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1.
DNA damage and repair in trypanosomatids impacts virulence, drug resistance and antigenic variation but, currently, little is known about DNA damage responses or cell cycle checkpoints in these divergent protozoa. One of the earliest markers of DNA damage in eukaryotes is γH2A(X), a serine phosphorylated histone H2A (variant). Here, we report the identification and initial characterization of γH2A in Trypanosoma brucei. We identified Thr(130) within the replication-dependent histone H2A as a candidate phosphorylation site and found that the abundance of this trypanosomal γH2A increased in vivo in response to DNA damage. Nuclear γH2A foci mark the sites of putative natural replication fork stalling, sites of meganuclease-induced DNA double strand breaks and sites of methyl methanesulphonate-induced DNA damage. Naturally occurring and meganuclease-induced γH2A and RAD51 double-positive repair foci are typically found in S-phase or G(2) nuclei. The results link trypanosomal γH2A, with an unusual histone modification motif, to DNA damage sensing and mitotic checkpoint signaling. 相似文献
2.
Turinetto V Orlando L Sanchez-Ripoll Y Kumpfmueller B Storm MP Porcedda P Minieri V Saviozzi S Accomasso L Rocchietti EC Moorwood K Circosta P Cignetti A Welham MJ Giachino C 《Stem cells (Dayton, Ohio)》2012,30(7):1414-1423
Phosphorylation of histone H2AX (γH2AX) is known to be the earliest indicator of DNA double-strand breaks. Recently, it has been shown that mouse embryonic stem cells (mESCs) have very high basal levels of γH2AX, even when they have not been exposed to genotoxic agents. As the specialized role of high basal γH2AX levels in pluripotent stem cells is still debated, we investigated whether H2AX phosphorylation is important in maintaining self-renewal of these cells. Here, we report that not only mESCs but also mouse-induced pluripotent stem cells (miPSCs), have high basal levels of γH2AX. We show that basal γH2AX levels decrease upon ESC and iPSC differentiation and increase when the cells are treated with self-renewal-enhancing small molecules. We observe that self-renewal activity is highly compromised in H2AX-/- cells and that it can be restored in these cells through reconstitution with a wild-type, but not a phospho-mutated, H2AX construct. Taken together, our findings suggest a novel function of H2AX that expands the knowledge of this histone variant beyond its role in DNA damage and into a new specialized biological function in mouse pluripotent stem cells. 相似文献
3.
《Mutation Research/DNA Repair》1997,383(1):71-80
The alkaline SCGE assay was evaluated for use with cryopreserved lymphocytes in order to obtain results similar to the freshly isolated ones. The induction of DNA damage as well as the repair capacity of γ-rays and H2O2 exposed cryopreserved human lymphocytes was found to be the same to that of the freshly isolated. Human lymphocytes (fresh or cryopreserved) responded differently to the effects of γ-irradiation if compared to the H2O2 treatment. The distribution of DNA damage among γ-irradiated lymphocytes was more homogeneous compared to H2O2, both in freshly isolated and in cryopreserved cells. 2.4 μg/ml phytohemagglutinin at the start of a 2-h incubation in RPMI of cryopreserved samples gave similar DNA repair and distribution patterns to the 2-h post-exposure incubation of freshly isolated lymphocytes. H2O2-induced DNA damage was not repaired completely. However, the repair of γ-rays-induced DNA damage was more efficient. These findings confirm the different mode of action of the two agents on the induction of DNA damage, as well as, the different response of the lymphocytes' DNA repair system. 相似文献
4.
