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Biomedical subjects

N Yoshida

Publications and source records attributed to N Yoshida.

755 records · Page 42Linked to original sources
PubMedDate not supplied

Immunoaffinity purification of aromatase cytochrome P-450 from human placental microsomes, metabolic switching from aromatization to 1 beta and 2 beta-monohydroxylation, and recognition of aromatase isozymes.

Microsomal estrogen synthetase (aromatase) cytochrome P-450 was purified from fresh human placental microsomes by monoclonal anti-aromatase P-450 antibody-Sepharose 4B chromatography. The purified P-450 showed a single band of 55 kDa on SDS-polyacrylamide gel electrophoresis and the aromatase specific activity on reconstitution was 70 nmol/min/mg protein. The purified P-450 was stable with a t 1/2 of approximately 2 years on storage at -90 degrees C and showed Km = 43 nM for androstenedione aromatization. However, it was unstable under spectral measurement conditions in the presence of sodium dithionite and carbon monoxide and the carbon monoxide difference spectra showed a maximum at 450 nm and a specific content of 9.1 nmol of P-450/mg protein, giving a turnover number of approximately 7.7 per min for the purified aromatase. The one-step immunochemical purification method gave a 490-fold increase of specific activity with 55% yield of aromatase activity of the original microsomes. Analysis of androgen metabolism by the purified aromatase and an apparent large kinetic isotope effect found at the secondary positions when using [19(-3)H3, 4(-14)C] androgens revealed metabolic switching from the first 19-hydroxylation to 1 beta- and 2 beta- monohydroxylation by aromatase. Substrate specificity for [19(-3)H3]androstenedione and testosterone was indicated by differences in the extent of metabolic switching (18% and 30%) and in the 2 beta/1 beta ratio (60/40 and 10/90, respectively). The mouse monoclonal antibody used for immunoaffinity purification suppresses aromatase activity of human placenta, but was totally ineffective for aromatase in goldfish brain and rat ovary. Rabbit polyclonal antibodies to human placental aromatase P-450 suppressed both human placental and rat ovarian aromatase but were ineffective for goldfish brain aromatase. The study indicates that they are isozymes of aromatase based on different structures of P-450.

Androstenedione↗
PubMedDate not supplied

Effects of stress on growth of transplanted hepatic tumours and immune responses.

Growth of transplanted hepatic tumours (T-9) was enhanced in immune rats under stress, compared with immune rats in an unstressed condition. Compared with unstressed immune rats, killer activity of mononuclear cells infiltrating the tumours against T-9 cells was significantly reduced in stressed immune rats. In contrast, killer activity of splenocytes obtained from stressed immune rats against T-9 cells was elevated compared with that from unstressed immune rats. In addition, natural killer cell activity of mononuclear cells infiltrating the tumours obtained from stressed immune rats was significantly reduced compared with that from unstressed immune rats. Cell populations infiltrating tumour tissues were identified by flow cytometric analysis. The percentage of CD8+ cells in mononuclear cells isolated from tumour tissues of stressed immune rats was reduced compared with that of unstressed immune rats. Furthermore, interleukin-2 responsiveness of splenocytes was suppressed in stressed immune rats, whereas T cell function as reflected by phytohaemagglutinin- or Concanavalin A-reactivity was unaffected by stress. Collectively, it is likely that stress suppressed the generation of cytotoxic cells from the spleen cells of immune rats.

Animals↗
PubMedDate not supplied

Heterologous expression of a Trypanosoma cruzi surface glycoprotein (gp82) in mammalian cells indicates the existence of different signal sequence requirements and processing.

Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82 kDa surface glycoprotein (gp82) that has been implicated in the mammalian cell invasion. When the non-infective epimastigote stage of the parasite was transfected with a vector containing the gp82 gene, an 82 kDa surface glycoprotein, which was indistinguishable from the metacyclic stage protein, was expressed. In contrast, when the same gene was expressed in transfected mammalian cells, although a large amount of protein was produced, it was not imported into the endoplasmic reticulum and glycosylated. This blockage in targeting and processing could be partially compensated for by the addition of a virus haemagglutinin signal peptide to the amino terminus of gp82. Thus, the requirements for membrane protein processing are distinct in mammals and T. cruzi, and an intrinsic feature of the gp82 prevents subsequent sorting to the mammalian cell surface. These results could be useful in the development of new DNA vaccines against T. cruzi employing parasite genes encoding immunodominant surface glycoproteins.

