Biomedical subjects

M Ueda

Publications and source records attributed to M Ueda.

At least 127 records · Page 7Linked to original sources

PubMed2001

Intelligent yeast strains with the ability to self-monitor the concentrations of intra- and extracellular phosphate or ammonium ion by emission of fluorescence from the cell surface.

Saccharomyces cerevisiae strains that respond to environmental changes and transmit the information by emission of fluorescence from the cell surface were constructed. The technique of cell surface engineering enabled the yeast cells to display enhanced cyan blue fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) on the surface under the control of promoters that sense environmental changes. Two model promoters were examined in this study. For monitoring the intra- and extracellular concentrations of phosphate ion, the PHO5 promoter was chosen to display ECFP. The MEP2 promoter was used to display EYFP to sense the concentrations of ammonium ion. Fluorescence was observed by fluorescence microscopy and immunofluorescence microscopy, and the intensity was measured by a flow cytometer. The relationship between ion concentration inside and outside the cells was evaluated by the change in the rate of fluorescence. This S. cerevisiae system enables environmental changes to be transmitted as intra- and extracellular information using a suitable promoter functioning at real time and in a non-invasive manner.

Bacterial Proteins↗

PubMed2001

Function of the prosequence for in vivo folding and secretion of active Rhizopus oryzae lipase in Saccharomyces cerevisiae.

The role of the prosequence of Rhizopus oryzae lipase (ROL) with a preprosequence was analyzed by an expression system using Saccharomyces cerevisiae. When the mature portion of ROL (mROL) fused to the pre-alpha-factor leader sequence was expressed, secretion of active mROL was not observed. However, when mROL was synthesized together with the prosequence in trans (individually and coincidentally), secretion of active mROL was observed. The results indicate that the prosequence of ROL helped correct folding of mROL and its subsequent secretion from the yeast cells, and that physical linkage (cis) of the prosequence to the mature region was not prerequisite. From the expression of the ROL mutants with deletions at the N-terminal end of the prosequence together with mROL in trans, the residues from 20 to 37 in the prosequence were essential for the secretion, and those from 38 to 57 were essential for the formation of the active ROL and might play a role as an intramolecular chaperone. The results using the fragment of the prosequence confirmed that these residues (20-57) were significant for in vivo folding and secretion of active mROL.

Amino Acid Sequence↗

PubMed2001

Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by fluorometric and confocal laser scanning microscopic analyses.

The number of foreign protein molecules expressed on the cell surface of the budding yeast Saccharomyces cerevisiae by cell surface engineering was quantitatively evaluated using enhanced green fluorescent protein (EGFP). The emission from EGFP on the cell surface was affected by changes in pH. The amount of EGFP on the cell surface, displayed as alpha-agglutinin-fusion protein under control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, was determined at the optimum pH of 7.0. The fluorometric analysis and the image analysis by confocal laser scanning microscopy (CLSM) showed a similar number of molecules displayed on the cell surface, demonstrating that 10(4)-10(5) molecules of alpha-agglutinin-fused molecules per cell were expressed. Furthermore, the amount of fluorescent protein expressed on cells harboring a multicopy plasmid was three to four times higher than that on cells harboring the gene integrated into the genome.

Arabidopsis↗

PubMed2001

Repression of fatty-acyl-CoA oxidase-encoding gene expression is not necessarily a determinant of high-level production of dicarboxylic acids in industrial dicarboxylic-acid-producing Candida tropicalis.

The synthesis of dicarboxylic acids (DCAs) in Candida tropicalis is thought to be induced by a decrease in fatty acyl-CoA-oxidase activity. However, in the present study we demonstrate that repression of the POX4 gene, encoding fatty acyl-CoA oxidase, does not directly lead to high-level production of DCAs. No fatty acyl-CoA-oxidase activity was detected if the POX4 gene of C. tropicalis strain 1098 (wild-type strain) was disrupted. Furthermore, introduction of the POX4 gene from C. tropicalis strain M1210A3, which is a mutant derived from strain 1098 and is used as an industrial DCA-producing strain, still exhibited low-level fatty acyl-CoA-oxidase activity. Nevertheless, production of DCA was not observed in either case. Furthermore, the increase in acyl-CoA-oxidase activity by expression of the POX4 gene in strain M1210A3 did not reduce high-level production of DCA. These results suggest that alterations in acyl-CoA-oxidase activity are not necessarily related to production of DCA in industrial DCA-producing C. tropicalis M1210A3.