S Ishizuka M Shiwaku M Hagio T Suzuki T Hira H Hara 《Biomedical research (Tokyo, Japan)》2012,33(3):159-165
Bile acids (BAs) are considered to be promotive factors in colorectal carcinogenesis. We investigated whether BAs in the cellular environment influence proliferation of intestinal epithelial cell lines. Some BAs induced proliferation in several epithelial cell lines. In the proliferation assay, significant increases in IEC-6 cell proliferation were observed in response to glycodeoxycholic acid or glycochenodeoxycholic acid (GCDCA). Among the glycine-conjugated derivatives of BAs, especially GCDCA reduced cAMP production in IEC-6 cells. Pertussis toxin completely inhibited the GCDCA-induced increase in IEC-6 proliferation, suggesting GCDCA-induced proliferation required Gαi activation and cAMP reduction. Treatment with 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, also suppressed GCDCA-induced IEC-6 proliferation. We confirmed an increase in MEK1/2 phosphorylation in GCDCA-treated IEC-6 cells, and inhibition of MEK1/2 by U0126 clearly suppressed GCDCA-induced IEC-6 cell proliferation. A significant increase was observed in the phosphorylation of histone H2AX in GCDCA-treated IEC-6 cells after exposure to γ-rays. Cell cycle analysis revealed that GCDCA increased the proportion of cells in S phase only after γ-ray exposure. These results indicate that glycine-conjugated BAs in the cellular environment are potent inducers of cell proliferation accompanied by genomic instability in intestinal epithelia. 相似文献
5.
Ryoichi Miyashita Li Chen Hisashi Oshiro Hiroyuki Uchino Futoshi Shibasaki 《Neuroscience letters》2012
We have previously shown that when siRNA against Int6 (siRNA-Int6) was used, hypoxia-inducible factor 2α (HIF2α) activity was stabilized even under normoxic conditions, and the expression of several angiogenic factors was increased. In neuronal tissues, the mechanism underlying angiogenesis remains largely unknown. In the current study, we investigate the role of the tumor suppressor Int6/eIF3e in the regulation of the expression of angiogenic factors in neuronal cells. In addition, we test whether siRNA-Int6 reduces cold-induced brain damage in rats. We used human neuroblastoma SHSY5Y cells transfected with either siRNA-Int6, or a negative control siRNA. Real-time PCR and supersensitive multiplex assay were used to detect gene and protein expression of several angiogenic factors after transfection. For the animal studies, Wistar rats were subjected to brain damage by cold injury, and 50 μg siRNA-Int6, 100 μg siRNA-Int6, or negative control was administrated. At day 7 post-treatment, brain sections were stained and image analysis system was used to determine the damaged area. Our experiments using SHSY5Y cells revealed a significant effect of siRNA-Int6 on the expression of HIF2α but not HIF1α, both at 8 and 24 h after transfection. The siRNA-Int6 led to significant up-regulation of angiogenic factors, including vascular endothelial growth factor and platelet-derived growth factor-B, both at the mRNA and protein levels. Furthermore, our animal studies revealed significantly reduced area of cold-induced damage in rats receiving siRNA-Int6, compared to negative controls. Our findings indicate that Int6 act as a hypoxia-independent master switch of angiogenesis in neuronal cells, and that inhibition of Int6 by siRNA may be an effective therapeutic strategy in treating ischemic diseases such as brain ischemia and injury. 相似文献
6.
《Mutation Research/DNAging》1991,256(1):59-66
A reduction in the amount of UV-induced unscheduled DNA synthesis (UDS), and reduced cell survival and host-cell reactivation against UV exposure in Hutchinson-Gilford progeria syndrome cell strains were shown. UV-induced UDS in 4 progeria cell strains was 33–50% of the normal level. A similar reduction in the UV-induced UDS in normal cells was caused by γ-ray irradiation to the cells before UV irradiation. The dose of γ-rays required to cause a reduction in UDS of normal cells to the level of progeria cells was 40 Gy and the reduction was reversible after 2 days. In progeria cells, γ-ray irradiation further reduced UDS with a lower γ-ray dose required than in normal cells, and the reduction was also reversible but with less relative recovery than in normal cells. The presence of a ‘built-in’ defect in progeria cells responsible for the reduced DNA-repair capacity was suggested, and such defect may share a common mechanism with the reduction of UV-induced UDS in normal cells caused by γ-ray irradiation. 相似文献
7.
Rodneva S. M. Osipov A. A. Guryev D. V. Tsishnatti A. A. Fedotov Y. А. Yashkina E. I. Vorobyova N. Y. Maksimov A. A. Kochetkov O. A. Samoylov A. S. Osipov A. N. 《Bulletin of experimental biology and medicine》2021,172(2):245-249
Bulletin of Experimental Biology and Medicine - We compared the formation of γH2AX foci (marker of DNA double-strand breaks) in human lung fibroblasts (MRC-5 line) during their 24-h incubation... 相似文献
8.
