[Effect of hypergravity on X-ray-induced cell cycle arrest in mouse m5S cells].
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Animals↗
Biomedical subjects
M Ikenaga
Publications and source records attributed to M Ikenaga.
Expression of a mammalian DNA photolyase confers light-dependent repair activity and reduces mutations of UV-irradiated shuttle vectors in xeroderma pigmentosum cells.
Photoreactivation is one of the DNA repair mechanisms to remove UV lesions from cellular DNA with a function of the DNA photolyase and visible light. Two types of photolyase specific for cyclobutane pyrimidine dimers (CPD) and for pyrimidine (6-4) pyrimidones (6-4PD) are found in nature, but neither is present in cells from placental mammals. To investigate the effect of the CPD-specific photolyase on killing and mutations induced by UV, we expressed a marsupial DNA photolyase in DNA repair-deficient group A xeroderma pigmentosum (XP-A) cells. Expression of the photolyase and visible light irradiation removed CPD from cellular DNA and elevated survival of the UV-irradiated XP-A cells, and also reduced mutation frequencies of UV-irradiated shuttle vector plasmids replicating in XP-A cells. The survival of UV-irradiated cells and mutation frequencies of UV-irradiated plasmids were not completely restored to the unirradiated levels by the removal of CPD. These results suggest that both CPD and other UV damage, probably 6-4PD, can lead to cell killing and mutations.
Base Sequence↗
A genetic effect of altered gravity: mutations induced by simulated hypogravity and hypergravity in microsatellite sequences of human tumor cells.
To determine the possible genetic effects of gravity alterations, we analyzed mutation induction in microsatellite sequences of human tumor cells treated with simulated hypogravity provided by a clinostat or hypergravity by a centrifuge. Microsatellite mutations were detected as changes in the size of polymerase chain reaction (PCR)-amplified allelic markers. The frequencies of mutant clones in cultures treated with simulated hypogravity for 24 or 48 h were almost the same as those of controls, but after 72 h of treatment, the mutant frequencies had increased significantly in all three microsatellite loci examined. Significantly higher mutant frequencies were similarly detected in cultures treated for 72 h with a hypergravity condition as low as 18xg, but not detected in 24 or 48 h treated cultures. These findings clearly show that gravity alterations that last for 3 days can induce microsatellite mutations in human cells. A genetic effect of gravity change, therefore, is established for the first time. Moreover, high frequencies of microsatellite mutations were induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) which activates protein kinase C-mediated signal transduction pathways and causes genetic instability. These findings suggest that gravity change induces microsatellite mutations by modulating the pattern of gene expression involved in signal transduction pathways.
DNA Primers↗
DCRY is a Drosophila photoreceptor protein implicated in light entrainment of circadian rhythm.
BACKGROUND: Light is the major environmental signal for the entrainment of circadian rhythms. In Drosophila melanogaster, the period(per) and timeless (tim) genes are required for circadian behavioural rhythms and their expression levels undergo circadian fluctuations. Light signals can entrain these rhythms by shifting their phases. However, little is known about the molecular mechanism for the perception and transduction of the light signal. The members of the photolyase/cryptochrome family contain flavin adenine dinucleotide (FAD) as chromophore and are involved in two diverse functions, DNA repair and photoreception of environmental light signals. RESULTS: We report the cloning of a new member of this family, dcry, from Drosophila. Northern blot analysis shows that this gene is expressed in various tissues. The dcry mRNA is expressed in a circadian manner in adult heads, while such rhythmic fluctuation is abolished in the clock-defective per0 and tim0 mutants. The circadian expression is dampened down in constant darkness. The over-expression of the dcry gene alters the light-induced phase delay in the locomotor activity rhythms of flies. CONCLUSION: These results suggest that DCRY is a circadian photoreceptor and that its expression is regulated by circadian clock genes.
Age Factors↗
[Neurological adverse effects of morphine].
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Analgesics, Opioid↗
[STS-95 space experiment: analysis of mutations induced in human tumor cells].
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Cell Physiological Phenomena↗
Specificity of mutations in the PMS2-deficient human tumor cell line HEC-1-A.
