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Biomedical subjects

T Takeuchi

Publications and source records attributed to T Takeuchi.

At least 541 records · Page 30Linked to original sources
PubMed1997

Renal primitive neuroectodermal tumor: a morphologic, cytogenetic, and molecular analysis with the establishment of two cultured cell lines.

We report two patients with renal primitive neuroectodermal tumor (PNET) in whom the diagnosis was established by both a cytogenetic and a molecular analysis. Histologically, both renal tumors were composed of uniform immature round cells with a positive immunoreactivity for O13 (p30/32 MIC2). The cytogenetic analysis with in situ hybridization (chromosome painting) demonstrated reciprocal translocation t(11;22)(q24;q12) specific to PNET in the cultured cells derived from each tumor. The reverse transcriptase-polymerase chain reaction (RT-PCR) in both tumors demonstrated EWS/ FLI-1 fusion transcripts, representing the molecular equivalent of t(11;22). A Southern blot analysis also confirmed EWS gene rearrangement in both renal tumors. In addition, the authors also established two new cell lines (designated as FU-RPNT-1 and FU-RPNT-2) from renal PNETs. When transplanted into athymic mice, FU-RPNT-1 and FU-RPNT-2 reproduced and maintained the morphologic and molecular characteristics of the original tumors. In conclusion, the detection of t(11;22) and EWS/FLI-1 fusion transcripts is considered to provide a novel adjunctive method for diagnosing renal PNET. These newly established cell lines thus may be used to investigate the biologic behavior related to renal PNETs.

Adult↗
PubMed1997

Modified formalin and methanol fixation methods for molecular biological and morphological analyses.

Several simplified fixation methods were examined to determine their suitability for both molecular biological analyses and morphological study. Fixation with 10% v/v formalin alone at 4 degrees C and containing 5 mmol/L ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) at room temperature preserved significantly more high-molecular-weight DNA than 10% v/v formalin fixation at room temperature. The morphological differences between tissues fixed using these modified formalin fixation methods and conventional 10% v/v formalin fixation were negligible. Of the dehydration fixatives tested, 100% methanol did not cause regional differences due to artificial tissue shrinkage and the morphology of sections prepared by methanol fixation was preserved consistently better than that of acetone- or ethanol-fixed sections. All three dehydration fixatives preserved relatively higher-molecular-weight DNA and RNA, compared with formalin. Cold formalin, formalin containing EDTA at room temperature and 100% methanol are recommended as standard and additional fixatives routine clinicopathological laboratory use.

Breast↗
PubMed1997

Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences.

Pseudomonas syringae pv. phaseolicola, which causes halo blight on various legumes, and pv. actinidiae, responsible for canker or leaf spot on actinidia plants, are known as phaseolotoxin producers, and the former possesses phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) which confers resistance to the toxin. We confirmed that the latter is also resistant to phaseolotoxin and possesses ROCT, and we compared the two pathovars by using sequence data of the ROCT gene and the intergenic spacer region located between the 16S and 23S rRNA genes (16S-23S spacer region) as an index. It was found that the identical ROCT gene (argK) is contained not only in bean isolates of P. syringae pv. phaseolicola in Mexico and the United States but also in bean isolates in Japan and Canada, and that it is also distributed in the kudzu (Pueraria lobata) isolates of P. syringae pv. phaseolicola. Moreover, the kiwifruit and tara vine isolates of P. syringae pv. actinidiae were also found to possess the identical argK. On the contrary, the 16S-23S spacer regions showed a significant level of sequence variation between P. syringae pv. actinidiae and pv. phaseolicola, suggesting that these two pathovars evolved differently from each other in the phylogenetic development. The fact that even synonymous substitution has not occurred in argK among these strains despite their extreme differences in phylogenetic evolution and geographical distribution suggests that it was only recently in evolutionary time that argK was transferred from its origin to P. syringae pv. actinidiae and/or pv. phaseolicola.

Base Sequence↗
PubMed1997

Preparation of a monoclonal antibody specific for Entamoeba dispar and its ability to distinguish E. dispar from E. histolytica.

