Biomedical subjects

A Yasui

Publications and source records attributed to A Yasui.

At least 55 records · Page 3Linked to original sources

PubMed1997

Suppression of pulmonary metastasis in murine B16 melanoma cells by transfection of a sialidase cDNA.

A cytosolic sialidase cDNA was transfected into a highly metastatic and invasive cell line, B16-BL6, derived from the murine B16 melanoma. Stable transfection of a cytosolic sialidase expression vector yielded 4 transfectants with high content of the exogenous sialidase protein as well as enzyme activity. These transfectants exhibited markedly decreased experimental pulmonary metastasis, invasiveness in collagen gels and cell motility on colloidal gold-coated glass plates but no change in cell attachment to fibronectin, collagen type VI or laminin. To cast light on the underlying mechanisms, cellular constituents of the transfectants were analyzed. Sialidase over-expression did not lead to any significant changes in cell surface carbohydrates or intracellular glycoproteins, as revealed by lectin flow cytometry and lectin blotting, respectively. Thin layer chromatography of intracellular glycolipids, however, revealed decreased ganglioside GM3 and increased lactosylceramide as major changes.

Animals↗

PubMed1997

Cloning and characterization of mammalian 8-hydroxyguanine-specific DNA glycosylase/apurinic, apyrimidinic lyase, a functional mutM homologue.

8-Hydroxyguanine (8-OH-G) is one of the major DNA oxidation products implicated in mutagenesis induced by oxygen radical-forming agents, including ionizing radiation. It is also believed to be involved in spontaneous mutation induced by metabolically produced oxygen radicals. A mammalian homologue of 8-OH-G glycosylase/apurinic, apyrimidinic lyase (mutM homologue, MMH) has been identified in the EST database (for expressed sequence tags) through a homology search with yeast OGG1 protein. The human MMH protein (hMMH), 34% identical to the yeast OGG1 protein, is a member of the DNA repair protein superfamily. The hMMH gene was composed of seven exons, with the alternate last exon, exon 8, producing three major alternative splicing isoforms, because splicing of the sixth intron was optional. The hMMH protein expressed in Escherichia coli revealed the glycosylase activity and apurinic, apyrimidinic lyase activity on duplex DNA containing 8-OH-G. The hMMH protein can rescue a spontaneous mutator strain of E. coli lacking mutM and mutY. By the introduction of recombinant hMMH, the rate of mutation, the formation of rifampicin-resistant revertants, was reduced by 4-7 fold. Genomic structure analysis showed that 3' exons of the hMMH gene are transcribed on the antisense strand of the calcium-dependent calmodulin kinase 1 gene.

Alternative Splicing↗

PubMed1997

Characterization of the alternative excision repair pathway of UV-damaged DNA in Schizosaccharomyces pombe.

Schizosaccharomyces pombe cells deficient in nucleotide excision repair (NER) are still able to remove photoproducts from cellular DNA, showing that there is a second pathway for repair of UV damage in this organism. We have characterized this repair pathway by cloning and disruption of the genomic gene encoding UV damage endonuclease (UVDE). Although uvde gene disruptant cells are only mildly UV sensitive, a double disruptant of uvde and rad13 (a S. pombe mutant defective in NER) was synergistically more sensitive than either single disruptant and was unable to remove any photoproducts from cellular DNA. Analysis of the kinetics of photoproduct removal in different mutants showed that the UVDE-mediated pathway operates much more rapidly than NER. In contrast to a previous report, our genetic analysis showed that rad12 and uvde are not the same gene. Disruption of the rad2 gene encoding a structure- specific flap endonuclease makes cells UV sensitive, but much of this sensitivity is not observed if the uvde gene is also disrupted. Further genetic and immunochemical analyses suggest that DNA incised by UVDE is processed by two separate mechanisms, one dependent and one independent of flap endonuclease.

Cloning, Molecular↗

PubMed1997

Unstable expression of the multi-drug-resistant phenotype in Chinese hamster ovary cells resistant to okadaic acid.

A Chinese hamster ovary cell line resistant to okadaic acid (OA), OAR2-3 has a mutation of the protein phosphatase (PP) 2A alpha gene and expresses a multi-drug resistance (MDR) phenotype. In the present work, we isolated two additional OA-resistant variants, also showing MDR with a cross-resistance profile similar to that of OAR2-3, and with increased and decreased expressions of the P-glycoprotein (Pgp) and DNA topoisomerase (topo) II protein, respectively. Unlike OAR2-3, however, they had no mutation in the same region of the PP2A alpha gene. Except for OA-resistance in OAR2-3, the MDR was found to decrease in the absence of OA, and this decrease was again associated with changes in topo II- and Pgp-expressions. Thus, we conclude that 1) OA regulates the expressions of Pgp and topo II positively and negatively, respectively, resulting in reversible expression of MDR irrespective of genetic changes and 2) in OAR2-3, the mutation in the PP2A alpha gene confers stable resistance to OA. The MDR was also linked with collateral sensitivity to some drugs, like cisplatin and nitrogen mustard.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Recovery of portal blood flow after percutaneous transhepatic biliary drainage in patients with obstructive jaundice.