Amino Acid Sequence↗
PubMedDate not supplied

Telomerase activity and expression of telomerase RNA component and catalytic subunits in precancerous and cancerous colorectal lesions.

To investigate the role of telomerase activity in colorectal adenoma-carcinomas, telomerase activity, human telomerase RNA component (hTERC) and human telomerase reverse transcriptase (hTERT) mRNA were quantitatively analyzed in human cancerous and precancerous colorectal tissues. Sixty-six colorectal tumor specimens, including 10 invasive carcinomas, 6 mucosal carcinomas and 50 adenomas were evaluated. Ten specimens of normal tissue were also included in the study. Telomerase activity was assayed by semiquantitative fluorescence using the TRAP-eze(TM) telomerase detection kit. Analysis of the expression of each telomerase subunit gene was performed by real-time PCR amplification. There was a positive correlation between histological atypia and telomerase activity (rho = 0.700, p < 0.0001), hTERT mRNA expression (rho = 0.603, p < 0.0001), and hTERC expression (rho = 0.290, p < 0.05). There was also a positive correlation between the levels of hTERT mRNA and telomerase activity (r = 0.455, p < 0.01). Significant differences in the levels of hTERT mRNA were shown between normal tissues and the adenomas (p < 0.05) and between the mucosal carcinomas and invasive carcinomas (p < 0.05). The values of hTERC expression in neoplastic tissues were significantly higher than in the normal tissues; however, there were no significant differences between the adenomas and the carcinomas. In summary, although upregulation of hTERC expression is an early event in adenoma development, hTERT mRNA expression is gradually upregulated during the adenoma-carcinoma sequence and may be a rate-limiting determinant of telomerase activity.

Adenocarcinoma, Mucinous↗
PubMedDate not supplied

Cellular immune response of humans to the circumsporozoite protein of Plasmodium vivax.

The cellular immune response to the circumsporozoite (CS) protein of Plasmodium vivax of individuals from malaria-endemic areas of Brazil was studied. We examined the in vitro proliferative response of the peripheral blood mononuclear cells (PBMC) of 22 individuals when stimulated with a CS recombinant protein (rPvCS-2) and two other synthetic peptides based on the sequence of the P. vivax CS protein. Seven of the individuals from malaria-endemic area displayed an antigen-specific in vitro proliferative response to the recombinant protein PvCS-2 and one out of 6, proliferative response to the peptide 308-320. In contrast, none of the individuals displayed a proliferative response when stimulated with the D/A peptide which represent some of the repeated units present in this CS protein. Our study, therefore, provides evidence for the presence, within the major surface antigen of P. vivax sporozoites, of epitopes capable to induce proliferation of human PBMC.

Adult↗
PubMedDate not supplied

Analysis of thrombopoietin and c-mpl expression in a child with essential thrombocythemia.

The pathogenesis of the increased number of megakaryocytes and thrombocytosis in essential thrombocythemia (ET) is still unknown. We examined the expression of c-mpl, a receptor of thrombopoietin (TPO), and its signaling molecules in a patient with ET. An 8-year-old girl showed a high platelet count and an increased number of bone marrow megakaryocytes. Neither chromosomal abnormalities nor myelofibrosis was observed. Following the diagnosis of ET, aspirin therapy was begun for the patient, with only modest improvement of symptoms. Her platelet count ranged from 1,200,000/microL to 2,200,000/microL for more than 2 years. In the analyses, the serum TPO level in the patient was 420 attomoles/mL (normal, 760 +/- 320). The level of c-mpl expression in bone marrow mononuclear cells was higher in the patient than in healthy children, while there was no difference in the level of c-mpl expression in CD34+ cells, indicating an expanded pool of megakaryocytic lineage cells. The level of Janus kinase 2 (Jak2) expression was lower in the patient than in a healthy child. These findings indicate that the signal pathway mediated by c-Mpl after binding to TPO may be impaired in ET. Further analysis is needed to clarify the mechanism underlying the development of thrombocytosis in ET patients.

Child↗
PubMedDate not supplied

TNF alpha, IL-1 beta and IL-6 production by peripheral blood monocytes in patients with renal cell carcinoma.

