A modified flow cytometric method for counting very low numbers of white cells in platelet concentrates.
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Blood Platelets↗
Biomedical subjects
T Seno
Publications and source records attributed to T Seno.
Incubation at the nonpermissive temperature induces deficiencies in UV resistance and mutagenesis in mouse mutant cells expressing a temperature-sensitive ubiquitin-activating enzyme (E1).
In temperature-sensitive (ts) mutants of mouse FM3A cells, the levels of mutagenesis and survival of cells treated with DNA-damaging agents have been difficult to assess because they are killed after their mutant phenotypes are expressed at the nonpermissive temperature. To avoid this difficulty, we incubated the ts mutant cells at the restrictive temperature, 39 degrees C, for only a limited period after inducing DNA damage. We used ts mutants defective in genes for ubiquitin-activating enzyme (E1), DNA polymerase alpha, and p34(cdc2) kinase. Whereas the latter two showed no effect, E1 mutants were sensitized remarkably to UV light if incubated at 39 degrees C for limited periods after UV exposure. Eighty-five percent of the sensitization occurred within the first 12 h of incubation at 39 degrees C, and more than 36 h at 39 degrees C did not produce any further sensitization. Moreover, while the 39 degrees C incubation gave E1 mutants a moderate spontaneous mutator phenotype, the same treatment significantly diminished the level of UV-induced 6-thioguanine resistance mutagenesis and extended the time necessary for expression of the mutation phenotype. These characteristics of E1 mutants are reminiscent of the defective DNA repair phenotypes of Saccharomyces cerevisiae rad6 mutants, which have defects in a ubiquitin-conjugating enzyme (E2), to which E1 is known to transfer ubiquitin. These results demonstrate the involvement of E1 in eukaryotic DNA repair and mutagenesis and provide the first direct evidence that the ubiquitin-conjugation system contributes to DNA repair in mammalian cells.
Animals↗
A ubiquitin-conjugating enzyme in fission yeast that is essential for the onset of anaphase in mitosis.
A cDNA encoding a ubiquitin-conjugating enzyme designated UbcP4 in fission yeast was isolated. Disruption of its genomic gene revealed that it was essential for cell viability. In vivo depletion of the UbcP4 protein demonstrated that it was necessary for cell cycle progression at two phases, G2/M and metaphase/anaphase transitions. The G2 arrest of UbcP4-depleted cells was dependent upon chk1, which mediates checkpoint pathway. UbcP4-depleted cells arrested at metaphase had condensed chromosomes but were defective in separation. However, septum formation and cytokinesis were not restrained during the metaphase arrest. Overexpression of UbcP4 specifically rescued the growth defect of cut9ts cells at a restrictive temperature. cut9 encodes a component of the anaphase-promoting complex (APC) which is required for chromosome segregation at anaphase and moreover is defined as cyclin-specific ubiquitin ligase. Cdc13, a mitotic cyclin in fission yeast, was accumulated in the UbcP4-depleted cells. These results strongly suggested that UbcP4 is a ubiquitin-conjugating enzyme working in conjunction with APC and mediates the ubiquitin pathway for degradation of "sister chromatid holding protein(s)" at the onset of anaphase and possibly of mitotic cyclin at the exit of mitosis.
Amino Acid Sequence↗
Genotyping for RhC/c and RhE/e by PCR using allele-specific oligonucleotide primers.
The Rh blood group system has five major antigens D, C/c, and E/e. These antigens are encoded in RHD and RHCE genes. In this report, we describe a systemic method for RhC/c and RhE/e genotyping by PCR using allele-specific oligonucleotide primers (ASO-PCR). The ASO-PCR was carried out to determine the RhC/c and RhE/e genotypes in DNA samples from 513 Japanese donors. Genotypes of RhC, RhE, and Rhe were in full concordance with serological phenotypes in 511 donors. However, in two cases with the phenotype of ccdee, the C-specific ASO-PCR product was also detected in addition to the c-specific one. This method is simple and quite useful for the RhC/c and RhE/c genotyping, although further investigation on the 2 exceptional ccdee cases is needed.
Base Sequence↗
A randomized trial comparing ftorafur alone with ftorafur plus tamoxifen in postoperative adjuvant therapy for breast cancer. Kinki Area Research Group for Postoperative Adjuvant Therapy for Breast Cancer.