Acyl-CoA Oxidase↗

PubMed2001

Spacer-mediated display of active lipase on the yeast cell surface.

We have constructed a Saccharomyces cerevisiae strain displaying an active lipase on the cell surface by cell surface engineering. The gene encoding Rhizopus oryzae lipase (ROL) was fused with the genes encoding the pre-alpha-factor leader sequence and the C-terminal half of alpha-agglutinin including the glycosylphosphatidylinositol-anchor attachment signal. The constructed gene was overexpressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. Linker peptides (spacers) consisting of the Gly/Ser repeat sequence were inserted at the C-terminal portion of ROL to enhance lipase activity by preserving the conformation of the active site near the C-terminal portion. Localization of the expressed ROL on the cell surface was confirmed by immunofluorescence microscopy. The ROL displayed on the yeast cell wall exhibited activity toward soluble 2,3-dimercaptopropan-1-ol tributyl ester (BALB) and insoluble triolein. The insertion of linker peptides effected the activity towards BALB, thereby demonstrating that the optimal length of linker peptides was present. The activity towards triolein was higher in lipases with longer linker peptides. ROL displayed on the cell wall exhibited a comparable and/or higher activity towards triolein than the secreted form of the enzyme. This is the first report of an active lipase displayed on the cell surface. Furthermore, insertion of a linker peptide of the appropriate length as a spacer may be an improved method to effectively display enzymes, especially those having the active region at the C-terminal portion, on the cell surface.

Agglutinins↗

PubMed2001

Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production.

Yeast whole-cell biocatalysts for lipase-catalyzed reactions were constructed by intracellularly overproducing Rhizopus oryzae lipase (ROL) in Saccharomvces cerevisiae MT8-1. The gene encoding lipase from R. orvzae IFO4697 was cloned, and intracellular overproduction systems of a recombinant ROL with a pro-sequence (rProROL) were constructed. When rProROL from R. oryzae IFO4697 was produced under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL) at 30 degrees C for 98 h by two-stage cultivation using SDC medium (SD medium with 2% casamino acids) containing 2.0% and 0.5% glucose, intracellular lipase activity reached levels up to 474.5 IU/l. These whole-cell biocatalysts were permeabilized by air-drying and used for the synthesis of methyl esters (MEs), a potential biodiesel fuel, from plant oil and methanol in a solvent-free and water-containing system. The ME content in the reaction mixture was 71 wt% after a 165-h reaction at 37 degrres C with stepwise addition of methanol. These results indicate that an efficient whole-cell biocatalyst can be prepared by intracellular overproduction of lipase in yeast cells and their permeabilization.

Amino Acid Sequence↗

PubMed2001

Creation of cell surface-engineered yeast that display different fluorescent proteins in response to the glucose concentration.

We have successfully created a novel yeast strain able to monitor changes in environmental conditions by displaying either green fluorescent protein (GFP) from Aequorea victoria or blue fluorescent protein (BFP), a variant of GFP, on its cell surface as a visible reporter. For the display of these fluorescent proteins on the cell surface of Saccharomyces cerevisiase, our cell-surface-engineering system was utilized. The GAPDH promoter, which is active in the presence of glucose, and the UPR-ICL promoter from Candida tropicalis, which starts to function in the presence of a reduced level of glucose, were employed simultaneously to express the GFP-encoding gene and the BFP-encoding gene, respectively. This cell-surface-engineered yeast strain emitted green fluorescence from the cell surface when sufficient glucose was present in the medium, and blue fluorescence from the same cell surface when the glucose in the medium was consumed. The fluorescent proteins displayed on the cell surface using the different promoters enabled us to monitor the concentrations of intra- and/or extracellular glucose that regulated activation or inactivation of the promoters. This novel yeast strain could facilitate the computerized control of various bioprocesses measuring emitted fluorescence.

