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Biomedical subjects

M Ueda

Publications and source records attributed to M Ueda.

At least 307 records · Page 17Linked to original sources
PubMed1999

Correlation of the expression of human telomerase subunits with telomerase activity in normal skin and skin tumors.

BACKGROUND: Telomerase activation is thought to be crucial for the continued growth or progression of cancer cells. Three major subunits of human telomerase, human telomerase RNA (hTR), telomerase-associated protein (TP1), and human telomerase catalytic subunit (hTERT) have been identified recently. In the current study, the expression of these telomerase subunits was examined in various human skin tumors and in normal skin samples, and the correlation of their expression with telomerase activity was evaluated. METHODS: Forty-five skin tumors, including benign, premalignant, and malignant tumors, and 19 samples of normal skin were analyzed by reverse transcriptase-polymerase chain reaction for the expression of telomerase subunits and by telomeric repeat amplification protocol assay for telomerase activity. RESULTS: TP1 expression was found in 43 of 45 skin tumors (95.6%) and in all normal skin samples analyzed. hTR expression was found in 42 of 45 skin tumors (93.3%) and in 18 of 19 normal skin samples (94.7%). In contrast, hTERT expression was detected in 33 of 45 skin tumors (73.3%) and in only 3 of 19 normal skin samples (15.8%). Telomerase activity was positive in 33 of 45 skin tumors (73.3%) and in 3 of 19 normal skin samples (15.8%). All together, 53 of 64 cases (82.8%) were concordant for both results (P < 0.001), i.e., telomerase activity positive and hTERT positive, or telomerase activity negative and hTERT negative. CONCLUSIONS: Activation of telomerase activity in skin tumors is strongly correlated with hTERT expression.

Catalytic Domain↗
PubMed1999

Role of saccharides on lung preservation.

BACKGROUND: Saccharides are considered to play a role as osmotic impermeants and serve as an energy source for the organ during ischemia. However, previous studies on the effectiveness of saccharides on organ preservation have yielded conflicting results. We compared the preservative effects of a monosaccharide (glucose), disaccharides (trehalose, maltose, sucrose), and a trisaccharide (raffinose) to investigate whether the effects of saccharides on lung preservation depend on their molecular weight, energy-level maintenance, and cytoprotective effects. METHODS: We used an ex vivo rat lung model using homologous blood as the perfusate. In the fresh group, the lungs were reperfused immediately after flush. In the other groups, the lungs were flushed with one of the solutions containing glucose, trehalose, maltose, sucrose, or raffinose and preserved for 14 hours. RESULTS: The results of the trehalose group were comparable to those of the fresh group. The glucose, maltose, and raffinose groups showed significantly higher levels of shunt fraction, pulmonary artery pressure, and peak inspiratory pressure compared with the fresh and trehalose groups. There were no differences among the groups in the levels of total adenine nucleotides, adenosine triphosphate of the lung after flush, and preservation. However, after reperfusion, levels of total adenine nucleotides became significantly lower in the glucose, sucrose, maltose, and raffinose groups. Ultrastructural examination revealed endothelial cell injury in the glucose, sucrose, maltose, and raffinose groups. CONCLUSIONS: These results show that the effects of saccharides may depend on their cytoprotective effect rather than on impermeant activity or energy-level maintenance of the preserved lung. Trehalose proved to be superior to the other saccharides.

Adenine Nucleotides↗
PubMed1999

Analysis of cells in cerebrospinal fluid from patients with neurocysticercosis by means of flow cytometry.

