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Repair Capacity (repair + capacity)
Kinds of Repair Capacity Selected AbstractsLight-dependent mutagenesis by benzo[a]pyrene is mediated via oxidative DNA damageENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 3 2005Su-Ryang Kim Abstract Benzo[a]pyrene (B[a]P) is an environmental carcinogenic polycyclic aromatic hydrocarbon (PAH). Mammalian enzymes such as cytochrome P-450s and epoxide hydrase convert B[a]P to reactive metabolites that can covalently bind to DNA. However, some carcinogenic compounds that normally require metabolic activation can also be directly photoactivated to mutagens. To examine whether B[a]P is directly mutagenic in the presence of light, we exposed Salmonella typhimurium strains with different DNA repair capacities to B[a]P and white fluorescent light at wavelengths of 370,750 nm. B[a]P plus light significantly enhanced the number of His+ revertants. Mutagenesis was completely light-dependent and required no exogenous metabolic activation. The order of mutability of strains with different DNA repair capacities was strain YG3001 (uvrB, mutMST) , strain TA1535 (uvrB) > strain YG3002 (mutMST) > strain TA1975. The uvrB gene product is involved in the excision repair of bulky DNA adducts, and the mutMST gene encodes 8-oxoguanine (8-oxoG) DNA glycosylase, which removes 8-oxoG from DNA. Introduction of a plasmid carrying the mOgg1 gene that is the mouse counterpart of mutMST substantially reduced the light-mediated mutagenicity of B[a]P in strain YG3001. B[a]P plus light induced predominantly G:C , T:A and G:C , C:G transversions. We propose that B[a]P can directly induce bulky DNA adducts if light is present, and that the DNA adducts induce oxidative DNA damage, such as 8-oxoG, when exposed to light. These findings have implications for the photocarcinogenicity of PAHs. Environ. Mol. Mutagen., 2005. © 2005 Wiley-Liss, Inc. [source] Enhanced sensitivity to DNA damage induced by cooking oil fumes in human OGG1 deficient cellsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2008Mei Wu Abstract Cooking oil fumes (COFs) have been implicated as an important nonsmoking risk factor of lung cancer in Chinese women. However, the molecular mechanism of COFs-induced carcinogenicity remains unknown. To understand the molecular basis underlying COFs-induced cytotoxicity and genotoxicity as well as the roles of hOGG1 in the repair of COFs-induced DNA damage, a human lung cancer cell line with hOGG1 deficiency, A549-R was established by using a ribozyme gene targeting technique that specifically knockdowned hOGG1 in A549 lung adenocarcinoma cells. MTT and comet assays were employed to examine cell viability and DNA damage/repair, respectively, in A549-R and A549 cell lines treated with COF condensate (COFC). RT-PCR and Western blot results showed that the expression of hOGG1 in A549-R cell line was significantly decreased compared with that in A549 cell line. The concentration of COFC that inhibited cell growth by 50% (the IC50) in the A549-R cell line was much lower than that in the A549 cell line, and more COFC-induced DNA damage was detected in the A549-R cell line. The time course study of DNA repair demonstrated delayed repair kinetics in the A549-R cell line, suggesting a decreased cellular damage repair capacity. Our results showed that hOGG1 deficiency enhanced cellular sensitivity to DNA damage caused by COFC. The results further indicate that hOGG1 plays an important role in repairing COF-induced DNA damage. Our study suggests that COFs may lead to DNA damage that is subjected to hOGG1 -mediated repair pathways, and oxidative DNA damage may be involved in COF-induced carcinogenesis. Environ. Mol. Mutagen., 2008. © 2008 Wiley-Liss, Inc. [source] DNA damage and repair capacity in lymphocytes from obstructive sleep apnea patientsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 9 2007Konstantina Kontogianni Abstract Obstructive sleep apnea (OSA) syndrome is a respiratory disease that is linked to heart attacks and high blood pressure. In the present study, we used the Comet assay to compare basal DNA damage and DNA damage induction by hydrogen peroxide, ethanol, and ,-irradiation in lymphocytes from 35 OSA patients and 35 controls. We also measured the apoptosis and necrosis produced by these agents and the ability of the lymphocytes to repair the induced DNA