Sample records for deoxynucleotidyl transferase tdt-mediated
-
Generation of Active Bovine Terminal Deoxynucleotidyl Transferase (TdT in E.coli
Directory of Open Access Journals (Sweden)
Wee Liang Kuan
2010-08-01
Full Text Available A synthetic gene encoding bovine terminal deoxynucleotidyl transferase (TdT was generated, cloned into an expression vector and expressed in E.coli. The effects of altering culture and induction conditions on the nature of recombinant protein production were investigated. This led to the expression of active recombinant bovine TdT in E.coli. After purification and characterisation, the activity of the enzyme was assessed in a biological assay for apoptosis. The process described in this report enables the economical production of TdT for high throughput applications.
-
Generation of Active Bovine Terminal Deoxynucleotidyl Transferase (TdT in E.coli
Directory of Open Access Journals (Sweden)
Wee Liang Kuan
2010-01-01
Full Text Available A synthetic gene encoding bovine terminal deoxynucleotidyl transferase (TdT was generated, cloned into an expression vector and expressed in E.coli. The effects of altering culture and induction conditions on the nature of recombinant protein production were investigated. This led to the expression of active recombinant bovine TdT in E.coli. After purification and characterisation, the activity of the enzyme was assessed in a biological assay for apoptosis. The process described in this report enables the economical production of TdT for high throughput applications.
-
Terminal deoxynucleotidyl transferase in the diagnosis of leukemia and malignant lymphoma.
Kung, P C; Long, J C; McCaffrey, R P; Ratliff, R L; Harrison, T A; Baltimore, D
1978-05-01
Neoplastic cells from 253 patients with leukemia and 46 patients with malignant lymphoma were studied for the presence of terminal deoxynucleotidyl transferase (TdT) by biochemical and fluorescent antibody technics. TdT was detected in circulating blast cells from 73 of 77 patients with acute lymphoblastic leukemia, 24 of 72 patients with chronic myelogenous leukemia examined during the blastic phase of the disorder and in cell suspensions of lymph nodes from nine of nine patients with diffuse lymphoblastic lymphoma. Blast cells from six of 10 patients with acute undifferentiated leukemia were TdT positive, but the enzyme was found in only two of 55 patients with acute myeloblastic leukemia. TdT was not detected in other lymphocytic or granulocytic leukemias or in other types of malignant lymphomas. The fluorescent antibody assay for TdT permits rapid and specific identification of the enzyme in single cells. The TdT assay is clinically useful in confirming the diagnosis of acute lymphoblastic leukemia, evaluating patients with blastic chronic myelogenous leukemia, and distinguishing patients with lymphoblastic lymphoma, whose natural history includes rapid extranodal dissemination, from patients with other poorly differentiated malignant lymphomas.
-
International Nuclear Information System (INIS)
Yoshida, S.; Masaki, S.; Nakamura, H.; Morita, T.
1981-01-01
The amount of DNA synthesis in vitro with the ultraviolet-irradiated poly(dT).oligo(rA) template initiators catalysed by DNA polymerase α (Masaki, S. and Yoshida, S., Biochim. Biophys. Acta 521, 74-88) decreased with the dose of ultraviolet-irradiation. The ultraviolet irradiation to the template, however, did not affect the rate of incorporation of incorrect deoxynucleotides into the newly synthesized poly(dA). The addition of terminal deoxynucleotidyl transferase to this system enhanced the DNA synthesis to a level which is comparable to that of the control and it concomitantly increased the incorporation of the mismatched deoxynucleotide into the newly synthesized poly(dA) strands. On the other hand, with an unirradiated template initiator, the misincorporation was only slightly enhanced by the addition of terminal deoxynucleotidyl transferase. The sizes of newly synthesized DNA measured by the sedimentation velocities were found to be smaller with the ultraviolet-irradiated templates but they increased to the control level with the addition of terminal deoxynucleotidyl transferase to the systems. These results suggest that terminal deoxynucleotidyl transferase can help DNA polymerase α to bypass thymine dimers in vitro by the formation of mismatched regions at the positions opposite to pyrimidine dimers on the template. (Auth.)
-
Terminal deoxynucleotidyl transferase activity in the regenerating thymus of X-irradiated mice
International Nuclear Information System (INIS)
Daculsi, R.; Astier, T.; Legrand, E.; Duplan, J.F.
1982-01-01
The distribution of terminal deoyxnucleotidyl transferase (TdT) enzyme activity (EU per 10 8 cells) between peaks I and II was followed for a period of 42 days in regenerating thymus of lethally irradiated (9 Gy) C3H mice restored with 10 6 (C3H x AKR) F1 bone marrow cells. The detection of Thy-1.1 and Thy-1.2 surface antigens allowed for the discrimination between host and donor cells, and the main subpopulations of thymic cells were characterized by their sensitivity to H-2sup(k) antiserum and to dexamethazone. Two peaks of TdT activity could be detected on phosphocellulose chromatographic separation. The distribution of TdT activity between these two peaks was followed during the two periods of thymic endo- and exoregeneration. Peak I TdT activity was closely correlated with the variation in the percentage of the high H-2 population. Peak II activity was mostly related to low H-2 cells. The per cell content of both peak I and peak II activities exceeded the norm in rapidly expanding populations. Finally between days 10 and 14 the TdT activity of the endoregenerating population was apparently not different from that of the exoregenerating population between days 14 and 22. (Auth.)
-
Hu, Yufang; Zhang, Qingqing; Xu, Lihua; Wang, Jiao; Rao, Jiajia; Guo, Zhiyong; Wang, Sui
2017-11-01
Electrochemical methods allow fast and inexpensive analysis of enzymatic activity. Here, a simple and yet efficient "signal-on" electrochemical assay for sensitive, label-free detection of DNA-related enzyme activity was established on the basis of terminal deoxynucleotidyl transferase (TdT)-mediated extension strategy. TdT, which is a template-independent DNA polymerase, can catalyze the sequential addition of deoxythymidine triphosphate (dTTP) at the 3'-OH terminus of single-stranded DNA (ssDNA); then, the TdT-yield T-rich DNA nanowires can be employed as the synthetic template of copper nanoclusters (CuNCs). Grown DNA nanowires-templated CuNCs (noted as DNA-CuNCs) were attached onto graphene oxide (GO) surface and exhibited unique electrocatalytic activity to H 2 O 2 reduction. Under optimal conditions, the proposed biosensor was utilized for quantitatively monitoring TdT activity, with the observed LOD of 0.1 U/mL. It also displayed high selectivity to TdT with excellent stability, and offered a facile, convenient electrochemical method for TdT-relevant inhibitors screening. Moreover, the proposed sensor was successfully used for BamHI activity detection, in which a new 3'-OH terminal was exposed by the digestion of a phosphate group. Ultimately, it has good prospects in DNA-related enzyme-based biochemical studies, disease diagnosis, and drug discovery. Graphical Abstract Extraordinary TdT-generated DNA-CuNCs are synthesized and act as a novel electrochemical sensing platform for sensitive detection of TdT and BamHI activity in biological environments.
-
Yang, Jing-Feng; Gao, Rong-Chun; Wu, Hai-Tao; Li, Peng-Fei; Hu, Xian-Shu; Zhou, Da-Yong; Zhu, Bei-Wei; Su, Yi-Cheng
2015-11-04
The sea cucumber body wall melting phenomenon occurs under certain circumstances, and the mechanism of this phenomenon remains unclear. This study investigated the apoptosis in the ultraviolet (UV)-induced sea cucumber melting phenomenon. Fresh sea cucumbers (Stichopus japonicus) were exposed to UV radiation for half an hour at an intensity of 0.056 mW/cm(2) and then held at room temperature for melting development. The samples were histologically processed into formalin-fixed paraffin-embedded tissues. The apoptosis of samples was analyzed with the terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) assay and cleaved caspase-3 immunohistochemistry. The emergence of TUNEL-positive cells speeds up between 0.5 and 2 h after UV irradiation. Cleaved caspase-3 positive cells were obviously detected in sample tissues immediately after the UV irradiation. These results demonstrated that sea cucumber melting induced by UV irradiation was triggered by the activation of caspase-3 followed by DNA fragmentation in sea cucumber tissue, which was attributed to apoptosis but was not a consequence of autolysis activity.
-
International Nuclear Information System (INIS)
Farrar, Y.J.K.; Evans, R.K.; Beach, C.M.; Coleman, M.S.
1991-01-01
Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32 P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32 P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp 221 -Lys 231 (peptide B8) and Cys 234 -Lys 249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an α-helical array of 39 angstrom which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase β that has been implicated in the binding of DNA template
-
International Nuclear Information System (INIS)
Mehnati, P.; Keshtkar, A.; Mesbahi, A.; Sasaki, H.
2006-01-01
The fatal effect of ionizing radiation on cells depends on Linear Energy Transfer level. The distribution of ionizing radiation is sparse and homogeneous for low Linear Energy Transfer radiations such as X or y, but it is dense and concentrated for high Linear Energy Transfer radiation such as heavy-ions radiation. Materials and Methods: Chinese hamster ovary cells (CHO-K1) were exposed to 4 Gy Fe-ion 2000 keV/μm. The Cr-39 is a special and sensitive plastic used to verify exact position of heavy-ions traversal. Terminal deoxynucleotidyl transferase is an enzyme labeled with [3 H ] d ATP for detection of cellular DNA damage by autoradiography assay. Results: The track of heavy ions traversals presented by pit size was almost similar for all different doses of radiation. No pits to show the track of traversal were found in 20% of the cell nuclei of the irradiation. Apparently these fractions of cells wave not hit by heavy ions. Conclusion: This study indicated the possible usefulness of both the Cr-39 plastics and DNA labeling with Terminal deoxynucleotidyl transferase method for evaluating the biological effect of heavy-ions in comparison with low Linear Energy Transfer ionizing radiation
-
Post-irradiation regeneration of early B-lymphocyte precursor cells in mouse bone marrow
International Nuclear Information System (INIS)
Park, Y.-H.; Osmond, D.G.
1989-01-01
To examine the sequential development of early B-cell precursors in mouse bone marrow, B-lineage cells have been examined during a wave of post-irradiation regeneration. Cell phenotypes have been defined for (i) terminal deoxynucleotidyl transferase (TdT); (ii) B220 glycoprotein, (iii) μ heavy chains in the cytoplasm (cμ) and at the cell surface (sμ). Three populations of μ - cells (TdT + 14.8 - ; TdT + 14.8 + ; TdT - 14.8 + ) have been proposed to be early B-cell precursors which would give rise to cμ + sμ - pre-B cells and to sμ + B lymphocytes. The timing, cell-size shifts and progressive amplification of the waves of regeneration accord with a dynamic model in which the TdT + 14.8 - , TdT + 14.8 + and TdT - 14.8 + cells form three successive stages in B-cell differentiation before the expression of μ chains, presumptively including the stage of μ chain gene rearrangement. In addition, the results provide an experimental system for the enrichment of early B-cell precursors in mouse bone marrow. (author)
-
Transonic Dynamics Tunnel (TDT)
Federal Laboratory Consortium — The Transonic Dynamics Tunnel (TDT) is a continuous flow wind-tunnel facility capable of speeds up to Mach 1.2 at stagnation pressures up to one atmosphere. The TDT...
-
Directory of Open Access Journals (Sweden)
Yusuke Takezawa
2016-06-01
Full Text Available A metal-mediated base pair, composed of two ligand-bearing nucleotides and a bridging metal ion, is one of the most promising components for developing DNA-based functional molecules. We have recently reported an enzymatic method to synthesize hydroxypyridone (H-type ligand-bearing artificial DNA strands. Terminal deoxynucleotidyl transferase (TdT, a template-independent DNA polymerase, was found to oligomerize H nucleotides to afford ligand-bearing DNAs, which were subsequently hybridized through copper-mediated base pairing (H–CuII–H. In this study, we investigated the effects of a metal cofactor, MgII ion, on the TdT-catalyzed polymerization of H nucleotides. At a high MgII concentration (10 mM, the reaction was halted after several H nucleotides were appended. In contrast, at lower MgII concentrations, H nucleotides were further appended to the H-tailed product to afford longer ligand-bearing DNA strands. An electrophoresis mobility shift assay revealed that the binding affinity of TdT to the H-tailed DNAs depends on the MgII concentration. In the presence of excess MgII ions, TdT did not bind to the H-tailed strands; thus, further elongation was impeded. This is possibly because the interaction with MgII ions caused folding of the H-tailed strands into unfavorable secondary structures. This finding provides an insight into the enzymatic synthesis of longer ligand-bearing DNA strands.
-
A Mitochondria-Dependent Pathway Mediates the Apoptosis of GSE-Induced Yeast
Cao, Sishuo; Xu, Wentao; Zhang, Nan; Wang, Yan; Luo, YunBo; He, Xiaoyun; Huang, Kunlun
2012-01-01
Grapefruit seed extract (GSE), which has powerful anti-fungal activity, can induce apoptosis in S. cerevisiae. The yeast cells underwent apoptosis as determined by testing for apoptotic markers of DNA cleavage and typical chromatin condensation by Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) and 4,6'-diaminidino-2-phenylindole (DAPI) staining and electron microscopy. The changes of ΔΨmt (mitochondrial transmembrane potential) and ROS (reactive oxygen species) ...
-
TdT activity in acute myeloid leukemias defined by monoclonal antibodies.
San Miguel, J F; González, M; Cañizo, M C; Anta, J P; Portero, J A; López-Borrasca, A
1986-09-01
Blast cells from eight out of 71 patients diagnosed with acute myeloid leukemia (AML) by morphological, cytochemical, and immunological criteria showed TdT activity. Their distribution according to the FAB classification was one M1, one M2, one M4, two M5a, one M5b, one M6, and one undifferentiated case. The TdT+ AML cases did not show major clinical and hematological differences when compared with the classical TdT- AML patients. Other phenotypical aberrations in the expression of membrane antigens, apart from the presence of nuclear TdT, were not observed in these TdT+ cases after study with a large panel of monoclonal antibodies. A higher incidence of TdT+ cases was found among the monocytic variants of AML (M4 and M5)--four cases--than in the granulocytic variants (M1, M2, and M3)--2 cases. These TdT+ cases should be distinguished from mixed leukemias by double labeling techniques, assessing in the TdT+ AML the coexpression of TdT and myeloid markers in individual cells as shown in four of our cases.
-
Jensen, Michael A; Davis, Ronald W
2018-03-27
There is a growing demand for sustainable methods in research and development, where instead of hazardous chemicals, an aqueous medium is chosen to perform biological reactions. In this Perspective, we examine the history and current methodology of using enzymes to generate artificial single-stranded DNA. By using traditional solid-phase phosphoramidite chemistry as a metric, we also explore criteria for the method of template-independent enzymatic oligonucleotide synthesis (TiEOS). As its key component, we delve into the biology of one of the most enigmatic enzymes, terminal deoxynucleotidyl transferase (TdT). As TdT is found to exponentially increase antigen receptor diversity in the vertebrate immune system by adding nucleotides in a template-free manner, researchers have exploited this function as an alternative to the phosphoramidite synthesis method. Though TdT is currently the preferred enzyme for TiEOS, its random nucleotide incorporation presents a barrier in synthesis automation. Taking a closer look at the TiEOS cycle, particularly the coupling step, we find it is comprised of additions > n+1 and deletions. By tapping into the physical and biochemical properties of TdT, we strive to further elucidate its mercurial behavior and offer ways to better optimize TiEOS for production-grade oligonucleotide synthesis.
-
A robust TDT-type association test under informative parental missingness.
Chen, J H; Cheng, K F
2011-02-10
Many family-based association tests rely on the random transmission of alleles from parents to offspring. Among them, the transmission/disequilibrium test (TDT) may be considered to be the most popular statistical test. The TDT statistic and its variations were proposed to evaluate nonrandom transmission of alleles from parents to the diseased children. However, in family studies, parental genotypes may be missing due to parental death, loss, divorce, or other reasons. Under some missingness conditions, nonrandom transmission of alleles may still occur even when the gene and disease are not associated. As a consequence, the usual TDT-type tests would produce excessive false positive conclusions in association studies. In this paper, we propose a novel TDT-type association test which is not only simple in computation but also robust to the joint effect of population stratification and informative parental missingness. Our test is model-free and allows for different mechanisms of parental missingness across subpopulations. We use a simulation study to compare the performance of the new test with TDT and point out the advantage of the new method. Copyright © 2010 John Wiley & Sons, Ltd.
-
Shumak, K H; Baker, M A; Taub, R N; Coleman, M S
1980-11-01
Blast cells were obtained from 17 patients with acute undifferentiated leukemia and 13 patients with chronic myelogenous leukemia in blast crisis. The blasts were tested with anti-i serum in cytotoxicity tests and with antisera to myeloblastic leukemia-associated antigens in immunofluorescence tests. The terminal deoxynucleotidyl transferase (TDT) content of the blasts was also measured. Lymphoblasts react strongly with anti-i, do not react with anti-myeloblast serum, and have high levels of TDT; myeloblasts react weakly with anti-i, do not react with anti-myeloblast serum, and have very low levels of TDT. Of the 17 patients with acute undifferentiated leukemia, there were six with blasts which reacted like lymphoblasts, six with blasts which reacted like myeloblasts, and five with blasts bearing different combinations of these lymphoblastic and myeloblastic markers. Eight of the 11 patients with lymphoblastic or mixed lymphoblastic-myeloblastic markers, but only one of the six with myeloblastic markers, achieved complete or partial remission in response to therapy. Thus, in acute undifferentiated leukemia, classification of blasts with these markers may be of prognostic value. Of the 13 patients with chronic myelogenous leukemia in blast crises, the markers were concordant (for myeloblasts) in only two cases. Three of the 13 patients had TDT-positive blasts, but the reactions of these cells with anti-i and with anti-myeloblast serum differed from those seen with lymphoblasts from patients with acute lymphoblastic leukemia. Although the cell involved in "lymphoid" blast crisis of chronic myelogenous leukemia is similar in many respects to that involved in acute lymphoblastic leukemia, these cells are not identical.
-
Effect of GuiXiong Xiaoyi Wan in Treatment of Endometriosis on Rats
Directory of Open Access Journals (Sweden)
Zhixing Jin
2015-01-01
Full Text Available Objective. To evaluate the effect of GuiXiong Xiaoyi Wan (GXXYW on the development of endometriosis in a rat model. Methods. Sprague-Dawley rats with surgically induced endometriosis were randomly treated with low-dose GXXYW, high-dose GXXYW, or vehicle (negative control for 28 days. Immunohistochemistry was used to assess cell proliferation in the lesions. The terminal deoxynucleotidyl transferase- (TdT- mediated dUTP biotin nick end labelling (TUNEL method was performed to analyse the apoptosis induced by GuiXiong Xiaoyi Wan. The percentages of CD3+ lymphocytes, CD4+ lymphocytes, and CD8+ lymphocytes in the spleens of the rats were evaluated using flow cytometric analysis. Results. Treatment with GXXYW significantly decreased the lesion size, inhibited cell proliferation, and induced apoptosis in endometriotic tissue. The spleens of GXXYW-treated rats also demonstrated a significant increase in the percentage of CD4+ lymphocytes and a significant decrease in the percentage of CD8+ lymphocytes. Conclusions. These results suggest that, in a rat model, GXXYW may be effective in the suppression of the growth of endometriosis, possibly through the inhibition of cell proliferation, the induction of apoptosis of endometriotic cells, and the regulation of the immune system.
-
Ghannoum, M; Isham, N; Henry, W; Kroon, H-A; Yurdakul, S
2012-05-01
TDT 067 is a novel, carrier-based dosage form of terbinafine in Transfersome (1.5%) formulated for topical delivery of terbinafine to the nail, nail bed, and surrounding tissue. We examined the effects of TDT 067 and conventional terbinafine on the morphology of dermatophytes. Trichophyton rubrum hyphae were exposed to TDT 067 or terbinafine (15 mg/ml) and examined under white light, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Subungual debris from patients treated with TDT 067 in a clinical trial was also examined. Exposure of T. rubrum hyphae to TDT 067 led to rapid and extensive ultrastructural changes. Hyphal distortion was evident as early as 4 h after exposure to TDT 067. After 24 h, there was complete disruption of hyphal structure with few intact hyphae remaining. Exposure to terbinafine resulted in morphological alterations similar to those seen with TDT 067; however, the effects of TDT 067 were more extensive, whereas a portion of hyphae remained intact after 24 h of exposure to terbinafine. Lipid droplets were observed under TEM following 30 min of exposure to TDT 067, which after 24 h had filled the intracellular space. These effects were confirmed in vivo in subungual debris from patients with onychomycosis who received topical treatment with TDT 067. The Transfersome in TDT 067 may potentiate the action of terbinafine by delivering terbinafine more effectively to its site of action inside the fungus. Our in vivo data confirm that TDT 067 can enter fungus in the nail bed of patients with onychomycosis and exert its antifungal effects.
-
Matsumoto, M; Imagawa, M; Aoki, Y
2000-01-01
Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did no...
-
Matsumoto, M; Imagawa, M; Aoki, Y
2000-07-01
Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGF alpha induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression.
-
Ghannoum, Mahmoud; Isham, Nancy; Herbert, Jacqueline; Henry, William; Yurdakul, Sam
2011-01-01
TDT 067 is a novel carrier-based dosage form (liquid spray) of 15 mg/ml of terbinafine in Transfersome that has been developed to deliver terbinafine to the nail bed to treat onychomycosis. In this study, we report the in vitro activities of TDT 067 against dermatophytes, compared with those of the Transfersome vehicle, naked terbinafine, and commercially available terbinafine (1%) spray. The MICs of TDT 067 and comparators against 25 clinical strains each of Trichophyton rubrum, T. mentagrophytes, and Epidermophyton floccosum were determined according to the CLSI M38–A2 susceptibility method (2008). Minimum fungicidal concentrations (MFCs) were determined by subculturing visibly clear wells from the MIC microtiter plates. TDT 067 demonstrated potent activity against the dermatophyte strains tested, with an MIC range of 0.00003 to 0.015 μg/ml. Overall, TDT 067 MIC50 values (defined as the lowest concentrations to inhibit 50% of the strains tested) were 8-fold and 60-fold lower than those of naked terbinafine and terbinafine spray, respectively. The Transfersome vehicle showed minimal inhibitory activity. TDT 067 demonstrated lower MFC values for T. rubrum and E. floccosum than naked terbinafine and terbinafine spray. TDT 067 has more potent antifungal activity against dermatophytes that cause nail infection than conventional terbinafine preparations. The Transfersome vehicle appears to potentiate the antifungal activity of terbinafine. Clinical investigation of TDT 067 for the topical treatment of onychomycosis is warranted. PMID:21411586
-
Ghannoum, Mahmoud; Isham, Nancy; Herbert, Jacqueline; Henry, William; Yurdakul, Sam
2011-05-01
TDT 067 is a novel carrier-based dosage form (liquid spray) of 15 mg/ml of terbinafine in Transfersome that has been developed to deliver terbinafine to the nail bed to treat onychomycosis. In this study, we report the in vitro activities of TDT 067 against dermatophytes, compared with those of the Transfersome vehicle, naked terbinafine, and commercially available terbinafine (1%) spray. The MICs of TDT 067 and comparators against 25 clinical strains each of Trichophyton rubrum, T. mentagrophytes, and Epidermophyton floccosum were determined according to the CLSI M38-A2 susceptibility method (2008). Minimum fungicidal concentrations (MFCs) were determined by subculturing visibly clear wells from the MIC microtiter plates. TDT 067 demonstrated potent activity against the dermatophyte strains tested, with an MIC range of 0.00003 to 0.015 μg/ml. Overall, TDT 067 MIC(50) values (defined as the lowest concentrations to inhibit 50% of the strains tested) were 8-fold and 60-fold lower than those of naked terbinafine and terbinafine spray, respectively. The Transfersome vehicle showed minimal inhibitory activity. TDT 067 demonstrated lower MFC values for T. rubrum and E. floccosum than naked terbinafine and terbinafine spray. TDT 067 has more potent antifungal activity against dermatophytes that cause nail infection than conventional terbinafine preparations. The Transfersome vehicle appears to potentiate the antifungal activity of terbinafine. Clinical investigation of TDT 067 for the topical treatment of onychomycosis is warranted.
-
Chromosome abnormalities in the acute phase of CML
Energy Technology Data Exchange (ETDEWEB)
Rowley, J D
1978-01-01
Additional chromosome changes are superimposed on the Ph/sup 1/ positive cell line in approximately 80% of patients in the acute phase of chronic myelogenous leukemia (CML). These changes may precede the onset of blast crisis by several months. They are nonrandom and frequently involve an extra No. 8, an isochromosome for the long arm of No. 17, an extra No. 19, and a second Ph/sup 1/ chromosome. Since such changes may occur in combination, modal numbers frequently range between 47 and 57 chromosomes. Although present evidence suggests that abnormal clones originate, or at least proliferate, in the spleen, similar changes have been observed in patients who underwent splenectomy during the chronic phase of their disease. The question of particular clinical-chromosomal correlations has been discussed in only one study. It appeared that patients whose karyotype did not change might have a longer median survival than those whose karyotype showed additional abnormalities. Tests for levels of terminal deoxynucleotidyl transferase (TDT) and response to anti-acute lymphoblastic leukemia (ALL) serum suggest that some, but not all patients react as do patients with ALL. Those who are similar to ALL have high levels of TDT and are anti-ALL serum-positive; the others have low levels of TDT and are anti-ALL serum-negative. In the future, correlations of these more sophisticated tests with the blast morphology, clinical course, and karyotype pattern should provide significant new insights into the acute phase of CML.
-
Sigurgeirsson, Bárdur; Ghannoum, Mahmoud
2012-10-01
Current topical treatments for onychomycosis are unsatisfactory. New topical agents that offer efficacy without the potential adverse effects of oral antifungal therapy would benefit patients with this condition and encourage a greater treatment rate. Currently available topical therapies are reviewed, and new approaches for enhancing delivery of the established antifungal terbinafine through the nail are summarized. We focus on the use of ultra-deformable lipid vesicles to facilitate delivery of terbinafine to the nail and surrounding tissue. TDT 067 (terbinafine in Transfersome) is the only such therapy in development for onychomycosis, and we review published preclinical and clinical studies on this formulation. TDT 067 offers the use of new technology to deliver an established antifungal, terbinafine. Preclinical data suggest that the Transfersome accelerates entry of terbinafine released from TDT 067 into fungi and potentiates its antifungal effects, resulting in enhanced activity, compared with conventional terbinafine. This translated into high rates of mycological cure and evidence of clinical effect in a study of TDT 067 administered twice daily for 12 weeks in patients with onychomycosis. An ongoing Phase-III trial involving more than 700 patients treated for 48 weeks is investigating the efficacy and safety of TDT 067.
-
CNS wound healing is severely depressed in metallothionein I- and II-deficient mice
DEFF Research Database (Denmark)
Penkowa, M; Carrasco, J; Giralt, M
1999-01-01
. In contrast to normal mice, at 20 dpl no wound healing had occurred. The rate of apoptosis, as determined by using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, was drastically increased in neurons of ipsilateral cortex of the MT-I+II null mice. Our results demonstrate that MT...
-
Directory of Open Access Journals (Sweden)
Jeffrey B. Driban
2011-01-01
Full Text Available We used our voluntary rat model of reaching and grasping to study the effect of performing a high-repetition and high-force (HRHF task for 12 weeks on wrist joints. We also studied the effectiveness of ibuprofen, administered in the last 8 weeks, in attenuating HRHF-induced changes in these joints. With HRHF task performance, ED1+ and COX2+ cells were present in subchondral radius, carpal bones and synovium; IL-1alpha and TNF-alpha increased in distal radius/ulna/carpal bones; chondrocytes stained with Terminal deoxynucleotidyl Transferase- (TDT- mediated dUTP-biotin nick end-labeling (TUNEL increased in wrist articular cartilages; superficial structural changes (e.g., pannus and reduced proteoglycan staining were observed in wrist articular cartilages. These changes were not present in normal controls or ibuprofen treated rats, although IL-1alpha was increased in reach limbs of trained controls. HRHF-induced increases in serum C1,2C (a biomarker of collagen I and II degradation, and the ratio of collagen degradation to synthesis (C1,2C/CPII; the latter a biomarker of collage type II synthesis were also attenuated by ibuprofen. Thus, ibuprofen treatment was effective in attenuating HRHF-induced inflammation and early articular cartilage degeneration.
-
Dennehy, Cornelius J.
2010-01-01
The NASA Engineering and Safety Center (NESC), initially formed in 2003, is an independently funded NASA Program whose dedicated team of technical experts provides objective engineering and safety assessments of critical, high risk projects. The GN&C Technical Discipline Team (TDT) is one of fifteen such discipline-focused teams within the NESC organization. The TDT membership is composed of GN&C specialists from across NASA and its partner organizations in other government agencies, industry, national laboratories, and universities. This paper will briefly define the vision, mission, and purpose of the NESC organization. The role of the GN&C TDT will then be described in detail along with an overview of how this team operates and engages in its objective engineering and safety assessments of critical NASA projects. This paper will then describe selected recent experiences, over the period 2007 to present, of the GN&C TDT in which they directly performed or supported a wide variety of NESC assessments and consultations.
-
Galler, Gunther R; Mundt, Cornelia; Parker, Mathew; Pelanda, Roberta; Mårtensson, Inga-Lill; Winkler, Thomas H
2004-06-07
Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre-B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible muHC transgene in Rag2-/- pro-B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-alpha. Furthermore, in wild-type mice and in mice lacking either lambda5, VpreB1/2, or the entire SLC, the TdT protein is down-regulated in muHC+LC- pre-B cells. Surprisingly, muHC without LC is expressed on the surface of pro-/pre-B cells from lambda5-/-, VpreB1-/-VpreB2-/-, and SLC-/- mice. Thus, SLC or LC is not required for muHC cell surface expression and signaling in these cells. Therefore, these findings offer an explanation for the occurrence of HC allelic exclusion in mice lacking SLC components.
-
Saito, Y.
2017-12-01
Large rivers in continents have a characteristic of slow rise and fall in water levels during floods or the wet season due to a wide drainage basin. A gentle river gradient and large water discharge have relatively large tidal ranges at the river mouth, resulting in large backwater effects further upstream. The result of the Mekong River survey (386 riverbed sediments, river topography, CTD, and biofacies) shows that the distributary channels of the Mekong River delta in Vietnam are divided into two parts: the landward river-dominated tract (RDT) and seaward tide-dominated tract (TDT). The RDT is characterized by a highly variable and deepening trend in water depth and coarse-grained sediments with a fining trend downstream. The TDT is characterized by a shallowing trend in water depth with river-widening, smooth riverbeds, a straight shape, and heterolithic f- to vf-sand and mud alternation (tidal thythmite). The boundary of both tracts is sharply identified by sediment facies and river morphology. Sediment facies indicates that the dominant sedimentary process of bottom sediments is "bedload" in the RDT and "suspension" in the TDT. Daily tidal changes are observed through the year, while water-level changes during the flood/wet season are limited in the TDT. Saltwater intrusion is limited within the seaward part of the TDT alone ( 50 km), close to final bifurcation points. However, brackish-water biofacies is observed in the TDT mainly due to diluted brackish water and/or tolerance to the freshwater environment. These characteristics are also found in the Yangtze; the distance of the TDT/RDT boundary from the river mouth is ca. 100 km in the Mekong, and 200 km in the Yangtze. The preservation potential of sediments in a TDT is low in a progradational system, and high in abandoned channels. The early Holocene transgressive estuary system in the incised valley of the Yangtze formed during the Last Glacial Maximum was composed of 20 m-thick fine-grained heterolithic
-
DEFF Research Database (Denmark)
Brasch-Andersen, Charlotte; Christiansen, L; Tan, Q
2004-01-01
-S-transferase (GST) involved in the antioxidant defense were tested for association to asthma using 246 Danish atopic families in a family-based transmission disequilibrium test (TDT) design. A real-time PCR assay for relative quantification of gene copy number of GSTM1 and GSTT1 was developed. The assay made......Asthma is a complex genetic disorder characterized by chronic inflammation in the airways. As oxidative stress is a key component of inflammation, variations in genes involved in antioxidant defense could therefore be likely candidates for asthma. Three enzymes from the superfamily glutathione...
-
Interactividad, dividendo digital e información en la implementación de la TDT, estudio de Ecuador
Directory of Open Access Journals (Sweden)
A Suing
2014-08-01
Full Text Available Introducción. La investigación busca establecer por qué la interactividad no está presente en emisión de televisión digital terrestre (TDT en Ecuador, conocer el empleo del dividendo digital; y, determinar cuánto conoce la población sobre la implementación de TDT. Metodología. Revisión de documentación oficial, entrevistas a representantes de sectores involucrados y encuestas. Resultados. Aplicaciones interactivas vinculadas con servicios públicos están desarrollándose en universidades pero no son emitidas. El dividendo digital será empleado para banda ancha móvil. Falta promover conocimiento de TDT. Conclusiones. En Ecuador hay capacidades para crear aplicaciones interactivas; los beneficios de interactividad están limitados a la aceptación universal del lenguaje del middleware. La asignación del dividendo digital para su explotación por empresas privadas y el desarrollo de IPTV dejan al usuario ante un futuro incierto sobre formas de consumo. Faltan definir responsabilidades y mecanismos de cooperación entre Estado y empresas para promover transición a TDT entre la comunidad.
-
Directory of Open Access Journals (Sweden)
Xiuling Wang
2017-02-01
Full Text Available Lychee seed is a traditional Chinese medicine and possesses many activities, including hypoglycemia, liver protection, antioxidation, antivirus, and antitumor. However, its effect on neuroprotection is still unclear. The present study investigated the effects of lychee seed saponins (LSS on neuroprotection and associated mechanisms. We established a rat model of Alzheimer’s disease (AD by injecting Aβ25–35 into the lateral ventricle of rats and evaluated the effect of LSS on spatial learning and memory ability via the Morris water maze. Neuronal apoptosis was analyzed by hematoxylin and eosin stain and terminal deoxynucleotidyl transferase (Tdt-mediated dUTP nick-end labeling analysis, and mRNA expression of caspase-3 and protein expressions of Bax and Bcl-2 by reverse transcription-polymerase chain reaction (RT-PCR and Western blotting, respectively. The results showed that LSS remarkably improved cognitive function and alleviated neuronal injury by inhibiting apoptosis in the hippocampus of AD rats. Furthermore, the mRNA expression of caspase-3 and the protein expression of Bax were downregulated, while the protein expression of Bcl-2 and the ratio of Bcl-2/Bax were increased by LSS. We demonstrate that LSS significantly improves cognitive function and prevent neuronal injury in the AD rats via regulation of the apoptosis pathway. Therefore, LSS may be developed as a nutritional supplement and sold as a drug for AD prevention and/or treatment.
-
A Bibliography of Transonic Dynamics Tunnel (TDT) Publications
Doggett, Robert V.
2016-01-01
The Transonic Dynamics Tunnel (TDT) at the National Aeronautics and Space Administration's (NASA) Langley Research Center began research operations in early 1960. Since that time, over 600 tests have been conducted, primarily in the discipline of aeroelasticity. This paper presents a bibliography of the publications that contain data from these tests along with other reports that describe the facility, its capabilities, testing techniques, and associated research equipment. The bibliography is divided by subject matter into a number of categories. An index by author's last name is provided.
-
International Nuclear Information System (INIS)
Singhal, Sharad S.; Singh, Sharda P.; Singhal, Preeti; Horne, David; Singhal, Jyotsana; Awasthi, Sanjay
2015-01-01
4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxides and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes — higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article. - Highlights: • GSTs are the major
-
The TCA cycle transferase DLST is important for MYC-mediated leukemogenesis.
Anderson, N M; Li, D; Peng, H L; Laroche, F J F; Mansour, M R; Gjini, E; Aioub, M; Helman, D J; Roderick, J E; Cheng, T; Harrold, I; Samaha, Y; Meng, L; Amsterdam, A; Neuberg, D S; Denton, T T; Sanda, T; Kelliher, M A; Singh, A; Look, A T; Feng, H
2016-06-01
Despite the pivotal role of MYC in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) and many other cancers, the mechanisms underlying MYC-mediated tumorigenesis remain inadequately understood. Here we utilized a well-characterized zebrafish model of Myc-induced T-ALL for genetic studies to identify novel genes contributing to disease onset. We found that heterozygous inactivation of a tricarboxylic acid (TCA) cycle enzyme, dihydrolipoamide S-succinyltransferase (Dlst), significantly delayed tumor onset in zebrafish without detectable effects on fish development. DLST is the E2 transferase of the α-ketoglutarate (α-KG) dehydrogenase complex (KGDHC), which converts α-KG to succinyl-CoA in the TCA cycle. RNAi knockdown of DLST led to decreased cell viability and induction of apoptosis in human T-ALL cell lines. Polar metabolomics profiling revealed that the TCA cycle was disrupted by DLST knockdown in human T-ALL cells, as demonstrated by an accumulation of α-KG and a decrease of succinyl-CoA. Addition of succinate, the downstream TCA cycle intermediate, to human T-ALL cells was sufficient to rescue defects in cell viability caused by DLST inactivation. Together, our studies uncovered an important role for DLST in MYC-mediated leukemogenesis and demonstrated the metabolic dependence of T-lymphoblasts on the TCA cycle, thus providing implications for targeted therapy.
-
Glutathione transferase-mediated benzimidazole-resistance in Fusarium graminearum.
Sevastos, A; Labrou, N E; Flouri, F; Malandrakis, A
2017-09-01
Fusarium graminearum laboratory mutants moderately (MR) and highly (HR) benzimidazole-resistant, carrying or not target-site mutations at the β 2 -tubulin gene were utilized in an attempt to elucidate the biochemical mechanism(s) underlying the unique BZM-resistance paradigm of this fungal plant pathogen. Relative expression analysis in the presence or absence of carbendazim (methyl-2-benzimidazole carbamate) using a quantitative Real Time qPCR (RT-qPCR) revealed differences between resistant and the wild-type parental strain although no differences in expression levels of either β 1 - or β 2 -tubulin homologue genes were able to fully account for two of the highly resistant phenotypes. Glutathione transferase (GST)-mediated detoxification was shown to be -at least partly- responsible for the elevated resistance levels of a HR isolate bearing the β 2 -tubulin Phe200Tyr resistance mutation compared with another MR isolate carrying the same mutation. This benzimidazole-resistance mechanism is reported for the first time in F. graminearum. No indications of detoxification involved in benzimidazole resistance were found for the rest of the isolates as revealed by GST and glutathione peroxidase (GPx) activities and bioassays using monoxygenase and hydrolase detoxification enzyme inhibiting synergists. Interestingly, besides the Phe200Tyr mutation-carrying HR isolate, the remaining highly-carbendazim resistant phenotypes could not be associated with any of the target site modification/overproduction, detoxification or reduced uptake-increased efflux mechanisms. Copyright © 2016 Elsevier B.V. All rights reserved.
-
Conaghan, Philip G; Bijlsma, Johannes W; Kneer, Werner; Wise, Elspeth; Kvien, Tore K; Rother, Matthias
2014-04-01
Many patients with osteoarthritis (OA) experience side effects with available systemic therapies, some of which can be life threatening. The widespread use of nonsteroidal anti-inflammatory drugs (NSAIDs), often without prescription, is concerning given their potential risks. New treatments for OA are therefore required. This review discusses evidence supporting the use of TDT 064, a drug-free, topical gel containing ultra-deformable phospholipid vesicles (Sequessome * vesicles), for OA-associated pain. Preclinical and clinical studies investigating TDT 064 in patients with OA-associated knee pain were identified in searches of PubMed and congress abstracts. The ultra-deformable phospholipid vesicles (sequessome vesicles) in TDT 064 pass through the skin intact to reach the synovial space within the joint. The mechanism of action is not yet certain, but the phospholipid-based structure of these ultra-deformable phospholipid vesicles, and the observation that they localize to the cartilage surface, support biolubrication as a possible mechanism of action of TDT 064. Data from randomized, phase III studies in OA knee pain in which TDT 064 was used as the drug-free vehicle control for IDEA-033 (ketoprofen in ultra-deformable phospholipid vesicles) demonstrate a marked and consistent response to TDT 064 in terms of pain, stiffness, and function. In a 12 week study of >1300 patients, the effects of TDT 064 on pain and function were statistically noninferior to those of oral celecoxib, and superior to oral placebo. TDT 064 was well tolerated in all studies, and adverse events were typically mild-to-moderate effects on the skin. Evidence from clinical studies supports the use of TDT 064 as a drug-free topical treatment for patients with OA. Further experience with TDT 064, particularly among patients with comorbidities or NSAID contraindications, will provide more information on its potential use.
-
Performance evaluation of TDT soil water content and watermark soil water potential sensors
This study evaluated the performance of digitized Time Domain Transmissometry (TDT) soil water content sensors (Acclima, Inc., Meridian, ID) and resistance-based soil water potential sensors (Watermark 200, Irrometer Company, Inc., Riverside, CA) in two soils. The evaluation was performed by compar...
-
Directory of Open Access Journals (Sweden)
Mukund Seshadri
2007-02-01
Full Text Available The acute effects of the vascular-disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA were investigated in vivo using intravital microscopy (IVM and magnetic resonance imaging (MRI. Changes in vascular permeability and blood flow of syngeneic CT-26 murine colon adenocarcinomas were assessed at 4 and 24 hours after DMXAA treatment (30 mg/kg, i.p. and correlated with induction of tumor necrosis factor-α (TNF-α, endothelial damage [CD31/terminal deoxynucleotidyl transferase (TdT], and treatment outcome. Intravital imaging revealed a marked increase in vascular permeability 4 hours after treatment, consistent with increases in intratumoral mRNA and protein levels of TNF-α. Parallel contrast-enhanced MRI studies showed a ~ 4-fold increase in longitudinal relaxation rates (ΔR1, indicative of increased contrast agent accumulation within the tumor. Dualimmunostained tumor sections (CD31/TdT revealed evidence of endothelial apoptosis at this time point. Twenty-four hours after treatment, extensive hemorrhage and complete disruption of vascular architecture were observed with IVM, along with a significant reduction in ΔR1 and virtual absence of CD31 immunostaining. DMXAA-induced tumor vascular damage resulted in significant long-term (60-day cures compared to untreated controls. Multimodality imaging approaches are useful in visualizing the effects of antivascular therapy in vivo. Such approaches allow cross validation and correlation of findings with underlying molecular changes contributing to treatment outcome.
-
SILENE and TDT: A code for collision probability calculations in XY geometries
International Nuclear Information System (INIS)
Sanchez, R.; Stankovski, Z.
1993-01-01
Collision probability methods are routinely used for cell and assembly multigroup transport calculations in core design tasks. Collision probability methods use a specialized tracking routine to compute neutron trajectories within a given geometric object. These trajectories are then used to generate the appropriate collision matrices in as many groups as required. Traditional tracking routines are based on open-quotes globalclose quotes geometric descriptions (such as regular meshes) and are not able to cope with the geometric detail required in actual core calculations. Therefore, users have to modify their geometry in order to match the geometric model accepted by the tracking routine, introducing thus a modeling error whose evaluation requires the use of a open-quotes referenceclose quotes method. Recently, an effort has been made to develop more flexible tracking routines either by directly adopting tracking Monte Carlo techniques or by coding of complicated geometries. Among these, the SILENE and TDT package is being developed at the Commissariat a l' Energie Atomique to provide routine as well as reference calculations in arbitrarily shaped XY geometries. This package combines a direct graphical acquisition system (SILENE) together with a node-based collision probability code for XY geometries (TDT)
-
Dennehy, Cornelius J.
2008-01-01
This paper will briefly define the vision, mission, and purpose of the NESC organization. The role of the GN&C TDT will then be described in detail along with an overview of how this team operates and engages in its objective engineering and safety assessments of critical NASA projects. This paper will then describe key issues and findings from several of the recent GN&C-related independent assessments and consultations performed and/or supported by the NESC GN&C TDT. Among the examples of the GN&C TDT s work that will be addressed in this paper are the following: the Space Shuttle Orbiter Repair Maneuver (ORM) assessment, the ISS CMG failure root cause assessment, the Demonstration of Autonomous Rendezvous Technologies (DART) spacecraft mishap consultation, the Phoenix Mars lander thruster-based controllability consultation, the NASA in-house Crew Exploration Vehicle (CEV) Smart Buyer assessment and the assessment of key engineering considerations for the Design, Development, Test & Evaluation (DDT&E) of robust and reliable GN&C systems for human-rated spacecraft.
-
Dennehy, Cornelius J.
2011-01-01
The NASA Engineering and Safety Center (NESC) is an independently funded NASA Program whose dedicated team of technical experts provides objective engineering and safety assessments of critical, high risk projects. NESC's strength is rooted in the diverse perspectives and broad knowledge base that add value to its products, affording customers a responsive, alternate path for assessing and preventing technical problems while protecting vital human and national resources. The Guidance Navigation and Control (GN&C) Technical Discipline Team (TDT) is one of fifteen such discipline-focused teams within the NESC organization. The TDT membership is composed of GN&C specialists from across NASA and its partner organizations in other government agencies, industry, national laboratories, and universities. This paper will briefly define the vision, mission, and purpose of the NESC organization. The role of the GN&C TDT will then be described in detail along with an overview of how this team operates and engages in its objective engineering and safety assessments of critical NASA.
-
Chen, Jinyuan; Liu, Zhoujie; Peng, Huaping; Zheng, Yanjie; Lin, Zhen; Liu, Ailin; Chen, Wei; Lin, Xinhua
2017-12-15
Previously reported electrochemical DNA biosensors based on in-situ polymerization approach reveal that terminal deoxynucleoside transferase (TdTase) has good amplifying performance and promising application in the design of electrochemical DNA biosensor. However, this method, in which the background is significantly affected by the amount of TdTase, suffers from being easy to produce false positive result and poor stability. Herein, we firstly present a novel electrochemical DNA biosensor based on grafting-to mode of TdTase-mediated extension, in which DNA targets are polymerized in homogeneous solution and then hybridized with DNA probes on BSA-based DNA carrier platform. It is surprising to find that the background in the grafting-to mode of TdTase-based electrochemical DNA biosensor have little interference from the employed TdTase. Most importantly, the proposed electrochemical DNA biosensor shows greatly improved detection performance over the in-situ polymerization approach-based electrochemical DNA biosensor. Copyright © 2017 Elsevier B.V. All rights reserved.
-
A mitochondria-dependent pathway mediates the apoptosis of GSE-induced yeast.
Directory of Open Access Journals (Sweden)
Sishuo Cao
Full Text Available Grapefruit seed extract (GSE, which has powerful anti-fungal activity, can induce apoptosis in S. cerevisiae. The yeast cells underwent apoptosis as determined by testing for apoptotic markers of DNA cleavage and typical chromatin condensation by Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL and 4,6'-diaminidino-2-phenylindole (DAPI staining and electron microscopy. The changes of ΔΨmt (mitochondrial transmembrane potential and ROS (reactive oxygen species indicated that the mitochondria took part in the apoptotic process. Changes in this process detected by metabonomics and proteomics revealed that the yeast cells tenaciously resisted adversity. Proteins related to redox, cellular structure, membrane, energy and DNA repair were significantly increased. In this study, the relative changes in the levels of proteins and metabolites showed the tenacious resistance of yeast cells. However, GSE induced apoptosis in the yeast cells by destruction of the mitochondrial 60 S ribosomal protein, L14-A, and prevented the conversion of pantothenic acid to coenzyme A (CoA. The relationship between the proteins and metabolites was analyzed by orthogonal projections to latent structures (OPLS. We found that the changes of the metabolites and the protein changes had relevant consistency.
-
A mitochondria-dependent pathway mediates the apoptosis of GSE-induced yeast.
Cao, Sishuo; Xu, Wentao; Zhang, Nan; Wang, Yan; Luo, YunBo; He, Xiaoyun; Huang, Kunlun
2012-01-01
Grapefruit seed extract (GSE), which has powerful anti-fungal activity, can induce apoptosis in S. cerevisiae. The yeast cells underwent apoptosis as determined by testing for apoptotic markers of DNA cleavage and typical chromatin condensation by Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) and 4,6'-diaminidino-2-phenylindole (DAPI) staining and electron microscopy. The changes of ΔΨmt (mitochondrial transmembrane potential) and ROS (reactive oxygen species) indicated that the mitochondria took part in the apoptotic process. Changes in this process detected by metabonomics and proteomics revealed that the yeast cells tenaciously resisted adversity. Proteins related to redox, cellular structure, membrane, energy and DNA repair were significantly increased. In this study, the relative changes in the levels of proteins and metabolites showed the tenacious resistance of yeast cells. However, GSE induced apoptosis in the yeast cells by destruction of the mitochondrial 60 S ribosomal protein, L14-A, and prevented the conversion of pantothenic acid to coenzyme A (CoA). The relationship between the proteins and metabolites was analyzed by orthogonal projections to latent structures (OPLS). We found that the changes of the metabolites and the protein changes had relevant consistency.
-
Perera, Varahenage R.; Lapek, John D.; Newton, Gerald L.; Gonzalez, David J.; Pogliano, Kit
2018-01-01
Bacillithiol is a low molecular weight thiol found in Firmicutes that is analogous to glutathione, which is absent in these bacteria. Bacillithiol transferases catalyze the transfer of bacillithiol to various substrates. The S-transferase-like (STL) superfamily contains over 30,000 putative members, including bacillithiol transferases. Proteins in this family are extremely divergent and are related by structural rather than sequence similarity, leaving it unclear if all share the same biochemical activity. Bacillus subtilis encodes eight predicted STL superfamily members, only one of which has been shown to be a bacillithiol transferase. Here we find that the seven remaining proteins show varying levels of metal dependent bacillithiol transferase activity. We have renamed the eight enzymes BstA-H. Mass spectrometry and gene expression studies revealed that all of the enzymes are produced to varying levels during growth and sporulation, with BstB and BstE being the most abundant and BstF and BstH being the least abundant. Interestingly, several bacillithiol transferases are induced in the mother cell during sporulation. A strain lacking all eight bacillithiol transferases showed normal growth in the presence of stressors that adversely affect growth of bacillithiol-deficient strains, such as paraquat and CdCl2. Thus, the STL bacillithiol transferases represent a new group of proteins that play currently unknown, but potentially significant roles in bacillithiol-dependent reactions. We conclude that these enzymes are highly divergent, perhaps to cope with an equally diverse array of endogenous or exogenous toxic metabolites and oxidants. PMID:29451913
-
The acute monocytic leukemias: multidisciplinary studies in 45 patients.
Straus, D J; Mertelsmann, R; Koziner, B; McKenzie, S; de Harven, E; Arlin, Z A; Kempin, S; Broxmeyer, H; Moore, M A; Menendez-Botet, C J; Gee, T S; Clarkson, B D
1980-11-01
The clinical and laboratory features of 37 patients with variants of acute monocytic leukemia are described. Three of these 37 patients who had extensive extramedullary leukemic tissue infiltration are examples of true histiocytic "lymphomas." Three additional patients with undifferentiated leukemias, one patient with refractory anemia with excess of blasts, one patient with chronic myelomonocytic leukemia, one patient with B-lymphocyte diffuse "histiocytic" lymphoma and one patient with "null" cell, terminal deoxynucleotidyl transferase-positive lymphoblastic lymphoma had bone marrow cells with monocytic features. Another patient had dual populations of lymphoid and monocytoid leukemic cells. The true monocytic leukemias, acute monocytic leukemia (AMOL) and acute myelomonocytic leukemia (AMMOL), are closely related to acute myelocytic leukemia (AML) morphologically and by their response to chemotherapy. like AML, the leukemic cells from the AMMOL and AMOL patients form leukemic clusters in semisolid media. Cytochemical staining of leukemic cells for nonspecific esterases, presence of Fc receptor on the cell surface, phagocytic ability, low TdT activity, presence of surface "ruffles" and "ridges" on scanning EM, elevations of serum lysozyme, and clinical manifestations of leukemic tissue infiltration are features which accompanied monocytic differentiation in these cases.
-
Neville, Claire; Murphy, Alan; Kavanagh, Kevin; Doyle, Sean
2005-04-01
Aspergillus fumigatus is a significant human pathogen. Non-ribosomal peptide (NRP) synthesis is thought to be responsible for a significant proportion of toxin and siderophore production in the organism. Furthermore, it has been shown that 4'-phosphopantetheinylation is required for the activation of key enzymes involved in non-ribosomal peptide synthesis in other species. Here we report the cloning, recombinant expression and functional characterisation of a 4'-phosphopantetheinyl transferase from A. fumigatus and the identification of an atypical NRP synthetase (Afpes1), spanning 14.3 kb. Phylogenetic analysis has shown that the NRP synthetase exhibits greatest identity to NRP synthetases from Metarhizium anisolpiae (PesA) and Alternaria brassicae (AbrePsy1). Northern hybridisation and RT-PCR analysis have confirmed that both genes are expressed in A. fumigatus. A 120 kDa fragment of the A. fumigatus NRP synthetase, containing a putative thiolation domain, was cloned and expressed in the baculovirus expression system. Detection of a 4'-phosphopantetheinylated peptide (SFSAMK) from this protein, by MALDI-TOF mass spectrometric analysis after coincubation of the 4'-phosphopantetheinyl transferase with the recombinant NRP synthetase fragment and acetyl CoA, confirms that it is competent to play a role in NRP synthetase activation in A. fumigatus. The 4'-phosphopantetheinyl transferase also activates, by 4'-phosphopantetheinylation, recombinant alpha-aminoadipate reductase (Lys2p) from Candida albicans, a key enzyme involved in lysine biosynthesis.
-
Inhibition of histone deacetylases prevents cytokine-induced toxicity in beta cells
DEFF Research Database (Denmark)
Larsen, L; Tonnesen, M; Ronn, S G
2007-01-01
B (NFkappaB) is a critical signalling molecule in inflammation and is required for expression of the gene encoding inducible NO synthase (iNOS) and of pro-apoptotic genes. NFkappaB has recently been shown to associate with chromatin-modifying enzymes histone acetyltransferases and histone...... by immunoblotting and by immunoblotting combined with electrophoretic mobility shift assay, respectively. Viability was analysed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and apoptosis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and histone...
-
Transferases in Polymer Chemistry
van der Vlist, Jeroen; Loos, Katja; Palmans, ARA; Heise, A
2010-01-01
Transferases are enzymes that catalyze reactions in which a group is transferred from one compound to another. This makes these enzymes ideal catalysts for polymerization reactions. In nature, transferases are responsible for the synthesis of many important natural macromolecules. In synthetic
-
Hibiscus cannabinus feruloyl-coa:monolignol transferase
Wilkerson, Curtis; Ralph, John; Withers, Saunia; Mansfield, Shawn D.
2016-11-15
The invention relates to isolated nucleic acids encoding a feruloyl-CoA:monolignol transferase and feruloyl-CoA:monolignol transferase enzymes. The isolated nucleic acids and/or the enzymes enable incorporation of monolignol ferulates into the lignin of plants, where such monolignol ferulates include, for example, p-coumaryl ferulate, coniferyl ferulate, and/or sinapyl ferulate. The invention also includes methods and plants that include nucleic acids encoding a feruloyl-CoA:monolignol transferase enzyme and/or feruloyl-CoA:monolignol transferase enzymes.
-
Kim, Su-Mi; Rhee, Yun-Hee; Kim, Jong-Soo
2017-11-01
We investigated the effect of photodynamic therapy (PDT) using radachlorin on invasion, vascular formation and apoptosis by targeting epidermal growth factor receptor (EGFR)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathways in the HEC-1-A endometrial adenocarcinoma cell line. To investigate the apoptotic pathway, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, and western blot analysis. We also evaluated the effects of PDT on tubular capillary formation in and invasion by HEC-1-A cells with a tube formation assay, invasion assay, prostaglandin E2 (PGE2) assay, and western blot analysis. PDT had anticancer effects on HEC-1-A through activation of the intrinsic pathway of apoptosis via caspase-9 and poly-(ADP-ribose) polymerase (PARP). PDT also inhibited tubular capillary formation in and invasion by HEC-1-A under VEGF pretreatment, that resulted from down-regulation of VEGFR2, EGFR, Ras homolog gene family/ member A (RhoA) and PGE2. These results are indicative of the specificity of radachlorin-mediated PDT to VEGF. The major advantage of radachlorin-mediated PDT is its selectivity for cancer tissue while maintaining adjacent normal endometrial tissue. Therefore, radachlorin-mediated PDT might offer high anticancer efficacy for endometrial adenocarcinoma and an especially useful modality for preserving fertility. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
-
Inflammation induction of Dickkopf-1 mediates chondrocyte apoptosis in osteoarthritic joint.
Weng, L-H; Wang, C-J; Ko, J-Y; Sun, Y-C; Su, Y-S; Wang, F-S
2009-07-01
Dysregulated Wnt signaling appears to modulate chondrocyte fate and joint disorders. Dickkopf-1 (DKK1) regulates the pathogenesis of skeletal tissue by inhibiting Wnt actions. This study examined whether DKK1 expression is linked to chondrocyte fate in osteoarthritis (OA). Articular cartilage specimens harvested from nine patients with knee OA and from six controls with femoral neck fracture were assessed for DKK1, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), Bad, Bax, Bcl2 and caspase-3 expression by real time-polymerase chain reaction (RT-PCR) and immunohistochemistry. Apoptotic chondrocytes were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labelling (TUNEL) and 4', 6-dianidino-2-phenylindole dihydrochloride (DAPI) staining. Human chondrocyte cultures were treated with recombinant IL-1beta and monoclonal DKK1 antibody to determine whether DKK1 impairs chondrocyte survival. Expression of DKK1 correlated with inflammatory cytokine levels (IL-1beta and TNF-alpha expressions), proapoptosis regulators (Bad and caspase-3 expressions) and TUNEL staining in OA cartilage tissues. The IL-1beta induced expressions of DKK1, Bax, Bad and caspase-3-dependent apoptosis of chondrocyte cultures. Neutralization of DKK1 by monoclonal DKK1 antibody significantly abrogated IL-1beta-mediated caspase-3 cleavage and apoptosis and reversed chondrocyte proliferation. Recombinant DKK1 treatment impaired chondrocyte growth and promoted apoptosis. By suppressing nuclear beta-catenin accumulation and Akt phosphorylation, DKK1 mediated IL-1beta promotion of chondrocyte apoptosis. Chondrocyte apoptosis correlates with joint OA. Expression of DKK1 contributes to cartilage deterioration and is a potent factor in OA pathogenesis. Attenuating DKK1 may reduce cartilage deterioration in OA.