Using an ultrasonic Doppler system, we prospectively studied the changes in portal venous flow (PVF) following percutaneous transhepatic biliary drainage (PTBD) and evaluated the correlation between PVF and liver function in 10 patients with obstructive jaundice. The patients were divided into two groups according to their rate of decrease in serum bilirubin ("b"). Group A comprised 5 patients with a "b" of less than -0.1, while group B consisted of 5 patients who did not meet this criterion. The mean PVF increased following PTBD (P < 0.01). The increase in PVF was due to an increase in the maximum velocity of the portal vein (Vmax). The rate of increase in the Vmax in group A was significantly higher than that in group B on both the 7th and 14th postdrainage days (P < 0.05). The rate of increase in the Vmax correlated significantly with the rate of decrease in the serum bilirubin concentration (P < 0.01). Based on the above findings, we conclude that measuring the Vmax by Doppler ultrasonography is useful in evaluating the liver function in patients with obstructive jaundice.

Aged↗

PubMed1997

Crystal structure of DNA photolyase from Anacystis nidulans.

The crystal structure at 1.8 A resolution of 8-HDF type photolyase from A. nidulans shows a backbone structure similar to that of MTHF type E. coli photolyase but reveals a completely different binding site for the light-harvesting cofactor.

Amino Acid Sequence↗

PubMed1996

Cloning, tissue expression, and mapping of a human photolyase homolog with similarity to plant blue-light receptors.

Enzymatic photoreactivation is a DNA repair mechanism that removes UV-induced pyrimidine dimer lesions by action of a single enzyme, photolyase, and visible light. Its presence has been demonstrated in a wide variety of organisms, ranging from simple prokaryotes to higher eukaryotes. We have isolated a human gene encoding a 66-kDa protein that shows clear overall homology to known bacterial photolyase genes. The human gene product is more similar to plant blue-light receptors within class I photolyases than to higher eukaryote class II photolyases. Northern blot analysis showed two transcripts with constitutive expression in all tissues examined and an elevated expression in testis. In situ hybridization with a cDNA-derived probe localized this human gene to chromosome 12q23-q24.1. Southern analysis of the cloned human gene suggests a wide distribution of the gene family in various species.

Amino Acid Sequence↗

PubMed1996

Enhanced UV sensitivity of yeast cells induced by overexpression of Mg(2+)-dependent protein phosphatase alpha (type 2C alpha).

The UV sensitivity of wild-type Saccharomyces cerevisiae cells was increased 2-fold when rat Mg(2+)-dependent protein phosphatase alpha (protein phosphatase type 2C alpha) was overexpressed in the cells. The overexpression of this enzyme rendered the rad 18 mutant (defective in postreplication repair) more UV-sensitive than was observed in the wild-type cells. However, this increase in UV sensitivity disappeared when the host cells had a rad 1 mutation (defective in excision repair). These results suggest that the Mg(2+)-dependent protein phosphatase overexpressed in the yeast cells inhibited their excision repair system.

Animals↗

PubMed1996

Characterization of a UV endonuclease gene from the fission yeast Schizosaccharomyces pombe and its bacterial homolog.

From the fission yeast Schizosaccharomyces pombe, a cDNA fragment was isolated, which confers UV resistance on repair deficient Escherichia coli host cells. The cloned cDNA encodes a protein of 68,815 Da, which has a 36.6% identity of amino acid sequence with the previously identified 74 kDa UV endonuclease of the filamentous fungus Neurospora crassa. Analysis of several truncated gene constructs shows that only the C-terminal two thirds region, which has 54% identity of amino acid sequence with the C-terminal region of the Neurospora homolog, is necessary for complementing activity of UV-sensitivity in the E. coli host cells. Purified recombinant protein from E. coli host cells incises both UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts at the sites immediately 5' to the DNA damage in the same fashion as the Neurospora protein. Furthermore, a bacterial homologous sequence was isolated from Bacillus subtilis and shows a similar complementing activity of UV sensitivity in E. coli host cells, indicating a wide distribution of this alternative excision repair mechanism in life.

Amino Acid Sequence↗

PubMed1996

Postprandial responses of liver blood flow prior to and following hepatectomy in conscious dogs.

