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.

[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.

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.

Design of oligonucleotides for sequence-specific triple-helix formation. Properties of oligonucleotides containing 2'-deoxyxanthosine.

Homopyrimidine-homopurine sequences are targeted by homopyrimidine oligodeoxynucleotides to form triple helix structure. Xanthine was introduced to the third strand of pyrimidine-rich 15-mer oligodeoxy-nucleotides to recognize a cytosine in the purine strand in a homopyrimidine-homopurine duplex target.

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.

A case of hepatocellular carcinoma with the sign of Leser-Trelat: a possible role of a cutaneous marker for internal malignancy.

A rare case of hepatocellular carcinoma who developed the complication of the sign of Leser-Trelat is reported. The patient, a 57-year-old male, visited our hospital with complaints of generalized malaise and anorexia. A diagnosis of hepatocellular carcinoma was made based on elevated alpha-fetoprotein measurement, ultrasonography, and hepatic arteriography findings.

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.

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.

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).

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.

Surveying cis-acting sequences of pre-mRNA by adding antisense 2'-O-methyl oligoribonucleotides to a splicing reaction.

We chemically synthesized antisense 12 mer 2'-O-methylribonucleotides and surveyed a scanning (signal-tracking) process as well as sequences within a beta-globin transcript acting in the splicing reaction in vitro. The pre-mRNA transcript contained the sequences of the first exon, first intron, and a major part of the second exon of the human beta-globin gene. We found that the antisense 2'-O-methylribonucleotides could anneal effectively to the target site in the pre-mRNA during the splicing reaction.

Crystal structure of an active form of RAS protein, a complex of a GTP analog and the HRAS p21 catalytic domain.

Normal RAS proteins play a key role of molecular switch in the transduction of the growth signal from extracellular to intracellular space. The state of the switch is "on" when GTP is bound and "off" when GDP is bound to the protein. The crystal structure of a complex between a nonhydrolyzable GTP analog and the catalytic domain of a RAS protein has been determined by a rotation-translation search method.

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.

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.

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.

c-Ha-ras down regulates the alpha-fetoprotein gene but not the albumin gene in human hepatoma cells.

We studied the effects of transfection of the normal c-Ha-ras gene, rasGly-12, and its oncogenic mutant, rasVal-12, on expression of the alpha-fetoprotein (AFP) and albumin genes in a human hepatoma cell line, HuH-7. The mutant and, to a lesser extent, the normal ras gene caused reduction of the AFP mRNA but not the albumin mRNA level in transfected HuH-7 cells. Cotransfection experiments with a rasVal-12 expression plasmid and a chloramphenicol acetyltransferase reporter gene fused to AFP regulatory sequences showed that rasVal-12 suppressed the activity of enhancer and promoter regions containing A + T-rich sequences (AT motif).

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.

Chemical synthesis of oligoribonucleotides for structural studies.

Large-scale synthesis of oligoribonucleotides has been performed successfully on a solid support by the phosphoramidite approach using levulinyl and tetrahydrofuranyl protection for the 5'- and 2'-hydroxyl groups respectively. A hexamer containing inosine and four fragments of a hammerhead-type ribozyme have been synthesized on a 10 mumol scale for structural studies by NMR spectroscopy and X-ray crystallography.

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study.

1H NMR spectra of a GDP/GTP-binding domain of human c-Ha-ras gene product (residues 1-171) in which glutamine-61 was replaced by leucine [ras(L61/1-171) protein] were analyzed. By one-dimensional and two-dimensional homonuclear Hartmann-Hahn spectroscopy and nuclear Overhauser effect (NOE) spectroscopy of the complex of the ras(L61/1-171) protein and GDP, the ribose H1', H2', H3', and H4' proton resonances of the bound GDP were identified. The guanine H8 proton resonance of the bound GDP was identified by substituting [8-2H]GDP for GDP.

Synthesis of a gene for the protein kinase domain of the epidermal growth factor receptor and its expression in Escherichia coli.

A gene encoding the protein kinase domain of the epidermal growth factor receptor has been chemically synthesised, cloned and expressed in Escherichia coli. The 942-base-pair gene was constructed by enzymatic ligation of 56 oligonucleotides and cloned into an expression vector downstream of the E. coli trp promoter.

Design of RNA enzymes distinguishing a single base mutation in RNA.

RNA enzymes (ribozymes) which can cleave RNA by recognizing sequences of 9-15 bases are described. Substrates must contain UX (X = U, C or A). A ribozyme consisting of two oligoribonucleotides (19 mer and 15 mer) was shown to cleave a ribo 11 mer catalytically with Km and kcat values of 0.

Microinjection of T4 endonuclease V produced by a synthetic denV gene stimulates unscheduled DNA synthesis in both xeroderma pigmentosum and normal cells.

A structural gene for T4 endonuclease V was constructed by ligating synthetic oligonucleotides. The endonuclease V was overproduced in E. coli under control of the E.