A randomized study was performed in 35 centers in the Kinki area of Japan to determine the effectiveness of ftrorafur (FT) plus tamoxifen (TAM) compared with FT monotherapy in postoperative adjuvant therapy for breast cancer. Patients were randomized by the envelope method to receive either FT 600 mg/day or FT 600 mg/day plus TAM 20 mg/day orally for 1 year, starting on day 7 after mastectomy. Between April 1982 and January 1985, 628 patients were assigned to treatment with FT alone and 626 to treatment with FT + TAM. Of these, 571 (90.9%) and 539 (86.1%) patients, respectively, met the eligibility requirements for this study. There were no significant differences in major background factors between the two groups of eligible patients. Five-year survival rates were 91.4% for FT alone and 91.1% for FT+TAM (not significantly different). Five-year disease-free survival rates showed a tendency towards a better prognosis (P = 0.090) in the FT + TAM group, with observed rates of 83.0% for FT alone and 86.7% for FT + TAM. Stratified analysis showed that disease-free survival with FT + TAM is better than with FT alone for patients aged 50 years or more ( P = 0.048) and for patients with from one to three positive nodes (P = 0.064).
Adult↗
A search for the Secretor gene nonsense mutation (G428 to A428) in Japanese nonsecretors.
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Base Sequence↗
Abnormal integrity of the nucleolus associated with cell cycle arrest owing to the temperature-sensitive ubiquitin-activating enzyme E1.
A mouse cell mutant, ts85, containing the temperature-sensitive ubiquitin-activating enzyme was arrested in G2 phase at the non-permissive temperature. In the arrested cells, azure C, a nucleolus-specific stain, revealed a U-shaped or ring-shaped arrangement of nucleolar lobes with an unstained region in the center. Silver staining of the nucleolar organizer region (NOR) and fluorescence in situ hybridization (FISH) with rDNA both gave signals in azure C-positive regions. Electron microscopic examination revealed a cloud of unidentified electron-dense particles (diameter approximately 70 nm) in the azure C-negative center space. When the arrested cells were released into M-phase, we observed the association of NOR-bearing chromosomes with a pulverization-like abnormality. FISH with rDNA and NOR silver staining demonstrated that the pulverization-like abnormality was restricted to NORs. The frequent occurrence of persistent nucleolar material in prophase and prometaphase of the stressed cells after release indicated a delayed dissociation of the nucleolus that brought about the abnormal chromosomes in M-phase. ts85 cells transfected with the mouse E1 cDNA recovered growth at the non-permissive temperature and no longer showed abnormal nucleolar morphology. It seems that the ubiquitin system plays a role in the dissolution of the nucleolus, possibly involving the NOR-bearing chromosomes.
Animals↗
Ubiquitin-activating enzyme, E1, is phosphorylated in mammalian cells by the protein kinase Cdc2.
The ubiquitin-activating enzyme (E1) is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates. E1 was found to be phosphorylated in cells of a mouse mammary carcinoma cell line, FM3A. Peptide mapping of trypsin digests of labeled E1 indicated that two oligopeptides were mainly phosphorylated in vivo. The same oligopeptides were also labeled in vitro on Cdc2 kinase-mediated phosphorylation of E1, affinity-purified from the same cell line. The Cdc2 kinase is a key enzyme playing a pivotal role in G2/M transition in the cell cycle. The phosphorylation of one of the two oligopeptides was prominent at the G2/M phase of the cell cycle, and dependent upon the Cdc2 kinase activity in vivo since it was significantly reduced in tsFT210, a mutant cell line deficient in Cdc2 kinase. Mutation analysis indicated that the serine residue at the fourth position of the E1 enzyme was a phosphorylation site of Cdc2 kinase. These findings suggest that E1 is a target of Cdc2 kinase in the cell, implying that the ubiquitin system may be dynamically involved in cell cycle control through phosphorylation of this key enzyme.
Amino Acid Sequence↗
Serum levels of six pancreatic enzymes as related to the degree of renal dysfunction.
OBJECTIVES: Currently, serum total amylase, pancreatic isoamylase (P-amylase), lipase, trypsin(ogen), phospholipase A2 (PLA2), and elastase I are advocated to be useful in diagnosing pancreatic diseases. However, the most useful among the above six enzymes in patients with impaired renal function has not been fully clarified. We, therefore, studied the relation of the serum levels of the above enzymes and creatinine clearance (CrCl) in normal controls and patients with chronic renal insufficiency or failure. METHODS: PLA2 and elastase I were assayed by RIA, trypsin(ogen) by EIA and others by activity. Subjects were 24 healthy controls and 47 patients with impaired renal function and no apparent pancreatic diseases. RESULTS: 1) Elastase I was significantly elevated only in patients with a CrCl of 10 ml/min or less, whereas others were elevated already in patients with a CrCl below 40 ml/min; 2) in 12 patients with a CrCl between 13 and 39 ml/min, lipase tended to be less frequently raised than others, except elastase I, although the difference was statistically insignificant; 3) in 28 patients with a CrCl between 40 and 74 ml/min, lipase was less frequently elevated than others, except elastase I and PLA2; 4) in seven patients with a CrCl of 10 ml/min or below, elastase I tended to be less frequently elevated than others, although the difference was statistically insignificant; and 5) the degree of elevation was within 2.5 times the upper limits of reference values in all enzymes, except trypsin(ogen) (within 4.8 times). CONCLUSIONS: Elastase I was least vulnerable to impaired renal function followed by lipase. We, therefore, recommend combined assays of elastase I and lipase for detecting pancreatic diseases in patients with renal insufficiency. When cut-off levels are set at 2.5 times the upper limit of reference values, P-amylase or PLA2 can replace lipase.
