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H Tohda

Publications and source records attributed to H Tohda.

At least 55 records · Page 3Linked to original sources
PubMed1991

Phosphorothioate-containing RNAs show mRNA activity in the prokaryotic translation systems in vitro.

Phosphorothioate-containing RNAs were generated by transcription of coliphage T7 DNA using the Sp diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates) and T7 RNA polymerase. RNAs in which a single nucleotide was substituted by the corresponding nucleoside phosphorothioate functioned as mRNA in the cell-free translation systems prepared from Escherichia coli and from an extreme thermophilic bacterium, Thermus thermophilus. This substitution increased the efficiency of protein synthesis by stabilizing the mRNAs in these systems. As the proportion of substituted nucleotides was increased, their mRNA activity was decreased accordingly. As judged from the analysis by SDS-polyacrylamide gel-electrophoresis, the proteins synthesized using phosphorothioate-containing mRNAs as template were identical to those obtained with unsubstituted mRNAs. However, larger proteins which were barely detectable when unsubstituted mRNA was used were well represented when phosphorothioate-RNA was used instead. The advantages in using the phosphorothioate-mRNAs in the in vitro translation systems are discussed.

Cell-Free System↗
PubMed1991

Isolation of two chloroethylnitrosourea-sensitive Chinese hamster cell lines.

1-[(4-Amino-2-methylpyrimidin-5-yl)methyl]-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU), a cancer chemotherapeutic bifunctional alkylating agent, causes chloroethylation of DNA and subsequent DNA strand cross-linking through an ethylene bridge. We isolated and characterized two ACNU-sensitive mutants from mutagenized Chinese hamster ovary cells and found them to be new drug-sensitive recessive Chinese hamster mutants. Both mutants were sensitive to various monofunctional alkylating agents in a way similar to that of the parental cell lines CHO9. One mutant (UVS1) was cross-sensitive to UV and complemented the UV sensitivity of all Chinese hamster cell lines of 7 established complementation groups. Since UV-induced unscheduled DNA synthesis was very low, a new locus related to excision repair is thought to be defective in this cell line. Another ACNU-sensitive mutant, CNU1, was slightly more sensitive to UV than the parent cell line. CNU1 was cross-sensitive to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and slightly more sensitive to mitomycin C. No increased accumulation of ACNU and a low level of UV-induced unscheduled DNA synthesis in this cell as compared with the parental cell line suggest that there is abnormality in a repair response of this mutant cell to some types of DNA cross-links.

Animals↗
PubMed1991

Cell-free translation system using phosphorothioate-containing mRNA.

Phosphorothioate-containing RNAs were generated by transcription of template DNA using the Sp diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates) (NTP alpha S) and T7 RNA polymerase. The substitution of mRNA by phosphorothioate increased the efficiency of protein synthesis by stabilizing the mRNAs in prokaryotic cell-free translation systems. The substituted mRNAs were also shown to be applicable to the continuous cell-free translation system developed by Spirin and coworkers.

Escherichia coli↗
PubMed1991

Posteruptive changes in human dental fluorosis--a histological and ultrastructural study.

The aim of the present study was to describe the structural features characterizing the severe grades of human fluorotic enamel (TF scores 5-9) with particular emphasis on the posteruptive changes in severely fluorosed teeth. Dental fluorosis is a subsurface hypomineralized lesion deep to a well-mineralized outer enamel surface, which in severe cases breaks apart shortly after eruption. Early signs of posteruptive changes comprise small defects corresponding to the opening of striae of Retzius. The enamel pits which develop after eruption in more severe cases exhibit an increase in mineral content at their base which correspond to the exposed subsurface hypomineralized lesions. Likewise, the extensive removal of surface enamel in the most severe cases of human fluorosis results in a highly varying uptake of mineral into the exposed subsurface hypomineralized lesions. The uptake varies greatly within apparently similar degrees of hypomineralized lesions. In approximal abrasion facets, however, where the subsurface lesions are also exposed, no evidence of mineral uptake was found. At the ultrastructural level, the well-mineralized surface zone consists of large hexagonal enamel crystals separated by rather large intercrystalline spaces in which numerous irregular small crystals are observed. Moreover, the large crystals may exhibit central and peripheral dissolution. In addition, mineral appeared to be deposited into such defects as well as along the side of the crystals, often with the lattices being continuous from the original crystal into the apparently posteruptive formed crystal material. It is concluded that a substantial mineral uptake can take place in exposed porous hypomineralized fluorotic enamel after eruption, but is most likely to be associated with the presence of microbial deposits, the metabolic activity of which may play a keyrole in mineral exchange.