-
Sineh Sepehr, Koushan; Baradaran, Behzad; Mazandarani, Masoumeh; Yousefi, Bahman; Abdollahpour Alitappeh, Meghdad; Khori, Vahid
2014-12-01
Punica granatum L. var. granatum (Pomegranate), an herbaceous plant found in Iran, The aim of this study was to investigate the cytotoxic effects, induction of apoptosis, and the mechanism of cell death of ethanol extract from Punica granatum L. var. spinosa on the mouse fibrosarcoma cell line, WEHI-164. Various parts of the herbs were extracted from fruit using ethanol as the solvent, and the cytotoxicity and cell viability of the ethanolic extract were determined by the MTT assay. To determine whether necrosis or apoptosis is the predominant cause of cell death, cell death detection was performed using the ELISA method. The induction of apoptosis was confirmed using the terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) assay. Moreover, a sensitive immunoblotting technique was used to examine the production of Caspase-3 and Bcl2 proteins. Our findings suggested that the ethalonic extract of Punica granatum L. var. spinosa altered cell morphology, decreased cell viability, suppressed cell proliferation and induced cell death in a time- and dose-dependent manner in WEHI-164 cells (IC50 = 229.024μg/ml), when compared to a chemotherapeutic anticancer drug, Toxol (Vesper Pharmaceuticals), with increased nucleosome production from apoptotic cells. Induction of apoptosis by the plant extract was proved by the decrease of pro-Caspase-3 and Bcl2 proteins and quantitatively confirmed by Immunoblotting analysis. The results obtained from the present study have demonstrated the growth-inhibitory effect of Ethanol Extracts from Punica granatum L. var. spinosa, and clearly showed that apoptosis was the major mechanism of in-vitro cell death induced by the extract.
-
Energy Technology Data Exchange (ETDEWEB)
Champagne, Devin P., E-mail: devin.champagne@uvm.edu; Shockett, Penny E., E-mail: pshockett@selu.edu
2014-03-15
similar for both genes and consistent with results at the TCRβ locus. Non-templated (N) nucleotide insertions appear to increase between fetal and postnatal stages for Notch1, consistent with normal terminal deoxynucleotidyl transferase (TdT) activity; however, neonatal Bcl11b junctions contain elevated levels of N insertions. Finally, contrasting with results at the HPRT1 locus, we find no obvious age or gender bias in junctional processing, and inverted repeats at recessed coding ends (P{sub r} nucleotides) correspond mostly to single-base additions consistent with normal TdT activity.
-
Ultrasound-microbubble mediated cavitation of plant cells: effects on morphology and viability.
Qin, Peng; Xu, Lin; Zhong, Wenjing; Yu, Alfred C H
2012-06-01
The interaction between ultrasound pulses and microbubbles is known to generate acoustic cavitation that may puncture biological cells. This work presents new experimental findings on the bioeffects of ultrasound-microbubble mediated cavitation in plant cells with emphasis on direct observations of morphological impact and analysis of viability trends in tobacco BY-2 cells that are widely studied in higher plant physiology. The tobacco cell suspensions were exposed to 1 MHz ultrasound pulses in the presence of 1% v/v microbubbles (10% duty cycle; 1 kHz pulse repetition frequency; 70 mm between probe and cells; 1-min exposure time). Few bioeffects were observed at low peak negative pressures (cavitation presumably occurred. In contrast, at 0.9 MPa peak negative pressure (with more inertial cavitation activities according to our passive cavitation detection results), random pores were found on tobacco cell wall (observed via scanning electron microscopy) and enhanced exogenous uptake into the cytoplasm was evident (noted in our fluorescein isothiocyanate dextran uptake analysis). Also, instant lysis was observed in 23.4% of cells (found using trypan blue staining) and programmed cell death was seen in 23.3% of population after 12 h (determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling [TUNEL]). These bioeffects generally correspond in trend with those for mammalian cells. This raises the possibility of developing ultrasound-microbubble mediated cavitation into a targeted gene transfection paradigm for plant cells and, conversely, adopting plant cells as experimental test-beds for sonoporation-based gene therapy in mammalian cells. Copyright © 2012 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.
-
Passantino, L; Ostillio, A; Cianciotta, A; Russo, C; Carrassi, M; Patruno, R; Dhaskali, L; Passantino, G F; Passantino, A
2011-06-01
Until now a few studies have been carried out on the gut lymphoid system in fish despite its protective role in the host. Here, we have evaluated the effects of Candida albicans (Ca) and lipopolysaccaride (LPS) on the pyloric and terminal segments of gut in the rainbow trout Oncorhynchus mykiss. In particular, data show that both Ca and LPS are able to cause apoptosis of intestinal lymphoid cells as detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) procedure. These findings suggest a further modality of gut response in fish to environmental antigens.
-
Apoptosis induction of human endometriotic epithelial and stromal cells by noscapine
Directory of Open Access Journals (Sweden)
Mohammad Rasoul Khazaei
2016-09-01
Full Text Available Objective(s: Endometriosis is a complex gynecologic disease with unknown etiology. Noscapine has been introduced as a cancer cell suppressor. Endometriosis was considered as a cancer like disorder, The aim of present study was to investigate noscapine apoptotic effect on human endometriotic epithelial and stromal cells in vitro. Materials and Methods:In this in vitro study, endometrial biopsies from endometriosis patients (n=9 were prepared and digested by an enzymatic method (collagenase I, 2 mg/ml. Stromal and epithelial cells were separated by sequential filtration through a cell strainer and ficoll layering. The cells of each sample were divided into five groups: control (0, 10, 25, 50 and 100 micromole/liter (µM concentration of noscapine and were cultured for three different periods of times; 24, 48 and 72 hr. Cell viability was assessed by colorimetric assay. Nitric oxide (NO concentration was measured by Griess reagent. Cell death was analyzed by Acridine Orange (AO–Ethidium Bromide (EB double staining and Terminal deoxynucleotidyl transferase (TdT dUTP Nick-End Labeling (TUNEL assay. Data were analyzed by one-way ANOVA. Results: Viability of endometrial epithelial and stromal cells significantly decreased in 10, 25, 50 and 100 µM noscapine concentration in 24, 48, 72 hr (P
-
Dietary curcumin prevents ocular toxicity of naphthalene in rats.
Pandya, U; Saini, M K; Jin, G F; Awasthi, S; Godley, B F; Awasthi, Y C
2000-06-05
Administration of naphthalene is known to cause cataract formation in rats and rabbits and naphthalene-initiated cataract is frequently used as a model for studies on senile cataract in humans. Oxidative stress has been implicated in the mechanism of naphthalene-induced cataract. Curcumin, a constituent of turmeric, a spice used in Indian curry dishes, is an effective antioxidant and is known to induce the enzymes of glutathione-linked detoxification pathways in rats. During the present studies, we have examined whether low levels of dietary curcumin could prevent naphthalene-induced opacification of rat lens. The presence of apoptotic cells in lens epithelial cells was also examined by catalytically incorporating labeled nucleotide to DNA with either Klenow fragment of DNA polymerase or by terminal deoxynucleotidyl transferase (TdT), which forms polymeric tail using the principle of TUNEL assay. The results of these studies demonstrated that the rats treated with naphthalene and kept on a diet supplemented with only 0.005% (w/w) curcumin had significantly less opacification of lenses as compared to that observed in rats treated only with naphthalene. Our studies also demonstrate, for the first time, that naphthalene-initiated cataract in lens is accompanied and perhaps preceded by apoptosis of lens epithelial cells and that curcumin attenuates this apoptotic effect of naphthalene.
-
La crisis de la TDT local pública en España: el caso de Cataluña
Directory of Open Access Journals (Sweden)
Josep-Àngel Guimerá-i-Orts
2011-01-01
Full Text Available El artículo describe y analiza el estado de la televisión digital terrestre local (TDT-L pública en Cataluña justo después del apagón analógico de abril de 2010. Los resultados muestran que sólo 12 de los 37 canales públicos previstos estaban emitiendo y que sólo cinco más podían hacerlo a medio plazo. Estos datos indican que la televisión pública local vive una crisis en el contexto de la migración digital, ya que estos canales sufren graves limitaciones para nacer y el apagón implica la desaparición de emisoras analógicas históricas. El artículo se basa en una investigación cualitativa en el transcurso de la cual se ha entrevistado en profundidad a representantes de los 37 canales previstos. Los objetivos del artículo son, por una parte, describir el modelo de TDT-L vigente y su estado de implantación en Cataluña en mayo de 2010. Por otra parte, aportar elementos interpretativos que expliquen la situación de crisis identificada. Finalmente, aportar conocimiento que permita formular hipótesis sobre el estado del sector en el resto de España. Los resultados ponen de manifiesto que los problemas de la TDT-L pública tienen su origen en las políticas de implantación digital de los gobiernos español y catalán, que no han tenido en cuenta la realidad analógica preexistente –sobre todo el primero. En este sentido, el caso catalán aporta indicios sobre la situación en el conjunto de España.
-
Energy Technology Data Exchange (ETDEWEB)
Carino, Emily V.; Staszak-Jirkovsky, Jakub; Assary, Rajeev S.; Curtiss, Larry A.; Markovic, Nenad M.; Brushett, Fikile R.
2016-03-24
We describe an electrochemically mediated interaction between Li+ and a promising active material for nonaqueous redox flow batteries (RFBs), 1,2,3,4-tetrahydro-6,7-dimethoxy-1,1,4,4-tetramethylnaphthalene (TDT), and the impact of this structural interaction on material stability during voltammetric cycling. TDT could be an advantageous organic positive electrolyte material for nonaqueous RFBs due to its high oxidation potential, 4.21 V vs Li/Li+, and solubility of at least 1.0 M in select electrolytes. Although results from voltammetry suggest TDT displays Nernstian reversibility in many nonaqueous electrolyte solutions, bulk electrolysis reveals significant degradation in all electrolytes studied, the extent of which depends on the electrolyte solution composition. Results of subtractively normalized in situ Fourier transform infrared spectroscopy (SNIFTIRS) confirm that TDT undergoes reversible structural changes during cyclic voltammetry in propylene carbonate and 1,2-dimethoxyethane solutions containing Li+ electrolytes, but irreversible degradation occurs when tetrabutylammonium (TBA+) replaces Li+ as the electrolyte cation in these solutions. By combining the results from SNIFTIRS experiments with calculations from density functional theory, solution-phase active species structure and potential-dependent interactions can be determined. We find that Li+ coordinates to the Lewis basic methoxy groups of neutral TDT and, upon electrochemical oxidation, this complex dissociates into the radical cation TDT•+ and Li+. The improved cycling stability in the presence of Li+ relative to TBA+ suggests that the structural interaction reported herein may be advantageous to the design of energy storage materials based on organic molecules.
-
Shin, Eun-Joo; Duong, Chu Xuan; Nguyen, Xuan-Khanh Thi; Li, Zhengyi; Bing, Guoying; Bach, Jae-Hyung; Park, Dae Hun; Nakayama, Keiichi; Ali, Syed F; Kanthasamy, Anumantha G; Cadet, Jean Lud; Nabeshima, Toshitaka; Kim, Hyoung-Chun
2012-06-15
This study examined the role of protein kinase C (PKC) isozymes in methamphetamine (MA)-induced dopaminergic toxicity. Multiple-dose administration of MA did not significantly alter PKCα, PKCβI, PKCβII, or PKCζ expression in the striatum, but did significantly increase PKCδ expression. Gö6976 (a co-inhibitor of PKCα and -β), hispidin (PKCβ inhibitor), and PKCζ pseudosubstrate inhibitor (PKCζ inhibitor) did not significantly alter MA-induced behavioral impairments. However, rottlerin (PKCδ inhibitor) significantly attenuated behavioral impairments in a dose-dependent manner. In addition, MA-induced behavioral impairments were not apparent in PKCδ knockout (-/-) mice. MA-induced oxidative stress (i.e., lipid peroxidation and protein oxidation) was significantly attenuated in rottlerin-treated mice and was not apparent in PKCδ (-/-) mice. Consistent with this, MA-induced apoptosis (i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells) was significantly attenuated in rottlerin-treated mice. Furthermore, MA-induced increases in the dopamine (DA) turnover rate and decreases in tyrosine hydroxylase (TH) activity and the expression of TH, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) were not significantly observed in rottlerin-treated or PKCδ (-/-) mice. Our results suggest that PKCδ gene expression is a key mediator of oxidative stress and dopaminergic damage induced by MA. Thus, inhibition of PKCδ may be a useful target for protection against MA-induced neurotoxicity. Copyright © 2012 Elsevier B.V. All rights reserved.
-
Singhal, Sharad S; Singh, Sharda P; Singhal, Preeti; Horne, David; Singhal, Jyotsana; Awasthi, Sanjay
2015-12-15
4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxides and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes - higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article. Copyright © 2015 Elsevier Inc. All rights
-
La TDT: El gran contenedor infantil del futuro
Directory of Open Access Journals (Sweden)
María Dolores MORENO RODRÍGUEZ
2009-08-01
Full Text Available RESUMEN: Los expertos en edu-comunicación apuestan por la tematización que favorece la proliferación de canales infantiles al amparo de las nuevas frecuencias disponibles a través de la TDT. Reconocen, asimismo, la interactividad como un valor añadido aplicable a la producción televisiva infantil en tanto que elemento clave en la estimulación de la creatividad y recepción participativa por parte del niño. En cambio, los especialistas rechazan los sistemas de control parental por resultar ineficaces y evidenciar la insuficiente implicación de las familias en la educación televisiva de sus hijos. Así lo revela la investigación de la que damos cuenta en este artículo y que ha sido desarrollada mediante la configuración de un panel de treinta expertos que se han pronunciado sobre la TDT, sus nuevos usos y servicios de aplicación en la programación infantil. La obtención de información se ha llevado a cabo mediante la aplicación del Método Delphi lo que ha permitido la aportación deslocalizada y asincrónica de expertos procedentes de distintas áreas geográficas y científicas. Siendo de este modo que hemos reunido la opinión de especialistas en pedagogía, didáctica, periodismo, televisión educativa y televisión infantil, así como de representantes de observatorios audiovisuales, consejos, revistas especializadas, y asociaciones de consumidores.ABSTRACT: Experts in edu-communication are in favour of the thematic specialization that encourages the proliferation of children’s channels under the protection of the new frequencies available through DTTV. They also recognize the interactivity available as a value added applicable to children’s television production as a key element in the stimulation of children’s creativity and participatory reception. However, the specialists reject the systems of parental control because they are ineffective and demonstrate the insufficient involvement of the families in the televised
-
Energy Technology Data Exchange (ETDEWEB)
Kawakatsu, Miho [Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Goto, Shinji, E-mail: sgoto@nagasaki-u.ac.jp [Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Yoshida, Takako; Urata, Yoshishige; Li, Tao-Sheng [Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan)
2011-08-12
Highlights: {yields} Nuclear translocation of GST{pi} is abrogated by the deletion of the last 16 amino acid residues in the carboxy-terminal region, indicating that residues 195-208 of GST{pi} are required for nuclear translocation. {yields} The lack of a contiguous stretch of positively charged amino acid residues within the carboxy-terminal region of GST{pi}, suggests that the nuclear translocation of GST{pi} is mediated by a non-classical nuclear localization signal. {yields} An in vitro transport assay shows that the nuclear translocation of GST{pi} is dependent on cytosolic factors and ATP. -- Abstract: Glutathione S-transferase {pi} (GST{pi}), a member of the GST family of multifunctional enzymes, is highly expressed in human placenta and involved in the protection of cellular components against electrophilic compounds or oxidative stress. We have recently found that GST{pi} is expressed in the cytoplasm, mitochondria, and nucleus in some cancer cells, and that the nuclear expression of GST{pi} appears to correlate with resistance to anti-cancer drugs. Although the mitochondrial targeting signal of GST{pi} was previously identified in the amino-terminal region, the mechanism of nuclear translocation remains completely unknown. In this study, we find that the region of GST{pi}195-208 is critical for nuclear translocation, which is mediated by a novel and non-classical nuclear localization signal. In addition, using an in vitro transport assay, we demonstrate that the nuclear translocation of GST{pi} depends on the cytosolic extract and ATP. Although further experiments are needed to understand in depth the precise mechanism of nuclear translocation of GST{pi}, our results may help to establish more efficient anti-cancer therapy, especially with respect to resistance to anti-cancer drugs.
-
Renal cell apoptosis in human lupus nephritis: a histological study
DEFF Research Database (Denmark)
Faurschou, M; Penkowa, Milena; Andersen, C B
2009-01-01
Nuclear autoantigens from apoptotic cells are believed to drive the immunological response in systemic lupus erythematosus (SLE). Conflicting data exist as to the possible renal origin of apoptotic cells in SLE patients with nephritis. We assessed the level of renal cell apoptosis in kidney...... biopsies from 35 patients with lupus nephritis by means of terminal deoxynucleotidyl-transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick end labeling (TUNEL). Five samples of normal kidney tissue served as control specimens. We did not observe apoptotic glomerular cells in any...... cells constitute a quantitatively important source of auto-antibody-inducing nuclear auto-antigens in human lupus nephritis....
-
Upregulation of Fas-Fas-L (CD95/CD95L)-mediated epithelial apoptosis--a putative role in pouchitis?
LENUS (Irish Health Repository)
Coffey, J C
2012-02-03
INTRODUCTION: Ileal pouch-anal anastomosis (IPAA) remains the gold standard for patients with refractory ulcerative colitis. Pouchitis causes considerable morbidity in 40% of patients with IPAA. This study examined the role of increased epithelial apoptosis in the etiology of pouchitis. METHODS: Following ethical approval pouch biopsies taken from patients with a history of pouchitis were compared with age-matched controls from patients who were pouchitis free. Apoptosis was detected immunohistochemically using a monoclonal antibody (M30) and terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin end labeling (TUNEL). Villous atrophy was assessed histologically and correlated with levels of apoptosis. Epithelial Fas-ligand (L) was also assessed immunohistochemically. RESULTS: A significant increase in TUNEL staining was seen at the epithelial but not at the lamina propria level for known pouchitis patients versus controls (0.091 vs 0.035; P < 0.01). Similarly, epithelial M30 immunoreactivity (0.225 vs 0.082; P < 0.05) and villous atrophy (0.035 vs 0.10; P < 0.05) were significantly increased in pouches with previous pouchitis when compared with normal pouches. Upregulation of Fas-L expression was characteristic of this epithelium. Mononuclear cells were strongly positive for Fas-L. Increased epithelial levels of apoptosis correlated with increased levels of villous atrophy. CONCLUSIONS: Our data suggest a role for elevated Fas-Fas-L (CD95-CD95L)-mediated epithelial apoptosis in the etiology of pouchitis. Increased levels of villous atrophy may result from increased apoptosis and thereby predispose to infection by otherwise apathogenic organisms.
-
Acrolein-detoxifying isozymes of glutathione transferase in plants.
Mano, Jun'ichi; Ishibashi, Asami; Muneuchi, Hitoshi; Morita, Chihiro; Sakai, Hiroki; Biswas, Md Sanaullah; Koeduka, Takao; Kitajima, Sakihito
2017-02-01
Acrolein is a lipid-derived highly reactive aldehyde, mediating oxidative signal and damage in plants. We found acrolein-scavenging glutathione transferase activity in plants and purified a low K M isozyme from spinach. Various environmental stressors on plants cause the generation of acrolein, a highly toxic aldehyde produced from lipid peroxides, via the promotion of the formation of reactive oxygen species, which oxidize membrane lipids. In mammals, acrolein is scavenged by glutathione transferase (GST; EC 2.5.1.18) isozymes of Alpha, Pi, and Mu classes, but plants lack these GST classes. We detected the acrolein-scavenging GST activity in four species of plants, and purified an isozyme showing this activity from spinach (Spinacia oleracea L.) leaves. The isozyme (GST-Acr), obtained after an affinity chromatography and two ion exchange chromatography steps, showed the K M value for acrolein 93 μM, the smallest value known for acrolein-detoxifying enzymes in plants. Peptide sequence homology search revealed that GST-Acr belongs to the GST Tau, a plant-specific class. The Arabidopsis thaliana GST Tau19, which has the closest sequence similar to spinach GST-Acr, also showed a high catalytic efficiency for acrolein. These results suggest that GST plays as a scavenger for acrolein in plants.
-
Regulatory effect of neuroglobin in the recovery of spinal cord injury.
Dai, Ji-Lin; Lin, Yun; Yuan, Yong-Jian; Xing, Shi-Tong; Xu, Yi; Zhang, Qiang-Hua; Min, Ji-Kang
2017-11-16
The present study was aimed to investigate the therapeutic potential of neuroglobin in the recovery of spinal cord injury. The male albino Wistar strain rats were used as an experimental model, and adeno associated virus (AAV) was administered in the T12 section of spinal cord ten days prior to the injury. Basso Beattie Bresnahan (BBB) locomotor rating scale was used to determine the recovery of the hind limb during four weeks post-operation. Malondialdehyde (MDA), catalase and superoxide dismutase (SOD) were determined in the spinal cord tissues. Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was carried out to determine the presence of apoptotic cells. Immunofluorescence analysis was carried out to determine the neuroglobin expression. Western blot analysis was carried out to determine the protein expressions of caspase-3, cytochrome c, bax and bcl-2 in the spinal cord tissues. Experimental results showed that rats were recovered from the spinal cord injury due to increased neuroglobin expression. Lipid peroxidation was reduced, whereas catalase and SOD activity were increased in the spinal cord tissues. Apoptosis and lesions were significantly reduced in the spinal cord tissues. Caspase-3, cytochrome c and bax levels were significantly reduced, whereas bcl-2 expression was reduced in the spinal cord tissues. Taking all these data together, it is suggested that the increased neuroglobin expression could improve the locomotor function.
-
Shaofu Zhuyu Decoction Regresses Endometriotic Lesions in a Rat Model
Directory of Open Access Journals (Sweden)
Guanghui Zhu
2018-01-01
Full Text Available The current therapies for endometriosis are restricted by various side effects and treatment outcome has been less than satisfactory. Shaofu Zhuyu Decoction (SZD, a classic traditional Chinese medicinal (TCM prescription for dysmenorrhea, has been widely used in clinical practice by TCM doctors to relieve symptoms of endometriosis. The present study aimed to investigate the effects of SZD on a rat model of endometriosis. Forty-eight female Sprague-Dawley rats with regular estrous cycles went through autotransplantation operation to establish endometriosis model. Then 38 rats with successful ectopic implants were randomized into two groups: vehicle- and SZD-treated groups. The latter were administered SZD through oral gavage for 4 weeks. By the end of the treatment period, the volume of the endometriotic lesions was measured, the histopathological properties of the ectopic endometrium were evaluated, and levels of proliferating cell nuclear antigen (PCNA, CD34, and hypoxia inducible factor- (HIF- 1α in the ectopic endometrium were detected with immunohistochemistry. Furthermore, apoptosis was assessed using the terminal deoxynucleotidyl transferase (TdT deoxyuridine 5′-triphosphate (dUTP nick-end labeling (TUNEL assay. In this study, SZD significantly reduced the size of ectopic lesions in rats with endometriosis, inhibited cell proliferation, increased cell apoptosis, and reduced microvessel density and HIF-1α expression. It suggested that SZD could be an effective therapy for the treatment and prevention of endometriosis recurrence.
-
Song, Zhaojun; Wang, Zhigang; Shen, Jieliang; Xu, Shengxi; Hu, Zhenming
2017-01-01
Background Spinal cord injuries (SCIs) can cause severe disability or death. Treatment options include surgical intervention, drug therapy, and stem cell transplantation. However, the efficacy of these methods for functional recovery remains unsatisfactory. Purpose This study was conducted to explore the effect of ultrasound (US)-mediated destruction of poly(lactic-co-glycolic acid) (PLGA) nanobubbles (NBs) expressing nerve growth factor (NGF) (NGF/PLGA NBs) on nerve regeneration in rats following SCI. Materials and methods Adult male Sprague Dawley rats were randomly divided into four treatment groups after Allen hit models of SCI were established. The groups were normal saline (NS) group, NGF and NBs group, NGF and US group, and NGF/PLGA NBs and US group. Histological changes after SCI were observed by hematoxylin and eosin staining. Neuron viability was determined by Nissl staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining was used to examine cell apoptosis. NGF gene and protein expressions were detected by quantitative reverse transcription polymerase chain reaction and Western blotting. Green fluorescent protein expression in the spinal cord was examined using an inverted fluorescence microscope. The recovery of neural function was determined using the Basso, Beattie, and Bresnahan test. Results NGF therapy using US-mediated NGF/PLGA NBs destruction significantly increased NGF expression, attenuated histological injury, decreased neuron loss, inhibited neuronal apoptosis in injured spinal cords, and increased BBB scores in rats with SCI. Conclusion US-mediated NGF/PLGA NBs destruction effectively transfects the NGF gene into target tissues and has a significant effect on the injured spinal cord. The combination of US irradiation and gene therapy through NGF/PLGA NBs holds great promise for the future of nanomedicine and the development of noninvasive treatment options for SCI and other diseases. PMID:28280337
-
Hayashi, Hiromitsu; Kuroki, Hideyuki; Higashi, Takaaki; Takeyama, Hideaki; Yokoyama, Naomi; Okabe, Hirohisa; Nitta, Hidetoshi; Beppu, Toru; Takamori, Hiroshi; Baba, Hideo
2017-07-01
Liver is an amazing organ that can undergo regenerative and atrophic changes inversely, depending on blood flow conditions. Although the regenerative mechanism has been extensively studied, the atrophic mechanism remains to be elucidated. To assess the molecular mechanism of liver atrophy due to reduced portal blood flow, we analyzed the gene expressions between atrophic and hypertrophic livers induced by portal vein embolization in three human liver tissues using microarray analyses. Thrombospondin (TSP)-1 is an extracellular protein and a negative regulator of liver regeneration through its activation of the transforming growth factor-β/Smad signaling pathway. TSP-1 was extracted as the most upregulated gene in atrophic liver compared to hypertrophic liver due to portal flow obstruction in human. Liver atrophic and hypertrophic changes were confirmed by HE and proliferating cell nuclear antigen staining and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling. In an in vivo model with portal ligation, TSP-1 and phosphorylated Smad2 expression were continuously induced at 6 h and thereafter in the portal ligated liver, whereas the induction was transient at 6 h in the portal non-ligated liver. Indeed, while cell proliferation represented by proliferating cell nuclear antigen expression at 48 h was induced in the portal ligated liver, the sinusoidal dilatation and hepatocyte cell death with terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling was detectable at 48 h in the portal ligated liver. Obstructed portal flow induces persistent TSP-1 expression and transforming growth factor-β/Smad signal activation in atrophic liver. Thrombospondin-1 may be implicated in the liver atrophic change due to obstructed portal flow as a pro-atrophic factor. © 2016 The Japan Society of Hepatology.
-
Directory of Open Access Journals (Sweden)
Budiasih Wahyuntari
2005-12-01
Full Text Available Cyclodextrin glycosyl transferase (CGT-ase is mainly produced by Bacilli. Systematical name of the enzyme is E.C. 2.4.1.19 a-1,4 glucan-4-glycosyl transferase. The enzyme catalyzes hydrolysis of starch intramolecular, and intermolecular transglycosylation of a-1,4, glucan chains. Cyclodextrins are a-1,4 linked cyclic oligosaccharides resulting from enzymatic degradation of starch by cyclodextrin glycosyl transferase through untramolecular transglycosylation. The major cyclodextrins are made up of 6, 7 and 8 glucopyranose units which are known as a-, b-, and y-cyclodextrin. All CGT-ase catalyze three kinds of cyclodextrins, the proportion of the cyclodextrins depends on the enzyme source and reaction conditions. The intermolecular transglycosylation ability of the enzyme has been applied in transfering glycosyl residues into suitable acceptor. Transglycosylation by the enzymes have been tested to improve solubility of some flavonoids and to favor precipitation ci some glycosides.
-
Directory of Open Access Journals (Sweden)
Yuanwei Zhang
2016-04-01
Full Text Available Finely tuned changes in cytosolic free calcium ([Ca2+]c mediate numerous intracellular functions resulting in the activation or inactivation of a series of target proteins. Palmitoylation is a reversible post-translational modification involved in membrane protein trafficking between membranes and in their functional modulation. However, studies on the relationship between palmitoylation and calcium signaling have been limited. Here, we demonstrate that the yeast palmitoyl transferase ScAkr1p homolog, AkrA in Aspergillus nidulans, regulates [Ca2+]c homeostasis. Deletion of akrA showed marked defects in hyphal growth and conidiation under low calcium conditions which were similar to the effects of deleting components of the high-affinity calcium uptake system (HACS. The [Ca2+]c dynamics in living cells expressing the calcium reporter aequorin in different akrA mutant backgrounds were defective in their [Ca2+]c responses to high extracellular Ca2+ stress or drugs that cause ER or plasma membrane stress. All of these effects on the [Ca2+]c responses mediated by AkrA were closely associated with the cysteine residue of the AkrA DHHC motif, which is required for palmitoylation by AkrA. Using the acyl-biotin exchange chemistry assay combined with proteomic mass spectrometry, we identified protein substrates palmitoylated by AkrA including two new putative P-type ATPases (Pmc1 and Spf1 homologs, a putative proton V-type proton ATPase (Vma5 homolog and three putative proteins in A. nidulans, the transcripts of which have previously been shown to be induced by extracellular calcium stress in a CrzA-dependent manner. Thus, our findings provide strong evidence that the AkrA protein regulates [Ca2+]c homeostasis by palmitoylating these protein candidates and give new insights the role of palmitoylation in the regulation of calcium-mediated responses to extracellular, ER or plasma membrane stress.
-
Population control of resident and immigrant microglia by mitosis and apoptosis
DEFF Research Database (Denmark)
Wirenfeldt, Martin; Dissing-Olesen, Lasse; Babcock, Alicia
2007-01-01
microglia often occurred in clusters, some having recently incorporated bromodeoxyuridine, showing that proliferation had occurred. Annexin V labeling and staining for activated caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling showed that apoptotic mechanisms participate...... in dissolution of the microglial response. Using bone marrow chimeric mice, we found that the lesion-induced proliferative capacity of resident microglia superseded that of immigrant microglia, whereas lesion-induced kinetics of apoptosis were comparable. Microglial numbers and responses were severely reduced...... in bone marrow chimeric mice. These results broaden our understanding of the microglial response to neural damage by demonstrating that simultaneously occurring mitosis and apoptosis regulate expansion and reduction of both resident and immigrant microglial cell populations....
-
Janossy, G; Coustan-Smith, E; Campana, D
1989-03-01
microplate assay for membrane antigens, followed by double staining for intracellular antigens such as terminal deoxynucleotidyl transferase, cCD3, cCD22, c mu heavy chain, and T cell receptor beta, it is possible to safely establish the lineage affiliation and subgrouping of virtually all acute leukemias. Among these cases are those with aberrant combinations of markers, including 14% of B-lineage ALL (cCD22+,CD19+,TdT+) and a single case T-ALL (cCD3+,CD7+,TdT+), which exhibit CD13 and/or CD33 antigens, cases with mixtures of ALL and AML blasts, and 1.2% of acute leukemias which lack lineage affiliation and can be regarded as acute undifferentiated leukemia.
-
Capture and sequencing of NAD-capped RNA sequences with NAD captureSeq
Czech Academy of Sciences Publication Activity Database
Winz, M. L.; Cahová, Hana; Nübel, G.; Frindert, J.; Höfer, K.; Jäschke, A.
2017-01-01
Roč. 12, č. 1 (2017), s. 122-149 ISSN 1754-2189 Institutional support: RVO:61388963 Keywords : terminal deoxynucleotidyl transferase * dynamic N-1-methyladenosine methylome * protein binding sites Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 10.032, year: 2016
-
National Aeronautics and Space Administration — The MATMCPTDT or tavgM_3d_tdt_Cp data product is the MERRA Data Assimilation System 3-Dimensional temperature tendencies that is time averaged on pressure levels at...
-
Seppan, Prakash; Muhammed, Ibrahim; Mohanraj, Karthik Ganesh; Lakshmanan, Ganesh; Premavathy, Dinesh; Muthu, Sakthi Jothi; Wungmarong Shimray, Khayinmi; Sathyanathan, Sathya Bharathy
2018-02-15
To study the effect of ethanolic seed extract of Mucuna pruriens on damaged dorsal nerve of the penis (DNP) in aged rat in relation to penile erection. The rats were divided into four groups Young (3 months), Aged (24 - 28 months), Aged + M. pruriens, and Young + M. pruriens (200 mg/kg b.w/60 days) and were subjected to the hypophysial - gonadal axis, nerve conduction velocity (NCV), and penile reflex. DNP sections were stained with nitric oxide synthase (nNOS), nicotinamide adenine dinucleotide phosphate (NaDPH) diaphorase, androgen receptor (AR), and osmium tetroxide. Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) staining, electron microscopy(EM) and histometric analyses were done. Significant disturbance in hypophysial - gonadal axis was noted in aged rat. With reduced number of myelinated fibers, diameter, vacuolization, indentation of the myelin sheath, and degeneration. nNOS and its cofactor (NaDPH diaphorase) were reduced in aged rat DNP. NCV was slow in aged rats and concomitant poor penile reflex was also noted. AR showed reduced expression in aged rat DNP when compared to young and control groups. TUNEL positive cells were increased in aged rat DNP. These pathological changes were remarkably reduced or recovered in M. pruriens treated aged rats. The results indicate a multi-factorial therapeutic activity in penile innervations towards sustaining the penile erection in the presence of the extract in aged rats and justifying the claim of traditional usage.
-
21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.
2010-04-01
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alanine amino transferase (ALT/SGPT) test system... Test Systems § 862.1030 Alanine amino transferase (ALT/SGPT) test system. (a) Identification. An alanine amino transferase (ALT/SGPT) test system is a device intended to measure the activity of the...
-
Das, Anindita; Xi, Lei; Kukreja, Rakesh C
2005-04-01
We investigated the effect of sildenafil in protection against necrosis or apoptosis in cardiomyocytes. Adult mouse ventricular myocytes were treated with sildenafil (1 or 10 microM) for 1 h before 40 min of simulated ischemia (SI). Necrosis was determined by trypan blue exclusion and lactate dehydrogenase release following SI alone or plus 1 or 18 h of reoxygenation (RO). Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay and mitochondrial membrane potential measured using a fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Sildenafil reduced necrosis as indicated by decrease in trypan blue-positive myocytes and leakage of lactate dehydrogenase compared with untreated cells after either SI or SI-RO. The number of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive myocytes or loss of JC-1 fluorescence following SI and 18 h of RO was attenuated in the sildenafil-treated group with concomitant inhibition of caspase 3 activity. An early increase in Bcl-2 to Bax ratio with sildenafil treatment was also observed in myocytes after SI-RO. The increase of Bcl-2 expression by sildenafil was inhibited by nitric-oxide synthase (NOS) inhibitor, L-nitro-amino-methyl-ester. The drug also enhanced mRNA and protein content of inducible NOS (iNOS) and endothelial NOS (eNOS) in the myocytes. Sildenafil-induced protection against necrosis and apoptosis was absent in the myocytes derived from iNOS knock-out mice and was attenuated in eNOS knock-out myocytes. The up-regulation of Bcl-2 expression by sildenafil was also absent in iNOS-deficient myocytes. Reverse transcription-PCR, Western blots, and immunohistochemical assay confirmed the expression of phosphodiesterase-5 in mouse cardiomyocytes. These data provide strong evidence for a direct protective effect of sildenafil against necrosis and apoptosis through NO signaling pathway. The results may have possible
-
José Rafael Uzcátegui; José Francisco Torres; Nelson Pérez García; Luís Duque; Zeldívar Bruzual
2010-01-01
En octubre 2009, la República Bolivariana de Venezuela adoptó el estándar ISDB-Tb (Integrated Services Digital Broadcasting – Terrestrial Built On), de origen japonés, y con las mejoras introducidas por Brasil, como base para el sistema de Televisión Digital Terrestre (TDT) en Venezuela. Con la elección del estándar se inicia el camino hacia la solución de transcendentales aspectos de carácter técnico y legal que permitan la prestación de un servicio de Televisión Digital Terrestre cónsono co...
-
International Nuclear Information System (INIS)
Hossain, Quazi Sohel; Ulziikhishig, Enkhbaatar; Lee, Kang Kwang; Yamamoto, Hideyuki; Aniya, Yoko
2009-01-01
We recently reported that the glutathione transferase in rat liver mitochondrial membranes (mtMGST1) is activated by S-glutathionylation and the activated mtMGST1 contributes to the mitochondrial permeability transition (MPT) pore and cytochrome c release from mitochondria [Lee, K.K., Shimoji, M., Quazi, S.H., Sunakawa, H., Aniya, Y., 2008. Novel function of glutathione transferase in rat liver mitochondrial membrane: role for cytochrome c release from mitochondria. Toxcol. Appl. Pharmacol. 232, 109-118]. In the present study we investigated the effect of reactive oxygen species (ROS), generator gallic acid (GA) and GST inhibitors on mtMGST1 and the MPT. When rat liver mitochondria were incubated with GA, mtMGST1 activity was increased to about 3 fold and the increase was inhibited with antioxidant enzymes and singlet oxygen quenchers including 1,4-diazabicyclo [2,2,2] octane (DABCO). GA-mediated mtMGST1 activation was prevented by GST inhibitors such as tannic acid, hematin, and cibacron blue and also by cyclosporin A (CsA). In addition, GA induced the mitochondrial swelling which was also inhibited by GST inhibitors, but not by MPT inhibitors CsA, ADP, and bongkrekic acid. GA also released cytochrome c from the mitochondria which was inhibited completely by DABCO, moderately by GST inhibitors, and somewhat by CsA. Ca 2+ -mediated mitochondrial swelling and cytochrome c release were inhibited by MPT inhibitors but not by GST inhibitors. When the outer mitochondrial membrane was isolated after treatment of mitochondria with GA, mtMGST1 activity was markedly increased and oligomer/aggregate of mtMGST1 was observed. These results indicate that mtMGST1 in the outer mitochondrial membrane is activated by GA through thiol oxidation leading to protein oligomerization/aggregation, which may contribute to the formation of ROS-mediated, CsA-insensitive MPT pore, suggesting a novel mechanism for regulation of the MPT by mtMGST1
-
Sun, Xiao-Qin; Zhang, Mao-Xin; Yu, Jing-Ya; Jin, Yu; Ling, Bing; Du, Jin-Ping; Li, Gui-Hua; Qin, Qing-Ming; Cai, Qing-Nian
2013-01-01
Plants have evolved complex processes to ward off attacks by insects. In parallel, insects have evolved mechanisms to thwart these plant defenses. To gain insight into mechanisms that mediate this arms race between plants and herbivorous insects, we investigated the interactions between gramine, a toxin synthesized by plants of the family Gramineae, and glutathione S transferase (GST), an enzyme found in insects that is known to detoxify xenobiotics. Here, we demonstrate that rice (Oryza sati...
-
Pettersson, Eva U; Ljunggren, Erland L; Morrison, David A; Mattsson, Jens G
2005-01-01
The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase.
-
Liu, Ruiling; Li, Boqiang; Qin, Guozheng; Zhang, Zhanquan; Tian, Shiping
2017-01-01
Acidity plays an important role in flavor and overall organoleptic quality of fruit and is mainly due to the presence of organic acids. Understanding the molecular basis of organic acid metabolism is thus of primary importance for fruit quality improvement. Here, we cloned a putative tonoplast dicarboxylate transporter gene ( SlTDT ) from tomato, and submitted it to the NCBI database (GenBank accession number: KC733165). SlTDT protein contained 13 putative transmembrane domains in silico analysis. Confocal microscopic study using green fluorescent fusion proteins revealed that SlTDT was localized on tonoplast. The expression patterns of SlTDT in tomato were analyzed by RT-qPCR. The results indicated that SlTDT expressed in leaves, roots, flowers and fruits at different ripening stages, suggesting SlTDT may be associated with the development of different tissues. To further explore the function of SlTDT , we constructed both overexpression and RNAi vectors and obtained transgenic tomato plants by agrobacterium-mediated method. Gas chromatography-mass spectrometer (GC-MS) analysis showed that overexpression of SlTDT significantly increased malate content, and reduced citrate content in tomato fruit. By contrast, repression of SlTDT in tomato reduced malate content of and increased citrate content. These results indicated that SlTDT played an important role in remobilization of malate and citrate in fruit vacuoles.
-
Carnitine palmityl transferase I deficiency
Al-Aqeel, A. I.; Rashed, M. S.; Ruiter, J. P.; Al-Husseini, H. F.; Al-Amoudi, M. S.; Wanders, R. J.
2001-01-01
Carnitine palmityl transferase I is the key enzyme in the carnitine dependent transport of long chain fatty acids across the mitochondrial inner membrane and its deficiency results in a decrease rate of fatty acids beta-oxidation with decreased energy production. We reported a family of 3 affected
-
Acceso y participación en la era digital: caso TDT en Argentina
Directory of Open Access Journals (Sweden)
Ornela Carboni
2011-03-01
Full Text Available El proceso de implementación la Televisión Digital Terrestre (TDT en Argentina se inició en septiembre de 2009/ luego de la publicación del Decreto 1148/09 por el cual se creó el Sistema Argentino de Televisión Digital Terrestre (SATVD-T, basado en el estándar nipón-brasilero Integrated Services Digital Broadcasting Terrestrial (lSDB-TJ. La elección de la norma y las medidas adoptadas para la expansión de la tecnología digital se insertan dentro de un contexto socio-político particular del país caracterizado por un hiperdinamismo en materia de políticas de comunicación. El presente trabajo intentará repensar las nociones de acceso y participación enmarcándolo en la implantación de una nueva tecnología. En este sentido se definirán tres etapas históricas de análisis ligadas al desarrollo de los sistemas de medios, particularmente de la televisión que permitirán establecer las mutaciones de los ejes conceptuales del artículo.
-
The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.
Diccianni, M B; Imagawa, M; Muramatsu, M
1992-10-11
Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lack of a role for AP1 in GPEI mediated trans-activation in F9 cells, although endogenously present AP1 can influence GPEI in HeLa cells. Co-transfection of delta fosB with c-jun, which forms an inactive c-Jun/delta FosB heterodimer that binds TRE sequences, inhibits GPEI-mediated transcription in AP1-lacking F9 cells as well as AP1-containing HeLa cells. These data suggest novel factor(s) other than AP1 are influencing GPEI. Binding studies reveal multiple nucleoproteins bind to GPEI. These factors are likely responsible for the high level of GPEI-mediated transcription observed in the absence of AP1 and during hepatocarcinogenesis.
-
Metallothionein 1+2 protect the CNS during neuroglial degeneration induced by 6-aminonicotinamide
DEFF Research Database (Denmark)
Penkowa, Milena; Giralt, Mercedes; Camats, Jordi
2002-01-01
6-Aminonicotinamide (6-AN) is a niacin antagonist, which leads to degeneration of gray matter astrocytes. Metallothionein 1+2 (MT-1+2) are neuroprotective factors in the central nervous system (CNS), and to determine the roles for MT after 6-AN, we have examined transgenic mice overexpressing MT-1...... (NITT), and the number of terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridine triphosphate [dUTP]-digoxigenin nick end labeling-positive (TUNEL+), caspase-3+ apoptotic cells were significantly increased in the brainstem of normal mice after 6-AN. In the TgMTI* mice, the 6-AN-induced tissue...... damage was decreased in comparison to control mice, and they showed significantly reduced numbers of recruited macrophages and T lymphocytes, and a drastic reduction of oxidative stress and apoptotic cell death. In addition, the accompanying reactive astrogliosis was increased in the transgenic mice...
-
Apoptosis and microvessel density in gastric cancer: correlation with tumor stage and prognosis.
Aurello, Paolo; Bellagamba, Riccardo; Rossi Del Monte, Simone; D'Angelo, Francesco; Nigri, Giuseppe; Cicchini, Claudia; Ravaioli, Matteo; Ramacciato, Giovanni
2009-12-01
Gastric cancer remains one of the most common human malignancies with a poor prognosis. Apoptosis is known to be a programmed cell death and its inhibition is involved in the unregulated cellular growth that leads to neoplasms. Microvessel density (MVD) has been investigated as a promoting factor for angiogenesis with conflicting results about its relation to survival. The aim of our study was to search a correlation between these factors and some clinicopathological features and prognosis. Identification of apoptotic cells was performed applying the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique and recorded as apoptotic index (A.I.), whereas monoclonal antibodies were used for the study of MVD. A significant correlation was found between low and high A.I. and the subgroup of patients in Stages I and II (P stage (P = 0.036) and to a poorer 5-year overall survival (P gastric cancer.
-
Directory of Open Access Journals (Sweden)
Ju-Ha Kim
Full Text Available Though piperazine derivative BK10007S was known to induce apoptosis in pancreatic cancer xenograft model as a T-type CaV3.1 a1G isoform calcium channel blocker, its underlying antitumor mechanism still remains unclear so far. Thus, in the present study, the antitumor mechanism of BK10007S was elucidated in hepatocellular carcinoma cells (HCCs. Herein, BK10007S showed significant cytotoxicity by 3-[4,5-2-yl]-2,5-diphenyltetra-zolium bromide (MTT assay and anti-proliferative effects by colony formation assay in HepG2 and SK-Hep1 cells. Also, apoptotic bodies and terminal deoxynucleotidyl transferase (TdT dUTP Nick End Labeling (TUNEL positive cells were observed in BK10007S treated HepG2 and SK-Hep1 cells by 4',6-diamidino-2-phenylinodole (DAPI staining and TUNEL assay, respectively. Consistently, BK10007S increased sub G1 population in HepG2 and SK-Hep1 cells by cell cycle analysis. Furthermore, Western blotting revealed that BK10007S activated the caspase cascades (caspase 8, 9 and 3, cleaved poly (ADP-ribose polymerase (PARP, and downregulated the expression of cyclin D1, survivin and for CUG-binding protein 1 (CUGBP1 or CELF1 in HepG2 and SK-Hep1 cells. Conversely, overexpression of CUGBP1 reduced cleavages of PARP and caspase 3, cytotoxicity and subG1 population in BK10007S treated HepG2 cells. Overall, these findings provide scientific evidences that BK10007S induces apoptosis via inhibition of CUGBP1 and activation of caspases in hepatocellular carcinomas as a potent anticancer candidate.
-
Lu, Jun; Wu, Dong-mei; Zheng, Yuan-lin; Hu, Bin; Zhang, Zi-feng
2010-05-01
Purple sweet potato color (PSPC), a class of naturally occurring anthocyanins, protects brain function against oxidative stress induced by D-galactose (D-gal) (Sigma-Aldrich, St. Louis, MO, USA). Our data showed that PSPC enhanced open-field activity, decreased step-through latency, and improved spatial learning and memory ability in D-gal-treated old mice by decreasing advanced glycation end-products' (AGEs) formation and the AGE receptor (RAGE) expression, and by elevating Cu,Zn-superoxide dismutase (Cu,Zn-SOD) (Sigma-Aldrich) and catalase (CAT) expression and activity. Cleavage of caspase-3 and increased terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labeling (TUNEL)-positive cells in D-gal-treated old mice were inhibited by PSPC, which might be attributed to its antioxidant property. PSPC also suppressed the activation of c-Jun NH(2)-terminal kinase (JNK) and the release of cytochrome c from mitochondria that counteracted the onset of neuronal apoptosis in D-gal-treated old mice. Furthermore, it was demonstrated that phosphoinositide 3-kinase (PI3K) activation was required for PSPC to promote the neuronal survival accompanied with phosphorylation and activation of Akt and p44/42 mitogen-activated protein kinase (MAPK) by using PI3K inhibitor LY294002 (Cell Signaling Technology, Inc., Beverly, MA, USA), implicating a neuronal survival mechanism. The present results suggest that neuronal survival promoted by PSPC may be a potentially effective method to enhance resistance of neurons to age-related disease.
-
Dixit, Prachy; Mukherjee, Prasun K.; Ramachandran, V.; Eapen, Susan
2011-01-01
Background Cadmium (Cd) is a major heavy metal pollutant which is highly toxic to plants and animals. Vast agricultural areas worldwide are contaminated with Cd. Plants take up Cd and through the food chain it reaches humans and causes toxicity. It is ideal to develop plants tolerant to Cd, without enhanced accumulation in the edible parts for human consumption. Glutathione transferases (GST) are a family of multifunctional enzymes known to have important roles in combating oxidative stresses induced by various heavy metals including Cd. Some GSTs are also known to function as glutathione peroxidases. Overexpression/heterologous expression of GSTs is expected to result in plants tolerant to heavy metals such as Cd. Results Here, we report cloning of a glutathione transferase gene from Trichoderma virens, a biocontrol fungus and introducing it into Nicotiana tabacum plants by Agrobacterium-mediated gene transfer. Transgenic nature of the plants was confirmed by Southern blot hybridization and expression by reverse transcription PCR. Transgene (TvGST) showed single gene Mendelian inheritance. When transgenic plants expressing TvGST gene were exposed to different concentrations of Cd, they were found to be more tolerant compared to wild type plants, with transgenic plants showing lower levels of lipid peroxidation. Levels of different antioxidant enzymes such as glutathione transferase, superoxide dismutase, ascorbate peroxidase, guiacol peroxidase and catalase showed enhanced levels in transgenic plants expressing TvGST compared to control plants, when exposed to Cd. Cadmium accumulation in the plant biomass in transgenic plants were similar or lower than wild-type plants. Conclusion The results of the present study suggest that transgenic tobacco plants expressing a Trichoderma virens GST are more tolerant to Cd, without enhancing its accumulation in the plant biomass. It should be possible to extend the present results to crop plants for developing Cd tolerance and
-
Directory of Open Access Journals (Sweden)
Prachy Dixit
Full Text Available BACKGROUND: Cadmium (Cd is a major heavy metal pollutant which is highly toxic to plants and animals. Vast agricultural areas worldwide are contaminated with Cd. Plants take up Cd and through the food chain it reaches humans and causes toxicity. It is ideal to develop plants tolerant to Cd, without enhanced accumulation in the edible parts for human consumption. Glutathione transferases (GST are a family of multifunctional enzymes known to have important roles in combating oxidative stresses induced by various heavy metals including Cd. Some GSTs are also known to function as glutathione peroxidases. Overexpression/heterologous expression of GSTs is expected to result in plants tolerant to heavy metals such as Cd. RESULTS: Here, we report cloning of a glutathione transferase gene from Trichoderma virens, a biocontrol fungus and introducing it into Nicotiana tabacum plants by Agrobacterium-mediated gene transfer. Transgenic nature of the plants was confirmed by Southern blot hybridization and expression by reverse transcription PCR. Transgene (TvGST showed single gene Mendelian inheritance. When transgenic plants expressing TvGST gene were exposed to different concentrations of Cd, they were found to be more tolerant compared to wild type plants, with transgenic plants showing lower levels of lipid peroxidation. Levels of different antioxidant enzymes such as glutathione transferase, superoxide dismutase, ascorbate peroxidase, guiacol peroxidase and catalase showed enhanced levels in transgenic plants expressing TvGST compared to control plants, when exposed to Cd. Cadmium accumulation in the plant biomass in transgenic plants were similar or lower than wild-type plants. CONCLUSION: The results of the present study suggest that transgenic tobacco plants expressing a Trichoderma virens GST are more tolerant to Cd, without enhancing its accumulation in the plant biomass. It should be possible to extend the present results to crop plants for
-
International Nuclear Information System (INIS)
Nemeti, Balazs; Gregus, Zoltan
2009-01-01
Three cytosolic phosphorolytic/arsenolytic enzymes, (purine nucleoside phosphorylase [PNP], glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase) have been shown to mediate reduction of arsenate (AsV) to the more toxic arsenite (AsIII) in a thiol-dependent manner. With unknown mechanism, hepatic mitochondria also reduce AsV. Mitochondria possess ornithine carbamoyl transferase (OCT), which catalyzes phosphorolytic or arsenolytic citrulline cleavage; therefore, we examined if mitochondrial OCT facilitated AsV reduction in presence of glutathione. Isolated rat liver mitochondria were incubated with AsV, and AsIII formed was quantified. Glutathione-supplemented permeabilized or solubilized mitochondria reduced AsV. Citrulline (substrate for OCT-catalyzed arsenolysis) increased AsV reduction. The citrulline-stimulated AsV reduction was abolished by ornithine (OCT substrate inhibiting citrulline cleavage), phosphate (OCT substrate competing with AsV), and the OCT inhibitor norvaline or PALO, indicating that AsV reduction is coupled to OCT-catalyzed arsenolysis of citrulline. Corroborating this conclusion, purified bacterial OCT mediated AsV reduction in presence of citrulline and glutathione with similar responsiveness to these agents. In contrast, AsIII formation by intact mitochondria was unaffected by PALO and slightly stimulated by citrulline, ornithine, and norvaline, suggesting minimal role for OCT in AsV reduction in intact mitochondria. In addition to OCT, mitochondrial PNP can also mediate AsIII formation; however, its role in AsV reduction appears severely limited by purine nucleoside supply. Collectively, mitochondrial and bacterial OCT promote glutathione-dependent AsV reduction with coupled arsenolysis of citrulline, supporting the hypothesis that AsV reduction is mediated by phosphorolytic/arsenolytic enzymes. Nevertheless, because citrulline cleavage is disfavored physiologically, OCT may have little role in AsV reduction in vivo.
-
Analysis of glutathione S-transferase (M1, T1 and P1) gene ...
African Journals Online (AJOL)
Glutathione S-transferase enzymes are active in detoxifying a wide number of endogenous and exogenous chemical carcinogens and subsequently, are crucial in protecting the DNA. Several studies show some differences in association of glutathione S-transferase M1, T1 and P1 genetic polymorphisms with the risk of ...
-
The association between glutathione S-transferase P1 ...
African Journals Online (AJOL)
Mahmoud I. Mahmoud
2011-08-10
Aug 10, 2011 ... B-adrenergic receptor polymorphisms and response to salmeterol. Am J Respir Crit Care ... transferase Pi locus and association with susceptibility to bladder, testicular and prostate cancer. Carcinogenesis 1997;18(4):641–4.
-
Diversity and repertoire of IgW and IgM VH families in the newborn nurse shark.
Rumfelt, Lynn L; Lohr, Rebecca L; Dooley, Helen; Flajnik, Martin F
2004-05-06
Adult cartilaginous fish express three immunoglobulin (Ig) isotypes, IgM, IgNAR and IgW. Newborn nurse sharks, Ginglymostoma cirratum, produce 19S (multimeric) IgM and monomeric/dimeric IgM1gj, a germline-joined, IgM-related VH, and very low amounts of 7S (monomeric) IgM and IgNAR proteins. Newborn IgNAR VH mRNAs are diverse in the complementarity-determining region 3 (CDR3) with non-templated nucleotide (N-region) addition, which suggests that, unlike in many other vertebrates, terminal deoxynucleotidyl transferase (TdT) expressed at birth is functional. IgW is present in the lungfish, a bony fish sharing a common ancestor with sharks 460 million years ago, implying that the IgW VH family is as old as the IgM VH family. This nurse shark study examined the IgM and IgW VH repertoire from birth through adult life, and analyzed the phylogenetic relationships of these gene families. IgM and IgW VH cDNA clones isolated from newborn nurse shark primary and secondary lymphoid tissues had highly diverse and unique CDR3 with N-region addition and VDJ gene rearrangement, implicating functional TdT and RAG gene activity. Despite the clear presence of N-region additions, newborn CDR3 were significantly shorter than those of adults. The IgM clones are all included in a conventional VH family that can be classified into five discrete groups, none of which is orthologous to IgM VH genes in other elasmobranchs. In addition, a novel divergent VH family was orthologous to a published monotypic VH horn shark family. IgW VH genes have diverged sufficiently to form three families. IgM and IgW VH serine codons using the potential somatic hypermutation hotspot sequence occur mainly in VH framework 1 (FR1) and CDR1. Phylogenetic analysis of cartilaginous fish and lungfish IgM and IgW demonstrated they form two major ancient gene groups; furthermore, these VH genes generally diversify (duplicate and diverge) within a species. As in ratfish, sandbar and horn sharks, most nurse shark IgM VH
-
Huang, Hai-li; Wu, Ben-yan; You, Wei-di; Shen, Ming-shi; Wang, Wen-ju
2003-09-01
To study the relation between dendritic cell (DC) infiltration and clinicopathologic parameters, biologic characteristics and prognosis of progressing gastric cancer. The development of apoptotic cell death (apoptotic index, AI) in 61 progressing gastric carcinoma tissues was analyzed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) method. The PCNA labeling index (PCNA-LI), density of dendritic cells in the tumor were detected by immunohistochemical method by the LSAB kit using antibody against S-100 protein and PC-10. DC infiltration was negatively correlated with lymph node metastasis, clinical stage and PCNA-LI, but positively with AI. The DCs in gastric cancer groups with and without lymph node metastasis were (5.63 +/- 4.37)/HPF and (8.51 +/- 5.57)/HPF with difference significant (P stage lesions were (11.23 +/- 6.05)/HPF, (6.28 +/- 4.37)/HPF and (5.53 +/- 5.19)/HPF also with differences significant (P gastric carcinoma.
-
Detection of programmed cell death in plant embryos.
Filonova, Lada H; Suárez, María F; Bozhkov, Peter V
2008-01-01
Programmed cell death (PCD) is an integral part of embryogenesis. In plant embryos, PCD functions during terminal differentiation and elimination of the temporary organ, suspensor, as well as during establishment of provascular system. Embryo abortion is another example of embryonic PCD activated at pathological situations and in polyembryonic seeds. Recent studies identified the sequence of cytological events leading to cellular self-destruction in plant embryos. As in most if not all the developmental cell deaths in plants, embryonic PCD is hallmarked by autophagic degradation of the cytoplasm and nuclear disassembly that includes breakdown of the nuclear envelope and DNA fragmentation. The optimized setup of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) allows the routine in situ analysis of nuclear DNA fragmentation in plant embryos. This chapter provides step-by-step procedure of how to process embryos for TUNEL and how to combine TUNEL with immunolocalization of the protein of interest.
