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Biomedical subjects

H Ueno

Publications and source records attributed to H Ueno.

At least 343 records · Page 19Linked to original sources
PubMed1994

Comparison of the inhibitory action of natural rotenone and its stereoisomers with various NADH-ubiquinone reductases.

Two stereoisomers of natural rotenone (5'alpha-epirotenone and 5'beta-epirotenone) were synthesized to identify the stereochemical factor of rotenone required for the inhibition and also to probe the structure of the rotenone binding site. The inhibitory action of the stereoisomers was compared with that of rotenone using NADH-ubiquinone reductases from bovine heart submitochondrial particles (SMP), potato tubers (Solanum tuberosum L.) SMP and Escherichia coli (GR19N) membranes. With respect to bovine heart SMP, it was found that the bent form of rotenone is essential for the activity. The modification of the E-ring moiety also affected both the inhibitory potency and the pattern of inhibition. These results indicated that the rotenone-binding site recognizes the whole molecular structure (or shape) of rotenone in a strict sense. Rotenone and 5'beta-epirotenone inhibited the NADH-ubiquinone reductase of bovine heart SMP in a noncompetitive manner against exogenous quinones. In contrast, the inhibition pattern of 5'alpha-epirotenone varied from noncompetitive to competitive as the concentration of quinone increased. These results suggest that rotenone binds close to, but not at a site identical to, the location for ubiquinone in the ubiquinone-catalytic reaction site, whereas the 5'alpha-epirotenone-binding site overlaps that for ubiquinone due to a structural modification of E-ring moiety. Furthermore, the complex inhibition pattern of 5'alpha-epirotenone suggests that there are two quinone-binding sites in NADH-ubiquinone reductase. In contrast, the order of the inhibitory potencies of the three inhibitors with proton-pumping NADH-ubiquinone reductase of potato SMP was the same as that observed for the bovine enzyme. This suggests that the structure of rotenone-binding sites (or ubiquinone-binding sites) of these enzymes are similar. It was further demonstrated that 5'alpha-epirotenone inhibits quinone binding to both proton-pumping and non-proton-pumping NADH-ubiquinone reductases of potato SMP in a competitive manner. With respect to the proton-pumping NADH-ubiquinone reductase of the E. coli membrane, the sensitivity of the enzyme to the inhibitor was remarkably decreased and the difference in the inhibitory potencies of the three inhibitors became ambiguous. In addition, the inhibition pattern of the three inhibitors was competitive against quinone. These results indicated that, contrary to the mammalian enzyme, only part of the rotenone molecule is recognized by the quinone-binding site of this enzyme.

Animals↗
PubMed1994

[The measurement of insulin antibodies and insulin autoantibodies by enzyme-linked immunosorbent assay using recombinant human insulin antigen and its clinical application].

Insulin antibodies (IA) are detectable in the sera of most insulin-treated patients with diabetes mellitus. Antibodies to exogenous insulin sometimes cause clinical symptoms of insulin resistance, allergy, and local lipoatrophy. Although the frequency of these complications has diminished with the use of highly purified porcine insulin or recombinant human insulin, there are some patients with high titer of IA. Autoantibodies to insulin (IAA) are also described. IAA has been reported to be in association with both insulin-dependent diabetes mellitus (IDDM) and polyendocrine autoimmune disease. For many years these antibodies have been measured by radiobinding assay (RBA) in which the complexes are precipitated non-specifically by polyethylene glycol. In the present study we developed a rapid and quantitative enzyme-linked immunosorbent assay (ELISA) method for measuring IA and IAA using recombinant human insulin antigen. We applied this method to the samples obtained from patients with diabetes mellitus and autoimmune thyroid disease and then compared the results with those obtained from the RBA method. The calibration curve for ELISA was derived from the dilution curve of a single serum from a patient positive for insulin antibody, and the results were expressed arbitrarily as ELISA UNIT. The calibration curve was approximately linear on the log-log scale within the range of 0.1-2.0 at optical density (OD)450nm, (6.25-200 ELISA UNIT). The intra-assay (CV = 2.3-3.1%) and inter-assay (CV = 2.8-7.2%) precisions were acceptable. Recovery rate varied from 74.5% to 118.5% and dilution experiments showed good linearity. Specificity was demonstrated by substituting purified human IgG for the test serum and glucagon for insulin. Except for hemoglobin, coexisting substances in serum had almost no effect on ELISA. The range of ELISA UNIT (Mean +/- SD) of 83 normal sera was 12.7 +/- 4.6. Positivity for IA by ELISA (> normal Mean + 3SD) was 11 out of 58 (19.0%) and 26 out of 55 (47.3%) in patients with IDDM and with non-insulin-dependent diabetes mellitus (NIDDM) who were treated with insulin, respectively. Positivity for IAA by ELISA was 5 out of 173 (2.8%) and 1 out of 20 (5.0%) in patients with NIDDM without insulin therapy and hyperthyroidism due to Graves' disease, respectively. However, by RBA, we detected 4 other cases positive for IAA in NIDDM without insulin therapy and one case in Graves' disease. The present study demonstrates that the newly developed method of ELISA using recombinant human insulin antigen is clinically useful for measuring IA and IAA.