This study addresses the timing of gammaherpesviral episomal DNA replication with respect to the cell cycle. For the first time we analyzed a rhadinovirus, the prototype Herpesvirus saimiri (HVS), and compared it to the lymphocryptovirus Epstein-Barr virus (EBV). Newly synthesized DNA of latently infected B- or T-cells was first BrdU-labeled; then we sorted the cells corresponding to cell cycle phases G0/1, G2/M, and S (4 fractions S1-S4) and performed anti-BrdU chromatin immunoprecipitation. Next, DNA of different viral gene loci was quantitatively detected together with cellular control genes of known replication time. The sensitive technique is further enhanced by an internal coprecipitation standard for increased precision. Both gammaherpesviruses replicated very early in S-phase, together with cellular euchromatin. Our work suggests that early S-phase DNA replication is a general characteristic of episomal herpesviral genomes. 相似文献
9.
10.
L. P. Kosichenko 《Bulletin of experimental biology and medicine》1964,55(1):106-108
Summary A study was made of the effect produced by ionizing radiation (190 and 400 r) on the 24-hour periodicity of mitotic activity of the mouse corneal epithelium. As noted, the 24-hour mitotic rhythm was retained on the 5th postirradiation day (doses of 190 and 400 r being used). The time of the maximal (8 A.M.) and the minimal (8 P.M.) number of mitosis in the corneal epithelium of experimental mice coincided with control indices. The difference between the maximal and minimal mitotic activity in the two experiments was highly significant. The range of the 24-hour variations of the number of mitoses showed no significant change in the experimental animals. After irradiation of mice in a dose of 400 r there was a tendency to a rise of the mean 24-hour intensity of cellular multiplication.(Presented by Active Member AMN SSSR A. V. Lebedinskii) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 55, No. 1, pp. 114–117, January 1963 相似文献
11.
Adult newt retinal pigment epithelium (RPE) cells are mitotically quiescent in the physiological condition, but upon a traumatic injury of the neural retina (NR) they re-enter the cell-cycle and eventually regenerate the missing NR. Here, to understand the mechanism underlying the cell-cycle re-entry of RPE cells following NR injury, we first investigated changes in MEK–ERK signaling activity in RPE cells upon removal of the NR (retinectomy) from the eye of living animals, and found that ERK-mediated signaling activity is elevated quickly (in 30 min) upon retinectomy. In addition, we found, in in vitro analyses, that immediate early activation of MEK–ERK signaling may occur in RPE cells upon NR injury, intensifying the MEK–ERK signaling itself through up-regulation of the expression of constituent molecules in the pathway, and that 1-h blockade of such early MEK–ERK signaling interferes with the cell-cycle re-entry, which occurs 5–10 days later. Together, these results provide us with insight that elevation of MEK–ERK signaling activity upon NR injury may be a key process for mitotically quiescent RPE cells to re-enter the cell-cycle, leading to retinal regeneration. 相似文献
12.
Vorob’eva N. Yu. Kochetkov O. A. Pustovalova M. V. Grekhova A. K. Blokhina T. M. Yashkina E. I. Osipov A. A. Kabanov D. I. Surin P. P. Barchukov V. G. Osipov A. N. 《Bulletin of experimental biology and medicine》2018,166(1):178-181
Bulletin of Experimental Biology and Medicine - We performed a comparative study of the formation of γН2АХ foci (a marker of DNA doublestrand breaks) in human bone marrow... 相似文献
13.
