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Biomedical subjects

A Oikawa

Publications and source records attributed to A Oikawa.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1990

Increased UV sensitivity of Escherichia coli cells after introduction of foreign photolyase genes.

High-expression plasmids for photolyase (phr) genes from the bacteria Escherichia coli, Anacystis nidulans, Streptomyces griseus and Halobacterium halobium and the yeast Saccharomyces cerevisiae were constructed and introduced into E. coli phr recA cells. As previously reported, al introduced phr genes provided the host cells with photoreactivation-repair activity and the introduced E. coli phr gene rendered the host cells more UV-resistant in the dark. E. coli cells harboring foreign phr genes, however, were found to be more sensitive to UV light in the dark than cells containing the vector plasmid only. These differences in UV sensitivity in the dark disappeared when the host cells had an additional mutation, uvrA, suggesting that the foreign photolyases inhibited the E. coli excision-repair system.

Base Sequence↗
PubMed1989

Molecular characterization of a gene encoding a photolyase from Streptomyces griseus.

By using a synthetic DNA probe derived from an amino acid sequence in the most conserved region of three known photolyases (Escherichia coli, Anacystis nidulans and Saccharomyces cerevisiae), we isolated a DNA fragment containing two long open reading frames (ORFs) from a genomic DNA library of Streptomyces griseus. One ORF encodes a polypeptide of 455 amino acids (Mr 50594), which exhibits substantial similarities with the other three photolyases. Photoreactivation-repair deficient E. coli cells could be converted into photoreactivatable ones by introduction of plasmids harboring this ORF, indicating that this is the photolyase gene of S. griseus. The deduced aa sequence of Streptomyces photolyase was most similar to that of E. coli. The putative DNA binding site as well as cofactor binding regions were proposed.

Amino Acid Sequence↗
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

Expression of an Anacystis nidulans photolyase gene in Escherichia coli; functional complementation and modified action spectrum of photoreactivation.

The Anacystis nidulans photolyase gene inserted in an expression vector plasmid was introduced into Escherichia coli cells and the production of Anacystis photolyase protein was confirmed by reaction with antibodies raised against photolyase purified from A. nidulans cells. The Anacystis photolyase functioned in photoreactivation repair defective E. coli cells. The E. coli transformants exhibited an action spectrum with a maximum around 380 nm similar to that of E. coli photolyase in contrast with the action spectrum of A. nidulans cells which has a maximum at 437 nm. These results indicate that the Anacystis photolyase produced in E. coli cells has enzymatic activity in spite of the apparent lack of its intrinsic 8-hydroxy-5-deazaflavin cofactor.

Base Sequence↗
PubMed1989

Tandem arrangement of photolyase and superoxide dismutase genes in Halobacterium halobium.

A DNA fragment containing the photolyase gene was cloned from Halobacterium halobium. The deduced amino acid sequence is highly similar to those of four known photolyases from eubacteria and a eucaryote. The cloned gene expressed in Escherichia coli increased the survival of UV-irradiated host cells by photoreactivation. These results indicate that photolyases of eucaryotes, eubacteria, and archaebacteria are derived from a common origin. In this cloned DNA fragment, two additional open reading frames (ORFs), ORF 151 and ORF 200, were found in the 5' and 3' adjacent flanking regions of the photolyase gene. ORF 200 shows unequivocal amino acid sequence homology to all known manganese and iron superoxide dismutases. Northern (RNA) hybridization analysis of H. halobium RNA revealed the existence of three transcripts, one of which covered all three ORFs, indicating that photolyase and superoxide dismutase are partly cotranscribed in this bacterium.

Amino Acid Sequence↗
PubMed1988

Cloning and characterization of a photolyase gene from the cyanobacterium Anacystis nidulans.

A 2 kb fragment was isolated from an Anacystis nidulans genomic DNA library by hybridization with synthetic oligonucleotide probes derived from the N-terminal amino acid sequence of Anacystis photolyase. This fragment contains a 1452 bp-long open reading frame encoding a polypeptide of 484 amino acids (Mr 54475). Antibodies raised against purified Anacystis photolyase reacted with extracts of cells harboring fused genes between lacZ of Escherichia coli and this gene. A 40.7% similarity was found between the deduced amino acid sequences of Anacystis and E. coli photolyases, notwithstanding the difference in chromophore structure.

Amino Acid Sequence↗
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

An association of melanophores appearing at metamorphosis as vehicles of asymmetric skin color formation with pigment anomalies developed under hatchery conditions in the Japanese flounder, Paralichthys olivaceus.