The events of the cellular immune response in neurocysticercosis (NC) are not fully understood. Studies of the CD3, CD3/CD4, CD3/CD8, CD45/CD19, and CD45/CD56 molecules and activation-related CD69 molecule in cells from the cerebrospinal fluid (CSF) and peripheral blood (PB) of patients with NC may provide a better elucidation of the inflammatory and immunological events occurring in this disease. Seven patients with NC and 3 individuals with other disorders were evaluated by a three-color flow cytometric method. CD69 was detected in a higher percentage of cells in all CSF samples from patients, but not in PB or CSF from the control group. The percentage of CD3+ cells did not differ significantly in CSF and PB cells from patients and controls. The predominance of CD3+CD8+ cells was observed in CSF from one patient and in PB from 2 patients, who were in stage III of the disease (inflammatory process). The percentage of CD45+CD19+ cells was higher in CSF than in PB from patients who presented anti-cysticercus antibodies in CSF. The percentage of CD45+CD56+ cells in CSF was higher than in PB, but this rate was similar to reference values reported by other authors. Our data suggest that the cytometric method applied to a larger number of CSF samples may provide a better understanding of the cell-mediated immune response involved in NC.

Antigens, CD↗
PubMed1999

Human CYP2C-mediated stereoselective phenytoin hydroxylation in Japanese: difference in chiral preference of CYP2C9 and CYP2C19.

Regio- and stereoselective hydroxylation of phenytoin was determined in liver microsomes of nine extensive (EM) and three poor metabolizers (PM) of mephenytoin. Hydroxyphenytoins (HPPH) were isolated and quantified after separation into four regio- and stereoisomers. The total rates of microsomal phenytoin 4'- hydroxylation were approximately 3-fold higher than those of 3'-hydroxylation, and not significantly different in EM and PM. Formation of 4'-(R)-HPPH was 4.4-fold higher in EM than in PM, whereas no clear differences between EM and PM were detected in the formation of 4'-(S)-, 3'-(R)-, and 3'-(S)-HPPH. Cytochrome P450 (CYP)2C9, expressed in a fission yeast, Schizosaccharomyces pombe, catalyzed the formation of 4'-(R)- and 4'-(S)-HPPH stereoselectively, as observed with EM, in which predominantly 4'-(S)-HPPH was formed. Recombinant CYP2C19 was more stereoselective for 4'-(R)-HPPH formation. These results, in addition to inhibition experiments with anti-human CYP2C antibody, indicate that phenytoin hydroxylation is mainly catalyzed by CYP2C9. Furthermore, CYP2C19 showed limited contribution to phenytoin 4'-hydroxylation with a different chiral preference from CYP2C9.

Antibodies↗
PubMed1999

Cloning and characterization of the gene encoding mouse osteoclast differentiation factor.

Osteoclast differentiation factor (ODF), a ligand for osteoclastogenesis inhibitory factor (OCIF)/ osteoprotegerin (OPG), is a member of the membrane-associated tumor necrosis factor (TNF) family and induces osteoclast-like cell formation in vitro. In the present study, mouse ODF genomic clones were isolated and sequenced to determine their gene structure. The mouse ODF gene is a single copy gene consisting of five exons and spans approximately 40kb of the mouse genome. The first exon encodes the intracellular and transmembrane domains. The extracellular region of ODF containing the TNF homologous domain is encoded by exons 1 through 5. The translation-termination codon and six polyadenylation signal residues are present in exon 5. A major transcription-initiation site is present 143 nucleotides upstream of the initiation-ATG codon. This genomic organization is similar to that of other members of the TNF family, especially the CD40 ligand.

Animals↗
PubMed1999

Successful culture and sustainability in vivo of gene-modified human oral mucosal epithelium.

Human oral mucosal cells are an attractive site for tissue engineering because they are the most accessible cells in the body and easy to manipulate in vitro. They thus have possibilities for targeting by somatic gene therapy. We examined the efficiency of retrovirus-mediated gene transfer and the construction of mucosal epithelium in vivo. Human oral mucosal cells were transduced with a retroviral vector carrying the lacZ gene at high efficiency and constructed epithelium after G418 selection with 3T3 cells in vitro. The cultured oral mucosal epithelium membrane was then grafted onto immunodeficient mice. Beta-Gal expression was detected histochemically in vivo 5 weeks after grafting. Furthermore, we transduced factor IX cDNA into the mucosal epithelium membrane, and it was then transplanted into nude mice. Between 0.6 and 1.8 ng of human factor IX per milliliter was found in mouse plasma, and the production was continued for 23 days in vivo. These results confirmed that the oral mucosal epithelium is an ideal target tissue for gene therapy or tissue engineering.

