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Biomedical subjects

E Ohtsuka

Publications and source records attributed to E Ohtsuka.

At least 109 records · Page 6Linked to original sources
PubMed1994

Role of the Mg2+ ion in the Escherichia coli ribonuclease HI reaction.

To study the interaction and the role of the metal ion in the reaction catalyzed by Escherichia coli ribonuclease HI (E. coli RNase HI), substrate analogues containing a phosphorothioate linkage or 2'-modified nucleosides at the cleavage site were used. In the presence of Mg2+, Mn2+, Co2+, Zn2+, or Cd2+, the phosphorothioate linkage with the RP-configuration was cleaved, while the SP-isomer was not. Kinetic studies showed that Mn2+ and Cd2+ facilitated the cleavage of the phosphorothioate to only a small extent, which indicated the absence of an interaction between the metal ion and this phosphate residue. The interaction of the metal ion with the 2'-functional group was analyzed by Mg(2+)-titration experiments using the -OH, -NH2, and -F substrates. From Hill plots, it was found that the KMg values were almost the same. These results are evidence of an interaction between Mg2+ and the 2'-functional group by the formation of an outer-sphere complex with a water molecule. The Hill coefficient of 1.0 for the -OH substrate indicated that a single Mg2+ ion is required for the catalysis.

Bacterial Proteins↗
PubMed1994

[Synthesis and functional studies on nucleic acids. Problems related to synthetic ras genes].

Synthesis of a gene for an oncoprotein, ras p21 and its functions are described. Point mutations in hot spots of ras genes have been found in human cancer cells and produce activated p21 which result in transformation of cultured NIH3T3 cells. To produce normal and activated p21 in quantity for biochemical and structural studies, genes encoding these proteins were synthesized and expressed in E. coli. Normal and activated RAS proteins were tested for their GTPase activity and three dimensional structures were determined by X-ray crystallography. Transforming activities of the synthetic genes have been tested by transfecting their expression vectors to NIH3T3 cells and the synthetic activated genes were found to transform these cells indicating that the product of the activated gene is responsible for these malignant growth of the cells. These activities were proved to be inhibited by transfecting designed ribozyme genes. These synthetic genes were used to investigate mutagenesis of damaged bases such as 7,8-dihydro-8-oxoguanine and thymine photodimers, by introducing the damaged base in hot spots of the oncogene. These unnatural bases in the ras gene were found to be mutagenic and cause malignant growth of NIH3T3 cells.

Amino Acid Sequence↗
PubMed1994

Probucol-induced QT prolongation and syncope.

We report a patient who experienced a reversible prolongation of the QT interval and episodes of syncope while receiving probucol. A 64-year-old woman experienced syncopal attacks 8 and 11 weeks after beginning probucol treatment (500 mg twice daily). The pre-treatment ECG showed a slight prolongation of the corrected QT interval (QTc) (0.46 sec). Her QTc increased to 0.62 sec 12 weeks after beginning probucol treatment and decreased to about the baseline value (0.48 sec) 6 weeks after treatment was discontinued. Probucol is known to prolong the QT interval. A long QT interval has been linked to an increased risk of ventricular arrhythmias, syncope or sudden death. However, clinical reports which causally relate probucol treatment to syncope are very rare. Although an ECG during the episodes of syncope was not available, this patient's syncope might be due to ventricular tachyarrhythmia associated with probucol-induced QT prolongation. This case emphasizes the need for careful evaluation of the QT interval before and during probucol treatment.

Atrial Fibrillation↗
PubMed1994

Clinicopathological features of adult T-cell leukemia with CD30 antigen expression.

Recently, several cases of adult T-cell leukemia (ATL) with CD30 antigen have been reported, but its clinical significance remains unknown. Accordingly, we studied CD30 antigen expression in ATL cases and documented the clinicopathological characteristics of these cases. Immunohistochemical and clinical characteristics were studied in 46 patients with malignant lymphoma or benign lesions of lymphoid tissue, who had antibodies against human T-cell leukemia virus type I (HTLV-I). Monoclonal integration of HTLV-I provirus was demonstrated in the tumor cells in 36 (ATL) of the 46 cases. CD30 antigen expression was evident in seven of these 36 cases (19.4%), however it was not seen in any of the ten cases lacking the integration of HTLV-I provirus. A comparison of ATL cases with and without CD30 antigen expression revealed significantly larger numbers of abnormal lymphocytes in the peripheral blood and lower serum calcium levels in ATL expressing CD30 antigen.