A monoclonal antibody (MAb), MAb ED17 (immunoglobulin G2a [IgG2a]), prepared against trophozoites of Entamoeba dispar SAW1734RclAR cultured monoxenically with Crithidia fasciculata, reacted with 25 of 26 isolates of E. dispar by an indirect fluorescent-antibody test. In contrast, the MAb failed to react with any of 20 isolates of E. histolytica or other enteric protozoan parasites. Western blot (immunoblot) analysis showed that the molecular mass of the E. dispar antigen recognized by the MAb was 160 kDa under reduced conditions. Immunoelectron microscopy revealed that the antigen was mainly located on digested C. fasciculata, but not on undigested organisms. Double staining with a mixture of MAb ED17 and MAb 4G6 (an IgG1 MAb which reacts exclusively with E. histolytica), followed by incubation with a mixture of fluorescein isothiocyanate-labeled anti-mouse IgG2a and tetramethylrhodamine isothiocyanate-labeled anti-mouse IgG1 antibodies, simultaneously identified mixed populations of E. dispar and E. histolytica. This method may prove to be useful for the accurate identification of E. dispar and E. histolytica, even in mixed infections.

Animals↗
PubMed1997

Spontaneous establishment of an Epstein-Barr virus-infected fibroblast line from the synovial tissue of a rheumatoid arthritis patient.

An Epstein-Barr virus (EBV)-infected fibroblast line, designated DSEK, was spontaneously established from synovial tissue of a patient with rheumatoid arthritis (RA). DSEK cells expressed EBV nuclear antigens EBNA-1 and EBNA-2 and latent membrane protein LMP-1. Cell surface markers of DSEK cells were similar to those of EBV-negative fibroblast clones derived from synoviocytes and were negative for lymphocyte and macrophage markers. DSEK cells expressed CD44, CD58, and HLA-DR antigens and spontaneously produced interleukin-10 basic fibroblast growth factor and transforming growth factor beta1. These results indicate that rheumatoid synoviocytes can be a target for EBV infection and suggest that EBV may play a role in the pathogenesis of RA.

Arthritis, Rheumatoid↗
PubMed1997

Dietary diosgenin attenuates subacute intestinal inflammation associated with indomethacin in rats.

We investigated the effects of dietary diosgenin (Dio), a plant-derived sapogenin, on indomethacin (Indo)-induced intestinal inflammation and alterations in bile secretion in rats. In anesthetized rats, bile secretion, intestinal inflammation, and blood chemistry were assessed 3 days after two subcutaneous injections of Indo given 24 h apart. Dio (> 80 mg.kg-1.day-1) pretreatment significantly inhibited weight and food intake decreases and intestinal inflammation. This protective effect was confirmed by examination of gross and histological findings and intestinal myeloperoxidase activity. Dio significantly increased biliary cholesterol (Chol) output and prevented the decreases in bile flow, bile acid output, and biliary alpha-muricholic acid and the increases in biliary hyodeoxycholic acid, deoxycholic acid, and hydrophobicity index of bile. Significantly more biliary Chol and phospholipids were present in macromolecules separate from bile acids and Indo in Dio-treated rats. Dio significantly increased the elimination constant of Indo and reduced plasma Indo levels at 3 and 12 h but did not influence biliary secretion of Indo for 3.5 h after injection. Although Dio dose-dependently attenuated subacute intestinal inflammation and normalized bile secretion in this model, it may also compromise the anti-inflammatory action of indo.

Acute Disease↗
PubMed1997

Flow cytometric analysis of surface antigens on human conjunctival epithelial cells.

We analyzed the levels of expression of different surface adhesion molecules on normal human conjunctival epithelial cells with cytology and flow cytometry. The levels of beta 1 (CD29), VLA-1 (CD49a), VLA-2 (CD49b), and VLA-3 (CD49c) integrins were high, whereas those of VLA-5 (CD49e) and VLA-6 (CD49f) were faint, and that of VLA-4(CD49d) was undetectable. Among the beta 2 integrins, the levels of Mac-1 (CD11b) and p150.25(CD11c) were positive, while LFA-1 (CD11a) was not detectable. HLA class I and class II were also expressed. However, CR2 (CD21), CD57, CD44 and ICAM-1 (CD54) were not detected. Our study suggests that these techniques may aid in identifying changes in the expression levels of various surface antigens on conjunctival epithelial cells that could occur in ocular surface disease.

Adult↗
PubMed1997

[Inhibitory neuronal control of smooth muscle activity of the gastrointestinal tract].

