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Vitro Treatment (vitro + treatment)
Selected AbstractsRemoval 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] Reactive oxygen species induce RNA damage in human atherosclerosisEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 5 2004W. Martinet Abstract Background, Reactive oxygen species (ROS)-induced DNA damage has recently been identified in both human and experimental atherosclerosis. This study was undertaken to investigate whether RNA damage occurs in human atherosclerotic plaques and whether this could be related to oxidative stress. Materials and methods, The integrity of total RNA isolated from carotid endarterectomy specimens (n = 20) and nonatherosclerotic mammary arteries (n = 20) was analyzed using an Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA). Oxidative modifications of RNA were detected by immunohistochemistry. Results, Eleven out of 20 atherosclerotic plaques showed a significant reduction of the 18S/28S rRNA peaks and a shift in the RNA electropherogram to shorter fragment sizes. In contrast, all mammary arteries showed good-quality RNA with clear 18S and 28S rRNA peaks. Strong nuclear and cytoplasmic immunoreactivity for oxidative damage marker 7,8-dihydro-8-oxo-2,-guanosine (8-oxoG) could be detected in the entire plaque in smooth muscle cells (SMCs), macrophages and endothelial cells, but not in SMCs of adjacent normal media or in mammary arteries. Cytoplasmic 8-oxoG staining in the plaque clearly diminished when tissue sections were pretreated with RNase A, suggesting oxidative base damage of RNA. In vitro treatment of total RNA with ROS-releasing compounds induced RNA degradation. Conclusion, Both loss of RNA integrity and 8-oxoG oxidative modifications were found in human atherosclerotic plaques. Because RNA damage may affect in vitro transcript quantification, RT-PCR results must be interpreted cautiously if independent experimental validation (e.g. evaluation of RNA integrity) is lacking. [source] Reduced fertility of mouse epididymal sperm lacking Prss21/Tesp5 is rescued by sperm exposure to uterine microenvironmentGENES TO CELLS, Issue 10 2008Misuzu Yamashita Although the acrosome reaction and subsequent penetration of sperm through the egg zona pellucida (ZP) are essential for mammalian fertilization, the molecular mechanism is still controversial. We have previously identified serine protease Tesp5 identical to Prss21 on the mouse sperm surface as a candidate enzyme involved in sperm penetration through the ZP. Here we show that despite normal fertility of male mice lacking Prss21/Tesp5, the epididymal sperm penetrates the ZP only at a very low rate in vitro, presumably owing to the reduced ability to bind the ZP and undergo the ZP-induced acrosome reaction. The ability of Prss21-null sperm to fuse with the egg in vitro was also impaired severely. Intriguingly, the reduced fertility of Prss21-null epididymal sperm was rescued by exposure of the sperm to the uterine microenvironment and by in vitro treatment of the sperm with uterine fluids. These data suggest the physiological importance of sperm transport through the uterus. [source] Conjugated linoleic acid inhibits peritoneal metastasis in human gastrointestinal cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 3 2006Hiroki Kuniyasu Abstract The effect of conjugated linoleic acid (CLA) on peritoneal metastasis was examined by in vitro treatment of cancer cells and mouse peritoneal metastasis models. First, cell growth of MKN28 human gastric cancer cells and Colo320 human colon cancer cells was suppressed by CLA in a dose-dependent manner with an increment in apoptosis. CLA significantly inhibited invasion into type IV collagen-coated membrane of MKN28 and Colo320 cells (p < 0.05). CLA-induced growth inhibition was recovered by the exposure to antisense S-oligodeoxynucleotide for peroxisome proliferator-activated receptor (PPAR)-, in both cell lines. BALB/c nu-nu mice were inoculated with MKN28 and Colo320 cells into their peritoneal cavity, and administrated with CLA intraperitoneally (weekly, 4 times). CLA treatment did not affect food intake or weight gain of mice. CLA treatment significantly decreased metastatic foci of both cells in the peritoneal cavity (p < 0.005). Survival rate in mice inoculated with MKN28 or Colo320 cells was significantly recovered by CLA treatment (p = 0.0025 and 0.0052, respectively). Protein production in MKN28 and Colo320 cells treated with CLA showed