damage. It was found that lymphocytes isolated from OSA patients had higher basal levels of DNA damage and were more sensitive to the effects of the DNA-damaging agents than lymphocytes from controls. OSA patients also had a reduced capacity to repair the DNA damage induced by the three agents, but apoptosis and necrosis were similar in OSA patients and the controls. Environ. Mol. Mutagen., 2007. © 2007 Wiley-Liss, Inc. [source] Polymorphisms in the thymidylate synthase promoter and the DNA repair genes XRCC1 and XPD in a Brazilian populationENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 9 2006Renata Canalle Abstract Polymorphisms in genes responsible for maintaining genomic integrity are potential modifiers of disease risk. Since considerable interindividual and interethnic variation in DNA repair capacity has been associated with polymorphic alleles, we evaluated the frequency of the 2R/3R variants in the TS promoter, Arg194Trp and Arg399Gln in the XRCC1 gene, and Asp312Asn and Lys751Gln in the XPD gene in 364 healthy individuals from a Brazilian population separated by ethnicity (European ancestry and African ancestry). The genotypes were determined by PCR (TS) or by PCR-RFLP (XRCC1 and XPD). The frequency of the TS 3R allele was 0.56 for whites and 0.51 for nonwhites. In the case of the XRCC1 MspI polymorphism, the allele frequencies were 0.09 for 194Trp in both nonwhites and whites and 0.27 and 0.28 for 399Gln in nonwhites and whites, respectively. For the XPD 312Asn allele, we found a frequency of 0.25 in white individuals, which was significantly different (P = 0.025) from that seen in nonwhites (0.15). Similarly, the 751Gln polymorphic allele of the XPD gene was significantly more frequent (P < 0.002) in whites (0.30) than in nonwhites (0.20). The genotype frequencies were within Hardy,Weinberg equilibrium. We concluded that the genotype and allele frequencies of XPD gene polymorphism differed between white and nonwhite Brazilians, and that the frequencies of the XPD 312Asn and XRCC1 399Gln alleles in this Brazilian population showed ethnic variability when compared with those observed in other populations. Environ. Mol. Mutagen., 2006. © 2006 Wiley-Liss, Inc. [source] DNA damage and repair measurements from cryopreserved lymphocytes without cell culture,A reproducible assay for intervention studiesENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 7 2006Jyh-Lurn Chang Abstract Single-cell gel electrophoresis (the Comet assay) can be used to measure DNA damage and DNA repair capacity (DRC). However, to test DRC of cryopreserved lymphocytes, published methods include steps for cell culturing and phytohemagglutinin stimulation, which may limit use of this assay in intervention studies. We developed a modified Comet assay protocol that allows us to measure DRC from cryopreserved lymphocytes without these in vitro manipulations. Assay reproducibility was evaluated by performing the assay six times on different dates using six aliquots from one blood draw of one individual. The interindividual variation was assessed by performing the assay using one aliquot from six individuals. When ,-irradiation was used as the mutagen, intra-assay coefficients of variation (CVs.) for baseline DNA damage, damage after ,-irradiation exposure, and DRC,measured as tail moment,were 8, 31, and 10%, respectively. Interindividual CVs. were higher. When H2O2 was used as the mutagen, intra-assay CVs. for damage measurements were lower for a protocol modification that included damage and repair at 37°C (CVs. ranging from 8 to 35%) than for the more standard 4°C protocol. Analyzing moment arm,the average distance of DNA migration within the tail,yielded similar results. DNA repair was successfully detected in each experiment. Comparing freshly isolated lymphocytes to cryopreserved lymphocytes from the same individuals' blood draw indicated that DRC was highly correlated when determined using moment arm values. This modified protocol extends the use of the Comet assay to measuring DRC in intervention studies (e.g., dietary interventions) in that it assesses cellular response after cryopreservation without cell culture or other extensive manipulation. Environ. Mol. Mutagen., 2006. © 2006 Wiley-Liss, Inc. [source] Removal of benzo(a)pyrene diol epoxide (BPDE)-DNA adducts as a measure of DNA repair capacity in lymphoblastoid cell lines from sisters