Antigens↗
PubMed1994

Total synthesis of taxol.

Taxol, a substance originally isolated from the Pacific yew tree (Taxus brevifolia) more than two decades ago, has recently been approved for the clinical treatment of cancer patients. Hailed as having provided one of the most significant advances in cancer therapy, this molecule exerts its anticancer activity by inhibiting mitosis through enhancement of the polymerization of tubulin and consequent stabilization of microtubules. The scarcity of taxol and the ecological impact of harvesting it have prompted extension searches for alternative sources including semisynthesis, cellular culture production and chemical synthesis. The latter has been attempted for almost two decades, but these attempts have been thwarted by the magnitude of the synthetic challenge. Here we report the total synthesis of taxol by a convergent strategy, which opens a chemical pathway for the production of both the natural product itself and a variety of designed taxoids.

Models, Molecular↗
PubMed1994

Search for accumulation of p53 protein and detection of human papillomavirus genomes in sebaceous gland carcinoma of the eyelid.

Twenty-one Japanese patients with sebaceous carcinoma of the eyelid were investigated for tumour incorporation of human papillomavirus (HPV) types-6, 11, 16, 18, 31, and/or 33 DNA by in situ hybridization with fluorescein isothiocyanate-labelled DNA probes, and for p53 protein accumulation by immunohistochemical analysis with an antibody to p53 protein. Thirteen tumours (61.9%), including 9 cases of multiple infections, were positive for HPV DNA. Positive signal in the nucleus was observed not only in the cancer cells, but also in the cells of surrounding normal sebaceous glands and epidermis. Positive nuclear staining of cancer cells with the antibody to p53 protein was detected in 12 cases (57.1%). p53 protein accumulation was more frequently observed in the clinically advanced cases, occasionally in association with recurrence and/or metastasis. Among the 12 p53-positive cases, 7 were also positive for the presence of HPV DNA. HPV infections exist in a high percentage of sebaceous carcinomas of the eyelid in Japan; the overexpression of p53 protein may be important in both carcinogenesis and progression.

Aged↗
PubMed1994

Transient neutropenia after intravenous injection of vindesine in patients with lung cancer.

We have found transient circulating neutropenia and pulmonary sequestration of neutrophils after the intravenous injection of vindesine, a microtubule disruptor. Experiment 1 Ten patients with lung cancer were given a bolus intravenous injection of 3 mg.m-2 vindesine (Fildesine(r)). In all patients, total leukocyte and neutrophil counts in the venous blood fell to 65% and 47% of baseline values respectively within 30 min, and returned to baseline values within 6 h. In contrast, the lymphocyte count was stable. Experiment 2 Male Wistar rats were given saline or 0.08 mg.kg-1 vindesine intravenously and were sacrificed after 30 min. Vindesine produced a 58% reduction in the neutrophil count in the systemic circulation and a threefold increase in the neutrophil/erythrocyte ratio in the pulmonary microvasculature. Experiment 3 We studied the effects of vindesine in vitro on neutrophils and lymphocytes isolated from the venous blood of healthy volunteers. Vindesine (10(-5)-10(-8) mol.l-1) reduced neutrophil deformability (filterability) and induced neutrophil polarization, with reversibility of both effects after washout. These effects of vindesine were completely inhibited by cytochalasin B, an actin filament disrupter. Vindesine did not stimulate the neutrophil functions of adherence to polystyrene tubes, chemotaxis, or superoxide anion generation. The filterability and morphology of lymphocytes were not altered by vindesine. Thus, we conclude that a bolus injection of vindesine produces pulmonary sequestration of neutrophils, which produces circulatory neutropenia, and that it is primarily mediated by a decrease in neutrophil deformability that occurs without activation of the cells.

Animals↗
PubMed1994

Imaging features of maxillary osteoblastoma and its malignant transformation.