Summary Human cytomegalovirus (HCMV), a member of the virus familyHerpesviridae that is associated with extensive worldwide morbidity and mortality in immunocompromised hosts, inhibits interferon- (IFN)-mediated induction of human leukocyte antigen (HLA) class II antigens on endothelial cells. In this study, the ability of HCMV-infected endothelial cells to synthesize interferon- (IFN), and the role of IFN in HCMV-mediated inhibition of HLA class II induction, was investigated. As detemined by an encephalomyocarditis virus protection assay, HCMV-infected endothelial cell culture supernatants contained 240 IU/ml of IFN type I activity, of which 99.9% was IFN, as compared to the absence of IFN in mock-infected culture supernatants. UV-irradiated supernatants from HCMV-infected cultures inhibited induction of HLA class II in noninfected cultures by 24%. This inhibition could be abolished with 500 NU/ml of anti-IFN antibody. Addition of anti-IFN antibody directly to HCMV-infected cultures mitigated but did not abolish HLA class II antigen inhibition. Dual immunohistochemistry for HCMV and HLA DR demonstrated that infected cells, in contrast to noninfected cells, were rarely induced to express HLA class II even in the presence of anti-IFN antibody. These findings suggest that HCMV inhibits induction of HLA class II antigens by IFN dependent and independent mechanisms. 相似文献
14.
The induction of cyclooxygenase-2 by 17β-estradiol in endothelial cells is mediated through protein kinase C 总被引:5,自引:0,他引:5
P. Akarasereenont K. Techatraisak A. Thaworn S. Chotewuttakorn 《Inflammation research》2000,49(9):460-465
OBJECTIVE AND DESIGN: We investigated whether estrogen affected COX isoform expressed in human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: HUVEC were grown to confluence and replaced with fresh medium containing 17beta-estradiol (0.001, 0.01, 0.1 and 1 nM) or 17beta-estradiol (1 nM) plus staurosporine (0.1, 1 and 10 ng/ml) for 24 h, after which the supernatant medium was collected to measure 6-keto-PGF1alpha using enzyme immunoassay. To measure COX activity via exogenous substrates, the remaining cells were replaced with fresh medium containing arachidonic acid (10 microM for 10 min), and then the medium was removed to measure 6-keto-PGF1alpha. The COX isoform expressed in cells was detected by immunoblotting using specific antibody. RESULTS: 17beta-estradiol (0.001 to 1 nM) increased the production of 6-keto-PGF1alpha via either endogenous or exogenous substrate in a dose dependent manner. These increases were significantly inhibited when cells were coincubated with staurosporine. Interestingly, only COX-2 protein, but not COX-1 protein, was induced in 17beta-estradiol treated HUVEC and was also inhibited by staurosporine. CONCLUSION: Our data showed that 17beta-estradiol increased the release of PGI2 from HUVEC via the induction of COX-2 which was mediated through protein kinase C. The results suggested that COX-2 might have a role in the cardiovascular protective effect of estrogen. 相似文献
15.
Kwang Seok Kim Kyung Won Kang Suk-Hwan Baek Jae-Ryong Kim 《Mechanisms of ageing and development》2009,130(3):179-783
Cellular senescence is a stress-response phenomenon in which cells lose the ability to proliferate; it is induced by telomere shortening, activation of oncogenes or tumor suppressor genes, or exposure to a sub-lethal dose of DNA damaging agents or oxidative stresses. cDNA microarray analysis reveals that the levels of interferons (IFNs) and IFN-inducible genes were altered during replicative senescence in human umbilical vascular endothelial cells (HUVECs). However, the role of IFNs in cellular senescence of HUVECs remains unidentified. This study demonstrated that prolonged treatment with IFN-γ induced cellular senescence in HUVECs, as confirmed by G0/G1 cell cycle arrest, up-regulation of p53 and p21 protein levels, increased SA-β-gal staining, and the accumulation of phospho-H2AX foci. IFN-γ-induced cellular senescence was observed only in p16-knockdown cells or p16-null mouse embryonic fibroblasts (MEFs), but not in p53-knockdown cells or p53-null MEFs. IFN-γ treatment increased ROS production, and an antioxidant, N-acetylcysteine, inhibited IFN-γ-induced cellular senescence. Knockdown of ATM kinase or IFI16 rescued IFN-γ-induced cellular senescence. Therefore, these results suggest that IFN-γ might play an important role in cellular senescence through a p53-dependent DNA damage pathway and contribute to the pathogenesis of atherosclerosis via its pro-senescent activity. 相似文献
16.