The spectrum of mutations was determined at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in the human uterine tumor cell line HEC-1-A which is defective in the mismatch repair gene hPMS2. The mutation frequency at the hprt locus in HEC-1-A was about two orders higher than that in wild type repair-proficient cells. The fifty-eight mutations detected were exclusively point mutations, with frameshifts of one base deletion/addition predominating (66%) the remaining were base substitutions. All the frameshift mutations occurred at sites of monotonous repeating sequences, including six consecutive guanine bases site which was the hot spot for the addition of one G that contributed 60% of the total mutations. Although the observed specificity of mutations in HEC-1-A apparently resembled that of the hMLH1-deficient cell line HCT116 [Ohzeki, S., Tachibana, A., Tatsumi, T., Kato, T., 1997. Spectra of spontaneous mutations at the hprt locus in colorectal carcinoma cell lines defective in mismatch repair. Carcinogenesis, 18, 1127-1133.], the pronounced increase of +/-1 bp frameshifts and the reduced incidence of C-->T transitions at the CpG site suggest that the hPMS2 gene product may have an additional function in the mismatch repair process independent of it's role in the hMutLalpha heterodimer.
Adenosine Triphosphatases↗
Biomechanical characterization of a biodegradable calcium phosphate hydraulic cement: a comparison with porous biphasic calcium phosphate ceramics.
Biomechanical properties of a biodegradable calcium phosphate hydraulic cement (CPHC) were tested with rabbits. The cement was composed of beta-tricalcium phosphate (beta-TCP), monocalcium phosphate monohydrate (MCPM), and calcium sulfate hemihydrate (CSH), beta-TCP-MCPM-CSH cement. Cylinders of 4.7 mm in diameter and 10 mm in length were put into bone cavities created in the distal epiphysis of femurs in rabbits. Cylinders of the same size of porous biphasic calcium phosphate ceramics (BCPC, 75% hydroxyapatite and 25% beta-TCP) were implanted as references. Two, 4, 12, and 16 weeks after the operation, the rabbits were sacrificed. Histomorphometry showed that the cement was resorbed, leaving only 7.67 +/- 1.81% of bone cavity after 12 weeks. Newly formed bone occupied 34.59 +/- 4.00% of the cavity. Cylindrical bone-material composites were cut out with a small dental burr. Compressive force was applied to the specimens and compressive strength, elastic modulus, and toughness were calculated. The same tests were performed on cylinders of normal bone from the same site, which served as controls. The compressive strength and the toughness of the cement-bone composite were higher than those of normal bone and porous ceramics 12 weeks after the operation (p < 0.05). At 16 weeks the compressive strength and the toughness returned to the normal bone values. The elastic modulus of the porous ceramic-bone composite was higher than the normal bone at 4, 12, and 16 weeks after surgery (p < 0.05). We found that the beta-TCP-MCPM-CSH cement is replaced by new bone and that the cement-new bone composite has similar or better mechanical properties than normal bone within 16 weeks. This study suggests the usefulness of a particular cement for filling bone defects or for temporary fixation of orthopedic implants.
Animals↗
Cockayne syndrome without typical clinical manifestations including neurologic abnormalities.
Although patients with mild symptoms of atypical Cockayne syndrome (CS) have been described, there has not been a report of a patient with CS whose only clinical manifestation was cutaneous photosensitivity. Cells from patients with CS show UV sensitivity, reduced recovery of RNA synthesis, but normal UV-induced unscheduled DNA synthesis. On the other hand, the patients with UV-sensitive syndrome have only cutaneous photosensitivity and skin freckles, whereas those cells respond to UV radiation in a similar fashion to the CS cells. We describe a patient with CS who showed only photosensitivity without typical clinical manifestations of CS, but his cells showed UV sensitivity, reduced recovery of RNA synthesis, and normal unscheduled DNA synthesis after UV radiation similar to CS cells. Furthermore, the patient was assigned to complementation group B of CS on the basis of the results of complementation analysis. The present report suggests that CS has a wider spectrum than that considered previously.
Adolescent↗
Neoplastic transformation of hamster embyro cells by heavy ions.
We have studied the induction of morphological transformation of Syrian hamster embryo cells by low doses of heavy ions with different linear energy transfer (LET), ranging from 13 to 400 keV/micrometer. Exponentially growing cells were irradiated with 12C or 28Si ion beams generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC), inoculated to culture dishes, and transformed colonies were identified when the cells were densely stacked and showed a crisscross pattern. Over the LET range examined, the frequency of transformation induced by the heavy ions increased sharply at very low doses no greater than 5 cGy. The relative biological effectiveness (RBE) of the heavy ions relative to 250 kVp X-rays showed an initial increase with LET, reaching a maximum value of about 7 at 100 keV/micrometer, and then decreased with the further increase in LET. Thus, we confirmed that high LET heavy ions are significantly more effective than X-rays for the induction of in vitro cell transformation.
Animals↗
Assignment of three Chinese xeroderma pigmentosum patients to complementation group C and one to group E.
