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Biomedical subjects

E Ohtsuka

Publications and source records attributed to E Ohtsuka.

At least 163 records · Page 9Linked to original sources
PubMed1991

An endonuclease activity of Escherichia coli that specifically removes 8-hydroxyguanine residues from DNA.

An enzyme that specifically removes an 8-hydroxyguanine (8-OH-Gua) residue in DNA has been purified from Escherichia coli. To assay the enzymatic activity, a synthetic double-stranded DNA (dsDNA) containing 8-OH-Gua at a defined position was used as a substrate. The substrate DNA was simultaneously cleaved at 2 sites, i.e., the phosphodiester bonds 5' and 3' to 8-OH-Gua, leaving a phosphate at each of the neighboring deoxynucleosides. The cleavage was observed only in dsDNA, but not with single-stranded DNA containing 8-OH-Gua. This enzyme showed almost no activity on DNAs containing other kinds of modified bases such as 8-hydroxyadenine, O6-methylguanine and N7-methylguanine. Also DNAs containing mismatches (A/G or C/T) were not cleaved. Studies on several other properties of this enzyme indicate that it differs from endonucleases previously isolated from E. coli, indicating that it is likely to be an endonuclease which specifically recognizes 8-OH-Gua in dsDNA.

Base Composition↗
PubMed1991

Conformation of 2'GMP bound to a mutant ribonuclease T1 (Y45W) determined by X-ray diffraction and NMR methods.

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a specific inhibitor, 2'GMP, has been determined by X-ray diffraction and refined at 1.9 A resolution to a conventional R-factor of 0.164. The mode of recognition of the guanine base by the enzyme is similar to that found for the wild-type ribonuclease T1 complexed with 2'GMP. The binding of the guanine base is clearly enhanced by maximum overlapping of the indole ring of Trp45 and the base. The glycosyl torsion angle of the inhibitor is in the syn conformation and the sugar exhibits a C3'-endo type pucker, which differs from that observed in the crystal of the complex between the wild-type ribonuclease T1 and 2'GMP. Analysis of 500-MHZ NMR spectra has also indicated that the 2'GMP molecule as bound to the mutant enzyme in solution exhibits a C3'-endo type pucker, similar to that bound to the wild-type enzyme in solution [Inagaki, Shimada, & Miyazawa (1985) Biochemistry 24, 1013-1020].

Carbohydrate Conformation↗
PubMed1991

Hydrophobic effects on protein/nucleic acid interaction: enhancement of substrate binding by mutating tyrosine 45 to tryptophan in ribonuclease T1.

Hydrophobic effects on binding of ribonuclease T1 to guanine bases of several ribonucleotides have been proved by mutating a hydrophobic residue at the recognition site and by measuring the effect on binding. Mutation of a hydrophobic surface residue to a more hydrophobic residue (Tyr45----Trp) enhances the binding to ribonucleotides, including mononucleotide inhibitor and product, and a synthetic substrate-analog trinucleotide as well as the binding to dinucleotide substrates and RNA. Enhancements on binding to non-substrate ribonucleotides by the mutation have been observed with free energy changes ranging from -2.2 to -3.9 kJ/mol. These changes are in good agreement with that of substrate binding, -2.3 kJ/mol, which is calculated from Michaelis constants obtained from kinetic studies. It is shown, by comparing the observed and calculated changes in binding free energy with differences in the observed transfer free energy changes of the amino acid side chains from organic solvents to water, that the enhancement observed on guanine binding comes from the difference in the hydrophobic effects of the side chains of tyrosine and tryptophan. Furthermore, a linear relationship between nucleolytic activities and hydrophobicity of the residues (Ala, Phe, Tyr, Trp) at position 45 is observed. The mutation could not change substantially the base specificity of RNase T1, which exhibits a prime requirement for guanine bases of substrates.

Binding Sites↗
PubMed1991

Transforming activity of a synthetic c-Ha-ras gene containing O6-methylguanine in codon 12.

