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Biomedical subjects

M Ueda

Publications and source records attributed to M Ueda.

At least 271 records · Page 15Linked to original sources
PubMed2000

Enhancement of dedifferentiation and myoid differentiation of retinal pigment epithelial cells by platelet derived growth factor.

AIMS: To clarify factor(s) involved in morphological dedifferentiation of retinal pigment epithelial (RPE) cells in vitro from mitotically quiescent hexagonal cells to flattened cells that lack epithelial characteristics and concurrent myoid differentiation. METHODS: RPE cells which retained their differentiated hexagonal morphology were isolated from bovine eyes by mechanical pipetting. Dedifferentiation and myoid differentiation of RPE cells were examined by microscopic observation and immunohistochemical analysis using antibodies against cytokeratin, an epithelial marker, and alpha smooth muscle actin, a marker of myoid differentiation. The contractile ability of RPE cells was evaluated by collagen gel contraction assay. RESULTS: Platelet derived growth factor (PDGF) enhanced morphological changes in the RPE from hexagonal-shaped cells to flattened cells. Coincident with this morphological alteration, the expression of cytokeratin in RPE cells decreased and expression of alpha smooth muscle actin began and was increased in a time dependent manner. These alterations were completely blocked by collagen synthesis inhibitors. Interleukin 1beta, transforming growth factor beta1, insulin-like growth factor I, and basic fibroblast growth factor had little or no effect on the dedifferentiation. PDGF also potentiated the RPE induced collagen gel contraction. CONCLUSIONS: These results demonstrate that PDGF enhanced the dedifferentiation of RPE cells, the initial step of proliferative vitreoretinopathy (PVR), as well as myoid differentiation and collagen gel contraction. PDGF may have a versatile role in the pathogenesis of PVR involving collagen synthesis.

Actins↗
PubMed2000

Role of endothelial cell denudation and smooth muscle cell dedifferentiation in neointimal formation of human vein grafts after coronary artery bypass grafting: therapeutic implications.

OBJECTIVE: To provide better insights into the genesis of neointimal thickening in human vein grafts early after surgery. DESIGN: Retrospective study. SETTING: Tertiary referral centre. SUBJECTS: 18 distal anastomotic sites of patent grafts, obtained at necropsy from eight patients who died over differing periods (ranging from two days to nine months) after the procedure. MAIN OUTCOME MEASURES: Immunohistochemical evaluation of smooth muscle cell phenotype modulation in relation to proliferative activity. RESULTS: The earliest changes are characterised by loss of surface lining endothelial cells and insudation of blood corpuscular elements admixed with fibrin-platelet thrombus. At sites of injury vimentin positive and actin negative spindle shaped cells appear in the intima, while the related pre-existent media shows focal absence of actin positive smooth muscle cells. Proliferative activity colocalises at these sites. With time distinct neointimal thickening occurs, associated with disappearance of proliferative activity and a phenotypic shift of the smooth muscle cells. CONCLUSIONS: The observation that luminal endothelial cell denudation, with insudation of the intima with blood elements, occurs in the very early stages suggests that these phenomena are responsible for the observed dedifferentiation of pre-existent smooth muscle cells, known to be a prerequisite for cell proliferation and the evolution of intimal thickening. It is likely, therefore, that platelet released growth factors play a pivotal role, which thus may provide a target for preventive pharmacological intervention.

Aged↗
PubMed2000

Contribution of proliferating leukocytes to phenotypic change in smooth muscle cells during the development of coronary arteriosclerosis in transplanted hearts.

