Biomedical subjects

Y Urata

Publications and source records attributed to Y Urata.

At least 55 records · Page 3Linked to original sources

PubMed1996

[Occurrence of numerical aberrations of chromosomes in metachronous liver metastases of colorectal cancers].

To investigate the change of numerical aberrations of chromosome 17 or 18 during progression of Colorectal carcinoma, we applied FISH, using chromosome specific probes, to 3 colorectal primary carcinomas and a total of 4 metachronous metastatic lesions in the liver. We also investigated the relationship between the DNA content of whole nucleus and the number of chromosome 17 by multiparametric analysis using autostage cytofluorometry. When compared with the results for primary lesions, in 4 metastatic lesions, the population of tetrasomy 17 cells increased in 2 lesions, and that of monosomy 18 cells increased in 1 lesion. The nuclear DNA contents histograms for disomy 17 and aneusomy 17 cells analyzed individually for 3 primary and 4 metastatic lesions, showed almost no appreciable difference of peak DNA values. These results indicate that the numerical aberrations of chromosome 17 and 18 accumulate during the progression of colorectal carcinomas, and that the subpopulation of aneusomy 17 consist mainly of those cells that involve the numerical aberrations in a few chromosomes.

Chromosome Aberrations↗

PubMed1996

[Clonal analysis of hepatocellular carcinoma].

We investigated the cell clonality of 12 cases of female solitary hepatocellular carcinoma (HCC) that were associated with hepatitis virus infection. The clonal origin of HCC could be assessed by the method based on restriction fragment length polymorphism (RFLP) of X-chromosome-linked androgen receptor gene (AR) and phosphoglycerate kinase (PGK) gene, taking advantage of random inactivation of one of two X-chromosomes by methylation in females. We extracted DNA samples from both fresh and paraffin-embedded specimens of the same lesion as a source of DNA sample for polymerase chain reaction (PCR). Consequently, it was possible to use methylation-sensitive restriction enzymes and PCR to study differential methylation patterns among alleles of these genes for both DNA samples. The RFLPs of AR gene and PGK gene were found in eight of 12 cases and five of 12 cases, respectively. There were two cases which had no RFLPs in either AR gene or PGK gene. All cases of HCC which had RFLP in either AR gene or PGK gene demonstrated monoclonal origin of the tumor regardless of their histologic patterns.

Aged↗

PubMed1995

Augmentation of transport for cisplatin-glutathione adduct in cisplatin-resistant cancer cells.

We studied the outward transport of cisplatin (CDDP)-glutathione (GSH) adduct (DDP-GSH) in CDDP-resistant cancer cells. Incubating the cells in the presence of CDDP resulted in the formation of an adduct with GSH and subsequent transport outside the cells. We used human colonic cancer cells sensitive (HCT8) and resistant (HCT8DDP) to CDDP and human ovarian cancer cells sensitive (A2780) and resistant (A2780DDP) to CDDP as materials. The concentration of intracellular GSH was higher in the resistant cells (118.7 +/- 5.9 nmol/10(6) HCT8DDP versus 19.0 +/- 1.0 nmol/10(6) HCT8 and 24.1 +/- 1.2 nmol/10(6) A2780DDP versus 9.4 +/- 0.5 nmol/10(6) A2780, respectively). The activity of the GSH-synthesizing enzyme, gamma-glutamylcysteine synthetase (gamma-GCS) was higher in the CDDP-resistant cells (7.1 +/- 0.2 milliunits/10(6) HCT8DDP versus 2.2 +/- 0.1 milliunits/10(6) HCT8 and 2.9 +/- 0.1 milliunits/10(6) A2780DDP versus 1.4 +/- 0.1 milliunits/10(6) A2780, respectively). Furthermore, immunological levels of gamma-GCS and the expression of gamma-GCS mRNA were higher in these CDDP-resistant cells than those in the control cells, in accordance with the change in the concentration of GSH. DDP-GSH transport increased in the CDDP-resistant colonic cancer cells by 219% (324 +/- 12 fmol/10(6) HCT8DDP cells/min versus 148 +/- 11 fmol/10(6) HCT8 cells/min) and the CDDP-resistant ovarian cancer cells by 126% (127 +/- 7 fmol/10(6) A2780DDP cells/min versus 101 +/- 8 fmol/10(6) A2780 cells/min). DDP-GSH transport was also estimated using inside-out vesicles from these cells. The active transport of DDP-GSH was 243% of HCT8 in HCT8DDP and 121% of A2780 in A2780DDP. These data suggest that the acquisition of CDDP resistance in cancer cells is due partly to the increase in the transport of DDP-GSH outside the cells as well as the increase in the concentration of GSH. Immunological estimation of the membrane proteins against human erythrocyte glutathione S-conjugate-stimulated Mg(2+)-ATPase sera resulted in no apparent cross-reactivity, suggesting that there are several transport systems for DDP-GSH.