-
International Nuclear Information System (INIS)
Kim, J. S.; Yang, M.; Kim, J.; Lee, D.; Kim, J. C.; Shin, T.; Kim, S. H.; Moon, C.
2012-01-01
The present study compared the dose-response curves for the frequency of apoptosis in mouse hippocampal dentate gyrus (DG) and intestinal crypt using whole-body gamma irradiation. The incidence of gamma-ray-induced apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end-labelling (TUNEL) method. TUNEL-positive apoptotic nuclei in the DG and intestinal crypt were increased in a dose-dependent pattern (0-2 Gy). The dose-response curves were linear-quadratic, with a significant relationship between the appearance of apoptosis and irradiation dose. The slopes of the dose-response curves in the DG were much steeper (∼5-6-fold) than those in the intestinal crypt within the range of 0-1 Gy exposure. Hippocampal DG might be a more effective and sensitive evaluation structure than the intestinal crypt to estimate the degree of radiation exposure in damaged organs of adult mice exposed to low irradiation dose. copy; The Author 2011. Published by Oxford Univ. Press. All rights reserved. (authors)
-
Activation of calcium-sensing receptor accelerates apoptosis in hyperplastic parathyroid cells
International Nuclear Information System (INIS)
Mizobuchi, Masahide; Ogata, Hiroaki; Hatamura, Ikuji; Saji, Fumie; Koiwa, Fumihiko; Kinugasa, Eriko; Koshikawa, Shozo; Akizawa, Tadao
2007-01-01
Calcimimetic compounds inhibit not only parathyroid hormone (PTH) synthesis and secretion, but also parathyroid cell proliferation. The aim of this investigation is to examine the effect of the calcimimetic compound NPS R-568 (R-568) on parathyroid cell death in uremic rats. Hyperplastic parathyroid glands were obtained from uremic rats (subtotal nephrectomy and high-phosphorus diet), and incubated in the media only or the media which contained high concentration of R-568 (10 -4 M), or 10% cyclodextrin, for 6 h. R-568 treatment significantly suppressed medium PTH concentration compared with that of the other two groups. R-568 treatment not only increased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay-positive cells, but also induced the morphologic changes of cell death determined by light or electron microscopy. These results suggest that CaR activation by R-568 accelerates parathyroid cell death, probably through an apoptotic mechanism in uremic rats in vitro
-
International Nuclear Information System (INIS)
Hayes, J.D.; Gilligan, D.; Beckett, G.J.
1983-01-01
A purification scheme is described for six human hepatic glutathione S-transferases from a single liver. Five of the transferases comprised Ya monomers and had a molecular mass of 44000. The remaining enzyme comprised Yb monomers and had a molecular mass of 47000. Data are presented demonstrating that there are at least two distinct Ya monomers. A radioimmunoassay has been developed that has sufficient precision and sensitivity to allow direct measurement of glutathione S-transferase concentrations in unextracted plasma. A comparison of aminotransferase and glutathione S-transferase levels, in three patients who had taken a paracetamol overdose, indicated that glutathione S-transferase measurements provided a far more sensitive index of hepatocellular integrity than the more conventional aminotransferase measurements. (Auth.)
-
Energy Technology Data Exchange (ETDEWEB)
Hayes, J.D.; Gilligan, D.; Beckett, G.J. (Edinburgh Univ. (UK). Dept. of Clinical Chemistry); Chapman, B.J. (Royal Infirmary, Edinburgh (UK))
1983-10-31
A purification scheme is described for six human hepatic glutathione S-transferases from a single liver. Five of the transferases comprised Ya monomers and had a molecular mass of 44000. The remaining enzyme comprised Yb monomers and had a molecular mass of 47000. Data are presented demonstrating that there are at least two distinct Ya monomers. A radioimmunoassay has been developed that has sufficient precision and sensitivity to allow direct measurement of glutathione S-transferase concentrations in unextracted plasma. A comparison of aminotransferase and glutathione S-transferase levels, in three patients who had taken a paracetamol overdose, indicated that glutathione S-transferase measurements provided a far more sensitive index of hepatocellular integrity than the more conventional aminotransferase measurements.
-
Radchuk, Volodymyr; Weier, Diana; Radchuk, Ruslana; Weschke, Winfriede; Weber, Hans
2011-01-01
After fertilization, filial grain organs are surrounded by the maternal nucellus embedded within the integuments and pericarp. Rapid early endosperm growth must be coordinated with maternal tissue development. Parameters of maternal tissue growth and development were analysed during early endosperm formation. In the pericarp, cell proliferation is accomplished around the time of fertilization, followed by cell elongation predominantly in longitudinal directions. The rapid cell expansion coincides with endosperm cellularization. Distribution of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei reveals distinct patterns starting in the nucellus at anthesis and followed later by the inner cell rows of the pericarp, then spreading to the whole pericarp. The pattern suggests timely and spatially regulated programmed cell death (PCD) processes in maternal seed tissues. When the endosperm is coenocytic, PCD events are only observed within the nucellus. Thereby, remobilization of nucellar storage compounds by PCD could nourish the early developing endosperm when functional interconnections are absent between maternal and filial seed organs. Specific proteases promote PCD events. Characterization of the barley vacuolar processing enzyme (VPE) gene family identified seven gene members specifically expressed in the developing grain. HvVPE2a (known as nucellain) together with closely similar HvVPE2b and HvVPE2d might be involved in nucellar PCD. HvVPE4 is strongly cell specific for pericarp parenchyma. Correlative evidence suggests that HvVPE4 plays a role in PCD events in the pericarp. Possible functions of PCD in the maternal tissues imply a potential nutritive role or the relief of a physical restraint for endosperm growth. PCD could also activate post-phloem transport functions.
-
International Nuclear Information System (INIS)
Kasinathan, C.; Kirchberger, M.A.
1988-01-01
Sarcoplasmic reticulum (SR) and plasma membranes from canine left ventricle were used to evaluate the presence of the enzyme CDPdiglyceride-inositol transferase in these membranes. (K + ,-Ca 2+ )-ATPase activity, a marker for SR, was 79.2 +/- 5.0 (SE) and 11.2 +/- 2.0 μmol x mg -1 x h -1 in SR and plasma membrane preparations, respectively, and (Na + , K + )-ATPase activity, a marker for plasma membranes, was 5.6 +/- 1.2 and 99.2 +/- 8.0 μmol x mg -1 x h -1 , respectively. Contamination of SR and plasma membrane preparations by mitochondria was estimated to be 2% and 8%, respectively, and by Golgi membranes, 0.9% and 1.8%, respectively. The transferase activity detected in the plasma membrane preparation could be accounted for largely, but not entirely, by contaminating SR membranes. The pH optimum for the SR transferase activity was between 8.0 and 9.0. Ca 2+ inhibited the enzyme, half-maximal inhibition occurring at about 10 μM Ca 2+ . No loss of [ 3 H]PtdIns could be detected when membranes were incubated in the presence or absence of Ca 2+ . The Ca 2+ inhibition of the transferase was noncompetitive with respect to CDP-dipalmitin while that with respect to myo-inositol was slightly noncompetitive at low [Ca 2+ ] and became uncompetitive at higher [Ca 2+ ]. It is concluded that CDPdiglyceride-inositol transferase is present on SR membranes and is sensitive to micromolar Ca 2+ . The data are consistent with a putative role for the inhibition of the SR transferase by Ca 2+ and acidic pH in the protection of the SR against calcium overload in ischemic myocardium
-
Insecticide resistance and glutathione S-transferases in mosquitoes ...
African Journals Online (AJOL)
Mosquito glutathione S-transferases (GSTs) have received considerable attention in the last 20 years because of their role in insecticide metabolism producing resistance. Many different compounds, including toxic xenobiotics and reactive products of intracellular processes such as lipid peroxidation, act as GST substrates.
-
Heil, Alexander
2016-01-01
The current work contains two projects "Accumulation of L-malate and D-lactate in Arabidopsis thaliana" (A) "Laccase/HBT mediated delignification of Spartina alterniflora and Phragmites australis" (B). In project A, L-malate and D-lactate accumulated in A. thaliana plants. The accumulation of L-malate is carried out by modification of the plant metabolism with the enzymes PEPC, MDH and the tonoplast dicarboxylate transporter (TDT). Gene pepci2 (Hydrilla verticillata), mdh5 (Zea mays) and tdt ...
-
Rumfelt, L L; McKinney, E C; Taylor, E; Flajnik, M F
2002-08-01
Secondary lymphoid tissue and immunoglobulin (Ig) production in mammals is not fully developed at birth, requiring time postnatally to attain all features required for adaptive immune responses. The immune system of newborn sharks - the oldest vertebrate group having adaptive immunity - also displays immature characteristics such as low serum IgM concentration and high levels of IgM1gj, an innate-like Ig. Primary and secondary lymphoid tissues in sharks and other cartilaginous fish were identified previously, but their cellular organization was not examined in detail. In this study of nurse shark lymphoid tissue, we demonstrate that the adult spleen contains well-defined, highly vascularized white pulp (WP) areas, composed of a central T-cell zone containing a major histocompatibility complex (MHC) class II+ dendritic cell (DC) network and a small number of Ig+ secretory cells, surrounded by smaller zones of surface Ig+ (sIg+) B cells. In neonates, splenic WPs are exclusively B-cell zones containing sIgM+-MHC class IIlow B cells; thus compartmentalized areas with T cells and DCs, as well as surface Ig novel antigen receptor (sIgNAR)-expressing B cells are absent at birth. Not until the pups are 5 months old do these WP areas become adult-like; concomitantly, sIgNAR+ B cells are readily detectable, indicating that this Ig class requires a 'mature immune-responsive environment'. The epigonal organ is the major site of neonatal B lymphopoiesis, based on the presence of developing B cells and recombination-activating gene 1 (RAG1)/terminal deoxynucleotidyl transferase (TdT) expression, indicative of antigen receptor rearrangement; such expression persists into adult life, whereas the spleen has negligible lymphopoietic activity. In adults but not neonates, many secretory B cells reside in the epigonal organ, suggesting, like in mammals, that B cells home to this primary lymphoid tissue after activation in other areas of the body.
-
Glutathione transferase mimics : Micellar catalysis of an enzymic reaction
Lindkvist, Björn; Weinander, Rolf; Engman, Lars; Koetse, Marc; Engberts, Jan B.F.N.; Morgenstern, Ralf
1997-01-01
Substances that mimic the enzyme action of glutathione transferases (which serve in detoxification) are described. These micellar catalysts enhance the reaction rate between thiols and activated halogenated nitroarenes as well as alpha,beta-unsaturated carbonyls. The nucleophilic aromatic
-
Czech Academy of Sciences Publication Activity Database
Havranová-Vidláková, Pavlína; Špaček, Jan; Vítová, Lada; Hermanová, Monika; Daďová, Jitka; Raindlová, Veronika; Hocek, Michal; Fojta, Miroslav; Havran, Luděk
2018-01-01
Roč. 30, č. 2 (2018), s. 371-377 ISSN 1040-0397 R&D Projects: GA ČR GA15-08434S Grant - others:AV ČR(CZ) AP1501 Program:Akademická prémie - Praemium Academiae Institutional support: RVO:68081707 ; RVO:61388963 Keywords : terminal deoxynucleotidyl transferase * electrochemical detection Subject RIV: CG - Electrochemistry; CC - Organic Chemistry (UOCHB-X) OBOR OECD: Electrochemistry (dry cells, batteries, fuel cells, corrosion metals, electrolysis); Organic chemistry (UOCHB-X) Impact factor: 2.851, year: 2016
-
Bahmed, Karim; Messier, Elise M; Zhou, Wenbo; Tuder, Rubin M; Freed, Curt R; Chu, Hong Wei; Kelsen, Steven G; Bowler, Russell P; Mason, Robert J; Kosmider, Beata
2016-09-01
Cigarette smoke (CS) is a main source of oxidative stress and a key risk factor for emphysema, which consists of alveolar wall destruction. Alveolar type (AT) II cells are in the gas exchange regions of the lung. We isolated primary ATII cells from deidentified organ donors whose lungs were not suitable for transplantation. We analyzed the cell injury obtained from nonsmokers, moderate smokers, and heavy smokers. DJ-1 protects cells from oxidative stress and induces nuclear erythroid 2-related factor-2 (Nrf2) expression, which activates the antioxidant defense system. In ATII cells isolated from moderate smokers, we found DJ-1 expression by RT-PCR, and Nrf2 and heme oxygenase (HO)-1 translocation by Western blotting and immunocytofluorescence. In ATII cells isolated from heavy smokers, we detected Nrf2 and HO-1 cytoplasmic localization. Moreover, we found high oxidative stress, as detected by 4-hydroxynonenal (4-HNE) (immunoblotting), inflammation by IL-8 and IL-6 levels by ELISA, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in ATII cells obtained from heavy smokers. Furthermore, we detected early DJ-1 and late Nrf2 expression after ATII cell treatment with CS extract. We also overexpressed DJ-1 by adenovirus construct and found that this restored Nrf2 and HO-1 expression and induced nuclear translocation in heavy smokers. Moreover, DJ-1 overexpression also decreased ATII cell apoptosis caused by CS extract in vitro. Our results indicate that DJ-1 activates the Nrf2-mediated antioxidant defense system. Furthermore, DJ-1 overexpression can restore the impaired Nrf2 pathway, leading to ATII cell protection in heavy smokers. This suggests a potential therapeutic strategy for targeting DJ-1 in CS-related lung diseases.
-
Glutathione S - transferases class Pi and Mi and their significance in oncology
Directory of Open Access Journals (Sweden)
Zofia Marchewka
2017-06-01
Full Text Available In this article the current data, which shows that glutathione S-transferases (GST class Pi and Mi are interesting and promising biomarkers in acute and chronic inflammatory processes as well as in the oncology, were presented based on the review of the latest experimental and clinical studies. The article shows their characteristics, functions and participation (direct - GST Pi, indirect - GST Mi in the regulation of signaling pathways of JNK kinases, which are involved in cell differentiation. Overexpression of glutathione S-transferases class Pi and Mi in many cancer cells plays a key role in cancer treatment, making them resistant to chemotherapy. GST isoenzymes are involved in the metabolism of various types of xenobiotics and endogenous substrates, so their altered expression in cancer tissues as well as in serum and urine could be an important potential marker of the cancer and an indicator of oxidative stress. The study shows the role of glutathione S-transferases in redox homeostasis of tumor cells and in the mechanism of resistance to anticancer drugs.
-
Glutathione S - transferases class Pi and Mi and their significance in oncology.
Marchewka, Zofia; Piwowar, Agnieszka; Ruzik, Sylwia; Długosz, Anna
2017-06-19
In this article the current data, which shows that glutathione S-transferases (GST) class Pi and Mi are interesting and promising biomarkers in acute and chronic inflammatory processes as well as in the oncology, were presented based on the review of the latest experimental and clinical studies. The article shows their characteristics, functions and participation (direct - GST Pi, indirect - GST Mi) in the regulation of signaling pathways of JNK kinases, which are involved in cell differentiation. Overexpression of glutathione S-transferases class Pi and Mi in many cancer cells plays a key role in cancer treatment, making them resistant to chemotherapy. GST isoenzymes are involved in the metabolism of various types of xenobiotics and endogenous substrates, so their altered expression in cancer tissues as well as in serum and urine could be an important potential marker of the cancer and an indicator of oxidative stress. The study shows the role of glutathione S-transferases in redox homeostasis of tumor cells and in the mechanism of resistance to anticancer drugs.
-
Directory of Open Access Journals (Sweden)
Chin-Soon Chee
2014-01-01
Full Text Available Glutathione transferases (GST were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW of 23 kDa. 2-dimensional (2-D gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5 and GST2 (pI 6.2 with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase and F0KKB0 (glutathione S-transferase III of Acinetobacter calcoaceticus strain PHEA-2, respectively.
-
Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali
2014-01-01
Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084
-
A glutathione s-transferase confers herbicide tolerance in rice
Directory of Open Access Journals (Sweden)
Tingzhang Hu
2014-07-01
Full Text Available Plant glutathione S-transferases (GSTs have been a focus of attention due to their role in herbicide detoxification. OsGSTL2 is a glutathione S-transferase, lambda class gene from rice (Oryza sativa L.. Transgenic rice plants over-expressing OsGSTL2 were generated from rice calli by the use of an Agrobacterium transformation system, and were screened by a combination of hygromycin resistance, PCR and Southern blot analysis. In the vegetative tissues of transgenic rice plants, the over-expression of OsGSTL2 not only increased levels of OsGSTL2 transcripts, but also GST and GPX expression, while reduced superoxide. Transgenic rice plants also showed higher tolerance to glyphosate and chlorsulfuron, which often contaminate agricultural fields. The findings demonstrate the detoxification role of OsGSTL2 in the growth and development of rice plants. It should be possible to apply the present results to crops for developing herbicide tolerance and for limiting herbicide contamination in the food chain.
-
Bahmed, Karim; Messier, Elise M.; Zhou, Wenbo; Tuder, Rubin M.; Freed, Curt R.; Chu, Hong Wei; Kelsen, Steven G.; Bowler, Russell P.; Mason, Robert J.
2016-01-01
Cigarette smoke (CS) is a main source of oxidative stress and a key risk factor for emphysema, which consists of alveolar wall destruction. Alveolar type (AT) II cells are in the gas exchange regions of the lung. We isolated primary ATII cells from deidentified organ donors whose lungs were not suitable for transplantation. We analyzed the cell injury obtained from nonsmokers, moderate smokers, and heavy smokers. DJ-1 protects cells from oxidative stress and induces nuclear erythroid 2–related factor-2 (Nrf2) expression, which activates the antioxidant defense system. In ATII cells isolated from moderate smokers, we found DJ-1 expression by RT-PCR, and Nrf2 and heme oxygenase (HO)-1 translocation by Western blotting and immunocytofluorescence. In ATII cells isolated from heavy smokers, we detected Nrf2 and HO-1 cytoplasmic localization. Moreover, we found high oxidative stress, as detected by 4-hydroxynonenal (4-HNE) (immunoblotting), inflammation by IL-8 and IL-6 levels by ELISA, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in ATII cells obtained from heavy smokers. Furthermore, we detected early DJ-1 and late Nrf2 expression after ATII cell treatment with CS extract. We also overexpressed DJ-1 by adenovirus construct and found that this restored Nrf2 and HO-1 expression and induced nuclear translocation in heavy smokers. Moreover, DJ-1 overexpression also decreased ATII cell apoptosis caused by CS extract in vitro. Our results indicate that DJ-1 activates the Nrf2-mediated antioxidant defense system. Furthermore, DJ-1 overexpression can restore the impaired Nrf2 pathway, leading to ATII cell protection in heavy smokers. This suggests a potential therapeutic strategy for targeting DJ-1 in CS-related lung diseases. PMID:27093578
-
Steroid sulfatase and sulfuryl transferase activities in human brain tumors
Czech Academy of Sciences Publication Activity Database
Kříž, L.; Bičíková, M.; Mohapl, M.; Hill, M.; Černý, Ivan; Hampl, R.
2008-01-01
Roč. 109, č. 1 (2008), s. 31-39 ISSN 0960-0760 Institutional research plan: CEZ:AV0Z40550506 Keywords : dehydroepiandrosterone * steroid sulfatase * steroid sulfuryl transferase * brain Subject RIV: CC - Organic Chemistry Impact factor: 2.827, year: 2008
-
The α7-nicotinic acetylcholine receptor mediates the sensitivity of gastric cancer cells to taxanes.
Tu, Chao-Chiang; Huang, Chien-Yu; Cheng, Wan-Li; Hung, Chin-Sheng; Uyanga, Batzorig; Wei, Po-Li; Chang, Yu-Jia
2016-04-01
Gastric cancer is difficult to cure because most patients are diagnosed at an advanced disease stage. Systemic chemotherapy remains an important therapy for gastric cancer, but both progression-free survival and disease-free survival associated with various combination regimens are limited because of refractoriness and chemoresistance. Accumulating evidence has revealed that the homomeric α7-nicotinic acetylcholine receptor (A7-nAChR) promotes human gastric cancer by driving cancer cell proliferation, migration, and metastasis. Therefore, A7-nAChR may serve as a potential therapeutic target for gastric cancer. However, the role of A7-nAChR in taxane therapy for gastric cancer was unclear. Cells were subjected to A7-nAChR knockdown (A7-nAChR KD) using short interfering RNA (siRNA). The anti-proliferative effects of taxane were assessed via 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL), and cell cycle distribution assays. A7-nAChR-KD cells exhibited low resistance to docetaxel and paclitaxel treatment, as measured by the MTT assay. Following paclitaxel treatment, the proportion of apoptotic cells was higher among A7-nAChR-KD cells than among scrambled control cells, as measured by cell cycle distribution and TUNEL assays. Further molecular analyses showed a reduction in the pAKT levels and a dramatic increase in the Bad levels in paclitaxel-treated A7-nAChR-KD cells but not in scrambled control cells. Following paclitaxel treatment, the level of Bax was slightly increased in both cell populations, whereas Poly (ADP-ribose) polymerase (PARP) cleavage was increased only in A7-nAChR-KD cells. These findings indicate that A7-nAChR-KD cells are more sensitive to paclitaxel treatment. We conclude that A7-nAChR may be a key biomarker for assessing the chemosensitivity of gastric cancer cells to taxane.
-
Homogentisate solanesyl transferase (HST) cDNA’s in maize
Maize white seedling 3 (w3) has served as a model albino-seedling mutant since its discovery in 1923. We show that the w3 phenotype is caused by disruptions in homogentisate solanesyl transferase (HST), an enzyme that catalyzes the committed step in plastoquinone-9 (PQ9) biosynthesis. This reaction ...
-
Directory of Open Access Journals (Sweden)
Min Ji Choi
2015-06-01
Full Text Available Transforming growth factor-β1 (TGF-β1 has been identified as one of the most important fibrogenic cytokines associated with Peyronie's disease (PD. The mothers against decapentaplegic homolog 7 (SMAD7 is an inhibitory Smad protein that blocks TGF-β signaling pathway. The aim of this study was to examine the anti-fibrotic effect of the SMAD7 gene in primary fibroblasts derived from human PD plaques. PD fibroblasts were pretreated with the SMAD7 gene and then stimulated with TGF-β1. Treated fibroblasts were used for Western blotting, fluorescent immunocytochemistry, hydroxyproline determination, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. Overexpression of the SMAD7 gene inhibited TGF-β1-induced phosphorylation and nuclear translocation of SMAD2 and SMAD3, transdifferentiation of fibroblasts into myofibroblasts, and quashed TGF-β1-induced production of extracellular matrix protein and hydroxyproline. Overexpression of the SMAD7 gene decreased the expression of cyclin D1 (a positive cell cycle regulator and induced the expression of poly (ADP-ribose polymerase 1, which is known to terminate Smad-mediated transcription, in PD fibroblasts. These findings suggest that the blocking of the TGF-β pathway by use of SMAD7 may be a promising therapeutic strategy for the treatment of PD.
-
Current research on methamphetamine-induced neurotoxicity: animal models of monoamine disruption.
Kita, Taizo; Wagner, George C; Nakashima, Toshikatsu
2003-07-01
Methamphetamine (METH)-induced neurotoxicity is characterized by a long-lasting depletion of striatal dopamine (DA) and serotonin as well as damage to striatal dopaminergic and serotonergic nerve terminals. Several hypotheses regarding the mechanism underlying METH-induced neurotoxicity have been proposed. In particular, it is thought that endogenous DA in the striatum may play an important role in mediating METH-induced neuronal damage. This hypothesis is based on the observation of free radical formation and oxidative stress produced by auto-oxidation of DA consequent to its displacement from synaptic vesicles to cytoplasm. In addition, METH-induced neurotoxicity may be linked to the glutamate and nitric oxide systems within the striatum. Moreover, using knockout mice lacking the DA transporter, the vesicular monoamine transporter 2, c-fos, or nitric oxide synthetase, it was determined that these factors may be connected in some way to METH-induced neurotoxicity. Finally a role for apoptosis in METH-induced neurotoxicity has also been established including evidence of protection of bcl-2, expression of p53 protein, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), activity of caspase-3. The neuronal damage induced by METH may reflect neurological disorders such as autism and Parkinson's disease.
-
Tao, K; Chen, D; Tian, Y; Lu, X; Yang, X
2000-01-01
The relationship between the apoptosis and the expression of proliferating cell nuclear antigen (PCNA) and the clinical stages in gastric cancers was studied. By using terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique and PCNA immunohistochemical staining, the apoptosis and the expression of PCNA in tissue of gastric carcinoma were assayed in situ, the index of apoptosis (AI), index of PCNA (PI) and the rate of AI/PI were calculated. AI and PI in gastric cancer tissues were (6.5 +/- 3.7)% and (49.8 +/- 15.9)% respectively, and the rate of AI/PI was 0.13 +/- 0.05, which were obviously different from those of normal gastric mucosa in paragastric cancer (P stages of gastric carcinoma, the AI was decreased, PI was increased and the rate of AI/PI decreased in gastric carcinoma. There was significant difference in them between the gastric cancer tissues and normal gastric mucosa in pericarcinoma in TNM stage II to IV (P gastric carcinoma. The AI, PI and the rate of AI/PI would become the prognostic factors in advanced gastric carcinoma.
-
Bacterial reduction in genotoxicity of Direct Red 28 dye.
Bafana, Amit; Jain, Minakshi; Agrawal, Gaurav; Chakrabarti, Tapan
2009-03-01
Direct Red 28 (DR28) is a benzidine-based azo dye widely used in several countries. It has also been a subject of intense research for its anti-prion activity. Like other benzidine-based azo dyes, it is also carcinogenic and toxic. However, there are very few studies addressing its detoxification. In the present study, a Bacillus velezensis strain was used for detoxification of DR28. Toxicity was checked by a battery of highly sensitive genotoxicity assays like comet assay, DNA ladder formation, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and flow cytometric Annexin V binding assay. HL-60 cell line was used as the test system. All the assays showed an initial increase in toxicity upon biodegradation due to release of mutagenic products, like benzidine and 4-aminobiphenyl, from the dye. These intermediates caused significant DNA damage and induced apoptosis in HL-60 cells. Then the culture degraded these mutagenic intermediates, due to which the toxicity was reduced gradually, finally resulting in nearly complete detoxification.
-
Genome-wide mapping of DNA strand breaks.
Directory of Open Access Journals (Sweden)
Frédéric Leduc
Full Text Available Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP, uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.
-
Diversity and repertoire of IgW and IgM VH families in the newborn nurse shark
Directory of Open Access Journals (Sweden)
Dooley Helen
2004-05-01
Full Text Available Abstract Background Adult cartilaginous fish express three immunoglobulin (Ig isotypes, IgM, IgNAR and IgW. Newborn nurse sharks, Ginglymostoma cirratum, produce 19S (multimeric IgM and monomeric/dimeric IgM1gj, a germline-joined, IgM-related VH, and very low amounts of 7S (monomeric IgM and IgNAR proteins. Newborn IgNAR VH mRNAs are diverse in the complementarity-determining region 3 (CDR3 with non-templated nucleotide (N-region addition, which suggests that, unlike in many other vertebrates, terminal deoxynucleotidyl transferase (TdT expressed at birth is functional. IgW is present in the lungfish, a bony fish sharing a common ancestor with sharks 460 million years ago, implying that the IgW VH family is as old as the IgM VH family. This nurse shark study examined the IgM and IgW VH repertoire from birth through adult life, and analyzed the phylogenetic relationships of these gene families. Results IgM and IgW VH cDNA clones isolated from newborn nurse shark primary and secondary lymphoid tissues had highly diverse and unique CDR3 with N-region addition and VDJ gene rearrangement, implicating functional TdT and RAG gene activity. Despite the clear presence of N-region additions, newborn CDR3 were significantly shorter than those of adults. The IgM clones are all included in a conventional VH family that can be classified into five discrete groups, none of which is orthologous to IgM VH genes in other elasmobranchs. In addition, a novel divergent VH family was orthologous to a published monotypic VH horn shark family. IgW VH genes have diverged sufficiently to form three families. IgM and IgW VH serine codons using the potential somatic hypermutation hotspot sequence occur mainly in VH framework 1 (FR1 and CDR1. Phylogenetic analysis of cartilaginous fish and lungfish IgM and IgW demonstrated they form two major ancient gene groups; furthermore, these VH genes generally diversify (duplicate and diverge within a species. Conclusion As in ratfish
-
Tajima, Shogo; Yanagiya, Masahiro; Sato, Masaaki; Nakajima, Jun; Fukayama, Masashi
2015-01-01
Among human neoplasms, thymomas are well known for their association with paraneoplastic autoimmune diseases such as myasthenia gravis. However, regarding rare metaplastic thymoma, only one case of an association with myasthenia gravis has been reported. Here, we present the second case of a 44-year-old woman with metaplastic thymoma associated with myasthenia gravis. In metaplastic thymoma, intratumoral terminal deoxynucleotidyl transferase-positive T-cells (immature T-cells) are generally scarce, while they were abundant in the present case. We believe that these immature T-cells could be related to the occurrence of myasthenia gravis.
-
DEFF Research Database (Denmark)
Scott, Nichollas E; Nothaft, Harald; Edwards, Alistair V G
2012-01-01
. Interrogation of these data allowed the identification of a phosphoethanolamine (pEtN)-modified variant of the N-glycan that was attached to multiple proteins. The pEtN moiety was attached to the terminal GalNAc of the canonical N-glycan. Deletion of the pEtN transferase eptC removed all evidence of the p......, yet above background levels of pEtN-glycan were also observed in E. coli not expressing eptC, suggesting that endogenous E. coli pEtN transferases can mediate the addition of pEtN to N-glycans. The addition of pEtN must be considered in the context of glycoengineering and may alter C. jejuni glycan...
-
Uchida, Kenzo; Nakajima, Hideaki; Hirai, Takayuki; Yayama, Takafumi; Chen, Kebing; Guerrero, Alexander Rodriguez; Johnson, William Eustace; Baba, Hisatoshi
2012-12-15
The twy/twy mouse undergoes spontaneous chronic mechanical compression of the spinal cord; this in vivo model system was used to examine the effects of retrograde adenovirus (adenoviral vector [AdV])-mediated brain-derived neurotrophic factor (BDNF) gene delivery to spinal neural cells. To investigate the targeting and potential neuroprotective effect of retrograde AdV-mediated BDNF gene transfection in the chronically compressed spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. Several studies have investigated the neuroprotective effects of neurotrophins, including BDNF, in spinal cord injury. However, no report has described the effects of retrograde neurotrophic factor gene delivery in compressed spinal cords, including gene targeting and the potential to prevent neural cell apoptosis. AdV-BDNF or AdV-LacZ (as a control gene) was injected into the bilateral sternomastoid muscles of 18-week old twy/twy mice for retrograde gene delivery via the spinal accessory motor neurons. Heterozygous Institute of Cancer Research mice (+/twy), which do not undergo spontaneous spinal compression, were used as a control for the effects of such compression on gene delivery. The localization and cell specificity of β-galactosidase expression (produced by LacZ gene transfection) and BDNF expression in the spinal cord were examined by coimmunofluorescence staining for neural cell markers (NeuN, neurons; reactive immunology protein, oligodendrocytes; glial fibrillary acidic protein, astrocytes; OX-42, microglia) 4 weeks after gene injection. The possible neuroprotection afforded by retrograde AdV-BDNF gene delivery versus AdV-LacZ-transfected control mice was assessed by scoring the prevalence of apoptotic cells (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells) and immunoreactivity to active caspases -3, -8, and -9, p75, neurofilament 200 kD (NF), and for the oligodendroglial progenitor marker, NG2. RESULTS
-
Pettersson, Par L; Johansson, Ann-Sofie; Mannervik, Bengt
2002-08-16
A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.
-
Bennett, M W; O'connell, J; O'sullivan, G C; Roche, D; Brady, C; Kelly, J; Collins, J K; Shanahan, F
1999-02-01
Despite being immunogenic, gastric cancers overcome antitumour immune responses by mechanisms that have yet to be fully elucidated. Fas ligand (FasL) is a molecule that induces Fas receptor mediated apoptosis of activated immunocytes, thereby mediating normal immune downregulatory roles including immune response termination, tolerance acquisition, and immune privilege. Colon cancer cell lines have previously been shown to express FasL and kill lymphoid cells by Fas mediated apoptosis in vitro. Many diverse tumours have since been found to express FasL suggesting that a "Fas counterattack" against antitumour immune effector cells may contribute to tumour immune escape. To ascertain if human gastric tumours express FasL in vivo, as a potential mediator of immune escape in stomach cancer. Thirty paraffin wax embedded human gastric adenocarcinomas. FasL protein was detected in gastric tumours using immunohistochemistry; FasL mRNA was detected in the tumours using in situ hybridisation. Cell death was detected in situ in tumour infiltrating lymphocytes using terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). Prevalent expression of FasL was detected in all 30 resected gastric adenocarcinomas examined. In the tumours, FasL protein and mRNA were co-localised to neoplastic gastric epithelial cells, confirming expression by the tumour cells. FasL expression was independent of tumour stage, suggesting that it may be expressed throughout gastric cancer progression. TUNEL staining disclosed a high level of cell death among lymphocytes infiltrating FasL positive areas of tumour. Human gastric adenocarcinomas express the immune downregulatory molecule, FasL. The results suggest that FasL is a prevalent mediator of immune privilege in stomach cancer.
-
LENUS (Irish Health Repository)
Bennett, M W
2012-02-03
BACKGROUND: Despite being immunogenic, gastric cancers overcome antitumour immune responses by mechanisms that have yet to be fully elucidated. Fas ligand (FasL) is a molecule that induces Fas receptor mediated apoptosis of activated immunocytes, thereby mediating normal immune downregulatory roles including immune response termination, tolerance acquisition, and immune privilege. Colon cancer cell lines have previously been shown to express FasL and kill lymphoid cells by Fas mediated apoptosis in vitro. Many diverse tumours have since been found to express FasL suggesting that a "Fas counterattack" against antitumour immune effector cells may contribute to tumour immune escape. AIM: To ascertain if human gastric tumours express FasL in vivo, as a potential mediator of immune escape in stomach cancer. SPECIMENS: Thirty paraffin wax embedded human gastric adenocarcinomas. METHODS: FasL protein was detected in gastric tumours using immunohistochemistry; FasL mRNA was detected in the tumours using in situ hybridisation. Cell death was detected in situ in tumour infiltrating lymphocytes using terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). RESULTS: Prevalent expression of FasL was detected in all 30 resected gastric adenocarcinomas examined. In the tumours, FasL protein and mRNA were co-localised to neoplastic gastric epithelial cells, confirming expression by the tumour cells. FasL expression was independent of tumour stage, suggesting that it may be expressed throughout gastric cancer progression. TUNEL staining disclosed a high level of cell death among lymphocytes infiltrating FasL positive areas of tumour. CONCLUSIONS: Human gastric adenocarcinomas express the immune downregulatory molecule, FasL. The results suggest that FasL is a prevalent mediator of immune privilege in stomach cancer.
-
Lectin Domains of Polypeptide GalNAc Transferases Exhibit Glycopeptide Binding Specificity
DEFF Research Database (Denmark)
Pedersen, Johannes W; Bennett, Eric P; Schjoldager, Katrine T-B G
2011-01-01
UDP-GalNAc:polypeptide a-N-acetylgalactosaminyltransferases (GalNAc-Ts) constitute a family of up to 20 transferases that initiate mucin-type O-glycosylation. The transferases are structurally composed of catalytic and lectin domains. Two modes have been identified for the selection...... of glycosylation sites by GalNAc-Ts: confined sequence recognition by the catalytic domain alone, and concerted recognition of acceptor sites and adjacent GalNAc-glycosylated sites by the catalytic and lectin domains, respectively. Thus far, only the catalytic domain has been shown to have peptide sequence...... on sequences of mucins MUC1, MUC2, MUC4, MUC5AC, MUC6, and MUC7 as well as a random glycopeptide bead library, we examined the binding properties of four different lectin domains. The lectin domains of GalNAc-T1, -T2, -T3, and -T4 bound different subsets of small glycopeptides. These results indicate...
-
Development of isoform-specific sensors of polypeptide GalNAc-transferase activity
DEFF Research Database (Denmark)
Song, Lina; Bachert, Collin; Schjoldager, Katrine T
2014-01-01
sequence influenced their activity and required modification, which we carried out based on previous in vitro work. Significantly, the modified T2 and T3 sensors were activated only in cells lacking their corresponding isozymes. Thus, we have developed T2- and T3-specific sensors that will be valuable......Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms....... Surprisingly, molecular probes to monitor GalNAc-transferase activity are lacking and there exist no effective global or isoform-specific inhibitors. Here we describe the development of T2- and T3-isoform specific fluorescence sensors that traffic in the secretory pathway. Each sensor yielded little signal...
-
Poulsen, S M; Karlsson, M; Johansson, L B; Vester, B
2001-09-01
The pleuromutilin antibiotic derivatives, tiamulin and valnemulin, inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria. The action and binding site of tiamulin and valnemulin was further characterized on Escherichia coli ribosomes. It was revealed that these drugs are strong inhibitors of peptidyl transferase and interact with domain V of 23S RNA, giving clear chemical footprints at nucleotides A2058-9, U2506 and U2584-5. Most of these nucleotides are highly conserved phylogenetically and functionally important, and all of them are at or near the peptidyl transferase centre and have been associated with binding of several antibiotics. Competitive footprinting shows that tiamulin and valnemulin can bind concurrently with the macrolide erythromycin but compete with the macrolide carbomycin, which is a peptidyl transferase inhibitor. We infer from these and previous results that tiamulin and valnemulin interact with the rRNA in the peptidyl transferase slot on the ribosomes in which they prevent the correct positioning of the CCA-ends of tRNAs for peptide transfer.
-
Directory of Open Access Journals (Sweden)
Ting-Ting Xiao
Full Text Available Accumulating evidence indicates that oxymatrine (OMT possesses variously pharmacological properties, especially on the cardiovascular system. We previously demonstrated that activated calpain/apoptosis-inducing factor (AIF-mediated pathway was the key molecular mechanism in aldosterone (ALD induces cardiomyocytes apoptosis. In the present study, we extended the experimentation by investigating the effect of OMT on cardiomyocytes exposed to ALD, as compared to spironolactone (Spiro, a classical ALD receptor antagonist. Cardiomyocytes were pre-incubated with OMT, Spiro or vehicle for 1 h, and then, cardiomyocytes were exposed to ALD 24 h. The cell injury was evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and lactate dehydrogenase (LDH leakage ratio. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay, annexin V/PI staining, and relative caspase-3 activity assay. Furthermore, expression of pro-apoptotic proteins including truncated Bid (tBid, calpain and AIF were evaluated by western blot analysis. ALD stimulation increased cardiomyocytes apoptosis, caspase-3 activity and protein expression of calpain, tBid and AIF in the cytosol (p<0.05. Pre-incubated with cardiomyocytes injury and increased caspase-3 activity were significantly attenuated (p<0.05. Furthermore, OMT suppressed ALD-induced high expression of calpain and AIF. And these effects of OMT could be comparable to Spiro. These findings indicated that OMT might be a potential cardioprotective-agent against excessive ALD-induced cardiotoxicity, at least in part, mediated through inhibition of calpain/AIF signaling.
-
A novel plant glutathione S-transferase/peroxidase suppresses Bax lethality in yeast
DEFF Research Database (Denmark)
Kampranis, S C; Damianova, R; Atallah, M
2000-01-01
The mammalian inducer of apoptosis Bax is lethal when expressed in yeast and plant cells. To identify potential inhibitors of Bax in plants we transformed yeast cells expressing Bax with a tomato cDNA library and we selected for cells surviving after the induction of Bax. This genetic screen allows...... for the identification of plant genes, which inhibit either directly or indirectly the lethal phenotype of Bax. Using this method a number of cDNA clones were isolated, the more potent of which encodes a protein homologous to the class theta glutathione S-transferases. This Bax-inhibiting (BI) protein was expressed...... in Escherichia coli and found to possess glutathione S-transferase (GST) and weak glutathione peroxidase (GPX) activity. Expression of Bax in yeast decreases the intracellular levels of total glutathione, causes a substantial reduction of total cellular phospholipids, diminishes the mitochondrial membrane...
-
Mori, Yuki; Terauchi, Ryu; Shirai, Toshiharu; Tsuchida, Shinji; Mizoshiri, Naoki; Arai, Yuji; Kishida, Tsunao; Fujiwara, Hiroyoshi; Mazda, Osam; Kubo, Toshikazu
2017-09-01
Although advances in chemotherapy have improved the prognosis for osteosarcoma, some patients do not respond sufficiently to treatment. Heat shock protein 70 (Hsp70) is expressed at high levels in cancer cells and attenuates the therapeutic efficacy of anticancer agents, resulting in a poorer prognosis. This study investigated whether small interfering RNA (siRNA)-mediated inhibition of Hsp70 expression in an osteosarcoma cell line would enhance sensitivity to cisplatin. The expression of Hsp70 with cisplatin treatment was observed by using Western blotting and real-time reverse transcription polymerase chain reaction (RT-PCR). Changes in the IC 50 of cisplatin when Hsp70 was inhibited by siRNA were evaluated. Cisplatin's effectiveness in inducing apoptosis was assessed by assay of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), caspase-3 activity, and mitochondrial membrane potential. Up-regulation of Hsp70 expression was dependent on the concentration of cisplatin. Inhibition of Hsp70 expression significantly reduced the IC 50 of cisplatin. When cisplatin was added to osteosarcoma cells with Hsp70 expression inhibited, a significant increase in apoptosis was demonstrated in TUNEL, caspase-3, and mitochondrial membrane potential assays. Inhibition of Hsp70 expression induced apoptosis in cultured osteosarcoma cells, indicating that Hsp70 inhibition enhanced sensitivity to cisplatin. Inhibition of Hsp70 expression may provide a new adjuvant therapy for osteosarcoma.
-
Inhibitory effects of CP on the growth of human gastric adenocarcinoma BGC-823 tumours in nude mice.
Wang, Hai-Jun; Liu, Yu; Zhou, Bao-Jun; Zhang, Zhan-Xue; Li, Ai-Ying; An, Ran; Yue, Bin; Fan, Li-Qiao; Li, Yong
2018-05-01
Objective To investigate the potential antitumour effects of [2-(6-amino-purine-9-yl)-1-hydroxy-phosphine acyl ethyl] phosphonic acid (CP) against gastric adenocarcinoma. Methods Human BGC-823 xenotransplants were established in nude mice. Animals were randomly divided into control and CP groups, which were administered NaHCO 3 vehicle alone or CP dissolved in NaHCO 3 (200 µg/kg body weight) daily, respectively. Tumour volume was measured weekly for 6 weeks. Resected tumours were assayed for proliferative activity with anti-Ki-67 or anti-proliferating cell nuclear antigen (PCNA) antibodies. Cell apoptosis was examined using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assays and with caspase-3 immunostaining. Proteins were measured by Western blotting. Results There was a significant reduction in tumour volume and a reduced percentage of Ki-67-positive or PCNA-positive cells in the CP group compared with the control group. The percentage of TUNEL-positive or caspase 3-positive cells significantly increased following CP treatment compared with the control group. Tumours from the CP group had higher levels of phosphorylated-extracellular signal-regulated kinase (p-ERK) and phosphorylated-AKT (p-AKT) compared with control tumours. Conclusion CP treatment inhibited tumour growth and induced tumour cell apoptosis in a nude mouse model of BGC-823 gastric adenocarcinoma. Activation of the AKT and ERK signalling pathways may mediate this antitumour activity.
-
Human glutathione S-transferase P1-1 functions as an estrogen receptor α signaling modulator
Energy Technology Data Exchange (ETDEWEB)
Liu, Xiyuan [Department of Biological Science, Sookmyung Women’s University, Seoul (Korea, Republic of); An, Byoung Ha [Department of Food and Nutrition, College of Life Science, Sookmyung Women’s University, Seoul (Korea, Republic of); Kim, Min Jung; Park, Jong Hoon [Department of Biological Science, Sookmyung Women’s University, Seoul (Korea, Republic of); Kang, Young Sook [Department of Pharmacy, College of Pharmacy, Sookmyung Women’s University, Seoul (Korea, Republic of); Chang, Minsun, E-mail: minsunchang@sm.ac.kr [Department of Medical and Pharmaceutical Science, College of Science, Sookmyung Women’s University, Seoul (Korea, Republic of)
2014-09-26
Highlights: • GSTP induces the classical ERα signaling event. • The functional GSTP is a prerequisite for GSTP-induced ERα transcription activity. • The expression of RIP140, a transcription cofactor, was inhibited by GSTP protein. • We propose the novel non-enzymatic role of GSTP. - Abstract: Estrogen receptor α (ERα) plays a crucial role in estrogen-mediated signaling pathways and exerts its action as a nuclear transcription factor. Binding of the ligand-activated ERα to the estrogen response element (ERE) is a central part of ERα-associated signal transduction pathways and its aberrant modulation is associated with many disease conditions. Human glutathione S-transferase P1-1 (GSTP) functions as an enzyme in conjugation reactions in drug metabolism and as a regulator of kinase signaling pathways. It is overexpressed in tumors following chemotherapy and has been associated with a poor prognosis in breast cancer. In this study, a novel regulatory function of GSTP has been proposed in which GSTP modulates ERE-mediated ERα signaling events. Ectopic expression of GSTP was able to induce the ERα and ERE-mediated transcriptional activities in ERα-positive but GSTP-negative MCF7 human breast cancer cells. This inductive effect of GSTP on the ERE-transcription activity was diminished when the cells express a mutated form of the enzyme or are treated with a GSTP-specific chemical inhibitor. It was found that GSTP inhibited the expression of the receptor interacting protein 140 (RIP140), a negative regulator of ERα transcription, at both mRNA and protein levels. Our study suggests a novel non-enzymatic role of GSTP which plays a significant role in regulating the classical ERα signaling pathways via modification of transcription cofactors such as RIP140.
-
International Nuclear Information System (INIS)
Torres, Rodrigo; Lan, Benson; Latif, Yama; Chim, Nicholas; Goulding, Celia W.
2014-01-01
The crystal structures of Y. pestis RipA mutants were determined to provide insights into the CoA transferase reaction pathway. Yersinia pestis, the causative agent of bubonic plague, is able to survive in both extracellular and intracellular environments within the human host, although its intracellular survival within macrophages is poorly understood. A novel Y. pestis three-gene rip (required for intracellular proliferation) operon, and in particular ripA, has been shown to be essential for survival and replication in interferon γ-induced macrophages. RipA was previously characterized as a putative butyryl-CoA transferase proposed to yield butyrate, a known anti-inflammatory shown to lower macrophage-produced NO levels. RipA belongs to the family I CoA transferases, which share structural homology, a conserved catalytic glutamate which forms a covalent CoA-thioester intermediate and a flexible loop adjacent to the active site known as the G(V/I)G loop. Here, functional and structural analyses of several RipA mutants are presented in an effort to dissect the CoA transferase mechanism of RipA. In particular, E61V, M31G and F60M RipA mutants show increased butyryl-CoA transferase activities when compared with wild-type RipA. Furthermore, the X-ray crystal structures of E61V, M31G and F60M RipA mutants, when compared with the wild-type RipA structure, reveal important conformational changes orchestrated by a conserved acyl-group binding-pocket phenylalanine, Phe85, and the G(V/I)G loop. Binary structures of M31G RipA and F60M RipA with two distinct CoA substrate conformations are also presented. Taken together, these data provide CoA transferase reaction snapshots of an open apo RipA, a closed glutamyl-anhydride intermediate and an open CoA-thioester intermediate. Furthermore, biochemical analyses support essential roles for both the catalytic glutamate and the flexible G(V/I)G loop along the reaction pathway, although further research is required to fully
-
Energy Technology Data Exchange (ETDEWEB)
Torres, Rodrigo; Lan, Benson; Latif, Yama; Chim, Nicholas [UC Irvine, 2212 Natural Sciences I, Irvine, CA 92697 (United States); Goulding, Celia W., E-mail: celia.goulding@uci.edu [UC Irvine, 2212 Natural Sciences I, Irvine, CA 92697 (United States); UC Irvine, 2302 Natural Sciences I, Irvine, CA 92697 (United States)
2014-04-01
The crystal structures of Y. pestis RipA mutants were determined to provide insights into the CoA transferase reaction pathway. Yersinia pestis, the causative agent of bubonic plague, is able to survive in both extracellular and intracellular environments within the human host, although its intracellular survival within macrophages is poorly understood. A novel Y. pestis three-gene rip (required for intracellular proliferation) operon, and in particular ripA, has been shown to be essential for survival and replication in interferon γ-induced macrophages. RipA was previously characterized as a putative butyryl-CoA transferase proposed to yield butyrate, a known anti-inflammatory shown to lower macrophage-produced NO levels. RipA belongs to the family I CoA transferases, which share structural homology, a conserved catalytic glutamate which forms a covalent CoA-thioester intermediate and a flexible loop adjacent to the active site known as the G(V/I)G loop. Here, functional and structural analyses of several RipA mutants are presented in an effort to dissect the CoA transferase mechanism of RipA. In particular, E61V, M31G and F60M RipA mutants show increased butyryl-CoA transferase activities when compared with wild-type RipA. Furthermore, the X-ray crystal structures of E61V, M31G and F60M RipA mutants, when compared with the wild-type RipA structure, reveal important conformational changes orchestrated by a conserved acyl-group binding-pocket phenylalanine, Phe85, and the G(V/I)G loop. Binary structures of M31G RipA and F60M RipA with two distinct CoA substrate conformations are also presented. Taken together, these data provide CoA transferase reaction snapshots of an open apo RipA, a closed glutamyl-anhydride intermediate and an open CoA-thioester intermediate. Furthermore, biochemical analyses support essential roles for both the catalytic glutamate and the flexible G(V/I)G loop along the reaction pathway, although further research is required to fully
-
Rubila, Sundararaj; Ranganathan, Thottiam Vasudevan; Sakthivel, Kunnathur Murugesan
2016-12-01
The aim of the present investigation was to evaluate Zingiber officinale paste against Dalton's lymphoma ascites (DLA)-induced tumours in Swiss albino mice. Experimental animals received Z. officinale paste (low dose 100 mg/kg bw and high dose 500 mg/kg bw) orally for eight alternative days. Treatment with Z. officinale paste showed significant increase in haemoglobin level and decrease in aspartate amino transferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma glutamyl transferase (γ-GT) level. Z. officinale paste reduced the inflammatory mediators and cytokine levels, such as inducible nitric oxide (iNOS), tumour necrosis factor level (TNF-α) and interleukin-1β (IL-1β). Treatment with Z. officinale paste also significantly increased the antioxidant enzyme level, such as superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and glutathione transferase (GST), and decreased the lipid peroxidation. Treatment also increased the vitamin C and E levels in treated animals compared with the DLA-bearing host. Histopathological studies also confirmed the protective influence of Z. officinale paste against DLA. The present study suggested that Z. officinale paste could be used as natural spice and a potent antitumour agent.
-
Hussar, Piret; Tokin, Ivan; Hussar, Ulo; Filimonova, Galina; Suuroja, Toivo
2006-01-01
The aim of the present study was to determine the target site cells in the rat thymus after exposure to the synthetic glucocorticoid, dexamethasone, at therapeutic doses. The findings of histology and histochemistry (Feulgen, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling--TUNEL) with quantification by computerized histomorphometry are described. A quantified investigation of apoptotic and mitotic thymic lymphocytes in 36 young adult Wistar rats was performed at 1-7 days after a 3-day injection of dexamethasone (a total dose of 1.2 mg/rat intraperitoneally). At the first day after dexamethasone administration the moderate involution and atrophy of thymus histology were observed with simultaneous fall in cortical cellularity and mitotic activity of thymocytes. More rapid fall appeared in the inner cortex. The number of apoptotic (TUNEL-positive) cells was significantly increased. On the days 5 and 7 the expression of apoptosis and the cell proliferation were at almost normal level. The findings suggest that dexamethasone-induced apoptosis of cortical thymic lymphocytes, mainly correlated with synchronous inhibition of mitosis and cell number fall in thymus. The main target sites of dexamethasone injury were cells in the inner cortex of lobuli thymi.
-
Expression of caspase-3 predicts prognosis in advanced noncardia gastric cancer.
Amptoulach, Sousana; Lazaris, Andreas C; Giannopoulou, Ioanna; Kavantzas, Nikolaos; Patsouris, Efstratios; Tsavaris, Nikolaos
2015-01-01
There is strong evidence that tumor growth is not only a result of uncontrolled cell proliferation but also of decreased apoptosis. Caspase-3 is a member of interleukin-1 beta-converting enzyme which is involved in the induction of apoptosis. Data on the expression of caspase-3 in patients with gastric cancer and its association with patient outcome are somewhat contradictory. We aimed to investigate the potential relation of the expression of caspase-3 protein with response to therapy and overall survival in patients with advanced noncardia gastric cancer. Tumor tissue samples collected from 359 consecutive patients with gastric cancer stage IV were retrospectively analyzed for the expression of caspase-3 in the primary tumor. The DNA apoptotic index assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling method. All patients were followed up until death. Caspase-3 was expressed in 43.5 % of tumors. Caspase-3 expression compared to no expression was related with a higher DNA apoptotic index (p gastric cancer is a predictor of poor response to treatment and survival. Further studies are needed to fully elucidate the prognostic value of caspase-3 expression in these patients.
-
International Nuclear Information System (INIS)
Li, D.X.; Deng, T.Z.; Lv, J.; Ke, J.
2014-01-01
Diabetics have an increased prevalence of periodontitis, and diabetes is one of the causative factors of severe periodontitis. Apoptosis is thought to be involved in this pathogenic relationship. The aim of this study was to investigate apoptosis in human periodontal ligament (PDL) fibroblasts induced by advanced glycation end products (AGEs) and their receptor (RAGE). We examined the roles of apoptosis, AGEs, and RAGE during periodontitis in diabetes mellitus using cultured PDL fibroblasts that were treated by AGE-modified bovine serum albumin (AGE-BSA), bovine serum albumin (BSA) alone, or given no treatment (control). Microscopy and real-time quantitative PCR indicated that PDL fibroblasts treated with AGE-BSA were deformed and expressed higher levels of RAGE and caspase 3. Cell viability assays and flow cytometry indicated that AGE-BSA reduced cell viability (69.80±5.50%, P<0.01) and increased apoptosis (11.31±1.73%, P<0.05). Hoechst 33258 staining and terminal-deoxynucleotidyl transferase-mediated nick-end labeling revealed that AGE-BSA significantly increased apoptosis of PDL fibroblasts. The results showed that the changes in PDL fibroblasts induced by AGE-BSA may explain how AGE-RAGE participates in and exacerbates periodontium destruction
-
Ruvolo, Giovanni; Roccheri, Maria Carmela; Brucculeri, Anna Maria; Longobardi, Salvatore; Cittadini, Ettore; Bosco, Liana
2013-04-01
An observational clinical and molecular study was designed to evaluate the effects of the administration of recombinant human FSH on sperm DNA fragmentation in men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In the study were included 53 men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In all patients, sperm DNA fragmentation index (DFI), assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) in situ DNA nick end-labelling (TUNEL) assay, was evaluated before starting the treatment with 150 IU of recombinant human FSH, given three times a week for at least 3 months. Patients' semen analysis and DNA fragmentation index were re-evaluated after the 3-month treatment period. After recombinant human FSH therapy, we did not find any differences in terms of sperm count, motility and morphology. The average DNA fragmentation index was significantly reduced (21.15 vs 15.2, p15 %), while no significant variation occurred in the patients with DFI values ≤ 15 %. Recombinant human FSH administration improves sperm DNA integrity in hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia men with DNA fragmentation index value >15 % .
-
Institute of Scientific and Technical Information of China (English)
洪军; 崔建忠; 周云涛; 高俊玲
2002-01-01
Objective: To explore the correlation between cognition disorder and morphologic change of hippocampal neurons after traumatic brain injury (TBI). Methods: Wistar rat models with severe TBI were made by Marmarous method. The histopathological change of the neurons in the hippocampus area were studied with hematoxylin-eosin (HE) staining and terminal deoxynucleotidyl transferase-mediated X-dUPT nick end labeling (TUNEL), respectively. The cognitive function was evaluated with the Morris water maze test. Results: The comprehensive neuronal degeneration and necrosis could be observed in CA2-3 regions of hippocampus at 3 days after injury. Apoptotic positive neurons in CA2-4 regions of hippocampus and dentate gyrus increased in the injured group at 24 hours following TBI. They peaked at 7 days and then declined. Significant impairment of spatial learning and memory was observed after injury in the rats. Conclusions: The rats have obvious disorders in spatial learning and memory after severe TBI. Meanwhile, delayed neuronal necrosis and apoptosis can be observed in the neurons in the hippocampus area. It suggests that delayed hippocampal cell death may contribute to the functional deficit.