A mutagenic DNA-adduct, O6-methylguanine, was introduced into codon 12 of the synthetic c-Ha-ras gene by cassette mutagenesis. Transfection of this modified ras gene into normal NIH3T3 cells by the calcium phosphate procedure resulted in significant induction of focus formation. The ras gene inserted into the transformed cells was found to have a G to A transition at the position of the modified base. These results indicate that an O6-methylguanine residue in DNA may lead to a mutation and be one cause of activation of the ras gene.

3T3 Cells↗
PubMed1991

8-Hydroxyguanine, a DNA adduct formed by oxygen radicals: its implication on oxygen radical-involved mutagenesis/carcinogenesis.

Oxygen radicals have been suggested to be involved in mutation/carcinogenesis. The C-8 position of guanine residues in DNA is hydroxylated to produce 8-hydroxyguanine (8-OH-Gua) in DNA in vitro by various oxygen radical producing agents. The formation of 8-OH-Gua was also observed in cellular DNA in vivo by radiation or oxygen radical forming carcinogens. The 8-OH-Gua residue in DNA is often misread in the position of 8-OH-Gua residue itself but also at neighboring residues next to 8-OH-Gua. When second guanine in codon 12 was specifically replaced with 8-OH-Gua and transferred to NIH3T3, the recipient cells were transformed to malignant cell type. E. coli was found to contain an endonuclease which specifically recognizes 8-OH-Gua residue and cleave DNA strand before and after the modified base. The data obtained imply that 8-OH-Gua formed in DNA in vivo is recognized as an abnormal modified base which, if not repaired, play a role in the mediation of oxygen radical-involved mutation/carcinogenesis.

Animals↗
PubMed1991

Atomic structure of a pyrimidine-dimer specific excision-repair enzyme from bacteriophage T4.

T4 endonuclease V is an enzyme responsible for the first step of a pyrimidine-dimer specific excision-repair process. The three-dimensional structure of this enzyme has been determined at 1.6A resolution by X-ray crystallography. The enzyme consists of one single compact domain classified into the all alpha structure. This single domain possesses two distinct catalytic activities, a pyrimidine-dimer glycosylase and an apurinic/apyrimidinic endonuclease. The backbone of the enzyme represents a unique folding scheme incompatible with close packing of alpha-helices. The refined structure suggests possible residues that participate in interactions with DNA.

Amino Acid Sequence↗
PubMed1991

[Evaluation of two quantitative tests for salivary secretion--the chewing gum test and the Saxon test in normal subjects and in patients with Sjögren's syndrome].

A cooperative multicenter study was performed to evaluate two salivary secretion methods-the chewing gum test and the Saxon test by a crossover method. Twenty healthy volunteers and 27 patients with Sjögren's syndrome were examined. In healthy controls the amount of saliva collected per minute was larger in the Saxon test. Both tests revealed a significant decrease of salivary secretion in patients with Sjögren's syndrome, but the amount of saliva per minute was also larger in Saxon test. By repeated trials of both tests a notable increase of saliva was always observed at the second trial. Since this phenomenon may be due to the learning effect of the tests, one should perform at least one practice before starting formal tests. Saxon test could be performed in a patient having dental prostheses who was unable to masticate chewing gum. The Saxon test, thus, may be an efficient method, to evaluate the salivary secretory function.

Adult↗
PubMed1991

[Successful reinduction of complete remission in a patient with ALL associated with multiple liver abscesses who relapsed after 6 years].

We reported on a 35 year-old patient suffering from ALL with multiple liver abscesses (Journal of Kyusyu Hematological Society, Vol. 31, No. 3 & 4, Dec., 1983). He experienced six years of complete hematological remission from 1983 to 1989, with low fever, positive CRP, polyclonal gamma-globulinemia and elevated alkaline phosphatase. A relapse occurred in June of 1989. A subsequent biopsy revealed fibrosis of the liver attributed to inflammation. At present, he has returned to the complete remission stage with no exacerbation of the liver abscesses.

Adult↗
PubMed1990

Synthesis and characterization of a substrate for T4 endonuclease V containing a phosphorodithioate linkage at the thymine dimer site.