Antineoplastic Agents↗

PubMed1995

Weakened cellular scavenging activity against oxidative stress in diabetes mellitus: regulation of glutathione synthesis and efflux.

Glutathione functions to scavenge oxidants or xenobiotics by covalently binding them and transporting the resulting metabolites through an adenosine 5'-triphosphate-dependent transport system. It has been reported that the intracellular concentration of glutathione decreases in diabetes mellitus. In order to elucidate the physiological significance and the regulation of anti-oxidants in diabetic patients, changes in the activity of the glutathione-synthesizing enzyme, gamma-glutamylcysteine synthetase, and transport of thiol [S-(2,4-dinitrophenyl)glutathione] were studied in erythrocytes from patients with non-insulin-dependent diabetes and K562 cells cultured with 27 mmol/l glucose for 7 days. The activity of gamma-glutamylcysteine synthetase, the concentration of glutathione, and the thiol transport were 77%, 77% and 69%, respectively in erythrocytes from diabetic patients compared to normal control subjects. Treatment of patients with an antidiabetic agent for 6 months resulted in the restoration of gamma-glutamylcysteine synthetase activity, the concentration of glutathione, and the thiol transport. A similar impairment of glutathione metabolism was observed in K562 cells with high glucose levels. The cytotoxicity by a xenobiotic (1-chloro-2,4-dinitrobenzene) was higher in K562 cells with high glucose than in control subjects (50% of inhibitory concentration 300 +/- 24 mumol/l vs 840 +/- 29 mumol/l, p < 0.01). Expression of gamma-glutamylcysteine synthetase protein was augmented in K562 cells with high glucose, while enzymatic activity and expression of mRNA were lower than those in the control subjects. These results suggest that inactivation of glutathione synthesis and thiol transport in diabetic patients increases the sensitivity of the cells to oxidative stresses, and these changes may lead to the development of some complications in diabetes mellitus.

Aged↗

PubMed1995

A three-dimensional structural dissection of Drosophila polytene chromosomes.

We have analyzed the three-dimensional structural details of Drosophila melanogaster polytene chromosome bands and interbands using three-dimensional light microscopy and a novel method of sample preparation that does not involve flattening or stretching the chromosomes. Bands have been visualized in unfixed chromosomes stained with the DNA specific dye 4,6-Diamidino-2-phenylindole (DAPI). Interbands have been visualized using fixed chromosomes that have been immunostained with an antibody to RNA polymerase II. Additionally, these structures have been analyzed using in situ hybridization with probes from specific genetic loci (Notch and white). Bands are seen to be composed of approximately 36 substructural features that measure 0.2-0.4 micron in diameter. We suggest that these substructural features are in fact longitudinal fibers made up of bundles of chromatids. Band shape can be a reproducible characteristic of a particular band and is dependent on the spatial relationship of these bundles, varying from bands with a uniform distribution of bundles to bands with a peripheral concentration of chromatin. Interbands are composed of bundles of chromatids of a similar size and number as those seen in the bands. The distribution of bundles is similar between a band and the neighboring interband, implying that there is a long range organization to the DNA that includes both the coding and the noncoding portions of genes. Finally, we note that the polytene chromosome has a circular shape when viewed in cross section, whether there are one or two homologs present.

Animals↗

PubMed1995

Significance of glutathione depletion and oxidative stress in early embryogenesis in glucose-induced rat embryo culture.