We report two cases of osteoblastoma, one of them an unusual case in a 32-year-old woman in whom a maxillary tumor was confidently diagnosed as an osteoblastoma at the time of primary excision and subsequently transformed into an osteosarcoma 7 years after the onset of clinical symptoms. The other patient developed osteosarcoma arising in the maxilla, which was diagnosed 3 years after the primary excision and is very suggestive of malignant transformation in osteoblastoma. We present the radiological features, including computed tomographic and magnetic resonance imaging studies, of this unusual event of transformed tumor and compare imaging features of benign and dedifferentiated counterparts of this rare tumor complex.

Adult↗
PubMed1994

Percutaneous transgastric intestinal decompression: the management of malignant bowel obstruction without nasal intubation.

We inserted a long intestinal tube via the percutaneous transgastric route in three patients with an inoperable malignant bowel obstruction. The percutaneous endoscopic gastrostomy technique facilitated this method. The main purpose of our procedure is to achieve direct intestinal decompression without nasal intubation, which is not always possible by venting gastrostomy alone. Palliative intestinal decompression proved to be effective in all patients. This alternative therapeutic strategy for managing bowel obstruction in terminal cancer patients is herein presented.

Cecal Neoplasms↗
PubMed1994

ATP sensitive potassium channels are involved in adenosine A2 receptor mediated coronary vasodilatation in the dog.

OBJECTIVE: The aim was to determine a role of ATP sensitive potassium (KATP) channels in adenosine A2 receptor mediated coronary vasodilatation in anaesthetised dogs in vivo. METHODS: Coronary blood flow in the left circumflex coronary artery, aortic pressure, and left ventricular pressure were measured during intracoronary infusions of the drugs into the left circumflex artery. RESULTS: A non-selective A2 receptor agonist NECA (5'-N-ethylcarboxamidoadenosine) at 10(-10)-10(-8) mol.min-1 before and after an A1 receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) increased coronary blood flow in a dose dependent manner, without affecting other haemodynamic variables. Glibenclamide at 10 micrograms.kg-1.min-1, which did not alter baseline haemodynamic variables, markedly inhibited the increases in coronary blood flow caused by NECA alone and after DPCPX (p < 0.01). A non-selective adenosine receptor antagonist 8-phenyltheophylline abolished the NECA induced increases in coronary blood flow after DPCPX. These results suggest that A2 receptor mediated coronary vasodilatation was mediated largely by opening of KATP channels. Glibenclamide did not alter the increase in coronary blood flow evoked by forskolin or acetylcholine, suggesting that KATP channels may not be involved in coronary vasodilatation induced by activation of adenylate cyclase or guanylate cyclase. Furthermore, DPCPX increased basal coronary blood flow, which was blocked by 8-phenyltheophylline and by glibenclamide, suggesting that it may have unmasked A2 receptor mediated coronary vasodilatation by inhibiting the A1 receptor mediated vasoconstricting action of endogenous adenosine. CONCLUSIONS: Opening of KATP channels may be involved importantly in adenosine A2 receptor mediated coronary vasodilatation in canine hearts.

Acetylcholine↗
PubMed1994

Binding of serum albumin on tumor cells and characterization of the albumin binding protein.

The binding of bovine (BSA) and human serum albumin (HSA) to the surface of human and mouse tumor cells in vitro was examined by flow cytometry using monoclonal antibodies against these albumins. Both isologous and heterologous types of albumin bound firmly on several lines of human and mouse tumor cells. The cell-bound albumin was removable by protease (actinase) treatment or by culture in albumin-free medium for more than 2 days, but not by simple washing. The amount of albumin capable of binding to the tumor cell surface differed among the 9 tumor cell lines tested. As determined with radioiodinated BSA, about 2.6 x 10(6) BSA molecules/cell could bind to MDA-MB-453 human mammary cancer cells, which exhibit high binding capacity as to BSA. A unique peptide having a molecular weight of 18 kDa was detected on gel electrophoresis in extracts of tumor cells or normal aortic endothelial cells which had been treated with radioiodinated, photocross-linker-labeled BSA or HSA, followed by UV-irradiation. This peptide was also immuno-precipitated with an anti-BSA or anti-HSA monoclonal antibody. With both methods, the yield of the peptide was decreased by previous addition of an excess amount of unlabeled BSA or HSA. These findings indicated that the 18-kDa peptide expressed on both normal aortic endothelial cells and tumor cells is a principal serum albumin-binding protein, and that this protein binds both BSA and HSA.

Animals↗
PubMed1994

Epitope mapping of bovine serum albumin using monoclonal antibodies coupled with a photoreactive crosslinker.