BACKGROUND: It is well known that coronary arteriosclerosis after heart transplantation is concentric and rich in smooth muscle cells (SMCs); however, the role played by rejection in the intimal thickening caused by SMCs in coronary arteriosclerosis remains unclear. In this study, we examined the process of intimal hyperplasia caused by SMCs and evaluated the relationship between the differentiation state of SMCs and local inflammation caused by rejection. METHODS: Lewis rat hearts were heterotopically transplanted into F344 rats (allotransplantation group) or other Lewis rats (isotransplantation group). Cyclosporin A (5 mg/kg/day) was injected intramuscularly for 20 days after transplantation in both groups. The transplanted hearts were examined immunohistochemically using several monoclonal antibodies; namely, HHF-35, CGA7, vimentin, alpha-actin, HIS36, R73 and proliferating cell nuclear antigen (PCNA). To evaluate the degree of local immunological response caused by rejection, the anti-PCNA antibody was used. To reveal the subtypes of proliferating cells in the thickened intima, HIS36 and R73 antibodies were used. RESULTS: In the allotransplantation group, SMCs in the media began to undergo a phenotypic change toward a poorly differentiated state 30 days after transplantation. Intimal hyperplasia was observed 60 days after transplantation, the thickened intima being composed mainly of dedifferentiated SMCs with abundant PCNA(+), most of which were macrophages and T cells. The state of differentiation of SMCs in the thickened intima 90 days after transplantation varied from a dedifferentiated to a highly differentiated state. These changes were strongly correlated with the expression of PCNA. CONCLUSION: The expression of PCNA was strongly correlated with the differentiation state of SMCs. Thus, local inflammation caused by rejection may play an important role in the initiation of phenotypic change in SMCs.

Animals↗
PubMed2000

Effects of dibutyryl cyclic adenosine monophosphate on the ultrastructure of endothelial cells in rat lungs cold preserved for 15 hours.

BACKGROUND: Dibutyryl cyclic adenosine monophosphate (db-cAMP) has been shown to protect vascular endothelial cells by increasing the level of intracellular cAMP, and we have previously reported its effectiveness in lung preservation. Here, the effects of db-cAMP in lung preservation were ultrastructurally investigated, and the ultrastructural changes before reperfusion were correlated with pulmonary function after reperfusion. METHODS: The lungs of 17 Lewis rats were flushed with perfusate and prostaglandin E(1), and were then divided into three groups. In the fresh group (n = 6), the lungs were flushed with extracellular-type trehalose-containing (ET-K) solution and were reperfused immediately. In the control group (n = 6) and db-cAMP group (n = 5), the lungs were flushed with ET-K solution and ET-K solution plus db-cAMP (2 mM), respectively, and were reperfused after cold preservation at 4 degrees C for 15 h. Before reperfusion, tissue was sampled and ultrastructurally analyzed by transmission electron microscopy. RESULTS: In the endothelial cells of pulmonary arterioles, the incidence of protrusion was significantly lower in the fresh and db-cAMP groups than in the control group (p < 0.05). The incidence of detachment and microvillus formation were significantly lower in the fresh and db-cAMP groups than in the control group (p < 0.01). The ultrastructure of the alveoli did not allow separation of the control and db-cAMP groups. The shunt fraction and wet to dry weight ratio of the lung tissue after reperfusion were significantly lower in the fresh and db-cAMP groups than in the control group (p < 0.01). Positive correlations were found between the incidence of these ultrastructural changes in the endothelial cells of the pulmonary arterioles and pulmonary function after reperfusion. CONCLUSION: These findings suggest that db-cAMP might attenuate the lung injury caused by cold preservation and ischemia-reperfusion, partly by suppressing the acceleration of the structural changes in the endothelial cells in the pulmonary arterioles.

Animals↗
PubMed2000

Salivary growth factors in health and disease.

The salivary gland is considered to be a reservoir of many growth factors in rodents. In humans, the epidermal growth factor, basic fibroblast growth factor, and insulin and insulin-like growth factor family have also been detected in this gland, but their physiological role remains unclear. In this study, we focused on bFGF, which is a well-known mitogen for various types of cells, and is present in the salivary gland as well as in saliva. The roles of bFGF in the salivary gland were investigated by three different procedures. First, the effects of bFGF on the salivary gland cells were investigated with a monolayer culture of normal submandibular gland cells. The effects of different concentrations of bFGF on the second passage of these cultured cells were examined. In both human and rat cultured submandibular gland cells, bFGF accelerated the cell proliferation at a concentration of 100 ng/mL or higher. Next, an atrophic model of the rat submandibular gland was used to examine the ability of bFGF to accelerate tissue repair. Two weeks after ductal ligation, the ligature was removed, and various amounts of bFGF, isoproterenol, or saline were administered via a retrograde duct instillation. Both isoproterenol and bFGF increased acinar and ductal cell proliferation significantly. To determine the role of bFGF in saliva, we investigated its effect on the healing process of oral mucosal defects. Four-millimeter mucosal defects were made to the depth of the periosteum in the rat palate under anesthesia. bFGF or vehicle alone was applied once only at the time of surgery as a suspension. At days 3, 5, and 7 in the bFGF group, significant increases in the degree of re-epithelialization were found in treated groups. These results indicate that its action as a mitogen stimulus is the major effect of bFGF on salivary gland cells and mucosal epithelium.