Recent studies have demonstrated the protective effects of supplementing free oxygen radical scavenging enzymes against hyperglycemia-induced embryonic malformations. In this study, the glutathione (GSH)-dependent protection system in hyperglycemia-induced embryopathy was investigated. Rat embryos at the early head-fold stage (day 9.5) cultured in 66.7 mmol/l glucose for 48 h showed significant growth retardation and an increase in the frequency of malformations. The concentration of GSH and activity of the rate-limiting GSH-synthesizing enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), significantly decreased in embryos exposed to hyperglycemia compared with controls (7.9 +/- 0.6 vs. 12.5 +/- 0.9 nmol/mg protein, P < 0.01 and 13.3 +/- 1.9 vs. 22.6 +/- 1.1 microU/mg protein, P < 0.01, respectively). Decreased activity of gamma-GCS in embryos exposed to hyperglycemia was associated with decreased expression of gamma-GCS mRNA levels. However, the activities of superoxide dismutase and glutathione peroxidase did not significantly change in these embryos. Extracellular and intracellular free oxygen radical formations estimated by Lucigenin-dependent chemoluminescence and flow cytometric analysis using 2',7'-dichlorofluorescein diacetate increased in isolated embryonic cells taken from embryos cultured under hyperglycemia. Supplementation of 2 mmol/l GSH ester into the hyperglycemic culture nearly restored GSH concentration in these embryos (11.9 +/- 0.5 vs. 12.5 +/- 0.9 nmol/mg protein) and reduced the formation of free oxygen radical species leading to almost complete normalization of growth retardation and embryonic dysmorphogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

[B-lymphoma arising in the temporal muscle].

B cell lymphoma arising in skeletal muscle is described with the review of literature. A 72-year-old man visited our hospital on September 21st 1992, because of a right temporal mass which had grown gradually since one year previously. A CT scan and MRI showed a soft tissue mass adjacent to the temporal muscle expanding from the right temporal to infratemporal fossa. Physical examination on admission revealed that the mass at the right temporal region, was non-tender, elastic soft, and 5 x 2 cm in diameter. Some elastic hard masses at the right infraauricular region, approximately 1.5 x 1.2 cm in diameter, were also found. Histological examination of a biopsied specimen obtained from the temporal mass revealed B cell lymphoma, diffuse medium-sized cell type. Tests for surface markers using tumor cell suspension showed positive results for CD5, CD19, CD20, SmIg mu, delta, lambda, but negative for CD10. Chromosomal analysis revealed clonal aberrations, and the defining karyotype as 51, X, +X, -Y, +2, +3, +4, +8, +12. The patient achieved a complete remission after treatment with combination chemotherapy including doxorubicin, cyclophosphamide, vincristine, etoposide, vindesine, procarbazine, and prednisolone.

Aged↗

PubMed1993

gamma-Glutamylcysteine synthetase and active transport of glutathione S-conjugate are responsive to heat shock in K562 erythroid cells.

Effect of heat shock on a glutathione-synthesizing enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), and ATP-dependent outward transport of glutathione S-conjugate was characterized using K562 erythroid cells. When K562 cells grown at 37 degrees C were shifted to 42 degrees C for 2 h, an approximate 1.7-fold increase in the activity of gamma-GCS was observed. Treatment of K562 cells with erythropoietin (EP) for 12 h resulted in a decrease in the activity of gamma-GCS to 64% of the control. However, responsiveness of this enzyme activity in the cells treated with EP to heat shock was similar to that in untreated cells. Changes in the immunological activity of gamma-GCS were also observed in parallel with those in the enzymatic activity. On Northern blot analysis of total RNAs isolated from the cells with human cDNA for gamma-GCS, a substantial induction of mRNA level was found by heat shock and a reduction of EP. These changes were modest but correlated to the mRNA expression of a heat shock protein, HSP 70. Heat shock also had an effect of 1.8-fold stimulation on glutathione S-conjugate transport in K562 cells previously incubated with 1-chloro-2,4-dinitrobenzene. Treatment of the cells with EP resulted in a decrease in this transport by 62%. Similarly, the levels of glutathione S-conjugate-stimulated Mg(2+)-ATPase, which enzyme is thought to be involved in the transport of glutathione S-conjugate, were responsive to heat shock and EP. These results suggest that glutathione synthesis and transport process of glutathione metabolites are responsive to heat shock and play a role in the defense system against stresses. It is also suggested that the regulatory site of the expression of these enzymes by heat shock is independent of that by EP.