Genetic Engineering↗

PubMed2001

Development of novel whole-cell immunoadsorbents by yeast surface display of the IgG-binding domain.

The ZZ domain derived from Staphylococcus aureus, which binds to the Fc part of immunoglobulin G (IgG), was displayed on the cell surfaces of yeast Saccharomyces cerevisiae by cell-surface engineering using the C-terminal half of alpha-agglutinin under control of the 5'-upstream region of the isocitrate lyase gene from Candida tropicalis (UPR-ICL). Display of ZZ on the cell surface was confirmed by immunofluorescence microscopy. Enzyme-linked immunosorbent assay (ELISA) and sandwich ELISA using the S. cerevisiae cells displaying ZZ detected IgG and antigen (human serum albumin) down to a concentration of 1-10 ng/ml in both cases. The detection range covered by these assay systems was wide and could be varied by adjusting the amount of cells and reaction times with horseradish peroxidase (HRP) substrate. Moreover, yeast cells displaying ZZ were successfully used for repeated affinity purification of IgG from serum. These results indicate that S. cerevisiae displaying ZZ may constitute novel and genetically renewable whole-cell immunoadsorbents widely applicable to immunoassays and affinity purification.

Animals↗

PubMed2001

Cell surface-engineered yeast displaying a histidine oligopeptide (hexa-His) has enhanced adsorption of and tolerance to heavy metal ions.

A histidine oligopeptide (hexa-His) with the ability to chelate divalent heavy metal ions was displayed on the yeast cell surface for the purpose of enhanced adsorption of heavy metal ions. We genetically fused a hexa-His-encoding gene with the gene encoding the C-terminal half of alpha-agglutinin that includes a glycosylphosphatidylinositol anchor attachment signal sequence and attached the hexa-His peptide on the cell wall of Saccharomyces cerevisiae. This surface-engineered yeast adsorbed three to eight times more copper ions than the parent strain and was more resistant to copper (4 mM) than the parent (below 1 mM at pH 7.8). It was possible to recover about a half of the copper ions adsorbed by whole cells with EDTA treatment without disintegrating the cells. Thus, we succeeded in constructing a novel yeast cell with both tolerance to toxic contaminants and enhanced adsorption of metal ions onto the cell surface.

Adsorption↗

PubMed2001

Role of esophagectomy in treatment of esophageal carcinoma with clinical evidence of adjacent organ invasion.

With carcinoma of the thoracic esophagus, clinical evidence of invasion of adjacent organs (T4) indicates a highly advanced stage, and most surgeons avoid esophagectomy. Although the therapeutic strategy for such disease is generally selected based on preoperative evaluation and intraoperative inspection, their accuracy and the relation to survival outcomes after esophagectomy have seldom been analyzed on the basis of exact histopathologic evidence. We performed esophagectomy, with perioperative adjuvant therapy when possible, on patients with clinical-T4 tumors unless absolutely unresectable conditions were detected. Among the 500 patients who underwent esophagectomy, the 78 patients whose tumors were confirmed to be T4 pathologically were compared with patients whose tumors were assessed as T4 preoperatively or intraoperatively to evaluate the role of esophagectomy for clinical-T4 carcinoma. Esophagectomy was possible for 99% of the pathologic-T4 tumors preoperatively assessed as resectable, but the resection was grossly incomplete in 35%. The true-positive rates in tumors preoperatively and intraoperatively assessed as T4 were 51% and 84%, respectively. The hospital mortality rate in patients with pathologic-T4 tumors was 4%. The overall 5-year survival rate for patients with pathologic-T4 tumors was 14%, compared with 60% for those with tumors assessed as T4 intraoperatively but not pathologically. Esophagectomy with perioperative adjuvant therapy yielded occasional cure with an acceptable mortality rate for patients with pathologic-T4 tumors assessed as technically resectable. Preoperative assessment and intraoperative macroscopic inspection had limitations for predicting pathologic-T4 disease and incomplete resection. Only patients with definitive evidence of unresectability should be excluded from esophagectomy.