-
Chlorella vulgaris triggers apoptosis in hepatocarcinogenesis-induced rats*
Mohd Azamai, Emey Suhana; Sulaiman, Suhaniza; Mohd Habib, Shafina Hanim; Looi, Mee Lee; Das, Srijit; Abdul Hamid, Nor Aini; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum
2009-01-01
Chlorella vulgaris (CV) has been reported to have antioxidant and anticancer properties. We evaluated the effect of CV on apoptotic regulator protein expression in liver cancer-induced rats. Male Wistar rats (200~250 g) were divided into eight groups: control group (normal diet), CDE group (choline deficient diet supplemented with ethionine in drinking water to induce hepatocarcinogenesis), CV groups with three different doses of CV (50, 150, and 300 mg/kg body weight), and CDE groups treated with different doses of CV (50, 150, and 300 mg/kg body weight). Rats were sacrificed at various weeks and liver tissues were embedded in paraffin blocks for immunohistochemistry studies. CV, at increasing doses, decreased the expression of anti-apoptotic protein, Bcl-2, but increased the expression of pro-apoptotic protein, caspase 8, in CDE rats, which was correlated with decreased hepatoctyes proliferation and increased apoptosis as determined by bromodeoxy-uridine (BrdU) labeling and terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assay, respectively. Our study shows that CV has definite chemopreventive effect by inducing apoptosis via decreasing the expression of Bcl-2 and increasing the expression of caspase 8 in hepatocarcinogenesis-induced rats. PMID:19198018
-
Energy Technology Data Exchange (ETDEWEB)
Li, D.X.; Deng, T.Z.; Lv, J.; Ke, J. [Department of Stomatology, Air Force General Hospital PLA, Haidian District, Beijing (China)
2014-09-19
Diabetics have an increased prevalence of periodontitis, and diabetes is one of the causative factors of severe periodontitis. Apoptosis is thought to be involved in this pathogenic relationship. The aim of this study was to investigate apoptosis in human periodontal ligament (PDL) fibroblasts induced by advanced glycation end products (AGEs) and their receptor (RAGE). We examined the roles of apoptosis, AGEs, and RAGE during periodontitis in diabetes mellitus using cultured PDL fibroblasts that were treated by AGE-modified bovine serum albumin (AGE-BSA), bovine serum albumin (BSA) alone, or given no treatment (control). Microscopy and real-time quantitative PCR indicated that PDL fibroblasts treated with AGE-BSA were deformed and expressed higher levels of RAGE and caspase 3. Cell viability assays and flow cytometry indicated that AGE-BSA reduced cell viability (69.80±5.50%, P<0.01) and increased apoptosis (11.31±1.73%, P<0.05). Hoechst 33258 staining and terminal-deoxynucleotidyl transferase-mediated nick-end labeling revealed that AGE-BSA significantly increased apoptosis of PDL fibroblasts. The results showed that the changes in PDL fibroblasts induced by AGE-BSA may explain how AGE-RAGE participates in and exacerbates periodontium destruction.
-
Energy Technology Data Exchange (ETDEWEB)
Kitamura, Makiko; Itoh, Kyoko; Matsumoto, Akira; Hayashi, Yoshitake; Sasaki, Ryohei; Imai, Yukihiro; Itoh, Hiroshi [Kobe Univ. (Japan). School of Medicine
2001-04-01
Apoptosis induced by ionizing irradiation of the developing mouse brain was investigated by using histology, analysis of DNA fragmentation on agarose gel and electron microscopy. A TUNEL-labeled index (L.I.) was calculated from the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay in 4 specific regions, cortical plate, intermediate zone, subependymal zone, and subependymal germinal matrix. The kinetics of apoptosis associated protein was examined by western blotting and immunofluorescence. C57BL/6J mice pregnant on embryonic day 14 (E14) were exposed to a single dose of 1.5-Gy irradiation. Irradiaited fetal brains at E15 and E17 showed extensive apoptosis with morphological characteristics. In all 4 regions, L.I. was greater in irradiated brains than in control brains at E15 and E17. Most of TUNEL-labeled cells expressed a mature neuronal marker (NeuN) and Bax protein, which is up-regulated in irradiation-induced apoptosis. Ionizing radiation moderately enhanced expression of Bax, Bcl-xL, and Cpp32 proteins. Postnatal irradiated mice showed microencephaly as compared to age-matched mice and the weight of whole body including brain decreased moderately. (author)
-
International Nuclear Information System (INIS)
Kitamura, Makiko; Itoh, Kyoko; Matsumoto, Akira; Hayashi, Yoshitake; Sasaki, Ryohei; Imai, Yukihiro; Itoh, Hiroshi
2001-01-01
Apoptosis induced by ionizing irradiation of the developing mouse brain was investigated by using histology, analysis of DNA fragmentation on agarose gel and electron microscopy. A TUNEL-labeled index (L.I.) was calculated from the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay in 4 specific regions, cortical plate, intermediate zone, subependymal zone, and subependymal germinal matrix. The kinetics of apoptosis associated protein was examined by western blotting and immunofluorescence. C57BL/6J mice pregnant on embryonic day 14 (E14) were exposed to a single dose of 1.5-Gy irradiation. Irradiated fetal brains at E15 and E17 showed extensive apoptosis with morphological characteristics. In all 4 regions, L.I. was greater in irradiated brains than in control brains at E15 and E17. Most of TUNEL-labeled cells expressed a mature neuronal marker (NeuN) and Bax protein, which is up-regulated in irradiation-induced apoptosis. Ionizing radiation moderately enhanced expression of Bax, Bcl-xL, and Cpp32 proteins. Postnatal irradiated mice showed microencephaly as compared to age-matched mice and the weight of whole body including brain decreased moderately. (author)
-
Directory of Open Access Journals (Sweden)
Ruei-Tang Cheng
2010-01-01
Full Text Available When the vehicle-treated, sham-operated mice underwent heat stress, the fraction survival and core temperature at +4 h of body heating were found to be 5 of 15 and 34.4∘C±0.3∘C, respectively. Castration 2 weeks before the start of heat stress decreased the plasma levels of testosterone almost to zero, protected the mice from heat-induced death (fraction survival, 13/15 and reduced the hypothermia (core temperature, 37.3∘C. The beneficial effects of castration in ameliorating lethality and hypothermia can be significantly reduced by testosterone replacement. Heat-induced apoptosis, as indicated by terminal deoxynucleotidyl- transferase- mediatedαUDP-biotin nick end-labeling staining, were significantly prevented by castration. In addition, heat-induced neuronal damage, as indicated by cell shrinkage and pyknosis of nucleus, to the hypothalamus was also castration-prevented. Again, the beneficial effects of castration in reducing neuronal damage to the hypothalamus as well as apoptosis in multiple organs during heatstroke, were significantly reversed by testosterone replacement. The data indicate that testosterone depletion by castration may protect mice from heatstroke-induced multiple organ damage and lethality.
-
Genomic organization of plant aminopropyl transferases.
Rodríguez-Kessler, Margarita; Delgado-Sánchez, Pablo; Rodríguez-Kessler, Gabriela Theresia; Moriguchi, Takaya; Jiménez-Bremont, Juan Francisco
2010-07-01
Aminopropyl transferases like spermidine synthase (SPDS; EC 2.5.1.16), spermine synthase and thermospermine synthase (SPMS, tSPMS; EC 2.5.1.22) belong to a class of widely distributed enzymes that use decarboxylated S-adenosylmethionine as an aminopropyl donor and putrescine or spermidine as an amino acceptor to form in that order spermidine, spermine or thermospermine. We describe the analysis of plant genomic sequences encoding SPDS, SPMS, tSPMS and PMT (putrescine N-methyltransferase; EC 2.1.1.53). Genome organization (including exon size, gain and loss, as well as intron number, size, loss, retention, placement and phase, and the presence of transposons) of plant aminopropyl transferase genes were compared between the genomic sequences of SPDS, SPMS and tSPMS from Zea mays, Oryza sativa, Malus x domestica, Populus trichocarpa, Arabidopsis thaliana and Physcomitrella patens. In addition, the genomic organization of plant PMT genes, proposed to be derived from SPDS during the evolution of alkaloid metabolism, is illustrated. Herein, a particular conservation and arrangement of exon and intron sequences between plant SPDS, SPMS and PMT genes that clearly differs with that of ACL5 genes, is shown. The possible acquisition of the plant SPMS exon II and, in particular exon XI in the monocot SPMS genes, is a remarkable feature that allows their differentiation from SPDS genes. In accordance with our in silico analysis, functional complementation experiments of the maize ZmSPMS1 enzyme (previously considered to be SPDS) in yeast demonstrated its spermine synthase activity. Another significant aspect is the conservation of intron sequences among SPDS and PMT paralogs. In addition the existence of microsynteny among some SPDS paralogs, especially in P. trichocarpa and A. thaliana, supports duplication events of plant SPDS genes. Based in our analysis, we hypothesize that SPMS genes appeared with the divergence of vascular plants by a processes of gene duplication and the
-
International Nuclear Information System (INIS)
Edwards, Thomas E.; Bryan, Cassie M.; Leibly, David J.; Dieterich, Shellie H.; Abendroth, Jan; Sankaran, Banumathi; Sivam, Dhileep; Staker, Bart L.; Van Voorhis, Wesley C.; Myler, Peter J.; Stewart, Lance J.
2011-01-01
The pathogenic fungus C. immitis causes coccidioidomycosis, a potentially fatal disease. Here, apo and glutathione-bound crystal structures of a previously uncharacterized protein from C. immitis that appears to be a ζ-class glutathione S-transferase are presented. Coccidioides immitis is a pathogenic fungus populating the southwestern United States and is a causative agent of coccidioidomycosis, sometimes referred to as Valley Fever. Although the genome of this fungus has been sequenced, many operons are not properly annotated. Crystal structures are presented for a putative uncharacterized protein that shares sequence similarity with ζ-class glutathione S-transferases (GSTs) in both apo and glutathione-bound forms. The apo structure reveals a nonsymmetric homodimer with each protomer comprising two subdomains: a C-terminal helical domain and an N-terminal thioredoxin-like domain that is common to all GSTs. Half-site binding is observed in the glutathione-bound form. Considerable movement of some components of the active site relative to the glutathione-free form was observed, indicating an induced-fit mechanism for cofactor binding. The sequence homology, structure and half-site occupancy imply that the protein is a ζ-class glutathione S-transferase, a maleylacetoacetate isomerase (MAAI)
-
MGST2 and WNT2 are candidate genes for comitant strabismus susceptibility in Japanese patients
Directory of Open Access Journals (Sweden)
Jingjing Zhang
2017-10-01
Full Text Available Background/Aim Strabismus is a common condition with misalignment between two eyes that may lead to decrease of visual acuity, lack of binocularity, and diplopia. It is caused by heterogeneous environmental and genetic risk factors. Our previous research has identified new chromosomal susceptibility loci in 4q28.3 and 7q31.2 regions for comitant strabismus in Japanese families. We conducted a verification study by linkage analysis to narrow the chromosomal loci down to a single gene. Methods From Japanese and U.S. databases, 24 rsSNPs and 233 rsSNPs were chosen from the 4q28.3 and 7q31.2 region, respectively, and were typed in 108 affected subjects and 96 unaffected subjects of 58 families with primary and non-syndromic comitant strabismus. Three major analytical methods were used: transmission disequilibrium test (TDT, TDT allowing for errors (TDTae, and linkage analysis under dominant and recessive inheritance. Results The SNPs with significant P values in TDT and TDTae were located solely at the gene, microsomal glutathione S-transferase 2 (MGST2, on chromosome 4q28.3 locus. In contrast, significant SNPs were dispersed in a few genes, containing wingless-type MMTV integration site family member 2 (WNT2, on chromosome 7q31.2 locus. The distribution of significant SNPs on the 7q31.2 locus showed that only the ST7 to WNT2 region in the same big haplotype block contained significant SNPs for all three methods of linkage analysis. Conclusions This study suggests that MGST2 and WNT2 are potential candidates for comitant strabismus in Japanese population.
-
Calmes, Benoit; Morel-Rouhier, Mélanie; Bataillé-Simoneau, Nelly; Gelhaye, Eric; Guillemette, Thomas; Simoneau, Philippe
2015-06-18
Glutathione transferases (GSTs) represent an extended family of multifunctional proteins involved in detoxification processes and tolerance to oxidative stress. We thus anticipated that some GSTs could play an essential role in the protection of fungal necrotrophs against plant-derived toxic metabolites and reactive oxygen species that accumulate at the host-pathogen interface during infection. Mining the genome of the necrotrophic Brassica pathogen Alternaria brassicicola for glutathione transferase revealed 23 sequences, 17 of which could be clustered into the main classes previously defined for fungal GSTs and six were 'orphans'. Five isothiocyanate-inducible GSTs from five different classes were more thoroughly investigated. Analysis of their catalytic properties revealed that two GSTs, belonging to the GSTFuA and GTT1 classes, exhibited GSH transferase activity with isothiocyanates (ITC) and peroxidase activity with cumene hydroperoxide, respectively. Mutant deficient for these two GSTs were however neither more susceptible to ITC nor less aggressive than the wild-type parental strain. By contrast mutants deficient for two other GSTs, belonging to the Ure2pB and GSTO classes, were distinguished by their hyper-susceptibility to ITC and low aggressiveness against Brassica oleracea. In particular AbGSTO1 could participate in cell tolerance to ITC due to its glutathione-dependent thioltransferase activity. The fifth ITC-inducible GST belonged to the MAPEG class and although it was not possible to produce the soluble active form of this protein in a bacterial expression system, the corresponding deficient mutant failed to develop normal symptoms on host plant tissues. Among the five ITC-inducible GSTs analyzed in this study, three were found essential for full aggressiveness of A. brassicicola on host plant. This, to our knowledge is the first evidence that GSTs might be essential virulence factors for fungal necrotrophs.
-
Ávila, Felipe; Theoduloz, Cristina; López-Alarcón, Camilo; Dorta, Eva; Schmeda-Hirschmann, Guillermo
2017-01-01
The prevalence of cytoprotective mechanisms induced by polyphenols such as activation of intracellular antioxidant responses (ICM) and direct free radical scavenging was investigated in native Chilean species of strawberries, raspberries, and currants. Human gastric epithelial cells were co- and preincubated with polyphenolic-enriched extracts (PEEs) from Chilean raspberries ( Rubus geoides ), strawberries ( Fragaria chiloensis ssp. chiloensis f . chiloensis ), and currants ( Ribes magellanicum ) and challenged with peroxyl and hydroxyl radicals. Cellular protection was determined in terms of cell viability, glyoxalase I and glutathione s-transferases activities, and carboxymethyl lysine (CML) and malondialdehyde levels. Our results indicate that cytoprotection induced by ICM was the prevalent mechanism for Rubus geoides and F. chiloensis . This agreed with increased levels of glyoxalase I and glutathione S-transferase activities in cells preincubated with PEEs. ORAC index indicated that F. chiloensis was the most efficient peroxyl radical scavenger. Moreover, ICM mediated by F. chiloensis was effective in protecting cells from CML accumulation in contrast to the protective effects induced by free radical scavenging. Our results indicate that although both polyphenol-mediated mechanisms can exert protective effects, ICM was the most prevalent in AGS cells. These results suggest a potential use of these native berries as functional food.
-
We established a mouse model of developmental nonalcoholic steatohepatitis (NASH) by feeding a high polyunsaturated fat liquid diet to female glutathione-S-transferase 4-4 (Gsta4-/-)/peroxisome proliferator activated receptor a (Ppara-/-) double knockout 129/SvJ mice for 12 weeks from weaning. We us...
-
Directory of Open Access Journals (Sweden)
Xiao-Qin Sun
Full Text Available Plants have evolved complex processes to ward off attacks by insects. In parallel, insects have evolved mechanisms to thwart these plant defenses. To gain insight into mechanisms that mediate this arms race between plants and herbivorous insects, we investigated the interactions between gramine, a toxin synthesized by plants of the family Gramineae, and glutathione S transferase (GST, an enzyme found in insects that is known to detoxify xenobiotics. Here, we demonstrate that rice (Oryza sativa, a hydrophytic plant, also produces gramine and that rice resistance to brown planthoppers (Nilaparvata lugens, BPHs is highly associated with in planta gramine content. We also show that gramine is a toxicant that causes BPH mortality in vivo and that knockdown of BPH GST gene nlgst1-1 results in increased sensitivity to diets containing gramine. These results suggest that the knockdown of key detoxification genes in sap-sucking insects may provide an avenue for increasing their sensitivity to natural plant-associated defense mechanisms.
-
Sun, Xiao-Qin; Zhang, Mao-Xin; Yu, Jing-Ya; Jin, Yu; Ling, Bing; Du, Jin-Ping; Li, Gui-Hua; Qin, Qing-Ming; Cai, Qing-Nian
2013-01-01
Plants have evolved complex processes to ward off attacks by insects. In parallel, insects have evolved mechanisms to thwart these plant defenses. To gain insight into mechanisms that mediate this arms race between plants and herbivorous insects, we investigated the interactions between gramine, a toxin synthesized by plants of the family Gramineae, and glutathione S transferase (GST), an enzyme found in insects that is known to detoxify xenobiotics. Here, we demonstrate that rice (Oryza sativa), a hydrophytic plant, also produces gramine and that rice resistance to brown planthoppers (Nilaparvata lugens, BPHs) is highly associated with in planta gramine content. We also show that gramine is a toxicant that causes BPH mortality in vivo and that knockdown of BPH GST gene nlgst1-1 results in increased sensitivity to diets containing gramine. These results suggest that the knockdown of key detoxification genes in sap-sucking insects may provide an avenue for increasing their sensitivity to natural plant-associated defense mechanisms.
-
Hyperoxygenated hydrogen-rich solution suppresses shock- and resuscitation-induced liver injury.
Dang, Yangjie; Liu, Ting; Mei, Xiaopeng; Meng, Xiangzhong; Gou, Xingchun; Deng, Bin; Xu, Hao; Xu, Lixian
2017-12-01
It is not known whether simultaneous delivery of hydrogen and oxygen can reduce injury caused by hemorrhagic shock and resuscitation (HSR). This study investigated the therapeutic potential of hyperoxygenated hydrogen-rich solution (HHOS), a combined hydrogen/oxygen carrier, in a rat model of HSR-induced liver injury. Rats (n = 60) were randomly divided into 5 groups (n = 6 per group at each time point). One group underwent sham operation, and the others were subjected to severe hemorrhagic shock and then treated with lactated Ringer's solution (LRS), hydrogen-rich solution, hyperoxygenated solution, or HHOS. At 2 and 6 h after resuscitation, blood samples (n = 6) were collected from the femoral artery and serum concentrations of alanine aminotransferase and aspartate aminotransferase (AST) were measured. Rats were then sacrificed, and histopathological changes in the liver were evaluated by quantifying the percentage of apoptotic cells by caspase-3 immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick-end labeling. Inflammation was assessed by assessing malondialdehyde content and tumor necrosis factor-α, and interleukin (IL)-6 expression. Compared to lactated Ringer's solution, hydrogen-rich solution, or hyperoxygenated solution groups, serum AST and alanine aminotransferase levels and IL-6, tumor necrosis factor-α, and malondialdehyde expression in liver tissue were decreased by HHOS treatment. The number of caspase-3- and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells was decreased (P < 0.05) by HHOS treatment, 2 and 6 h after resuscitation. HHOS has protective effects against liver injury in a rat model of HSR. Copyright © 2017 Elsevier Inc. All rights reserved.
-
Directory of Open Access Journals (Sweden)
Xiaoyan Wang
2015-04-01
Full Text Available All types of small RNAs in plants, piwi-interacting RNAs (piRNAs in animals and a subset of siRNAs in Drosophila and C. elegans are subject to HEN1 mediated 3' terminal 2'-O-methylation. This modification plays a pivotal role in protecting small RNAs from 3' uridylation, trimming and degradation. In Arabidopsis, HESO1 is a major enzyme that uridylates small RNAs to trigger their degradation. However, U-tail is still present in null hen1 heso1 mutants, suggesting the existence of (an enzymatic activities redundant with HESO1. Here, we report that UTP: RNA uridylyltransferase (URT1 is a functional paralog of HESO1. URT1 interacts with AGO1 and plays a predominant role in miRNA uridylation when HESO1 is absent. Uridylation of miRNA is globally abolished in a hen1 heso1 urt1 triple mutant, accompanied by an extensive increase of 3'-to-5' trimming. In contrast, disruption of URT1 appears not to affect the heterochromatic siRNA uridylation. This indicates the involvement of additional nucleotidyl transferases in the siRNA pathway. Analysis of miRNA tailings in the hen1 heso1 urt1 triple mutant also reveals the existence of previously unknown enzymatic activities that can add non-uridine nucleotides. Importantly, we show HESO1 may also act redundantly with URT1 in miRNA uridylation when HEN1 is fully competent. Taken together, our data not only reveal a synergistic action of HESO1 and URT1 in the 3' uridylation of miRNAs, but also independent activities of multiple terminal nucleotidyl transferases in the 3' tailing of small RNAs and an antagonistic relationship between uridylation and trimming. Our results may provide further insight into the mechanisms of small RNA 3' end modification and stability control.
-
Association study on glutathione S-transferase omega 1 and 2 and familial ALS
van de Giessen, Elsmarieke; Fogh, Isabella; Gopinath, Sumana; Smith, Bradley; Hu, Xun; Powell, John; Andersen, Peter; Nicholson, Garth; Al Chalabi, Ammar; Shaw, Christopher E.
2008-01-01
Glutathione S-transferase omega 1 and 2 (GSTO1 and 2) protect from oxidative stress, a possible pathogenic mechanism underlying the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Significant association of age of onset in Alzheimer's
-
Pavlidi, N.; Tseliou, V.; Riga, M.; Nauen, R.; Van Leeuwen, T.; Labrou, N.E.; Vontas, J.
2015-01-01
The two-spotted spider mite Tetranychus urticae is one of the most important agricultural pests world-wide. It is extremely polyphagous and develops resistance to acaricides. The overexpression of several glutathione S-transferases (GSTs) has been associated with insecticide resistance. Here, we
-
Glutathione S-transferase P protects against cyclophosphamide-induced cardiotoxicity in mice
Energy Technology Data Exchange (ETDEWEB)
Conklin, Daniel J., E-mail: dj.conklin@louisville.edu [Diabetes and Obesity Center, University of Louisville, Louisville, KY 40292 (United States); Institute of Molecular Cardiology, University of Louisville, Louisville, KY 40292 (United States); Haberzettl, Petra; Jagatheesan, Ganapathy; Baba, Shahid [Diabetes and Obesity Center, University of Louisville, Louisville, KY 40292 (United States); Institute of Molecular Cardiology, University of Louisville, Louisville, KY 40292 (United States); Merchant, Michael L. [Diabetes and Obesity Center, University of Louisville, Louisville, KY 40292 (United States); Division of Nephrology, Department of Medicine, University of Louisville, Louisville, KY 40292 (United States); Prough, Russell A. [Diabetes and Obesity Center, University of Louisville, Louisville, KY 40292 (United States); Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, KY 40292 (United States); Williams, Jessica D. [University of Cincinnati College of Medicine, Internal Medicine, Cincinnati, OH 45267 (United States); Prabhu, Sumanth D. [Division of Cardiovascular Disease, University of Alabama-Birmingham, Birmingham, AL 35294 (United States); Bhatnagar, Aruni [Diabetes and Obesity Center, University of Louisville, Louisville, KY 40292 (United States); Institute of Molecular Cardiology, University of Louisville, Louisville, KY 40292 (United States); Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, KY 40292 (United States)
2015-06-01
High-dose chemotherapy regimens using cyclophosphamide (CY) are frequently associated with cardiotoxicity that could lead to myocyte damage and congestive heart failure. However, the mechanisms regulating the cardiotoxic effects of CY remain unclear. Because CY is converted to an unsaturated aldehyde acrolein, a toxic, reactive CY metabolite that induces extensive protein modification and myocardial injury, we examined the role of glutathione S-transferase P (GSTP), an acrolein-metabolizing enzyme, in CY cardiotoxicity in wild-type (WT) and GSTP-null mice. Treatment with CY (100–300 mg/kg) increased plasma levels of creatine kinase-MB isoform (CK·MB) and heart-to-body weight ratio to a significantly greater extent in GSTP-null than WT mice. In addition to modest yet significant echocardiographic changes following acute CY-treatment, GSTP insufficiency was associated with greater phosphorylation of c-Jun and p38 as well as greater accumulation of albumin and protein–acrolein adducts in the heart. Mass spectrometric analysis revealed likely prominent modification of albumin, kallikrein-1-related peptidase, myoglobin and transgelin-2 by acrolein in the hearts of CY-treated mice. Treatment with acrolein (low dose, 1–5 mg/kg) also led to increased heart-to-body weight ratio and myocardial contractility changes. Acrolein induced similar hypotension in GSTP-null and WT mice. GSTP-null mice also were more susceptible than WT mice to mortality associated with high-dose acrolein (10–20 mg/kg). Collectively, these results suggest that CY cardiotoxicity is regulated, in part, by GSTP, which prevents CY toxicity by detoxifying acrolein. Thus, humans with low cardiac GSTP levels or polymorphic forms of GSTP with low acrolein-metabolizing capacity may be more sensitive to CY toxicity. - Graphical abstract: Cyclophosphamide (CY) treatment results in P450-mediated metabolic formation of phosphoramide mustard and acrolein (3-propenal). Acrolein is either metabolized and
-
Glutathione S-transferase P protects against cyclophosphamide-induced cardiotoxicity in mice
International Nuclear Information System (INIS)
Conklin, Daniel J.; Haberzettl, Petra; Jagatheesan, Ganapathy; Baba, Shahid; Merchant, Michael L.; Prough, Russell A.; Williams, Jessica D.; Prabhu, Sumanth D.; Bhatnagar, Aruni
2015-01-01
High-dose chemotherapy regimens using cyclophosphamide (CY) are frequently associated with cardiotoxicity that could lead to myocyte damage and congestive heart failure. However, the mechanisms regulating the cardiotoxic effects of CY remain unclear. Because CY is converted to an unsaturated aldehyde acrolein, a toxic, reactive CY metabolite that induces extensive protein modification and myocardial injury, we examined the role of glutathione S-transferase P (GSTP), an acrolein-metabolizing enzyme, in CY cardiotoxicity in wild-type (WT) and GSTP-null mice. Treatment with CY (100–300 mg/kg) increased plasma levels of creatine kinase-MB isoform (CK·MB) and heart-to-body weight ratio to a significantly greater extent in GSTP-null than WT mice. In addition to modest yet significant echocardiographic changes following acute CY-treatment, GSTP insufficiency was associated with greater phosphorylation of c-Jun and p38 as well as greater accumulation of albumin and protein–acrolein adducts in the heart. Mass spectrometric analysis revealed likely prominent modification of albumin, kallikrein-1-related peptidase, myoglobin and transgelin-2 by acrolein in the hearts of CY-treated mice. Treatment with acrolein (low dose, 1–5 mg/kg) also led to increased heart-to-body weight ratio and myocardial contractility changes. Acrolein induced similar hypotension in GSTP-null and WT mice. GSTP-null mice also were more susceptible than WT mice to mortality associated with high-dose acrolein (10–20 mg/kg). Collectively, these results suggest that CY cardiotoxicity is regulated, in part, by GSTP, which prevents CY toxicity by detoxifying acrolein. Thus, humans with low cardiac GSTP levels or polymorphic forms of GSTP with low acrolein-metabolizing capacity may be more sensitive to CY toxicity. - Graphical abstract: Cyclophosphamide (CY) treatment results in P450-mediated metabolic formation of phosphoramide mustard and acrolein (3-propenal). Acrolein is either metabolized and
-
Cooke, R J; Björnestedt, R; Douglas, K T; McKie, J H; King, M D; Coles, B; Ketterer, B; Mannervik, B
1994-01-01
The glutathione transferases (GSTs) form a group of enzymes responsible for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobic region of the active site of the rat liver GST 1-1 and 2-2 isoenzymes (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity labelling was carried out using a version of S-(2-nitro-4-azidophenyl)glutathione tritiated in the arylazido ring. The labelling occurred with higher ...
-
DEFF Research Database (Denmark)
Egeberg, Rikke; Olsen, Anja; Autrup, Herman
2008-01-01
The aim of this study was to investigate whether polymorphisms in N-acetyl transferase 1 and 2 modify the association between meat consumption and risk of breast cancer. A nested case-control study was conducted among 24697 postmenopausal women included in the 'Diet, Cancer and Health' cohort study...... (1993-2000). Three hundred and seventy-eight breast cancer cases were identified and matched to 378 controls. The incidence rate ratio (95% confidence interval) for breast cancer was 1.09 (1.02-1.17) for total meat, 1.15 (1.01-1.31) for red meat and 1.23 (1.04-1.45) for processed meat per 25 g daily...... total meat intake and red meat intake and breast cancer risk were confined to intermediate/fast N-acetyl transferase 2 acetylators (P-interaction=0.03 and 0.04). Our findings support an association between meat consumption and breast cancer risk and that N-acetyl transferase 2 polymorphism has...
-
Directory of Open Access Journals (Sweden)
Ozge Korkmaz
Full Text Available ABSTRACT CONTEXT AND OBJECTIVE: The location of embolism is associated with clinical findings and disease severity in cases of acute pulmonary embolism. The level of gamma-glutamyl transferase increases under oxidative stress-related conditions. In this study, we investigated whether gamma-glutamyl transferase levels could predict the location of pulmonary embolism. DESIGN AND SETTING: Hospital-based cross-sectional study at Cumhuriyet University, Sivas, Turkey. METHODS : 120 patients who were diagnosed with acute pulmonary embolism through computed tomography-assisted pulmonary angiography were evaluated. They were divided into two main groups (proximally and distally located, and subsequently into subgroups according to thrombus localization as follows: first group (thrombus in main pulmonary artery; n = 9; second group (thrombus in main pulmonary artery branches; n = 71; third group (thrombus in pulmonary artery segmental branches; n = 34; and fourth group (thrombus in pulmonary artery subsegmental branches; n = 8. RESULTS : Gamma-glutamyl transferase levels on admission, heart rate, oxygen saturation, right ventricular dilatation/hypokinesia, pulmonary artery systolic pressure and cardiopulmonary resuscitation requirement showed prognostic significance in univariate analysis. The multivariate logistic regression model showed that gamma-glutamyl transferase level on admission (odds ratio, OR = 1.044; 95% confidence interval, CI: 1.011-1.079; P = 0.009 and pulmonary artery systolic pressure (OR = 1.063; 95% CI: 1.005-1.124; P = 0.033 remained independently associated with proximally localized thrombus in pulmonary artery. CONCLUSIONS : The findings revealed a significant association between increased existing embolism load in the pulmonary artery and increased serum gamma-glutamyl transferase levels.
-
A novel method for screening the glutathione transferase inhibitors
Directory of Open Access Journals (Sweden)
Węgrzyn Grzegorz
2009-03-01
Full Text Available Abstract Background Glutathione transferases (GSTs belong to the family of Phase II detoxification enzymes. GSTs catalyze the conjugation of glutathione to different endogenous and exogenous electrophilic compounds. Over-expression of GSTs was demonstrated in a number of different human cancer cells. It has been found that the resistance to many anticancer chemotherapeutics is directly correlated with the over-expression of GSTs. Therefore, it appears to be important to find new GST inhibitors to prevent the resistance of cells to anticancer drugs. In order to search for glutathione transferase (GST inhibitors, a novel method was designed. Results Our results showed that two fragments of GST, named F1 peptide (GYWKIKGLV and F2 peptide (KWRNKKFELGLEFPNL, can significantly inhibit the GST activity. When these two fragments were compared with several known potent GST inhibitors, the order of inhibition efficiency (measured in reactions with 2,4-dinitrochlorobenzene (CDNB and glutathione as substrates was determined as follows: tannic acid > cibacron blue > F2 peptide > hematin > F1 peptide > ethacrynic acid. Moreover, the F1 peptide appeared to be a noncompetitive inhibitor of the GST-catalyzed reaction, while the F2 peptide was determined as a competitive inhibitor of this reaction. Conclusion It appears that the F2 peptide can be used as a new potent specific GST inhibitor. It is proposed that the novel method, described in this report, might be useful for screening the inhibitors of not only GST but also other enzymes.
-
Directory of Open Access Journals (Sweden)
Sung-Kyun Park
2017-08-01
Full Text Available Modification of nucleocytoplasmic proteins with O-GlcNAc regulates a wide variety of cellular processes and has been linked to human diseases. The enzymes O-GlcNAc transferase (OGT and O-GlcNAcase (OGA add and remove O-GlcNAc, but the mechanisms regulating their expression remain unclear. Here, we demonstrate that retention of the fourth intron of OGT is regulated in response to O-GlcNAc levels. We further define a conserved intronic splicing silencer (ISS that is necessary for OGT intron retention. Deletion of the ISS in colon cancer cells leads to increases in OGT, but O-GlcNAc homeostasis is maintained by concomitant increases in OGA protein. However, the ISS-deleted cells are hypersensitive to OGA inhibition in culture and in soft agar. Moreover, growth of xenograft tumors from ISS-deleted cells is compromised in mice treated with an OGA inhibitor. Thus, ISS-mediated regulation of OGT intron retention is a key component in OGT expression and maintaining O-GlcNAc homeostasis.
-
K.W.A. Grinsven; S. Rosnowsky (Silke); S.W.H. van Weelden (Susanne); S. Pütz (Simone); M. van der Giezen (Mark); W. Martin (William); J.J. van Hellemond (Jaap); A.G.M. Tielens (Aloysius); K. Henze (Katrin)
2008-01-01
textabstractAcetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial
-
Shi, Hao; Munk, Alexander; Nielsen, Thomas S; Daughtry, Morgan R; Larsson, Louise; Li, Shize; Høyer, Kasper F; Geisler, Hannah W; Sulek, Karolina; Kjøbsted, Rasmus; Fisher, Taylor; Andersen, Marianne M; Shen, Zhengxing; Hansen, Ulrik K; England, Eric M; Cheng, Zhiyong; Højlund, Kurt; Wojtaszewski, Jørgen F P; Yang, Xiaoyong; Hulver, Matthew W; Helm, Richard F; Treebak, Jonas T; Gerrard, David E
2018-05-01
Given that cellular O-GlcNAcylation levels are thought to be real-time measures of cellular nutrient status and dysregulated O-GlcNAc signaling is associated with insulin resistance, we evaluated the role of O-GlcNAc transferase (OGT), the enzyme that mediates O-GlcNAcylation, in skeletal muscle. We assessed O-GlcNAcylation levels in skeletal muscle from obese, type 2 diabetic people, and we characterized muscle-specific OGT knockout (mKO) mice in metabolic cages and measured energy expenditure and substrate utilization pattern using indirect calorimetry. Whole body insulin sensitivity was assessed using the hyperinsulinemic euglycemic clamp technique and tissue-specific glucose uptake was subsequently evaluated. Tissues were used for histology, qPCR, Western blot, co-immunoprecipitation, and chromatin immunoprecipitation analyses. We found elevated levels of O-GlcNAc-modified proteins in obese, type 2 diabetic people compared with well-matched obese and lean controls. Muscle-specific OGT knockout mice were lean, and whole body energy expenditure and insulin sensitivity were increased in these mice, consistent with enhanced glucose uptake and elevated glycolytic enzyme activities in skeletal muscle. Moreover, enhanced glucose uptake was also observed in white adipose tissue that was browner than that of WT mice. Interestingly, mKO mice had elevated mRNA levels of Il15 in skeletal muscle and increased circulating IL-15 levels. We found that OGT in muscle mediates transcriptional repression of Il15 by O-GlcNAcylating Enhancer of Zeste Homolog 2 (EZH2). Elevated muscle O-GlcNAc levels paralleled insulin resistance and type 2 diabetes in humans. Moreover, OGT-mediated signaling is necessary for proper skeletal muscle metabolism and whole-body energy homeostasis, and our data highlight O-GlcNAcylation as a potential target for ameliorating metabolic disorders. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.
-
Glutathione s-transferase isoenzymes in relation to their role in detoxification of xenobiotics
Vos, R.M.E.
1989-01-01
The glutathione S-transferases (GST) are a family of isoenzymes serving a major part in the biotransformation of many reactive compounds. The isoenzymes from rat, man and mouse are divided into three classes, alpha, mu and pi, on the basis of similar structural and enzymatic
-
Directory of Open Access Journals (Sweden)
Felipe Ávila
2017-01-01
Full Text Available The prevalence of cytoprotective mechanisms induced by polyphenols such as activation of intracellular antioxidant responses (ICM and direct free radical scavenging was investigated in native Chilean species of strawberries, raspberries, and currants. Human gastric epithelial cells were co- and preincubated with polyphenolic-enriched extracts (PEEs from Chilean raspberries (Rubus geoides, strawberries (Fragaria chiloensis ssp. chiloensis f. chiloensis, and currants (Ribes magellanicum and challenged with peroxyl and hydroxyl radicals. Cellular protection was determined in terms of cell viability, glyoxalase I and glutathione s-transferases activities, and carboxymethyl lysine (CML and malondialdehyde levels. Our results indicate that cytoprotection induced by ICM was the prevalent mechanism for Rubus geoides and F. chiloensis. This agreed with increased levels of glyoxalase I and glutathione S-transferase activities in cells preincubated with PEEs. ORAC index indicated that F. chiloensis was the most efficient peroxyl radical scavenger. Moreover, ICM mediated by F. chiloensis was effective in protecting cells from CML accumulation in contrast to the protective effects induced by free radical scavenging. Our results indicate that although both polyphenol-mediated mechanisms can exert protective effects, ICM was the most prevalent in AGS cells. These results suggest a potential use of these native berries as functional food.
-
International Nuclear Information System (INIS)
Xing, Jiali; Wang, Gang; Zhao, Jichun; Wang, Eryin; Yin, Boxing; Fang, Dongsheng; Zhao, Jianxin; Zhang, Hao; Chen, Yong Q.; Chen, Wei
2016-01-01
Perfluorooctane sulfonate (PFOS) is a principal representative and the final degradation product of several commercially produced perfluorinated compounds. However, PFOS has a high bioaccumulation potential and therefore can exert toxicity on aquatic organisms, animals, and cells. Considering the widespread concern this phenomenon has attracted, we examined the acute and subchronic toxic effects of varying doses of PFOS on adult male C57BL/6 mice. The acute oral LD_5_0 value of PFOS in male C57BL/6J mice was 0.579 g/kg body weight (BW). Exposure to the subchronic oral toxicity of PFOS at 2.5, 5, and 10 mg PFOS/kg BW/day for 30 days disrupted the homeostasis of antioxidative systems, induced hepatocellular apoptosis (as revealed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay), triggered liver injury (as evidenced by the increased serum levels of aspartate aminotransferase, alanine amino transferase, alkaline phosphatase, and gamma-glutamyl transpeptidase and by the altered histology), and ultimately increased the liver size and relative weight of the mice. PFOS treatment caused liver damage but only slightly affected the kidneys and spleen of the mice. This study provided insights into the toxicological effects of PFOS. - Highlights: • The acute and subchronic toxicity of PFOS was systematically investigated. • The acute oral LD_5_0 value for PFOS in C57BL/6J mice was 0.579 g/kg body weight. • PFOS disrupted the homeostasis of antioxidative systems. • PFOS induced hepatocellular apoptosis and triggered liver injury. - PFOS disrupted the homeostasis of antioxidative systems, induced hepatocellular apoptosis, and triggered liver injury.
-
Interaction of Pleuromutilin Derivatives with the Ribosomal Peptidyl Transferase Center
Long, Katherine S.; Hansen, Lykke H.; Jakobsen, Lene; Vester, Birte
2006-01-01
Tiamulin is a pleuromutilin antibiotic that is used in veterinary medicine. The recently published crystal structure of a tiamulin-50S ribosomal subunit complex provides detailed information about how this drug targets the peptidyl transferase center of the ribosome. To promote rational design of pleuromutilin-based drugs, the binding of the antibiotic pleuromutilin and three semisynthetic derivatives with different side chain extensions has been investigated using chemical footprinting. The nucleotides A2058, A2059, G2505, and U2506 are affected in all of the footprints, suggesting that the drugs are similarly anchored in the binding pocket by the common tricyclic mutilin core. However, varying effects are observed at U2584 and U2585, indicating that the side chain extensions adopt distinct conformations within the cavity and thereby affect the rRNA conformation differently. An Escherichia coli L3 mutant strain is resistant to tiamulin and pleuromutilin, but not valnemulin, implying that valnemulin is better able to withstand an altered rRNA binding surface around the mutilin core. This is likely due to additional interactions made between the valnemulin side chain extension and the rRNA binding site. The data suggest that pleuromutilin drugs with enhanced antimicrobial activity may be obtained by maximizing the number of interactions between the side chain moiety and the peptidyl transferase cavity. PMID:16569865
-
Directory of Open Access Journals (Sweden)
Marija G Matic
Full Text Available OBJECTIVE: We investigated the role of the glutathione S-transferase A1, M1, P1 and T1 gene polymorphisms and potential effect modification by occupational exposure to different chemicals in Serbian bladder cancer male patients. PATIENTS AND METHODS: A hospital-based case-control study of bladder cancer in men comprised 143 histologically confirmed cases and 114 age-matched male controls. Deletion polymorphism of glutathione S-transferase M1 and T1 was identified by polymerase chain reaction method. Single nucleotide polymorphism of glutathione S-transferase A1 and P1 was identified by restriction fragment length polymorphism method. As a measure of effect size, odds ratio (OR with corresponding 95% confidence interval (95%CI was calculated. RESULTS: The glutathione S-transferase A1, T1 and P1 genotypes did not contribute independently toward the risk of bladder cancer, while the glutathione S-transferase M1-null genotype was overrepresented among cases (OR = 2.1, 95% CI = 1.1-4.2, p = 0.032. The most pronounced effect regarding occupational exposure to solvents and glutathione S-transferase genotype on bladder cancer risk was observed for the low activity glutathione S-transferase A1 genotype (OR = 9.2, 95% CI = 2.4-34.7, p = 0.001. The glutathione S-transferase M1-null genotype also enhanced the risk of bladder cancer among subjects exposed to solvents (OR = 6,5, 95% CI = 2.1-19.7, p = 0.001. The risk of bladder cancer development was 5.3-fold elevated among glutathione S-transferase T1-active patients exposed to solvents in comparison with glutathione S-transferase T1-active unexposed patients (95% CI = 1.9-15.1, p = 0.002. Moreover, men with glutathione S-transferase T1-active genotype exposed to pesticides exhibited 4.5 times higher risk in comparison with unexposed glutathione S-transferase T1-active subjects (95% CI = 0.9-22.5, p = 0.067. CONCLUSION: Null or low-activity genotypes of the
-
T-lineage blast crisis of chronic myelogenous leukemia: simple record of 4 cases
Directory of Open Access Journals (Sweden)
Kartika W. Taroeno-Hariadi
2005-09-01
Full Text Available Blast crisis (BC transformation in chronic myelogenous leukemia (CML can involve each differentiation lineage of the hematopoietic system, i.e. granulocyte, monocyte, erythrocyte, megakaryocyte, and lymphocyte lineage. The lymphoid blast crisis (BC leukemia cells usually belong to B-lineage, commonly having the phenotype of Pre-B stage of the B-lineage, in which cell-surface immunoglobulin (sIg is not yet expressed. In contrast, T-lineage BC of CML is extremely rare. The objective of this study is to describe the fenotype, fusion transcript of bcr-abl, TdT, and cytoplasmic CD3 in T-lineage BC CML cases. Case report study. This report shows a simple summary of 4 cases of T-lineage BC of CML which have been collected in the phenotypic and genotypic analysis study for 17 years (1987-2004. In all cases, the chromosomal analysis revealed the presence of t(9;22(q34;q11 at presentation. Cell surface analysis were done at diagnosis. Cases’ mononuclear cells stored as 10% DMSO were retrieved to be performed reverse transcription (RT PCR BCR-ABL multiplex to demonstrate the presence of the fusion transcript of bcr-abl. RT-PCR was also performed for detecting the expression of cytoplasmic CD3ε and terminal deoxynucleotydil transferase (TdT. The results of cell surface antigen (CSA at presentation showed that 1 case was CD7+, CD5-, and CD2-; 1 case CD7+, CD5+, and CD2-; and 2 cases CD7+, CD5+ and CD2+ indicating that all these T-lineage BC of CML cells show the phenotype of pre-(pro- thymic stage phenotype. In the present study, two cases showed b2a2, one e1a2, and one negative bcr-abl transcript. The RT-PCR revealed the presence of CD3ε mRNA in all cases, and TdT mRNA in only one case. These results can constitute a basis for the future analysis of T-lineage BC of CML from now on. (Med J Indones 2005; 14: 184-9Keywords: chronic myelogenous leukemia (CML, blastic crisis (BC, T-lineage, bcr-abl fusion gene, CDε, TdT
-
Ganoderma tsugae Hepatoprotection against Exhaustive Exercise-Induced Liver Injury in Rats
Directory of Open Access Journals (Sweden)
Wan-Teng Lin
2013-01-01
Full Text Available Several studies have been shown that accelerated apoptosis is involved in post-exercise lymphocytopenia and tissue damage after high-intensity exercise. Ganoderma tsugae (GT is one of the well-known medicinal mushrooms that possess various pharmacological functions. This mushroom has traditionally been used for health promotion purposes. This study investigates the hepatoprotective effects of GT on exhaustive exercise-induced liver damage. Twenty-four male Sprague-Dawley rats were randomly divided into four groups and designated as exhaustive exercise only (E, exhaustive exercise with low dosage (EL, medium dosage (EM and high dosage (EH GT at 0, 0.1875, 0.9375 and 1.875 g/kg/day, respectively. After 30 days all rats were euthanized immediately after an exhaustive running challenge on a motorized treadmill. The rat livers were immediately harvested. Evidence of apoptotic liver cell death was revealed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay and caspases mediated cascade events. DNA fragmentation, an apoptosis process, can be examined using TUNEL assay. A few TUNEL-positive hepatocytes, compared to the exercise only group, were observed in the livers from exhaustive animals supplemented with GT. Immunoblot analysis also showed that caspase-6-mediated specific cleavage of lamin A/C was increased significantly in the livers of group E, but was significantly decreased in the EM and EH groups. Our observations demonstrate that GT possesses anti-apoptotic and hepatoprotective potential after exhaustive exercise.
-
Ganoderma tsugae hepatoprotection against exhaustive exercise-induced liver injury in rats.
Huang, Chi-Chang; Huang, Wen-Ching; Yang, Suh-Ching; Chan, Chih-Chi; Lin, Wan-Teng
2013-01-29
Several studies have been shown that accelerated apoptosis is involved in post-exercise lymphocytopenia and tissue damage after high-intensity exercise. Ganoderma tsugae (GT) is one of the well-known medicinal mushrooms that possess various pharmacological functions. This mushroom has traditionally been used for health promotion purposes. This study investigates the hepatoprotective effects of GT on exhaustive exercise-induced liver damage. Twenty-four male Sprague-Dawley rats were randomly divided into four groups and designated as exhaustive exercise only (E), exhaustive exercise with low dosage (EL), medium dosage (EM) and high dosage (EH) GT at 0, 0.1875, 0.9375 and 1.875 g/kg/day, respectively. After 30 days all rats were euthanized immediately after an exhaustive running challenge on a motorized treadmill. The rat livers were immediately harvested. Evidence of apoptotic liver cell death was revealed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and caspases mediated cascade events. DNA fragmentation, an apoptosis process, can be examined using TUNEL assay. A few TUNEL-positive hepatocytes, compared to the exercise only group, were observed in the livers from exhaustive animals supplemented with GT. Immunoblot analysis also showed that caspase-6-mediated specific cleavage of lamin A/C was increased significantly in the livers of group E, but was significantly decreased in the EM and EH groups. Our observations demonstrate that GT possesses anti-apoptotic and hepatoprotective potential after exhaustive exercise.
-
The contribution of p53 and Y chromosome long arm genes to regulation of apoptosis in mouse testis.
Lech, Tomasz; Styrna, Józefa; Kotarska, Katarzyna
2018-03-01
Apoptosis of excessive or defective germ cells is a natural process occurring in mammalian testes. Tumour suppressor protein p53 is involved in this process both in developing and adult male gonads. Its contribution to testicular physiology is known to be modified by genetic background. The aim of this study was to evaluate the combined influence of the p53 and Y chromosome long arm genes on male germ cell apoptosis. Knockout of the transformation related protein 53 (Trp53) gene was introduced into congenic strains: B10.BR (intact Y chromosome) and B10.BR-Ydel (Y chromosome with a deletion in the long arm). The level of apoptosis in the testes of 19-day-old and 3-month-old male mice was determined using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick-end labelling (TUNEL) method. The study revealed that although p53 is involved in germ cell apoptosis in peripubertal testes, this process can also be mediated by p53-independent mechanisms. However, activation of p53-independent apoptotic pathways in the absence of the p53 protein requires engagement of the multicopy Yq genes and was not observed in gonads of B10.BR-Ydel-p53-/- males. The role of Yq genes in the regulation of testicular apoptosis seems to be restricted to the initial wave of spermatogenesis and is not evident in adult gonads. The study confirmed, instead, that p53 does participate in spontaneous apoptosis in mature testes.
-
Abramovitz, M; Ishigaki, S; Felix, A M; Listowsky, I
1988-11-25
Glutathione S-transferases containing Yb3 subunits are relatively uncommon forms that are expressed in a tissue-specific manner and have not been identified unequivocally or characterized. A cDNA clone containing the entire coding sequence of Yb3 glutathione S-transferase mRNA was incorporated into a pIN-III expression vector used to transform Escherichia coli. A fusion Yb3-protein containing 14 additional amino acid residues at its N terminus was purified to homogeneity. Recombinant Yb3 was enzymatically active with both 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates but lacked glutathione peroxidase activity. Substrate specificity patterns of recombinant Yb3 were more limited than those of glutathione S-transferase isoenzymes containing Yb1- or Yb2-type subunits. Peptides corresponding to unique amino acid sequences of Yb3 as well as a peptide from a region of homology with Yb1 and Yb2 subunits were synthesized. These synthetic peptides were used to raise antibodies specific to Yb3 and others that cross-reacted with all Yb forms. Immunoblotting was utilized to identify the natural counterpart of recombinant Yb3 among rat glutathione transferases. Brain and testis glutathione S-transferases were rich in Yb3 subunits, but very little was found in liver or kidney. Physical properties, substrate specificities, and binding patterns of the recombinant protein paralleled properties of the natural isoenzyme isolated from brain.
-
The role of glutathione S-transferase and claudin-1 gene polymorphisms in contact sensitization
DEFF Research Database (Denmark)
Ross-Hansen, K; Linneberg, A; Johansen, J D
2013-01-01
BACKGROUND: Contact sensitization is frequent in the general population and arises from excessive or repeated skin exposure to chemicals and metals. However, little is known about its genetic susceptibility. OBJECTIVES: To determine the role of polymorphisms of glutathione S-transferase (GST) genes...
-
Glutathione S-transferase genotype and p53 mutations in adenocarcinoma of the small intestine
DEFF Research Database (Denmark)
Pedersen, Lisbeth Nørum; Kaerlev, L; Stubbe Teglbjaerg, P
2003-01-01
Adenocarcinoma of the small intestine (ASI) is a rare disease of unknown aetiology. The glutathione S-transferase M1 (GSTM1) enzyme catalyses the detoxification of compounds involved in carcinogenesis of adenocarcinoma of the stomach, colon and lung, including constituents of tobacco smoke. We in...
-
A systematic review on sperm DNA fragmentation in male factor infertility: Laboratory assessment
Directory of Open Access Journals (Sweden)
Manesh Kumar Panner Selvam
2018-03-01
Full Text Available Objective: To review sperm DNA fragmentation (SDF testing as an important sperm function test in addition to conventional semen analysis. High SDF is negatively associated with semen quality, the fertilisation process, embryo quality, and pregnancy outcome. Over recent decades, different SDF assays have been developed and reviewed extensively to assess their applicability and accuracy as advanced sperm function tests. Amongst them, the standardisation of the terminal deoxynucleotidyl transferased UTP nick-end labelling (TUNEL assay with a bench top flow cytometer in clinical practice deserves special mention with a threshold value of 16.8% to differentiate infertile men with DNA damage from fertile men. Materials and methods: A systematic literature search was performed through the PubMed, Medline, and ScienceDirect databases using the keywords ‘sperm DNA fragmentation’ and ‘laboratory assessment’. Non-English articles were excluded and studies related to humans were only included. Results: Of the 618 identified, 87 studies (original research and reviews and in addition eight book chapters meeting the selection criteria were included in this review. In all, 366 articles were rejected in the preliminary screening and a further 165 articles related to non-human subjects were excluded. Conclusion: There are pros and cons to all the available SDF assays. TUNEL is a reliable technique with greater accuracy and as an additional diagnostic test in Andrology laboratories along with basic semen analysis can predict fertility outcome, and thus direct the choice of an assisted reproductive technology procedure for infertile couples. Also, the TUNEL assay can be used as a prognostic test and results are beneficial in deciding personalised treatment for infertile men. Keywords: Sperm DNA fragmentation (SDF, Terminal deoxynucleotidyl transferased UTP nick-end labelling (TUNEL, DNA damage, Sperm DNA fragmentation (SDF assay
-
Directory of Open Access Journals (Sweden)
Ayodele O. Kolawole
Full Text Available Fish hepatic glutathione transferases are connected with the elimination of intracellular pollutants and detoxification of organic micro-pollutants in their aquatic ecosystem. The two-substrate steady state kinetic mechanism of Silver catfish (Synodontis eupterus major hepatic glutathione transferases purified to apparent homogeneity was explored. The enzyme was dimeric enzyme with a monomeric size of 25.6 kDa. Initial-velocity studies and Product inhibition patterns by methyl glutathione and chloride with respect to GSH-CDNB; GSH-ρ-nitrophenylacetate; and GSH-Ethacrynic acid all conforms to a rapid equilibrium sequential random Bi Bi kinetic mechanism rather than steady state sequential random Bi Bi kinetic. α was 2.96 ± 0.35 for the model. The pH profile of Vmax/KM (with saturating 1-chloro-2,4-dinitrobenzene and variable GSH concentrations showed apparent pKa value of 6.88 and 9.86. Inhibition studies as a function of inhibitor concentration show that the enzyme is a homodimer and near neutral GST. The enzyme poorly conjugates 4-hydroxylnonenal and cumene hydroperoxide and may not be involved in oxidative stress protection. The seGST is unique and overwhelmingly shows characteristics similar to those of homodimeric class Pi GSTs, as was indicated by its kinetic mechanism, substrate specificity and inhibition studies. The rate- limiting step, probably the product release, of the reaction is viscosity-dependent and is consequential if macro-viscosogen or micro-viscosogen. Keywords: Silver catfish, Glutathione transferase, Steady-state, Kinetic mechanism, Inhibition
-
Directory of Open Access Journals (Sweden)
María de Medina-Redondo
Full Text Available BACKGROUND: The formation of the cell wall in Schizosaccharomyces pombe requires the coordinated activity of enzymes involved in the biosynthesis and modification of β-glucans. The β(1,3-glucan synthase complex synthesizes linear β(1,3-glucans, which remain unorganized until they are cross-linked to other β(1,3-glucans and other cell wall components. Transferases of the GH72 family play important roles in cell wall assembly and its rearrangement in Saccharomyces cerevisiae and Aspergillus fumigatus. Four genes encoding β(1,3-glucanosyl-transferases -gas1(+, gas2(+, gas4(+ and gas5(+- are present in S. pombe, although their function has not been analyzed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report the characterization of the catalytic activity of gas1p, gas2p and gas5p together with studies directed to understand their function during vegetative growth. From the functional point of view, gas1p is essential for cell integrity and viability during vegetative growth, since gas1Δ mutants can only grow in osmotically supported media, while gas2p and gas5p play a minor role in cell wall construction. From the biochemical point of view, all of them display β(1,3-glucanosyl-transferase activity, although they differ in their specificity for substrate length, cleavage point and product size. In light of all the above, together with the differences in expression profiles during the life cycle, the S. pombe GH72 proteins may accomplish complementary, non-overlapping functions in fission yeast. CONCLUSIONS/SIGNIFICANCE: We conclude that β(1,3-glucanosyl-transferase activity is essential for viability in fission yeast, being required to maintain cell integrity during vegetative growth.
-
Stefanidis, Lazaros; Scinto, Krystal V.; Strada, Monica I.; Alper, Benjamin J.
2018-01-01
Most biochemical transformations involve more than one substrate. Bisubstrate enzymes catalyze multiple chemical reactions in living systems and include members of the transferase, oxidoreductase, and ligase enzyme classes. Working knowledge of bisubstrate enzyme kinetic models is thus of clear importance to the practicing biochemist. However,…
-
DEFF Research Database (Denmark)
Beld, Joris; Sonnenschein, Eva; Vickery, Christopher R.
2013-01-01
Covering: up to 2013 Although holo-acyl carrier protein synthase, AcpS, a phosphopantetheinyl transferase (PPTase), was characterized in the 1960s, it was not until the publication of the landmark paper by Lambalot et al. in 1996 that PPTases garnered wide-spread attention being classified...... knowledge on this class of enzymes that post-translationally install a 4′-phosphopantetheine arm on various carrier proteins....
-
Interaction of pleuromutilin derivatives with the ribosomal peptidyl transferase center
DEFF Research Database (Denmark)
Long, K. S.; Hansen, L. K.; Jakobsen, L.
2006-01-01
Tiamulin is a pleuromutilin antibiotic that is used in veterinary medicine. The recently published crystal structure of a tiamulin-50S ribosomal subunit complex provides detailed information about how this drug targets the peptidyl transferase center of the ribosome. To promote rational design...... mutant strain is resistant to tiamulin and pleuromutilin, but not valnemulin, implying that valnemulin is better able to withstand an altered rRNA binding surface around the mutilin core. This is likely due to additional interactions made between the valnemulin side chain extension and the rRNA binding...
-
Genin, Michael J; Gonzalez Valcarcel, Isabel C; Holloway, William G; Lamar, Jason; Mosior, Marian; Hawkins, Eric; Estridge, Thomas; Weidner, Jeffrey; Seng, Thomas; Yurek, David; Adams, Lisa A; Weller, Jennifer; Reynolds, Vincent L; Brozinick, Joseph T
2016-06-23
To develop novel treatments for type 2 diabetes and dyslipidemia, we pursued inhibitors of serine palmitoyl transferase (SPT). To this end compounds 1 and 2 were developed as potent SPT inhibitors in vitro. 1 and 2 reduce plasma ceramides in rodents, have a slight trend toward enhanced insulin sensitization in DIO mice, and reduce triglycerides and raise HDL in cholesterol/cholic acid fed rats. Unfortunately these molecules cause a gastric enteropathy after chronic dosing in rats.