Biological Transport, Active↗

PubMed1993

[Image analysis on isolated colorectal tumor cells].

Image analysis of the nuclear morphology was performed on propidium iodide-stained isolated cells from 9 adenomas and 9 cancers of the colorectum. We analyzed DNA content, seven nuclear geometric features and four shape factors of tumor cells using an image cytometry system that has been developed in our laboratory. Nuclear breadth and the degree of contour irregularity of cancer cells were found to be significantly different from those of adenoma cells. The coefficients of variation (CV) of some nuclear features and the degree of contour irregularity were increased in cancer cells, while CV of the degree of circularity was increased in adenoma cells. Moreover, nuclear in G2 phase were found to be larger and more round than those in G1 phase. These results suggest that cell-cycle-related analysis of nuclear features would be a valid means to assess the nuclear morphology of colorectal tumors.

Adenocarcinoma↗

PubMed1993

[DNA-cytofluorometric, histopathological and immunohistochemical analyses of pancreatoduodenal cancers].

To study the cell characteristics of relatively small pancreatoduodenal cancers (less than 4 cm in the greatest dimension in histopathological examination), we analyzed the relation between the cell kinetics, and histopathological and immunohistochemical findings. Tumors were classified into t1 (0-2 cm in its greatest dimension) and t2 (2-4 cm). We analyzed DNA contents of five tumors of t1 and twelve of t2 by cytofluorometry. Histopathological and immunohistochemical (CA19-9 and CEA) examinations were also carried out. Nuclear DNA content histograms of the tumors in the t1 group showed all diploid pattern, while those in the t2 group exhibited 4 diploid, 7 euploid polyploid and 1 aneuploid patterns. Invasions to the lymphatic (ly), vein (v) and nerve (pn) were found in five cases of diploid tumors (3 of t1 and 2 of t2) and all (8 in t2) of non-diploid tumors. A medullary pattern of tumor growth was only noted in three tumors of t1. This pattern was not found in t2 (4 diploid, 8 non-diploid). These results suggest that the cellular DNA content increased in association with the tumor growth in size. In the histopathological findings, both the invasions (ly, v, pn) and fibrosis of the stroma more often appeared as the tumor size and polyploid and aneuploid cells increased. However, other histopathological findings and the immunohistochemical examination for CA19-9 and CEA had no relation to the tumor growth and the DNA ploidy pattern.

Aged↗

PubMed1992

Cloning and nucleotide sequence of the cDNA encoding human 2-oxoglutarate dehydrogenase (lipoamide).

2-Oxoglutarate dehydrogenase (lipoamide) (( OGDH: 2-oxoglutarate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-succinylating), EC 1.2.4.2 )) is a component enzyme of the 2-oxoglutarate dehydrogenase complex. We have cloned a human cDNA encoding OGDH from a fetal liver cDNA library by plaque hybridization with a mixture of oligonucleotide probes designed from the amino acid sequences of porcine OGDH. This cDNA spans 4156 bases and contains an open reading frame of 3009 nucleotides encoding a presequence of 40 amino acid residues and a mature protein of 963 amino acid residues (Mr = 108,642). The size of the mRNA is approximately 4.2 kilobases. Comparison of the deduced amino acid sequence of the human OGDH with experimentally determined segments of porcine OGDH comprising 308 amino acid residues shows 93% sequence identity. The human OGDH has 37% sequence identity with 933 amino acid residues of the Escherichia coli OGDH and 40% sequence identity with 1014 residues of the yeast OGDH.

Amino Acid Sequence↗

PubMed1992

Proteinase-catalyzed activation of porcine heart muscle pyruvate dehydrogenase and identification of its cleavage site.