The responses of the portal and hepatic arterial blood flows to various diets and nutrients were measured simultaneously in conscious dogs prior to and following hepatic resection. Prior to hepatectomy, the increase in the portal blood flow was significantly larger in response to an elemental diet, fats, or amino acids than to glucose or water. The peak increase was 60.2 +/- 14.4 ml for water, 144.7 +/- 22.1 ml for a 150-cal elemental diet, 168.5 +/- 16.1 ml for a 300-cal elemental diet, 86.7 +/- 14.0 ml for a glucose solution, 159.3 +/- 16.7 ml for an amino acid meal, and 188.5 +/- 25.3 ml for a fat meal. Following partial hepatectomy, fats and amino acids induced an increase in the portal blood flow similar to that prior to hepatectomy. Glucose and the elemental diet, on the other hand, induced a significantly larger increase in portal blood flow following the surgery although water did not. The peak increase was 144.4 +/- 27.8 ml for glucose (166% of the peak increase prior to hepatectomy) and 221.8 +/- 32.5 ml for the 300-cal elemental diet (132%). The postprandial response of the hepatic artery to every diet was quite different among the dogs and there were no significant changes both prior to and following surgery. The different response of the portal flow to intraluminal glucose following partial hepatectomy may be due to alterations in glucose metabolism following hepatectomy. We have shown that the postprandial response of the portal blood flow varies with the type of nutrient, and it can be altered by hepatectomy.

Animals↗

PubMed1996

Changes in liver blood flow after hepatectomy in conscious dogs.

Hepatic circulation after hepatectomy was investigated in conscious dogs under fasting and feeding conditions. After a 40% hepatectomy, both the hepatic arterial and portal blood flow were measured simultaneously using ultrasonic transit time flowmeters. During fasting, the total hepatic blood flow (i.e., the sum of arterial and portal blood flow) changed in a biphasic pattern after hepatectomy. The first peak (517.9 +/- 42.7 ml/min; 130.1% of preoperative flow) was seen on the 1st postoperative day (POD) and the second peak (444.8 +/- 25.6 ml/min; 112.7% of preoperative flow) occurred on the 7th POD. The portal flow demonstrated the same biphasic changes as the total hepatic flow, although the hepatic arterial flow showed only the first peak. A heart rate analysis suggested that the first peak was probably due to hyperdynamic circulatory conditions, as has been previously reported. In addition, the existence of the second peak was established by the present study. The postprandial hepatic blood flow decreased during the first 2 weeks postoperatively, but exceeded the presurgical levels on PODs 21 and 28.

Animals↗

PubMed1996

Molecular cloning of murine decay accelerating factor by immunoscreening.

Although the cDNA of human decay accelerating factor (DAF) which restricts complement activation on homologous cell membranes was cloned in 1987, all trials to detect the cDNA of mouse DAF by cross-hybridization were unsuccessful. However, by immunoscreening with a rabbit antiserum against purified mouse DAF, we successfully cloned the cDNA. It contains four typical short consensus repeats (SCR) similar to that in human and guinea pig DAF. The base sequence showed 63.7 and 63.8% identity to that of human and guinea pig DAF respectively. The deduced amino acid sequence identity to human and guinea pig DAF was 47.2 and 46.5% respectively. Mouse complement receptor related gene Y (Crry)/p65 function is comparable to DAF. SCR3 and SCR4 of mouse DAF showed 50% identity to SCR2 and SCR3 of Crry/p65 respectively. Identification of the mouse DAF gene should open a new approach for determining the actual in vivo role of DAF by analyzing autoimmune mice as well as generating DAF gene knockout mice using embryonic stem cells.

Amino Acid Sequence↗

PubMed1995

A eukaryotic gene encoding an endonuclease that specifically repairs DNA damaged by ultraviolet light.

Many eukaryotic organisms, including humans, remove ultraviolet (UV) damage from their genomes by the nucleotide excision repair pathway, which requires more than 10 separate protein factors. However, no nucleotide excision repair pathway has been found in the filamentous fungus Neurospora crassa. We have isolated a new eukaryotic DNA repair gene from N.crassa by its ability to complement UV-sensitive Escherichia coli cells. The gene is altered in a N.crassa mus-18 mutant and responsible for the exclusive sensitivity to UV of the mutant. Introduction of the wild-type mus-18 gene complements not only the mus-18 DNA repair defect of N.crassa, but also confers UV-resistance on various DNA repair-deficient mutants of Saccharomyces cerevisiae and a human xeroderma pigmentosum cell line. The cDNA encodes a protein of 74 kDa with no sequence similarity to other known repair enzymes. Recombinant mus-18 protein was purified from E.coli and found to be an endonuclease for UV-irradiated DNA. Both cyclobutane pyrimidine dimers and (6-4)photoproducts are cleaved at the sites immediately 5' to the damaged dipyrimidines in a magnesium-dependent, ATP-independent reaction. This mechanism, requiring a single polypeptide designated UV-induced dimer endonuclease for incision, is a substitute for the role of nucleotide excision repair of UV damage in N.crassa.