Aged↗
Molecular genetic analysis of the human Lewis histo-blood group system.
The expression of Lewis histo-blood group antigen is determined by the Lewis type alpha(1,3/1,4) fucosyltransferase encoded in Fuc-TIII gene (Le gene) on chromosome 19. Weak hemagglutination reactions are often observed in the Lewis blood typing to cause false-negative reactions. The level of CA19-9 tumor marker, defined as sialylated Le(a) (sLe(a)), is influenced by the Lewis blood phenotype. The sLe(a) antigen is also considered to play a role in hematogenous metastasis of tumor cells. The accurate genotyping of Fuc-TIII gene would be, therefore, necessary for solving above questions. In addition to the two missense mutations previously reported by us, i.e. the T59G and the G508A, further sequence analyses revealed a new single base substitution, the T1067A, changing the Ile356 to Lys in the catalytic region. Polymerase chain reaction-restriction fragment length polymorphism methods were developed for the detection of these three missense mutations and applied in 15 Lewis negative (le/le) and 65 Lewis positive individuals (Le/Le or Le/le). These methods were found to be useful for genetic diagnosis for the Lewis histo-blood typing. All le alleles had the T59G mutation, whereas none of the Le alleles did. The le alleles were divided in two types, le1, having the G508A mutation, and le2, having the T1067A mutation. The T1067A mutation reduced the enzyme activity less than 10%, whereas the G508A mutation in the catalytic domain made the enzyme completely inactive. The frequency of occurrence of Le, le1, and le2 in the Japanese population by random sampling study was found to be 66, 30, and 4%, respectively.
Alleles↗
A detection method for point mutation in the precore region of human hepatitis B virus (HBV)-DNA using mutation-site-specific assay.
In the natural progression of acute active hepatitis and chronic active hepatitis in human hepatitis B virus (HBV)-infected patients, inactive hepatitis develops by seroconversion, which can be explained by the disappearance of HBe antigen. However, it has been found that in some patients with hepatitis, alanine aminotransferase levels undergo fluctuation even though their serum is negative for HBe antigen. In these patients, HBV-DNA has been detected in the serum and the HBV-DNA so detected has been considered a cause of worsening liver function. Most HBV-DNA detected in these cases has a point mutation from G to A at the 83rd base in the precore region. As a result of this point mutation, HBV is unable to produce HBe antigen. We have devised a sensitive polymerase chain reaction (PCR) method, a mutation-site-specific assay, for the detection of point mutations at the 83rd base in the precore region using a specific mutation-trapped oligonucleotide primer for the mutant HBV genome.
Base Sequence↗
Conversion of group A red cells by deacetylation to ones that react with monoclonal antibodies specific for the acquired B phenotype.
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ABO Blood-Group System↗
Evaluation of cytology and tumor markers of pure pancreatic juice for the diagnosis of pancreatic cancer at early stages.
To evaluate the value of cytology of pure pancreatic juice (PPJ) and tumor marker determination in PPJ and serum for the diagnosis of early pancreatic cancer (EPC), PPJ was obtained endoscopically from 16 patients with EPC (< 20 mm, confined to pancreas), 16 patients with chronic pancreatitis (CP), and 20 controls. Cutoff levels of CEA, CA19-9, and POA in PPJ were set from ROC curves at 40 ng/ml, 7,500 U/ml, and 1.5 U/ml, respectively. For the differentiation of EPC from CP, the sensitivity of CEA, CA19-9, and POA was 71.4, 42.9, and 50%, respectively; specificity was 93.3, 46.7, and 80%, respectively; and diagnostic accuracy was 82.8, 44.8, and 65.5%, respectively. Determination of serum tumor markers was useless. Sensitivity of cytology was 75%, specificity was 93.8%, and diagnostic accuracy was 84.4%. Combined cytology and CEA determination in PPJ increased the diagnostic accuracy to 93.1%. The combination was useful in supporting and supplementing endoscopic retrograde cholangiopancreatography (ERCP) findings for the correct diagnosis in 11 and 4 patients, respectively, with EPC; in one patient EPC was correctly diagnosed on ERCP findings alone. One of 16 patients with CP showed false-positive results. We conclude that cytology and CEA determination in PPJ with ERCP is a useful combination for the diagnosis of pancreatic cancer even in early stages.