-
Energy Technology Data Exchange (ETDEWEB)
Yu, Mi Hye [Dept. of Radiology, Konkuk University Medical Center, Seoul (Korea, Republic of); Lee, Jae Young; Kim, Bo Ram; Park, Eun Joo; Kim, Hoe Suk; Han, Joon Koo [Dept. of Radiology, Seoul National University Hospital, Seoul (Korea, Republic of); Kim, Hae Ri [Dept. of Pre-Dentistry, Gangneung-Wonju National University College of Dentistry, Gangneung (Korea, Republic of); Choi, Byung Ihn [Dept. of Radiology, Chung-Ang University Hospital, Seoul (Korea, Republic of)
2016-09-15
To investigate whether high-intensity focused ultrasound (HIFU) combined with microbubbles enhances the therapeutic effects of chemotherapy. A pancreatic cancer xenograft model was established using BALB/c nude mice and luciferase-expressing human pancreatic cancer cells. Mice were randomly assigned to five groups according to treatment: control (n = 10), gemcitabine alone (GEM; n = 12), HIFU with microbubbles (HIFU + MB, n = 11), combined HIFU and gemcitabine (HIGEM; n = 12), and HIGEM + MB (n = 13). After three weekly treatments, apoptosis rates were evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay in two mice per group. Tumor volume and bioluminescence were monitored using high-resolution 3D ultrasound imaging and in vivo bioluminescence imaging for eight weeks in the remaining mice. The HIGEM + MB group showed significantly higher apoptosis rates than the other groups (p < 0.05) and exhibited the slowest tumor growth. From week 5, the tumor-volume-ratio relative to the baseline tumor volume was significantly lower in the HIGEM + MB group than in the control, GEM, and HIFU + MB groups (p < 0.05). Despite visible distinction, the HIGEM and HIGEM + MB groups showed no significant differences. High-intensity focused ultrasound combined with microbubbles enhances the therapeutic effects of gemcitabine chemotherapy in a pancreatic cancer xenograft model.
-
Energy Technology Data Exchange (ETDEWEB)
Yu, Mi Hye [Department of Radiology, Konkuk University Medical Center, Seoul 05030 (Korea, Republic of); Lee, Jae Young [Department of Radiology, Seoul National University Hospital, Seoul 03080 (Korea, Republic of); Kim, Hae Ri [Department of Pre-Dentistry, Gangneung-Wonju National University College of Dentistry, Gangneung 25457 (Korea, Republic of); Kim, Bo Ram; Park, Eun-Joo; Kim, Hoe Suk; Han, Joon Koo [Department of Radiology, Seoul National University Hospital, Seoul 03080 (Korea, Republic of); Choi, Byung Ihn [Department of Radiology, Chung-Ang University Hospital, Seoul 06973 (Korea, Republic of)
2016-11-01
To investigate whether high-intensity focused ultrasound (HIFU) combined with microbubbles enhances the therapeutic effects of chemotherapy. A pancreatic cancer xenograft model was established using BALB/c nude mice and luciferase-expressing human pancreatic cancer cells. Mice were randomly assigned to five groups according to treatment: control (n = 10), gemcitabine alone (GEM; n = 12), HIFU with microbubbles (HIFU + MB, n = 11), combined HIFU and gemcitabine (HIGEM; n = 12), and HIGEM + MB (n = 13). After three weekly treatments, apoptosis rates were evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay in two mice per group. Tumor volume and bioluminescence were monitored using high-resolution 3D ultrasound imaging and in vivo bioluminescence imaging for eight weeks in the remaining mice. The HIGEM + MB group showed significantly higher apoptosis rates than the other groups (p < 0.05) and exhibited the slowest tumor growth. From week 5, the tumor-volume-ratio relative to the baseline tumor volume was significantly lower in the HIGEM + MB group than in the control, GEM, and HIFU + MB groups (p < 0.05). Despite visible distinction, the HIGEM and HIGEM + MB groups showed no significant differences. High-intensity focused ultrasound combined with microbubbles enhances the therapeutic effects of gemcitabine chemotherapy in a pancreatic cancer xenograft model.
-
International Nuclear Information System (INIS)
Xia, D.Y.; Li, W.; Qian, H.R.; Yao, S.; Liu, J.G.; Qi, X.K.
2013-01-01
Sublethal ischemic preconditioning (IPC) is a powerful inducer of ischemic brain tolerance. However, its underlying mechanisms are still not well understood. In this study, we chose four different IPC paradigms, namely 5 min (5 min duration), 5×5 min (5 min duration, 2 episodes, 15-min interval), 5×5×5 min (5 min duration, 3 episodes, 15-min intervals), and 15 min (15 min duration), and demonstrated that three episodes of 5 min IPC activated autophagy to the greatest extent 24 h after IPC, as evidenced by Beclin expression and LC3-I/II conversion. Autophagic activation was mediated by the tuberous sclerosis type 1 (TSC1)-mTor signal pathway as IPC increased TSC1 but decreased mTor phosphorylation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and hematoxylin and eosin staining confirmed that IPC protected against cerebral ischemic/reperfusion (I/R) injury. Critically, 3-methyladenine, an inhibitor of autophagy, abolished the neuroprotection of IPC and, by contrast, rapamycin, an autophagy inducer, potentiated it. Cleaved caspase-3 expression, neurological scores, and infarct volume in different groups further confirmed the protection of IPC against I/R injury. Taken together, our data indicate that autophagy activation might underlie the protection of IPC against ischemic injury by inhibiting apoptosis
-
Takikawa, Megumi; Ishihara, Masayuki; Takabayashi, Yuki; Sumi, Yuki; Takikawa, Makoto; Yoshida, Ryuichi; Nakamura, Shingo; Hattori, Hidemi; Yanagibayashi, Satoshi; Yamamoto, Naoto; Kiyosawa, Tomoharu
2015-04-13
The purpose of this study was to evaluate the accelerating effects of platelet-rich plasma-containing (PRP&) fragmin/protamine microparticles (F/P MPs) for repairing mitomycin C-treated healing-impaired wounds. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL-staining) showed that apoptosis of dermal fibroblast cells (DFCs) and epidermal keratinocyte cells (EKCs) were significantly induced in the skin of the mitomycin C-treated rats. Full-thickness skin defects were made on the back of rats and mitomycin C was applied on the wounds to prepare a healing-impaired wound. After washing out the mitomycin C, saline (control), F/P MPs alone, PRP alone, and PRP&F/P MPs were injected around the wounds. The rats were later euthanised and histological sections of the wounds were then prepared at indicated time periods after the treatment. These results indicated the numbers of large, medium, and small capillary lumens 7 days after injection of PRP&F/P MPs were significantly higher than those after injection of PRP or F/P MPs alone. Furthermore, epithelium and granulation tissue formations were significantly stimulated in the healing-impaired wounds treated with PRP&F/P MPs 3, 7 and 14 days after injection of PRP&F/P MPs.
-
Ishida, M; Gomyo, Y; Ohfuji, S; Ikeda, M; Kawasaki, H; Ito, H
1997-05-01
To examine in vivo the validity of the results of experiments in vitro, we analyzed the relationship between p53 gene status and apoptotic cell death of human gastric intestinal-type adenocarcinomas. Surgical specimens were classified into two categories: 18 gastric cancers with nuclear p53 protein (A), and 17 gastric cancers without nuclear p53 protein (B). Polymerase chain reaction-single strand conformation polymorphism disclosed a shifted band that corresponded to a mutation in the p53 gene in 13 cases (72%) in category A and 3 cases (18%) in category B, the frequency being significantly higher in the former (P terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The TUNEL index [TI; (the number of TUNEL-positive apoptotic cells/the total number of tumor cells) x 100] was 3.8 +/- 1.4% in category A and 4.9 +/- 1.2% in category B, the value being significantly lower in the former (P gastric cancer, in accordance with the previous in vitro finding that p53 gene mutation provides a possible selective advantage for tumor cell proliferation, and (2) apoptosis is related not only to expression of p53 and the stage of the cell cycle, but also to p53-independent and cell cycle-independent events.
-
Shi, Hongguang; Liu, Xuefeng; Tang, Gusheng; Liu, Haiyan; Zhang, Yinghui; Zhang, Bo; Zhao, Xuezhi; Wang, Wanyin
2014-01-01
Acute hepatic injury causes high morbidity and mortality world-wide. Management of severe acute hepatic failure continues to be one of the most challenging problems in clinical medicine. In present study, carbon tetrachloride (CCl4) was used to induce acute liver damage in mice and the protective effects of ethanol extract of Portulaca Oleracea L. (PO) were examined. The aminotransferase activities were biochemical estimated and the liver damage was tested by morphological histological analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The role of PO on the activity of NF-κB was determined by luciferase reporter gene assay and immunohistochemistry. The level of p-p65 was tested by western blot. Our results showed that PO administration on mice would decrease the serum aminotransferase level and reduced the liver histological damage. We also found that nuclear translocation of p65 was enhanced in liver tissues of mice treated with PO compared with control animals. In addition, in cultured hepatic cells, PO increased the NF-κB luciferase reporter gene activity and upregulated the level of phosphorylation of p65, but had no effects on mice liver SOD activity and MDA level. Collectively, PO attenuated CCl4 induced mice liver damage by enhancement of NF-κB activity. PMID:25628785
-
Directory of Open Access Journals (Sweden)
Jinlong Zhang
2015-11-01
Full Text Available Background/Aims: Cardiac malfunction is a common complication in sepsis and significantly increases the mortality of patients in septic shock. However, no studies have examined whether andrographolide (And reduces LPS-induced myocardial malfunction. Methods: Left ventricular systolic and diastolic functions were examined using echocardiography. TNF-a and IL-1ß protein levels were detected by an enzyme-linked immunosorbent assay (ELISA. NO oxidation products were determined using Griess reagent. Protein expression levels of inhibitors of NF-κBa (IκB and phospho-IκB were determined via Western blot. Oxidative injury was determined by measuring myocardial lipid peroxidation and superoxide dismutase activity. Cardiac apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nickend-labeling (TUNEL and cardiac caspase 3/7 activity. Results: And blunted LPS-induced myocardial malfunctions in mice. LPS induced TNF-a, IL-1ß, and NO production as well as I-κB phosphorylation. Cardiac apoptosis was attenuated via incubation with And, but the extent of oxidative injury remained unaffected. Conclusion: And prevents LPS-induced cardiac malfunctions in mice by inhibiting TNF-a, IL-1ß, and NO production, IκB phosphorylation, and cardiac apoptosis, indicating that And may be a potential agent for preventing myocardial malfunction during sepsis.
-
Hwang, S-K; Jin, H; Kwon, J T; Chang, S-H; Kim, T H; Cho, C-S; Lee, K H; Young, M R; Colburn, N H; Beck, G R; Yang, H-S; Cho, M-H
2007-09-01
The long-term survival of lung cancer patients treated with conventional therapies remains poor and therefore the need for novel approaches remains high. This has led to the re-emergence of aerosol delivery as a therapeutic intervention. In this study, glucosylated polyethylenimine (GPEI) was used as carrier to investigate programmed cell death 4 (PDCD4) and PDCD4 mutant (D418A), an eIF4A-binding mutant, on PDCD4-related signaling and activator protein-1 (AP-1) activity in the lungs of AP-1 luciferase reporter mice. After confirming the efficiency of GPEI as a carrier in lungs, the effects of aerosol-delivered PDCD4 were investigated in AP-1 luciferase reporter mice. Aerosol delivery of GPEI/PDCD4 through a nose-only inhalation facilitated the apoptosis of lungs whereas aerosol PDCD4 mutant did not. Also, such aerosol delivery regulated proteins relevant to cell-cycle control and suppressed AP-1 activity. Results obtained by western blot analysis, immunohistochemistry, luciferase assay and deoxynucleotidyl-transferase-mediated nick end labeling study suggest that combined actions such as facilitating apoptosis, controlling cell cycle and suppression of AP-1 activity by PDCD4 may provide useful tool for designing lung tumor prevention and treatment by which PDCD4 functions as a transformation suppressor in the future.
-
High clusterin expression correlates with a poor outcome in stage II colorectal cancers.
LENUS (Irish Health Repository)
Kevans, David
2012-02-01
The role of clusterin in tumor growth and progression remains unclear. Overexpression of cytoplasmic clusterin has been studied in aggressive colon tumors; however, no correlation between clusterin expression and survival in colorectal cancer has been identified to date. We assessed levels of clusterin expression in a group of stage II colorectal cancer patients to assess its utility as a prognostic marker. The study included 251 patients with stage II colorectal cancer. Tissue microarrays were constructed and immunohistochemistry done and correlated with clinical features and long term outcome. Dual immunofluorescence and confocal microscopy were used with terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling probes and clusterin antibody to assess the degree of co localization. Percentage epithelial cytoplasmic staining was higher in tumor compared with nonadjacent normal mucosa (P < 0.001). Within the stromal compartment, percentage cytoplamic staining and intensity was lower in tumor tissue compared with normal nonadjacent mucosa (P < or = 0.001). Survival was significantly associated with percentage epithelial cytoplasmic staining (P < 0.001), epithelial cytoplasmic staining intensity (P < 0.001), percentage stromal cytoplasmic staining (P = 0.002), and stromal cytoplasmic staining intensity (P < 0.001). Clusterin levels are associated with poor survival in stage II colorectal cancer.
-
Overexpression of IL-7R alpha provides a competitive advantage during early T-cell development.
Laouar, Yasmina; Crispe, I Nicholas; Flavell, Richard A
2004-03-15
Critical checkpoints controlling early thymic T-cell development and homeostasis are set by the proper signaling function of the interleukin 7 receptor (IL-7R) and the pre-T-cell antigen receptor. Although alpha beta T-cell development is observed in IL-7- and IL-7R alpha-deficient mice, the number of thymocytes is significantly reduced, implying a role for the IL-7R in controlling the size of the thymic T-cell compartment. Here, we report the overexpression of IL-7R alpha that occurs in the early T-cell compartment from AKR/J mice, animals that are highly susceptible to the spontaneous development of thymoma. Increased IL-7R alpha was revealed by surface staining, and increased IL-7R alpha mRNA was documented by using reverse transcriptase-polymerase chain reaction (RT-PCR). This resulted in increased survival of AKR/J early thymocytes, shown by the decreased frequency of TUNEL(+) (terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate [dUTP]-fluorescein nick end labeling) cells. In an in vivo thymocyte repopulation model, AKR/J thymocytes had a selective advantage over healthy thymocytes. This advantage occurred at early stages of T-cell development. Our findings support the model that overexpression of growth factor receptors can contribute to proliferation and malignancy.
-
Population control of resident and immigrant microglia by mitosis and apoptosis.
Wirenfeldt, Martin; Dissing-Olesen, Lasse; Anne Babcock, Alicia; Nielsen, Marianne; Meldgaard, Michael; Zimmer, Jens; Azcoitia, Iñigo; Leslie, Robert Graham Quinton; Dagnaes-Hansen, Frederik; Finsen, Bente
2007-08-01
Microglial population expansion occurs in response to neural damage via processes that involve mitosis and immigration of bone marrow-derived cells. However, little is known of the mechanisms that regulate clearance of reactive microglia, when microgliosis diminishes days to weeks later. We have investigated the mechanisms of microglial population control in a well-defined model of reactive microgliosis in the mouse dentate gyrus after perforant pathway axonal lesion. Unbiased stereological methods and flow cytometry demonstrate significant lesion-induced increases in microglial numbers. Reactive microglia often occurred in clusters, some having recently incorporated bromodeoxyuridine, showing that proliferation had occurred. Annexin V labeling and staining for activated caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling showed that apoptotic mechanisms participate in dissolution of the microglial response. Using bone marrow chimeric mice, we found that the lesion-induced proliferative capacity of resident microglia superseded that of immigrant microglia, whereas lesion-induced kinetics of apoptosis were comparable. Microglial numbers and responses were severely reduced in bone marrow chimeric mice. These results broaden our understanding of the microglial response to neural damage by demonstrating that simultaneously occurring mitosis and apoptosis regulate expansion and reduction of both resident and immigrant microglial cell populations.
-
Shen, Zhaoliang; Zhou, Zipeng; Gao, Shuang; Guo, Yue; Gao, Kai; Wang, Haoyu; Dang, Xiaoqian
2017-08-01
The spinal cord is highly sensitive to spinal cord injury (SCI) by external mechanical damage, resulting in irreversible neurological damage. Activation of the Wnt/β-catenin signaling pathway can effectively reduce apoptosis and protect against SCI. Melatonin, an indoleamine originally isolated from bovine pineal tissue, exerts neuroprotective effects after SCI through activation of the Wnt/β-catenin signaling pathway. In this study, we demonstrated that melatonin exhibited neuroprotective effects on neuronal apoptosis and supported functional recovery in a rat SCI model by activating the Wnt/β-catenin signaling pathway. We found that melatonin administration after SCI significantly upregulated the expression of low-density lipoprotein receptor related protein 6 phosphorylation (p-LRP-6), lymphoid enhancer factor-1 (LEF-1) and β-catenin protein in the spinal cord. Melatonin enhanced motor neuronal survival in the spinal cord ventral horn and improved the locomotor functions of rats after SCI. Melatonin administration after SCI also reduced the expression levels of Bax and cleaved caspase-3 in the spinal cord and the proportion of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) positive cells, but increased the expression level of Bcl-2. These results suggest that melatonin attenuated SCI by activating the Wnt/β-catenin signaling pathway.
-
Polymeric micelles encapsulating fisetin improve the therapeutic effect in colon cancer.
Chen, Yishan; Wu, Qinjie; Song, Linjiang; He, Tao; Li, Yuchen; Li, Ling; Su, Weijun; Liu, Lei; Qian, Zhiyong; Gong, Changyang
2015-01-14
The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) was discovered to possess antitumor activity, revealing its potential value in future chemotherapy. However, its poor water solubility makes it difficult for intravenous administration. In this study, the monomethyl poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) copolymer was applied to prepare nanoassemblies of fisetin by a self-assembly procedure. The prepared fisetin micelles gained a mean particle size of 22 ± 3 nm, polydisperse index of 0.163 ± 0.032, drug loading of 9.88 ± 0.14%, and encapsulation efficiency of 98.53 ± 0.02%. Compared with free fisetin, fisetin micelles demonstrated a sustained and prolonged in vitro release behavior, as well as enhanced cytotoxicity, cellular uptake, and fisetin-induced apoptosis in CT26 cells. As for in vivo studies, fisetin micelles were more competent for suppressing tumor growth and prolonging survival time than free fisetin in the subcutaneous CT26 tumor model. Furthermore, histological analysis, terminal deoxynucleotidyl transferase-mediated nick-end labeling assay, immunohistochemical detection of Ki-67, and microvessel density detection were conducted, demonstrating that fisetin micelles gained increased tumor apoptosis induction, proliferation suppression, and antiangiogenesis activities. In conclusion, we have successfully produced a MPEG-PCL-based nanocarrier encapsulating fisetin with enhanced antitumor activity.
-
Hydroxysafflor Yellow A protects spinal cords from ischemia/reperfusion injury in rabbits
Directory of Open Access Journals (Sweden)
Shan Le-qun
2010-08-01
Full Text Available Abstract Background Hydroxysafflor Yellow A (HSYA, which is one of the most important active ingredients of the Chinese herb Carthamus tinctorius L, is widely used in the treatment of cerebrovascular and cardiovascular diseases. However, the potential protective effect of HSYA in spinal cord ischemia/reperfusion (I/R injury is still unknown. Methods Thirty-nine rabbits were randomly divided into three groups: sham group, I/R group and HSYA group. All animals were sacrificed after neurological evaluation with modified Tarlov criteria at the 48th hour after reperfusion, and the spinal cord segments (L4-6 were harvested for histopathological examination, biochemical analysis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL staining. Results Neurological outcomes in HSYA group were slightly improved compared with those in I/R group. Histopathological analysis revealed that HSYA treatment attenuated I/R induced necrosis in spinal cords. Similarly, alleviated oxidative stress was indicated by decreased malondialdehyde (MDA level and increased superoxide dismutase (SOD activity after HSYA treatment. Moreover, as seen from TUNEL results, HSYA also protected neurons from I/R-induced apoptosis in rabbits. Conclusions These findings suggest that HSYA may protect spinal cords from I/R injury by alleviating oxidative stress and reducing neuronal apoptosis in rabbits.
-
Directory of Open Access Journals (Sweden)
Hai-feng Li
2015-01-01
Full Text Available Lung injury is the main manifestation of paraquat poisoning. Few studies have addressed brain damage after paraquat poisoning. Ulinastatin is a protease inhibitor that can effectively stabilize lysosomal membranes, prevent cell damage, and reduce the production of free radicals. This study assumed that ulinastatin would exert these effects on brain tissues that had been poisoned with paraquat. Rat models of paraquat poisoning were intraperitoneally injected with ulinastatin. Simultaneously, rats in the control group were administered normal saline. Hematoxylin-eosin staining showed that most hippocampal cells were contracted and nucleoli had disappeared in the paraquat group. Fewer cells in the hippocampus were concentrated and nucleoli had disappeared in the ulinastatin group. Western blot assay showed that expressions of GRP78 and cleaved-caspase-3 were significantly lower in the ulinastatin group than in the paraquat group. Immunohistochemical findings showed that CHOP immunoreactivity was significantly lower in the ulinastatin group than in the paraquat group. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining showed that the number of apoptotic cells was reduced in the paraquat and ulinastatin groups. These data confirmed that endoplasmic reticular stress can be induced by acute paraquat poisoning. Ulinastatin can effectively inhibit this stress as well as cell apoptosis, thereby exerting a neuroprotective effect.
-
Jiang, Mei Hua; Lim, Ji Eun; Chi, Guang Fan; Ahn, Woosung; Zhang, Mingzi; Chung, Eunkyung; Son, Youngsook
2013-10-23
Previously, we have reported that substance P (SP) enhanced functional recovery from spinal cord injury (SCI) possibly by the anti-inflammatory modulation associated with the induction of M2-type macrophages at the injured lesion. In this study, we explored the cytokine expression profiles and apoptotic cell death in the lesion site of the SCI after an immediate intravenous injection of SP. SP injection increased the levels of interleukin-4 (IL-4), IL-6, and IL-10 at day 1 after the SCI approximately by 2-, 9-, and 10-folds when compared with the control SCI, respectively. On the basis of double immunofluorescence staining with IL-10 and CD11b, activated macrophages or microglia expressing IL-10 appeared in the margin of the lesion site at day 1 only after the SP injection. This SP-mediated alteration in the lesion microenvironment was shown to be associated with the lower cell death of neuronal cells at day 1 and oligodendrocytes at day 5 by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which was also accompanied by a decrease in caspase-3 activation. These findings suggest that SP may reduce the inflammation-induced secondary cell death, possibly through immune modulation at an early stage after the SCI.
-
Energy Technology Data Exchange (ETDEWEB)
Xia, D.Y. [Department of Neurology, Navy General Hospital of PLA, Beijing (China); Li, W. [General Hospital of Shenyang Military Command, Department of Neurology, Shenyang, China, Department of Neurology, General Hospital of Shenyang Military Command, Shenyang (China); Qian, H.R.; Yao, S.; Liu, J.G.; Qi, X.K. [Department of Neurology, Navy General Hospital of PLA, Beijing (China)
2013-08-10
Sublethal ischemic preconditioning (IPC) is a powerful inducer of ischemic brain tolerance. However, its underlying mechanisms are still not well understood. In this study, we chose four different IPC paradigms, namely 5 min (5 min duration), 5×5 min (5 min duration, 2 episodes, 15-min interval), 5×5×5 min (5 min duration, 3 episodes, 15-min intervals), and 15 min (15 min duration), and demonstrated that three episodes of 5 min IPC activated autophagy to the greatest extent 24 h after IPC, as evidenced by Beclin expression and LC3-I/II conversion. Autophagic activation was mediated by the tuberous sclerosis type 1 (TSC1)-mTor signal pathway as IPC increased TSC1 but decreased mTor phosphorylation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and hematoxylin and eosin staining confirmed that IPC protected against cerebral ischemic/reperfusion (I/R) injury. Critically, 3-methyladenine, an inhibitor of autophagy, abolished the neuroprotection of IPC and, by contrast, rapamycin, an autophagy inducer, potentiated it. Cleaved caspase-3 expression, neurological scores, and infarct volume in different groups further confirmed the protection of IPC against I/R injury. Taken together, our data indicate that autophagy activation might underlie the protection of IPC against ischemic injury by inhibiting apoptosis.
-
From glutathione transferase to pore in a CLIC
Cromer, B A; Morton, C J; Parker, M W; 10.1007/s00249-002-0219-1
2002-01-01
Many plasma membrane chloride channels have been cloned and characterized in great detail. In contrast, very little is known about intracellular chloride channels. Members of a novel class of such channels, called the CLICs (chloride intracellular channels), have been identified over the last few years. A striking feature of the CLIC family of ion channels is that they can exist in a water- soluble state as well as a membrane-bound state. A major step forward in understanding the functioning of these channels has been the recent crystal structure determination of one family member, CLIC1. The structure confirms that CLICs are members of the glutathione S- transferase superfamily and provides clues as to how CLICs can insert into membranes to form chloride channels. (69 refs).
-
Directory of Open Access Journals (Sweden)
Marc eDiederich
2014-07-01
Full Text Available Glutathione S-transferases (GSTs are phase II drug detoxifying enzymes that play an essential role in maintenance of cell integrity and protection against DNA damage by catalyzing the conjugation of glutathione to a wide variety of exo- and endogenous electrophilic substrates. GSTP1, the gene encoding the piclass GST is frequently inactivated by acquired somatic CpG island promoter hypermethylation in multiple cancer subtypes including prostate, breast, liver and blood cancers. Epigenetically mediated GSTP1 silencing is associated with enhanced cancer susceptibility by decreasing its caretaker gene function, which tends to promote neoplastic transformation allowing the cell to acquire additional alterations. Thus, this epigenetic alteration is now considered as a cancer biomarker but could as well play a driving role in multistep cancer development especially well documented in prostate cancer development.The present review discusses application of epigenetic alterations affecting GSTP1 in cancer medicine used alone or in combination with other biomarkers for cancer detection and diagnosis as well as for future targeted preventive and therapeutic interventions including by dietary agents.
-
International Nuclear Information System (INIS)
Cassidy, M.; Gregory, M.C.; Harley, E.H.
1980-01-01
Inherited enzyme deficiencies are found in a small proportion of patients with gout who produce an excess of uric acid. The clinical, biochemical and therapeutic aspects of a case of hyperuricaemia caused by an atypical mutant hypoxanthine-guanine phophoribosyl transferase are presented. Urate overproduction was moderate and controlled by allopurinol therapy
-
Antibiotic inhibition of the movement of tRNA substrates through a peptidyl transferase cavity
DEFF Research Database (Denmark)
Porse, B T; Rodriguez-Fonseca, C; Leviev, I
1996-01-01
The present review attempts to deal with movement of tRNA substrates through the peptidyl transferase centre on the large ribosomal subunit and to explain how this movement is interrupted by antibiotics. It builds on the concept of hybrid tRNA states forming on ribosomes and on the observed movem...
-
International Nuclear Information System (INIS)
Garcia, Wanius; Travensolo, Regiane F.; Rodrigues, Nathalia C.; Muniz, João R. C.; Caruso, Célia S.; Lemos, Eliana G. M.; Araujo, Ana Paula U.; Carrilho, Emanuel
2008-01-01
Glutathione S-transferase from X. fastidiosa (xfGST) has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.23 Å. Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (xfGST) was overexpressed in Escherichia coli and purified by conventional affinity chromatography. In this study, the crystallization and preliminary X-ray analysis of xfGST is described. The purified protein was crystallized by the vapour-diffusion method, producing crystals that belonged to the triclinic space group P1. The unit-cell parameters were a = 47.73, b = 87.73, c = 90.74 Å, α = 63.45, β = 80.66, γ = 94.55°. xfGST crystals diffracted to 2.23 Å resolution on a rotating-anode X-ray source
-
Energy Technology Data Exchange (ETDEWEB)
Garcia, Wanius, E-mail: wanius@if.sc.usp.br [Laboratório de Biofísica Molecular ‘Sérgio Mascarenhas’, Instituto de Física de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Travensolo, Regiane F. [Grupo de Bioanalítica, Microfabricação e Separações, Instituto de Química de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Rodrigues, Nathalia C.; Muniz, João R. C. [Laboratório de Biofísica Molecular ‘Sérgio Mascarenhas’, Instituto de Física de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Caruso, Célia S. [Grupo de Bioanalítica, Microfabricação e Separações, Instituto de Química de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Lemos, Eliana G. M. [Laboratório de Bioquímica de Microrganismos e de Plantas, Departamento de Tecnologia, UNESP, Jaboticabal (Brazil); Araujo, Ana Paula U. [Laboratório de Biofísica Molecular ‘Sérgio Mascarenhas’, Instituto de Física de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Carrilho, Emanuel, E-mail: wanius@if.sc.usp.br [Grupo de Bioanalítica, Microfabricação e Separações, Instituto de Química de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Laboratório de Biofísica Molecular ‘Sérgio Mascarenhas’, Instituto de Física de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil)
2008-02-01
Glutathione S-transferase from X. fastidiosa (xfGST) has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.23 Å. Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (xfGST) was overexpressed in Escherichia coli and purified by conventional affinity chromatography. In this study, the crystallization and preliminary X-ray analysis of xfGST is described. The purified protein was crystallized by the vapour-diffusion method, producing crystals that belonged to the triclinic space group P1. The unit-cell parameters were a = 47.73, b = 87.73, c = 90.74 Å, α = 63.45, β = 80.66, γ = 94.55°. xfGST crystals diffracted to 2.23 Å resolution on a rotating-anode X-ray source.
-
Dirven, H.A.A.M.; Ommen, B. van; Bladeren, P.J. van
1994-01-01
Alkylating agents can be detoxified by conjugation with glutathione (GSH). One of the physiological significances of this lies in the observation that cancer cells resistant to the cytotoxic effects of alkylating agents have higher levels of GSH and high glutathione S-transferase (GST) activity.
-
Willems, A.P.; Gundogdu, M.; Kempers, M.J.E.; Giltay, J.C.; Pfundt, R.P.; Elferink, M.; Loza, B.F.; Fuijkschot, J.; Ferenbach, A.T.; Gassen, K.L. van; Aalten, D.M.F. van; Lefeber, D.J.
2017-01-01
N-Acetylglucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential and dynamic post-translational modification. The O-GlcNAc modification is present on numerous nuclear and cytosolic proteins and has been implicated in essential cellular functions such as signaling and
-
Kop, D.A.M. van der; Schuyer, M.; Scheres, B.J.G.; Zaal, B.J. van der; Hooykaas, P.J.J.
1996-01-01
Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a λ clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions
-
Directory of Open Access Journals (Sweden)
Hao Shi
2018-05-01
Full Text Available Objective: Given that cellular O-GlcNAcylation levels are thought to be real-time measures of cellular nutrient status and dysregulated O-GlcNAc signaling is associated with insulin resistance, we evaluated the role of O-GlcNAc transferase (OGT, the enzyme that mediates O-GlcNAcylation, in skeletal muscle. Methods: We assessed O-GlcNAcylation levels in skeletal muscle from obese, type 2 diabetic people, and we characterized muscle-specific OGT knockout (mKO mice in metabolic cages and measured energy expenditure and substrate utilization pattern using indirect calorimetry. Whole body insulin sensitivity was assessed using the hyperinsulinemic euglycemic clamp technique and tissue-specific glucose uptake was subsequently evaluated. Tissues were used for histology, qPCR, Western blot, co-immunoprecipitation, and chromatin immunoprecipitation analyses. Results: We found elevated levels of O-GlcNAc-modified proteins in obese, type 2 diabetic people compared with well-matched obese and lean controls. Muscle-specific OGT knockout mice were lean, and whole body energy expenditure and insulin sensitivity were increased in these mice, consistent with enhanced glucose uptake and elevated glycolytic enzyme activities in skeletal muscle. Moreover, enhanced glucose uptake was also observed in white adipose tissue that was browner than that of WT mice. Interestingly, mKO mice had elevated mRNA levels of Il15 in skeletal muscle and increased circulating IL-15 levels. We found that OGT in muscle mediates transcriptional repression of Il15 by O-GlcNAcylating Enhancer of Zeste Homolog 2 (EZH2. Conclusions: Elevated muscle O-GlcNAc levels paralleled insulin resistance and type 2 diabetes in humans. Moreover, OGT-mediated signaling is necessary for proper skeletal muscle metabolism and whole-body energy homeostasis, and our data highlight O-GlcNAcylation as a potential target for ameliorating metabolic disorders. Keywords: O-GlcNAc signaling, Type 2 diabetes, N
-
DEFF Research Database (Denmark)
Poulsen, S M; Karlsson, M; Johansson, L B
2001-01-01
The pleuromutilin antibiotic derivatives, tiamulin and valnemulin, inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria. The action and binding site of tiamulin and valnemulin was further characterized on Escherichia coli ribosomes. It was revealed that these drugs...... centre and have been associated with binding of several antibiotics. Competitive footprinting shows that tiamulin and valnemulin can bind concurrently with the macrolide erythromycin but compete with the macrolide carbomycin, which is a peptidyl transferase inhibitor. We infer from these and previous...... results that tiamulin and valnemulin interact with the rRNA in the peptidyl transferase slot on the ribosomes in which they prevent the correct positioning of the CCA-ends of tRNAs for peptide transfer....
-
Inhibition of rat, mouse, and human glutathione S-transferase by eugenol and its oxidation products
Rompelberg, C.J.M.; Ploemen, J.H.T.M.; Jespersen, S.; Greef, J. van der; Verhagen, H.; Bladeren, P.J. van
1996-01-01
The irreversible and reversible inhibition of glutathione S-transferases (GSTs) by eugenol was studied in rat, mouse and man. Using liver cytosol of human, rat and mouse, species differences were found in the rate of irreversible inhibition of GSTs by eugenol in the presence of the enzyme
-
Bøsling, Jacob; Poulsen, Susan M; Vester, Birte; Long, Katherine S
2003-09-01
The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the peptidyl transferase center. Tiamulin-resistant Escherichia coli mutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon. Selection in a strain with all seven chromosomal rRNA operons yielded a mutant with an A445G mutation in the gene coding for ribosomal protein L3, resulting in an Asn149Asp alteration. Complementation experiments and sequencing of transductants demonstrate that the mutation is responsible for the resistance phenotype. Chemical footprinting experiments show a reduced binding of tiamulin to mutant ribosomes. It is inferred that the L3 mutation, which points into the peptidyl transferase cleft, causes tiamulin resistance by alteration of the drug-binding site. This is the first report of a mechanism of resistance to tiamulin unveiled in molecular detail.
-
Aralia elata inhibits neurodegeneration by downregulating O-GlcNAcylation of NF-κB in diabetic mice
Directory of Open Access Journals (Sweden)
Seong-Jae Kim
2017-08-01
Full Text Available AIM: To investigate the role of O-GlcNAcylation of nuclear factor-kappa B (NF-κB in retinal ganglion cell (RGC death and analysedthe effect of Aralia elata (AE on neurodegeneration in diabetic mice. METHODS: C57BL/6mice with streptozotocin-induced diabetes were fed daily with AE extract or control (CTL diet at the onset of diabetes mellitus (DM. Two months after injection of streptozotocin or saline, the degree of cell death and the expression of O-GlcNAc transferase (OGT, N-acetyl-b-D-glucosaminidase (OGA, O-GlcNAcylated proteins, and O-GlcNAcylation of NF-κB were examined. RESULTS: AE did not affect the metabolic status of diabetic mice. The decrease in the inner retinal thickness (P<0.001 vs CTL, P<0.01 vs DM and increases in RGCs with terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (P<0.001 vs CTL, P<0.0001 vs DM, glial activation, and active caspase-3 (P<0.0001 vs CTL, P<0.0001 vs DM were blocked in diabetic retinas of AE extract-fed mice. Expression levels of protein O-GlcNAcylation and OGT were increased in diabetic retinas (P<0.0001 vs CTL, and the level of O-GlcNAcylation of the NF-κB p65 subunit was higher in diabetic retinas than in controls (P<0.0001 vs CTL. AE extract downregulated O-GlcNAcylation of NF-κB and prevented neurodegeneration induced by hyperglycemia (P<0.0001 vs DM. CONCLUSION: O-GlcNAcylation of NF-κB is concerned in neuronal degeneration and that AE prevents diabetes-induced RGC apoptosis via downregulation of NF-κB O-GlcNAcylation. Hence, O-GlcNAcylation may be a new object for the treatment of DR, and AE may have therapeutic possibility to prevent diabetes-induced neurodegeneration.
-
Lack of Ach1 CoA-Transferase Triggers Apoptosis and Decreases Chronological Lifespan in Yeast
International Nuclear Information System (INIS)
Orlandi, Ivan; Casatta, Nadia; Vai, Marina
2012-01-01
ACH1 encodes a mitochondrial enzyme of Saccharomyces cerevisiae endowed with CoA-transferase activity. It catalyzes the CoASH transfer from succinyl-CoA to acetate generating acetyl-CoA. It is known that ACH1 inactivation results in growth defects on media containing acetate as a sole carbon and energy source which are particularly severe at low pH. Here, we show that chronological aging ach1Δ cells which accumulate a high amount of extracellular acetic acid display a reduced chronological lifespan. The faster drop of cell survival is completely abrogated by alleviating the acid stress either by a calorie restricted regimen that prevents acetic acid production or by transferring chronologically aging mutant cells to water. Moreover, the short-lived phenotype of ach1Δ cells is accompanied by reactive oxygen species accumulation, severe mitochondrial damage, and an early insurgence of apoptosis. A similar pattern of endogenous severe oxidative stress is observed when ach1Δ cells are cultured using acetic acid as a carbon source under acidic conditions. On the whole, our data provide further evidence of the role of acetic acid as cell-extrinsic mediator of cell death during chronological aging and highlight a primary role of Ach1 enzymatic activity in acetic acid detoxification which is important for mitochondrial functionality.
-
Liu, Xu-hua; Chen, Yu; Wang, Tai-ling; Lu, Jun; Zhang, Li-jie; Song, Chen-zhao; Zhang, Jing; Duan, Zhong-ping
2007-10-01
To establish a practical and reproducible animal model of human acute-on-chronic liver failure for further study of the pathophysiological mechanism of acute-on-chronic liver failure and for drug screening and evaluation in its treatment. Immunological hepatic fibrosis was induced by human serum albumin in Wistar rats. In rats with early-stage cirrhosis (fibrosis stage IV), D-galactosamine and lipopolysaccharide were administered. Mortality and survival time were recorded in 20 rats. Ten rats were sacrificed at 4, 8, and 12 hours. Liver function tests and plasma cytokine levels were measured after D-galactosamine/lipopolysaccharide administration and liver pathology was studied. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Most of the rats treated with human albumin developed cirrhosis and fibrosis, and 90% of them died from acute liver failure after administration of D-galactosamine/lipopolysaccharide, with a mean survival time of (16.1+/-3.7) hours. Liver histopathology showed massive or submassive necrosis of the regenerated nodules, while fibrosis septa were intact. Liver function tests were compatible with massive necrosis of hepatocytes. Plasma level of TNFalpha increased significantly, parallel with the degree of the hepatocytes apoptosis. Plasma IL-10 levels increased similarly as seen in patients with acute-on-chronic liver failure. We established an animal model of acute-on-chronic liver failure by treating rats with human serum albumin and later with D-galactosamine and lipopolysaccharide. TNFalpha-mediated liver cell apoptoses plays a very important role in the pathogenesis of acute liver failure.
-
Lei, Shan; Zhang, Pengbo; Li, Weisong; Gao, Ming; He, Xijing; Zheng, Juan; Li, Xu; Wang, Xiao; Wang, Ning; Zhang, Junfeng; Qi, Cunfang; Lu, Haixia; Chen, Xinlin; Liu, Yong
2014-01-01
Edaravone is clinically used for treatment of patients with acute cerebral infarction. However, the effect of double application of edaravone on neurogenesis in the hippocampus following ischemia remains unknown. In the present study, we explored whether pre- and posttreatment of edaravone had any effect on neural stem/progenitor cells (NSPCs) in the subgranular zone of hippocampus in a rat model of transient global cerebral ischemia and elucidated the potential mechanism of its effects. Male Sprague-Dawley rats were divided into three groups: sham-operated (n = 15), control (n = 15), and edaravone-treated (n = 15) groups. Newly generated cells were labeled by 5-bromo-2-deoxyuridine. Immunohistochemistry was used to detect neurogenesis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling was used to detect cell apoptosis. Reactive oxygen species (ROS) were detected by 2,7-dichlorofluorescien diacetate assay in NSPCs in vitro. Hypoxia-inducible factor-1α (HIF-1α) and cleaved caspase-3 proteins were quantified by western blot analysis. Treatment with edaravone significantly increased the number of NSPCs and newly generated neurons in the subgranular zone (p edaravone also decreased apoptosis of NSPCs (p edaravone significantly decreased ROS generation and inhibited HIF-1α and cleaved caspase-3 protein expressions. These findings indicate that pre- and posttreatment with edaravone enhances neurogenesis by protecting NSPCs from apoptosis in the hippocampus, which is probably mediated by decreasing ROS generation and inhibiting protein expressions of HIF-1α and cleaved caspase-3 after cerebral ischemia. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.
-
Directory of Open Access Journals (Sweden)
Shan Lei
2014-11-01
Full Text Available Edaravone is clinically used for treatment of patients with acute cerebral infarction. However, the effect of double application of edaravone on neurogenesis in the hippocampus following ischemia remains unknown. In the present study, we explored whether pre- and posttreatment of edaravone had any effect on neural stem/progenitor cells (NSPCs in the subgranular zone of hippocampus in a rat model of transient global cerebral ischemia and elucidated the potential mechanism of its effects. Male Sprague-Dawley rats were divided into three groups: sham-operated (n = 15, control (n = 15, and edaravone-treated (n = 15 groups. Newly generated cells were labeled by 5-bromo-2-deoxyuridine. Immunohistochemistry was used to detect neurogenesis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling was used to detect cell apoptosis. Reactive oxygen species (ROS were detected by 2,7-dichlorofluorescien diacetate assay in NSPCs in vitro. Hypoxia-inducible factor-1α (HIF-1α and cleaved caspase-3 proteins were quantified by western blot analysis. Treatment with edaravone significantly increased the number of NSPCs and newly generated neurons in the subgranular zone (p < .05. Treatment with edaravone also decreased apoptosis of NSPCs (p < .01. Furthermore, treatment with edaravone significantly decreased ROS generation and inhibited HIF-1α and cleaved caspase-3 protein expressions. These findings indicate that pre- and posttreatment with edaravone enhances neurogenesis by protecting NSPCs from apoptosis in the hippocampus, which is probably mediated by decreasing ROS generation and inhibiting protein expressions of HIF-1α and cleaved caspase-3 after cerebral ischemia.
-
Directory of Open Access Journals (Sweden)
Yao Chou Tsai
2016-09-01
Full Text Available Purpose The aim of this study was to analyze patterns of sensory protein expression and urothelial dysfunction in ketamine-related cystitis (KC in humans. Methods Biopsies of bladder mucosa were performed in 29 KC patients during cystoscopy. Then specimens were analyzed for tryptase, zonula occludens-1 (ZO-1, E-cadherin, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL with immunofluorescence staining and quantification. In addition, 10 healthy control bladder specimens were analyzed and compared with the KC specimens. Another 16 whole bladder specimens obtained from partial cystectomy were also analyzed for the muscarinic receptors M2 and M3, endothelial nitric oxide synthase (eNOS, inducible nitric oxide synthase (iNOS, β-3 adrenergic receptors (β3-ARs, and the P2X3 receptor by western blotting. In addition, 3 normal control bladder specimens were analyzed and compared with the KC specimens. Results The KC bladder mucosa revealed significantly less expression of ZO-1 and E-cadherin, and greater expression of TUNEL and tryptase activity than the control samples. The expression of M3 and β3-AR in the KC specimens was significantly greater than in the controls. The expression of iNOS, eNOS, M2, and P2X3 was not significantly different between the KC and control specimens. Conclusions The bladder tissue of KC patients revealed significant urothelial dysfunction, which was associated with mast-cell mediated inflammation, increased urothelial cell apoptosis, and increased expression of the M3 and β3-AR.
-
Directory of Open Access Journals (Sweden)
Harshavardhanan Vijayakumar
2016-07-01
Full Text Available Plants, as sessile organisms, can suffer serious growth and developmental consequences under cold stress conditions. Glutathione transferases (GSTs, EC 2.5.1.18 are ubiquitous and multifunctional conjugating proteins, which play a major role in stress responses by preventing oxidative damage by reactive oxygen species (ROS. Currently, understanding of their function(s during different biochemical and signaling pathways under cold stress condition remain unclear. In this study, using combined computational strategy, we identified 65 Brassica oleracea glutathione transferases (BoGST and characterized them based on evolutionary analysis into 11 classes. Inter-species and intra-species duplication was evident between BoGSTs and Arabidopsis GSTs. Based on localization analyses, we propose possible pathways in which GST genes are involved during cold stress. Further, expression analysis of the predicted putative functions for GST genes were investigated in two cold contrasting genotypes (cold tolerance and susceptible under cold condition, most of these genes were highly expressed at 6 h and 1 h in the cold tolerant (CT and cold susceptible (CS lines, respectively. Overall, BoGSTU19, BoGSTU24, BoGSTF10 are candidate genes highly expressed in B. oleracea. Further investigation of GST superfamily in B. oleracea will aid in understanding complex mechanism underlying cold tolerance in plants.
-
Changes in amino transferases and muscle proteins when treating pigmeat with ionising rays
Energy Technology Data Exchange (ETDEWEB)
Hamm, R; Hofmann, K; Gruenewald, T; Partmann, W [Bundesanstalt fuer Fleischforschung, Kulmbach (F.R. Germany). Inst. fuer Chemie und Physik; Bundesforschungsanstalt fuer Ernaehrung, Karlsruhe [F.R. Germany
1975-01-01
Slices of lean pigmeat were treated with electron beams doses of 0.2, 1.0 and 5.0 Mrad. Low irradiation doses led to an increase in the activity of aspartate amino transferase (GOT) and alanin amino transferase (GPT) in the tissue generally and in the sarcoplasm (juice expressed from the muscle). 5 Mrad caused a great reduction in the activity of GOT and GPT in the tissue and the sarcoplasm. It seems doubtful whether this inactivation is due to a destruction of enzyme sulphhydryl groups. Irradiating with 5 Mrad resulted in partial release of the mitochondrial GOT isozyme (GOTsub(M)) into the sarcoplasm. This indicates damage to the mitochondrial membranes by ionising radiation. Irradiating the pigmeat increased the pH of the tissue and lowered its water binding ability (increase in drip). Up to a dose of 1 Mrad the solubility of the sarcoplasmic proteins was not definitely affected, but 5 Mrad caused a considerable drop in protein solubility. Surprisingly a dose of even 5 Mrad did not change the total number of sulphhydryl groups present in the tissue. Sephadex thin layer electrophoresis showed that at 0.2 Mrad there was a drastic decrease in the myosin band and an increase in peptide fragments of low molecular weight, whilst actin was little changed.
-
Neta, Raimunda Nonata Fortes Carvalho; Torres, Audalio Rebelo
2017-11-01
In this work, we validate the glutathione-S-transferase and branchial lesions as biomarkers in catfish Sciades herzbergii to obtain a predictive model of the environmental impact effects in a harbor of Brazil. The catfish were sampled from a port known to be contaminated with heavy metals and organic compounds and from a natural reserve in São Marcos Bay, Maranhão. Two biomarkers, hepatic glutathione S-transferase (GST) activity and branchial lesions were analyzed. The values for GST activity were modeled with the occurrence of branchial lesions by fitting a third order polynomial. Results from the mathematical model indicate that GST activity has a strong polynomial relationship with the occurrence of branchial lesions in both the wet and the dry seasons, but only at the polluted port site. Our mathematic model indicates that when the GST ceases to act, serious branchial lesions are observed in the catfish of the contaminated port area.
-
Effects of curcumin on cytochrome P450 and glutathione S-transferase activities in rat liver.
Oetari, S.; Sudibyo, M.; Commandeur, J.N.M.; Samhoedi, R.; Vermeulen, N.P.E.
1996-01-01
The stability of curcumin, as well as the interactions between curcumin and cytochrome P450s (P450s) and glutathione S-transferases (GSTs) in rat liver, were studied. Curcumin is relatively unstable in phosphate buffer at pH 7.4. The stability of curcumin was strongly improved by lowering the pH or
-
DEFF Research Database (Denmark)
Rivas, Matilde De Las; Lira-Navarrete, Erandi; Daniel, Earnest James Paul
2017-01-01
The polypeptide GalNAc-transferases (GalNAc-Ts), that initiate mucin-type O-glycosylation, consist of a catalytic and a lectin domain connected by a flexible linker. In addition to recognizing polypeptide sequence, the GalNAc-Ts exhibit unique long-range N- A nd/or C-terminal prior glycosylation ...
-
Glutathione S-transferase M1 and T1, CYP1A2-2467T/delT ...
African Journals Online (AJOL)
The present study investigated the impact of metabolic gene polymorphisms in modulating lung cancer risk susceptibility. Gene polymorphisms encoding Cytochrome 1A2 (CYP1A2) and Glutathione-S-transferases (GSTT1 and GSTM1) are involved in the bioactivation and detoxification of tobacco carcinogens and may ...
-
Parsa, Kishore V. L.; Kain, Vasundhara; Behera, Soma; Suraj, Sashidhara Kaimal; Babu, Phanithi Prakash; Kar, Anand; Panda, Sunanda; Zhu, Yi-jun; Jia, Yuzhi; Thimmapaya, Bayar; Reddy, Janardan K.; Misra, Parimal
2013-01-01
PRIP-Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB-binding protein (CBP)/ E1A binding protein p300 (Ep300) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERK2 phosphorylates PIMT at Ser298 and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMTS298D) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMTS298D but not PIMTS298A augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U0126, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T4 hormone to rats activated ERK1/2 resulting in enhanced PIMT ser298 phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERK2-mediated phosphorylation of PIMT at Ser298 is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia. PMID:24358311
-
LENUS (Irish Health Repository)
Bennett, M W
2012-02-03
Various cancer cell lines express Fas ligand (FasL) and can kill lymphoid cells by Fas-mediated apoptosis in vitro. FasL expression has been demonstrated in several human malignancies in vivo. We sought to determine whether human esophageal carcinomas express FasL, and whether FasL expression is associated with increased apoptosis of tumor-infiltrating lymphocytes (TIL) in vivo, thereby contributing to the immune privilege of the tumor. Using in situ hybridization and immunohistochemistry, respectively, FasL mRNA and protein were colocalized to neoplastic esophageal epithelial cells in all esophageal carcinomas (squamous, n = 6; adenocarcinoma, n = 2). The Extent of FasL expression was variable, with both FasL-positive and FasL-negative neoplastic regions occurring within tumors. TIL were detected by immunohistochemical staining for the leukocyte common Ag, CD45. FasL expression was associated with a mean fourfold depletion of TIL when compared with FasL-negative areas within the same tumors (range 1.6- to 12-fold, n = 6,p < 0.05). Cell death of TIL was detected by dual staining of CD45 (immunohistochemistry) and DNA strand breaks (TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). There was a mean twofold increase in detectable cell death among TIL in FasL-positive areas compared with FasL-negative areas (range 1.6- to 2.4-fold, n = 6, p < 0.05). In conclusion, we demonstrate a statistically significant, quantitative reduction of TIL concomitant with significantly increased TIL apoptosis within FasL-expressing areas of esophageal tumors. Our findings suggest Fas-mediated apoptotic depletion of TIL in response to FasL expression by esophageal cancers, and provide the first direct, quantitative evidence to support the Fas counterattack as a mechanism of immune privilege in vivo in human cancer.
-
Oxidative stress is involved in Dasatinib-induced apoptosis in rat primary hepatocytes
Energy Technology Data Exchange (ETDEWEB)
Xue, Tao; Luo, Peihua; Zhu, Hong; Zhao, Yuqin [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Wu, Honghai; Gai, Renhua; Wu, Youping [Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China); Yang, Bo [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Yang, Xiaochun, E-mail: yangxiaochun@zju.edu.cn [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China); He, Qiaojun, E-mail: qiaojunhe@zju.edu.cn [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China)
2012-06-15
Dasatinib, a multitargeted inhibitor of BCR–ABL and SRC kinases, exhibits antitumor activity and extends the survival of patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL). However, some patients suffer from hepatotoxicity, which occurs through an unknown mechanism. In the present study, we found that Dasatinib could induce hepatotoxicity both in vitro and in vivo. Dasatinib reduced the cell viability of rat primary hepatocytes, induced the release of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in vitro, and triggered the ballooning degeneration of hepatocytes in Sprague–Dawley rats in vivo. Apoptotic markers (chromatin condensation, cleaved caspase-3 and cleaved PARP) were detected to indicate that the injury induced by Dasatinib in hepatocytes in vitro was mediated by apoptosis. This result was further validated in vivo using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Here we found that Dasatinib dramatically increased the level of reactive oxygen species (ROS) in hepatocytes, reduced the intracellular glutathione (GSH) content, attenuated the activity of superoxide dismutase (SOD), generated malondialdehyde (MDA), a product of lipid peroxidation, decreased the mitochondrial membrane potential, and activated nuclear factor erythroid 2-related factor 2 (Nrf2) and mitogen-activated protein kinases (MAPK) related to oxidative stress and survival. These results confirm that oxidative stress plays a pivotal role in Dasatinib-mediated hepatotoxicity. N-acetylcysteine (NAC), a typical antioxidant, can scavenge free radicals, attenuate oxidative stress, and protect hepatocytes against Dasatinib-induced injury. Thus, relieving oxidative stress is a viable strategy for reducing Dasatinib-induced hepatotoxicity. -- Highlights: ►Dasatinib shows potential hepatotoxicity both in vitro and in vivo. ►Apoptosis plays a vital role in Dasatinib
-
Prins, T.W.; Wagemakers, L.; Schouten, A.; Kan, van J.A.L.
2000-01-01
A gene was cloned from Botrytis cinerea that encodes a protein homologous to glutathione S-transferase (GST). The gene, denominated Bcgst1, is present in a single copy and represents the first example of such a gene from a filamentous fungus. The biochemical function of GSTs is to conjugate toxic
-
van Beek, J.H.D.A.; de Moor, M.H.M.; Geels, L.M.; Sinke, M.R.T.; de Geus, E.J.C.; Lubke, G.H.; Kluft, C.; Neuteboom, J.; Vink, J.M.; Willemsen, G.; Boomsma, D.I.
2014-01-01
Background: Blood levels of gamma-glutamyl transferase (GGT) are used as a marker for (heavy) alcohol use. The role of GGT in the anti-oxidant defense mechanism that is part of normal metabolism supposes a causal effect of alcohol intake on GGT. However, there is variability in the response of GGT
-
Directory of Open Access Journals (Sweden)
Gulhan Bourget
Full Text Available The Transmission Disequilibrium Test (TDT compares frequencies of transmission of two alleles from heterozygote parents to an affected offspring. This test requires all genotypes to be known from all members of the nuclear families. However, obtaining all genotypes in a study might not be possible for some families, in which case, a data set results in missing genotypes. There are many techniques of handling missing genotypes in parents but only a few in offspring. The robust TDT (rTDT is one of the methods that handles missing genotypes for all members of nuclear families [with one affected offspring]. Even though all family members can be imputed, the rTDT is a conservative test with low power. We propose a new method, Mendelian Inheritance TDT (MITDT-ONE, that controls type I error and has high power. The MITDT-ONE uses Mendelian Inheritance properties, and takes population frequencies of the disease allele and marker allele into account in the rTDT method. One of the advantages of using the MITDT-ONE is that the MITDT-ONE can identify additional significant genes that are not found by the rTDT. We demonstrate the performances of both tests along with Sib-TDT (S-TDT in Monte Carlo simulation studies. Moreover, we apply our method to the type 1 diabetes data from the Warren families in the United Kingdom to identify significant genes that are related to type 1 diabetes.
-
Directory of Open Access Journals (Sweden)
Jing Han
2012-01-01
Full Text Available In linkage analysis for mapping genetic diseases, the transmission/disequilibrium test (TDT uses the linkage disequilibrium (LD between some marker and trait loci for precise genetic mapping while avoiding confounding due to population stratification. The sib-TDT (S-TDT and combined-TDT (C-TDT proposed by Spielman and Ewens can combine data from families with and without parental marker genotypes (PMGs. For some families with missing PMG, the reconstruction-combined TDT (RC-TDT proposed by Knapp may be used to reconstruct missing parental genotypes from the genotypes of their offspring to increase power and to correct for potential bias. In this paper, we propose a further extension of the RC-TDT, called the reconstruction-combined transmission disequilibrium/heterogeneity (RC-TDH test, to take into account the identical-by-descent (IBD sharing information in addition to the LD information. It can effectively utilize families with missing or incomplete parental genetic marker information. An application of this proposed method to Genetic Analysis Workshop 14 (GAW14 data sets and extensive simulation studies suggest that this approach may further increase statistical power which is particularly valuable when LD is unknown and/or when some or all PMGs are not available.
-
Anak Ngadin , Andrew
2011-01-01
Phanerochaete chrysosporium is a ligninolytic fungus widely studied because of its capacities to degrade wood and xenobiotics through an extracellular enzymatic system. Its genome has been sequenced and has provided researchers with a complete inventory of the predicted proteins produced by this organism. This has allowed the description of many protein superfamilies. Among them, Glutathione S-transferases (GSTs) constitute a complex and widespread superfamily classified as enzymes of seconda...
-
CONTACT (NAME ONLY): Hassan El-Fawal Abstract Details PRESENTATION TYPE: Platform or Poster CURRENT CATEGORY: Neurodegenerative Disease | Biomarkers | Neurotoxicity, Metals KEYWORDS: Autoantibodies, Glutathione-S-Transferase, DATE/TIME LAST MODIFIED: DATE/TIME SUBMITTED: Abs...
-
Markers of potential malignancy in chronic hyperplastic candidiasis.
Darling, Mark R; McCord, Christina; Jackson-Boeters, Linda; Copete, Maria
2012-08-01
To examine the presence of markers associated with malignancy, including p53, p21 cyclin-dependent kinase inhibitor 1A, murine double minutes-2, and others, in chronic hyperplastic candidiasis. Immunohistochemical methods were used to examine the expression of p53, murine double minutes-2, p21 cyclin-dependent kinase inhibitor 1A, metallothionein, and proliferating cell nuclear antigen in 42 chronic hyperplastic candidiasis lesions and 11 non-infected control tissues. Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling was used to examine apoptosis, which was correlated with p53 expression. These markers were measured in lesions of chronic hyperplastic candidiasis that did not show any epithelial dysplasia or histological signs of malignancy. p53 scores were higher in chronic hyperplastic candidiasis than in controls (P = 0.0046). Murine double-minutes 2 levels were not elevated. p21 cyclin-dependent kinase inhibitor 1A was increased in parabasal (P candidiasis lesions showed a similar basal/parabasal metallothionein staining pattern to that seen in normal squamous epithelium. Proliferating cell nuclear antigen was increased (P = 0.0007), as was apoptosis (P = 0.0033). Increased p53 in oral chronic hyperplastic candidiasis suggests an increased potential for malignant change in the epithelium, above that of normal tissues. Further functional investigation is required, as well as clinical follow-up studies. © 2012 Blackwell Publishing Asia Pty Ltd.