Renal cell carcinoma (RCC) has been shown to be immunologically more labile than other types of cancer. In this study, we examined tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) production of peripheral blood monocytes in 38 RCC patients. Monocytes were isolated from peripheral blood mononuclear cells by adherence to a plastic dish and cultured with lipopolysaccharide for 24 hours. The culture supernatant was obtained, and the production of TNF alpha, IL-1 beta and IL-6 was measured by ELISA. As a result, TNF alpha and IL-1 beta production was significantly higher in the high stage patients compared to the control subjects and low stage patients. When the patients were divided according to serum C-reactive protein (CRP), TNF alpha, IL-1 beta and IL-6 production was significantly higher in the CRP-positive patients compared to the control subjects and the CRP-negative patients. Overexpression of these cytokines may therefore induce a hypermetabolic status that may be a cause of malnutrition and cancer cachexia.

Blood Cell Count↗
PubMedDate not supplied

Differing effects of two nitric oxide synthase inhibitors on experimental colitis.

BACKGROUND/AIMS: The aim of this study was to investigate the effects of two inhibitors of nitric oxide synthase on a rat model of colitis. METHODOLOGY: Colitis was induced by administration of an enema containing trinitrobenzene sulfonic acid. This colitis was treated everyday for one week with NG-nitro-L-arginine (10 mg/kg, i.v.), which is a non-selective inhibitor of both constitutive nitric oxide synthase and inducible nitric oxide synthase, or aminoguanidine (10 mg/kg, i.v.) which is an inhibitor of inducible nitric oxide synthase. Exposure to the trinitrobenzene sulfonic acid enema inhibited the increase in body weight of rats, and markedly increased the colonic damage scores, wet weight, thiobarbituric acid-reactive substances and myeloperoxidase activity. RESULTS: The inhibition of weight increase caused by trinitrobenzene sulfonic acid was significantly reduced by aminoguanidine treatment, whereas weight loss tended to be aggravated by NG-nitro-L-arginine treatment. The increases in the colonic damage scores, wet weight, thiobarbituric acid-reactive substances and myeloperoxidase activity in trinitrobenzene sulfonic acid-colitis were significantly inhibited by aminoguanidine treatment, although they tended to be aggravated by NG-nitro-L-arginine treatment. CONCLUSIONS: These results suggest that an inhibitor of inducible nitric oxide synthase, but not of constitutive nitric oxide synthase, was effective in treating experimental colitis in rats.

Animals↗
PubMedDate not supplied

Cytotoxic and DNA damage-inducing activities of low molecular weight phenols from rhubarb.

Six new phenol (anthraquinone or stilbene) glycosides with an acyl group at 6-position of the glucopyranose moiety were isolated from rhubarb (the roots of Rheum palmatum) cultivated in Japan, together with 22 known compounds. Most of these compounds were evaluated for cytotoxic activity against tumor and normal cells and for induction of DNA damage by spore rec-assay. Among them, emodin and aloe-emodin showed higher cytotoxic activities against human oral squamous cell carcinoma (HSC-2) and salivary gland tumor (HSG) cell lines than against normal human gingival fibroblasts (HGF). Chrysophanol 8-O-beta-(6'-acetyl)glucopyranoside, 4-(4'-hydroxyphenyl)-2-butanone 4'-O-beta-D-(2"-O-galloyl-6"-O-cinnamoyl) glucopyranoside, and 6"-O-(4'''-hydroxybenzoyl) resveratroloside exhibited relatively higher cytotoxic activities against all these cells. The other glycosides of anthraquinone or stilbene showed weaker cytotoxic activity against these tumor cell lines, but may be considered as cancer chemopreventive agents. Spore rec-assay with a recombination deficient mutant of Bacillus subtilis M45 demonstrated the DNA damage-inducing activity of emodin and aloe-emodin 15-O-beta-D-glucopyranoside among, rhubarb phenols.

Bacillus subtilis↗
PubMedDate not supplied

AS-4370, a novel gastrokinetic agent free of dopamine D2 receptor antagonist properties.