Recent findings suggest that nonadrenergic inhibitory responses of the smooth muscle of the gastrointestinal tract is mediated by nitric oxide and some intestinal peptides including VIP, PACAP and CGRP. Although nitric oxide was suggested to mediate the nonadrenergic relaxation in various regions of gastrointestinal tracts of many species, further careful studies revealed that nitric oxide participates in the relaxation in restricted regions, not throughout the tract. It was also found that the peptides work in extremely restricted regions of the tract. Importance of the role of nitric oxide in the relaxation varies with different regions, strains and species. Moreover, it significantly decreases with the age of the rat, especially between 4-8 weeks of age. Inhibitors of Ca(2+)-activated K+ channels inhibited the relaxation in almost all regions examined in rats, although the magnitude of the inhibition varied from region to region. The intracellular action mechanism(s) of nitric oxide was discussed in relation to changes in cyclic GMP level, intracellular Ca2+ level and membrane potentials of the smooth muscle cells.

Aging↗
PubMed1997

Nonadrenergic, noncholinergic relaxation in longitudinal muscle of rat jejunum.

The mediators of nonadrenergic, noncholinergic (NANC) relaxation in the longitudinal muscle of rat jejunum were studied in vitro. Electrical field stimulation (EFS) of segments of rat jejunum induced a rapid transient relaxation followed by a subsequent contraction in the presence of atropine and guanethidine. NG-Nitro-L-arginine (L-NOARG, 10 microM) inhibited the EFS-induced NANC relaxation by about 25%, and L-arginine (1 mM) completely reversed this inhibition. Exogenously added nitric oxide (0.1-10 microM) induced relaxation of the segment. Treatment of the segment with alpha-chymotrypsin resulted in about 50% inhibition of the EFS-induced relaxation. Several peptide candidates for the mediator of NANC relaxation were examined by using selective antagonists of their receptors or by a receptor-desensitization method. Results indicated that vasoactive intestinal peptide, pituitary adenylate cyclase activating peptide, peptide histidine isoleucine, atrial natriuretic peptide and neurotensin are not associated with NANC relaxation of the segments. On the other hand, apamin at 1 microM inhibited the EFS-induced relaxation by 74%. Inhibitory effects of L-NOARG and, apamin or alpha-chymotrypsin treatment on the EFS-induced relaxation were additive and almost complete. Exogenous nitric oxide-induced relaxation was not affected by apamin. Inhibitory junction potentials (i.j.p.'s) were recorded from longitudinal muscle cells of rat jejunum. Apamin at 200 nM abolished i.j.p.'s induced by two pulses of EFS. These results suggest that NANC relaxation in longitudinal muscle of rat jejunum involves two independent components: one is a nitric oxide-mediated minor component, and the other is an unknown substance-mediated apamin-sensitive major component that is inhibited by alpha-chymotrypsin treatment.

Adrenergic Fibers↗
PubMed1997

Muscarinic autoinhibition and modulatory role of protein kinase C in acetylcholine release from the myenteric plexus of guinea pig ileum.

The modulatory role of protein kinase C on phospholipase A2, activation of which had been suggested to result in acetylcholine release from cholinergic neurons, was studied in longitudinal muscle preparations with the myenteric plexus of guinea pig ileum. The relationship of muscarinic autoinhibition to the modulation was also examined. Phorbol-12,13-dibutyrate (PDBu), an activator of protein kinase C, dose-dependently increased spontaneous and electrical field stimulation-induced acetylcholine releases from the preparation. The inhibitors of protein kinase C, staurosporine and calphostin C, inhibited the stimulatory effects of PDBu, but neither inhibitor affected spontaneous or electrical field stimulation-induced acetylcholine release in the absence of PDBu. On the other hand, atropine significantly increased electrical field stimulation-induced release by blocking a muscarinic autoinhibitory mechanism. Under the auto-inhibition blocked condition, U73122, an inhibitor of phospholipase C, and staurosporine significantly inhibited the effect of atropine on electrical field stimulation-induced release. An inhibitor of phospholipase A2, mepacrine, inhibited PDBu-induced acetylcholine release and also inhibited the effect of atropine on electrical field stimulation-induced release. An activator of phospholipase A2, melittin, and a product of the phospholipase, arachidonic acid, increased the spontaneous and electrical field stimulation-induced releases. These results suggest that the phospholipase C-protein kinase C system modulates acetylcholine release from cholinergic neurons by activating phospholipase A2 in the myenteric plexus of guinea pig ileum, and the activation of muscarinic autoreceptor may negatively modulate acetylcholine release at a point upstream of the system.