Carcinoma, Squamous Cell↗

PubMed2001

MR imaging findings of uterine endometrial stromal sarcoma: differentiation from endometrial carcinoma.

Endometrial stromal sarcoma (ESS) is a rare malignant neoplasm of the uterus. We retrospectively analyzed pelvic MR imaging in 8 cases of ESS to determine the characteristic MR findings of ESS. Magnetic resonance images of 8 cases of ESS were evaluated for findings including the size, margin of the tumor, nodular lesions at the tumor margin, intramyometrial worm-like nodular extension, multiple nodular mass formation, hemorrhage, and necrosis in the tumor. The degree of contrast enhancement was also analyzed. The MR imaging findings of 21 consecutive cases of endometrial carcinoma (EC) were also evaluated and compared with those of ESS. The mean and the standard deviation of the maximum diameter of ESS and EC were 8.80 +/- 4.99 and 3.93 +/- 2.47 cm, respectively. Increased enhancement in at least a part of the tumor was observed in five of the six analyzed ESS cases and in 2 of 12 analyzed EC cases. The irregular margin, nodular lesions at the margin, intramyometrial nodular extension, and multiple nodular mass formation were more frequently seen in cases of ESS than in cases of EC. Magnetic resonance imaging can play a role in both diagnosing ESS as well as in differentiating ESS from EC.

Aged↗

PubMed2001

Indications for surgical treatment of funnel chest by chest radiograph.

Forty-seven children with funnel chest (FC) who underwent sternal elevation and 210 normal children were examined to determine the indications for surgical treatment using the vertebral index (VI) and frontosagittal index (FSI). In normal children VI gradually increased and FSI gradually decreased with age. Both indices changed significantly at 3 years of age. Although the VI of FC patients decreased significantly from 33.8 +/- 7.6 (n=40) to 24.4 +/- 3.9 (n=38) postoperatively (P < 0.0001), it was significantly larger than that of normal children over 3 years of age (20.2 +/- 2.2, n=150) (P < 0.0001), and although the FSI of FC patients increased significantly from 22.0 +/- 7.0 (n=40) to 34.5 +/- 6.5 (n=38) postoperatively (P < 0.0001), it was significantly smaller than that of normal children over 3 years of age (41.1 +/- 4.0, n=150) (P < 0.0001). Since many patients had a thin and flat chest despite excellent correction, their postoperative indices were not normal. There was a correlation between VI and FSI in normal children and a high degree of correlation between VI and FSI both before and after operation in FC patients. We conclude that a VI of more than 27 and/or a FSI of less than 29 are indications for surgical treatment based on the mean VI + 3SD and FSI - 3SD of normal children over 3 years of age. These values are almost equal to the mean VI - SD and FSI + SD of patients with physical, cosmetic, and/or psychological disturbances. However, it is not necessary to measure both indices simultaneously. Postoperative VI and FSI did not always reflect the degree of chest-wall depression in FC patients because of their flat chests.

Adolescent↗

PubMed2001

Living-donor liver transplantation for Caroli's disease with intrahepatic adenocarcinoma.

A 36-year-old woman who had Caroli's disease with refractory cholangitis and complicated intrahepatic cholangiocarcinoma was successfully treated with living-donor liver transplantation. Preoperative computed tomography and ultrasonography showed a small nodule in the dilated intrahepatic bile duct. In the resected liver specimen, a small papillary tumor was located in the dilated intrahepatic bile duct of the right lobe. The pathological finding revealed a well differentiated papillary adenocarcinoma without invasion to the parenchyma. The patient is currently doing well 2.5 years after transplantation, with no signs of recurrence of the disease. For Caroli's disease, we believe we can achieve good results with liver transplantation, not only for cholangitis but also for the carcinoma when it is localized in the liver and the patient is carefully followed up.

Adult↗

PubMed2001

Detection of IL-2 receptor gene expression in peripheral blood from renal transplant patients.