Porcine heart muscle pyruvate dehydrogenase (PDH, EC 1.2.4.1) with subunit composition alpha 2 beta 2 catalyzes the initial decarboxylation step of an oxidative decarboxylation sequence of pyruvate. Highly purified PDH, was further activated several-fold by limited digestion with trypsin, Staphylococcus aureus V8 proteinase (V8) or papain. The activation with these proteinases required about 10 min to attain a maximal level, lasted 1/2-2 h and thereafter decreased gradually. Addition of an inhibitor of each proteinase resulted in an immediate cessation of any further changes in the enzymatic activity. The optimal pH of the proteinase-activated PDH was not affected. Proteinases increased the maximum velocity and the apparent Km values for pyruvate, but the Hill coefficients for pyruvate were unchanged. Proteinase-activated PDH was capable of associating two other component enzymes to produce large unit resembling the native complex. The Coomassie brilliant blue stained gels after SDS-PAGE showed that the PDH alpha subunit (41 kDa) was cleaved by trypsin or V8 into two major fragments (31 and 10 kDa), whereas PDH beta was unaffected. By amino-terminal sequence analyses of these fragments the trypsin cleavage sites were identified as Arg-273 and Arg-282 and the V8 cleavage sites were Glu-277 and Glu-280.

Amino Acid Sequence↗

PubMed1992

Immunochemical detection of unrepaired cyclobutane-type pyrimidine dimers of DNAs extracted from human skin tumours.

Unrepaired cyclobutane-type pyrimidine dimers of DNA extracted from human skin tumours were examined by an immunoblotting method using polyclonal antibodies raised against UV-irradiated calf thymus DNA. A total of 40 DNA samples extracted from seven SCC lesions, two AK lesions, two lymphomas, one basal cell epithelioma, one eccrine poroma, one neurofibroma of Recklinghausen's disease, on verruca vulgaris, four femoral normal skins and white blood cells of 21 humans were studied by immunoblotting using this antibody. Two of the 40 DNAs examined, one from facial actinic keratosis (AK) and one from a squamous cell carcinoma (SCC) which developed form facial AK formed immunoprecipitates. It was found, using photoreactivation enzyme plus visible light, that both immunoprecipitates were cyclobutane-type pyrimidine dimers. In addition, immunofluorescent studies on AK tissue were positive in an immunoblotting assay and revealed that the unremoved photodamage in DNA remained in the nucleus of AK cells. These findings indicate that these tumour cells may be deficient in the enzyme function for repairing photoproduct damage. The unrepaired cyclobutane-type pyrimidine dimer in AK cells might reflect the genetic process in multistage carcinogenesis as well as in xeroderma pigmentosum.

Adult↗

PubMed1992

Molecular genetic aspects of human pyruvate dehydrogenase and its defect.

Genomic clones encompassing the entire genes for the human pyruvate dehydrogenase alpha and beta subunits (PDH alpha or beta) have been isolated by screening the leukocyte genomic libraries with a nick-translated human foreskin fibroblast PDH alpha or beta cDNA probe. These genomic clones were characterized by restriction enzyme analysis. extensive DNA sequencing and primer extension analysis. The PDH alpha gene spans 17.08 kilobases and is composed of 11 exons and 10 introns within its coding region. The 18-kilobase clone of PDH beta gene is composed of 10 exons and 9 introns. All intron-exon splice junctions of two genes follow the GT/AG rule. A total of seven Alu repeats in the PDH alpha gene were found in five introns and two Alu family in the PDH beta gene were found in intron 2 and 8. The 5'-flanking region of the PDH alpha gene contains typical CCAAT and TATA-like consensus promoter sequence and two Sp1 binding sequences. That of the PDH beta gene contains a TCAAT sequence but no TATA sequence. Primer extension analyses indicated that the PDH alpha and beta genes transcription start sites are thymine and adenine residues located 124 and 132 bases upstream from initiation codon in exon 1, respectively. Genomic DNA of patient, died 93 hours after birth with acidemia and defect of PDH activity, was isolated and all of the exons of PDH alpha and beta genes were amplified by PCR.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1992

[Vacuolar inclusions with multivesicular structure in leukemic multiple myeloma].