Antigenic determinants (epitopes) on bovine serum albumin (BSA) to anti-BSA monoclonal antibodies (mAbs) were assessed by means of photoaffinity labeling. Anti-BSA mAbs, AB3 and AB6, were modified with an 125I-labeled photoreactive crosslinker, sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD) and then bound with BSA. The complex yielded was irradiated with UV light to facilitate covalent binding of the modified mAb with BSA. Reduction of the product with 2-mercaptoethanol revealed that radioactivity of 125I-SASD was specifically introduced into BSA, probably near the mAb combining site(s). The radiolabeled or native BSA was digested with V8 protease and the peptides produced were assessed for their amino acid sequence, the radiolabeled amino acids contained, and their reactivity to mAb. The results indicated that AB3 mAb recognized an epitope in the region of Ala-537 to Asp-554 on a linear part of the BSA molecule, whereas AB6 mAb probably recognized an epitope of the assembled form present in a peptide of Asp-299 to Glu-338.

Amino Acid Sequence↗
PubMed1994

Identification of active site lysyl residues of phenylalanine dehydrogenase by chemical modification with methyl acetyl phosphate combined with site-directed mutagenesis.

A monoanionic acetylation reagent, methyl acetyl phosphate, was used to acetylate lysyl residues of the recombinant thermostable phenylalanine dehydrogenase from Thermoactinomyces intermedius. The enzyme was irreversibly inactivated with the reagent in a time- and dose-dependent manner. Simultaneous addition of substrate and coenzyme markedly protected the enzyme from inactivation. Acetylated lysyl residues presumably occurring at the active site were determined by differential modification; the enzyme was first modified with a cold reagent in the presence of both substrate and coenzyme and, after removal of the added substances by gel filtration, was then labeled with a radioactive reagent. At least 7 lysyl residues per enzyme subunit were radiolabeled by this method. To further specify the lysyl residue(s) whose modification results in inactivation of the enzyme, 5 lysyl residues highly conserved in various amino acid dehydrogenase sequences were replaced with Ala by site-directed mutagenesis. Although all of the single mutant enzymes were inactivated with the reagent as effectively as the wild-type enzyme, a double mutant enzyme in which both Lys-69 and Lys-81 were replaced with Ala was found to be inactivated very slowly. These results suggest that the reagent can acetylate both of these lysyl residues and inactivate the enzyme. Kinetic analyses of the single Lys-69 and Lys-81 mutant enzymes revealed that they are involved in substrate binding and catalysis, respectively, like the corresponding residues in the homologous leucine dehydrogenase.

Amino Acid Oxidoreductases↗
PubMed1994

Seroepidemiological survey of Coxiella burnetii in domestic cats in Japan.

Cats are assumed to be one of the most important reservoirs of causative agent of human Q fever especially in urban areas. There is no evidence of Coxiella burnetii infection in cats in Japan prior to this. Sera from 100 cats, collected in various parts of Japan, were examined for antibody against C. burnetii. Sixteen out of the 100 samples contained antibodies against C. burnetii. The prevalence of the antibody decreased from the northeastern to the southwestern part of Japan. A high prevalence of the antibodies was observed in sera from cats of more than four years of age. It is difficult to deny that cats would be one of the important sources of human Q fever in Japan.

Age Factors↗
PubMed1994

Verrucous carcinoma of the face with a massive neutrophil infiltrate. Analysis of leucocyte chemotactic activity in the tumour extract.

An unusual case of verrucous carcinoma of the face with a massive infiltration of neutrophils was found in a 60-year-old Japanese man who presented with a scaly, crusty tumour studded with pustules of 3 years duration on the left cheek. Histological examination showed a proliferation of deceptively benign-looking epidermal cells with an intact basement membrane, accompanied by a dermal infiltration of neutrophils and mononuclear cells and formation of microabscesses containing multinucleate giant cells, suggesting deep fungal infection or blastomycosis-like pyoderma. The lesion, however, was unresponsive to antifungal or antibacterial treatments and ultimately attained a considerable size in the following months. Based on the findings of repeat biopsy conducted 4 months later indicating further deeper invasion, a diagnosis of verrucous carcinoma of the face was made, and a total excision of the tumour and left cervical lymph node dissection were finally carried out. It can be speculated that aberrant production of leucocyte chemotactic cytokines such as interleukin-8 by dysplastic keratinocytes, subsequent neutrophil infiltration/serum permeation, generation of leucotactic anaphylatoxin C5a from serum through complement activation and of lipid chemotactic factors (leukotriene B4 and 12-hydroxy-eicosatetraenoic acid) by infiltrating leucocytes and/or dysplastic keratinocytes provoked the characteristic accumulation of neutrophils in the verrucous carcinoma of this case.