discordant for breast cancerENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2002Grazyna Motykiewicz Abstract The mutagen sensitivity assay is one of the approaches used to investigate individual DNA repair capacity. This method is based on the premise that after in vitro treatment with a test mutagen, DNA from subjects with defective repair will be more damaged than DNA from those with an efficient repair system. However, very little is known about unmeasured processes that occur between cell treatment and final assessment of DNA damage. To develop a more precise assay, we modified the traditional mutagen sensitivity assay to also include measurement of DNA damage after culturing cells in the absence of mutagen. First, we treated apparently normal and xeroderma pigmentosum lymphoblastoid cell lines with various doses of benzo(a)pyrene diol epoxide (BPDE) and harvested cells at different time points. A polyclonal antiserum against BPDE-DNA was used to quantitate levels of adducts by immunoslot-blot and immunohistochemistry. Selected conditions included treatment with 10 ,M BPDE, a 4-hr culture in mutagen-free medium, and immunohistochemical measurement of BPDE-DNA adducts. The method was then applied in a pilot study to 50 lymphoblastoid lines from sisters discordant for breast cancer. There was no significant difference between cases and controls in the level of BPDE-DNA adducts in lymphoblasts harvested immediately after BPDE treatment. However, after a 4-hr culture in mutagen-free medium, the level of adducts was significantly higher (P = 0.006) among cases than in controls. There was a two-fold increase in mean adduct removal in lines from nonaffected as compared to affected sisters (44% and 22% decrease, respectively). DNA repair capacity was predictive of case status (P = 0.04) in logistic regression analysis. This method, which can be easily applied to large numbers of samples, should be useful in studies to investigate the role of DNA repair in cancer risk. Environ. Mol. Mutagen. 40:93,100, 2002. © 2002 Wiley-Liss, Inc. [source] Oxidative stress as a multiple effector in Fanconi anaemia clinical phenotypeEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 2 2005Giovanni Pagano Abstract:, Fanconi anaemia (FA) is a genetic disease characterised by bone marrow failure with excess risk of myelogenous leukaemia and solid tumours. A widely accepted notion in FA research invokes a deficiency of response to DNA damage as the fundamental basis of the ,crosslinker sensitivity' observed in this disorder. However, such an isolated defect cannot readily account for the full cellular and clinical phenotype, which includes a number of other abnormalities, such as malformations, endocrinopathies, and typical skin spots. An extensive body of evidence pointing toward an involvement of oxidative stress in the FA phenotype includes the following: (i) In vitro and ex vivo abnormalities in a number of redox status endpoints; (ii) the functions of several FA proteins in protecting cells from oxidative stress; (iii) redox-related toxicity mechanisms of the xenobiotics evoking excess toxicity in FA cells. The clinical features in FA and the in vivo abnormalities of redox parameters are here reconsidered in view of the pleiotropic clinical phenotype and known biochemical and molecular links to an in vivo prooxidant state, which causes oxidative damage to biomolecules, resulting in an excessive number of acquired abnormalities that may overwhelm the cellular repair capacity rather than a primary deficiency in DNA repair. FA may thus represent a unique model disease in testing the integration between the acquisition of macromolecular damage as a result of oxidative stress and the ability of the mammalian cell to respond effectively to such damage. [source] Single nucleotide polymorphisms 5, upstream the coding region of the NEIL2 gene influence gene transcription levels and alter levels of genetic damageGENES, CHROMOSOMES AND CANCER, Issue 11 2008Carla J. Kinslow NEIL2 (EC 4.2.99.18), a mammalian DNA glycosylase and ortholog of the bacterial Fpg/Nei, excises oxidized DNA lesions from bubble or single-stranded structures, suggesting its involvement in transcription-coupled DNA repair. Because base excision repair (BER) proteins act collectively and in a progressive fashion, their proper balance is essential for optimal repair. Thus, inter-individual