-
Yu, Di; Fan, Changfeng; Zhang, Weiyan; Wen, Zhongyuan; Hu, Liang; Yang, Lei; Feng, Yu; Yin, Ke-Jie; Mo, Xuming
2015-10-15
Nicorandil exerts a protective effect on ischemia-reperfusion (I/R) injury in the brain and kidney through anti-apoptotic mechanisms. However, the mechanism by which nicorandil protects against I/R injury induced by deep hypothermic low flow (DHLF) remains unclear. We used a cerebral I/R model induced by DHLF to determine the neuroprotective effects and possible mechanisms of nicorandil. Hematoxylin-eosin (HE) staining and in situ terminal deoxynucleotidyl transferase UTP nick end labeling (TUNEL) assay were used to detect changes in cell morphology and the number of apoptotic cells in hippocampus, respectively. The apoptotic regulators including Bcl-2, Bax, Akt, and p-Akt (the active, phosphorylated form of Akt) were examined by Western blot (WB). Histopathological findings showed that nicorandil significantly alleviated morphological damage in hippocampal and reduced the number of TUNEL-positive nuclei induced by DHLF. Nicorandil also increased the expression of Bcl-2 and decreased the expression of Bax, while increasing p-Akt level. Consistent with these results, nicorandil-mediated neuroprotection was reduced in the Akt1+/- mutant mice and inhibited by LY294002, a PI3K inhibitor. These findings showed that nicorandil provides a neuroprotective role in DHLF-induced I/R injury by inhibiting apoptosis via activation of the PI3K/Akt1 signaling pathway. Copyright © 2015 Elsevier B.V. All rights reserved.
-
International Nuclear Information System (INIS)
Su, Chen-Ming; Wang, Shih-Wei; Lee, Tzong-Huei; Tzeng, Wen-Pei; Hsiao, Che-Jen; Liu, Shih-Chia; Tang, Chih-Hsin
2013-01-01
Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress. - Highlights: • Trichodermin induces chondrosarcoma apoptosis. • ER stress is involved in trichodermin-induced cell death. • Trichodermin induces chondrosarcoma death in vivo.
-
Energy Technology Data Exchange (ETDEWEB)
Su, Chen-Ming [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Wang, Shih-Wei [Department of Medicine, Mackay Medical College, New Taipei City, Taiwan (China); Lee, Tzong-Huei [Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan (China); Tzeng, Wen-Pei [Graduate Institute of Sports and Health, National Changhua University of Education, Changhua, Taiwan (China); Hsiao, Che-Jen [School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Liu, Shih-Chia [Department of Orthopaedics, Mackay Memorial Hospital, Taipei, Taiwan (China); Tang, Chih-Hsin, E-mail: chtang@mail.cmu.edu.tw [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan (China); Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan (China)
2013-10-15
Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress. - Highlights: • Trichodermin induces chondrosarcoma apoptosis. • ER stress is involved in trichodermin-induced cell death. • Trichodermin induces chondrosarcoma death in vivo.
-
Beltrán-Frutos, E; Seco-Rovira, V; Ferrer, C; Madrid, J F; Sáez, F J; Canteras, M; Pastor, L M
2016-04-01
The aim of this study was to evaluate the cellular changes that occur in the hamster testicular interstitium in two very different physiological situations involving testicular involution: ageing and exposure to a short photoperiod. The animals were divided into an 'age group' with three subgroups - young, adult and old animals - and a 'regressed group' with animals subjected to a short photoperiod. The testicular interstitium was characterised by light and electron microscopy. Interstitial cells were studied histochemically with regard to their proliferation, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end labelling (TUNEL+) and testosterone synthetic activity. We identified two types of Leydig cell: Type A cells showed a normal morphology, while Type B cells appeared necrotic. With ageing, pericyte proliferation decreased but there was no variation in the index of TUNEL-positive Leydig cells. In the regressed group, pericyte proliferation was greater and TUNEL-positive cells were not observed in the interstitium. The testicular interstitium suffered few ultrastructural changes during ageing and necrotic Leydig cells were observed. In contrast, an ultrastructural involution of Leydig cells with no necrosis was observed in the regressed group. In conclusion, the testicular interstitium of Mesocricetus auratus showed different cellular changes in the two groups (age and regressed), probably due to the irreversible nature of ageing and the reversible character of changes induced by short photoperiod.
-
Directory of Open Access Journals (Sweden)
Barbara Dariš
2018-02-01
Full Text Available Background. To determine the relationship between sperm morphological abnormalities, DNA fragmentation and fertilization rate in IVF and ICSI. Methods. Sperm samples from 10 IVF and 20 ICSI cycles were analyzed. Morphology was assessed according to strict criteria, and DNA fragmentation was measured by terminal deoxynucleotidyl transferase (TdT-mediated fluorescein-dUTP nick end labelling (TUNEL using a flow cytometry. Results. There was a significant difference in the amount of morphological abnormalities between sperm samples with low (< 20 % and high (≥ 20 % degree of DNA fragmentation. The percentages of amorphous heads (10 vs. 4 % and overall head abnormalities (42 vs. 30 % were significantly higher in sperm samples with elevated degree of DNA fragmentation. No correlation was found between sperm DNA fragmentation and fertilization rate after IVF and ICSI. When the predominant morphological abnormality in sperm samples was determined, a negative correlation was found between the percentage of spermatozoa with elongated heads and fertilization rate in ICSI (r = –0.45, P < 0.05. The fertilization rate after IVF was lower in the case of acrosomal abnormalities (35.3 %, compared to the cases of other predominant morphological abnormalities. Conclusions. Head abnormalities, especially amorphous heads, are related to elevated degree of DNA fragmentation. Predominant abnormal form in sperm samples, such as elongated heads and acrosomal abnormalities, may affect fertilization in ART.
-
Mirella da Silva, P; Villalba, Antonio; Sunila, Inke
2006-06-12
An experiment to evaluate differences in growth, mortality and disease susceptibility among Ostrea edulis stocks was performed. Five families were produced from each of 4 oyster populations (Irish, Greek and 2 Galician). The spat were transferred to a raft in the Ria de Arousa (Galicia, Spain) for grow-out. Monthly samples of each family were histologically processed from 2001 to 2003. One of the pathological conditions discovered by this study was the occurrence of extensive branchial lesions characterized by haemocytic infiltration and loss of branchial architecture. Furthermore, abundant atypical cells occurred among the haemocytes in the lesions in the branchial connective and epithelial tissues, but rarely in the mantle. These cells were contracted in size with nuclei showing chromatin condensation and fragmentation. Some nuclear chromatin aggregated under the nuclear membranes into crescent shapes, whereas others were uniformly dense. Those characteristics suggested that the cells were apoptotic haemocytes, which was confirmed by transmission electron microscopy (TEM) and by a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) assay using the Apoptag Kit on paraffin sections. A low prevalence of gill lesions was detected in some, but not all, families of every origin peaking in July 2002 and April 2003. No etiologic agent was identified by either histology or TEM; thus, the cause of the abundance of apoptotic cells remains unclear.
-
Yang, Yang; Yang, Ming; Ai, Fen; Huang, Congxin
2017-06-01
The present study was undertaken to evaluate the cardioprotection potential and underlying molecular mechanism afforded by a selenium (Se) polysaccharide (Se-AVP) from Aloe vera in the ischemia-reperfusion (I/R) model of rats in vivo. Myocardial I/R injury was induced by occluding the left anterior descending coronary artery (LAD) for 30 min followed by 2-h continuous reperfusion. Pretreatment with Se-AVP (100, 200, and 400 mg/kg) attenuated myocardial damage, as evidenced by reduction of the infarct sizes, increase in serum and myocardial endogenous antioxidants (superoxide dismutase (SOD), glutathione peroxidase (GSH), and catalase (CAT)), and decrease in the malondialdehyde (MDA) level in the rats suffering I/R injury. This cardioprotective activity afforded by Se-AVP is further supported by the decreased levels of cardiac marker enzymes creatine kinase (CK) and lactate dehydrogenase (LDH), as well as the rise of myocardial Na + -K + -ATPase and Ca 2+ -Mg 2+ -ATPase activities in I/R rats. Additionally, cardiomyocytic apoptosis was measured by terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining and the result showed that the percent of TUNEL-positive cells in myocardium of Se-AVP-treated groups was lower than I/R rats. In conclusion, we clearly demonstrated that Se-AVP had a protective effect against myocardial I/R injury in rats by augmenting endogenous antioxidants and protecting rat hearts from oxidative stress-induced myocardial apoptosis.
-
International Nuclear Information System (INIS)
Ji Xinxin; Hou Wugang; Zhao Jie; Zhao Yong; Li Wei; Zhang Yuanqiang
2007-01-01
The aim of the study was to investigate the relationships between apoptosis induced by radiation of electromagnetic pulses (EMP) and the expression of Smad4 in mouse spermatogenic cells. 40 adult Balb/c mice were used, and 20 were irradiated with whole-body 400kV/m EMP. The mice were sacrificed and specimens were harvested at 1, 7, 14, 21 and 28 days after the irradiation. Histological changes were observed through Hematoxylin-Eosin staining (H-E staining), the apoptosis of spermatogenic cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method (TUNEL method) and the Smad4 expression was observed using immunohistochemistry SABC methods. Severe injuries were observed 1 day after the radiation and seminiferous epithelium was mostly recovered 28d after the radiation. The localization of smad4 was significantly different in EMP group compared to the control group, and the expression densities of smad4 decreased significantly at 7, 14 and 21d after irradiation (p<0.05). TUNEL assays demonstrated that there was a significant increase in the mean apoptotic index (AI) in irradiation groups than that of control groups (p<0.01). The results suggested that Smad4 and TGF-13/Smad signal pathway might play an important role in spermatogenic cells apoptosis induced by radiation of EMP. (authors)
-
International Nuclear Information System (INIS)
Tsuji, Takayuki; Kato, Akihiko; Yasuda, Hideo; Miyaji, Takehiko; Luo, Jinghui; Sakao, Yukitoshi; Ito, Hideaki; Fujigaki, Yoshihide; Hishida, Akira
2009-01-01
Dimethylthiourea (DMTU), a potent hydroxyl radical scavenger, affords protection against cisplatin (CDDP)-induced acute renal failure (ARF). Since the suppression of oxidative stress and the enhancement of heat shock proteins (HSPs) are both reported to protect against CDDP-induced renal damage, we tested whether increased HSP expression is involved in the underlying mechanisms of the DMTU-induced renal protection. We examined the effect of DMTU treatment on the expression of HSPs in the kidney until day 5 following a single injection of CDDP (5 mg/kg BW). DMTU significantly inhibited the CDDP-induced increments of serum creatinine, the number of 8-hydroxyl-2'-deoxyguanosine (8-OHdG)- and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL)-positive tubular cells, and tubular damage score (p < 0.05). CDDP significantly increased renal abundances of HO-1, HSP60, HSP72 and HSP90 at days 1, 3, and 5. DMTU significantly augmented only the expression of HSP60 expression mainly in the cytoplasm of the proximal tubular cells at days 1 and 3 in CDDP-induced ARF. DMTU also inhibited the CDDP-induced increment of Bax, a pro-apoptotic protein, in the fraction of organelles/membranes at day 3. The findings suggest that DMTU may afford protection against CDDP-induced ARF, partially through the early induction of cytoplasmic HSP60, thereby preventing the Bax-mediated apoptosis in renal tubular cells
-
Directory of Open Access Journals (Sweden)
Fanren Tang
2017-09-01
Full Text Available Background/Aims: Neurite outgrowth and synaptogenesis are critical steps for functional recovery after stroke. Resveratrol promotes neurite outgrowth and synaptogenesis, but the underlying mechanism is not well understood, although the Sonic hedgehog (Shh signaling pathway may be involved. Given that resveratrol activates sirtuin (Sirt1, the present study examined whether this is mediated by Shh signaling. Methods: Primary cortical neuron cultures were pretreated with drugs before oxygen-glucose deprivation/reoxygenation (OGD/R. Cell viability and apoptosis were evaluated with Cell Counting Kit 8 and by terminal deoxynucleotidyl transferase dUTP nick end labeling, respectively. Neurite outgrowth and synaptogenesis were assessed by immunocytochemistry and western blotting, which was also used to examine the expression of Sirt1 and Shh signaling proteins. Results: Resveratrol and the Smoothened (Smo agonist purmophamine, which activates Shh signaling, increased viability, reduced apoptosis, and stimulated neurite outgrowth after OGD/R injury. Moreover, the expression of growth-associated protein(GAP-43, synaptophysin, Shh, Patched (Ptc-1, Smo, glioma-associated oncogene homolog (Gli-1, and Sirt1 were upregulated under these conditions. These effects were reversed by treatment with the Smo inhibitor cyclopamine, whereas the Sirt1 inhibitor sirtinol reduced the levels of Shh, Ptc-1, Smo, and Gli-1. Conclusions: Resveratrol reduces neuronal injury following OGD/R injury and enhances neurite outgrowth and synaptogenesis by activating Shh signaling, which in turn induces Sirt1.
-
Changes in amino transferases and muscle proteins when treating pigmeat with ionising rays
International Nuclear Information System (INIS)
Hamm, R.; Hofmann, K.; Gruenewald, T.; Partmann, W.
1975-01-01
Slices of lean pigmeat were treated with electron beams doses of 0.2, 1.0 and 5.0 Mrad. Low irradiation doses led to an increase in the activity of aspartate amino transferase (GOT) and alanin amino transferase (GPT) in the tissue generally and in the sarcoplasm (juice expressed from the muscle). 5 Mrad caused a great reduction in the activity of GOT and GPT in the tissue and the sarcoplasm. It seems doubtful whether this inactivation is due to a destruction of enzyme sulphhydryl groups. Irradiating with 5 Mrad resulted in partial release of the mitochondrial GOT isozyme (GOTsub(M)) into the sarcoplasm. This indicates damage to the mitochondrial membranes by ionising radiation. Irradiating the pigmeat increased the pH of the tissue and lowered its water binding ability (increase in drip). Up to a dose of 1 Mrad the solubility of the sarcoplasmic proteins was not definitely affected, but 5 Mrad caused a considerable drop in protein solubility. Surprisingly a dose of even 5 Mrad did not change the total number of sulphhydryl groups present in the tissue. Sephadex thin layer electrophoresis showed that at 0.2 Mrad there was a drastic decrease in the myosin band and an increase in peptide fragments of low molecular weight, whilst actin was little changed. (orig.) [de
-
DEFF Research Database (Denmark)
Palner, Mikael; McCormick, Patrick; Parkes, Jun
2010-01-01
R-[(11)C]-SKF 82957 is a high-affinity and potent dopamine D(1) receptor agonist radioligand, which gives rise to a brain-penetrant lipophilic metabolite. In this study, we demonstrate that systemic administration of catechol-O-methyl transferase (COMT) inhibitors blocks this metabolic pathway, f...
-
The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.
Diccianni, M B; Imagawa, M; Muramatsu, M
1992-01-01
Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lac...
-
Glutathione-binding site of a bombyx mori theta-class glutathione transferase.
Directory of Open Access Journals (Sweden)
M D Tofazzal Hossain
Full Text Available The glutathione transferase (GST superfamily plays key roles in the detoxification of various xenobiotics. Here, we report the isolation and characterization of a silkworm protein belonging to a previously reported theta-class GST family. The enzyme (bmGSTT catalyzes the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy-propane, and 4-nitrophenethyl bromide. Mutagenesis of highly conserved residues in the catalytic site revealed that Glu66 and Ser67 are important for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTT and into the metabolism of exogenous chemical agents.
-
Directory of Open Access Journals (Sweden)
Katharine S Dobb
Full Text Available Antifungal drugs acting via new mechanisms of action are urgently needed to combat the increasing numbers of severe fungal infections caused by pathogens such as Candida albicans. The phosphopantetheinyl transferase of Aspergillus fumigatus, encoded by the essential gene pptB, has previously been identified as a potential antifungal target. This study investigated the function of its orthologue in C. albicans, PPT2/C1_09480W by placing one allele under the control of the regulatable MET3 promoter, and deleting the remaining allele. The phenotypes of this conditional null mutant showed that, as in A. fumigatus, the gene PPT2 is essential for growth in C. albicans, thus fulfilling one aspect of an efficient antifungal target. The catalytic activity of Ppt2 as a phosphopantetheinyl transferase and the acyl carrier protein Acp1 as a substrate were demonstrated in a fluorescence transfer assay, using recombinant Ppt2 and Acp1 produced and purified from E.coli. A fluorescence polarisation assay amenable to high-throughput screening was also developed. Therefore we have identified Ppt2 as a broad-spectrum novel antifungal target and developed tools to identify inhibitors as potentially new antifungal compounds.
-
Combined glutathione S transferase M1/T1 null genotypes is associated with type 2 diabetes mellitus
POROJAN, MIHAI D.; BALA, CORNELIA; ILIES, ROXANA; CATANA, ANDREEA; POPP, RADU A.; DUMITRASCU, DAN L.
2015-01-01
Background Due to new genetic insights, a considerably large number of genes and polymorphic gene variants are screened and linked with the complex pathogenesis of type 2 diabetes (DM). Our study aimed to investigate the association between the two isoforms of the glutathione S-transferase genes (Glutathione S transferase isoemzyme type M1- GSTM1 and Glutathione S transferase isoemzyme type T1-GSTT1) and the prevalence of DM in the Northern Romanian population. Methods We conducted a cross-sectional, randomized, case-control study evaluating the frequency of GSTM1 and GSTT1 null alleles in patients diagnosed with DM. A total of 106 patients diagnosed with DM and 124 healthy controls were included in the study. GSTM1 and GSTT1 null alleles genotyping was carried out using Multiplex PCR amplification of relevant gene fragments, followed by gel electrophoresis analysis of the resulting amplicons. Results Molecular analysis did not reveal an increased frequency of the null GSTM1 and GSTT1 alleles (mutant genotypes) respectively in the DM group compared to controls (p=0.171, OR=1.444 CI=0.852–2.447; p=0.647, OR=0.854, CI=0.436–1.673). Nevertheless, the combined GSTM1/GSTT1 null genotypes were statistically significantly higher in DM patients compared to control subjects (p=0.0021, OR=0.313, CI=0.149–0.655) Conclusions The main finding of our study is that the combined, double GSTM1/GSTT1 null genotypes are to be considered among the polymorphic genetic risk factors for type 2 DM. PMID:26528065
-
Oh, Mi Mi; Kim, Jin Wook; Park, Min Gu; Kim, Je Jong; Yoo, Kee Hwan; Moon, Du Geon
2012-03-01
We assessed the role of therapeutic delay time (TDT) in acute renal cortical scintigraphic lesion (ASL) and ultimate scar formation (USF) in children with first febrile UTI and whether it is affected by the presence of vesico-ureteral reflux (VUR). 230 children, 90 girls and 140 boys with first febrile UTI were included. Radiologic (USG, DMSA, and VCUG), clinical (age, gender, peak fever, therapeutic delay time) and laboratory (CBC with differential count, ANC (absolute neutrophil count), BUN, Creatinine, urine analysis, gram stain, culture, CRP and ESR) variables were analysed. DMSA was performed within 5 days and after six months. VCUG was performed after acute phase of UTI. The differences in TDT according to the presence of ASL, USF and VUR were assessed. And the correlation between ASL or USF with the duration of TDT was assessed. Of 230 patients enrolled, 142 patients had refluxing UTI and 88 patients had non-refluxing UTI. TDT was the risk factor associated with ASL and USF along with presence of VUR. TDT was longer in ASL positive group compared with the ASL negative group. Also USF group showed longer TDT compared with those without USF in both refluxing UTI and non refluxing UTI. The TDT was significantly shorter in USF group with the presence of VUR. Positive linear association was noted between prevalence of ASL and USF and duration of TDT. In conclusion, the impact of UTI on formation of USF may be enhanced by the presence of VUR with shorter duration of TDT.
-
DEFF Research Database (Denmark)
Wandall, Hans H; Irazoqui, Fernando; Tarp, Mads Agervig
2007-01-01
Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificit...
-
Directory of Open Access Journals (Sweden)
Lineu Cesar Werneck
1983-12-01
Full Text Available Relato dos casos de dois irmãos, que desde a infância apresentavam dores musculares após exercício prolongado ou exposição ao frio, com diminuição da força, à medida que o exercício continuava. Um deles desenvolveu mioglobinúria recorrente e, em um episódio apresentou insuficiência renal aguda, necessitando de diálise peritonial. A investigação laboratorial intercrise foi normal, mas durante o episódio de mioglobinúria, apresentou grande aumento da creatinafosfoquinase. A eletromiografia foi sugestiva de processo de denervação. Teste de produção de lactato durante isquemia foi normal. Biópsias musculares mostraram discreto aumento dos lipídios nas fibras musculares e maior atividade da desidrogenase succínica na histoquímica. O estudo bioquímico do tecido muscular dos dois pacientes, revelou importante redução da atividade da carnitina-palmitil-transferase, com atividade normal da carnitina-octanoil-transferase e carnitina-acetil-transferase. São discutidas as vias metabólicas, sua importância na manutenção da energia muscular durante o exercício prolongado e o papel dos ácidos graxos como fonte energética muscular durante condições normais e patológicas.
-
Digital Repository Service at National Institute of Oceanography (India)
Patra, J.K.; Rath, S.K.; Jena, K.B.; Rathod, V.K.; Thatoi, H.
In the present study, the free radical scavenging potentials (DPPH radical and hydroxyl radical), inhibition of lipid peroxidation, and glutathione-S-transferase and antimicrobial properties of Sargassum sp. extract were investigated. The tested...
-
Middelberg, R.P.S.; Benyamin, B.; de Moor, M.H.M.; Warrington, N.M.; Gordon, S.; Henders, A.K.; Medland, S.E.; Nyholt, DR; de Geus, E.J.C.; Hottenga, J.J.; Willemsen, G.; Beilin, L.J.; Mori, T.A.; Wright, M.J.; Heath, A.C.; Madden, P.A.F.; Boomsma, D.I.; Pennell, C.E.; Montgomery, G.W.; Martin, N.G.; Whitfield, J.B.
2012-01-01
Serum gamma-glutamyl transferase (GGT) activity is a marker of liver disease which is also prospectively associated with the risk of all-cause mortality, cardiovascular disease, type 2 diabetes and cancers. We have discovered novel loci affecting GGT in a genome-wide association study (rs1497406 in
-
Zhou, Chen-hui; Wang, Chun-xi; Xie, Guang-bin; Wu, Ling-yun; Wei, Yong-xiang; Wang, Qiang; Zhang, Hua-sheng; Hang, Chun-hua; Zhou, Meng-liang; Shi, Ji-xin
2015-12-10
Early brain injury (EBI) determines the unfavorable outcomes after subarachnoid hemorrhage (SAH). Fisetin, a natural flavonoid, has anti-inflammatory and neuroprotection properties in several brain injury models, but the role of fisetin on EBI following SAH remains unknown. Our study aimed to explore the effects of fisetin on EBI after SAH in rats. Adult male Sprague-Dawley rats were randomly divided into the sham and SAH groups, fisetin (25mg/kg or 50mg/kg) or equal volume of vehicle was given at 30min after SAH. Neurological scores and brain edema were assayed. The protein expression of toll-like receptor 4 (TLR 4), p65, ZO-1 and bcl-2 was examined by Western blot. TLR 4 and p65 were also assessed by immunohistochemistry (IHC). Enzyme-linked immunosorbent assay (ELISA) was performed to detect the production of pro-inflammatory cytokines. Terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL) was perform to assess neural cell apoptosis. High-dose (50mg/kg) fisetin significantly improved neurological function and reduced brain edema at both 24h and 72h after SAH. Remarkable reductions of TLR 4 expression and nuclear factor κB (NF-κB) translocation to nucleus were detected after fisetin treatment. In addition, fisetin significantly reduced the productions of pro-inflammatory cytokines, decreased neural cell apoptosis and increased the protein expression of ZO-1 and bcl-2. Our data provides the evidence for the first time that fisetin plays a protective role in EBI following SAH possibly by suppressing TLR 4/NF-κB mediated inflammatory pathway. Copyright © 2015 Elsevier B.V. All rights reserved.
-
Increased Dickkopf-1 expression accelerates bone cell apoptosis in femoral head osteonecrosis.
Ko, Jih-Yang; Wang, Feng-Sheng; Wang, Ching-Jen; Wong, To; Chou, Wen-Yi; Tseng, Shin-Ling
2010-03-01
Intensive bone cell apoptosis contributes to osteonecrosis of femoral head (ONFH). Dickkopf-1 (DKK1) reportedly mediates various types of skeletal disorders. This study investigated whether DKK1 was linked to the occurrence of ONFH. Thirty-nine patients with various stages of ONFH were recruited. Bone specimens were harvested from 34 ONFH patients underwent hip arthroplasty, and from 10 femoral neck fracture patients. Bad, Bcl2 TNFalpha, DKK1, Wnt3a, LRP5, and Axin1 expressions were analyzed by quantitative RT-PCR and ELISA. Apoptotic cells were assayed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labelling (TUNEL). Primary bone-marrow mesenchymal cells were treated with DKK1 RNA interference and recombinant DKK1 protein. ONFH patients with the histories of being administrated corticosteroids and excessive alcohol consumption had significantly higher Bad and DKK1 mRNA expressions in bone tissue and DKK1 abundances in serum than femoral neck fracture patients. Bone cells adjacent to osteonecrotic bone displayed strong DKK1 immunoreactivity and TUNEL staining. Increased DKK1 expression in bone tissue and serum correlated with Bad expression and TUNEL staining. Serum DKK1 abundance correlated with the severity of ONFH. The DKK1 RNA interference and recombinant DKK1 protein regulated Bad expression and apoptosis of primary bone-marrow mesenchymal cells. Knock down of DKK1 reduced dexamethasone-induced apoptosis of mesenchymal cells. Taken together, promoted DKK1 expression was associated with bone cell apoptosis in the occurrence of ONFH patients with the histories of corticosteroid and alcohol intake and progression of ONFH. DKK1 expression in injured tissue provides new insight into ONFH pathogenesis.
-
Pirger, Zsolt; Rácz, Boglárka; Kiss, Tibor
2009-02-01
PCD (programmed cell death) is a common mechanism to remove unwanted and excessive cells from organisms. In several exocrine cell types, PCD mode of release of secretory products has been reported. The molecular mechanism of the release, however, is largely unknown. Our aim was to study the molecular mechanism of saliva release from cystic cells, the specific cell type of snail SGs (salivary glands). SG cells in active feeding animals revealed multiple morphological changes characteristic of PCD. Nerve stimulation and DA (dopamine) increased the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive cells both in inactive and feeding animals. The DA-induced PCD was prevented by TEA (tetraethylammonium chloride) and eticlopride, emphasizing the role of K channels and D2 receptors in the PCD of cystic cells. DA enhanced cyto-c (cytochrome c) translocation into the cytosol and methyl-beta-cyclodextrin prevented it, suggesting apoptosome formation and ceramide involvement in the PCD linking of the surface DA receptor to mitochondria. Western blot analysis revealed that the release of cyto-c was under the control of Bcl-2 and Bad. DA also increased the active caspase-3 in gland cells while D2 receptor antagonists and TEA attenuated it. Our results provide evidence for a type of transmitter-mediated pathway that regulates the PCD of secretory cells in a mitochondrial-caspase-dependent manner. The activation of specific molecules, such as K channels, DA receptors, cyto-c, ceramide, Bcl-2 proteins and caspase-3, but not caspase-8, was demonstrated in cells involved in the DA-induced PCD, suggesting that PCD is a physiological method for the release of saliva from SG cells.
-
Directory of Open Access Journals (Sweden)
Yan-Der Hsuuw
2013-01-01
Full Text Available Ochratoxin A (OTA, a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, both in vitro and in vivo. In the present study, we explored the cytotoxic effects exerted by OTA on the blastocyst stage of mouse embryos, on subsequent embryonic attachment, on outgrowth in vitro, and following in vivo implantation via embryo transfer. Mouse blastocysts were incubated with or without OTA (1, 5, or 10 μM for 24 h. Cell proliferation and growth were investigated using dual differential staining; apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay; and embryo implantation and post-implantation development were assessed by examination of in vitro growth and the outcome of in vivo embryo transfer, respectively. Blastocysts treated with 10 μM OTA displayed a significantly increased level of apoptosis and a reduction in total cell number. Interestingly, we observed no marked difference in implantation success rate between OTA-pretreated and control blastocysts either during in vitro embryonic development (following implantation in a fibronectin-coated culture dish or after in vivo embryo transfer. However, in vitro treatment with 10 μM OTA was associated with increased resorption of post-implantation embryos by the mouse uterus, and decreased fetal weight upon embryo transfer. Our results collectively indicate that in vitro exposure to OTA triggers apoptosis and retards early post-implantation development after transfer of embryos to host mice. In addition, OTA induces apoptosis-mediated injury of mouse blastocysts, via reactive oxygen species (ROS generation, and promotes mitochondrion-dependent apoptotic signaling processes that impair subsequent embryonic development.
-
Liu, D; Liao, M; Zuo, J; Henner, W D; Fan, F
2001-03-01
To investigate mechanisms of rat glutathione S-transferase P1 gene (rGSTP1) expression regulation during chemical carcinogenesis. we studied enhancer elements located in the region between -2.5 kb to -2.2 kb. The region was upstream from the start site of transcription and was divided into two major fragments, GPEI and GPEII. The GPEII fragment was further divided into two smaller fragments, GPEII- I and GPEII-2. Using a luciferase reporter system, we identified a strong enhancer of GPEI and a weak enhancer of GPEII in HeLa and a rat hepatoma cell line CBRH79 19 cell. The enhancer of GPEII was located within the GPEII-I region. Chemical stimulation by glycidyl methatylate (GMA) and phorbol 12-o-tetradecanoate 13-acetate (TPA) analysis revealed that induction of rGSTP1 expression was mainly through GPEI. Although H2O2 could enhance GPEII enhancer activity, the enhancement is not mediated by the NF-kappaB factor that bound the NF-kappaB site in GPEII. Using electrophoretic mobility shift assays (EMSA) and the UV cross-linking assays, we found that HeLa and CBRH7919 cells had proteins that specifically bound GPEI core sequence and a 64 kDa protein that interacted with GPEII-1. The cells from normal rat liver did not express the binding proteins. Therefore, the trans-acting factors seem to be closely related to GPEI, GPEII enhancer activities and may play an important role in high expression of rGSTPI gene.
-
International Nuclear Information System (INIS)
Achilonu, Ikechukwu; Gildenhuys, Samantha; Fisher, Loren; Burke, Jonathan; Fanucchi, Sylvia; Sewell, B. Trevor; Fernandes, Manuel; Dirr, Heini W.
2010-01-01
The role of a topologically conserved isoleucine in the structure of glutathione transferase was investigated by replacing the Ile71 residue in human GSTA1-1 by alanine or valine. The common fold shared by members of the glutathione-transferase (GST) family has a topologically conserved isoleucine residue at the N-terminus of helix 3 which is involved in the packing of helix 3 against two β-strands in domain 1. The role of the isoleucine residue in the structure, function and stability of GST was investigated by replacing the Ile71 residue in human GSTA1-1 by alanine or valine. The X-ray structures of the I71A and I71V mutants resolved at 1.75 and 2.51 Å, respectively, revealed that the mutations do not alter the overall structure of the protein compared with the wild type. Urea-induced equilibrium unfolding studies using circular dichroism and tryptophan fluorescence suggest that the mutation of Ile71 to alanine or valine reduces the stability of the protein. A functional assay with 1-chloro-2,4-dinitrobenzene shows that the mutation does not significantly alter the function of the protein relative to the wild type. Overall, the results suggest that conservation of the topologically conserved Ile71 maintains the structural stability of the protein but does not play a significant role in catalysis and substrate binding
-
Antioxidant enzyme gene delivery to protect from HIV-1 gp120-induced neuronal apoptosis.
Agrawal, L; Louboutin, J-P; Reyes, B A S; Van Bockstaele, E J; Strayer, D S
2006-12-01
Human immunodeficiency virus-1 (HIV-1) infection in the central nervous system (CNS) may lead to neuronal loss and progressively deteriorating CNS function: HIV-1 gene products, especially gp120, induce free radical-mediated apoptosis. Reactive oxygen species (ROS), are among the potential mediators of these effects. Neurons readily form ROS after gp120 exposure, and so might be protected from ROS-mediated injury by antioxidant enzymes such as Cu/Zn-superoxide dismutase (SOD1) and/or glutathione peroxidase (GPx1). Both enzymes detoxify oxygen free radicals. As they are highly efficient gene delivery vehicles for neurons, recombinant SV40-derived vectors were used for these studies. Cultured mature neurons derived from NT2 cells and primary fetal neurons were transduced with rSV40 vectors carrying human SOD1 and/or GPx1 cDNAs, then exposed to gp120. Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Transduction efficiency of both neuron populations was >95%, as assayed by immunostaining. Transgene expression was also ascertained by Western blotting and direct assays of enzyme activity. Gp120 induced apoptosis in a high percentage of unprotected NT2-N. Transduction with SV(SOD1) and SV(GPx1) before gp120 challenge reduced neuronal apoptosis by >90%. Even greater protection was seen in cells treated with both vectors in sequence. Given singly or in combination, they protect neuronal cells from HIV-1-gp120 induced apoptosis. We tested whether rSV40 s can deliver antioxidant enzymes to the CNS in vivo: intracerebral injection of SV(SOD1) or SV(GPx1) into the caudate putamen of rat brain yielded excellent transgene expression in neurons. In vivo transduction using SV(SOD1) also protected neurons from subsequent gp120-induced apoptosis after injection of both into the caudate putamen of rat brain. Thus, SOD1 and GPx1 can be delivered by SV40 vectors in vitro or in vivo. This approach may merit consideration for
-
Directory of Open Access Journals (Sweden)
Young-Hee Jeong
Full Text Available The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC compared with the human umbilical vein endothelial cells (HUVEC. Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative
-
Directory of Open Access Journals (Sweden)
Hai-Yan Shi
Full Text Available Two genes encoding putative glutathione S-transferase proteins were isolated from pear (Pyrus pyrifolia and designated PpGST1 and PpGST2. The deduced PpGST1 and PpGST2 proteins contain conserved Glutathione S-transferase N-terminal domain (GST_N and Glutathione S-transferase, C-terminal domain (GST_C. Using PCR amplification technique, the genomic clones corresponding to PpGST1 and PpGST2 were isolated and shown to contain two introns and a singal intron respectively with typical GT/AG boundaries defining the splice junctions. Phylogenetic analysis clearly demonstrated that PpGST1 belonged to Phi class of GST superfamilies and had high homology with apple MdGST, while PpGST2 was classified into the Tau class of GST superfamilies. The expression of PpGST1 and PpGST2 genes was developmentally regulated in fruit. Further study demonstrated that PpGST1 and PpGST2 expression was remarkably induced by glucose, salicylic acid (SA and indole-3-aceticacid (IAA treatments in pear fruit, and in diseased fruit. These data suggested that PpGST1 and PpGST2 might be involved in response to sugar, SA, and IAA signaling during fruit development of pear.
-
DEFF Research Database (Denmark)
Porse, B T; Garrett, R A
1995-01-01
Random mutations were generated in the lower half of the peptidyl transferase loop in domain V of 23 S rRNA from Escherichia coli using a polymerase chain reaction (PCR) approach, a rapid procedure for identifying mutants and a plasmid-based expression system. The effects of 21 single-site mutati...
-
Studying the host-associated butyrate-producing bacterial community is important because butyrate is essential for colonic homeostasis and gut health. Previous research has identified the butyryl-coA:acetate transferase (2.3.8.3) as a the main gene for butyrate production in intestinal ecosystems; h...
-
Energy Technology Data Exchange (ETDEWEB)
Sakaidani, Yuta [Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan); Ichiyanagi, Naoki [Department of Applied Molecular Biosciences, Nagoya University Graduate School of Bioagricultural Sciences, Furo-cho, Chikusa-ku, Nagoya 464-8601 (Japan); Saito, Chika; Nomura, Tomoko [Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan); Ito, Makiko [Department of Applied Molecular Biosciences, Nagoya University Graduate School of Bioagricultural Sciences, Furo-cho, Chikusa-ku, Nagoya 464-8601 (Japan); Nishio, Yosuke [Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan); Nadano, Daita; Matsuda, Tsukasa [Department of Applied Molecular Biosciences, Nagoya University Graduate School of Bioagricultural Sciences, Furo-cho, Chikusa-ku, Nagoya 464-8601 (Japan); Furukawa, Koichi [Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan); Okajima, Tetsuya, E-mail: tokajima@med.nagoya-u.ac.jp [Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan); Department of Applied Molecular Biosciences, Nagoya University Graduate School of Bioagricultural Sciences, Furo-cho, Chikusa-ku, Nagoya 464-8601 (Japan)
2012-03-02
Highlights: Black-Right-Pointing-Pointer We characterized A130022J15Rik (Eogt1)-a mouse gene homologous to Drosophila Eogt. Black-Right-Pointing-Pointer Eogt1 encodes EGF domain O-GlcNAc transferase. Black-Right-Pointing-Pointer Expression of Eogt1 in Drosophila rescued the cell-adhesion defect in the Eogt mutant. Black-Right-Pointing-Pointer O-GlcNAcylation reaction in the secretory pathway is conserved through evolution. -- Abstract: O-linked-{beta}-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-GlcNAcylated by an atypical O-GlcNAc transferase, EOGT, in Drosophila. However, no study has yet shown whether O-GlcNAcylation of extracellular proteins is limited to insects such as Drosophila or whether it occurs in other organisms, including mammals. Here, we report the characterization of A130022J15Rik, a mouse gene homolog of Drosophila Eogt (Eogt 1). Enzymatic analysis revealed that Eogt1 has a substrate specificity similar to that of Drosophila EOGT, wherein the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domains is modified. This observation is supported by the fact that the expression of Eogt1 in Drosophila rescued the cell-adhesion defect caused by Eogt downregulation. In HEK293T cells, Eogt1 expression promoted modification of Notch1 EGF repeats by O-GlcNAc, which was further modified, at least in part, by galactose to generate a novel O-linked-N-acetyllactosamine structure. These results suggest that Eogt1 encodes EGF domain O-GlcNAc transferase and that O-GlcNAcylation reaction in the secretory pathway is a fundamental biochemical process conserved through evolution.
-
International Nuclear Information System (INIS)
Sakaidani, Yuta; Ichiyanagi, Naoki; Saito, Chika; Nomura, Tomoko; Ito, Makiko; Nishio, Yosuke; Nadano, Daita; Matsuda, Tsukasa; Furukawa, Koichi; Okajima, Tetsuya
2012-01-01
Highlights: ► We characterized A130022J15Rik (Eogt1)—a mouse gene homologous to Drosophila Eogt. ► Eogt1 encodes EGF domain O-GlcNAc transferase. ► Expression of Eogt1 in Drosophila rescued the cell-adhesion defect in the Eogt mutant. ► O-GlcNAcylation reaction in the secretory pathway is conserved through evolution. -- Abstract: O-linked-β-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-GlcNAcylated by an atypical O-GlcNAc transferase, EOGT, in Drosophila. However, no study has yet shown whether O-GlcNAcylation of extracellular proteins is limited to insects such as Drosophila or whether it occurs in other organisms, including mammals. Here, we report the characterization of A130022J15Rik, a mouse gene homolog of Drosophila Eogt (Eogt 1). Enzymatic analysis revealed that Eogt1 has a substrate specificity similar to that of Drosophila EOGT, wherein the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domains is modified. This observation is supported by the fact that the expression of Eogt1 in Drosophila rescued the cell-adhesion defect caused by Eogt downregulation. In HEK293T cells, Eogt1 expression promoted modification of Notch1 EGF repeats by O-GlcNAc, which was further modified, at least in part, by galactose to generate a novel O-linked-N-acetyllactosamine structure. These results suggest that Eogt1 encodes EGF domain O-GlcNAc transferase and that O-GlcNAcylation reaction in the secretory pathway is a fundamental biochemical process conserved through evolution.
-
Flavahan, Sheila; Flavahan, Nicholas A
2014-08-15
Endothelium of fetal or newborn arteries is atypical, displaying actin stress fibers and reduced nitric oxide (NO)-mediated dilatation. This study tested the hypothesis that Rho/Rho kinase signaling, which promotes endothelial stress fibers and inhibits endothelial dilatation, contributed to this phenotype. Carotid arteries were isolated from newborn [postnatal day 1 (P1)], P7, and P21 mice. Endothelial dilatation to acetylcholine (pressure myograph) was minimal at P1, increased at P7, and further increased at P21. Inhibition of Rho (C3 transferase) or Rho kinase (Y27632, fasudil) significantly increased dilatation to acetylcholine in P1 arteries but had no effect in P7 or P21 arteries. After inhibition of NO synthase (N(G)-nitro-l-arginine methyl ester), Rho kinase inhibition no longer increased acetylcholine responses in P1 arteries. Rho kinase inhibition did not affect dilatation to the NO donor DEA-NONOate. The endothelial actin cytoskeleton was labeled with phalloidin and visualized by laser-scanning microscopy. In P1 arteries, the endothelium had prominent transcytoplasmic stress fibers, whereas in P7 and P21 arteries, the actin fibers had a significantly reduced intensity and were restricted to cell borders. Phosphorylation of myosin light chains, a Rho kinase substrate, was highest in P1 endothelium and significantly reduced in P7 and P21 endothelium (laser-scanning microscopy). In P1 arteries, inhibition of Rho (C3 transferase) or Rho kinase (Y27632) significantly reduced the intensity of actin fibers, which were restricted to cell borders. Similarly, in P1 arteries, Rho inhibition significantly reduced endothelial levels of phosphorylated myosin light chains. These results indicate that the atypical function and morphology of newborn endothelium is mediated by Rho/Rho kinase signaling. Copyright © 2014 the American Physiological Society.
-
Directory of Open Access Journals (Sweden)
Devesh eShukla
2014-11-01
Full Text Available The unique physico-chemical properties of gold nanoparticles (AuNPs find manifold applications in diagnostics, medicine and catalysis. Chemical synthesis produces reactive AuNPs and generates hazardous by-products. Alternatively, plants can be utilized to produce AuNPs in an eco-friendly manner. To better control the biosynthesis of AuNPs, we need to first understand the detailed molecular response induced by AuCl4- In this study, we carried out global transcriptome analysis in root tissue of Arabidopsis grown for 12- hours in presence of gold solution (HAuCl4 using the novel unbiased Affymetrix exon array. Transcriptomics analysis revealed differential regulation of a total of 704 genes and 4900 exons. Of these, 492 and 212 genes were up- and downregulated, respectively. The validation of the expressed key genes, such as glutathione-S-transferases, auxin responsive genes, cytochrome P450 82C2, methyl transferases, transducin (G protein beta subunit, ERF transcription factor, ABC, and MATE transporters, was carried out through quantitative RT-PCR. These key genes demonstrated specific induction under AuCl4- treatment relative to other heavy metals, suggesting a unique plant-gold interaction. GO enrichment analysis reveals the upregulation of processes like oxidative stress, glutathione binding, metal binding, transport, and plant hormonal responses. Changes predicted in biochemical pathways indicated major modulation in glutathione mediated detoxification, flavones and derivatives, and plant hormone biosynthesis. Motif search analysis identified a highly significant enriched motif, ACGT, which is an abscisic acid responsive core element (ABRE, suggesting the possibility of ABA- mediated signaling. Identification of abscisic acid response element (ABRE points to the operation of a predominant signaling mechanism in response to AuCl4- exposure. Overall, this study presents a useful picture of plant-gold interaction with an identification of
-
DNA fragmentation in spermatozoa
DEFF Research Database (Denmark)
Rex, A S; Aagaard, J.; Fedder, J
2017-01-01
Sperm DNA Fragmentation has been extensively studied for more than a decade. In the 1940s the uniqueness of the spermatozoa protein complex which stabilizes the DNA was discovered. In the fifties and sixties, the association between unstable chromatin structure and subfertility was investigated....... In the seventies, the impact of induced DNA damage was investigated. In the 1980s the concept of sperm DNA fragmentation as related to infertility was introduced as well as the first DNA fragmentation test: the Sperm Chromatin Structure Assay (SCSA). The terminal deoxynucleotidyl transferase nick end labelling...... (TUNEL) test followed by others was introduced in the nineties. The association between DNA fragmentation in spermatozoa and pregnancy loss has been extensively investigated spurring the need for a therapeutic tool for these patients. This gave rise to an increased interest in the aetiology of DNA damage...
-
Enzymatic production of single-molecule FISH and RNA capture probes.
Gaspar, Imre; Wippich, Frank; Ephrussi, Anne
2017-10-01
Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed. © 2017 Gaspar et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
-
DEFF Research Database (Denmark)
Rogers, A J; Brasch-Andersen, C; Ionita-Laza, I
2009-01-01
BACKGROUND: The glutathione S-transferase M1 (GSTM1)-null variant is a common copy number variant associated with adverse pulmonary outcomes, including asthma and airflow obstruction, with evidence of important gene-by-environment interactions with exposures to oxidative stress. OBJECTIVE: To exp...
-
Directory of Open Access Journals (Sweden)
Ya Lin Zhang
2013-03-01
Full Text Available Previous investigations have implicated glutathione S-transferases (GSTs as one of the major reasons for insecticide resistance. Therefore, effectiveness of new candidate compounds depends on their ability to inhibit GSTs to prevent metabolic detoxification by insects. Cantharidin, a terpenoid compound of insect origin, has been developed as a bio-pesticide in China, and proves highly toxic to a wide range of insects, especially lepidopteran. In the present study, we test cantharidin as a model compound for its toxicity, effects on the mRNA transcription of a model Helicoverpa armigera glutathione S-transferase gene (HaGST and also for its putative inhibitory effect on the catalytic activity of GSTs, both in vivo and in vitro in Helicoverpa armigera, employing molecular and biochemical methods. Bioassay results showed that cantharidin was highly toxic to H. armigera. Real-time qPCR showed down-regulation of the HaGST at the mRNA transcript ranging from 2.5 to 12.5 folds while biochemical assays showed in vivo inhibition of GSTs in midgut and in vitro inhibition of rHaGST. Binding of cantharidin to HaGST was rationalized by homology and molecular docking simulations using a model GST (1PN9 as a template structure. Molecular docking simulations also confirmed accurate docking of the cantharidin molecule to the active site of HaGST impeding its catalytic activity.
-
S-Nitrosation destabilizes glutathione transferase P1-1.
Balchin, David; Stoychev, Stoyan H; Dirr, Heini W
2013-12-23
Protein S-nitrosation is a post-translational modification that regulates the function of more than 500 human proteins. Despite its apparent physiological significance, S-nitrosation is poorly understood at a molecular level. Here, we investigated the effect of S-nitrosation on the activity, structure, stability, and dynamics of human glutathione transferase P1-1 (GSTP1-1), an important detoxification enzyme ubiquitous in aerobes. S-Nitrosation at Cys47 and Cys101 reduces the activity of the enzyme by 94%. Circular dichroism spectroscopy, acrylamide quenching, and amide hydrogen-deuterium exchange mass spectrometry experiments indicate that the loss of activity is caused by the introduction of local disorder at the active site of GSTP1-1. Furthermore, the modification destabilizes domain 1 of GSTP1-1 against denaturation, smoothing the unfolding energy landscape of the protein and introducing a refolding defect. In contrast, S-nitrosation at Cys101 alone introduces a refolding defect in domain 1 but compensates by stabilizing the domain kinetically. These data elucidate the physical basis for the regulation of GSTP1-1 by S-nitrosation and provide general insight into the consequences of S-nitrosation on protein stability and dynamics.
-
Energy Technology Data Exchange (ETDEWEB)
Osterman, Ilya A.; Khabibullina, Nelli F.; Komarova, Ekaterina S.; Kasatsky, Pavel; Kartsev, Victor G.; Bogdanov, Alexey A.; Dontsova, Olga A.; Konevega, Andrey L.; Sergiev, Petr V.; Polikanov, Yury S. (InterBioScreen); (UIC); (MSU-Russia); (Kurchatov)
2017-05-13
The emergence of multi-drug resistant bacteria is limiting the effectiveness of commonly used antibiotics, which spurs a renewed interest in revisiting older and poorly studied drugs. Streptogramins A is a class of protein synthesis inhibitors that target the peptidyl transferase center (PTC) on the large subunit of the ribosome. In this work, we have revealed the mode of action of the PTC inhibitor madumycin II, an alanine-containing streptogramin A antibiotic, in the context of a functional 70S ribosome containing tRNA substrates. Madumycin II inhibits the ribosome prior to the first cycle of peptide bond formation. It allows binding of the tRNAs to the ribosomal A and P sites, but prevents correct positioning of their CCA-ends into the PTC thus making peptide bond formation impossible. We also revealed a previously unseen drug-induced rearrangement of nucleotides U2506 and U2585 of the 23S rRNA resulting in the formation of the U2506•G2583 wobble pair that was attributed to a catalytically inactive state of the PTC. The structural and biochemical data reported here expand our knowledge on the fundamental mechanisms by which peptidyl transferase inhibitors modulate the catalytic activity of the ribosome.
-
Niezen-Koning, K. E.; Wanders, R. J.; Ruiter, J. P.; IJlst, L.; Visser, G.; Reitsma-Bierens, W. C.; Heymans, H. S.; Reijngoud, D. J.; Smit, G. P.
1997-01-01
We describe the clinical symptoms and biochemical findings of a patient with succinyl-CoA:acetoacetate transferase deficiency who presented in the neonatal period and review the current literature on this subject. Our patient was initially suspected to have distal renal tubular acidosis, and
-
Niezen-Koning, K E; Wanders, R J; Ruiter, J P; Ijlst, L; Visser, G; Reitsma-Bierens, W C; Heijmans, Hugo; Reijngoud, D J; Smit, G P
1997-01-01
UNLABELLED: We describe the clinical symptoms and biochemical findings of a patient with succinyl-CoA:acetoacetate transferase deficiency who presented in the neonatal period and review the current literature on this subject. Our patient was initially suspected to have distal renal tubular acidosis,
-
Niezen-Koning, K E; Wanders, R J; Ruiter, J P; Ijlst, L; Visser, G; Reitsma-Bierens, W C; Heijmans, Hugo; Reijngoud, D J; Smit, G P
UNLABELLED: We describe the clinical symptoms and biochemical findings of a patient with succinyl-CoA:acetoacetate transferase deficiency who presented in the neonatal period and review the current literature on this subject. Our patient was initially suspected to have distal renal tubular acidosis,
-
Yang, Guanghong; Zhou, Zhiwei; Cen, Yanli; Gui, Xiaolin; Zeng, Qibing; Ao, Yunxia; Li, Qian; Wang, Shiran; Li, Jun; Zhang, Aihua
2015-01-01
Persistent organic pollutants in drinking water impose a substantial risk to the health of human beings, but the evidence for liver toxic effect and the underlying mechanism is scarce. This study aimed to examine the liver toxicity and elucidate the molecular mechanism of organic pollutants in drinking water in normal human liver cell line L02 cells and rats. The data showed that organic extraction from drinking water remarkably impaired rat liver function, evident from the increase in the serum level of alanine aminotransferase, aspartate aminotransferase, and cholinesterase, and decrease in the serum level of total protein and albumin. Organic extraction dose-dependently induced apoptotic cell death in rat liver and L02 cells. Administration of rats with organic extraction promoted death receptor signaling pathway through the increase in gene and protein expression level of Fas and FasL. Treatment of rats with organic extraction also induced mitochondria-mediated apoptosis via increasing the expression level of proapoptotic protein, Bax, but decreasing the expression level of antiapoptotic protein, Bcl-2, resulting in an upregulation of cytochrome c and activation of caspase cascade at both transcriptional and post-transcriptional levels. Moreover, organic extraction enhanced rat liver glutathione S-transferases activity and reactive oxygen species generation, and upregulated aryl hydrocarbon receptor and glutathione S-transferase A1 at both transcriptional and translational levels. Collectively, the results indicate that organic extraction from drinking water impairs liver function, with the involvement of death receptor and mitochondria-mediated apoptosis in rats. The results provide evidence and molecular mechanisms for organic pollutants in drinking water-induced liver dysfunction, which may help prevent and treat organic extraction-induced liver injury.
-
Yang, Guanghong; Zhou, Zhiwei; Cen, Yanli; Gui, Xiaolin; Zeng, Qibing; Ao, Yunxia; Li, Qian; Wang, Shiran; Li, Jun; Zhang, Aihua
2015-01-01
Persistent organic pollutants in drinking water impose a substantial risk to the health of human beings, but the evidence for liver toxic effect and the underlying mechanism is scarce. This study aimed to examine the liver toxicity and elucidate the molecular mechanism of organic pollutants in drinking water in normal human liver cell line L02 cells and rats. The data showed that organic extraction from drinking water remarkably impaired rat liver function, evident from the increase in the serum level of alanine aminotransferase, aspartate aminotransferase, and cholinesterase, and decrease in the serum level of total protein and albumin. Organic extraction dose-dependently induced apoptotic cell death in rat liver and L02 cells. Administration of rats with organic extraction promoted death receptor signaling pathway through the increase in gene and protein expression level of Fas and FasL. Treatment of rats with organic extraction also induced mitochondria-mediated apoptosis via increasing the expression level of proapoptotic protein, Bax, but decreasing the expression level of antiapoptotic protein, Bcl-2, resulting in an upregulation of cytochrome c and activation of caspase cascade at both transcriptional and post-transcriptional levels. Moreover, organic extraction enhanced rat liver glutathione S-transferases activity and reactive oxygen species generation, and upregulated aryl hydrocarbon receptor and glutathione S-transferase A1 at both transcriptional and translational levels. Collectively, the results indicate that organic extraction from drinking water impairs liver function, with the involvement of death receptor and mitochondria-mediated apoptosis in rats. The results provide evidence and molecular mechanisms for organic pollutants in drinking water-induced liver dysfunction, which may help prevent and treat organic extraction-induced liver injury. PMID:26316710
-
Arsenic induced apoptosis in rat liver following repeated 60 days exposure
International Nuclear Information System (INIS)
Bashir, Somia; Sharma, Yukti; Irshad, M.; Nag, T.C.; Tiwari, Monica; Kabra, M.; Dogra, T.D.
2006-01-01
Background: Accumulation of the wide spread environmental toxin arsenic in liver results in hepatotoxcity. Exposure to arsenite and other arsenicals has been previously shown to induce apoptosis in certain tumor cell lines at low (1-3 μM) concentration. Aim: The present study was focused to elucidate the role of free radicals in arsenic toxicity and to investigate the nature of in vivo sodium arsenite induced cell death in liver. Methods: Male wistar rats were exposed to arsenite at three different doses of 0.05, 2.5 and 5 mg/l for 60 days. Oxidative stress in liver was measured by estimating pro-oxidant and antioxidant activity in liver. Histopathological examination of liver was carried out by light and transmission electron microscopy. Analysis of DNA fragmentation by gel electrophoresis was used to identify apoptosis after the exposure. Terminal deoxy-nucleotidyl transferase mediated dUTP Nick end-labeling (TUNEL) assay was used to qualify and quantify apoptosis. Results: A significant increase in cytochrome-P450 and lipid peroxidation accompanied with a significant alteration in the activity of many of the antioxidants was observed, all suggestive of arsenic induced oxidative stress. Histopathological examination under light and transmission electron microscope suggested a combination of ongoing necrosis and apoptosis. DNA-TUNEL showed an increase in apoptotic cells in liver. Agarose gel electrophoresis of DNA of hepatocytes resulted in a characteristic ladder pattern. Conclusion: Chronic arsenic administration induces a specific pattern of apoptosis called post-mitotic apoptosis
-
Detection of radiation-induced apoptosis using the comet assay
International Nuclear Information System (INIS)
Wada, Seiichi; Kobayashi, Yasuhiko; Funayama, Tomoo; Yamamoto, Kazuo; Khoa, Tran Van; Natsuhori, Masahiro; Ito, Nobuhiko
2003-01-01
The electrophoresis pattern of apoptotic cells detected by the comet assay has a characteristic small head and spread tail. This image has been referred to as an apoptotic comet, but it has not been previously proven to be apoptotic cells by any direct method. In order to identify this image obtained by the comet assay as corresponding to an apoptotic cell, the frequency of appearance of apoptosis was examined using CHO-K1 and L5178Y cells which were exposed to gamma irradiation. As a method for detecting apoptosis, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was used. When the frequency of appearance of apoptotic cells following gamma irradiation was observed over a period of time, there was a significant increase in appearance of apoptosis when using the TUNEL assay. However, there was only a slight increase when using the comet assay. In order to verify the low frequency of appearance of apoptosis when using the comet assay, we attempted to use the TUNEL assay to satin the apoptotic comets detected in the comet assay. The apoptotic comets were TUNEL positive and the normal comets were TUNEL negative. This indicates that the apoptotic comets were formed from DNA fragments with 3'-hydroxy ends that are generated as cells undergo apoptosis. Therefore, it was understood that the characteristic pattern of apoptotic comets detected by the comet assay corresponds to cells undergoing apoptosis. (author)
-
Park, Mi-Sook; Oh, Hyean-Ae; Ko, Il-Gyu; Kim, Sung-Eun; Kim, Sang-Hoon; Kim, Chang-Ju; Kim, Hyun-Bae; Kim, Hong
2014-06-01
Traumatic brain injury (TBI) is a leading cause of neurological deficit in the brain, which induces short- and long-term brain damage, cognitive impairment with/without structural alteration, motor deficits, emotional problems, and death both in children and adults. In the present study, we evaluated whether mild TBI in childhood causes persisting memory impairment until adulthood. Moreover, we investigated the influence of mild TBI on memory impairment in relation with hippocampal apoptosis. For this, step-down avoidance task, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and immunohistochemistry for caspase-3 were performed. Male Sprague-Dawley rats were used in the experiments. The animals were randomly divided into two groups: sham-operation group and TBI-induction group. The mild TBI model was created with an electromagnetic contusion device activated at a velocity of 3.0 m/sec. The results showed that mild TBI during the pediatric stage significantly decreased memory retention. The numbers of TUNEL-positive and caspase-3-positive cells were increased in the TBI-induction group compared to those in the sham-operation group. Defective memory retention and apoptosis sustained up to the adult stage. The present results shows that mild TBI induces long-lasting cognitive impairment from pediatric to adult stages in rats through the high level of apoptosis. The finding of this study suggests that children with mild TBI may need intensive treatments for the reduction of long-lasting cognitive impairment by secondary neuronal damage.