Animals↗
PubMed1999

Susceptibility to neuroleptic malignant syndrome in Parkinson's disease.

OBJECTIVE: To determine susceptibility to neuroleptic malignant syndrome (NMS) in patients with PD in relation to central monoamine metabolism. METHODS: CSF levels of homovanillic acid (HVA), 3-methoxy-4-hydroxy phenyletilene glycol (MHPG), and 5-hydroxyindole acetic acid (5-HIAA) were assayed in 98 PD patients (mean age, 77.2 years), including 11 patients with a prior NMS-like episode, by high-performance liquid chromatography with electrochemical detection. RESULTS: Patients with a previous NMS-like episode had worse parkinsonian disability as measured by Hoehn & Yahr scale (3.7 +/- 0.8 versus 3.0 +/- 1.1; p = 0.038) and lower CSF HVA levels (20.9 +/- 17.3 versus 44.7 +/- 22.2 ng/mL; p = 0.001) compared to those without, despite similar age, disease duration, and daily dosages of antiparkinsonian drugs between groups. Logistic regression analysis showed that the CSF HVA level (p = 0.008), but not 5-HIAA level (p = 0.621), was significantly and independently related to NMS, and that the MHPG level (p = 0.070) was tendentially associated with the disorder. Odds ratios (95% confidence intervals) corresponding to 10 ng/mL increment in CSF HVA, MHPG, and 5-HIAA levels were 0.30 (0.13 to 0.73), 4.03 (0.89 to 18.2) and 1.29 (0.47 to 3.58), respectively. CONCLUSIONS: Central dopaminergic and possible noradrenergic activity contributes to NMS development in an elderly population of PD patients. Measuring CSF levels of monoamine metabolites may provide a means for identifying NMS susceptibility in PD patients.

Aged↗
PubMed1999

Intraluminal increase of superoxide anion following transient focal cerebral ischemia in rats.

Using a modification of Karnovsky's Mn2+/diaminobenzizine (DAB) technique, we examined the production of superoxide anion (.O-2) in the vascular lumen following transient occlusion and reperfusion of the left middle cerebral artery (MCA) in Sprague-Dawley rats. The MCA was occluded for 2 h using an intraluminal suture method. Zero, 15, 30, and 60 min after reperfusion, animals were perfused transcardially with buffer containing Mn2+ and DAB, and brain samples were prepared for light and electron microscopic examination. The amber reaction deposits of.O-2 were observable to the naked eye along the major cerebral vessels of the ischemic hemisphere after each reperfusion period. Upon microscopic examination the deposits were revealed to be within arterial, capillary, and venular lumen. The amount of reaction deposits in the ischemic hemisphere corresponded to the duration of reperfusion. The formation of.O-2 was suppressed when the perfusate contained superoxide dismutase and when either Mn2+ or DAB was omitted, confirming that the reaction products produced are due to the enhanced production of.O-2. These results show that there is a progressive increase in intraluminal. O-2 during reperfusion following an ischemic insult which may participate in the aggravation of cerebral damage.

Animals↗
PubMed1999

High-level expressing YAC vector for transgenic animal bioreactors.

The position effect is one major problem in the production of transgenic animals as mammary gland bioreactors. In the present study, we introduced the human growth hormone (hGH) gene into 210-kb human alpha-lactalbumin position-independent YAC vectors using homologous recombination and produced transgenic rats via microinjection of YAC DNA into rat embryos. The efficiency of producing transgenic rats with the YAC vector DNA was the same as that using plasmid constructs. All analyzed transgenic rats had one copy of the transgene and produced milk containing a high level of hGH (0.25-8.9 mg/ml). In transgenic rats with the YAC vector in which the human alpha-lactalbumin gene was replaced with the hGH gene, tissue specificity of hGH mRNA was the same as that of the endogenous rat alpha-lactalbumin gene. Thus, the 210-kb human alpha-lactalbumin YAC is a useful vector for high-level expression of foreign genes in the milk of transgenic animals.