Antigens, Neoplasm↗
Conditional resistance to thymineless death predominantly selects DNA synthesis-deficient mutants of mammalian cells.
Temperature-sensitive growth mutants of the mouse mammary carcinoma cell line FM3A were isolated by selecting survivors of thymidylate starvation for a limited time at the restrictive temperature (39.5 degrees C). Nineteen lines of independent isolates were established and all were found to be deficient in DNA synthesis. Cell-cell hybridization with authentic mutant lines of FM3A demonstrated that the mutants fell into three complementation groups, which were deficient in DNA polymerase alpha or ubiquitin-activating enzyme E1 or both.
Animals↗
Cloning and sequence of a functionally active cDNA encoding the mouse ubiquitin-activating enzyme E1.
A cDNA encoding the ubiquitin-activating enzyme, E1, was isolated from the mouse mammary carcinoma cell line, FM3A, and shown to complement mutant mouse cells deficient in the enzyme. The 3495-bp cDNA encodes 1058 amino acids (aa), and shares extensive homology with the human E1 enzyme at both the nucleotide and aa sequence levels.
Amino Acid Sequence↗
Intron 1 and the 5'-flanking region of the human thymidylate synthase gene as a regulatory determinant of growth-dependent expression.
We have determined the regulatory regions responsible for the growth-dependent expression of the human thymidylate synthase (TS) gene, using a set of minigenes constructed from segments of the human TS gene and the cDNA clone. Each construct was introduced stably into a TS-negative mutant of rat fibroblast 3Y1 cells. By serum-restricted synchronization of the cloned transformant cells, we found that a minigene with the genomic 5'-flanking region and intron 1 without other introns were sufficient for the normal extent and pattern of S-phase specific expression at the levels of both mRNA and enzymatic activity. In contrast, a TS cDNA clone driven by an SV40-based expression vector showed constitutive expression. Insertion of intron 1 into the cDNA clone in the normal location, or replacement of the viral 5'-promoter region of the cDNA clone by the genomic 5'-flanking sequence converted the constitutive expression to the S-phase dependent one, but only partly, that is, coexistence of the two regions were required for the normal expression. Results obtained by nuclear run-on assay suggested that posttranscriptional controls are also involved in this regulation in consistent with our previous results with the bona fide human TS gene.
Animals↗
tsBN75 and tsBN423, temperature-sensitive x-linked mutants of the BHK21 cell line, can be complemented by the ubiquitin-activating enzyme E1 cDNA.
tsBN75 and tsBN423 are independently isolated temperature-sensitive (ts) mutants of the BHK21 cell line for cell growth. Both tsBN75 and tsBN423 belong to the same complementation group and show G2 block at the nonpermissive temperature. Both were efficiently transformed to ts+ cells with the mouse and human cDNA encoding the ubiquitin-activating enzyme, E1. While no transformants of tsBN423 cells had a DNA content greater than the parental 2C, several ts+ transformants of tsBN75 cells acquired a multiploid DNA content. These data thus demonstrate the function of the human and mouse E1 cDNAs and further suggest that E1 functions in more than one step in cell cycle progression.
Animals↗
Regulatory sequences clustered at the 5' end of the first intron of the human thymidylate synthase gene function in cooperation with the promoter region.
A human thymidylate synthase (TS) minigene containing 5'- and 3'-flanking sequences, all the exons, and only intron 1 showed a normal frequency of stable transformation when transfected into TS-negative mutant cells, whereas minigenes in which intron 1 was replaced by intron 2 or deleted in the above construct showed only a few percent of the above frequency. Introduction of intron 1 into the above intronless or intron 2 minigene restored the transforming activities regardless of its position and orientation. Deletion analysis revealed two positive and one negative regulatory sequences in the 5' end of intron 1, each of which seemed to bind specific proteins as shown by gel shift analysis. Intron 1 also stimulated expression of a TS promoter-CAT gene construct but not that of an SV40 promoter-CAT gene construct. These results indicate that the multiple regulatory sequences clustered in intron 1 stimulate TS gene expression in concert with the 5'-flanking sequences.
Gene Expression Regulation, Enzymologic↗