Crystallography↗
PubMed1990

Hypoxanthine enhances hydroxyurea-induced sister-chromatid exchanges in Chinese hamster ovary cells.

Treatment with 1 mM hydroxyurea (HU) for 12 h induced sister-chromatid exchange (SCE) in CHO-K1 cells. The induced SCE frequency was always higher in cells grown in Ham's F12 medium than in those grown in RPMI 1640 medium. It was shown that hypoxanthine (Hyp), a component of Ham's F12, was to a great extent responsible for producing a higher level of HU-induced SCEs: HU-induced SCEs were synergistically enhanced when Hyp was added to RPMI 1640 medium supplemented with dialyzed fetal bovine serum at a concentration of 30 microM, which is the concentration in Ham's F12 medium. The radioactivity of [14C]Hyp was readily incorporated into DNA in either the presence or the absence of HU. The greater part was in the forms of dGMP and dAMP. It was not clear whether Hyp was incorporated in the form of dIMP or not. Deoxyguanosine (dGuo), but not deoxyadenosine (dAdo) reversed both the incorporation of radioactivity into DNA and the SCE-enhancing effect of Hyp. Our results indicate that incorporation of Hyp into the dNTP pools and into the DNA, together with perturbation of dGuo metabolism under abnormal conditions during and after HU treatment, is involved in the enhancement by Hyp of HU-induced SCEs.

Animals↗
PubMed1989

Sister chromatid exchange-related characteristics of excision repair-proficient xeroderma pigmentosum cells.

A high incidence of skin cancer characterizes patients with xeroderma pigmentosum (XP). XP patients have hereditary defects in repair mechanisms of ultraviolet light (UV)-induced damage to DNA. Progress in elucidating the pathogenesis of cutaneous cancers can be expected by analysis of the biologic defects of cultured cells from XP patients. Such information may also contribute, at least in part, to an understanding of carcinogenesis in general.

Adult↗
PubMed1989

High resolution electron microscopy of enamel crystals in cases of human dental fluorosis.

Surface enamel from human subjects with dental fluorosis was studied by means of high-resolution transmission electron microscopy. Immediately below the relatively highly mineralized outermost surface enamel layer was an extensive hypomineralized area. The highly mineralized layer was composed of many large elongated hexagonal crystals and extremely small hexagonal crystals. Frequently the small crystals were attached to the periphery of the large crystals. In the hypomineralized area, large crystals were sparsely arranged; and a few small crystals were seen. Some large crystals showed either perforated centers or defects on their peripheries. These findings suggest that the hypomineralized area undergoes caries-like changes in terms of crystal dissolution and that the highly mineralized surface layer is either formed or modified by remineralization.

Adult↗
PubMed1988

Characterization of the enhancing effect of caffeine on sister-chromatid exchanges induced by ultraviolet radiation in excision-proficient xeroderma pigmentosum lymphoblastoid cells.

Cells of some excision-proficient xeroderma pigmentosum (XP) cell lines are highly sensitive to post-UV caffeine treatment in terms of sister-chromatid exchange (SCE) induction as well as cell lethality. In the present study, we conducted a detailed investigation of the enhancing effect of caffeine on SCE frequency induced by UV in excision-proficient XP cells, and obtained the following results. (1) Continuous post-UV treatment with 1 mM caffeine markedly enhances UV-induced SCEs and such enhanced SCEs occur with similar frequency during either the 1st or the 2nd cell cycle in the presence of caffeine and 5-bromodeoxyuridine (BrdUrd). (2) The high sensitivity of the cells to post-UV caffeine treatment persists for at least 2 days after UV when irradiated cells are held in either the proliferating or the nonproliferating state prior to the addition of BrdUrd. (3) Caffeine exerts its effect on cells in S phase. (4) Neither BrdUrd in the medium nor the incorporated 5-bromodeoxyuridine monophosphate (BrdUMP) in DNA plays an appreciable role in the expression of the enhancing effect of caffeine. The most likely explanation for our findings is as follows. In excision-proficient XP cells, the cause of SCE formation such as UV-induced lesions or resulting perturbations of DNA replication persists until the 2nd round or more of post-UV DNA replication. If caffeine is given as post-UV treatment, such abnormalities may be amplified, resulting in a synergistic increase in SCE frequency.

Adult↗
PubMed1987

Intracrystalline structure of enamel crystals affected by caries.