a decrease in epidermal growth factor receptor and transforming growth factor-, and an increase in Bax. These findings suggest that CLA inhibits metastasis of human gastric and colon cancer cells. © 2005 Wiley-Liss, Inc. [source] Non-specific immune response of turbot, Scophthalmus maximus (L.), experimentally infected with a pathogenic Vibrio pelagiusJOURNAL OF FISH DISEASES, Issue 6 2003L Villamil Abstract The effect of a pathogenic Vibrio pelagius, isolated during a mass mortality of turbot larvae, on the non-specific immune response of turbot, Scophthalmus maximus (L.), macrophages was studied both in vitro and in vivo. The in vitro treatment of head kidney (HK) macrophages with viable V. pelagius caused a significant inhibition of the chemiluminescence (CL) response in comparison with untreated macrophages, while incubation with heat-killed bacteria did not affect this response. In vivo, the intraperitoneal injection of V. pelagius resulted in a significant inhibition of the CL response in infected fish at days 1 and 4 post-infection compared with the control fish response. The HK macrophage nitric oxide (NO) production was enhanced by in vitro incubation with intermediate doses of viable V. pelagius (5 × 103 and 5 × 104 bacteria mL,1) and higher doses of the heat-killed bacteria (5 × 104,5 × 106 bacteria mL,1). In both cases, the NO inhibitorN- , -nitro-L-arginine was capable of down-regulating the specific NO induction caused by incubation with the bacterial treatments. In contrast, incubation with ECPs at higher doses caused a reduction in NO production. In vivo, a significant enhancement in NO production was also observed in macrophage supernatants at day 10 post-infection. Lysozyme concentration in the serum was also significantly increased in the experimentally infected fish at days 4 and 10 post-injection. In addition, viable V. pelagius and its ECPs significantly reduced HK macrophage viability in vitro, whereas no significant differences in viability were observed during the incubation with heat-killed bacteria. As NO production was enhanced in the experimentally infected fish, the inhibitory effect of the NO donor, S-nitroso-acetyl-penicillamine (SNAP), was tested in vitro in a cell-free assay. The results showed that growth of V. pelagius was significantly inhibited using SNAP at a high concentration (1 mm). [source] Intervention with polyphenol-rich fruit juices results in an elevation of glutathione S -transferase P1 (hGSTP1) protein expression in human leucocytes of healthy volunteersMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 12 2006Thomas Hofmann Abstract Polyphenols are probably antigenotoxic on account of their antioxidant activities and might alter phase I and II enzymes in a way that results in chemoprotection. We investigated the hypothesis that polyphenols enhance expression of glutathione S -transferases (GSTs), which increases carcinogen detoxification and thereby provides protection against oxidative stress. HGSTP1 protein expression and GST polymorphisms were determined in leucocytes obtained during an intervention study with healthy subjects consuming two fruit juices in an 8 wk trial (polyphenol-free run in phase, juice intervention phase, washout phase, second juice intervention phase, each treatment regime lasted for 2 wk). The study had originally shown that juice intervention significantly reduced oxidative DNA damage in leucocytes at week 8 (Bub, A., Watzl, B., Blockhaus, M., Briviba, K. et al., J. Nutr. Biochem. 2003, 14, 90,98). We reanalysed the levels of DNA damage based on GST genotypes. We also treated leucocytes in vitro with mixtures of polyphenols and determined cytotoxicity and expression of 96 genes related to drug metabolism. Key results with leucocytes of the intervention study were that the initial content of hGSTP1 protein was first suppressed at weeks 4 and 6. At week 8, however, hGSTP1 protein expression was significantly increased. HGSTP1 protein levels and DNA damage were inversely correlated (p = 0.005), but there was no difference for cells obtained from subjects with hGSTM1*1 and hGSTM1*0 genotypes, nor was there any difference between cells from subjects consuming the two different juices. The treatment of leucocytes with polyphenol mixtures in vitro did not result in modulated GST gene expression or total GST activity, but in an up-regulation of other biotransformation enzymes (e. g., members of the cytochrom P450 and the sulphotransferase family). In conclusion, in