Four Chinese patients with xeroderma pigmentosum (XP), who had different degrees of skin symptoms, were tested for their genetic complementation groups. Skin fibroblasts obtained from the patients were used for complementation analysis done by a cell-fusion technique. Three of the patients belonged to group C and one, who had the mildest cutaneous manifestations, to group E. This is the first report of a group E XP patient in China. Our present findings together with previous reports suggest that group C XP is more common in China, similar to the distribution among Caucasian XP patients but markedly different from the Japanese distribution.
Adolescent↗
Three-dimensional visualization of ultraviolet-induced DNA damage and its repair in human cell nuclei.
The two major forms of DNA damage produced by 254 nm UV light are cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct (6-4PP). Both photolesions are repaired in normal human cells by nucleotide excision repair; however, little is known about where CPD or 6-4PP are repaired in relation to the various subnuclear structures. This study aimed to produce a three-dimensional demonstration of UV-induced DNA damage and its repair in human cell nuclei. We first investigated the repair kinetics of CPD and 6-4PP using an enzyme-linked immunosorbent assay with damage-specific monoclonal antibodies in normal human and xeroderma pigmentosum complementation group C cells. We also examined the kinetics of repair DNA synthesis (unscheduled DNA synthesis) using a quantitative immunofluorescence method with anti-5-bromo-2'-deoxyuridine antibodies. We confirmed the normal repair in normal human cells and the impaired repair in xeroderma pigmentosum complementation group C cells. Then, using laser scanning confocal microscopy, we succeeded in forming a three-dimensional visualization of the nuclear localization of CPD, 6-4PP, and unscheduled DNA synthesis in individual human cells. The typical three-dimensional images of photolesions or unscheduled DNA synthesis at various repair times reflected the repair kinetics obtained by enzyme-linked immunosorbent assay or immunofluorescence very well. The important finding is that the punctate, not diffusely spread, nuclear localization of unrepaired 6-4PP was found 2 h after irradiation. Similarly, the focal nuclear localization of unscheduled DNA synthesis was observed during both the first and the second 3 h repair periods. The present results suggest that both 6-4PP and CPD are nonrandomly repaired from nuclei in normal human cells.
Cell Nucleus↗
The relative biological effectiveness of accelerated carbon ions with different LET for inducing mitotic crossing over and intragenic reversion of the white-ivory allele in Drosophila larvae.
PURPOSE: To evaluate the relative biological effectiveness (RBE) of accelerated carbon ions generated with a synchrotron for inducing mutations as a function of linear energy transfer (LET), using the loss of heterozygosity for wing-hair mutations and the reversion of the mutant white-ivory eye-colour in Drosophila melanogaster. MATERIALS AND METHODS: The measurements were made using a combined mutation assay system so that induced mutant wing-hair clones as well as revertant eye-colour clones can be detected simultaneously in the same fly. Larvae were irradiated at the age of 72+/-6 h post-oviposition with X-rays or carbon ions with LET values of 13, 60 and 95 keV/microm. RESULTS: The RBE of carbon ions for producing wing-hair mosaic spots increased with increasing LET values. The RBE for the induction of eye-colour mutants did not change with LET. The estimated RBE values were found to be in the range 2 to 6.5 for the wing-hair and nearly unity for the eye-colour mosaic spot mutations. CONCLUSIONS: RBE-LET relationships were obtained for the induction of wing-hair and eye-colour mosaic spots. These relationships suggest that more complex types of DNA damage, such as nonrejoinable strand breaks that increase with LET, may be responsible for inducing the wing-hair mutation, while more simple forms of molecular damage induce reversion in the white-ivory allele.
Animals↗
Low levels of apoptosis and proliferative activity in colorectal villous tumors: comparison with tubular tumors.
In order to clarify the cell kinetics of colorectal villous tumors (VT), 21 villous adenomatous areas and 12 carcinomatous areas within villous adenomas were investigated for proliferative activity and apoptosis and compared with a series of 41 tubular tumors (TT), demonstrating elements of intramucosal carcinomas as well as tubular adenomas (so-called carcinoma in tubular adenoma). Proliferation was estimated in terms of Ki-67 labeling indices and mitotic indices, and apoptosis was assessed by DNA nick-end labeling to give apoptotic indices. Apoptotic indices of villous adenomatous and carcinomatous regions were significantly lower than the values for their tubular counterparts. Ki-67 labeling indices were also significantly lower for adenoma components. Apoptotic indices, Ki-67 labeling indices and mitotic indices increased with atypia raised in tubular adenoma components. Correlations of mitotic indices with apoptotic indices, Ki-67 labeling indices with apoptotic indices and mitotic indices with Ki-67 labeling indices were found for each villous tumor group and tubular tumor group, and the apoptosis and proliferation ratios for villous tumors were relatively low, suggesting a tendency for greater growth due to less cell deletion. Although this is only one of the biological features of villous tumor groups, it might play a major role in generation of malignancy.