Animals↗
PubMed1999

Analysis of control elements for position-independent expression of human alpha-lactalbumin YAC.

A major problem in the production of transgenic animal bioreactors using microinjections is the low production rate of high-expressing transgenic animals due to the position effect. We previously reported that transgenic rats carrying the 210 kb yeast artificial chromosome (YAC) including the human alpha-lactalbumin gene express the transgene in a position-independent manner. The 210 kb YAC was thought to have all the elements necessary for position-independent expression. In this paper, we constructed fragmented YAC clones and a cosmid clone, and produced transgenic rats to analyze these elements. Transgenic rats with both the 50 kb upstream and downstream regions of the alpha-lactalbumin gene had position-independent expression. Transgenic rats with the 20 kb upstream and downstream regions, however, had position-dependent expression. Therefore, all the elements necessary for position-independent expression are thought to be located in the 50 kb upstream to 50 kb downstream region of the alpha-lactalbumin gene. Furthermore, we replaced the human alpha-lactalbumin promoter with the bovine alphaS1-casein promoter in the 210 kb YAC and produced transgenic rats. Position-dependent expression was observed. The elements required for position-independent expression of the bovine alphaS1-casein gene are different from those required for the human alpha-lactalbumin gene, despite the fact that the two genes have the same tissue and developmental specificity.

Animals↗
PubMed1999

Effects of timing of oocyte cryopreservation on in vitro development of nuclear-transferred bovine zygotes.

The utility of cryopreserved bovine oocytes as recipient cytoplasts for nuclear transfer (NT) was examined. In vitro-matured (IVM), metaphase-II oocytes were enucleated by mechanical suction and activated parthenogenetically. The cytoplasts were fused with blastomeres of in vitro-produced day-5 morulae by a DC electropulse, and then cultured up to 8 days (non-frozen controls; group I). Oocytes were frozen-thawed in 1.5-M ethylene glycol and 0.1-M sucrose before enucleation (group II), after enucleation (group III), after enucleation and aging culture (group IV), or after activation (group V). In group I, 91% of IVM oocytes could be used for NT and 89% of them fused successfully. Finally, 36% of the fused zygotes developed into blastocysts. The proportions of morphologically normal oocytes after thawing in groups IV and V (70 and 69%, respectively) were higher than in group III (56%), and the proportion of IVM oocytes used for NT in group IV (56%) was higher than those in groups II, III, and V (33%, 35%, and 38%, respectively). Fusion rates of the NT zygotes in groups III, IV, and V (90%, 88%, and 88%, respectively) were higher than the rate in group II (75%). Rates of development into blastocysts of the fused zygotes in groups II, III, IV, and V were 0%, 3%, 2%, and 6%, respectively (P < 0.05, group II vs. groups III, IV, and V). Developmental kinetics and cell numbers of the blastocysts were similar among the groups. It was suggested that timing of oocyte cryopreservation is among the factors influencing efficiency of production of cloned embryos in cattle.

Animals↗
PubMed1999

Autoimmune diseases developed in athymic nude mice grafted with embryonic thymus of xenogeneic origin.