Replacement therapy, using subcutaneous infusions of -globulin, is being applied increasingly for antibody-deficient patients, as this form of treatment has been found to be related to a very low frequency of adverse systemic reactions. However, the uptake of IgG from subcutaneous tissue may be low, owing to degradation locally, especially for the IgG3 molecule. Therefore, the kinetics of IgG and IgG-subclass concentrations in the sera of 23 patients with common variable immunodeficiency was investigated during 18 months of subcutaneous infusions of -globulin (100 mg/kg/week). Seventeen patients were previously treated with intramuscular injections or intravenous infusions. The mean serum IgG level increased twice in the previously treated patients and four times in the previously untreated patients. A steady state was reached after 6 months if the subcutaneous infusions were given weekly and after 1 week if the patients were given daily infusions for 5 consecutive days and, thereafter, weekly infusions. The fractional catabolic rate of IgG (4.1–5.9% per day) was found to be at the lower limit reported for normal controls, if 100% bioavailability of the infused IgG was assumed. The fractional contents of IgG subclasses in the patients' serum IgG resembled the physiological pattern, with the exception of IgG4, which was not present in the -globulin preparations used. Significantly increased levels of IgG1 and -2 were seen in both previously treated and untreated patients during the treatment. 相似文献
17.
Background
Skeletal uptake of 99mTc labelled methylene diphosphonate (99mTc-MDP) is used for producing images of pathological bone uptake due to its incorporation to the sites of active bone turnover. This study was done to validate bone turnover markers using total skeletal uptake (TSU) of 99mTc-MDP.Methods
22 postmenopausal women (52–80 years) volunteered to participate. Scintigraphy was performed by injecting 520 MBq of 99mTc-MDP and taking whole body images after 3 minutes, and 5 hours. TSU was calculated from these two images by taking into account the urinary loss and soft tissue uptake. Bone turnover markers used were bone specific alkaline phosphatase (S-Bone ALP), three different assays for serum osteocalcin (OC), tartrate resistant acid phosphatase 5b (S-TRACP5b), serum C-terminal cross-linked telopeptides of type I collagen (S-CTX-I) and three assays for urinary osteocalcin (U-OC).Results
The median TSU of 99mTc-MDP was 23% of the administered activity. All bone turnover markers were significantly correlated with TSU with r-values from 0.52 (p = 0.013) to 0.90 (p < 0.001). The two resorption markers had numerically higher correlations (S-TRACP5b r = 0.90, S-CTX-I r = 0.80) than the formation markers (S-Total OC r = 0.72, S-Bone ALP r = 0.66), but the difference was not statistically significant. TSU did not correlate with age, weight, body mass index or bone mineral density.Conclusion
In conclusion, bone turnover markers are strongly correlated with total skeletal uptake of 99mTc-MDP. There were no significant differences in correlations for bone formation and resorption markers. This should be due to the coupling between formation and resorption. 相似文献18.
19.
Many solid cancers respond to chemo or radiotherapy with a type 2 form of programmed cell death. This requires direct participation of mitochondria with release of cytochrome c and other factors that activate the 'execution' phase of the process. It is believed that as solid cancers progress, less differentiated clones containing fewer mitochondria evolve. Consequently, the mitochondrial 'switch' that activates the type 2 process will become less effective, as the number of elements available and their mass-effect declines. The opportunity for successful therapy, considered to depend upon the ability to activate programmed cell death, therefore becomes progressively less probable. 相似文献
20.
Ursula Holzer Katharina ReinhardtPeter Lang Rupert HandgretingerNadja Fischer 《Human immunology》2013
The T cell subsets involved in inflammatory reactions are mainly the IFN-γ secreting Th1 cells and IL17-producing Th17 cells. Although Th17 cells are primed in the thymus, there is evidence that Th17 cells can be generated from effector memory CD4+ T cells. Cytokines as IL-6, TGF-β, IL-21 and IL-23 involved in development of Th17 cells are well described. Here we analyzed the impact of a mutation in the IFN-γ receptor 2 (IFN-γR2) on the induction of Th17 cells. By isolation of T cells and monocytes of a patient with this mutation we could demonstrate an inhibitory role of IFN-γ signaling as IFN-γR2-deficient monocytes induce a higher percentage of IL-17+ cells from both healthy and IFN-γR2-deficient CD4+ T cells. This data confirm the interference of these two T helper subsets and points to a balance of Th1 and Th17 cells obtained by their own cytokine production and their interplay with APCs. 相似文献