Acetylcholine↗
PubMed1997

Inhibitory effect of alginic acids on hyaluronidase and on histamine release from mast cells.

The effects of various types of alginic acid consisting of L-guluronic acids (G) and D-mannuronic acids (M) on hyaluronidase and mast cell degranulation were examined. Alginic acid with an M/G ratio of 1.0 exhibited the strongest inhibition of both activities, the higher molecular weight alginic acids of 150 to 370 kDa being preferable in both cases. Esterification of the carboxyl residue enhanced the latter activity.

Alginates↗
PubMed1997

Purification and characterization of extracellular poly(beta-D-1,4-mannuronide) lyase from Dendryphiella salina IFO 32139.

An extracellular endo poly(beta-D-1,4-mannuronide) lyase of Dendryphiella salina IF 32139 was purified to homogeneity by Q Sepharose FF and Sephacryl S-200 HR column chromatographies. The purified enzyme had a molecular weight of 35,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 3.65 by isoelectric focusing. The optimum pH and temperature for enzyme activity were pH 5.0 and 45 degrees C, respectively. The enzyme was stable from pH 4 to 10 and at temperature below 40 degrees C. Some divalent cations, Ca2+, Mn2+, and Zn2+, increased the enzyme activity. Hg2+ and NBS strongly inhibited the activity. This enzyme susceptibly degraded poly-M, produced a wide range of 4,5-unsaturated oligomannuronic acids, and further degraded these unsaturated oligomannuronic acids to produce the unsaturated monomer and dimer as final products.

Amino Acid Sequence↗
PubMed1997

Characterization of antimuscarinic effect of cimetropium bromide in guinea pig ileum.

Pharmacological characteristics of cimetropium bromide (cimetropium), a muscarinic receptor antagonist, were studied in longitudinal muscle preparations with myenteric plexus of guinea-pig ileum. Cimetropium was shown to have more potent antimuscarinic effect than butylscopolamine in inhibition of contraction of the preparations. Interestingly, when the inhibitory effects of cimetropium were compared in respect of relative potency to atropine between its effects on electrical field stimulation or nicotine-, and exogenous ACh-induced contraction, it has a more potent effect on the former contraction than that on the latter one. In the superfusion experiments of the preparation which had been preloaded with labelled choline, cimetropium decreased the labelled ACh release induced by electrical field stimulation under the muscarinic autoinhibition blocked-condition. From these findings, two pharmacologically characteristic effects of cimetropium in addition to post-synaptic muscarinic receptor antagonism were suggested: one is a weak effect on muscarinic autoreceptors in comparison to atropine and the other is an inhibitory effect on the ACh release.

Acetylcholine↗
PubMed1997

Cryofibrinogenemia with polyarthralgia, Raynaud's phenomenon and acral ulcer in a patient with Graves' disease treated with methimazole.

Cryofibrinogenemia is a cryopathy in which hypersensitivity to cold is a prominent feature. Cryofibrinogenemia developed in an 18-year-old Japanese female patient during methimazole therapy for Graves' disease. She developed cryopathy (livedo reticularis, Raynaud's phenomenon and acral ulcer) and polyarthralgia during methimazole therapy, and we detected cryofibrinogen in her plasma. Her symptoms resolved after administration of prostaglandins and anticoagulants. Several reports indicate that methimazole therapy induces autoantibody-related disease. In the present case, we cannot exclude the possibility that methimazole therapy contributed to the cryofibrinogenemia.

Adolescent↗
PubMed1997

Proprotein-processing endoprotease furin controls growth of pancreatic beta-cells.