Reconstitution of T cell functions in athymic BALB / c nude mice was investigated after transplantation of embryonic thymus grafts under the renal capsule (TG nude mice). Thymi were obtained from mice, rats, rabbits, hamsters, guinea pigs, swine and cows. All of the grafted thymi grew and formed proper thymic structures with host CD90(+) cells. The TCRalpha beta expression pattern of the lymphocytes in the grafted thymi was quite similar to that of normal mouse thymocytes. Sufficient CD4(+) populations were observed in the peripheral lymphoid organs of all groups of TG nude mice. Plaque-forming cell assay revealed that the TG nude mice had acquired considerable helper T cell functions. Histological and serological studies, however, showed multiple organ-localized inflammatory diseases in TG nude mice of all xenogeneic thymus groups, but not with syngeneic or allogeneic thymi. The organs affected were the thyroid, the lacrimal, submandibular and sublingual glands, the eyes, stomach and ovaries. The incidence of lesions was variable depending on the species of origin of the grafted thymus. Lesions from TG nude mice were successfully transferred into naive nude mice by host CD4(+) cells. These results together suggested that the microenvironment of grafted thymi, even if xenogeneic, is able to educate host T cell precursors. However, this reconstitution of functions does not always induce tolerance to certain autoantigens, resulting in development of multiple autoimmune lesions.

Animals↗
PubMed1999

Visualization of a specific sequence on a single large DNA molecule using fluorescence microscopy based on a new DNA-stretching method.

A method for analyzing large DNA which makes it possible to obtain spatial information on the positions of specific sequences along a DNA molecule has been developed. Making use of the fact that large DNA molecules are stably elongated under an alternating-current field in a concentrated linear polymer solution, the direct observation of elongated individual lambda DNA molecules with fluorescence probes was carried out using fluorescence microscopy. The spatial positions of the fluorescent spots of the probe (fluorescence-labeled restriction endonuclease EcoRI) on DNA molecules were determined by image analysis. As expected, fluorescent spots of EcoRI were observed at certain positions on lambda DNA, where sequences to which EcoRI binds are located. Finally, the potential application of single large DNA molecule analysis using this DNA-stretching method is discussed.

Bacteriophage lambda↗
PubMed1999

Expression of the SNF1 gene from candida tropicalis is required for growth on various carbon sources, including glucose

SNF1 of Saccharomyces cerevisiae is an essential gene for the derepression of glucose repression. A homolog of SNF1 (CtSNF1) was isolated from an n-alkane-assimilating diploid yeast, Candida tropicalis. CtSNF1 could complement the snf1 mutant of S. cerevisiae. The previously published method for introducing the exogenous DNA into C. tropicalis was employed to construct SNF1/ snf1 heterozygote and snf1/snf1 homozygote strains. The successfully constructed SNF1/snf1 heterozygote was named KO-1. Disruption of the second CtSNF1 allele was unsuccessful, suggesting that CtSNF1 might be essential for cell viability. Therefore, in order to control the expression of CtSNF1, a strain (named KO-1G) in which the promoter region of CtSNF1 was replaced with the GAL10 promoter of C. tropicalis was constructed, and the growth of strains KO-1 and KO-1G was compared with that of the parental strain. The growth of strain KO-1 on glucose, sucrose, or acetate did not differ from the growth of the parental strain, but strain KO-1 showed a slight growth retardation on n-alkane. The growth of strain KO-1G on galactose was normal, but the cells stopped growing when transferred to glucose-, acetate-, or n-alkane-containing medium. Northern blot analysis against mRNA from the n-alkane-grown KO-1G strain demonstrated a close relationship between the presence of CtSNF1 mRNA and the growth of the cells, indicating that CtSNF1 is essential for cell viability. Moreover, mRNA levels of isocitrate lyase, which is localized in peroxisomes of C. tropicalis, were significantly affected by the level of CtSNF1 mRNA.

Journal Article↗
PubMed1999

Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface.

The construction of a whole-cell biocatalyst with its sequential reaction has been performed by the genetic immobilization of two amylolytic enzymes on the yeast cell surface. A recombinant strain of Saccharomyces cerevisiae that displays glucoamylase and alpha-amylase on its cell surface was constructed and its starch-utilizing ability was evaluated. The gene encoding Rhizopus oryzae glucoamylase, with its own secretion signal peptide, and a truncated fragment of the alpha-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast alpha factor, respectively, were fused with the gene encoding the C-terminal half of the yeast alpha-agglutinin. The constructed fusion genes were introduced into the different loci of chromosomes of S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The glucoamylase and alpha-amylase activities were not detected in the culture medium, but in the cell pellet fraction. The transformant strain co-displaying glucoamylase and alpha-amylase could grow faster on starch as the sole carbon source than the transformant strain displaying only glucoamylase.