variability in transcription levels of NEIL2 may predispose to compromised DNA repair capacity and genomic instability by altering the balance of critical BER proteins. In a study of lymphocytes of 129 healthy subjects, using absolute quantitative reverse transcription PCR, we found that NEIL2 transcription varied significantly (up to 63 fold) and that this variability was influenced by certain single nucleotide polymorphisms (SNPs) located 5, of the start site. Using the mutagen sensitivity assay to characterize the biological significance of these SNPs, we observed a significant increase in mutagen-induced genetic damage associated with two SNPs in the promoter region of the NEIL2 gene. To characterize the functional significance of these SNPs, we engineered luciferase-reporter constructs of the NEIL2 promotor with mutations corresponding to these SNPs. We transfected these constructs into MRC-5 cells and evaluated their impact on NEIL2 expression levels. Our results indicate that NEIL2 expression was significantly reduced by over 50% (P < 0.01) in the presence of two SNPs, ss74800505 and rs8191518, located near the NEIL2 start site, which were in significant linkage disequilibrium (D, = 73%; P < 0.05). This first report on in vivo variability in NEIL2 expression in humans identifies SNPs in the NEIL2 promoter region that have functional effects. © 2008 Wiley-Liss, Inc. [source] ,93G,A polymorphism of hMLH1 and risk of primary lung cancerINTERNATIONAL JOURNAL OF CANCER, Issue 4 2004Sun Ha Park Abstract Polymorphisms in DNA repair genes may be associated with differences in the repair capacity of DNA damage and may thereby influence an individual's susceptibility to smoking-related cancer. We investigated the association between the ,93G,A polymorphism in the hMLH1 gene and the risk of lung cancer in a Korean population. The hMLH1 ,93G,A polymorphism was typed in 372 lung cancer patients and 371 healthy controls that were frequency-matched for age and sex. There was no significant association between the hMLH1 ,93G,A genotype and the risk for adenocarcinoma or small cell carcinoma. However, the AA genotype was associated with a significantly increased risk for squamous cell carcinoma compared with both the GG genotype (adjusted OR = 2.02; 95% CI = 1.15,3.55; p = 0.014) and the combined GG and GA genotype (adjusted OR = 1.83; 95% CI = 1.24,2.71; p = 0.003). When the subjects were stratified by smoking exposure, the AA genotype was associated with a significantly increased risk for squamous cell carcinoma in lighter smokers (, 39 pack-years; adjusted OR = 1.95; 95% CI = 1.03,3.66; p = 0.039) compared with the combined GG and GA genotype, whereas there was no significant association in heavier smokers (> 39 pack-years; adjusted OR = 1.47; 95% CI = 0.82,2.61). These results suggest that the hMLH1 ,93G,A polymorphism could be used as a marker of genetic susceptibility to squamous cell carcinoma of the lung. © 2004 Wiley-Liss, Inc. [source] Progenitor cells in vascular diseaseJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 3 2005Neil Roberts Abstract Stem cell research has the potential to provide solutions to many chronic diseases via the field of regeneration therapy. In vascular biology, endothelial progenitor cells (EPCs) have been identified as contributing to angiogenesis and hence have therapeutic potential to revascularise ischaemic tissues. EPCs have also been shown to endothelialise vascular grafts and therefore may contribute to endothelial maintenance. EPC number has been shown to be reduced in patients with cardiovascular disease, leading to speculation that atherosclerosis may be caused by a consumptive loss of endothelial repair capacity. Animal experiments have shown that EPCs reendothelialise injured vessels and that this reduces neointimal formation, confirming that EPCs have an atheroprotective effect. Smooth muscle cell accumulation in the neointimal space is characteristic of many forms of atherosclerosis, however the source of these cells is now thought to be from smooth muscle progenitor cells (SMPCs) rather than the adjacent media. There is evidence for the presence of SMPCs in the adventitia of animals and that SMPCs circulate in human blood. There is also data to support SMPCs contributing to neointimal formation but their origin remains unknown. This article will review the roles of EPCs and SMPCs in the