vitro treatment of leucocytes led to a modulated mRNA expression of selected genes, not directly related to oxidative defence systems. In vivo, however, we observed a delayed enhancement of hGSTP1, which could be associated with an initial repression of oxidative DNA damage in leucocytes from human subjects, consuming juices with high levels of polyphenols. [source] Identification of discriminant factors after treatment of resistant and susceptible banana leaves with Fusarium oxysporum f. sp. cubense culture filtratesPLANT BREEDING, Issue 1 2005B. Companioni Abstract Among the most important crops in developing countries are banana and plantain. However, the production is threatened by increasingly virulent forms of Fusarium wilt, and therefore, intensive breeding programmes are being carried out worldwide. As conventional field studies of banana resistance to this disease are time-consuming and destructive, an easy-to-do procedure was previously developed to differentiate field-grown resistant and susceptible banana cultivars at leaf level. Such a procedure involved the in vitro treatment of fungal culture filtrates on to field-grown adult leaves and the measurement of lesion areas 48 h later. The present report includes measurements of other indicators such as biochemical compounds. The cultivar ,Gross Michel' (susceptible) and cv. ,FHIA-01' (resistant) leaves were treated with Fusarium oxysporum f. sp. cubense race 1 culture filtrates. Evaluations were performed 48 h after leaf treatment. Compared with culture medium-treated leaves (control treatment), fungal metabolites produced leaf lesions, decreased freephenolic contents and increased protein levels in both cultivars. In ,FHIA-01', the culture filtrate increased contents of cell wall-linked phenolics and the pool of aldehydes (except malondialdehyde). Fungal metabolites did not cause variations in peroxidase activity, chlorophyll pigment contents or malondialdehyde level in any cultivar. The use of Fisher's linear discriminant analysis to differentiate resistant and susceptible banana cultivars in breeding programmes is also a novel aspect of this report. Such an estimation was performed from a data matrix that included the effects of the fungal metabolites (leaf lesion area and levels of free and cell wall-linked phenolics, aldehydes, except malondialdehyde, and proteins) on banana leaves of seven cultivars (four susceptible and three resistant). [source] SHORT COMMUNICATION: Increased Expression of Glutathione by Estradiol, Tumor Necrosis Factor-Alpha, and Interleukin 1-Beta in Endometrial Stromal CellsAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 6 2009Sa Ra Lee Problem, The intracellular antioxidant system, based on glutathione (GSH), plays a key role in endometrial detoxification reactions and has been proposed to be involved in the pathogenesis endometriosis. This study was designed to evaluate whether estradiol (E2) and proinflammatory cytokines have any effects on expression of glutathione in endometrial stromal cells (ESCs). Method of study, Glutathione levels were measured utilizing high-performance liquid chromatography following in vitro culture and treatment of ESCs with estradiol, tumor necrosis factor-alpha (TNF-,) and interleukin 1-beta (IL-1,). Results, The GSH level in E2 (10,8 m) treatment group was significantly higher than in the control group at 48 h (P < 0.05). In vitro treatment of ESCs with TNF-, 10 ng/mL as well as E2 (10,8 m) plus TNF-, 10 ng/mL for 48 hr also led to a significant increase in GSH level (P < 0.05; P < 0.05, respectively). Both IL-1, 10 ng/mL and E2 (10,8 m) plus IL-1, 10 ng/mL for 48 hr increased GSH level significantly (P < 0.05; P < 0.05, respectively) as well. Conclusions, These findings might suggest that increased production of estradiol and proinflammatory cytokines in the peritoneal cavity possibly leads to the establishment of endometriosis through increased level of GSH. [source] Alterations in sperm protein phosphorylation in male infertilityANDROLOGIA, Issue 5 2001M. L. Hortas Summary. Protein phosphorylation is involved in sperm capacitation, so the effect of protein phosphatase inhibitors on the capacitation of spermatozoa of males with unexplained infertility was investigated. d -mannose ligand specific receptor expression in fresh, living spermatozoa, capacitated or treated with calyculin A (an inhibitor of protein phosphatases 1 and 2A), was studied in three groups of men: pre-vasectomy (fertile) males, males in