The gastrokinetic effects of AS-4370, 4-amino-5-chloro-2-ethoxy-N-([4-(4-fluorobenzyl)-2-morpholinyl] methyl) benzamide citrate, were compared with those of metoclopramide in experimental animals. In rats, AS-4370 increased the gastric emptying of a semi-solid meal and of resin pellets at dose ranges of 0.03-30 mg/kg and 1-10 mg/kg p.o., respectively. The minimal effective doses were 3-10 times lower than those of metoclopramide. Gastric emptying, delayed by gastroduodenal surgical intervention, was improved with AS-4370 (0.3-3 mg/kg p.o.). In conscious dogs with strain gauges implanted, AS-4370 (0.5 and 1.0 mg/kg i.v.) enhanced antral and duodenal motility without affecting ileal and colonic motility, indicating a selective enhancing effect on upper gastrointestinal motility. AS-4370 (10(-7) - 3 x 10(-5) M) increased electrically evoked, cholinergically mediated contractions in isolated guinea-pig ileum. AS-4370 (3 mg/kg i.v.) was without effect on gastric acid secretion in anesthetized rats. Unlike metoclopramide, AS-4370 neither depressed the active avoidance response in mice nor the food-reinforced lever pressing response in rats, even at 100 mg/kg p.o. Moreover, AS-4370 (10(-4) M) showed no affinity for D2, alpha 1, alpha 2, 5-HT1 and 5-HT2 binding sites in rat brain synaptic membranes. These results suggest that AS-4370 is a new and potent gastrokinetic agent that lacks dopamine D2 receptor antagonist properties.

Animals↗
PubMedDate not supplied

Effect of metoclopramide and ranitidine on acetylcholine release from isolated rat stomach.

Metoclopramide and ranitidine (10(-6)-10(-4) M) enhanced the electrical field stimulation-evoked contractions of isolated rat fundus and increased the gastric emptying in conscious rats. The enhancement of the fundus contractions by metoclopramide and ranitidine was abolished by atropine, but not by yohimbine, hexamethonium, propranolol or methysergide. The electrical field stimulation-evoked [3H]outflow from rat fundus strips, which has been preincubated with [3H]choline, was reduced by tetrodotoxin (10(-6) M) or in calcium-free medium, and potentiated by 4-amino-pyridine (3 X 10(-4) M), an acetylcholine (ACh)-releasing agent. Metoclopramide and ranitidine (10(-6)-10(-4) M) did not increase the [3H]outflow from the strips, in spite of causing a significant enhancement of their contractile response. However, both agents caused an increase in the ratio of [3H]acetylcholine/[3H]choline released into the superfusate during electrical field-stimulation. In rat fundus homogenates, metoclopramide and ranitidine showed a significant cholinesterase inhibition. These results seem to cast a doubt on the generally held ACh release hypothesis for the action mechanism of metoclopramide on one hand, and suggest, on the other hand, that cholinesterase inhibition contributes to some extent to the gastrokinetic effects of metoclopramide and ranitidine.

Acetylcholine↗
PubMedDate not supplied

Analysis of 16,16-dimethylprostaglandin E2-induced diarrhea in cecectomized rats.

The 16,16-dimethylprostaglandin E2 (dmPGE2)-induced diarrhea was analyzed in cecectomized rats prepared by resecting the cecum and its vasculature without disturbing the ileocecal junction. dmPGE2 (0.1-1.0 mg/kg, p.o.) dose-dependently increased the number of defecation episodes and induced a soft and watery stool in cecectomized rats. At 0.3 mg/kg, the diarrhea-inducing effects of dmPGE2 were more pronounced in cecectomized than in control rats. When given i.p., dmPGE2 (0.3 mg/kg) induced a watery stool in cecectomized and control rats with the same efficacy, although these effects were short-lasting as compared to oral administration. Castor oil (4 ml/kg, p.o.) also induced diarrhea, but did not produce a watery stool in cecectomized rats. There were no differences between cecectomized and control rats in basal small intestinal transits or in dmPGE2 (0.3 mg/kg, p.o.)-induced enhancements. Moreover, the basal and dmPGE2-induced jejunal net fluid transfers were the same in cecectomized and in control rats. On the other hand, the enhanced secretion of colonic fluid by dmPGE2, given intraluminally, was only half of that in control rats, whereas the colonic transit-enhancing effect of dmPGE2 in cecectomized rats was more pronounced than in control rats at 15 but not at 30 min after its administration. The basal colonic fluid contents and transits were the same in cecectomized and in control rats. Loperamide and morphine (0.1 and 1.0 mg/kg, s.c.) inhibited the dmPGE2 (0.3 mg/kg, p.o.)-induced diarrhea in cecectomized rats. N-methyllevallorphan (5 mg/kg, s.c.) completely antagonized the inhibitory effect of loperamide and partly antagonized the effect of morphine. These results suggest that oral administration of dmPGE2 induces a more pronounced secretory diarrhea in cecectomized than in control rats, probably due to the lack of the reservoir function of the cecum in the operated animals. This secretory diarrhea model is suitable for evaluating the antidiarrheal activity of drugs.

16,16-Dimethylprostaglandin E2↗