development of vascular disease by examining experimental data from in vitro studies, animal models of atherosclerosis and clinical studies. [source] Hedgehog signaling maintains hair follicle stem cell phenotype in young and aged human skinAGING CELL, Issue 6 2009Laure Rittié Summary Skin hair follicles (HF) contain bulge stem cells (SC) that regenerate HFs during hair cycles, and repair skin epithelia following injury. As natural aging is associated with decreased skin repair capacity in humans, we have investigated the impact of age on human scalp HF bulge cell number and function. Here, we isolated human bulge cells, characterized as CD200+/KRT15+/KRT19+ cells of the HF, by dissection-combined CD200 selection in young and aged human skin. Targeted transcriptional profiling indicates that KRT15, KRT19, Dkk3, Dkk4, Tcf3, S100A4, Gas1, EGFR and CTGF/CCN2 are also preferentially expressed by human bulge cells, compared to differentiated HF keratinocytes (KC). Our results demonstrate that aging does not alter expression or localization of these HF SC markers. In addition, we could not detect significant differences in HF density or bulge cell number between young and aged human scalp skin. Interestingly, hedgehog (Hh) signaling is activated in human bulge cells in vivo, and down-regulated in differentiated HF KCs, both in young and aged skin. In addition, activation of Hh signaling by lentivirus-mediated overexpression of transcription factor Gli1 induces transcription of HF SC markers KRT15, KRT19, and Gas1, in cultured KCs. Together with previously reported knock-out mouse results, these data suggest a role for Hh signaling in maintaining bulge cell phenotype in young and aged human skin. [source] Is the modulation of retinoid and retinoid-associated signaling a future therapeutic strategy in neurological trauma and neurodegeneration?JOURNAL OF NEUROCHEMISTRY, Issue 3 2008Andrea Malaspina Abstract The complex molecular pathways that mediate the effects of vitamin A and its derivatives, are increasingly recognized as a component of the repair capacity that could be activated to induce protection and regeneration in the mature nervous tissue. Retinoid and retinoid-associated signaling plays an essential role in normal neurodevelopment and appears to remain active in the adult CNS. In this paper, we review evidence which supports the hypothesis of an activation of retinoid-associated signaling molecular pathways in the mature nervous tissue and its significance in the context of neurodegenerative, trauma-induced and psychiatric disorders, at spinal and supra-spinal levels. Finally, we summarize the potential therapeutic avenues based on the modulation of retinoid targets undergoing reactivation under conditions of acute injury and chronic degeneration in the central nervous system, and discuss some of the unresolved issues linked to this treatment strategy. [source] The effect of skeletal maturity on the regenerative function of intrinsic ACL cellsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2010Ashley N. Mastrangelo Abstract Anterior cruciate ligament (ACL) injuries are an important clinical problem, particularly for adolescent patients. The effect of skeletal maturity on the potential for ACL healing is as yet unknown. In this study, we hypothesized that fibroblastic cells from the ACLs of skeletally immature animals would proliferate and migrate more quickly than cells from adolescent and adult animals. ACL tissue from skeletally immature, adolescent, and adult pigs and sheep were obtained and cells obtained using explant culture. Cell proliferation within a collagen,platelet scaffold was measured at days 2, 7, and 14 of culture using AM MTT assay. Cellular migration was measured at 4 and 24 h using a modified Boyden chamber assay, and cell outgrowth from the explants also measured at 1 week. ACL cells from skeletally immature animals had higher proliferation between 7 and 14 days (p,<,0.01 for all comparisons) and higher migration potential at all time points in both species (p,<,0.01 for all comparisons). ACL cells from skeletally immature animals have greater cellular proliferation and migration potential than cells from adolescent or adult animals. These experiments suggest that skeletal maturity may influence the biologic repair capacity of intrinsic ACL cells. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:644,651, 2010 [source] INTERACTIONS BETWEEN UV-B EXPOSURE AND PHOSPHORUS NUTRITION.JOURNAL OF PHYCOLOGY, Issue 6 2005The interactive effects of P starvation and exposure to UV radiation (UVR) on rates of damage (k) and repair (r), modeled from exposure response curves (ERCs), in the chlorophyte microalga Dunaliella tertiolecta Butcher were investigated. When nutrient-replete cells were exposed to the UVR during growth, k and r both increased by approximately 62% and 100%, respectively. However, when cells were starved of phosphorus, k increased by a similar amount as observed in replete cells, but r decreased by about 70%, explaining the increased susceptibility of cells to UVR-induced inhibition of photosynthesis under P starvation. Although not specifically investigated in this study, it is argued that the decreased repair capacity under P starvation is due to a decline in nucleotides such as ATP and GTP, which are necessary for protein repair. [source] Elevation of XPA protein level in testis tumor cells without increasing resistance to cisplatin or UV radiationMOLECULAR CARCINOGENESIS, Issue 8 2008Beate Köberle Abstract Most testicular germ cell tumors are curable using cisplatin-based chemotherapy, and cell lines from these tumors are unusually sensitive to cisplatin and other DNA-damaging agents. It has been suggested that this might be caused by a lower-than normal nucleotide excision repair (NER) activity. Previous studies found that cell lines from testicular germ cell tumors have on average about one-third the level of the NER protein XPA in comparison to cell lines from other tumors. We asked whether over-expression of XPA protein would alleviate the cellular sensitivity and increase the DNA repair capacity of a testis tumor cell line. Increasing XPA levels in 833K cells by 10-fold did not increase resistance to UV irradiation. XPA was localized to the cell nucleus in all cell lines, before and after exposure to UV-radiation. 833K cells were proficient in removing UV radiation-induced photoproducts from the genome and increased XPA did not enhance the rate of removal. Further, over-expressing functional XPA protein did not correlate with increased resistance of 833K testis tumor cells to cisplatin. Thus, although the amount of XPA in this testis tumor cell line is lower than normal, it is sufficient for NER in vivo. The relative sensitivity of testis tumor cells to cisplatin, UV radiation, and other DNA damaging agents is likely related not to NER capacity, but to other factors such as the integrity of the p53 pathway in these cells. © 2008 Wiley-Liss, Inc. [source] Polymorphisms in DNA repair genes XPD and XRCC1 and p53 mutations in lung carcinomas of never-smokersMOLECULAR CARCINOGENESIS, Issue 11 2006Wei-Min Gao Abstract The etiology of lung cancer in population with little or no tobacco exposure is not well understood. Individual genetic susceptibility factors have been suggested to contribute to lung cancer risk in this population. Mutations in the p53 tumor suppressor gene are implicated in the development of lung cancer as they are frequently found in lung tumors from both smokers and never-smokers. In order to determine whether genetic polymorphisms affecting DNA repair capacity modulate p53 mutations in lung tumors from never-smokers, we compared p53 mutations with genotypes of XPD 312, XPD 751, and XRCC1 399 in lung tumors from 43 lifetime never-smokers. p53 mutations were identified in 10 (23%) cases and consisted mostly of G/C to A/T transitions. No statistically significant association was found between p53 mutations and genotypes of XPD 312 or XPD 751. However, patients with the XRCC1 399 Gln allele, that results in a lower base excision repair capacity, were more likely to have p53 mutations, compared with patients the wild-type Arg allele (P,=,0.03). In addition, the p53 mutation frequency increased with an increasing number of combined genotypes associated with a lower DNA repair capacity of XPD 312, XPD 751, and XRCC1 399 (P,=,0.02). These results suggest that individuals who never smoked and had XRCC1 399 Gln allele may be at a greater risk of p53 mutations, especially if combined with the genotypes of XPD 312 and XPD 751 that may result in a lower DNA repair capacity. © 2006 Wiley-Liss, Inc. [source] Developmental and light effects on the accumulation of FtsH protease in Arabidopsis chloroplasts , implications for thylakoid