couples with male infertility, and males in couples with infertility of unknown aetiology. Flow cytometry showed significant differences between infertile couples with a male factor and fertile couples (P < 0.05), both after capacitation and after treatment with calyculin A. In the group of couples with infertility of unknown aetiology (n = 15), d -mannose receptor expression was diminished in six cases after classical capacitation. However, when the spermatozoa of these six men were treated with calyculin A, five showed an increased specific d -mannose receptor expression. From these results it is suggested that in vitro treatment of spermatozoa with inhibitors of protein phosphatases may be of great value in some cases of unexplained infertility. [source] Breed difference and regulation of the porcine adipose triglyceride lipase and hormone sensitive lipase by TNF,ANIMAL GENETICS, Issue 6 2009T. Shan Summary Adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) are major novel triglyceride lipases in animals. The aim of this study was to determine if there are differences in the porcine ATGL (pATGL) and HSL genes between Jinhua pigs (a fatty breed) and Landrace pigs (a leaner breed). In addition, the effect of TNF, and pATGL-specific siRNA (pATGL-siRNA) on the expression of pATGL and HSL in porcine adipocytes was also examined. Compared with Landrace pigs, the body weight (BW) of Jinhua pigs was lower (P < 0.01), while intramuscular fat content (in the longissimus dorsi muscle), as well as the back fat thickness and body fat content were higher (P < 0.01). The expression of pATGL and HSL mRNA in Jinhua pigs was lower (P < 0.01) in subcutaneous adipose tissue, and greater (P < 0.01) in longissimus dorsi muscle compared with Landrace pigs. In vitro treatment of porcine adipocytes with TNF, decreased (P < 0.01) the glycerol release and the gene expression of pATGL, HSL and PPAR, in porcine adipocytes. Furthermore, transfection with pATGL-siRNA significantly decreased (P < 0.01) the expression of pATGL, while it had no effect on the expression of HSL. Treatment with 25 ng/ml TNF, in conjunction with pATGL-siRNA significantly decreased (P < 0.01) the expression of pATGL and HSL in cultured porcine adipocytes. These results provide useful information to further the understanding of the function of pATGL and HSL in porcine lipid metabolism, which should be applicable to the regulation of fat deposition and improvement of meat quality. [source] Promotion of central nervous system remyelination by induced differentiation of oligodendrocyte precursor cells,ANNALS OF NEUROLOGY, Issue 3 2009Sha Mi PhD Objective Repair of demyelinated axons in diseases such as multiple sclerosis requires activation of the myelination program in existing or newly recruited oligodendrocyte precursor cells (OPCs). The control of OPC differentiation and initiation of myelination during repair is poorly understood. In this study, we test the ability of anti,LINGO-1 reagents to promote myelination in vitro and remyelination in the rodent adult central nervous system in vivo. Methods The effects of LINGO-1 antagonists on the differentiation of OPCs and the promotion of myelination has been assayed using a combination of coculture and slice culture preparations. Using three different animal models of demyelination and remyelination, we morphologically and functionally assessed the effects of LINGO-1 antagonists on OPC differentiation and myelin repair. Results The data indicate that in vitro treatment with antagonists of LINGO-1 promote OPC differentiation and myelination, whereas in vivo remyelination is accelerated in lysophosphatidylcholine- or cuprizone-induced demyelination. This remyelination is associated with enhanced OPC differentiation and functional recovery of conduction velocities in demyelinated axons. Interpretation Our studies demonstrate that LINGO-1 antagonism promotes OPC differentiation and remyelination, and suggest LINGO-1 functions as an inhibitor of OPC differentiation to retard central nervous system remyelination. Ann Neurol 2009;65:304,315 [source] MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responsesARTHRITIS & RHEUMATISM, Issue 9 2009Shigeru Miyaki Objective MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue-specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA-140 (miR-140). Methods To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR-140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin-1, (IL-1,) on miR-140 