Adult↗
PubMed1994

Suppression of splenic enzyme activities by administration of aminopeptidase N (CD13) inhibitors: relationship between actions in vivo and in vitro.

The enzymatic changes in murine spleen caused by the administration for 20 successive days of various inhibitors of aminopeptidase N (leucocyte antigen CD13) have been compared. When compared with the control (saline), most of the inhibitors significantly suppressed splenic enzyme activities including those of ectoenzymes. A multivariate study indicated that the in vivo effects of the inhibitors were closely related to their inhibitory actions in vitro.

Amino Acids↗
PubMed1994

[Problems concerned with adult T-cell leukemia. 1) Causes of death and early diagnosis of cytomegalovirus pneumonia in adult T-cell leukemia. 2) Clinicopathological features of CD30 positive adult T-cell leukemia].

1) We studied the causes of death confirmed by autopsy or necropsy in 23 adult T-cell leukemia (ATL) patients. Of them eight showed involvement of tumor (35%), eleven infectious disease (47%) (nine cytomegalovirus (CMV) pneumonia, one varicella-zostervirus pneumonia, one pseudomonas aeruginosa pneumonia), and four others (17%). In seven recent cases treated with a new chemotherapy regimen in combination with G-CSF administration, survival was longer than in previous cases and tumor involvement as a cause of death decreased (one case, 14%). However, CMV pneumonia inclined to increase (six cases, 86%). Therefore, we tried to retrospectively detect CMV DNA in the serum of ATL patients using a nested polymerase chain reaction (PCR). CMV pneumonia was reliably diagnosed in eleven ATL patients, whereas CMV DNA was detected in all patients at the time of clinical onset of pneumonia and CMV DNA was detected only in eight patients from 7-35 days before the onset of pneumonia. These findings suggest that the nested PCR assay is a useful tool to early diagnosis CMV pneumonia in ATL patients. 2) Recently, several cases of ATL with CD30 antigen have been reported, but its clinical relevance remains unknown. Accordingly, we studied CD30 antigen expression in 36 ATL patients who had monoclonal integration of HTLV-I provirus in the tumor cells and demonstrated the immunohistochemical and clinical characteristics of these patients. CD30 antigen expression was evident in seven of these 36 patients (19.4%). A comparison of ATL cases with and without CD30 antigen expression revealed significantly large numbers of abnormal lymphocytes in the peripheral blood and lower serum calcium levels in CD30 antigen positive ATL.

Adult↗
PubMed1994

Efficacy of additive DMARD therapy in patients with rheumatoid arthritis. Double blind controlled trial using bucillamine and placebo with maintenance doses of gold sodium thiomalate.

OBJECTIVE: To examine the efficacy of the addition of small doses of additional disease modifying antirheumatic drugs (DMARD) to ongoing DMARD treatment [additive DMARD therapy (ADT)]. METHODS: A 3-month prospective, double blind, randomized, placebo controlled study was performed using either 100 mg/day of bucillamine (Buc) or an inactive placebo (P1). Two groups of 12 patients each who had experienced an insufficient benefit from gold sodium thiomalate (GSTM) alone were enrolled in the study. RESULTS: The addition of Buc proved more beneficial than P1 regarding improvement in disease activity (p = 0.0032) and drug usefulness (p = 0.0025). A significant within group improvement was observed in joint swelling count, the Lansbury activity index, erythrocyte sedimentation rate and C-reactive protein. However, the difference in the clinical variables between the 2 groups was minimal. CONCLUSION: The usefulness of ADT was suggested by this trial; however, further confirmation by additional studies is still needed.

Aged↗
PubMed1993

Binding of nucleic acids to E. coli RNase HI observed by NMR and CD spectroscopy.