Enamel crystals in the demineralized zones in early caries lesions of human teeth were observed by high-resolution electron microscopy. The enamel crystals frequently exhibited perforations in their centers and defects of various sizes on their lateral surfaces. There were a number of small electron-lucent spots, suggesting that the dissolution of crystals had taken place there. These spots were in especially large numbers near the central dark line. The central perforations, the lateral defects, and the small spots had a common habit which formed regularly along the crystalline a- and b-axes. In many cases, when the central dark line was seen, the perforations were located a few unit cells away from the line. The perforations seem to result from a fusion of small spots, which enlarge by involving other small spots. The lateral defect seemed to enlarge by removal of unit cells and progression along the a- and b-axes. In the regions where the small spots were present, however, the enlargement of the defects also progressed involving the spots. The central dark line seems to be rather resistant to dissolution. One of the main factors for the central perforation of the crystals is thought to be the presence there of especially large numbers of defective sites.

Adult↗
PubMed1986

Proliferation-dependent reduction of sister-chromatid exchange frequency induced by mitomycin C in human lymphoblastoid cells and its suppression by inhibitors of DNA replication.

A high frequency of sister-chromatid exchange (SCE) induced in cells of a human lymphoblastoid cell line, NL3, by 2-h treatment with 1 microM mitomycin C (MMC) was maintained after holding the treated cells in a nonproliferating state for 48 h before cells were transferred into the BrdUrd-containing medium for SCE assay. The same was observed in cells treated with 4-nitroquinoline 1-oxide (4NQO) or ethyl methanesulfonate (EMS). In contrast, when MMC-treated cells were transferred into a growth medium and allowed to proliferate for various periods of time before SCE assay, MMC-induced SCE frequency decreased with time and reached near control level after 48 h. The reduction in SCE was also observed in 4NQO-treated cells, though to a lesser extent, but not in EMS-treated cells. When hydroxyurea or 1-beta-D-arabinofuranosylcytosine was given as a post-MMC treatment during this recovery process, such a reduction of SCE frequency was suppressed and the extent of the suppression appears to be roughly parallel to their ability to inhibit DNA replication. Cycloheximide and 5-azacytidine also exerted a similar inhibitory effect on the reduction of SCE. Benzamide and caffeine had no appreciable effect. Our results indicate that the SCE-forming lesions induced by MMC can be eliminated only in proliferating cells, probably during DNA replication.

4-Nitroquinoline-1-oxide↗
PubMed1986

Novel responses of peripheral lymphocytes of cancer patients to chemical induction of sister chromatid exchanges.

Sensitivities to sister chromatid exchange (SCE) induction by chemicals of peripheral lymphocytes from 26 cancer patients were estimated under conditions identical to those for healthy humans which had been reported (Cancer Res., 43: 439-442, 1983). The sensitive individual was defined as one whose cells give a mean induced SCE frequency more than 2 standard deviation units above the population mean of induced SCEs in cells from the healthy humans. When cells were treated with 3-amino-1-methyl-5H-pyrido[4, 3-b]indole in the presence of rat liver S9 mix, 8 in 10 stomach cancer patients, 4 in 4 colon cancer patients, 3 in 9 lung cancer patients, 0 in 3 patients bearing other cancers, and 0 in 9 non-cancerous individuals were sensitive. The corresponding frequency of individuals in the healthy population, reported previously, was 1 in 33 persons. Thus, the frequency of sensitive individuals in the combined group of stomach and colon cancer patients was very significantly higher than were frequencies in control groups. Three in 10 patients with stomach cancer and 4 in 16 patients with other cancers were sensitive to induction of SCE by methyl methanesulfonate. Six in these 7 methyl methanesulfonate-sensitive patients were also 3-amino-1-methyl-5H-pyrido[4,3-b]indole sensitive. The frequency of methyl methanesulfonate-sensitive individuals in the healthy populations was 2 in 50. There was no patient who was sensitive to SCE induction by 4-nitroquinoline 1-oxide. The frequency was not significantly different from the healthy population, in which 3 in 50 persons were sensitive. These results suggest that a particular cancer correlates with the sensitivity of peripheral lymphocytes to SCE induction by particular chemicals.

4-Nitroquinoline-1-oxide↗
PubMed1985

Clinical and biological studies of 26 cases of xeroderma pigmentosum in northeast district of Japan.