Animals↗
PubMed2000

Epoxide hydrolase affects estrogen production in the human ovary.

To investigate the mechanisms of ovarian cell differentiation, we raised a new monoclonal antibody, HCL-3, which reacted with human luteal cells. It also reacted with human and porcine hepatocytes. The immunoaffinity-purified HCL-3 antigen from human corpora lutea (CL) was shown to be a 46-kDa protein. The N-terminal 22 amino acids of the 46-kDa protein from porcine liver exhibited high homology (82%) to human microsomal epoxide hydrolase (mEH). The purified HCL-3 antigen from human CL or porcine liver showed EH enzyme activity, confirming that HCL-3 antigen is identical to mEH, which is reported to detoxify the toxic substrates in the liver. In human follicles, mEH was immunohistochemically detected on granulosa and theca interna cells. In the menstrual and pregnant CL, mEH was also expressed on large and small luteal cells. A competitive inhibitor of EH, 1,2-epoxy-3,3,3-trichloropropane, inhibited the conversion of estradiol from testosterone by granulosa cells cultured in vitro, indicating the involvement of mEH in ovarian estrogen production. Because anticonvulsant sodium valproate and its analogues were reported to inhibit EH enzyme activity, these findings provide a new insight into the etiology of endocrine disorders that are frequently observed among epileptic patients taking anticonvulsant drugs.

Adolescent↗
PubMed2000

Adenosine-induced coronary flow reserve in Watanabe heritable hyperlipidemic rabbits.

The Watanabe heritable hyperlipidemic (WHHL) rabbit develops coronary atherosclerosis and hypercholesterolemia because of a genetic deficiency of low-density lipoprotein receptors and is therefore a good animal model for studying the relationships of coronary atherosclerosis, hypercholesterolemia and coronary flow reserve. The aim of the present study was to assess myocardial perfusion at baseline and during adenosine infusion (0.2 mg x kg(-1) x min(-1)) in 8 WHHL rabbits (13.8+/-0.5 months) with 13N-ammonia, small-animal positron emission tomography (PET) and colored microspheres. Results were compared with those from 6 age-matched Japanese white rabbits. Plaque distribution was also examined in the extramural coronary arteries. All 8 WHHL rabbits had coronary plaques, with 6 showing multiple plaques. Mean global myocardial blood flow (ml x min(-1) x g(-1)) did not differ significantly between control and WHHL groups both at baseline (3.67+/-0.72 vs 4.26+/-1.12 ml x min(-1) x g(-1), p=NS) and with adenosine (7.92+/-2.00 vs 9.27+/-2.91 ml x min(-1) x g(-1), p=NS), nor did coronary flow reserve (2.16+/-0.37 vs 2.18+/-0.41, p=NS). None showed evidence of regional perfusion abnormalities by visual and semiquantitative analyses of PET images. It was concluded that WHHL rabbits preserve adenosine-induced coronary flow reserve despite coronary atherosclerosis and hypercholesterolemia, suggesting that a compensatory mechanism develops in this animal model.

Adenosine↗
PubMed2000

Production of transgenic rabbits using centrifuged pronuclear zygotes.

