O2- release from HHM phi. These results may suggest the probable contribution of PGE2 in regulating HHM phi mediator release in vivo.

Cells, Cultured↗

PubMed1992

Mutations in the envelope protein of Japanese encephalitis virus affect entry into cultured cells and virulence in mice.

The nucleotide sequences of the envelope protein of the Kamiyama 1 strain of Japanese encephalitis (JE) virus and a passaged mutant (Kamiyama 2 strain) were determined. Two amino acid differences, Ser-Phe at residue 364 and Asn-Ile at residue 367, distinguished Kamiyama 2 from Kamiyama 1. Six neutralization-resistant variants were selected from these two strains using a JE species-specific monoclonal antibody with neutralization and hemagglutination-inhibition reactivities. All variants had a single amino acid substitution at residue 52 and significantly reduced reactivity with other JE species-specific monoclonal antibodies. The variants derived from Kamiyama 2 strain showed reduced virulence in 3-week-old mice after peripheral inoculation but were as virulent as the parent virus when inoculated intracranially. These variants also showed altered early virus-cell interaction but not replication and reproduction in Vero cells. These findings indicate that the mutations at residues 52, 364, and 367 affect early virus-cell interaction in Vero cells and virulence in mice.

Amino Acid Sequence↗