Cytokine monitoring is a potentially useful tool in the management of transplant patients. This study was conducted to analyze interleukin (IL)-1, tumor necrosis factor (TNF), IL-2, and IL-2 receptor gene expression by reverse transcriptase-polymerase chain reaction (RT-PCR) in peripheral whole blood from healthy volunteers and patients who underwent laparotomy or renal transplantation. In the patients who underwent laparotomy, IL-1 and TNF expression was detected postoperatively, but IL-2 and IL-2 receptor expression was not. IL-1, TNF, and IL-2 receptor gene expression was detected in all the renal transplant patients, including those with signs of rejection. No cytokine gene expression was detected preoperatively, pretransplant, or in the healthy volunteers. We were able to discriminate transplantation from laparotomy by the cytokine profile. These results indicate that our method may be a useful tool for the immunological monitoring of transplant patients.

Adult↗

PubMed2001

Heparin and the nonanticoagulant N-acetyl heparin attenuate capillary no-reflow after normothermic ischemia of the lung.

BACKGROUND: Ischemia-reperfusion injury of the lung frequently occurs after cardiopulmonary bypass, after pulmonary thromboendarterectomy, and especially after lung transplantation. Heparin is known to be protective in ischemia-reperfusion injury, but the risk for bleeding disorders may restrict its use in a variety of diseased conditions. Therefore, we tested the efficiency of nonanticoagulant N-acetyl (NA) heparin to protect from postischemic reperfusion injury of the lung. METHODS: Pentobarbital-anesthetized, mechanically ventilated Lewis rats were heparinized (100 IU/kg) before insertion of catheters. Additionally, animals received either heparin (200 IU/kg; n = 7), NA heparin (1.1 mg/kg; n = 7), or saline (control, n = 7) before ischemia. After normothermic ischemia for 50 minutes, the left lung was reperfused for 120 minutes, or until the death of the animal. The nonischemic right lung was excluded after 10 minutes of reperfusion. RESULTS: Survival rate at 120 minutes of reperfusion was 7 of 7 and 6 of 7 in the heparin and the NA-heparin group, but 0 in 7 in the control group (p < 0.01). At 30 minutes of reperfusion, PaO2, blood flow through the ascending aorta and mean systemic blood pressure were also significantly higher in the heparin and the NA-heparin group when compared with the control group (p < 0.05). Pulmonary vascular resistance was significantly lower in the heparin and the NA-heparin groups, and histologic examination of the lungs from these groups confirmed reperfusion of nutritive alveolar capillaries by the presence of red blood cells. Lack of red blood cells in the alveolar capillaries of lung specimens from the control group indicated failure of capillary reperfusion. CONCLUSIONS: Heparin and NA heparin exert similar protection against capillary no-reflow after normothermic ischemia of the lung. This implies that the protective effect of heparin is mediated by properties different from its anticoagulant activity. Thus the nonanticoagulant N-acetyl heparin may pose a safe new therapeutic approach in lung ischemia-reperfusion injury without increasing the risk of hemorrhagic complications.

Animals↗

PubMed2001

Thiazolidinediones, peroxisome proliferator-activated receptor gamma agonists, regulate endothelial cell growth and secretion of vasoactive peptides.