To clarify the mechanism by which the RNA portion of a DNA/RNA hybrid is specifically hydrolyzed by ribonuclease H (RNase H), the binding of a DNA/RNA hybrid, a DNA/DNA duplex, or an RNA/RNA duplex to RNase HI from Escherichia coli was investigated by 1H-15N heteronuclear NMR. Chemical shift changes of backbone amide resonances were monitored while the substrate, a hybrid 9-mer duplex, a DNA/DNA 12-mer duplex, or an RNA/RNA 12-mer duplex was titrated. The amino acid residues affected by the addition of each 12-mer duplex were almost identical to those affected by the substrate hybrid binding, and resided close to the active site of the enzyme. The results reveal that all the duplexes, hybrid-, DNA-, and RNA-duplex, bind to the enzyme. From the linewidth analysis of the resonance peaks, it was found that the exchange rates for the binding were different between the hybrid and the other duplexes. The NMR and CD data suggest that conformational changes occur in the enzyme and the hybrid duplex upon binding.

Base Sequence↗
PubMed1993

Identification of important bases in a single-stranded region (SSrC) of the hepatitis delta (delta) virus ribozyme.

Models for the secondary structure of genomic and antigenomic self-cleaving RNAs of human hepatitis delta (delta) virus (HDV) have been proposed by several groups. Our recent results support a pseudoknot structure and have allowed us to identify functionally important nucleotides in single-stranded regions [nucleotides 726-731 (SSrA) and nucleotides 762-766 (SSrB)]. For the identification of the important residues in the remaining single-stranded region, nucleotides 708-715 (SSrC), of the genomic HDV ribozyme, we made derivatives with a single-base substitution in the SSrC region. To screen inactive mutants rapidly, we use a simplified in-vitro selection method. Among the various base substitutions in mutants in the SSrC, U708A, C709(A/G/U) and G713C variants had less than 10% of the cleavage activity of the wild-type SSrC (HDV86). By analyzing the self-cleavage activities of various mutants, we determined the base requirements for SSrC as 5'-(U/C/G)-C-N-N-(C/A/G)-(G/A/U)-N-N-3'.

Base Sequence↗
PubMed1993

Evidence for two DNA repair enzymes for 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) in human cells.

Two DNA repair enzymes for 8-hydroxyguanine (also known as 7,8-dihydro-8-oxoguanine; OH8Gua, oxo8Gua) have been identified in human HeLa cell nuclear extract. One is OH8Gua-glycosylase and the other is OH8Gua-endonuclease that lacks OH8Gua-glycosylase activity. They were separated by heparin-Sepharose column chromatography and characterized by endonuclease nicking assay or by measuring the OH8Gua released from substrate DNA using high pressure liquid chromatography-electrochemical detection. Both OH8Gua repair enzymes act only on the OH8Gua-containing strand of the duplex substrate DNA containing OH8Gua/C, OH8Gua/T, or OH8Gua/G. DNA containing OH8Gua/A base pair was very poor substrate for either enzymes. OH8Gua-endonuclease simultaneously cleaves phosphodiester bonds on both sides of the OH8Gua residue, leaving 5'-hydroxy and 3'-hydroxy groups.

Base Sequence↗
PubMed1993

Mutation-spectrum of a true abasic site in codon 12 of a c-Ha-ras gene in mammalian cells.

An abasic site is postulated to be a premutagenic lesion. We previously reported that a c-Ha-ras gene with an abasic site analogue at either the first or the second position of codon 12 induced the formation of foci by point-mutational activation of the gene [Kamiya et al. (1992) Nucleic Acids Res. 20, 4409-4415]. In this study we constructed c-Ha-ras genes with a true abasic site in codon 12. The ras genes were found to be activated in NIH3T3 cells by a mutation to A at the modified and flanking positions, and the flanking mutations were detected more frequently.

3T3 Cells↗
PubMed1993

Development of a recombinant RNA technique for the construction of chimeric RNA with a long poly(C) tract.