-
Prognostic significance of Fas and Fas ligand system-associated apoptosis in gastric cancer.
Ohno, S; Tachibana, M; Shibakita, M; Dhar, D K; Yoshimura, H; Kinugasa, S; Kubota, H; Masunaga, R; Nagasue, N
2000-12-01
Previous studies indicate that gastric carcinomas express Fas ligand and down-regulate Fas to escape from the host immune attack; however, the prognostic importance of Fas/FasL expression in this tumor is yet to be evaluated. Specimens from 87 gastric carcinoma patients of different stages treated in a defined period with curative intent were evaluated for apoptosis, Fas, FasL, and CD8 expression using an immunohistochemical method. The percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic cells expressed as apoptotic index (AI) was higher in 43 patients when the cut-off value was set at the median value. There were no significant correlations between AI and clinicopathologic parameters. Thirty-nine patients showed a high number of CD8+ cells within cancer nests. Positive FasL and Fas expression was seen in 53 and 72 patients, respectively. CD8 and FasL expressions were related only to patients' age. Fas expression had significant correlations with tumor invasion and Lauren classification. There were significant direct correlations between AI and number of nest CD8+ cells and between AI and grade of Fas expression. Apoptotic index, pT stage, CD8 expression, and Fas expression were identified as independent prognostic factors. Spontaneous apoptosis in gastric carcinoma may be an independent prognosticator for survival and is significantly influenced by tumor Fas expression and number of nest CD8 + cells.
-
Inhibition of miR-155 Protects Against LPS-induced Cardiac Dysfunction and Apoptosis in Mice
Directory of Open Access Journals (Sweden)
Hui Wang
2016-01-01
Full Text Available Sepsis-induced myocardial dysfunction represents a major cause of death in intensive care units. Dysregulated microRNAs (miR-155 has been implicated in multiple cardiovascular diseases and miR-155 can be induced by lipopolysaccharide (LPS. However, the role of miR-155 in LPS-induced cardiac dysfunction is unclear. Septic cardiac dysfunction in mice was induced by intraperitoneal injection of LPS (5 mg/kg and miR-155 was found to be significantly increased in heart challenged with LPS. Pharmacological inhibition of miR-155 using antagomiR improved cardiac function and suppressed cardiac apoptosis induced by LPS in mice as determined by echocardiography, terminal deoxynucleotidyl transferase nick-end labeling (TUNEL assay, and Western blot for Bax and Bcl-2, while overexpression of miR-155 using agomiR had inverse effects. Pea15a was identified as a target gene of miR-155, mediating its effects in controlling apoptosis of cardiomyocytes as evidenced by luciferase reporter assays, quantitative real time-polymerase chain reaction, Western blot, and TUNEL staining. Noteworthy, miR-155 was also found to be upregulated in the plasma of patients with septic cardiac dysfunction compared to sepsis patients without cardiac dysfunction, indicating a potential clinical relevance of miR-155. The receiver-operator characteristic curve indicated that plasma miR-155 might be a biomarker for sepsis patients developing cardiac dysfunction. Therefore, inhibition of miR-155 represents a novel therapy for septic myocardial dysfunction.
-
Directory of Open Access Journals (Sweden)
Junghyun Kim
2016-09-01
Full Text Available Retinal capillary cell loss is a hallmark of early diabetic retinal changes. Advanced glycation end products (AGEs are believed to contribute to retinal microvascular cell loss in diabetic retinopathy. In this study, the protective effects of Aster koraiensis extract (AKE against damage to retinal vascular cells were investigated in streptozotocin (STZ-induced diabetic rats. To examine this issue further, AGE accumulation, nuclear factor-kappaB (NF-κB and inducible nitric oxide synthase (iNOS were investigated using retinal trypsin digests from streptozotocin-induced diabetic rats. In the diabetic rats, TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling-positive retinal microvascular cells were markedly increased. Immunohistochemical studies revealed that AGEs were accumulated within the retinal microvascular cells, and this accumulation paralleled the activation of NF-κB and the expression of iNOS in the diabetic rats. However, AKE prevented retinal microvascular cell apoptosis through the inhibition of AGE accumulation and NF-κB activation. Moreover, to determine the active compounds of AKE, two major compounds, chlorogenic acid and 3,5-di-O-caffeoylquinic acid, were tested in an in vitro assay. Among these compounds, chlorogenic acid significantly reduced AGE formation as well as AGE/RAGE (receptor for AGEs binding activity. These results suggest that AKE, particularly chlorogenic acid, is useful in inhibiting AGE accumulation in retinal vessels and exerts a preventive effect against the injuries of diabetic retinal vascular cells.
-
Tavares, R S; Silva, A F; Lourenço, B; Almeida-Santos, T; Sousa, A P; Ramalho-Santos, J
2013-11-01
Sperm chromatin/DNA damage can be measured by a variety of assays. However, it has been reported that these tests may lose prognostic value in Assisted Reproductive Technology (ART) cycles when assessed in post-prepared samples, possibly due to the normalizing effect promoted by sperm preparation procedures. We have recently implemented a modified version of the Diff-Quik staining assay that allows for the evaluation of human sperm chromatin status in native samples, together with standard sperm morphology assessment. However, the value of this parameter in terms of predicting in vitro fertilization (IVF) and Intracytoplasmic sperm injection (ICSI) outcomes after sperm selection is unknown. In this study, data from 138 couples undergoing in vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI) treatments showed that sperm chromatin integrity was significantly improved after density gradient centrifugation and swim up (p embryo development rates (p > 0.05). However, sperm samples presenting lower percentages of damaged chromatin were associated with better quality (Grade I) embryos in both ART procedures (p selection may occur; but not in ICSI, where sperm selection is operator dependent. This quick and low-cost assay is suggested as an alternative method to detect sperm chromatin status in minimal clinical settings, when no other well-established and robust assays (e.g. Sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling) are available. © 2013 American Society of Andrology and European Academy of Andrology.
-
Wang, Pu; Zhang, Hao; Hou, Haoli; Wang, Qing; Li, Yingnan; Huang, Yan; Xie, Liangfu; Gao, Fei; He, Shibin; Li, Lijia
2016-07-01
Epigenetic modifications play crucial roles in the regulation of chromatin architecture and are involved in cell cycle progression, including mitosis and meiosis. To explore the relationship between epigenetic modifications and the cell cycle, we treated maize (Zea mays) seedlings with six different epigenetic modification-related inhibitors and identified the postsynthetic phase (G2 ) arrest via flow cytometry analysis. Total H4K5ac levels were significantly increased and the distribution of H3S10ph signalling was obviously changed in mitosis under various treatments. Further statistics of the cells in different periods of mitosis confirmed that the cell cycle was arrested at preprophase. Concentrations of hydrogen peroxide were relatively higher in the treated plants and the antioxidant thiourea could negate the influence of the inhibitors. Moreover, all of the treated plants displayed negative results in the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) and γ-H2AX immunostaining assays after exposure for 3 d. Additionally, the expression level of topoisomerase genes in the treated plants was relatively lower than that in the untreated plants. These results suggest that these inhibitors of epigenetic modifications could cause preprophase arrest via reactive oxygen species formation inhibiting the expression of DNA topoisomerase genes, accompanied by changes in the H4K5ac and H3S10ph histone modifications. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.
-
Hydrogen-rich saline ameliorates the severity of L-arginine-induced acute pancreatitis in rats
International Nuclear Information System (INIS)
Chen, Han; Sun, Yan Ping; Li, Yang; Liu, Wen Wu; Xiang, Hong Gang; Fan, Lie Ying; Sun, Qiang; Xu, Xin Yun; Cai, Jian Mei; Ruan, Can Ping; Su, Ning; Yan, Rong Lin; Sun, Xue Jun; Wang, Qiang
2010-01-01
Molecular hydrogen, which reacts with the hydroxyl radical, has been considered as a novel antioxidant. Here, we evaluated the protective effects of hydrogen-rich saline on the L-arginine (L-Arg)-induced acute pancreatitis (AP). AP was induced in Sprague-Dawley rats by giving two intraperitoneal injections of L-Arg, each at concentrations of 250 mg/100 g body weight, with an interval of 1 h. Hydrogen-rich saline (>0.6 mM, 6 ml/kg) or saline (6 ml/kg) was administered, respectively, via tail vein 15 min after each L-Arg administration. Severity of AP was assessed by analysis of serum amylase activity, pancreatic water content and histology. Samples of pancreas were taken for measuring malondialdehyde and myeloperoxidase. Apoptosis in pancreatic acinar cell was determined with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique (TUNEL). Expression of proliferating cell nuclear antigen (PCNA) and nuclear factor kappa B (NF-κB) were detected with immunohistochemistry. Hydrogen-rich saline treatment significantly attenuated the severity of L-Arg-induced AP by ameliorating the increased serum amylase activity, inhibiting neutrophil infiltration, lipid oxidation and pancreatic tissue edema. Moreover, hydrogen-rich saline treatment could promote acinar cell proliferation, inhibit apoptosis and NF-κB activation. These results indicate that hydrogen treatment has a protective effect against AP, and the effect is possibly due to its ability to inhibit oxidative stress, apoptosis, NF-κB activation and to promote acinar cell proliferation.
-
Giriş, Murat; Erbil, Yeşim; Depboylu, Bilge; Mete, Ozgür; Türkoğlu, Umit; Abbasoğlu, Semra Doğru; Uysal, Müjdat
2010-12-01
The exact pathogenesis of hepatic dysfunction in hyperthyroidism is still unknown. We aimed to investigate the pathogenesis of liver dysfunction caused by hyperthyroidism through inducing heme oxygenase-1 (HO-1) expression, which has antioxidant and anti-apoptotic properties. Rats were divided into six groups: untreated (group 1), treated with zinc protoporphyrin (ZnPP) (group 2), treated with hemin (group 3), treated with tri-iodothyronine (T3) (group 4), treated with T3 and ZnPP (group 5), and treated with T3 and hemin (group 6). After 22 d, oxidative stress and antioxidant enzymes and the expression of HO-1, mitochondrial permeability transition, cytochrome c, Bax, Bcl-2, caspase-3, caspase-8, and caspase-3 activity, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay were examined. Hyperthyroidism induced oxidative stress of liver tissue was ameliorated by HO-1 induction. Administration of hemin (HO-1 inducer) increased Bcl-2 expression. Decreased expression of cytochrome c was accompanied by a decrease in caspase-3, caspase-8, Bax expression, and caspase-3 activity. The apoptotic activity and oxidative damage were found to be increased by the administration of ZnPP (HO-1 inhibitor). Immunohistochemistry findings supported these results. HO-1 induction plays a protective role in the pathogenesis of the liver dysfunction in hyperthyroidism. This effect is dependent on modulation of the antiapoptotic and antioxidative pathways by HO-1 expression. Copyright © 2010 Elsevier Inc. All rights reserved.
-
Directory of Open Access Journals (Sweden)
Kuen-Daw Tsai
2012-01-01
Full Text Available Curcumin (CUR has been shown to possess a preventive effect against various cancers and interfere with multiple-cell signaling pathways. We evaluated the protective effects of CUR in regression of UVB-induced skin tumor formation in SKH-1 hairless mice and its underlying early molecular biomarkers associated with carcinogenesis. Mice irradiated with UVB at 180 mJ/cm2 twice per week elicited 100% tumor incidence at 20 weeks. Topical application of CUR prior to UVB irradiation caused delay in tumor appearance, multiplicity, and size. Topical application of CUR prior to and immediately after a single UVB irradiation (180 mJ/cm2 resulted in a significant decrease in UVB-induced thymine dimer-positive cells, expression of proliferative cell nuclear antigen (PCNA, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and apoptotic sunburn cells together with an increase in p53 and p21/Cip1-positive cell population in epidermis. Simultaneously, CUR also significantly inhibited NF-κB, cyclooxygenase-2 (COX-2, prostaglandin E2 (PGE2, and nitric oxide (NO levels. The results suggest that the protective effect of CUR against photocarcinogenesis is accompanied by downregulation of cell proliferative controls, involving thymine dimer, PCNA, apoptosis, transcription factors NF-κB, and of inflammatory responses involving COX-2, PGE2, and NO, while upregulation of p53 and p21/Cip1 to prevent DNA damage and facilitate DNA repair.
-
Pepper, Andrew R; Bruni, Antonio; Pawlick, Rena; Wink, John; Rafiei, Yasmin; Gala-Lopez, Boris; Bral, Mariusz; Abualhassan, Nasser; Kin, Tatsuya; Shapiro, A M James
2017-10-01
Islet transplantation is an effective therapy in type 1 diabetes and recalcitrant hypoglycemia. However, there is an ongoing need to circumvent islet loss posttransplant. We explore herein the potential of the pan-caspase inhibitor F573 to mitigate early apoptosis-mediated islet death within portal and extrahepatic portal sites in mice. Mouse or human islets were cultured in standard media ±100 μM F573 and subsequently assessed for viability and apoptosis via terminal deoxynucleotidyl transferase dUTP nick end labeling staining and caspase-3 activation. Diabetic mice were transplanted with syngeneic islets placed under the kidney capsule (KC) or into the subcutaneous deviceless (DL) site at a marginal islet dose (150 islets), or into the portal vein (PV) at a full dose (500 islets). Human islets were transplanted under the KC of diabetic immunodeficient mice at a marginal dose (500 islet equivalents). Islets were cultured in the presence of F573, and F573 was administered subcutaneously on days 0 to 5 posttransplant. Control mice were transplanted with nontreated islets and were injected with saline. Graft function was measured by nonfasting blood glucose and glucose tolerance testing. F573 markedly reduced human and mouse islet apoptosis after in vitro culture (P islet function when transplanted under the KC (P islet marginal KC transplants. Conversely, F573 significantly improved mouse islet engraftment in the PV and DL site (P islet apoptosis and improves engraftment most effectively in the portal and DL subcutaneous sites.
-
Kobayashi, Yoshinori; Mori, Masaaki; Naruto, Takuya; Kobayashi, Naoki; Sugai, Toshiyuki; Imagawa, Tomoyuki; Yokota, Shumpei
2004-12-01
In the process of apoptosis, it is known that the transition of cytochrome c from mitochondria into the cytosol occurs, and tumor necrosis factor (TNF)-alpha is one of the molecules responsible for this event. But in the state of hypercytokine induced by D-galactosamine (D-GaIN)/Lipopolysaccharide (LPS), the localization of cytochrome c is little known. Rats were administrated with D-GaIN(700 mg/kg)/LPS(200 microg/kg). Blood and tissue samples were collected and examined for levels of pro-inflammatory cytokines, the apoptosis of liver cells, and the localization of cytochrome c. Before administration of D-GaIN/LPS, cytochrome c was definitely localized in the mitochondria. At 2 h after simultaneous administration of D-GaIN/LPS, cytochrome c had accumulated in the cytosol following abrupt increases of plasma TNF-alpha. Massive cell destruction due to apoptosis proved by Terminal deoxynucleo-tidyl transferase-mediated dUTP nick end labeling staining was observed in liver tissue 4 h later and markedly increased levels of cytochrome c were detected in the plasma 12 h after D-GaIN/LPS administration. Liver injury induced by simultaneous administration of D-GaIN/LPS was closely associated with the production of TNF-alpha, and also with the dynamic movement of cytochrome c from the mitochondria into the cytosol, and then into the systemic circulation. The detection of plasma cytochrome c levels may be a useful clinical tool for the detection of apoptosis in vivo.
-
Directory of Open Access Journals (Sweden)
G. Perrone
2010-09-01
Full Text Available The human equilibrative nucleoside transporter 1 (hENT1 is the major means by which gemcitabine enters human cells; recent evidence exists that hENT1 is expressed in carcinoma of the ampulla of Vater and that it should be considered as a molecular prognostic marker for patients with resected ampullary cancer. Aim of the present study is to evaluate the variations of hENT1 expression in ampullary carcinomas and to correlate such variations with histological subtypes and clinicopathological parameters. Forty-one ampullary carcinomas were histologically classified into intestinal, pancreaticobiliary and unusual types. hENT1 and Ki67 expression were evaluated by immunohistochemistry, and apoptotic cells were identified by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labelling (TUNEL method. hENT1 overexpression was detected in 63.4% ampullary carcinomas. A significant difference in terms of hENT1 and Ki67 expression was found between intestinal vs. pancreaticobiliary types (P=0.03 and P=0.009 respectively. Moreover, a significant statistical positive correlation was found between apoptotic and proliferative Index (P=0.036, while no significant correlation was found between hENT1 and apoptosis. Our results on hENT1 expression suggest that classification of ampullary carcinoma by morphological subtypes may represent an additional tool in prospective clinical trials aimed at examining treatment efficacy; in addition, data obtained from Ki67 and TUNEL suggest a key role of hENT1 in tumour growth of ampullary carcinoma.
-
Li, Jianru; Chen, Jingsen; Mo, Hangbo; Chen, Jingyin; Qian, Cong; Yan, Feng; Gu, Chi; Hu, Qiang; Wang, Lin; Chen, Gao
2016-05-01
Minocycline has beneficial effects in early brain injury (EBI) following subarachnoid hemorrhage (SAH); however, the molecular mechanisms underlying these effects have not been clearly identified. This study was undertaken to determine the influence of minocycline on inflammation and neural apoptosis and the possible mechanisms of these effects in early brain injury following subarachnoid hemorrhage. SAH was induced by the filament perforation model of SAH in male Sprague-Dawley rats. Minocycline or vehicle was given via an intraperitoneal injection 1 h after SAH induction. Minocycline treatment markedly attenuated brain edema secondary to blood-brain barrier (BBB) dysfunction by inhibiting NLRP3 inflammasome activation, which controls the maturation and release of pro-inflammatory cytokines, especially interleukin-1β (IL-1β). Minocycline treatment also markedly reduced the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells. To further identify the potential mechanisms, we demonstrated that minocycline increased Bcl2 expression and reduced the protein expression of P53, Bax, and cleaved caspase-3. In addition, minocycline reduced the cortical levels of reactive oxygen species (ROS), which are closely related to both NLRP3 inflammasome and P53 expression. Minocycline protects against NLRP3 inflammasome-induced inflammation and P53-associated apoptosis in early brain injury following SAH. Minocycline's anti-inflammatory and anti-apoptotic effect may involve the reduction of ROS. Minocycline treatment may exhibit important clinical potentials in the management of SAH.
-
Yao, Yao; Jiang, Cheng-Shuai; Sun, Na; Li, Wei-Qi; Niu, Yang; Han, Huai-Qin; Miao, Zhen-Hua; Zhao, Xun-Xia; Zhao, Jing; Li, Juan
2017-01-10
Chemical investigation of Tamarix ramosissima Ledeb, a traditional herbal medicine used for rheumatoid arthritis (RA) treatment in northwest China, led to the discovery of a new phenolic aromatic rings substituted lactam, tamaractam ( 1 ), together with the previously reported compounds cis - N -feruloyl-3- O -methyldopamine ( 2 ) and trans - N -feruloyl-3- O -methyldopamine ( 3 ). The structures of the compounds were determined by high resolution electrospray ionization mass spectroscopy (HRESIMS) and 1D and 2D-NMR experiments, as well as comparison with the literature data. The effects of the three compounds on the viability of RA fibroblast-like synoviocytes (RA-FLS) were assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2- H -tetrazolium bromide (MTT) assay. Pro-apoptosis effect of compound 1 in RA-FLS was further investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, activated caspase-3/7 level assessment using luminescence assay, and sub-G₁ fraction measurement using flow cytometry. It was found that these three compounds displayed variable proliferation inhibitory activity in RA-FLS, and compound 1 exhibited the strongest effect. Compound 1 could remarkably induce cellular apoptosis of RA-FLS, increase activated caspase-3/7 levels, and significantly increase sub-G₁ fraction in the cell cycle. The results suggested that compound 1 may inhibit the proliferation of RA-FLS through apoptosis-inducing effect, and these compounds may contribute to the anti-RA effect of T. ramosissima .
-
Directory of Open Access Journals (Sweden)
Yao Yao
2017-01-01
Full Text Available Chemical investigation of Tamarix ramosissima Ledeb, a traditional herbal medicine used for rheumatoid arthritis (RA treatment in northwest China, led to the discovery of a new phenolic aromatic rings substituted lactam, tamaractam (1, together with the previously reported compounds cis-N-feruloyl-3-O-methyldopamine (2 and trans-N-feruloyl-3-O-methyldopamine (3. The structures of the compounds were determined by high resolution electrospray ionization mass spectroscopy (HRESIMS and 1D and 2D-NMR experiments, as well as comparison with the literature data. The effects of the three compounds on the viability of RA fibroblast-like synoviocytes (RA-FLS were assessed by 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide (MTT assay. Pro-apoptosis effect of compound 1 in RA-FLS was further investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay, activated caspase-3/7 level assessment using luminescence assay, and sub-G1 fraction measurement using flow cytometry. It was found that these three compounds displayed variable proliferation inhibitory activity in RA-FLS, and compound 1 exhibited the strongest effect. Compound 1 could remarkably induce cellular apoptosis of RA-FLS, increase activated caspase-3/7 levels, and significantly increase sub-G1 fraction in the cell cycle. The results suggested that compound 1 may inhibit the proliferation of RA-FLS through apoptosis-inducing effect, and these compounds may contribute to the anti-RA effect of T. ramosissima.
-
Chen, Songjie; Hu, Hui; Miao, Shushu; Zheng, Jiayong; Xie, Zhijian; Zhao, Hui
2017-05-01
Oral squamous cell carcinoma is one of the most common neoplasm in the world. Despite the improvements in diagnosis and treatment, the outcome is still poor now. Thus, the development of novel therapeuticapproaches is needed. The aim of this study is to assess the synergistic anti-tumor effect of andrographolide with cisplatin (DDP) in oral squamous cell carcinoma CAL-27 cells in vitro and in vivo. We performed Cell Counting Kit-8 proliferation assay, apoptosis assay, and western blotting on CAL-27 cells treated with andrographolide, DDP or the combination in vitro. In vivo, we also treated CAL-27 xenografts with andrographolide or the combination, and performed terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay and immunohistochemical analysis of Ki-67. The results showed the combination of andrographolide and DDP synergistically inhibited CAL-27 cell proliferation in vitro and caused tumor regression in vivo in the CAL-27 xenografts. In addition, the synergistic anti-tumor effect of andrographolide with synergistic was due to an enhanced apoptosis. Moreover, the combination therapy upregulated the expression level of p-p53 in vitro and decreased Ki-67 expression in vivo. Our data indicate that the combination treatment of andrographolide and DDP results in synergistic anti-tumor growth activity against oral squamous cell carcinoma CAL-27 in vitro and in vivo. These results demonstrated that combination of andrographolide with DDP was likely to represent a potential therapeutic strategy for oral squamous cell carcinoma.
-
Ji, Qianqian; Zhu, Kongju; Liu, Zhiguo; Song, Zhenwei; Huang, Yuankai; Zhao, Haijing; Chen, Yaosheng; He, Zuyong; Mo, Delin; Cong, Peiqing
2013-03-15
Trichostain A (TSA), an inhibitor of histone deacetylases, improved developmental competence of SCNT embryos in many species, apparently by improved epigenetic reprogramming. The objective of the present study was to determine the effects of TSA-induced apoptosis in cloned porcine embryos. At various developmental stages, a comet assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were used to detect apoptosis, and real-time polymerase chain reaction was used to assess expression of genes related to apoptosis and pluripotency. In this study, TSA significantly induced apoptosis (in a dose-dependent manner) at the one-, two-, and four-cell stages. However, in blastocyst stage embryos, TSA decreased the apoptotic index (P < 0.05). Expression levels of Caspase 3 were higher in TSA-treated versus control embryos at the two-cell stage (not statistically significant). The expression ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of antiapoptotic potential, was higher in TSA-treated groups at the one-, two-, and four-cell and blastocyst stages. Furthermore, expression levels of pluripotency-related genes, namely, Oct4 and Nanog, were elevated at the morula stage (P < 0.05) in TSA treatment groups. We concluded that inducing apoptosis might be a mechanism by which TSA promotes development of reconstructed embryos. At the initial stage of apoptosis induction, abnormal cells were removed, thereby enhancing proliferation of healthy cells and improving embryo quality. Copyright © 2013 Elsevier Inc. All rights reserved.
-
Pushparajah, Daphnee S; Umachandran, Meera; Plant, Kathryn E; Plant, Nick; Ioannides, Costas
2007-02-28
The principal objective was to ascertain whether precision-cut tissue slices can be used to evaluate the potential of chemicals to induce CYP1, epoxide hydrolase and glutathione S-transferase activities, all being important enzymes involved in the metabolism of polycyclic aromatic hydrocarbons. Precision-cut rat liver and lung slices were incubated with a range of benzo[a]pyrene concentrations for various time periods. A rise in the O-deethylation of ethoxyresorufin was seen in both liver and lung slices exposed to benzo[a]pyrene, which was accompanied by increased CYP1A apoprotein levels. Pulmonary CYP1B1 apoprotein levels and hepatic mRNA levels were similarly enhanced. Elevated epoxide hydrolase and glutathione S-transferase activities were also observed in liver slices following incubation for 24h; similarly, a rise in apoprotein levels of both enzymes was evident, peak levels occurring at the same time point. When mRNA levels were monitored, a rise in the levels of both enzymes was seen as early as 4h after incubation, but maximum levels were attained at 24 h. In lung slices, induction of epoxide hydrolase by benzo[a]pyrene was observed after a 24-h incubation, and at a concentration of 1 microM; a rise in apoprotein levels was seen at this time point. Glutathione S-transferase activity was not inducible in lung slices by benzo[a]pyrene but a modest increase was observed in hepatic slices. Collectively, these studies confirmed CYP1A induction in rat liver slices and established that CYP1B1 expression, and epoxide hydrolase and glutathione S-transferase activities are inducible in precision-cut tissue slices.
-
DEFF Research Database (Denmark)
Schjoldager, Katrine Ter-Borch Gram; Vester-Christensen, Malene B; Bennett, Eric Paul
2010-01-01
immediately C-terminal (TT(226)). We developed an in vivo model system in CHO ldlD cells that was used to show that O-glycosylation in the processing site blocked processing of ANGPTL3. Genome-wide SNP association studies have identified the polypeptide GalNAc-transferase gene, GALNT2, as a candidate gene...... for low HDL and high triglyceride blood levels. We hypothesized that the GalNAc-T2 transferase performed critical O-glycosylation of proteins involved in lipid metabolism. Screening of a panel of proteins known to affect lipid metabolism for potential sites glycosylated by GalNAc-T2 led to identification...
-
DEFF Research Database (Denmark)
Buchard, Anders; Eefsen, Martin; Semb, Synne
2012-01-01
The aim of this study was to assess if genetic variants in the glutathione-S-transferase genes GST-T1, M1, and P1 reflect risk factors in acetaminophen (APAP)-poisoned patients assessed by investigation of the relation to prothrombin time (PT), which is a sensitive marker of survival in these pat...
-
Cuevas, Carlos; Huenchuguala, Sandro; Muñoz, Patricia; Villa, Monica; Paris, Irmgard; Mannervik, Bengt; Segura-Aguilar, Juan
2015-04-01
U373MG cells are able to take up aminochrome that induces glutathione transferase M2-2 (GSTM2) expression in a concentration-dependent manner where 100 µM aminochrome increases GSTM2 expression by 2.1-fold (P protects SH-SY5Y cells incubated with 10 µM aminochrome. The significant protection provided by U373MG-conditioned medium in SH-SY5Y cells incubated with aminochrome was dependent on GSTM2 internalization into SH-SY5Y cells as evidenced by (i) uptake of (14)C-GSTM2 released from U373MG cells into SH-SY5Y cells, a process inhibited by anti-GSTM2 antiserum; (ii) lack of protection of U373MG-conditioned medium in the presence of anti-GSTM2 antiserum on SH-SY5Y cells treated with aminochrome; and (iii) lack of protection of conditioned medium from U373MGsiGST6 that expresses an siRNA directed against GSTM2 on SH-SY5Y cells treated with aminochrome. In conclusion, our results demonstrated that U373MG cells protect SH-SY5Y cells against aminochrome neurotoxicity by releasing GSTM2 into the conditioned medium and subsequent internalization of GSTM2 into SH-SY5Y cells. These results suggest a new mechanism of protection of dopaminergic neurons mediated by astrocytes by releasing GSTM2 into the intersynaptic space and subsequent internalization into dopaminergic neuron in order to protect these cells against aminochrome neurotoxicity.
-
Resistance to the Peptidyl Transferase Inhibitor Tiamulin Caused by Mutation of Ribosomal Protein L3
Bøsling, Jacob; Poulsen, Susan M.; Vester, Birte; Long, Katherine S.
2003-01-01
The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the peptidyl transferase center. Tiamulin-resistant Escherichia coli mutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon. Selection in a strain with all seven chromosomal rRNA operons yielded a mutant with an A445G mutation in the gene coding for ri...
-
Chimalapati, S.; Cohen, J.M.; Camberlein, E.; Macdonald, N.; Durmort, C.; Vernet, T.; Hermans, P.W.M.; Mitchell, T.; Brown, J.S.
2012-01-01
Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the
-
Directory of Open Access Journals (Sweden)
Loida López-Fernández
Full Text Available With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity.
-
López-Fernández, Loida; Ruiz-Roldán, Carmen; Pareja-Jaime, Yolanda; Prieto, Alicia; Khraiwesh, Husam; Roncero, M. Isabel G.
2013-01-01
With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity. PMID:24416097
-
Khan, M.I.; Micheal, S.; Akhtar, F.; Ahmed, W.; Ijaz, B.; Ahmed, A.; Qamar, R.
2010-01-01
PURPOSE: The aim of the present study was to investigate the association of glutathione S-transferase GSTT1 and GSTM1 genotypes with pseudoexfoliative glaucoma (PEXG) in a group of Pakistani patients. METHODS: Multiplex polymerase chain reaction was used to study the GSTT1 and GSTM1 polymorphisms in
-
Lin28 Mediates Cancer Chemotherapy Resistance via Regulation of miRNA Signaling.
Xu, Chaoyang; Xie, Shuduo; Song, Chunjiao; Huang, Liming; Jiang, Zhinong
2014-06-01
Chemotherapy resistance is one of the major obstacles limiting the success of cancer drug treatment. Among the mechanisms of resistance to chemotherapy treatment, there are those closely related to P-Glycoprotein, multidrug resistance-related protein, glutathione S-transferase pi and topoisomerase-II. Lin28 is a highly conserved RNA-binding protein, it consists of a cold shock domain and retroviral-type (CCHC) zinc finger motifs. In previous preclinical and clinical studies, positive Lin28 expression in cancer cells was correlated with decreased sensitivity to chemotherapy. And Lin28 could mediate cancer chemotherapy resistance via regulation of miR107 and Let-7 MiRNA. This article reviews current knowledge on predictive value of Lin28 in response to chemotherapy. Better understanding of its role may facilitate patient's selection of therapeutic regimen and lead to optimal clinical outcome.
-
Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.
Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R
2007-08-01
INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.
-
Glutathione Transferase GSTπ In Breast Tumors Evaluated By Three Techniques
Directory of Open Access Journals (Sweden)
Rafael Molina
1993-01-01
Full Text Available The glutathione transferases are involved in intracellular detoxification reactions. One of these, GSTπ, is elevated in some breast cancer cells, particularly cells selected for resistance to anticancer agents. We evaluated GSTπ expression in 60 human breast tumors by three techniques, immunohistochemistry, Northern hybridization, and Western blot analysis. There was a significant positive correlation between the three methods, with complete concordance seen in 64% of the tumors. There was strong, inverse relationship between GSTπ expression and steroid receptor status with all of the techniques utili zed. [n addition, there was a trend toward higher GSTπ expression in poorly differentiated tumors, but no correlation was found between tumor GSTπ content and DNA ploidy or %S-phase. GSTπ expression was also detected in adjacent benign breast tissue as well as infiltrating lymphocytes; this expression may contribute to GSTπ measurements using either Northern hybridization or Western blot analysis. These re sults suggest that immunohistochemistry is the method of choice for measuring GSTπ in breast tumors.
-
Isozyme-specific fluorescent inhibitor of glutathione s-transferase omega 1.
Son, Junghyun; Lee, Jae-Jung; Lee, Jun-Seok; Schüller, Andreas; Chang, Young-Tae
2010-05-21
Recently, the glutathione S-transferase omega 1 (GSTO1) is suspected to be involved in certain cancers and neurodegenerative diseases. However, profound investigation on the pathological roles of GSTO1 has been hampered by the lack of specific methods to determine or modulate its activity in biological systems containing other isoforms with similar catalytic function. Here, we report a fluorescent compound that is able to inhibit and monitor the activity of GSTO1. We screened 43 fluorescent chemicals and found a compound (6) that binds specifically to the active site of GSTO1. We observed that compound 6 inhibits GSTO1 by covalent modification but spares other isoforms in HEK293 cells and demonstrated that compound 6 could report the activity of GSTO1 in NIH/3T3 or HEK293 cells by measuring the fluorescence intensity of the labeled amount of GSTO1 in SDS-PAGE. Compound 6 is a useful tool to study GSTO1, applicable as a specific inhibitor and an activity reporter.
-
International Nuclear Information System (INIS)
Lee, Kang Kwang; Shimoji, Manami; Hossain, Quazi Sohel; Sunakawa, Hajime; Aniya, Yoko
2008-01-01
Microsomal glutathione transferase (MGST1) is activated by oxidative stress. Although MGST1 is found in mitochondrial membranes (mtMGST1), there is no information about the oxidative activation of mtMGST1. In the present study, we aimed to determine whether mtMGST1 also undergoes activation and about its function. When rats were treated with galactosamine/lipopolysaccharide (GalN/LPS), mtMGST1 activity was significantly increased, and the increased activity was reduced by the disulfide reducing agent dithiothreitol. In mitochondria from GalN/LPS-treated rats, disulfide-linked mtMGST1 dimer and mixed protein glutathione disulfides (glutathionylation) were detected. In addition, cytochrome c release from mitochondria isolated from GalN/LPS-treated rats was observed, and the release was inhibited by anti-MGST1 antibodies. Incubation of mitochondria from control rats with diamide and diamide plus GSH in vitro resulted in dimer- and mixed disulfide bond-mediated activation of mtMGST1, respectively. The activation of mtMGST1 by diamide plus GSH caused cytochrome c release from the mitochondria, and the release was prevented by treatment with anti-MGST1 antibodies. In addition, diamide plus GSH treatment caused mitochondrial swelling accompanied by cytochrome c release, which was inhibited by cyclosporin A (CsA) and bongkrekic acid (BKA), inhibitors of the mitochondrial permeability transition (MPT) pore. Furthermore, mtMGST1 activity was also inhibited by CsA and BKA. These results indicate that mtMGST1 is activated through mixed disulfide bond formation that contributes to cytochrome c release from mitochondria through the MPT pore
-
Yang, Jun; Sun, Xiao-Qin; Yan, Shu-Ying; Pan, Wen-Jun; Zhang, Mao-Xin; Cai, Qing-Nian
2017-07-01
Plant phenolics are crucial defense phytochemicals against herbivores and glutathione S-transferase (GST) and carboxylesterase (CarE) in herbivorous insects are well-known detoxification enzymes for such xenobiotics. To understand relationship between a plant phenolic and herbivore GST or CarE genes, we evaluated the relationship between a rice phenolic ferulic acid and resistance to brown planthopper (BPH, Nilaparvata lugens), and investigated the interaction of ferulic acid with GST or CarE genes in BPH. The results indicate that ferulic acid content in tested rice varieties was highly associated with resistance to BPH. Bioassays using artificial diets show that the phenolic acid toxicity to BPH was dose dependent and the LC 25 and LC 50 were 5.81 and 23.30 μg/ml at 72 hr, respectively. Activities of the enzymes BPH GST and CarE were increased at concentrations below the LC 50 of ferulic acid. Moreover, low ferulic acid concentrations (gene silencing (DIGS) of GST or CarE, it was shown that suppressed expression levels of NlGSTD1, NlGSTE1 and NlCE were 14.6%-21.2%, 27.8%-34.2%, and 10.5%-19.8%, respectively. Combination of NlGSTD1, NlGSTE1 or NlCE knockdown with ferulic acid increased nymph mortality by 92.9%, 119.9%, or 124.6%, respectively. These results suggest that depletion of detoxification genes in herbivorous insects by plant-mediated RNAi technology might be a new potential resource for improving rice resistance to BPH.
-
Differential roles of tau class glutathione S-transferases in oxidative stress
DEFF Research Database (Denmark)
Kilili, Kimiti G; Atanassova, Neli; Vardanyan, Alla
2004-01-01
The plant glutathione S-transferase BI-GST has been identified as a potent inhibitor of Bax lethality in yeast, a phenotype associated with oxidative stress and disruption of mitochondrial functions. Screening of a tomato two-hybrid library for BI-GST interacting proteins identified five homologous...... Tau class GSTs, which readily form heterodimers between them and BI-GST. All six LeGSTUs were found to be able to protect yeast cells from prooxidant-induced cell death. The efficiency of each LeGSTU was prooxidant-specific, indicating a different role for each LeGSTU in the oxidative stress......-response mechanism. The prooxidant protective effect of all six proteins was suppressed in the absence of YAP1, a transcription factor that regulates hydroperoxide homeostasis in Saccharomyces cerevisiae, suggesting a role for the LeGSTUs in the context of the YAP1-dependent stress-responsive machinery...
-
Reiner, David J; Yu, Seong-Jin; Shen, Hui; He, Yi; Bae, Eunkyung; Wang, Yun
2014-04-01
Methamphetamine (MA) is a drug of abuse as well as a dopaminergic neurotoxin. 9-Cis retinoic acid (9cRA), a biologically active derivative of vitamin A, has protective effects against damage caused by H(2)O(2) and oxygen-glucose deprivation in vitro as well as infarction and terminal deoxynucleotidyl transferase-mediated dNTP nick-end labeling (TUNEL) labeling in ischemic brain. The purpose of this study was to examine if there was a protective role for 9cRA against MA toxicity in nigrostriatal dopaminergic neurons. Primary dopaminergic neurons, prepared from rat embryonic ventral mesencephalic tissue, were treated with MA. High doses of MA decreased tyrosine hydroxylase (TH) immunoreactivity while increasing TUNEL labeling. These toxicities were significantly reduced by 9cRA. 9cRA also inhibited the export of Nur77 from nucleus to cytosol, a response that activates apoptosis. The interaction of 9cRA and MA in vivo was next examined in adult rats. 9cRA was delivered intracerebroventricularly; MA was given (5 mg/kg, 4×) one day later. Locomotor behavior was measured 2 days after surgery for a period of 48 h. High doses of MA significantly reduced locomotor activity and TH immunoreactivity in striatum. Administration of 9cRA antagonized these changes. Previous studies have shown that 9cRA can induce bone morphogenetic protein-7 (BMP7) expression and that administration of BMP7 attenuates MA toxicity. We demonstrated that MA treatment significantly reduced BMP7 mRNA expression in nigra. Noggin (a BMP antagonist) antagonized 9cRA-induced behavioral recovery and 9cRA-induced normalization of striatal TH levels. Our data suggest that 9cRA has a protective effect against MA-mediated neurodegeneration in dopaminergic neurons via upregulation of BMP.
-
Li, Bo; He, Yue; Xu, Liang; Hu, Qin; Tang, Junjia; Chen, Yujie; Tang, Jiping; Feng, Hua; Zhang, John H
2015-08-01
Progranulin has been reported to have neuroprotective actions in cultured neurons. This study investigated the effect of recombinant rat progranulin on early brain injury after subarachnoid hemorrhage. Controlled in vivo laboratory study. Animal research laboratory. Two hundred thirty adult male Sprague-Dawley rats weighing 280-320 g. Subarachnoid hemorrhage was induced in rats by endovascular perforation. Rat recombinant progranulin (1 and 3 ng) was administrated intracerebroventricularly at 1.5 hours after subarachnoid hemorrhage. Progranulin small interfering RNA was administrated by intracerebroventricularly at 1 day before subarachnoid hemorrhage induction. Subarachnoid hemorrhage grade, neurologic score, and brain water content were measured at 24 and 72 hours after subarachnoid hemorrhage. Neural apoptosis was evaluated by double immunofluorescence staining using terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick-end labeling and neuronal nuclei. For mechanistic study, the expression of progranulin, phosphorylated Akt, Akt, p-Erk, Erk, Bcl-2, and cleaved caspase-3 were analyzed by Western blot at 24 hours after subarachnoid hemorrhage. siRNA for sortilin 1 (a progranulin receptor) was used to intervene the downstream pathway. The expression of progranulin decreased and reached the lowest point at 24 hours after subarachnoid hemorrhage. Administration of rat recombinant progranulin decreased brain water content and improved neurologic functions at both 24 and 72 hours after subarachnoid hemorrhage, while knockdown of endogenous progranulin aggravated neurologic deficits after subarachnoid hemorrhage. Rat recombinant progranulin treatment reduced neuronal apoptosis, while progranulin deficiency promoted neuronal apoptosis at 24 hours after subarachnoid hemorrhage. Rat recombinant progranulin promoted Akt activation, increased Bcl-2 level, but reduced caspase-3 level. Knockdown of progranulin binding factor sortilin 1
-
Cytokine-induced activation of glial cells in the mouse brain is enhanced at an advanced age.
Deng, X-H; Bertini, G; Xu, Y-Z; Yan, Z; Bentivoglio, M
2006-08-25
Numerous neurological diseases which include neuroinflammatory components exhibit an age-related prevalence. The aging process is characterized by an increase of inflammatory mediators both systemically and in the brain, which may prime glial cells. However, little information is available on age-related changes in the glial response of the healthy aging brain to an inflammatory challenge. This problem was here examined using a mixture of the proinflammatory cytokines interferon-gamma and tumor necrosis factor-alpha, which was injected intracerebroventricularly in young (2-3.5 months), middle-aged (10-11 months) and aged (18-21 months) mice. Vehicle (phosphate-buffered saline) was used as control. After a survival of 1 or 2 days (all age groups) or 4 days (young and middle-aged animals), immunohistochemically labeled astrocytes and microglia were investigated both qualitatively and quantitatively. In all age groups, astrocytes were markedly activated in periventricular as well as in deeper brain regions 2 days following cytokine treatment, whereas microglia activation was already evident at 24 h. Interestingly, cytokine-induced activation of both astrocytes and microglia was significantly more marked in the brain of aged animals, in which it included numerous ameboid microglia, than of younger age groups. Moderate astrocytic activation was also seen in the hippocampal CA1 field of vehicle-treated aged mice. FluoroJade B histochemistry and the terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling technique, performed at 2 days after cytokine administration, did not reveal ongoing cell death phenomena in young or aged animals. This indicated that glial cell changes were not secondary to neuronal death. Altogether, the findings demonstrate for the first time enhanced activation of glial cells in the old brain, compared with young and middle-aged subjects, in response to cytokine exposure. Interestingly, the results also suggest that such enhancement
-
Ota, Shinichi; Geschwind, Jean-Francois H; Buijs, Manon; Wijlemans, Joost W; Kwak, Byung Kook; Ganapathy-Kanniappan, Shanmugasundaram
2013-06-01
Studies in animal models of cancer have demonstrated that targeting tumor metabolism can be an effective anticancer strategy. Previously, we showed that inhibition of glucose metabolism by the pyruvate analog, 3-bromopyruvate (3-BrPA), induces anticancer effects both in vitro and in vivo. We have also documented that intratumoral delivery of 3-BrPA affects tumor growth in a subcutaneous tumor model of human liver cancer. However, the efficacy of such an approach in a clinically relevant orthotopic tumor model has not been reported. Here, we investigated the feasibility of ultrasound (US) image-guided delivery of 3-BrPA in an orthotopic mouse model of human pancreatic cancer and evaluated its therapeutic efficacy. In vitro, treatment of Panc-1 cells with 3-BrPA resulted in a dose-dependent decrease in cell viability. The loss of viability correlated with a dose-dependent decrease in the intracellular ATP level and lactate production confirming that disruption of energy metabolism underlies these 3-BrPA-mediated effects. In vivo, US-guided delivery of 3-BrPA was feasible and effective as demonstrated by a marked decrease in tumor size on imaging. Further, the antitumor effect was confirmed by (1) a decrease in the proliferative potential by Ki-67 immunohistochemical staining and (2) the induction of apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphospate nick end labeling staining. We therefore demonstrate the technical feasibility of US-guided intratumoral injection of 3-BrPA in a mouse model of human pancreatic cancer as well as its therapeutic efficacy. Our data suggest that this new therapeutic approach consisting of a direct intratumoral injection of antiglycolytic agents may represent an exciting opportunity to treat patients with pancreas cancer.
-
Enhancement of P53-Mutant Human Colorectal Cancer Cells Radiosensitivity by Flavonoid Fisetin
International Nuclear Information System (INIS)
Chen Wenshu; Lee Yijang; Yu Yichu; Hsaio Chinghui
2010-01-01
Purpose: The aim of this study was to investigate whether fisetin is a potential radiosensitizer for human colorectal cancer cells, which are relatively resistant to radiotherapy. Methods and Materials: Cell survival was examined by clonogenic survival assay, and DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The effects of treatments on cell cycle distribution and apoptosis were examined by flow cytometry. Western blot analysis was performed to ascertain the protein levels of γ-H2AX, phospho-Chk2, active caspase-3, PARP cleavage, phospho-p38, phospho-AKT, and phospho-ERK1/2. Results: Fisetin pretreatment enhanced the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells but not human keratocyte HaCaT cells; it also prolonged radiation-induced G 2 /M arrest, enhanced radiation-induced cell growth arrest in HT-29 cells, and suppressed radiation-induced phospho-H2AX (Ser-139) and phospho-Chk2 (Thr-68) in p53-mutant HT-29 cells. Pretreatment with fisetin enhanced radiation-induced caspase-dependent apoptosis in HT-29 cells. Fisetin pretreatment augmented radiation-induced phosphorylation of p38 mitogen-activated protein kinase, which is involved in caspase-mediated apoptosis, and SB202190 significantly reduced apoptosis and radiosensitivity in fisetin-pretreated HT-29 cells. By contrast, both phospho-AKT and phospho-ERK1/2, which are involved in cell proliferation and antiapoptotic pathways, were suppressed after irradiation combined with fisetin pretreatment. Conclusions: To our knowledge, this study is the first to provide evidence that fisetin exerts a radiosensitizing effect in p53-mutant HT-29 cells. Fisetin could potentially be developed as a novel radiosensitizer against radioresistant human cancer cells.
-
International Nuclear Information System (INIS)
LaCourse, E. James; Hernandez-Viadel, Mariluz; Jefferies, James R.; Svendsen, Claus; Spurgeon, David J.; Barrett, John; John Morgan, A.; Kille, Peter; Brophy, Peter M.
2009-01-01
The earthworm Lumbricus rubellus (Hoffmeister, 1843) is a terrestrial pollution sentinel. Enzyme activity and transcription of phase II detoxification superfamily glutathione transferases (GST) is known to respond in earthworms after soil toxin exposure, suggesting GST as a candidate molecular-based pollution biomarker. This study combined sub-proteomics, bioinformatics and biochemical assay to characterise the L. rubellus GST complement as pre-requisite to initialise assessment of the applicability of GST as a biomarker. L. rubellus possesses a range of GSTs related to known classes, with evidence of tissue-specific synthesis. Two affinity-purified GSTs dominating GST protein synthesis (Sigma and Pi class) were cloned, expressed and characterised for enzyme activity with various substrates. Electrospray ionisation mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) following SDS-PAGE were superior in retaining subunit stability relative to two-dimensional gel electrophoresis (2-DE). This study provides greater understanding of Phase II detoxification GST superfamily status of an important environmental pollution sentinel organism. - This study currently provides the most comprehensive view of the Phase II detoxification enzyme superfamily of glutathione transferases within the important environmental pollution sentinel earthworm Lumbricus rubellus.
-
Energy Technology Data Exchange (ETDEWEB)
LaCourse, E. James, E-mail: james.la-course@liverpool.ac.u [Institute of Biological, Environmental, and Rural Sciences, Aberystwyth University, Aberystwyth SY23 3DA (United Kingdom); Hernandez-Viadel, Mariluz; Jefferies, James R. [Institute of Biological, Environmental, and Rural Sciences, Aberystwyth University, Aberystwyth SY23 3DA (United Kingdom); Svendsen, Claus; Spurgeon, David J. [Centre for Ecology and Hydrology, Huntingdon PE28 2LS (United Kingdom); Barrett, John [Institute of Biological, Environmental, and Rural Sciences, Aberystwyth University, Aberystwyth SY23 3DA (United Kingdom); John Morgan, A.; Kille, Peter [Biosciences, University of Cardiff, Cardiff CF10 3TL (United Kingdom); Brophy, Peter M. [Institute of Biological, Environmental, and Rural Sciences, Aberystwyth University, Aberystwyth SY23 3DA (United Kingdom)
2009-08-15
The earthworm Lumbricus rubellus (Hoffmeister, 1843) is a terrestrial pollution sentinel. Enzyme activity and transcription of phase II detoxification superfamily glutathione transferases (GST) is known to respond in earthworms after soil toxin exposure, suggesting GST as a candidate molecular-based pollution biomarker. This study combined sub-proteomics, bioinformatics and biochemical assay to characterise the L. rubellus GST complement as pre-requisite to initialise assessment of the applicability of GST as a biomarker. L. rubellus possesses a range of GSTs related to known classes, with evidence of tissue-specific synthesis. Two affinity-purified GSTs dominating GST protein synthesis (Sigma and Pi class) were cloned, expressed and characterised for enzyme activity with various substrates. Electrospray ionisation mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) following SDS-PAGE were superior in retaining subunit stability relative to two-dimensional gel electrophoresis (2-DE). This study provides greater understanding of Phase II detoxification GST superfamily status of an important environmental pollution sentinel organism. - This study currently provides the most comprehensive view of the Phase II detoxification enzyme superfamily of glutathione transferases within the important environmental pollution sentinel earthworm Lumbricus rubellus.
-
Ridder, L.; Rietjens, I.M.C.M.; Vervoort, J.J.M.; Mulholland, A.J.
2002-01-01
Glutathione S-transferases (GSTs) play an important role in the detoxification of xenobiotics in mammals. They catalyze the conjugation of glutathione to a wide range of electrophilic compounds. Phenanthrene 9,10-oxide is a model substrate for GSTs, representing an important group of epoxide
-
Energy Technology Data Exchange (ETDEWEB)
Stoddard, Ethan G. [Chemical Biology and Exposure; Killinger, Bryan J. [Chemical Biology and Exposure; Nair, Reji N. [Chemical Biology and Exposure; Sadler, Natalie C. [Chemical Biology and Exposure; Volk, Regan F. [Chemical Biology and Exposure; Purvine, Samuel O. [Chemical Biology and Exposure; Shukla, Anil K. [Chemical Biology and Exposure; Smith, Jordan N. [Chemical Biology and Exposure; Wright, Aaron T. [Chemical Biology and Exposure
2017-11-01
Glutathione S-transferases (GSTs) comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity-based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “H site”. Therefore, we developed (1) a glutathione-based photoaffinity probe (GSH-ABP) to target the “G site”, and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. The GST-ABP is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competitors in liver homogenates. Herein, we present robust tools for the novel characterization of enzyme- and active site-specific GST activity in mammalian model systems.
-
International Nuclear Information System (INIS)
Casalino, Elisabetta; Sblano, Cesare; Calzaretti, Giovanna; Landriscina, Clemente
2006-01-01
Acute cadmium intoxication affects glutathione S-transferase (GST) in rat liver. It has been found that 24 h after i.p. cadmium administration to rats, at a dose of 2.5 mg CdCl 2 kg -1 body weight, the activity of this enzyme in liver cytosol increased by 40%. A less stimulatory effect persisted till 48 h and thereafter the enzyme activity normalized. Since, GST isoenzymes belong to different classes in mammalian tissues, we used quantitative immunoassays to verify which family of GST isoenzymes is influenced by this intoxication. Only alpha-class glutathione S-transferase (α-GST) proteins were detected in rat liver cytosol and their level increased by about 25%, 24 h after cadmium treatment. No pi-GST isoforms were found in liver cytosol from either normal or cadmium-treated rats. Co-administration of actinomycin D with cadmium normalized both the protein level and the activity of α-GST, suggesting that some effect occurs on enzyme transcription of these isoenzymes by this metal. On the other hand, it seems unlikely that the stimulatory effect is due to the high level of peroxides caused by lipid peroxidation, since Vitamin E administration strongly reduced the TBARS level, but did not cause any GST activity decrease
-
Preconditioning Strategies for Solving Elliptic Difference Equations on a Multiprocessor.
1982-01-01
CALLING MA31C. C TD - TIME REQUIRED BY MA31C TO PERFORM THE C FACTORIZATION. 160 C TDT - TOTAL TIME REQUIRED BY SUBROUTINE FACTOR. C TDT-T IM3-T IMI...TPD-TIM2-TIM1 TD -TIM3-TIM2 165 C WRITE(LP,70) TDT,TPD, TD 70 FORMAT(7H TDT - ,F6.3,7H TPD " F6.3,6H TD ,F6.3) C WRITE(LP, 85) NTYPE,NVERN 170 85 FORMAT...INTEGER INI(IAI) ,INJ(IAJ) ,IK(NN,4) INTEGER NU(3000) C COMMON/EA 4BD /PRVT(4),IPRVT(6) 15 COMMON/MA31I/DD,LP,MP COMMON/MA31J/LROW,LCOL,NCP,ND, IPD
-
The O-GlcNAc Transferase Intellectual Disability Mutation L254F Distorts the TPR Helix.
Gundogdu, Mehmet; Llabrés, Salomé; Gorelik, Andrii; Ferenbach, Andrew T; Zachariae, Ulrich; van Aalten, Daan M F
2018-05-17
O-linked β-N-acetyl- D -glucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential post-translational modification that is abundant in the brain. Recently, OGT mutations have been associated with intellectual disability, although it is not understood how they affect OGT structure and function. Using a multi-disciplinary approach we show that the L254F OGT mutation leads to conformational changes of the tetratricopeptide repeats and reduced activity, revealing the molecular mechanisms contributing to pathogenesis. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.
-
Serum glutathione transferase does not respond to indole-3-carbinol: A pilot study
Directory of Open Access Journals (Sweden)
Daniel R McGrath
2010-05-01
Full Text Available Daniel R McGrath1, Hamid Frydoonfar2, Joshua J Hunt3, Chris J Dunkley3, Allan D Spigelman41Ipswich Hospital, Ipswich, UK; 2Hunter Pathology Service, New South Wales; 3Royal Newcastle Centre, Newcastle; 4St Vincent’s Clinical School, Sydney, AustraliaBackground: Despite the well recognized protective effect of cruciferous vegetables against various cancers, including human colorectal cancers, little is known about how this effect is conferred. It is thought that some phytochemicals found only in these vegetables confer the protection. These compounds include the glucosinolates, of which indole-3-carbinol is one. They are known to induce carcinogen-metabolizing (phase II enzymes, including the glutathione S-transferase (GST family. Other effects in humans are not well documented. We wished to assess the effect of indole-3-carbinol on GST enzymes.Methods: We carried out a placebo-controlled human volunteer study. All patients were given 400 mg daily of indole-3-carbinol for three months, followed by placebo. Serum samples were tested for the GSTM1 genotype by polymerase chain reaction. Serum GST levels were assessed using enzyme-linked immunosorbent assay and Western Blot methodologies.Results: Forty-nine volunteers completed the study. GSTM1 genotypes were obtained for all but two volunteers. A slightly greater proportion of volunteers were GSTM1-positive, in keeping with the general population. GST was detected in all patients. Total GST level was not affected by indole-3-carbinol dosing compared with placebo. Although not statistically significant, the GSTM1 genotype affected the serum GST level response to indole-3-carbinol.Conclusion: Indole-3-carbinol does not alter total serum GST levels during prolonged dosing.Keywords: pilot study, colorectal cancer, glutathione transferase, human, indole-3-carbinol
-
International Nuclear Information System (INIS)
Dixit, Prachy; Mukherjee, Prasun K.; Sherkhane, Pramod D.; Kale, Sharad P.; Eapen, Susan
2011-01-01
Highlights: → Transgenic plants expressing a TvGST gene were tested for tolerance, uptake and degradation of anthracene. → Transgenic plants were more tolerant to anthracene and take up more anthracene from soil and solutions compared to control plants. → Using in vitro T 1 seedlings, we showed that anthracene-a three fused benzene ring compound was phytodegraded to naphthalene derivatives, having two benzene rings. → This is the first time that a transgenic plant was shown to have the potential to phytodegrade anthracene. - Abstract: Plants can be used for remediation of polyaromatic hydrocarbons, which are known to be a major concern for human health. Metabolism of xenobiotic compounds in plants occurs in three phases and glutathione transferases (GST) mediate phase II of xenobiotic transformation. Plants, although have GSTs, they are not very efficient for degradation of exogenous recalcitrant xenobiotics including polyaromatic hydrocarbons. Hence, heterologous expression of efficient GSTs in plants may improve their remediation and degradation potential of xenobiotics. In the present study, we investigated the potential of transgenic tobacco plants expressing a Trichoderma virens GST for tolerance, remediation and degradation of anthracene-a recalcitrant polyaromatic hydrocarbon. Transgenic plants with fungal GST showed enhanced tolerance to anthracene compared to control plants. Remediation of 14 C uniformly labeled anthracene from solutions and soil by transgenic tobacco plants was higher compared to wild-type plants. Transgenic plants (T 0 and T 1 ) degraded anthracene to naphthalene derivatives, while no such degradation was observed in wild-type plants. The present work has shown that in planta expression of a fungal GST in tobacco imparted enhanced tolerance as well as higher remediation potential of anthracene compared to wild-type plants.