Superovulation of female rabbits was induced by subcutaneous injection(s) of porcine FSH. Zygotes were recovered 17 to 19 hr after hCG injection and were classified into two categories under a microscope equipped with Nomarski interference-contrast optics at x 200 magnification: (A) zygotes with clearly visible pronuclei, or (B) zygotes with visualized pronuclei after 10 min centrifugation at 12,000 x g. No significant difference between strains was found in the proportion of category-A zygotes (JW 72.6% vs NZW 79.3%). Pronuclei of category-A zygotes were located in the center of the cytoplasm, and the pronuclei of category-B zygotes were slightly moved by centrifugation toward the mass of cytoplasmic lipid droplets. Exogenous DNA solution (5 microg/ml of fusion gene composed of bovine alphaS1-casein promoter and human growth hormone structural gene) was microinjected into the pronucleus of the JW zygotes. The pronucleus of category-A zygotes with a mean volume of 7.4 pl swelled up to 16.6 pl (132% increase), while that of category-B zygotes with a mean volume of 6.1 pl swelled up to 15.9 pl (148% increase). Nevertheless, similar proportions of category-A and category-B zygotes developed into offspring after transfer to recipient females (11.1 and 11.2%, respectively). The efficiency to produce hGH-carrying transgenic rabbits was 0.9% (2/235) from category-A zygotes and 0.5% (1/215) from category-B zygotes (P>0.05). To date, transgenic rabbits have been produced without centrifugation of pronuclear zygotes. However approximately 25% of fertilized rabbit zygotes can be used for DNA microinjection after they have been centrifuged to visualize their pronuclei.

Animals↗
PubMed2000

Nuclear transfer in cattle : effect of linoleic acid-albumin on freezing sensitivity of enucleated oocytes.

The effect of linoleic acid-albumin (LAA) supplementation to the media for IVM, enucleation, and activation on the developmental potential of bovine embryos produced by nuclear transfer (NT) into frozen-thawed cytoplasts was investigated. Blastomeres derived from morulae was placed in the perivitelline space of frozen-thawed cytoplasts, which were then fused by a DC pulse. The proportion of fused embryos was similar between groups with and without LAA (87 vs. 90%). The proportion of development to blastocysts of NT embryos derived from the media with LAA (14%) was higher than that without LAA (4%), indicating that LAA treatment of bovine oocytes during IVM, enucleation and activation can improve the ability of such cytoplasts after freezing and thawing to develop into blastocysts after NT.

Albumins↗
PubMed2000

Sorting of peroxisomal and mitochondrial carnitine acetyltransferase isozymes in the diploid yeast, Candida tropicalis.

Peroxisomal and mitochondrial carnitine acetyltransferase (CAT; EC 2.3.1.7) isozymes are synthesized from the first and second ATG codons of the open reading frame of one gene, Candida tropicalis CAT. Primer extension analysis and RNase protection assay revealed that the peroxisomal CAT, initiating at the second AUG codon of the transcripts, was synthesized by a translational readthrough of the first AUG codon of the open reading frame. When C. tropicalis CAT was introduced into the other yeast, Saccharomyces cerevisiae, 5' ends of transcripts were similar to those observed in C. tropicalis. Peroxisomal and mitochondrial CAT isozymes were strongly suggested to occur by the alternative initiation of translation, chiefly dependent on the structure or sequence context of the region from the 5' end to the second AUG codon; their transcripts harbored sufficient information to bring about alternative initiation of translation in both yeasts. Sorting of peroxisomal and mitochondrial CAT isozymes to their own compartment was carried out by their own targeting sequences, but, before transportation to their destination, their biosyntheses were regulated by alternative initiation of translation.

Amino Acid Sequence↗
PubMed2000

Genetic evaluation of peroxisomal and cytosolic acetoacetyl-CoA thiolase isozymes in n-alkane-assimilating diploid yeast, Candida tropicalis.

The n-alkane-assimilating diploid yeast, Candida tropicalis, possesses two acetoacetyl-CoA thiolase (Thiolase I) isozymes encoded by one allele: peroxisomal and cytosolic Thiolase Is encoded by both CT-T1A and CT-T1B. To clarify the function of peroxisomal and cytosolic Thiolase Is, the site-directed mutation leading Thiolase I delta C6 without a putative C-terminal peroxisomal targeting signal was introduced on CT-T1A locus in the ct-t1b delta-null mutant. The C-terminus-truncated Thiolase I was active and solely present in the cytosol. Although the ct-t1a delta/t1b delta-null mutants showed mevalonate auxotrophy, the mutants having the C-terminus-truncated Thiolase I did not require mevalonate for growth, as did the strains having cytosolic Thiolase I. These results demonstrate that the presence of Thiolase I in the cytoplasm is indispensable for the sterol synthesis in this yeast. It is of greater interest that peroxisomal and cytosolic Thiolase I isozymes, products of the same genes, play different roles in the respective compartments, although further investigations will be necessary to analyze how to be sorted into peroxisomes and the cytosol.