A case of leukemic multiple myeloma with IgG-lambda type, which plasma cells in the peripheral blood and the bone marrow had large vacuolar inclusions is reported. A 67-year-old male was admitted because of bone pain. A diagnosis of leukemic multiple myeloma of IgG-lambda type was established, based on Bence Jones proteinuria (1.5 g/day), marked plasmacytosis in peripheral blood (63%) and bone marrow (90%), serum M-component (IgG-lambda type, 6.0 g/dl) and multiple osteolytic lesions. Most plasma cells in the bone marrow as well as in the blood had vacuolar inclusions in the cytoplasm which were 1-8 microns across and were negative with PAS and myeloperoxidase staining. Acid phosphatase reaction was distributed densely around vacuolar inclusions and sparsely within them in the form of fine granules. Ultrastructurally, most of the vacuolar inclusions were electron-lucent cytoplasmic spaces, encircled in a distinct limiting membrane, in which inner microvesicles were distributed diffusely. A few vacuoles showed fibrillary structures. These findings suggested that the lysosomal system might play a major role in the vacuolation of these plasma cells.

Aged↗

PubMed1992

[Progress in microscopic image analysis and the trend of image cytometry].

Image cytometry has recently developed prominently based on the digital imaging of cell morphology brought about by great advances in microcomputer hardware and software, electronics etc. This method is characterized by the quantitation of both the amount of intracellular bioactive materials and cell morphology, and is expected to further advance many cell analysis techniques involving automated cytology, chromosome image analysis, automated histopathology, viable cell analysis, 3D image construction, etc. The present use is still minimal, and advances in both hardware and software for cyto-histologic recognition are essentially necessary. Perhaps by the end of this century, more sophisticated and faster machines for automated cyto-histologic analysis will appear, replacing many microscopic techniques of presently subjective observation in the biomedical field.

Cell Cycle↗

PubMed1992

[An unusual case of localized form of primary macroglobulinemia developing from a nodular primary pulmonary amyloidosis].

The authors reported here a case of primary pulmonary amyloidosis, which developed into a localized form of primary macroglobulinemia (PMG) 10 years later. A nodular shadow was pointed out on routine chest x-ray films of a 61-year-old Japanese male in 1977. In 1981, a diagnosis of nodular primary pulmonary amyloidosis was made by percutaneous lung biopsy. At that time, he suffered from signs and symptoms of chronic cold agglutinin disease (CCAD). Cold agglutinins were IgM-kappa antibodies. In 1986, serum immunoelectrophoresis demonstrated the presence of a small amount of monoclonal IgM-kappa. In 1987, the patient was readmitted because of pleural effusions. In the pleural effusion, the IgM level was 3,341 mg/dl and the titer of cold agglutinin was 32,000. Cytological examinations of pleural effusion showed the proliferation of lymphocytes, lymphoid cells and plasma cells. These cells showed the monoclonality of IgM-kappa by the peroxidase-antiperoxidase method. At post-mortem examination in March 1988, PA and PMG were found, but both lesions were localized only in the thoracic cavity. It is suggested that primary amyloidosis (PA) as well as CCAD and monoclonal gammopathy of undetermined significance may be one of the pre-neoplastic conditions of PMG.

Aged↗

PubMed1992

[Analysis of cell proliferation kinetics and the effects of cisplatin on the cell cycle of human gastric cancer cells by autostage cytofluorometry].

Analysis of both cell proliferation kinetics and effects of cis-diamminedichloroplatinum (CDDP) on cell cycle in human gastric cancer cell line (HGC-Y2) by measuring the contents of nuclear DNA, RNA and the Ki-67 antigen using autostage cytofluorometry system was described. In HGC-Y2 cells, RNA content increased during the cell cycle and reached to the maximum at G2/M phase. The results of pulse treatment with CDDP on these cells demonstrated a prolongation of S phase and G2 arrest with increasing of RNA content of these cells. We classified the cells by intranuclear distribution pattern of Ki-67 antigen and thus could identified the cells at G0 and M phases from these classification. The content of Ki-67 antigen was moderate grade at G1 phase and it decreased in the early S phase, then increased gradually during S phase and at the late S phase. It increased rapidly, reaching to the maximum at G2/M phase. After CDDP treatment, the content of Ki-67 antigen increased in the cells in prolonged S phase and in the cells arrested at G2 phase. It was also found that the syntheses of both Ki-67 antigen and RNA were not inhibited by CDDP. These results suggest that the method using autostage cytofluorometry system was useful for the research, on the mechanism of cancer therapy because of making possible to analyze precisely the cell cycle and the influence of anticancer drugs.

Cell Cycle↗