Agglutinins↗
PubMed1999

Independent production of two molecular forms of a recombinant Rhizopus oryzae lipase by KEX2-engineered strains of Saccharomyces cerevisiae.

A mixture of rProROL having the full-length prosequence (97 amino acids) for a recombinant lipase of Rhizopus oryzae (rROL) and r28ROL having 28 amino acids of the same prosequence has been produced as active forms by Saccharomyces cerevisiae [Takahashi et al. (1998) J Ferment Bioeng 86: 164-168]. However, the separation of rProROL and r28ROL has not been successful due to their identical behavior on column chromatographs, presumably because of the similarity of their surface properties. The independent production of two different molecular forms of rROL was carried out using KEX2-engineered strains of S. cerevisiae, since r28ROL was predicted to be a product from rProROL by a Kex2-like protease. rProROL was successfully obtained by expression of the ROL gene in the S. cerevisiae kex2 strain in which the KEX2 gene encoding Kex2p was disrupted, while r28ROL was obtained by co-expression of the gene (KEX2 delta 613) encoding the soluble form of the C-terminal truncated Kex2 protease (sKex2p). The specific lipase activities of rProROL and r28ROL were 92.9 U/mg and 140 U/mg, respectively. rProROL was stable at pH 2.2-8.0, and showed the optimal reaction temperature to be 30-35 degrees C with a T50 of 55 degrees C (T50 is the temperature resulting in 50% loss of activity). The values for r28ROL were pH 3.0-10.0, 25-30 degrees C, and 40 degrees C, respectively. rProROL was an N-linked glycosylated form, but r28ROL was not. The enhanced thermostability of rProROL did not seem to be due to the N-linked glycosylation, as judged by the results of the Endo H treatment. rProROL had the highest esterase activity toward p-nitrophenyl laurate (C12), whereas r28ROL had the highest esterase activity toward p-nitrophenyl caprylate (C8) and stearate (C18). These results suggest that the distinct properties of these two forms of lipase are caused by the different length of the ROL prosequence.

Bacterial Proteins↗
PubMed1999

Induction of smooth muscle cells in the fibrous capsule of human hepatocellular carcinoma but not in the septa of hepatic cirrhosis.

We examined the expression of smooth muscle cytoskeleton in spindle-shaped cells in the capsule of hepatocellular carcinoma (HCC) and the septa of liver cirrhosis (LC). Serial sections of livers resected from 11 patients were stained with monoclonal antibodies against vimentin, desmin, smooth muscle actin (1A4, HHF35, CGA7) and smooth muscle myosin heavy chain isoforms (SM1, SM2). Capsular spindle-shaped cells exhibited a cytoskeletal feature indicative of intermediately differentiated smooth muscle cells. Computer-assisted morphometry revealed that the proportions of 1A4-, HHF35-, CGA7- and SM1- positive areas to vimentin-positive area were 88.0+/-11.0%, 50.8+/-17.4%, 25.3+/-16.4% and 19.4+/-12.4% (n=11) in main tumours and 86.6+/-9.4%, 50.9+/-18.7%, 21.1+/-12.3% and 17.6+/-9.7% (n=12) in daughter tumours, indicating that spindle-shaped cells are heterogeneous in cytoskeletal expression. Septal spindle-shaped cells in LC lacked the cytoskeletal proteins specific to differentiated smooth muscle cells (CGA7, SM1, SM2 and desmin). Electron microscopically, capsular spindle-shaped cells contained more microfilaments and less rough endoplasmic reticulum than do septal cells. Intermediately differentiated smooth muscle cells are induced in the capsule of HCC but not in the septa of LC, suggesting a role for stromal interaction by tumour cells in the induction of smooth muscle cells.

Actins↗