Acetyl-CoA C-Acetyltransferase↗
PubMed2000

Gender-related difference in relationship between insulin resistance and serum leptin level in Japanese type 2 diabetic and non-diabetic subjects.

It remains controversial whether or not a correlation exists between serum leptin levels and insulin resistance, and, if such a correlation does exist, whether it is independent of adiposity. To investigate the possible existence of an independent correlation, we have assessed serum leptin levels and insulin resistance in Japanese diabetic and non-diabetic subjects by means of Homeostatic Model Assessment (HOMA-R). Sixty-four Japanese patients with Type 2 diabetes mellitus (DM) (33 men and 31 women) and 53 sex-, age-, and body mass index (BMI)-matched non-diabetic adults (29 men and 24 women) were enrolled. The fasting plasma level of glucose (FPG) and the fasting serum levels of immunoreactive insulin (FIRI) and leptin were determined. Multiple linear regression analysis demonstrated that, in both male diabetic and male non-diabetic subjects, HOMA-R and BMI were independently correlated with serum leptin levels. In females, BMI, but not HOMA-R, was correlated to the serum levels of leptin in both groups. There was no statistically significant difference in the partial regression coefficients between male diabetic and male non-diabetic subjects. These results suggest that the correlation of HOMA-R to the serum levels of leptin in females is dependent on BMI. In males, the relationship between serum leptin levels and the insulin resistance was not affected by the extent of glucose intolerance.

Adult↗
PubMed2000

Generation of transgenic rats with YACs and BACs: preparation procedures and integrity of microinjected DNA.

The aim of the present study was to investigate differences in the methods for preparing a large DNA fragment to be used for making transgenic rats from the standpoint of transgenic production efficiency and integrity of the introduced gene. In yeast artificial chromosome (YAC) transgenesis, three methods for preparing DNA for microinjection were compared: amplification of YAC in yeast (AMP), amplification of YAC in yeast and removal of the amplification element (AMP/RE), and no amplification of the YAC in yeast (AMP-). Production efficiency per microinjected ovum with DNA by the AMP method was four times higher than that by the AMP/RE and AMP-. Based on these results, we favor the AMP method in spite of the thymidine kinase gene-induced male sterility. In bacterial artificial chromosome (BAC) transgenesis, linear DNA fragments for microinjection prepared by three kinds of purification procedures were compared: Not I digestion and CsCl gradient ultra-centrifugation (Prep. 1), CsCl gradient ultra-centrifugation, Not I digestion, gel electrophoresis, and beta-agarase digestion (Prep. 2), and CsCl gradient ultra-centrifugation, Not I digestion, pulse field gel electrophoresis, and beta-agarase digestion (Prep. 3). Although the efficiency of producing transgenic rats was similar with all these three DNA preparations, integration of the intact DNA fragment only occurred with the Prep. 3 procedure. We therefore favor the Prep. 3 method for preparing BAC DNA fragments. These results indicate that the method used to prepare a large DNA fragment such as YAC and BAC DNAs is important in order to produce transgenic rats with an intact transgene.

Animals↗
PubMed2000

Clinical evaluation of vascularized bone grafts and osseointegrated implants.

We evaluated mandibular rehabilitation using vascularized bone graft and osseointegrated implants. Questionnaires were used to evaluate the masticatory function, and we measured the occlusal force in each patient. In addition, we measured the height of grafted bone to assess the possible relationship between masticatory rehabilitation and the change in bony height. Five of 13 patients showed over 12.0% increase in bony height after superstructure fabrication. Most of the patients who underwent tongue resection scored low points on the questionnaire. Also, most patients with resection, including resection of the angles of the mandible, showed a lower occlusal force than those without.

Adult↗
PubMed2000

Effects of exercise experienced in the life stages on climacteric symptoms for females.