expression. Double-stranded miR-140 (ds,miR-140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA. Results Microarray analysis showed that miR-140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR-140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR-140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1, suppressed miR-140 expression. Transfection of chondrocytes with ds,miR-140 down-regulated IL-1,,induced ADAMTS5 expression and rescued the IL-1,,dependent repression of AGGRECAN gene expression. Conclusion This study shows that miR-140 has a chondrocyte differentiation,related expression pattern. The reduction in miR-140 expression in OA cartilage and in response to IL-1, may contribute to the abnormal gene expression pattern characteristic of OA. [source] ,, T cells assist ,, T cells in the adoptive transfer of contact hypersensitivity to para-phenylenediamineCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2001H. Yokozeki Para-phenylenediamine (PPD) is known to be a common sensitizer of allergic contact dermatitis and contact urticaria. To clarify the mechanism of contact hypersensitivity (CHS) to PPD, we established a mouse model of PPD-induced CHS. BALB/c mice were immunized for 3 consecutive days by painting topically a 2·5% PPD solution on their shaved abdominal skin. On days 5, 7 or 9 after the initial application, the mice were challenged by applications of a 2·5% PPD solution. Maximal ear swelling was determined at 24 h but another statistically significant and smaller ear swelling was observed 1 h after challenge with PPD in a hapten-specific manner. Adoptive cell transfer experiments demonstrated that the ear swelling of the adoptive cell transferred mice displayed an early response at 6 h and a late response from 12 h to 24 h when the recipient mice were challenged immediately after transfer. Both MoAbs and complement treatment of the transferred cells demonstrated that the phenotype of the early response cells which elicited a response at 6 h after challenge was Thy1+, B220+, ,, TCR, ,, TCR, CD3, CD4, CD5+ and CD8. The in vitro treatment of effector cells with MoAbs against not only ,, TCR but also ,, TCR, together with complement, was found to diminish substantially the late response, elicited 12,24 h after challenge. ,, T cells reconstituted the ability of ,, T cells to transfer 24 h CHS responsiveness. The phenotype of the ,, T cells that assist CHS effector ,, T cells was CD3+, CD4 and CD8+ and these regulatory ,, T cells were neither Ag-specific nor MHC-restricted. Furthermore, ,, T cells from normal spleen could also assist ,, T cells in adoptive transfer of the 24 h CHS response in a non-MHC-restricted manner. RT-PCR demonstrated that ,, T cells strongly expressed mRNA IFN- ,, whereas ,, T cells expressed not only IFN- , but also IL-4 and IL-10. These data indicate that not only early response cells and ,, T cells but also Th2 type ,, T cells may play an important role in the elicitation of CHS to PPD. [source] Expression and modulation of ghrelin O -acyltransferase in cultured chondrocytesARTHRITIS & RHEUMATISM, Issue 6 2009Rodolfo Gómez Objective To use reverse transcription,polymerase chain reaction to detect ghrelin O -acyltransferase (GOAT) transcripts in both murine and human chondrocytes, to evaluate the effect of pharmacologic in vitro treatments with lipopolysaccharide (LPS), growth hormone, ghrelin, and dexamethasone on GOAT messenger RNA (mRNA) expression, and to study the GOAT mRNA profile during chondrocyte differentiation. Methods Murine and human GOAT and ghrelin mRNA levels were determined by the SYBR Green,based quantitative real-time polymerase chain reaction method. Results GOAT mRNA was expressed in murine cartilage explants as well as in the cultured murine chondrogenic ATDC-5 cell line. GOAT was also expressed in human immortalized chondrocyte cell lines and in human cultured primary chondrocytes. In addition, GOAT mRNA expression in differentiating ATDC-5 cells was lower at the early stage of differentiation (days 3,7), whereas GOAT mRNA levels increased progressively at the late stages. Finally, among the drugs and hormones tested, only LPS was able to strongly decrease GOAT mRNA expression. Conclusion These data indicate that chondrocytes are equipped with biochemical machinery for the synthesis of acylated ghrelin and suggest a novel role of the ghrelin axis in prehypertrophic and hypertrophic chondrocyte differentiation during endochondral ossification. [source] | |||||||||||||