A dodecadeoxyribonucleotide containing a cis-syn thymine dimer with a phosphorodithioate linkage was synthesized on a solid support using a dinucleotide coupling unit prepared by UV-irradiation of dithymidine monophosphorodithioate followed by S- and 5'-O-protection and 3'-phosphitylation. A photodimer-containing dodecamer without phosphate modification was also synthesized. The dodecamers were hybridized to the complementary sequence, and the duplexes used as substrates for T4 endonuclease V. This enzyme cleaved the phosphate-modified substrate more slowly than the unmodified duplex with the same dissociation constant.

Base Sequence↗
PubMed1990

Crystallization and preliminary X-ray investigation of non-specific complexes of a mutant ribonuclease T1 (Y45W) with 2'AMP and 2'UMP.

We have succeeded in crystallizing complexes of a mutant ribonuclease T1 (Y45W) with the non-cognizable ribonucleotides 2'AMP and 2'UMP by macroscopic seeding of microcrystals of the mutant enzyme complexed with 2'GMP, which is the cognizable nucleotide inhibitor. The mutant enzyme has a tryptophan residue instead of Tyr45 of the wild-type enzyme and thus this mutation enhances the binding of ribonucleotides to the enzyme. The space group is P212121 with unit cell dimensions a = 49.40 A, b = 46.71 A, c = 41.02 A for the complex with 2'AMP and a = 48.97, b = 46.58 A, c = 40.97 A for the complex with 2'UMP, both of which are poorly isomorphous to the mother crystals. Diffraction data for the complexes with 2'AMP and 2'UMP were collected on a diffractometer at 1.7 A and 2.4 A resolution, respectively. The present studies show that crystallization of non-specific complexes of other protein-ligand systems with the dissociation constants around 10(-3) M, or even larger, could be feasible by application of the seeding technique. A comparison of the crystal structures of the complexes with that with 2'GMP may serve as a structural basis for the determination of differences between the specific and non-specific interactions of the enzyme.

Adenosine Monophosphate↗
PubMed1990

Secondary structure in formylmethionine tRNA influences the site-directed cleavage of ribonuclease H using chimeric 2'-O-methyl oligodeoxyribonucleotides.

In order to cleave RNA at specific positions in Escherichia coli formylmethionine tRNA, RNase H and complementary chimeric oligonucleotides consisting of DNA and 2'-O-methyl-RNA (Inoue et al. (1987) FEBS Lett. 215, 327] were used. Specific cleavages in the D loop, anticodon loop, T psi C loop, anticodon stem, and acceptor stem were investigated. Virtually unique hydrolyses with RNase H were observed at the T psi C loop, anticodon stem, and acceptor stem when relatively longer chimeric oligonucleotides (20-mer) were used. An efficient cleavage at the anticodon was obtained with a chimeric 13-mer when the higher structure of the tRNA was broken by hybridization with a 20-mer at the acceptor as well as the T psi C stem region. It was found that stabilities of hybrids with chimeric oligonucleotides and the presence of minor nucleosides affect the cleavage of tRNA by this approach.

Anticodon↗
PubMed1990

In vitro and in vivo activities of T4 endonuclease V mutants altered in the C-terminal aromatic region.

Genes encoding mutants of the thymine photodimer repair enzyme from bacteriophage T4 (T4 endonuclease V) having an amino acid substitution (T127M, W128A, W128S, Y129A, K130L, Y131A, Y132A) were constructed by use of a previously obtained synthetic gene and expressed in Escherichia coli under the control of the E. coli tryptophan promoter. An in vitro assay of partially fractionated mutant proteins for glycosylase activity was performed with chemically synthesized substrates containing a thymine photodimer. T127M and K130L showed almost the same activity as the wild-type protein. Although W128S, Y131A, and Y132A were slightly active, W128A and Y129A lost activity. The results indicated that the aromatic amino acids around position 130 may be important for the glycosylase activity. Mutant T127M was purified, and the Km value was found to be of the same order as that of the wild type (10(-8) M). In vivo activities for all mutants were characterized with UV-sensitive E. coli. The results showed that substitution of Thr-127 with Met or Lys-130 with Leu did not have an effect on the survival of the bacteria but substitution of aromatic amino acids (128-132) had various effects on survival.

Amino Acid Sequence↗
PubMed1990

Changes in HBsAg carrier rate in Goto Islands, Nagasaki Prefecture, Japan.