Carcinoma, Verrucous↗
PubMed1994

Antigenic cross-reactivity between human T lymphotropic virus type I (HTLV-I) and retinal antigens recognized by T cells.

To investigate the relationship between uveitis and HTLV-I infection, we examined the cross-reactivity between HTLV-I antigens and retinal antigens recognized by T cells in B10.BR mice immunized with human HTLV-I-infected MT-2 cells. We found that T cells obtained from MT-2 immune mouse spleen responded not only to HTLV-I antigens but also to retinal antigens of various species. However, they did not respond to HTLV-I-negative lymphoid cell lines. Furthermore, established T cell lines from MT-2 immune spleen cells also responded to both HTLV-I and retinal antigens. The phenotype of the immune cells that responded to both HTLV-I and retinal antigens was CD4+, CD8-, and CD3+. The proliferative response of T cell lines to HTLV-I as well as various retinal antigens, was clearly blocked by addition of anti-CD3, anti-CD4, or anti-I-Ak MoAbs, but not by anti-CD8 antibody. The established T cell lines from HTLV-I immune spleen cells were all found to be CD3+, TCR beta +, CD4+, CD8- cells by flow cytometric analysis. These results indicate that an epitope of HTLV-I antigens is cross-reactive to an epitope of retinal antigens extracted from either human retinoblastoma or normal murine, rat, and bovine retinae at a T cell recognition level.

Animals↗
PubMed1994

Relationship between mean arterial pressure and muscle cell pH during forearm ischaemia after sustained handgrip.

Recent evidence suggests that there is a close correlation between the physiological responses to muscle chemoreflex and the decrease in intracellular pH during ischaemia after handgrip. This study evaluated whether the relationship is linear or has an apparent threshold. We measured muscle cellular pH through phosphorous nuclear magnetic resonance spectroscopy (31P-NMR), mean arterial blood pressure (MAP) and heart rate (HR) during ischaemia after sustained handgrip exercise at 50% of maximum voluntary contraction (MVC). Contraction was sustained for 15, 30, 45 and 60 s, followed by 2 min of circulatory arrest, respectively. Muscular pH during the ischaemia decreased linearly with increasing contraction time, from the base-line level of 7.11 +/- 0.03 units (U) to 6.98 +/- 0.03, 6.90 +/- 0.04, 6.72 +/- 0.06 and 6.54 +/- 0.06 U after 15-, 30, 45-, and 60-s contractions, respectively. The MAP was 86 +/- 2 mmHg at rest and did not change during the ischaemia after 15- and 30-s contractions. However, it significantly increased to 95 +/- 2 and 107 +/- 2 mmHg, after 45- and 60-s contractions, respectively. These data indicate that the relationship between MAP and pH is not a single linear relationship, showing one breaking point around the pH of 6.90 units. It suggests that the muscle chemoreflex has a clear threshold around 6.90 units of muscle pH, and below this pH, MAP increased linearly with decreasing muscle cellular pH.

Adolescent↗
PubMed1994

Effects of erythropoietin treatment on thyroid dysfunction in hemodialysis patients with renal anemia.

The thyroid function was evaluated before and after 6 months of recombinant human erythropoietin (rhEPO) treatment (1,500-9,000 U/week) in 22 hemodialysis patients with hematocrit levels < 25%. Based upon the changes in hematocrit following rhEPO treatment, the patients were divided into two groups: 11 patients with an increase of the hematocrit level > 5% (group I) and 11 patients with an increase < 5% (group II). Before rhEPO administration, the levels of thyroid hormones, especially free thyroxine (T4) and free triiodothyronine (T3), were below the normal range despite normal thyrotropin values in most of the patients (low T4:7 cases in group I and 9 in group II; low T3:10 cases in group I and 10 in group II). RhEPO treatment significantly increased both total amount and free fractions of thyroid hormones in group I, whereas it did not affect these values in group II. Consequently, the pretreatment low T4 or low T3 status was resolved in a substantial number of the patients in group I (low T4:5 cases, low T3:4 cases). In addition, there was a significant correlation between the increases in hematocrit and free T3 in all studied subjects (r = 0.603; p < 0.05). These results suggest that anemia may participate to some extent in the pathogenesis of thyroid dysfunction in hemodialysis patients with renal anemia.

Adult↗