The mechanisms for asymmetric skin color formation in the Japanese flounder are studied with particular concerns to causes for pigment disorder (hypomelanosis) occurring under hatchery conditions. For an analysis of normal pigmentation, fish were raised with wild zooplanktons in an indoor hatchery, whilst for hypomelanosis, they were raised with Brazilian Artemia nauplii, a diet used in the hatcheries. Morphological observations, counting of melanophores, histochemical assay of DOPA-positive immature cells (melanoblasts), and radiometric estimation of tyrosinase activities in skins of developing larvae and juveniles indicate that 1) the structural plan for pigmentation in this species is bilaterally symmetric until metamorphosis, utilizing large-sized melanophores (hence larval melanophores) as main vehicles, and 2) an asymmetric coloration characteristic to metamorphosed juveniles is formed by an intensive development of smaller-sized melanophores (hence adult-type melanophores) appearing selectively in the ocular side at the later stages of metamorphosis and by an absence of it in the blind. These findings apparently indicate that 1) two types of melanophores occur in this species which differ with respect to morphological properties and developmental fate, and 2) selective differentiation of adult type melanophores in the ocular side of the body at or after metamorphosis is primarily responsible for an asymmetric coloration of its adult form. The similar assays on the fish fed with Artemia nauplii indicate that defective development of adult-type melanophores results in hypomelanosis in their ocular-sided skins, yielding a pigmentary pattern seen in the blind side of the metamorphosed juveniles with normal pigmentation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1987

Electron microscopic evidence for stimulation of melanosomal maturation by lysosomotropic agents and monensin in cultured B16 mouse melanoma cells.

Mouse melanoma cells, B16-C2M in monolayer culture were treated with either lysosomotropic agent, 10 mM NH4Cl, or 20 microM chloroquine, an ionophore, or 10 microns monensin for 3 h at 37 degrees C, and examined with regard to the site of melanin deposition and numbers of melanized (type 1) and unmelanized (type 2) melanosomes under a transmission electron microscope. The numbers of these two types of melanosomes were counted on electron micrographs of thin sections of 20 to 40 cells for each experimental group and expressed in terms of number per unit area of sectioned cytoplasm. Although most melanosomes were largely swollen in monensin-treated cells, melanin deposition was apparently confined in melanosomes in all experimental groups. The compound melanosomes were scarcely found. The mean population density (number per unit cytoplasmic area) of type 1 melanosomes was highest in the NH4Cl-treated cell group followed by monensin-treated, chloroquine-treated, and control cell groups. When the relative abundance of type 1 melanosomes was expressed as a fraction of total number of type 1 and 2 melanosomes (melanosomal maturation index, MMI), the differences were much more evident. Type 1 melanosomes were found in every cell (MMI not equal to 0) of the groups treated with NH4Cl and chloroquine only, which suggested the existence of a subpopulation of cells responsive to lysosomotropic agents but not to monensin in regard to melanosome maturation. All these findings indicate that the stimulation of melanogenesis proceeds mainly through maturation of preexisting melanosomes under these conditions.

Ammonium Chloride↗
PubMed1987

[Sister chromatid exchange and detection of cancer predisposition].

Based on a result suggesting that the frequency of the Trp-P-2-sensitive persons in patients suffering from cancers at digestive tract is higher than in healthy persons when the sensitive person is defined as the person whose SCE frequency in peripheral lymphocytes induced by in vitro treatment with Trp-P-2 is more than 2 sigma above the mean of the SCEs of healthy population under the identical assay conditions. The possibility to detect the cancer predisposition is discussed.

Bloom Syndrome↗
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↗
PubMed1985

Stimulation by ionophores of tyrosinase activity of mouse melanoma cells in culture.

The hypothesis that the melanosome is an acidic vesicle in which the tyrosinase action is suppressed under the ordinary culture conditions was examined with a variety of ionophores added in cultures of mouse melanoma cell line B16-C2M. In the presence of monensin or nigericin, which exchange H+ for Na+ or K+, respectively, through bio-membrane, the tyrosinase activity of cells in culture was more than 10 times that in the control culture. This stimulation was observed without delay after addition of the chemicals and was not inhibited by cycloheximide. The enzyme activity of sonicated cell-free extracts, in which melanosomes were disrupted, was not stimulated by these ionophores. The tyrosinase activity was stimulated to a lesser extent by a proton ionophore, p-trifluoromethoxyphenylhydrazone (FCCP). The activity was also stimulated by kryptofix 221, valinomycin (Na+ and K+ carrier, respectively), and tetraethylammonium ions (permeant cations) but only in the presence of a limited concentration of FCCP. N-Ethylmaleimide and N, N'-dicyclohexylcarbodiimide, inhibitors of lysosomal proton pump, stimulated tyrosinase activity of cells in the presence of FCCP. These facts are consistent with the hypothesis described above.

Animals↗