Adenocarcinoma↗
Relative biological effectiveness of accelerated heavy ions for induction of morphological transformation in Syrian hamster embryo cells.
Syrian hamster embryo cells were used to study the morphological transformation induced by accelerated heavy ions with different linear energy transfer (LET) ranging from 13 to 400 keV/micron. Exponentially growing cells were irradiated with 12C or 28Si ion beams generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC), then inoculated to culture dishes. Morphologically altered colonies were scored as transformants. Over the LET range examined, the frequency of transformation induced by the heavy ions increased sharply at very low doses no greater than 5 cGy. The relative biological effectiveness (RBE) of the heavy ions relative to X-rays first increased with LET, reached a maximum value of about 7 at 100 keV/micron, then decreased with the further increase of LET. Our findings confirmed that high LET heavy ions are much more effective than X-rays for the induction of in vitro cell transformation.
Animals↗
[Development of a model which makes it possible to test injectable bone substitutes and evaluation of a calcium phosphate cement].
New percutaneous filling techniques are beginning to be used in bone tumor pathology. The purpose of this study was to develop a reproducible model for testing new injectable bone substitutes. A closed cancellous bone defect was created in the distal femoral condyles of rabbit. Bone defect was filled by a percutaneous injection. Several situations were tested: spontaneous evolution of unfilled cavities, evolution of cavities filled either with a conventional orthopedic cement (PMMA), either with a mixed collagen-hydroxyapatite material, either with a biomaterial derived from the vegetal protein zein, either with a calcium phosphate cement brushite type. The new bone formation was quantified in the defect for each group in which several delays were tested. A bone defect of reproducible size was obtained consistently. A partial bone formation was observed in the unfilled group. The best bone formation rate was obtained in the phosphate cement group. This model showed the interest of injectable biomaterials and more precisely the interest of the phosphate calcium cements as brushite.
Animals↗
Binding and catalytic properties of Xenopus (6-4) photolyase.
Xenopus (6-4) photolyase binds with high affinity to DNA bearing a (6-4) photoproduct and repairs it in a light-dependent reaction. To clarify its repair mechanism of (6-4) photolyase, we determined its binding and catalytic properties using synthetic DNA substrate which carries a photoproduct at a single location. The (6-4) photolyase binds to T[6-4]T in double-stranded DNA with high affinity (KD = 10(-9)) and to T[6-4]T in single-stranded DNA and T[Dewar]T in double- and single-stranded DNA although with slightly lower affinity (KD = approximately 2 x 10(-8)). Majority of the T[6-4]T-(6-4) photolyase complex dissociates very slowly (koff = 2.9 x 10(-5) s-1). Its absolute action spectrum without a second chromophore in the 350-600 nm region closely matches the absorption spectrum of the enzyme. The quantum yield (phi) of repair is approximately 0.11. The fully reduced form (E-FADH-) of (6-4) photolyase is catalytically active. Direct analysis of the photoreactivated product showed that (6-4) photolyase restores the original pyrimidines. These findings demonstrate that cis, syn-cyclobutane pyrimidine dimer photolyase and (6-4) photolyase are quite similar, but they are different with regard to the binding properties.
Animals↗
Flavin adenine dinucleotide as a chromophore of the Xenopus (6-4)photolyase.
Two types of enzyme utilizing light from the blue and near-UV spectral range (320-520 nm) are known to have related primary structures: DNA photolyase, which repairs UV-induced DNA damage in a light-dependent manner, and the blue light photoreceptor of plants, which mediates light-dependent regulation of seedling development. Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)photoproducts] are the two major photoproducts produced in DNA by UV irradiation. Two types of photolyases have been identified, one specific for CPDs (CPD photolyase) and another specific for (6-4)photoproducts [(6-4)photolyase]. (6-4)Photolyase activity was first found in Drosophila melanogaster and to date this gene has been cloned only from this organism. The deduced amino acid sequence of the cloned gene shows that (6-4)photolyase is a member of the CPD photolyase/blue light photoreceptor family. Both CPD photolyase and blue light photoreceptor are flavoproteins and bound flavin adenine dinucleotides (FADs) are essential for their catalytic activity. Here we report isolation of a Xenopus laevis(6-4)photolyase gene and show that the (6-4)photolyase binds non- covalently to stoichiometric amounts of FAD. This is the first indication of FAD as the chromophore of (6-4)photolyase.
Animals↗