Annual mass examinations in an area where hepatitis B virus (HBV) infection is very prevalent revealed that 12.1% of inhabitants born during 1946-50 were positive for hepatitis B surface antigen (HBsAg), compared with only 0.6% of those born during 1971-75. To find out why the HBV carrier rate has fallen, changes in the modes of HBV infection were examined. The HBsAg positivity rate among mothers who gave birth to HBV carrier children in 1965 and before was 26.8% and that for such mothers whose babies were born in 1966 and after was 66.7%, whereas the HBsAg positivity rate among children born to HBV carrier mothers in 1965 and before and in 1966 and after were 30.8% and 28.3%, respectively. None of the 503 inhabitants who had no HBV markers in 1976 had become carriers by 1981. These findings indicate that the decrease in the prevalence of HBV carriage is caused mainly by the reduction in occurrence of horizontal transmission of HBV in infancy.

Adolescent↗
PubMed1990

Identification of the amino acid residues involved in an active site of Escherichia coli ribonuclease H by site-directed mutagenesis.

The amino acid residues essential for the catalytic activity of ribonuclease H (RNase H) from Escherichia coli (E. coli) were identified by site-directed mutagenesis. It has been proposed by computer analysis that E. coli RNase H has homologous amino acid sequence with the RNase H domains of various retroviral reverse transcriptases (RTs) (Johnson, M. S., McClure, M. A., Feng, D. F., Gray, J., and Doolittle, R. F. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7648--7652). Of the eight highly conserved residues examined, Asp10, Glu48, and Asp70 were found to be crucial for RNase H activity. Determination of the kinetic parameters for the mutated enzymes using the chemically synthesized nonanucleotide duplex as a substrate demonstrated that these residues are involved in the catalytic site rather than the substrate-binding site. These residues are fully conserved in the amino acid sequences of not only retroviral RTs but also hepadnaviral, plant viral and retrotransposon RTs. This strongly suggests that they are also involved in the active site of these RTs and RT related enzymes.

Amino Acid Sequence↗
PubMed1990

27 amino acid residues can be deleted from the N-terminus of human lymphotoxin without impairment of its cytotoxic activity.

In order to study the relationship between activity and structure of human lymphotoxin (hLT, 171 aa), we synthesized the gene (519 bp) for hLT and expressed it in Escherichia coli. Purification of the recombinant hLT from crude extracts was difficult because of the low level of expression of the gene. To improve the yield of the recombinant protein, we prepared five truncated genes for mutant proteins in which 25, 26, 27, 28 and 37 amino acid residues, respectively, were missing from the N-terminus. All of the genes were efficiently expressed and adequate amounts of mutant proteins were synthesized. The proteins were recovered mainly in the supernatant fractions after disruption of cells, with the exception of LT delta 37N, in which 37 residues were absent from the N-terminal region. Cytotoxic activities against mouse fibroblast L929 cells were detected in supernatant fractions that contained these mutant proteins, except in the case of LT delta 28N, which lacks the first amino acid residue conserved in both hLT and human tumour necrosis factor (hTNF). LT delta 27N, which is the smallest of the active proteins, was purified to homogeneity, and its cytotoxic activity was found to be similar to that of recombinant hTNF.

Amino Acid Sequence↗
PubMed1990

The specificity of antiglobulin autoantibodies in patients with primary Sjögren's syndrome.

Antiglobulin autoantibodies have already been demonstrated in the sera of patients with primary Sjögren's syndrome (primary SS). In our study, the specificity of primary SS antiglobulins for different regions of IgG molecules was examined by employing both direct binding and competitive inhibition enzyme-linked immunosorbent assay. We found that a considerable amount of total antiglobulins in primary SS was specific for the Fab portion, although the remainder was specific for the Fc portion, namely rheumatoid factor (RF). In contrast, most of the antiglobulins in RA were specific for the Fc portion of IgG. These results indicate that in primary SS, antiglobulins directed against epitopes different from those of RF are produced. These antiglobulins may prove to have a different role in primary SS than that ascribed to RF in RA.

Antibodies, Anti-Idiotypic↗