formation and photosystem II maintenanceTHE PLANT JOURNAL, Issue 5 2005Adi Zaltsman Summary The chloroplast ATP-dependent metalloprotease FtsH is involved in the degradation of unassembled proteins, the repair of photosystem II (PSII) from photoinhibition, and, apparently, the formation of thylakoids. In Arabidopsis, it is encoded by a family of 12 genes. However, the products of only four of them, FtsH1, 2, 5 and 8, have been found in chloroplasts to date. Mutations in two of these, FtsH2 and 5, demonstrate a visible phenotype of variegated leaves, with the phenotype of the FtsH2 mutant being more pronounced. Moreover, the degree of variegation appears to be dependent on developmental stage and environmental factors, suggesting an intricate relationship between the different gene products. To explore this, developmental and light effects on the accumulation of FtsH protease were studied in wild-type (WT) and FtsH2-mutant plants. Whereas cotyledons of the mutant were indistinguishable from those of the WT, the first true leaves were almost completely white. Subsequent leaves contained increasing proportions of green sectors. Analysis of the mRNA of the four FtsH genes, in cotyledons, first and second leaves of WT and mutant plants, revealed that: (i) transcript level increases during development, and (ii) transcript level in the mutant is higher than in the WT. FtsH protein level in the mutant was ca. 50% of that found in the WT, whereas the levels of other thylakoid proteins were the same. In individual leaves, the level of FtsH protein increased during development as well. Exposure of seedlings to different light intensities did not affect the degree of variegation, suggesting that it is due to a defect in chloroplast development rather than photobleaching. Examination of FtsH protein during exposure to high light revealed a decrease in its level, concomitant with a decrease in PSII potential, suggesting that the kinetics of photoinhibition reflects not only photodamage to PSII and induction of protective mechanisms, but also a decrease in repair capacity due to a reduction in the level of FtsH protease. [source] Plasticity of clonal populations of dedifferentiated adult human articular chondrocytesARTHRITIS & RHEUMATISM, Issue 5 2003Andrea Barbero Objective To investigate whether adult human articular chondrocytes (AHACs), dedifferentiated by monolayer expansion, can differentiate toward diverse mesenchymal lineages and, if so, whether this ability is regulated by growth factors during monolayer expansion. Methods AHACs were expanded as multiclonal or clonal populations in medium without (control) or with factors enhancing cell dedifferentiation (transforming growth factor ,1, fibroblast growth factor 2, and platelet-derived growth factor type BB [TFP]). Cells were then cultured under conditions promoting chondrogenic, osteogenic, or adipogenic differentiation, and the acquired phenotypes were assessed histologically, biochemically, and by real-time reverse transcriptase,polymerase chain reaction. Results Multiclonal populations of both control- and TFP-expanded AHACs differentiated toward the chondrogenic, osteogenic, and adipogenic lineages. Compared with control-expanded AHACs, TFP-expanded cells displayed enhanced chondrogenic differentiation capacity (2.4-fold higher glycosaminoglycan/DNA content and 2,500-fold higher up-regulation of type II collagen) and osteogenic differentiation capacity (9.4-fold higher increase in alkaline phosphatase activity and 12.4-fold higher up-regulation of bone sialoprotein), but reduced formation of adipocytes (5.2-fold lower oil red O,positive cells/area). Clonal populations of AHACs could be efficiently expanded in TFP, but not in control medium. Most TFP-expanded clones were able to redifferentiate only into chondrocytes (7 of 20) or were unable to differentiate (6 of 20). However, some clones (2 of 20) differentiated toward all of the lineages investigated, thus displaying characteristics of mesenchymal progenitor cells. Conclusion Dedifferentiated AHACs exhibit differentiation plasticity, which is modulated by growth factors used during monolayer expansion and is highly heterogeneous across different clones. Clonal culture of AHACs in the presence of regulatory molecules could lead to the identification of AHAC subpopulations with enhanced cartilage repair capacity. [source] | |||||||||||||