Twenty-six patients with xeroderma pigmentosum (XP), who live in the Northeast (Tohoku) District of Japan, were examined for the clinical characteristics of UV-induced DNA synthesis (unscheduled DNA synthesis, UDS) and UV sensitivity of skin fibroblasts or lymphoblastoid cells, or both. A history of consanguineous marriage within two generations was found in 19 of 26 cases (73%). Two pairs of siblings showed similar manifestations and almost the same levels of UDS and of UV sensitivity. Squamous cell carcinoma, basal cell carcinoma, or both were observed on the exposed skin in 14 patients, but no malignant melanoma was found. Cancer had developed in approximately 71% (10/14) of the cancer-bearing patients by the age of 20, and 8 of them belonged to the UDS-deficient group. Neurological manifestations were associated with nine patients, including 3 with typical de Sanctis-Cacchione syndrome (DSC), and most of the cells derived from these patients had a UDS level less than 10% of that of the normal cells. A clear correlation between the levels of UDS and UV sensitivity, on the one hand, and the severity of clinical manifestations on the other could not be detected, but it seems that the UDS-deficient group is generally much more sensitive to UV in terms of cell killing and the induction of sister chromatid exchange (SCE) than the UDS-proficient group. After a photosensitivity test, one patient with mild skin manifestations showed distinct skin tanning without preceding erythema.

Adolescent↗
PubMed1984

Antagonizing effect of 3-aminoharman on induction of sister-chromatid exchanges by mutagens.

3-Aminoharman (3AH, 3-amino-1-methyl-9H-pyrido[3,4-b]indole), which has been reported as a novel substance with an antagonistic effect on induction of sister-chromatid exchange (SCE) by polycyclic mutagens in the presence of the metabolic activation system, was examined with a cultured human lymphoblastoid cell line, NL3, for its effect on SCE induction by direct-acting mutagens such as mitomycin C (MMC), nitrogen mustard N-oxide (NMO), methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline 1-oxide (4NQO) and 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (OH-Trp-P-2), and also by ultraviolet light (UV) irradiation. The results obtained on simultaneous treatment with 3AH and mutagens were as follows: (1) 3AH suppressed more than 50% of SCEs induced by MMC, NMO and OH-Trp-P-2; (2) 4NQO- and MNNG-induced SCEs were also suppressed by 3AH but to a lesser degree; (3) MMS-induced SCEs were not, however, altered by 3AH; and (4) the suppression of SCE by 3AH was dose-dependent. Treatment of cells with 3AH for 2 h immediately before MMC exposure suppressed SCE induction to a significant degree similar to the simultaneous treatment, but post-treatment with 3AH was much less effective. 3AH inhibited SCE induction by NMO when 3AH treatment was carried out either before or after NMO treatment, to an extent similar to the simultaneous treatment. Treatments with 3AH either before or after UV exposure did not change the UV-induced SCEs. Results with these direct-acting mutagens ruled out the relevance of metabolic activation as a necessary step for the antagonizing effect of 3AH.

Biotransformation↗
PubMed1983

Differential features of sister-chromatid exchange responses to ultraviolet radiation and caffeine in xeroderma pigmentosum lymphoblastoid cell lines.

Sister-chromatid exchange (SCE) induced by ultraviolet (UV) irradiation and viability after UV irradiation were studied in lymphoblastoid cell lines derived from 7 patients with xeroderma pigmentosum (XP) and 6 normal donors. UV irradiation caused significant increases of SCEs in both XP and normal cells. In 3 XP cell lines, which were deficient in unscheduled DNA synthesis (UDS) and sensitive to the killing effect of UV, very high SCE frequencies were observed after UV irradiation. Cells from a patient with the De Sanctis-Cacchione syndrome were the most sensitive to UV in terms of both SCE induction and cell killing. In 2 of 4 UDS-proficient XP cell lines tested, the incidences of UV-induced SCEs were similar to those in normal cell lines, but in 2 other UDS-proficient lines from 2 XP patients with skin cancer, the frequencies of UV-induced SCEs were significantly higher than in normal cells. Continuous post-UV treatment with 1 mM caffeine markedly enhanced UV-induced SCEs in 3 of 4 UDS-proficient XP cell lines but had only slight effects on cells from the 4th UDS-proficient XP patient and from normal individuals.

Caffeine↗
PubMed1983

Actions of amino-beta-carbolines on induction of sister-chromatid exchanges.

Synthetic 3-aminoharman and 3-aminonorharman (amino-beta-carbolines) caused slight but definite induction of sister-chromatid exchanges (SCEs) in human lymphoblastoid cells NL3 and Chinese hamster cells CHO-K1. These amino-beta-carbolines are ranked between 2-amino-alpha-carboline and 2-amino-6-methyl-9a-aza-delta-carboline (Glu-P-2) and much lower than 3-amino-gamma-carbolines (Trp-P-1 and 2) in inductive activity. 1-Amino-beta-carboline, harman and norharman had very weak, if any, SCE-inducer activity. Norharman had a synergistic effect with aromatic amines such as Trp-P-2 and aniline on SCE induction, while 3-aminoharman suppressed SCE induction by more potent inducers such as Trp-P-2 and benzo[a]pyrene.

Animals↗