We have previously reported that in the well-differentiated beta-cell line MIN6 cells, the beta-cell-specific differentiated characteristics, such as insulin content, expression of prohormone convertases PC2 and PC3, and glucose-regulated insulin secretion, diminished when the proprotein-processing endoprotease furin was highly expressed. Since furin converts many growth-related protein precursors to their bioactive forms, we compared the four pancreatic islet cell lines RINm5F, betaTC3, betaHC9, and MIN6 with respect to cell growth rate, furin expression, endoprotease activity, and insulin content. RINm5F cells exhibited the strongest furin expression, higher furin-type endoprotease activity, and the fastest cell growth, but had the least insulin content. In contrast, MIN6 cells exhibited only a weak furin expression, little furin-type endoprotease activity, and the slowest cell growth, but had the highest insulin content. To test whether furin-expressing cells secrete growth-promoting factors cleaved by furin, we prepared conditioned media from RINm5F and furin cDNA-introduced MIN6 (MIN6-F) cells. The conditioned media from RINm5F and MIN6-F induced increased DNA synthesis and promoted the growth of normal MIN6 cells, compared with the medium from the empty vector-introduced MIN6-0 cells. We then examined the effect of the protease inhibitors alpha1-antitrypsin and its variants by infecting their vaccinia recombinants to the four cell lines. All conditioned media from each cell line expressing the furin-specific alpha1-antitrypsin variant exhibited the least DNA synthetic capacity on normal MIN6 cells. Furthermore, all three sublines of MIN6-F grew faster than MIN6-0 and MIN6. Thus, we suggest that the islet cells with higher furin expression may induce increased production of growth factors, which result in an increase in cell growth, through an autocrine/paracrine mechanism.

Animals↗
PubMed1997

Identification of mutations in the hepatocyte nuclear factor (HNF)-1 alpha gene in Japanese subjects with IDDM.

One form of maturity-onset diabetes of the young, MODY3, is characterized by a severe insulin secretory defect, compared with MODY2, a glucokinase-deficient diabetes. It has recently been shown that mutations of the gene encoding the transcription factor hepatocyte nuclear factor (HNF)-1 alpha cause MODY3. Because of the rapid progress to overt diabetes and the high prevalence of required insulin treatment in patients with MODY3, we screened the HNF-1 alpha gene for mutations in Japanese subjects with IDDM. Ten exons and flanking introns of the HNF-1 alpha gene in these subjects were amplified by polymerase chain reaction and direct sequencing of the products. Mutations were identified in three (5.5%) of the 55 unrelated subjects with IDDM. A missense mutation of R272H (replacement of Arg by His in codon 272) in the DNA binding domain of HNF-1 alpha was found in a subject who developed IDDM 1 year after diagnosis of NIDDM at 8 years of age. A frameshift mutation of P291 fsinsC (insertion of a C in a polyC tract around codon 291 for Pro), which would generate a mutant truncated protein of 340 amino acids, was found in a subject who started insulin treatment when hyperglycemia and ketonuria were noticed at 13 years of age. A missense mutation of R583G (replacement of Arg by Gly in codon 583) in the transactivation domain of HNF-1 alpha was found in a subject with sudden-onset IDDM at 20 years of age. None of these mutations were present in 100 nondiabetic subjects (200 normal chromosomes). These results indicate that the HNF-1 alpha gene defects could lead to the development of not only early-onset NIDDM but also IDDM, implicating the importance of subclassification of HNF-1 alpha-deficient IDDM from a classical type of autoimmune-based IDDM in Japanese.

Adolescent↗
PubMed1997

Effect of growth hormone suppression on the serum levels of ovarian and adrenal sex steroid hormones in normal women and in women with polycystic ovary syndrome.

We investigated the acute effect on the serum levels of ovarian and adrenal sex steroid hormones of the suppression of growth hormone during oral glucose tolerance test (OGTT). A standard 75 g OGTT was performed in 11 healthy women and eight women with polycystic ovary syndrome (PCOS). Another five controls were given a sham loading of oral distilled water. Blood samples were obtained immediately before and at 30-minute intervals after glucose or sham loading. Significant progressive declines in testosterone, estradiol, dehydroepiandrosterone sulfate (DHEAS) and growth hormone levels were observed during OGTT in both groups. In the PCOS and normal groups, respectively, at 120 min, testosterone levels were 75.8% and 64.4% of the baseline (0-time) value, estradiol levels were 83.4% and 83.1%, DHEAS levels were 79.3% and 79.1%, and growth hormone levels were 33.9% and 21.2%. Significant positive correlations were observed between the level of growth hormone and each of the testosterone, estradiol and DHEAS levels in both the groups. The area under the curve for growth hormone was significantly smaller in the PCOS group than in the normal group. Gonadotropins were not changed at any time during OGTT. It appears that growth hormone may modify ovarian and adrenal sex steroidogenic responses to tropic hormones directly or via local insulin-like growth factor-I. Women with PCOS may be relatively deficient in growth hormone, a deficiency which may play a role in the pathophysiology of ovulatory disturbance.

Adult↗