The purpose of this study is to investigate the effects of exercise experienced in the life stages on climacteric symptoms for females after menopause. Four-hundred and eight postmenopausal women completed a questionnaire. The results were as follows: (1) Mean age at menopause +/- standard deviation was 50.1 +/- 0.5 and did not show a significant relationship with the degree of exercise in the life stages. (2) The degree of climacteric symptoms had a significant relationship, or a tendency toward a significant relationship, with the degree of exercise in and after the 40's; and the greater the degree of exercise, the lesser the degree of climacteric symptoms. (3) Kupperman's index was found to be, or tended to be, significantly related to the degree of exercise in and after the 30's. Those who exercised heavily in their 30's showed a significantly lower Kupperman's index. Those who answered that they had exercised "moderately" in their "40's to menopause" and "menopause to 60 years old" tended to have the lowest index. (4) Exercise experience in the life stages was negatively correlated, in particular, to psychosomatic symptoms among the 3 climacteric symptom categories. This negative correlation tended to be higher in those who answered that they had done "less exercise" in and after their 30's. (5) A significant relationship was noted between the degree of exercise in the 30's and "weakness" in Kupperman's index, and between exercise in and after the 40's and "nervousness" and "melancholia". Therefore, it is suggested that exercising "moderately" from the subjective viewpoint in the climacteric period may alleviate psychosomatic symptoms.

Adult↗
PubMed2000

Correlation between erythropoietin and lactate in humans during altitude exposure.

The plasma concentrations of both immunoreactive erythropoietin (EPO) and lactate were determined in four healthy untrained subjects at sea level and on the 2nd or 3rd day at altitudes (1,300 and 3,500 m). The mean plasma EPO (18.8 +/- 1.6 mU/ml at sea level) increased significantly on the 3rd day at 1,300 m (25.5 +/- 2.0 mU/ml, p < 0. 05) and showed an almost three-fold increase on the 2nd day at 3,500 m (53.5 +/- 3.7 mU/ml, p < 0.001). Likewise, the mean plasma lactate at 3,500 m (3.98 +/- 0.27 mmol/l) was 3.6 times as high as that at sea level (1.11 +/- 0.05 mmol/l) (p < 0.001). The plasma EPO concentrations were found to correlate well with the lactate concentrations at sea level and altitudes (r = 0.86, p < 0.01). These results are consistent with the well-known EPO/lactate response to altitudes and suggest that the circulating EPO concentration as well as blood lactate concentration can be used as an index of anaerobic condition.

Adult↗
PubMed2000

mRna expression and transport characterization of conditionally immortalized rat brain capillary endothelial cell lines; a new in vitro BBB model for drug targeting.

Brain capillary endothelial cell lines (TR-BBB) were established from a recently developed transgenic rat harboring temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat) and used to characterize the endothelial marker, transport activity, and mRNA expression of transporters and tight-junction strand proteins at the blood-brain barrier (BBB). These cell lines expressed active large T-antigen and grew well at 33 degrees C with a doubling-time of about 22-31 hr, but did not grow at 39 degrees C. TR-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated low-density lipoprotein uptake activity. Although the gamma-glutamyltranspeptidase activity in TR-BBBs was approximately 13% of that of the brain capillary fraction of a normal rat, it was localized in the apical side, suggesting that it reflects the functional polarity of the in vivo BBB. The mRNA of tight-junction strand proteins such as claudine-5, occludin, and junctional adhesion molecule are expressed in TR-BBB13. Drug efflux transporter, P-glycoprotein, with a molecular weight of 170 kDa was expressed in all TR-BBBs and mdr 1a, mdr 1b, and mdr 2 mRNA were detected in TR-BBBs using RT-PCR. Moreover, mrp1 mRNA was expressed in all TR-BBBs. Influx transporter, GLUT-1, expressed at 55 kDa was revealed by Western blot analysis. It had 3-O-methyl-D-glucose (3-OMG) uptake activity which was concentration-dependent with a Michaelis-Menten constant of 9.86 +/- 1.20 mM. The mRNA of large neutral amino acid transporter, which consists of LAT-1 and 4F2hc was expressed in TR-BBBs. In conclusion, the conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB) had endothelial makers, expressed mRNA for tight-junction strand proteins and the influx and efflux transporters and produced GLUT-1, which is capable of 3-OMG transport activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