Amino Acid Sequence↗

PubMed1994

Construction of a human B cell line, TKHMY, suitable for production of stable human hybridomas.

In order to produce stable hybridomas for generation of human monoclonal antibodies (HMAbs), an attempt was made to construct a suitable human parental B cell line with double selection markers, the strategy being to produce HMAbs by fusing a parental B cell line with EBV-transformed B cells producing specific antibodies. The resultant TKHMY cell line, constructed by transfecting the Blasticidin S resistant (bsr) gene into the HAT sensitive human B cell line LTR 228 could be used as a fusion partner for very efficient production of human hybridomas. Secretion of IgG monoclonal antibodies to antigens on lung cancer cells by one such hybridoma was very stable, indicating that this TKHMY cell line has practical advantage in the area of antibody production.

Aminohydrolases↗

PubMed1994

A new class of DNA photolyases present in various organisms including aplacental mammals.

DNA photolyase specifically repairs UV light-induced cyclobutane-type pyrimidine dimers in DNA through a light-dependent reaction mechanism. We have obtained photolyase genes from Drosophila melanogaster (fruit fly), Oryzias latipes (killifish) and the marsupial Potorous tridactylis (rat kangaroo), the first photolyase gene cloned from a mammalian species. The deduced amino acid sequences of these higher eukaryote genes show only limited homology with microbial photolyase genes. Together with the previously cloned Carassius auratus (goldfish) gene they form a separate group of photolyase genes. A new classification for photolyases comprising two distantly related groups is proposed. For functional analysis P.tridactylis photolyase was expressed and purified as glutathione S-transferase fusion protein from Escherichia coli cells. The biologically active protein contained FAD as light-absorbing cofactor, a property in common with the microbial class photolyases. Furthermore, we found in the archaebacterium Methanobacterium thermoautotrophicum a gene similar to the higher eukaryote photolyase genes, but we could not obtain evidence for the presence of a homologous gene in the human genome. Our results suggest a divergence of photolyase genes in early evolution.

Amino Acid Sequence↗

PubMed1994

Chinese hamster ovary cells resistant to okadaic acid express a multidrug resistant phenotype.

Two Chinese hamster ovary cell clones resistant to okadaic acid (OA) were isolated. The OA-resistance was associated with resistance to colchicine, Vinca alkaloids and inhibitors of DNA topoisomerase (topo) II. Drug accumulation assays showed that the intracellular levels of OA, vinblastine and vincristine, but not the topo II inhibitor etoposide, were significantly lowered in the OA-resistant mutants than in the parental cells. These results, together with the finding of an increased level of P-glycoprotein (P-gp) in the mutant cells, indicate that the resistances to OA, Vinca alkaloids and colchicine are due to a P-gp-mediated mechanism. Resistance to topo II inhibitors, however, was associated with reduced activity of topo II. Thus, at least two events, overexpression of P-gp and reduction of topo II activity, occurred in a single OA-resistant cell line, contributing to expression of the MDR phenotype.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

A case of Crohn's disease limited to the appendix, showing a portentous ultrasonographic finding.

A case of Crohn's disease limited to the appendix is reported. A preoperative ultrasonic study of the lower right abdomen revealed an interesting appearance that we named the "milky way sign." Microscopic findings in the resected specimen confirmed that the sign represented adipose tissue of the mesoappendix that had become involved by transmural inflammation. This seems to be highly suggestive of Crohn's disease.

Adipose Tissue↗

PubMed1994

Evaluation of tracheal mucosal blood flow during an extended radical operation for esophageal carcinoma: clinical and experimental studies.

Using the hydrogen gas clearance method, a plate-type probe was attached to the surface of the cuff of an endotracheal tube in order to measure the ischemic changes in the tracheal mucosa produced by peritracheal lymph node dissection. In eight human subjects with intrathoracic esophageal carcinoma, the tracheal mucosal blood flow was 40.1 ml/100 g per minute after they had undergone a right thoracotomy and then decreased to 13.0 ml/100 g per minute after they had further received an extended radical operation preserving the right bronchial artery. No tracheal ulcers were seen. The same ischemic changes in the tracheal mucosa were also measured in dogs. The right bronchial artery contributes about one-third of the total blood flow to the trachea and this was thus calculated to be about 10-14 ml/100 g per minute.

Animals↗