Insulin resistance has been highlighted as a common causal factor for glucose intolerance, hypertension and dyslipidemia, all of which are cardiovascular risk factors. A new class of antidiabetic agents, thiazolidinediones (TZDs), has been developed and demonstrated to improve insulin sensitivity. TZDs are high affinity ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), the crucial transcription factor for adipocytes. Recent studies showed that PPARgamma is also expressed in monocytes/macrophages and is suggested to be involved in atherosclerosis. We could detect PPARgamma gene transcript in several cultured endothelial cells (human aortic endothelial cells (HAoECs), human coronary artery endothelial cells (HCAECs), human umbilical vein endothelial cells (HUVECs) and bovine carotid artery endothelial cells (BAECs)) as well as human coronary arteries we examined. Since endothelial dysfunction is critical for atherosclerosis, we investigated the effects of TZDs, troglitazone (TRO) and pioglitazone (PIO), on endothelial cell growth and secretion of C-type natriuretic peptide (CNP), which we demonstrated as a novel endothelium-derived relaxing peptide, and endothelin (ET), a potent vasoconstrictor, using HAoECs, HCAECs, HUVECs and BAECs. When all these cultured endothelial cells were daily treated with TRO and PIO for 5 days, both TRO and PIO (10(-8)M) significantly stimulated (3)H-thymidine incorporation of all these endothelial cells. In contrast, higher dose of TRO and PIO (10(-5)M) significantly suppressed DNA synthesis. TRO and PIO also exerted the compatible effect on the increase of cell numbers. TRO and PIO significantly enhanced CNP secretion from BAECs. In contrast, ET secretion from BAECs was suppressed by both TRO and PIO in a dose-dependent manner. The results of the present study suggest that TZDs modulate endothelial functions, including regulation of endothelial cell growth and secretion of endothelium-derived vasoactive substances, which affect vascular tone and remodeling in the process of atherosclerosis.

Animals↗

PubMed2001

Low-dose ultraviolet B radiation synergizes with TNF-alpha to induce apoptosis of keratinocytes.

High-dose ultraviolet B (UVB) irradiation is known to induce apoptosis of keratinocytes, but low-dose UVB dose not. In this paper we present evidence that low-dose UVB can induce TNF-alpha-dependent apoptosis of keratinocytes. In our study, 5 mJ/cm(2) doses of UVB were not sufficient by themselves to induce apoptosis of cultured human keratinocytes, but 20 mJ/cm(2) doses of UVB were. The combination of 5 mJ/cm(2) doses of UVB and exogenous TNF-alpha (15 ng/ml) induced significant apoptosis of keratinocytes, although exogenous TNF-alpha without UVB did not. This phenomenon was accompanied by enhanced clustering of tumor necrosis factor receptor 1 (TNFR1). TNF-alpha's promotion of the induction of apoptosis by low-dose UVB was seen until 30 min after irradiation but not at 1 h. We confirmed this finding using a skin organ culture system. UVB (20 mJ/cm(2)), which did not induce transformation of epidermal keratinocytes into sunburn cells, induced apoptosis when TNF-alpha was added to the culture medium. These results suggest that one of the possible mechanisms of inducing keratinocyte apoptosis by low-dose UVB and TNF-alpha is that low-dose UVB augments ligand-binding-induced TNFR1 clustering, resulting in increased apoptotic cell death.

Antigens, CD↗

PubMed2001

Analysis of alloreactivity and intragraft cytokine profiles in living donor liver transplant recipients with graft acceptance.

Although some previous studies have indicated the possibility of immunosuppression withdrawal in clinical liver transplantation, the mechanism of graft acceptance is not clear. The aim of this study is to elucidate the alloreactivity against the donor and intragraft cytokine profiles in living donor liver transplant (LDLT) recipients with graft acceptance. In October 1999, we had 23 patients who survived without immunosuppression after LDLT with a median drug-free period of 25 months (range: 3-69 months). They consisted of six patients who were electively weaned by an elective weaning protocol and 17 either forcibly or accidentally weaned patients due to various causes but mainly due to infection. We evaluated the alloreactivity against the donor in these patients by a mixed lymphocyte reaction and intragraft cytokine profiles by real-time reverse transcriptase-polymerase chain reaction. The development of donor-specific hyporeactivity was observed in the patients with graft acceptance. The cytokine pattern in the supernatant of the culture medium revealed a down regulation of T helper (Th) 1 cytokine INF gamma against the donor while no significant difference was seen in Th2 cytokine IL-10. Regarding the intragraft cytokine profiles, we could find no amplification of Thl cytokines (IL-2, INF y) and IL-4 while some of the patients revealed a gene expression of IL-10 with no significant difference from that of the normal, untransplanted liver specimen. In addition, no difference was observed in any other cytokines (IL-1beta, IL-8, IL-15, TNFalpha) compared with those of the normal controls. We propose that the down regulation of Th1 cytokine is one possible mechanism of graft acceptance in LDLT recipients.

Administration, Oral↗