The murine cardioviruses and bovine aphthoviruses are distinguished from other (+) strand RNA viruses by their long poly(C) tract in the 5'-noncoding region. The presence of this poly(C) tract has long hampered the construction of full-length cDNA with the complete poly(C) tract, because long poly(dC-dG) homopolymer-containing plasmids are difficult to amplify in bacterial systems. To overcome this problem, we constructed a chimeric RNA by joining the poly(C) region of the viral RNA to the 5'-truncated RNA transcript of the encephalomyocarditis (EMC) virus cDNA. The non-chimeric, recombinant EMC virus with a short poly(C) tract produces recombinant progeny virus, but this is not pathogenic in vivo. On the other hand, the EMC viral RNA chimera with the complete poly(C) tract produces recombinant progeny virus that is pathogenic in vivo. This method of viral RNA construction will be invaluable for functional studies of other cardioviruses and aphthoviruses, as well as for recombinant RNA manipulations.

Animals↗
PubMed1993

Cyclobutane thymine dimers in a ras proto-oncogene hot spot activate the gene by point mutation.

ras proto-oncogenes with a cyclobutane-type thymine photodimer (cis-syn or trans-syn isomer) were constructed by replacement of a portion of the gene with a chemically synthesized fragment. When the genes were transfected by the calcium phosphate method into mouse NIH3T3 cells, they induced focus-formation, indicating that both photoproducts were mutagenic in mammalian cells. Sequence analysis of the ras gene fragments derived from the transformed cells showed that the genes were activated by a point mutation. The mutations detected most frequently were 3'-T-->A for the cis-syn isomer and 5'-T-->A for the trans-syn isomer. In contrast, a different trend of mutations was observed when a primer on a DNA template with a cis-syn dimer was extended in vitro by either DNA polymerase beta or alpha.

3T3 Cells↗
PubMed1993

A 2-thiouridine derivative in tRNAGlu is a positive determinant for aminoacylation by Escherichia coli glutamyl-tRNA synthetase.

Early investigations into the interaction between Escherichia coli glutamyl-tRNA synthetase (GluRS) and tRNAGlu have implicated the modified nucleoside 5-[(methylamino)methyl]-2-thiouridine in the first position of the anticodon as an important contact for efficient aminoacylation. However, the experimental methods employed were not sufficient to determine whether the interaction was dependent on the presence of the modification or simply involved other anticodon loop-nucleotides, now occluded from interaction with the synthetase. Unmodified E. coli tRNA(Glu), derived by in vitro transcription of the corresponding gene, is a poor substrate for GluRS, exhibiting a 100-fold reduction in its specificity constant (kcat/KM) compared to that of tRNA(Glu) prepared from an overproducing strain. Through the use of recombinant RNA technology, we created several hybrid tRNAs which combined sequences from the in vitro transcript with that of the native tRNA, resulting in tRNA molecules differing in modified base content. By in vitro aminoacylation of these hybrid tRNA molecules and of tRNAs with base substitutions at positions of nucleotide modification, we show conclusively that the modified uridine at position 34 in tRNA(Glu) is required for efficient aminoacylation by E. coli GluRS. This is only the second example of a tRNA modification acting as a positive determinant for interaction with its cognate aminoacyl-tRNA synthetase.

Base Sequence↗
PubMed1993

Human nucleotide excision nuclease incises synthetic double-stranded DNA containing a pyrimidine dimer at the fourth phosphodiester linkage 3' to the pyrimidine dimer.

Linear 75mer double-stranded DNA containing a single pyrimidine dimer at a unique site was used to investigate pyrimidine dimer-dependent endonuclease activities from human cells. HeLaS3 cell extract incised the target DNA at the fourth phosphodiester linkage 3' to the pyrimidine dimer. However, incision of the DNA at 5' side of the pyrimidine dimer was not detected. The incision was also detected in cell extracts prepared from other excision repair-proficient cell lines. Incision was detected only on the DNA strand containing a pyrimidine dimer in the presence of poly(dI-dC)-poly(dI- dC) double strand. The reaction required Mg2+ but not ATP. The extract prepared from excision repair-deficient xeroderma pigmentosum (XP) cells belonging to the complementation group A was unable to incise the DNA. Extracts from the complementation groups C, D, and G incised the DNA very weakly at the third phosphodiester linkage 3' to the pyrimidine dimer, a site different from that incised by normal human cell extract. These results suggest that the observed incision reaction is associated with excision repair in human cells.

Adenosine Triphosphate↗