-
The Genetic Architecture of Murine Glutathione Transferases.
Directory of Open Access Journals (Sweden)
Lu Lu
Full Text Available Glutathione S-transferase (GST genes play a protective role against oxidative stress and may influence disease risk and drug pharmacokinetics. In this study, massive multiscalar trait profiling across a large population of mice derived from a cross between C57BL/6J (B6 and DBA2/J (D2--the BXD family--was combined with linkage and bioinformatic analyses to characterize mechanisms controlling GST expression and to identify downstream consequences of this variation. Similar to humans, mice show a wide range in expression of GST family members. Variation in the expression of Gsta4, Gstt2, Gstz1, Gsto1, and Mgst3 is modulated by local expression QTLs (eQTLs in several tissues. Higher expression of Gsto1 in brain and liver of BXD strains is strongly associated (P < 0.01 with inheritance of the B6 parental allele whereas higher expression of Gsta4 and Mgst3 in brain and liver, and Gstt2 and Gstz1 in brain is strongly associated with inheritance of the D2 parental allele. Allele-specific assays confirmed that expression of Gsto1, Gsta4, and Mgst3 are modulated by sequence variants within or near each gene locus. We exploited this endogenous variation to identify coexpression networks and downstream targets in mouse and human. Through a combined systems genetics approach, we provide new insight into the biological role of naturally occurring variants in GST genes.
-
Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas
DEFF Research Database (Denmark)
Mandel, U; Hassan, H; Therkildsen, M H
1999-01-01
GalNAc-T1, -T2, and -T3. Application of this panel of novel antibodies revealed that GalNAc- transferases are differentially expressed in different cell lines, in spermatozoa, and in oral mucosa and carcinomas. For example, GalNAc-T1 and -T2 but not -T3 were highly expressed in WI38 cells, and Gal......NAc-T3 but not GalNAc-T1 or -T2 was expressed in spermatozoa. The expression patterns in normal oral mucosa were found to vary with cell differentiation, and for GalNAc-T2 and -T3 this was reflected in oral squamous cell carcinomas. The expression pattern of GalNAc-T1 was on the other hand changed...
-
Peran Kedelai (Glycine max L. dalam Pencegahan Apoptosis pada Cedera Jaringan Hati
Directory of Open Access Journals (Sweden)
Maya Tejasari
2013-02-01
Full Text Available Abstrak Pada liver injury akibat berbagai sebab, terjadi apoptosis sel yang sangat banyak yang dapat memengaruhi fungsi metabolik hati. Isoflavon kedelai telah diketahui dapat mencegah apoptosis sel pada folikel ovarium dan osteoblas. Penelitian ini bertujuan untuk mengetahui pengaruh kedelai pada pencegahan apoptosis sel pada jaringan hati mencit yang diinduksi CCl4. Penelitian dilakukan menggunakan 30 ekor mencit jantan galur DDY berumur 8─10 minggu yang dibagi dalam 6 kelompok perlakuan. Kelompok 1 merupakan kontrol positif yang hanya diberi makanan pelet standar selama 3 minggu kemudian diberi 0,2 mL larutan CCl4 per oral selama 4 hari. Kelompok 2 merupakan kontrol negatif yang hanya diberi makanan pelet standar dan tidak diberi CCl4, sedangkan kelompok 3─6 merupakan kelompok uji yang selain diberi makanan pelet standar juga diberi kedelai dengan kadar berturut-turut 145,6 mg/hari, 218,4 mg/hari, 291,2 mg/hari dan 364 mg/hari selama 3 minggu kemudian diberi 0,2 mL larutan CCl4 peroral selama 4 hari. Seluruh kelompok kemudian dikorbankan dan diambil organ hatinya untuk dilakukan pemeriksaan histokimia terminal deoxynucleotidyl transferase-mediated dUTP Nick end labeling (TUNEL. Parameter yang diukur adalah jumlah apoptosis sel pada sayatan jaringan hati mencit menggunakan mikroskop cahaya. Data disajikan dan dianalisis secara statistik menggunakan uji analysis of varians (ANOVA untuk menganalisis perbedaan antar kelompok. Hasil penelitian memperlihatkan bahwa dari hasil pemeriksaan imunohistokimia TUNEL tampak jumlah sel yang mengalami apoptosis pada kelompok yang diberi kedelai lebih sedikit dibandingkan dengan kelompok yang tidak diberi kedelai. Analisis uji ANOVA antara kelompok tersebut menunjukan perbedaaan yang signifikan dengan nilai p<0,05. Simpulan, bahwa pemberian kedelai dapat mencegah apoptosis sel pada jaringan hati mencit yang diinduksi CCl4. Kata kunci: Apoptosis, CCl4, isoflavon, kedelai, liver injury, TUNEL
-
DEFF Research Database (Denmark)
Hersoug, Lars-Georg; Brasch-Andersen, Charlotte; Husemoen, Lise-Lotte
2012-01-01
Introduction: Exposure to particulate matter (PM) may induce inflammation and oxidative stress in the airways. Carriers of null polymorphisms of glutathione S-transferases (GSTs), which detoxify reactive oxygen species, may be particularly susceptible to the effects of PM. Objectives: To investig....... The relationship of glutathione-S-transferases copy number variation and indoor air pollution to symptoms and markers of respiratory disease. Clin Respir J 2011; DOI:10.1111/j.1752-699X.2011.00258.x.......: To investigate whether deletions of GSTM1 and GSTT1 modify the potential effects of exposure to indoor sources of PM on symptoms and objective markers of respiratory disease. Methods: We conducted a population-based, cross-sectional study of 3471 persons aged 18-69 years. Information about exposure to indoor......: We found that none of the symptoms and objective markers of respiratory disease were significantly associated with the GST null polymorphisms. An increasing number of positive alleles of the GSTM1 polymorphism tended to be associated lower prevalence of wheeze, cough, and high forced expiratory...
-
International Nuclear Information System (INIS)
Kuwabara, Hiroko; Yoneda, Masahiko; Hayasaki, Hana; Nakamura, Toshiya; Mori, Hiroshi
2006-01-01
The receptor for hyaluronan mediated motility (RHAMM), which is a hyaluronan-binding protein, is a centrosomal and microtubal protein. Here, we have identified two RHAMM-binding proteins, glucose regulated protein (GRP) 78 and GRP75, using co-immunoprecipitation analysis. These two proteins directly bound to glutathione-S-transferase-RHAMM fusion proteins. By double immunostaining, GRP78 and GRP75 colocalized with RHAMM in interphase microtubules, but were separated in mitotic spindles. Prevention of microtubule polymerization by TN-16 and vincristine sulfate induced RHAMM overexpression without a significant change in GRP78/75. Taken together, GRP78/75 and RHAMM complexes may stabilize microtubules in the interphase, associated with a downregulation of RHAMM. These results reveal a new biochemical activity of RHAMM
-
LENUS (Irish Health Repository)
Flanagan, J M
2010-02-01
Classical or transferase-deficient galactosaemia is an inherited metabolic disorder caused by mutation in the human Galactose-1-phosphate uridyl transferase (GALT) gene. Of some 170 causative mutations reported, fewer than 10% are observed in more than one geographic region or ethnic group. To better understand the population history of the common GALT mutations, we have established a haplotyping system for the GALT locus incorporating eight single nucleotide polymorphisms and three short tandem repeat markers. We analysed haplotypes associated with the three most frequent GALT gene mutations, Q188R, K285N and Duarte-2 (D2), and estimated their age. Haplotype diversity, in conjunction with measures of genetic diversity and of linkage disequilibrium, indicated that Q188R and K285N are European mutations. The Q188R mutation arose in central Europe within the last 20 000 years, with its observed east-west cline of increasing relative allele frequency possibly being due to population expansion during the re-colonization of Europe by Homo sapiens in the Mesolithic age. K285N was found to be a younger mutation that originated in Eastern Europe and is probably more geographically restricted as it arose after all major European population expansions. The D2 variant was found to be an ancient mutation that originated before the expansion of Homo sapiens out of Africa.
-
Comparing endophenotypes in adult-onset primary torsion dystonia.
LENUS (Irish Health Repository)
Bradley, David
2012-02-01
Adult-onset primary torsion dystonia (AOPTD) has an autosomal dominant pattern of inheritance with markedly reduced penetrance; the genetic causes of most forms of AOPTD remain unknown. Endophenotypes, markers of sub-clinical gene carriage, may be of use detecting non-manifesting gene carriers in relatives of AOPTD patients. The aim of this study was to compare the utility of the spatial discrimination threshold (SDT) and temporal discrimination threshold (TDT) as potential endophenotypes in AOPTD. Data on other published candidate endophenotypes are also considered. Both SDT and TDT testing were performed in 24 AOPTD patients and 34 of their unaffected first degree relatives; results were compared with normal values from a control population. Of the 24 AOPTD patients 5 (21%) had abnormal SDTs and 20 (83%) had abnormal TDTs. Of the 34 first degree relatives 17 (50%) had abnormal SDTs and 14 (41%) had abnormal TDTs. Discordant results on SDT and TDT testing were found in 16 (67%) AOPTD patients and 21 (62%) first degree relatives. TDT testing has superior sensitivity compared to SDT testing in AOPTD patients; although false positive TDTs are recognised, the specificity of TDT testing in unaffected relatives is not determinable. The high level of discordance between the two tests probably relates methodological difficulties with SDT testing. The SDT is an unreliable AOPTD endophenotype; TDT testing fulfils criteria for a reliable endophenotype with a high sensitivity.
-
Glutathione S-transferase gene polymorphisms in presbycusis.
Ateş, Nurcan Aras; Unal, Murat; Tamer, Lülüfer; Derici, Ebru; Karakaş, Sevim; Ercan, Bahadir; Pata, Yavuz Selim; Akbaş, Yücel; Vayisoğlu, Yusuf; Camdeviren, Handan
2005-05-01
Glutathione and glutathione-related antioxidant enzymes are involved in the metabolism and detoxification of cytotoxic and carcinogenic compounds as well as reactive oxygen species. Reactive oxygen species generation occurs in prolonged relative hypoperfusion conditions such as in aging. The etiology of presbycusis is much less certain; however, a complex genetic cause is most likely. The effect of aging shows a wide interindividual range; we aimed to investigate whether profiles of (glutathione S-transferase (GST) M1, T1 and P1 genotypes may be associated with the risk of age-related hearing loss. We examined 68 adults with presbycusis and 69 healthy controls. DNA was extracted from whole blood, and the GSTM1, GSTT1 and GSTP1 polymorphisms were determined using a real-time polymerase chain reaction and fluorescence resonance energy transfer with a Light-Cycler Instrument. Associations between specific genotypes and the development of presbycusis were examined by use of logistic regression analyses to calculate odds ratios and 95% confidence intervals. Gene polymorphisms at GSTM1, GSTT1, and GSTP1 in subjects with presbycusis were not significantly different than in the controls (p > 0.05). Also, the combinations of different GSTM1, GSTT1, and GSTP1 genotypes were not an increased risk of presbycusis (p > 0.05). We could not demonstrate any significant association between the GSTM1, GSTT1, and GSTP1 polymorphism and age-related hearing loss in this population. This may be because of our sample size, and further studies need to investigate the exact role of GST gene polymorphisms in the etiopathogenesis of the presbycusis.
-
Glutathione transferases are structural and functional outliers in the thioredoxin fold.
Atkinson, Holly J; Babbitt, Patricia C
2009-11-24
Glutathione transferases (GSTs) are ubiquitous scavengers of toxic compounds that fall, structurally and functionally, within the thioredoxin fold suprafamily. The fundamental catalytic capability of GSTs is catalysis of the nucleophilic addition or substitution of glutathione at electrophilic centers in a wide range of small electrophilic compounds. While specific GSTs have been studied in detail, little else is known about the structural and functional relationships between different groupings of GSTs. Through a global analysis of sequence and structural similarity, it was determined that variation in the binding of glutathione between the two major subgroups of cytosolic (soluble) GSTs results in a different mode of glutathione activation. Additionally, the convergent features of glutathione binding between cytosolic GSTs and mitochondrial GST kappa are described. The identification of these structural and functional themes helps to illuminate some of the fundamental contributions of the thioredoxin fold to catalysis in the GSTs and clarify how the thioredoxin fold can be modified to enable new functions.
-
International Nuclear Information System (INIS)
Scharmach, E.; Hessel, S.; Niemann, B.; Lampen, A.
2009-01-01
The human colon adenocarcinoma cell line Caco-2 is frequently used to study human intestinal metabolism and transport of xenobiotica. Previous studies have shown that both Caco-2 cells and human colon cells constitutively express the multigene family of detoxifying enzymes glutathione S-transferases (GSTs), particularly GST alpha and GST pi. GSTs may play a fundamental role in the molecular interplay between phase I, II enzymes and ABC-transporters. The gut fermentation product, butyrate, can modulate the potential for detoxification. The aim of this study was to investigate the basal expression of further cytosolic GSTs in Caco-2 cells during cell differentiation. In addition, a comparison was made with expression levels in MCF-7 and HepG2, two other cell types with barrier functions. Finally, the butyrate-mediated modulation of gene and protein expression was determined by real time PCR and western blot analysis. In Caco-2, gene and protein expression levels of GST alpha increased during cell differentiation. High levels of GSTO1 and GSTP1 were constantly expressed. No expression of GSTM5 and GSTT1 was detected. HepG2 expressed GSTO1 and MCF-7 GSTZ1 most intensively. No expression of GSTA5, GSTM5, or GSTP1 was detected in either cell. Incubation of Caco-2 cells with butyrate (5 mM) significantly induced GSTA1 and GSTM2 in proliferating Caco-2 cells. In differentiated cells, butyrate tended to increase GSTO1 and GSTP1. The results of this study show that a differentiation-dependent expression of GSTs in Caco-2 cells may reflect the in vivo situation and indicate the potential of butyrate to modify intestinal metabolism. GSTA1-A4 have been identified as good markers for cell differentiation. The Caco-2 cell line is a useful model for assessing the potential of food-related substances to modulate the GST expression pattern.
-
Scharmach, E; Hessel, S; Niemann, B; Lampen, A
2009-11-30
The human colon adenocarcinoma cell line Caco-2 is frequently used to study human intestinal metabolism and transport of xenobiotica. Previous studies have shown that both Caco-2 cells and human colon cells constitutively express the multigene family of detoxifying enzymes glutathione S-transferases (GSTs), particularly GST alpha and GST pi. GSTs may play a fundamental role in the molecular interplay between phase I, II enzymes and ABC-transporters. The gut fermentation product, butyrate, can modulate the potential for detoxification. The aim of this study was to investigate the basal expression of further cytosolic GSTs in Caco-2 cells during cell differentiation. In addition, a comparison was made with expression levels in MCF-7 and HepG2, two other cell types with barrier functions. Finally, the butyrate-mediated modulation of gene and protein expression was determined by real time PCR and western blot analysis. In Caco-2, gene and protein expression levels of GST alpha increased during cell differentiation. High levels of GSTO1 and GSTP1 were constantly expressed. No expression of GSTM5 and GSTT1 was detected. HepG2 expressed GSTO1 and MCF-7 GSTZ1 most intensively. No expression of GSTA5, GSTM5, or GSTP1 was detected in either cell. Incubation of Caco-2 cells with butyrate (5 mM) significantly induced GSTA1 and GSTM2 in proliferating Caco-2 cells. In differentiated cells, butyrate tended to increase GSTO1 and GSTP1. The results of this study show that a differentiation-dependent expression of GSTs in Caco-2 cells may reflect the in vivo situation and indicate the potential of butyrate to modify intestinal metabolism. GSTA1-A4 have been identified as good markers for cell differentiation. The Caco-2 cell line is a useful model for assessing the potential of food-related substances to modulate the GST expression pattern.
-
Energy Technology Data Exchange (ETDEWEB)
Dixit, Prachy; Mukherjee, Prasun K.; Sherkhane, Pramod D.; Kale, Sharad P. [Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Eapen, Susan, E-mail: eapenhome@yahoo.com [Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400085 (India)
2011-08-15
Highlights: {yields} Transgenic plants expressing a TvGST gene were tested for tolerance, uptake and degradation of anthracene. {yields} Transgenic plants were more tolerant to anthracene and take up more anthracene from soil and solutions compared to control plants. {yields} Using in vitro T{sub 1} seedlings, we showed that anthracene-a three fused benzene ring compound was phytodegraded to naphthalene derivatives, having two benzene rings. {yields} This is the first time that a transgenic plant was shown to have the potential to phytodegrade anthracene. - Abstract: Plants can be used for remediation of polyaromatic hydrocarbons, which are known to be a major concern for human health. Metabolism of xenobiotic compounds in plants occurs in three phases and glutathione transferases (GST) mediate phase II of xenobiotic transformation. Plants, although have GSTs, they are not very efficient for degradation of exogenous recalcitrant xenobiotics including polyaromatic hydrocarbons. Hence, heterologous expression of efficient GSTs in plants may improve their remediation and degradation potential of xenobiotics. In the present study, we investigated the potential of transgenic tobacco plants expressing a Trichoderma virens GST for tolerance, remediation and degradation of anthracene-a recalcitrant polyaromatic hydrocarbon. Transgenic plants with fungal GST showed enhanced tolerance to anthracene compared to control plants. Remediation of {sup 14}C uniformly labeled anthracene from solutions and soil by transgenic tobacco plants was higher compared to wild-type plants. Transgenic plants (T{sub 0} and T{sub 1}) degraded anthracene to naphthalene derivatives, while no such degradation was observed in wild-type plants. The present work has shown that in planta expression of a fungal GST in tobacco imparted enhanced tolerance as well as higher remediation potential of anthracene compared to wild-type plants.
-
Xie, Yiyue; Dahlin, Jayme L; Oakley, Aaron J; Casarotto, Marco G; Board, Philip G; Baell, Jonathan B
2018-05-10
Early stage drug discovery reporting on relatively new or difficult targets is often associated with insufficient hit triage. Literature reviews of such targets seldom delve into the detail required to critically analyze the associated screening hits reported. Here we take the enzyme glutathione transferase omega-1 (GSTO1-1) as an example of a relatively difficult target and review the associated literature involving small-molecule inhibitors. As part of this process we deliberately pay closer-than-usual attention to assay interference and hit quality aspects. We believe this Perspective will be a useful guide for future development of GSTO1-1 inhibitors, as well serving as a template for future review formats of new or difficult targets.
-
Nies, E; Almar, M M; Hermenegildo, C; Monsalve, E; Romero, F J
1991-01-01
1. The glutathione S-transferase activity in hepatopancreas of the American red crayfish Procambarus clarkii after 15 days' acclimatization in tap water aquaria was measured in specimens collected monthly for a whole year, and shows seasonal variation. 2. Previous data on the environmental pollution of Lake Albufera suggest a possible correlation with the activity tested in the different seasons of the year considering the results of non-acclimatized animals.
-
Energy Technology Data Exchange (ETDEWEB)
Takako, Furukawa; Tsuneo, Saga; Yasuhisa, Fujibayashi [Institute of Radiological Sciences, Dept. of Diagnostic Imaging, Molecular Imaging Center, Nationa, Chiba (Japan); Takako, Furukawa; Takeshi, Tanaka; Yasuhisa, Fujibayashi [Fukui Univ., Biomedica Imaging Research Center (Japan)
2006-07-01
Intratumoral distribution of [Cu-64]Cu-di-acetyl-bis (N4-methyl-thio-semi-carbazone ) ({sup 64}Cu-ATSM) and fluorine-18 2-fluoro-2-deoxyglucose ({sup 18}FDG) in mice bearing tumors of four different origins, LLC1 (Lewis lung carcinoma), Meth-A (sarcoma), B16 (melanoma) and colon26 (adenocarcinoma), were compared to the immunohistochemical staining for proliferating cells (Ki67), blood vessels (CD34 or von Willebrand Factor (vWF)) and apoptotic cells (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method). With all the cell lines, {sup 64}Cu-ATSM and {sup 18}FDG were distributed to different regions of the tumor mass. The immunohistochemical study demonstrated that the high {sup 64}Cu-ASTM uptake regions were hypo-vascular and consisted of tumor cells arrested in cell cycle, while the high {sup 18}FGD uptake regions were hyper-vascular and consisted of proliferating cells. Through our study, it was revealed that one tumor mass contains two regions of different characteristics, which can be distinguished by {sup 64}Cu-ATSM and {sup 18}FDG. Since hypoxia and cell cycle arrest are critical factors to reduce the sensitivity of tumor to radiation and conventional chemotherapy, regions with such characteristics in tumor should be treated intensively as one of the primary targets. {sup 64}Cu-ATSM which can delineate hypoxic and cell cycle arrested regions in tumors can provide valuable information for cancer treatment as well as the possibility to treat such regions directly as an internal radiotherapy reagent. (author)
-
Tang, Zhibin; Li, Chun; Chen, Zhiwei
2015-03-01
To explore the effect of baicalein on the proliferation and invasion of osteosarcoma cells and its related mechanism. Osteosarcoma MG-63 cells that were cultured in vitro were respectively treated with 20 μL culture medium (control group), dehydrated alcohol (0 μmol/L baicalein group), 100 and 200 μmol/L baicalein solution for 48 hours. Cell proliferation was analyzed by MTT assay. The cell invasion ability was detected using Transwell(TM) invasion assay. The expression of ezrin mRNA was examined by real-time quantitative PCR. The expressions of ezrin protein and p-ezrin protein were measured using Western blotting. Apoptosis index (AI) was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The inhibitory rates of cell proliferation significantly increased in 100 and 200 μmol/L baicalein groups as compared with 0 μmol/L baicalein group. Moreover, that was higher in 200 μmol/L baicalein group than in 100 μmol/L baicalein group. In comparison with control and 0 μmol/L baicalein groups, the mean cell numbers of permeated membrane and levels of ezrin mRNA, ezrin protein and p-ezrin protein gradually decreased, but AI was gradually elevated with the increase of baicalein concentrations, whereas there was no significant difference in these indicators between 0 μmol/L baicalein group and control group. Baicalein can inhibit the proliferation and invasion of osteosarcoma MG-63 cells. The mechanism may be associated with the inhibited expression and activity of ezrin protein and the promoted tumor cell apoptosis.
-
Institute of Scientific and Technical Information of China (English)
张新霞; 崔长琮; 李江; 崔翰斌; 徐仓宝; 朱参战
2002-01-01
Objective To investigate the effect of gelatin coated Platinium-Iridium stent absorbed c-myc antisense oligodeoxynucleotide (ASODN) on smooth muscle cells apoptosis in a normal rabbit carotid arteries. Methods Gelatin coated Platinium-Iridium stents were implanted in the right carotid arteries of 32 rabbits under vision. Animals were randomly divided into control group and treated group receiving c-myc ASODN (n=16, respectively). On 7, 14, 30 and 90 days following the stenting procedure ,morphometry for caculation of neointimal area and mean neointimal thickness were performed.The expression of c-myc protein was detected by immunohistochemical method. Apoptotic smooth muscle cells was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL). Results At 7 and 14 days after stenting,there were no detectable apoptotic cells in both groups. The apoptotic cells occurred in the neointima 30 and 90 days after stenting, and the number of apoptotic cells at 30 days were less [4.50±1.29 vs 25.75±1.89 (number/0.1mm2)] than that at 90 days [13.50±1.91 vs 41.50±6.46 (number/0.1mm2)]. Meanwhile c-myc ASODN induced more apoptotic cells than the control group(P<0.0001). c-myc protein expression was weak positive or negative in treated group and positive in control group.Conclusion c-myc ASODN can induce smooth muscle cells apoptosis after stenting in normal rabbit carotid arteries,and it can be used to prevent in-stent restenosis.
-
Antiangiogenic Effects of Noscapine Enhance Radioresponse for GL261 Tumors
International Nuclear Information System (INIS)
Newcomb, Elizabeth W.; Lukyanov, Yevgeniy; Alonso-Basanta, Michelle; Esencay, Min; Smirnova, Iva; Schnee, Tona; Shao Yongzhao; Devitt, Mary Louise; Zagzag, David; McBride, William; Formenti, Silvia C.
2008-01-01
Purpose: To assess the effects of noscapine, a tubulin-binding drug, in combination with radiation in a murine glioma model. Methods and Materials: The human T98G and murine GL261 glioma cell lines treated with noscapine, radiation, or both were assayed for clonogenic survival. Mice with established GL261 hind limb tumors were treated with noscapine, radiation, or both to evaluate the effect of noscapine on radioresponse. In a separate experiment with the same treatment groups, 7 days after radiation, tumors were resected and immunostained to measure proliferation rate, apoptosis, and angiogenic activity. Results: Noscapine reduced clonogenic survival without enhancement of radiosensitivity in vitro. Noscapine combined with radiation significantly increased tumor growth delay: 5, 8, 13, and 18 days for control, noscapine alone, radiation alone, and the combination treatment, respectively (p < 0.001). To assess the effect of the combination of noscapine plus radiation on the tumor vasculature, tubule formation by the murine endothelial 2H11 cells was tested. Noscapine with radiation significantly inhibited tubule formation compared with radiation alone. By immunohistochemistry, tumors treated with the combination of noscapine plus radiation showed a decrease in BrdU incorporation, an increase in apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, and a decrease in tumor vessel density compared with tumors treated with radiation alone. Conclusion: Noscapine enhanced the sensitivity of GL261 glioma tumors to radiation, resulting in a significant tumor growth delay. An antiangiogenic mechanism contributed to the effect. These findings are clinically relevant, particularly in view of the mild toxicity profile of this drug
-
International Nuclear Information System (INIS)
Nakasu, Satoshi; Fukami, Tadateru; Matsuda, Masayuki; Nakasu, Yoko
2003-01-01
The effect of ionizing radiation on the expression of immunohistochemical proliferation markers was examined in the rat pituitary gland. Rats were irradiated in the pituitary region with a dose of 40 Gy, or were sham-irradiated as controls. Bromodeoxyuridine (BrdU) was given to the rats after one week, either one hour (Br-1 group) or 17 hours (Br-17 group) before perfusion fixation. Immunohistochemical staining for BrdU, topoisomerase II-alpha (TopoII), Ki-67 (MIB-5), p21 WAF1/CiP1 (p21), and p27 Kip1 (p27) was performed. Apoptotic cells were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling method. The mean BrdU labeling index (LI) and MIB-5 LI were significantly higher in the irradiated rats than in the sham rats in the Br-1 group. TopoII LI was higher in the irradiated rats than in the sham rats, although not significantly. p27-positive cells decreased in irradiated rats, but p21-positive cells increased more than in the sham rats. The number of apoptotic cells increased significantly after radiation. BrdU LIs were lower in the irradiated rats than in the sham rats in the Br-17 group. A few small BrdU-positive fragments with apoptotic features were phagocytosed in the anterior lobe cells. These results indicate that some ''immunohistochemically proliferating cells'' subsequently undergo apoptosis in the irradiated pituitary gland. The values of proliferative indices should be cautiously interpreted after irradiation of tissue. (author)
-
Directory of Open Access Journals (Sweden)
Duncan RS
2011-04-01
Full Text Available R. Scott Duncan1,*, Hua Xin1,*, Daryl L Goad1, Kent D Chapman2,3, Peter Koulen1,31Vision Research Center and Departments of Ophthalmology and Basic Medical Science, School of Medicine, University of Missouri, Kansas City, MO, USA; 2Department of Biological Sciences, University of North Texas, Denton, TX, USA; 3Center for Plant Lipid Research, University of North Texas, Denton, TX, USA *Authors contributed equallyAbstract: Retinal ganglion cell (RGC death is a hallmark of neurodegenerative diseases and disease processes of the eye, including glaucoma. The protection of RGCs has been an important strategy for combating glaucoma, but little clinical success has been reported to date. One pathophysiological consequence of glaucoma is excessive extracellular glutamate subsequently leading to excitotoxicity in the retina. Endocannabinoids, such as the N-acylethanolamine (NAE, arachidonylethanolamine (NAE 20:4, exhibit neuroprotective properties in some models of neurodegenerative disease. The majority of NAEs, however, are not cannabinoids, and their physiological function is not clear. Here, we determined whether the noncannabinoid NAE, linoleoylethanolamine (NAE18:2, protects neurons in the RGC layer against glutamate excitotoxicity in ex-vivo retina cultures. Using a terminal deoxynucleotidyl transferase-mediated dUTP (2´-deoxyuridine 5´-triphosphate nick-end labeling (TUNEL assay, we determined that NAE18:2 reduces the number of apoptotic RGC layer neurons in response to glutamate and conclude that NAE18:2 is a neuroprotective compound with potential for treating glaucomatous retinopathy.Keywords: neuroprotection, glutamate, calcium signaling, immunocytochemistry, eye, vision, glaucoma.
-
Sclerotic effect of oxytetracycline on the submandibular gland: An experimental model.
Güçlü, Oğuz; Muratli, Asli; Arik, Deniz; Tekin, Kazım; Erdogan, Halil; Dereköy, Fevzi Sefa
2016-12-01
Oxytetracycline has been suggested as an alternate therapy for chronic recurrent sialadenitis and sialorrhea. We conducted an experimental study to investigate the sclerotic effect of this drug on the submandibular gland by histopathologic methods. Our subjects were 20 New Zealand white rabbits, which were divided into two groups of 10. The right submandibular gland of the rabbits in the active-treatment group was injected with 0.3 ml of oxytetracycline (100 mg/ml), and that of the controls was injected with saline. Four weeks after the injections, all the glands were removed. Histopathologic studies, including hematoxylin and eosin and Masson trichrome staining, were carried out. The glands were evaluated for tissue inflammation, congestion, fibrosis, edema, lipomatosis, and atrophy. To investigate apoptosis, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) immunohistochemical staining was used. In the study group, inflammation (n = 9), congestion (n = 9), fibrosis (n = 6), edema (n = 6), and lipomatosis (n = 4) were observed; in the sham group, only lipomatosis was seen (n = 5). The TUNEL assay results for acinar cells were 4.51 ± 1.41% in the oxytetracycline group and 2.08 ± 1.76% in the control group (p = 0.006); the corresponding figures for the duct cells were 7.05 ± 0.87% and 3.10 ± 2.26% (p = 0.001). Based on our findings, we conclude that oxytetracycline might be a viable alternative for the treatment of chronic recurrent sialadenitis and sialorrhea. However, more research in this area is needed.
-
Does sucralfate prevent apoptosis occurring in the ischemia/reperfusion-induced intestinal injury?
Sencan, A; Yilmaz, O; Ozer, E; Günşar, C; Genç, K; Ulukuş, C; Taneli, C; Mir, E
2003-08-01
We have shown in a previous study that sucralfate is beneficial in the prophylaxis and treatment of hypoxia/reoxygenation-induced intestinal injury. The aim of this study is to investigate whether sucralfate has any effect on the prevention of apoptosis in the ischemia/reperfusion (I/R)-induced intestinal injury. Rats were randomized into three groups. Group 1 and 2 were subjected to I/R. Group 1 (treatment group) received sucralfate while group 2 (treatment control group) did not. Group 3 served as a normal control group (sham group). The terminal ileum was harvested for histopathologic investigation by light microscopy. The presence of apoptotic enterocytes (DNA fragmentation in cell nuclei) was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) reaction. In treatment control group, 3 of 7 rats had severe inflammation. None of the sucralfate-treated rats showed severe inflammation, 6 of them only showed mild inflammatory changes (p < 0.05). The apoptotic percentage was found to be 37.1 +/- 9.4 in the sucralfate-treated group (group 1), whereas it was 45.4 +/- 3.9 in the untreated group (group 2) (p < 0.05). The sham group had a completely normal intestinal architecture. The present study shows that 1) the experimental model of I/R-induced intestinal injury induces enterocyte apoptosis; 2) sucralfate decreases enterocyte apoptosis in the experimental model of I/R-induced intestinal injury which may play a key role in the pathophysiological events leading to failure of the intrinsic gut barrier defense mechanisms.
-
Agmatine protects against cell damage induced by NMDA and glutamate in cultured hippocampal neurons
Wang, Wei-Ping; Iyo, Abiye H.; Miguel-Hidalgo, Javier; Regunathan, Soundar; Zhu, Meng-Yang
2010-01-01
Agmatine is a polyamine and has been considered as a novel neurotransmitter or neuromodulator in the central nervous system. In the present study, the neuroprotective effect of agmatine against cell damage caused by N-methyl-d-aspartate (NMDA) and glutamate was investigated in cultured rat hippocampal neurons. Lactate dehydrogenase (LDH) activity assay, β-tubulin III immunocytochemical staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay were conducted to detect cell damage. Exposure of 12-day neuronal cultures of rat hippocampus to NMDA or glutamate for 1 h caused a concentration-dependent neurotoxicity, as indicated by the significant increase in released LDH activities. Addition of 100 µM agmatine into media ablated the neurotoxicity induced by NMDA or glutamate, an effect also produced by the specific NMDA receptor antagonist dizocilpine hydrogen maleate (MK801). Arcaine, an analog of agmatine with similar structure as agmatine, fully prevented the NMDA- or glutamate-induced neuronal damage. Spermine and putrescine, the endogenous polyamine and metabolic products of agmatine without the guanidine moiety of agmatine, failed to show this effect, indicating a structural relevance for this neuroprotection. Immunocytochemical staining and TUNEL assay confirmed the findings in the LDH measurement. That is, agmatine and MK801 markedly attenuated NMDA-induced neuronal death and significantly reduced TUNEL-positive cell numbers induced by exposure of cultured hippocampal neurons to NMDA. Taken together, these results demonstrate that agmatine can protect cultured hippocampal neurons from NMDA- or glutamate-induced excitotoxicity, through a possible blockade of the NMDA receptor channels or a potential anti-apoptotic property. PMID:16546145
-
Kapucu, Burak; Cekin, Engin; Erkul, Bulent Evren; Cincik, Hakan; Gungor, Atila; Berber, Ufuk
2012-09-01
The purpose of this study was to compare the apoptotic responses to systemic, topical, and intrapolyp injection of glucocorticoid with no treatment in nasal polyps. Prospective, randomized controlled study. Tertiary training hospital. The study was performed on 48 patients with nasal polyposis in the Department of Otorhinolaryngology between 2008 and 2009. Patients were assigned to 1 of 4 groups of 12 patients. Group A was treated with oral methylprednisolone 1 mg/kg/d, and the dose was tapered gradually. Group B received 0.3 mL triamcinolone acetonide (40 mg/mL), which was injected into polyp tissue. Group C was treated with topical 55 µg triamcinolone acetonide 2 times daily for 1 month. Group D received no medication. Samples were collected endoscopically after the seventh day for groups A and B, the first month for group C, and the first visit for group D. Apoptotic indexes were determined using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. Statistically significant differences in apoptotic index were found between each steroid-medicated group and the control group (P (D-A) = .0001; P (D-B) = .003; P (D-C) = .026) and between groups A and C (P (A-C) = .012). Group B did not differ significantly from either group A or C (P (A-B) = .11; P (B-C) = .75). The apoptotic index in nasal polyps treated with systemic, topical, and intrapolyp injection forms of glucocorticoids was higher than that in the control group. Systemic steroid treatment induced the most apoptosis.
-
Zhang, Qin; Huang, Wei-Dong; Lv, Xue-Ying; Yang, Yun-Mei
2012-05-01
Oxidative stress has been implicated as a major mechanism underlying the pathogenesis of neurodegenerative disorders. ROS (reactive oxygen species) can cause cell death via apoptosis. NGF (nerve growth factor) differentiated rat PC12 cells have been extensively used to study the differentiation and apoptosis of neurons. This study has investigated the protective effects of puerarin in H2O2-induced apoptosis of differentiated PC12 cells, and the possible molecular mechanisms involved. Differentiated PC12 cells were incubated with 700 μM H2O2 in the absence or presence of different doses of puerarin (4, 8 and 16 μM). Apoptosis was assessed by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) analysis and Annexin V-PI (propidium iodide) double staining flow cytometry. Protein levels of phospho-Akt and phospho-BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) were assayed by Western blotting. After stimulation with H2O2 for 18 h, the viability of differentiated PC12 cells decreased significantly and a large number of cells underwent apoptosis. Differentiated PC12 cells were rescued from H2O2-induced apoptosis at different concentrations of puerarin in a dose-dependent manner. This was through increased production of phospho-Akt and phospho-BAD, an effect that could be reversed by wortmannin, an inhibitor of PI3K (phosphoinositide 3-kinase). The results suggest that puerarin may have neuroprotective effect through activation of the PI3K/Akt signalling pathway.
-
Directory of Open Access Journals (Sweden)
Guerin Jean F
2011-06-01
Full Text Available Abstract Background The objective of the present study is to assess viability tests and to evaluate follicle ovarian tissue quality after freezing-thawing procedures. Methods Ewe's ovaries were harvested at the slaughterhouse, after dissection each ovarian specimen was divided into two groups: fresh tissue (control group and frozen tissue. In the first part of the study, the follicles viability was assessed by trypan blue staining, calcein AM/ethidium homodimer-1 staining (LIVE/DEAD viability/cytotoxicity kit, Molecular Probes and morphology in the two groups. In the second part of the study the quality of the whole ovarian tissue was evaluated by the quantification of the release of lactate dehydrogenase measurement (Cytotoxicity Detection kit ROCHE, DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL in primordial and primary follicles (ApopDETEK Kit system Enzo and morphology in the two groups. 100 Follicles (primordial and primary were counted on both fresh and frozen hemiovary to assess this various tests. Results Ovarian follicle viability assessment was similar using trypan blue or calcein/ethidium staining. Follicles showed a decreased viability after freezing-thawing. After cryopreservation, a significant correlation between the percentage of normal follicles and viability rate was found using trypan blue (r = 0.82, p Conclusion We suggest the use of trypan blue staining for the histological assessment of viability, the use of LDH assay for the cytotoxicity assessement and finally the use of DNA fragmentation assessment to valid different freezing-thawing protocols.
-
Hu, Xianwen; Wang, Jingxian; Zhang, Li; Zhang, Qiquan; Duan, Xiaowen; Zhang, Ye
2018-06-02
Hemorrhage shock could initiate endoplasmic reticulum stress (ERS) and then induce neuronal apoptosis. The aim of this study was to investigate whether sevoflurane postconditioning could attenuate brain injury via suppressing apoptosis induced by ERS. Seventy male rats were randomized into five groups: sham, shock, low concentration (sevo1, 1.2%), middle concentration (sevo2, 2.4%) and high concentration (sevo3, 3.6%) of sevoflurane postconditioning. Hemorrhage shock was induced by removing 40% of the total blood volume during an interval of 30 min. 1h after the completion of bleeding, the animals were reinfused with shed blood during the ensuing 30 min. The spatial learning and memory ability of rats were measured by Morris water maze (MWM) test three days after the operation. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells in the hippocampus CA1 region were assessed after the MWM test. The expression of C/EBP-homologousprotein (CHOP) and glucose-regulated protein 78 (GRP78) in the hippocampus were measured at 24h after reperfusion. We found that sevoflurane postconditioning with the concentrations of 2.4% and 3.6% significantly ameliorated the spatial learning and memory ability, decreased the TUNEL-positive cells, and reduced the GRP78 and CHOP expression compared with the shock group. These results suggested that sevoflurane postconditioning with the concentrations of 2.4% and 3.6% could ameliorate spatial learning and memory deficit after hemorrhage shock and resuscitation injury via suppressing apoptosis induced by ERS. Copyright © 2018. Published by Elsevier B.V.
-
Ding, Mei-Li; Ma, Hui; Man, Yi-Gang; Lv, Hong-Yan
2017-12-01
Epigallocatechin-3-gallate (EGCG), a polyphenol in green tea, is an effective antioxidant and possesses neuroprotective effects. Brain-derived neurotrophic factor (BDNF) and cyclic AMP response element-binding protein (CREB) are crucial for neurogenesis and synaptic plasticity. In this study, we aimed to assess the protective effects of EGCG against sevoflurane-induced neurotoxicity in neonatal mice. Distinct groups of C57BL/6 mice were given EGCG (25, 50, or 75 mg/kg body weight) from postnatal day 3 (P3) to P21 and were subjected to sevoflurane (3%; 6 h) exposure on P7. EGCG significantly inhibited sevoflurane-induced neuroapoptosis as determined by Fluoro-Jade B staining and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Increased levels of cleaved caspase-3, downregulated Bad and Bax, and significantly enhanced Bcl-2, Bcl-xL, xIAP, c-IAP-1, and survivin expression were observed. EGCG induced activation of the PI3K/Akt pathway as evidenced by increased Akt, phospho-Akt, GSK-3β, phospho-GSK-3β, and mTORc1 levels. Sevoflurane-mediated downregulation of cAMP/CREB and BDNF/TrkB signalling was inhibited by EGCG. Reverse transcription PCR analysis revealed enhanced BDNF and TrkB mRNA levels upon EGCG administration. Improved performance of mice in Morris water maze tests suggested enhanced learning and memory. The study indicates that EGCG was able to effectively inhibit sevoflurane-induced neurodegeneration and improve learning and memory retention of mice via activation of CREB/BDNF/TrkB-PI3K/Akt signalling.
-
Cytosolic labile zinc: a marker for apoptosis in the developing rat brain.
Lee, Joo-Yong; Hwang, Jung Jin; Park, Mi-Ha; Koh, Jae-Young
2006-01-01
Cytosolic zinc accumulation was thought to occur specifically in neuronal death (necrosis) following acute injury. However, a recent study demonstrated that zinc accumulation also occurs in adult rat neurons undergoing apoptosis following target ablation, and in vitro experiments have shown that zinc accumulation may play a causal role in various forms of apoptosis. Here, we examined whether intraneuronal zinc accumulation occurs in central neurons undergoing apoptosis during development. Embryonic and newborn Sprague-Dawley rat brains were double-stained for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) detection of apoptosis and immunohistochemical detection of stage-specific neuronal markers, such as nestin, proliferating cell nuclear antigen (PCNA), TuJ1 and neuronal nuclear specific protein (NeuN). The results revealed that apoptotic cell death occurred in neurons of diverse stages (neural stem cells, and dividing, young and adult neurons) throughout the brain during the embryonic and early postnatal periods. Further staining of brain sections with acid fuchsin or zinc-specific fluorescent dyes showed that all of the apoptotic neurons were acidophilic and contained labile zinc in their cell bodies. Cytosolic zinc accumulation was also observed in cultured cortical neurons undergoing staurosporine- or sodium nitroprusside (SNP)-induced apoptosis. In contrast, zinc chelation with CaEDTA or N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) reduced SNP-induced apoptosis but not staurosporine-induced apoptosis, indicating that cytosolic zinc accumulation does not play a causal role in all forms of apoptosis. Finally, the specific cytosolic zinc accumulation may have a practical application as a relatively simple marker for neurons undergoing developmental apoptosis.
-
Tumor-specific apoptotic gene targeting overcomes radiation resistance in esophageal adenocarcinoma
International Nuclear Information System (INIS)
Chang, Joe Y.; Zhang Xiaochun; Komaki, Ritsuko; Cheung, Rex; Fang Bingliang
2006-01-01
Purpose: To overcome radiation resistance in esophageal adenocarcinoma by tumor-specific apoptotic gene targeting using tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Methods and Materials: Adenoviral vector Ad/TRAIL-F/RGD with a tumor-specific human telomerase reverse transcription promoter was used to transfer TRAIL gene to human esophageal adenocarcinoma and normal human lung fibroblastic cells (NHLF). Activation of apoptosis was analyzed by Western blot, fluorescent activated cell sorting, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate labeling (TUNEL) assay. A human esophageal adenocarcinoma mouse model was treated with intratumoral injections of Ad/TRAIL-F/RGD plus local radiotherapy. Results: The combination of Ad/TRAIL-F/RGD and radiotherapy increased the cell-killing effect in all esophageal adenocarcinoma cell lines but not in NHLF cells. This combination also significantly reduced clonogenic formation (p < 0.05) and increased sub-G1 deoxyribonucleic acid accumulation in cancer cells (p < 0.05). Activation of apoptosis by Ad/TRAIL-F/RGD plus radiotherapy was demonstrated by activation of caspase-9, caspase-8, and caspase-3 and cleaved poly (adenosine diphosphate-ribose) polymerase in vitro and TUNEL assay in vivo. Combined Ad/TRAIL-F/RGD and radiotherapy dramatically inhibited tumor growth and prolonged mean survival in the esophageal adenocarcinoma model to 31.6 days from 16.7 days for radiotherapy alone and 21.5 days for Ad/TRAIL-F/RGD alone (p < 0.05). Conclusions: The combination of tumor-specific TRAIL gene targeting and radiotherapy enhances the effect of suppressing esophageal adenocarcinoma growth and prolonging survival
-
Kaden, Jürgen; May, Gottfried; Völp, Andreas; Wesslau, Claus
2009-01-01
In organ grafts donor-specific sensitization is initiated immediately after revascularization. Therefore, in 1990 we introduced the intra-operative single high-dose ATG-Fresenius (ATG-F) induction in addition to standard triple drug therapy (TDT) consisting of steroids, azathioprine and cyclosporin. A total of 778 first renal transplantations from deceased donors, performed between 1987 and 1998, were included in this evaluation. This retrospective analysis of clinic records and electronic databases presents data of all recipients of first kidney grafts who received two different ATG-F inductions (1(st) group: 9 mg/kg body weight as single high-dose intra-operatively, n=484; 2(nd) group: 3 mg/kg body weight on 7 or 8 consecutive days as multiple-dose starting also intra-operatively, n=78) and standard TDT alone (3(rd) group: TDT alone, n=216). The 10-year patient survival rates were 72.6+/-2.6% (TDT + ATG-F single high-dose), 79.5+/-5.1% (TDT + ATG-F multiple-dose) and 67.2+/-3.7%% (TDT alone; Kaplan-Meier estimates with standard errors; ATG-F vs TDT alone, p=0.001). The 10-year graft survival rates with censoring of patients that died with a functioning graft were 73.8+/-2.4%, 57.7+/-5.8% and 58.4+/-3.6% (Kaplan-Meier estimates with standard errors; 1(st) vs 2(nd )and 3(rd) group, respectively, p<0.001) and the 10-year graft survival rates with patient death counted as graft failure were 58.3+/-2.7%, 55.7+/-5.8% and 48.2+/-3.5% (Kaplan-Meier estimates with standard errors; ATG-F single high-dose vs TDT, p=0.023). In pre-sensitized recipients there were also significant differences in favour of ATG-F, more notably in the single high-dose ATG-F induction. A total of 69% of the patients in the two cohorts receiving ATG-F did not experience any transplant rejections compared to 56% in patients undergoing TDT alone (p=0.018). The incidence of infectious complications was comparable across all groups. According to evidence obtained from the routine documentation of 778
-
Transmission/disequilibrium tests incorporating unaffected offspring.
Directory of Open Access Journals (Sweden)
Qinyu Wei
Full Text Available We propose a new method for family-based tests of association and linkage called transmission/disequilibrium tests incorporating unaffected offspring (TDTU. This new approach, constructed based on transmission/disequilibrium tests for quantitative traits (QTDT, provides a natural extension of the transmission/disequilibrium test (TDT to utilize transmission information from heterozygous parents to their unaffected offspring as well as the affected offspring from ascertained nuclear families. TDTU can be used in various study designs and can accommodate all types of independent nuclear families with at least one affected offspring. When the study sample contains only case-parent trios, the TDTU is equivalent to TDT. Informative-transmission disequilibrium test (i-TDT and generalized disequilibrium test(GDT are another two methods that can use information of both unaffected offspring and affected offspring. In contract to i-TDT and GDT, the test statistic of TDTU is simpler and more explicit, and can be implemented more easily. Through computer simulations, we demonstrate that power of the TDTU is slightly higher compared to i-TDT and GDT. All the three methods are more powerful than method that uses affected offspring only, suggesting that unaffected siblings also provide information about linkage and association.
-
Wilson, K; Auer, M; Binnie, M; Zheng, X; Pham, N T; Iredale, J P; Webster, S P; Mole, D J
2016-04-14
Kynurenine 3-monooxygenase (KMO) is a critical regulator of inflammation. The preferred KMO substrate, kynurenine, is converted to 3-hydroxykynurenine (3HK), and this product exhibits cytotoxicity through mechanisms that culminate in apoptosis. Here, we report that overexpression of human KMO with orthotopic localisation to mitochondria creates a metabolic environment during which the cell exhibits increased tolerance for exogenous 3HK-mediated cellular injury. Using the selective KMO inhibitor Ro61-8048, we show that KMO enzyme function is essential for cellular protection. Pan-caspase inhibition with Z-VAD-FMK confirmed apoptosis as the mode of cell death. By defining expression of pathway components upstream and downstream of KMO, we observed alterations in other key kynurenine pathway components, particularly tryptophan-2,3-dioxygenase upregulation, through bidirectional nonlinear feedback. KMO overexpression also increased expression of inducible nitric oxide synthase (iNOS). These changes in gene expression are functionally relevant, because siRNA knockdown of the pathway components kynureninase and quinolinate phosphoribosyl transferase caused cells to revert to a state of susceptibility to 3HK-mediated apoptosis. In summary, KMO overexpression, and importantly KMO activity, have metabolic repercussions that fundamentally affect resistance to cell stress.
-
Directory of Open Access Journals (Sweden)
Reto Müller
Full Text Available The O-GlcNAc transferase Eogt modifies EGF repeats in proteins that transit the secretory pathway, including Dumpy and Notch. In this paper, we show that the Notch ligands Delta and Serrate are also substrates of Eogt, that mutation of a putative UDP-GlcNAc binding DXD motif greatly reduces enzyme activity, and that Eogt and the cytoplasmic O-GlcNAc transferase Ogt have distinct substrates in Drosophila larvae. Loss of Eogt is larval lethal and disrupts Dumpy functions, but does not obviously perturb Notch signaling. To identify novel genetic interactions with eogt, we investigated dominant modification of wing blister formation caused by knock-down of eogt. Unexpectedly, heterozygosity for several members of the canonical Notch signaling pathway suppressed wing blister formation. And importantly, extensive genetic interactions with mutants in pyrimidine metabolism were identified. Removal of pyrimidine synthesis alleles suppressed wing blister formation, while removal of uracil catabolism alleles was synthetic lethal with eogt knock-down. Therefore, Eogt may regulate protein functions by O-GlcNAc modification of their EGF repeats, and cellular metabolism by affecting pyrimidine synthesis and catabolism. We propose that eogt knock-down in the wing leads to metabolic and signaling perturbations that increase cytosolic uracil levels, thereby causing wing blister formation.
-
Directory of Open Access Journals (Sweden)
Edward Rockenstein
2015-01-01
Full Text Available Neuronal stem cell (NSC grafts have been investigated as a potential neuro-restorative therapy in Parkinson's disease (PD but their use is compromised by the death of grafted cells. We investigated the use of Cerebrolysin (CBL, a neurotrophic peptide mixture, as an adjunct to NSC therapy in the α-synuclein (α-syn transgenic (tg model of PD. In vehicle-treated α-syn tg mice, there was decreased survival of NSCs. In contrast, CBL treatment enhanced the survival of NSCs in α-syn tg groups and ameliorated behavioral deficits. The grafted NSCs showed lower levels of terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells in the CBL-treated mice when compared with vehicle-treated α-syn tg mice. No evidence of tumor growth was detected. Levels of α-syn were similar in the vehicle in CBL-treated tg mice. In conclusion, CBL treatment might be a potential adjuvant for therapeutic NSC grafting in PD.
-
Evaluation of Dying Vocal Fold Epithelial Cells by Ultrastructural Features and TUNEL Method
Novaleski, Carolyn K.; Mizuta, Masanobu; Rousseau, Bernard
2016-01-01
Cell death is a regulated mechanism of eliminating cells to maintain tissue homeostasis. This study described two methodological procedures for evaluating cell death in the epithelium of immobilized, approximated, and vibrated vocal folds from 12 New Zealand white breeder rabbits. The gold standard technique of transmission electron microscopy evaluated high-quality ultrastructural criteria of cell death and a common immunohistochemical marker, terminal deoxynucleotidyl transferase dUTP nick end labeling method, to confirm cell death signaling. Results revealed that ultrastructural characteristics of apoptotic cell death, specifically condensed chromatin and apoptotic bodies, were observed after vocal fold vibration and approximation. Although episodes of necrotic cell death were rare, few enlarged cell nuclei were present after vibration and approximation. The vocal fold expresses an immunohistochemical marker for apoptosis along the apical surface of the epithelium. This study provides a solid foundation for future investigations regarding the role of cell death in vocal fold health and disease. PMID:27537846
-
Meller, R; Schindler, C K; Chu, X P; Xiong, Z G; Cameron, J A; Simon, R P; Henshall, D C
2003-05-01
Seizure-induced neuronal death may involve engagement of the BCL-2 family of apoptosis-regulating proteins. In the present study we examined the activation of proapoptotic BAD in cultured hippocampal neurons following seizures induced by removal of chronic glutamatergic transmission blockade. Kynurenic acid withdrawal elicited an increase in seizure-like electrical activity, which was inhibited by blockers of AMPA (CNQX) and NMDA (MK801 and AP5) receptor function. However, only NMDA receptor antagonists inhibited calcium entry as assessed by fura-2, and cell death of hippocampal neurons. Seizures increased proteolysis of caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) of cells. Seizure-like activity induced dephosphorylation of BAD and the disruption of its constitutive interaction with 14-3-3 proteins. In turn, BAD dimerized with antiapoptotic BCL-Xl after seizures. However, the absence of neuroprotective effects of pathway intervention suggests that BAD may perform a reinforcement rather than instigator role in cell death following seizures in vitro.
-
George, Suja; Venkataraman, Gayatri; Parida, Ajay
2010-03-01
Plant growth and productivity are adversely affected by various abiotic stress factors. In our previous study, we used Prosopis juliflora, a drought-tolerant tree species of Fabaceae, as a model plant system for mining genes functioning in abiotic stress tolerance. Large-scale random EST sequencing from a cDNA library obtained from drought-stressed leaves of 2-month-old P. juliflora plants resulted in identification of three different auxin-inducible glutathione S-transferases. In this paper, we report the cellular localization and the ability to confer drought tolerance in transgenic tobacco of one of these GSTs (PjGSTU1). PjGSTU1 was overexpressed in Escherichia coli and GST and GPX activities in total protein samples were assayed and compared with controls. The results indicated that PjGSTU1 protein forms a functional homo-dimer in recombinant bacteria with glutathione transferase as well as glutathione peroxidase activities. PjGSTU1 transgenic tobacco lines survived better under conditions of 15% PEG stress compared with control un-transformed plants. In vivo localization studies for PjGSTU1 using GFP fusion revealed protein localization in chloroplasts of transgenic plants. The peroxidase activity of PjGSTU1 and its localization in the chloroplast indicates a possible role for PjGSTU1 in ROS removal. Copyright 2009 Elsevier GmbH. All rights reserved.
-
Directory of Open Access Journals (Sweden)
Fucini Paola
2004-04-01
Full Text Available Abstract Background The bacterial ribosome is a primary target of several classes of antibiotics. Investigation of the structure of the ribosomal subunits in complex with different antibiotics can reveal the mode of inhibition of ribosomal protein synthesis. Analysis of the interactions between antibiotics and the ribosome permits investigation of the specific effect of modifications leading to antimicrobial resistances. Streptogramins are unique among the ribosome-targeting antibiotics because they consist of two components, streptogramins A and B, which act synergistically. Each compound alone exhibits a weak bacteriostatic activity, whereas the combination can act bactericidal. The streptogramins A display a prolonged activity that even persists after removal of the drug. However, the mode of activity of the streptogramins has not yet been fully elucidated, despite a plethora of biochemical and structural data. Results The investigation of the crystal structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with the clinically relevant streptogramins quinupristin and dalfopristin reveals their unique inhibitory mechanism. Quinupristin, a streptogramin B compound, binds in the ribosomal exit tunnel in a similar manner and position as the macrolides, suggesting a similar inhibitory mechanism, namely blockage of the ribosomal tunnel. Dalfopristin, the corresponding streptogramin A compound, binds close to quinupristin directly within the peptidyl transferase centre affecting both A- and P-site occupation by tRNA molecules. Conclusions The crystal structure indicates that the synergistic effect derives from direct interaction between both compounds and shared contacts with a single nucleotide, A2062. Upon binding of the streptogramins, the peptidyl transferase centre undergoes a significant conformational transition, which leads to a stable, non-productive orientation of the universally conserved U2585. Mutations of this r
-
Glycoproteins and sialyl transferase of human B lymphoblastoid cell lines
International Nuclear Information System (INIS)
Lui, S.W.L.; Ng, M.H.
1980-01-01
We used two radiolabeling methods to study glycoproteins on the surface of lymphoblastoid cells. One of the methods affects tritiation of residues which are oxidized with galactose oxidase and the other causes tritiation of neuraminic acid residues. This approach was shown to allow a better resolution of cell surface glycoproteins than if either method were used alone. Glycoproteins of B 1 - 19 cells which harbor the Epstein-Barr virus genomes were compared with those of its parental cell line, BJAB, which does not harbor the viral genomes. These studies did not reveal a unique viral protein. A 28,000 mol. wt. glycoprotein was found to be the most prominent neuraminic acidlabeled product of B 1 - 19 cells and also of the two other cell lines, Raji and Ly38, which harbor the EBV genomes. A similar molecular weight species from BJAB cells identified by galactose oxidase labeling might be deficient in neuraminic acid residues as it was poorly labeled by the periodate oxidation method. The neuraminic acid content and level of sialyl transferase of BJAB cells were found to be lower than those of the other cell lines studied. (auth.)
-
Arbildi, Paula; La-Rocca, Silvana; Lopez, Veronica; Da-Costa, Natalia; Fernandez, Veronica
2017-01-01
In the cestode parasite Echinococcus granulosus, three phylogenetically distant cytosolic glutathione transferases (GSTs) (EgGST1, 2 and 3) were identified. Interestingly, the C-terminal domains of EgGST3 and EgGST2 but not EgGST1, exhibit all amino acids involved in Sigma-class GST dimerization. Here, we provide evidence indicating that EgGST2 and EgGST3 naturally form a heterodimeric structure (EgGST2-3), and also we report the enzymatic activity of the recombinant heterodimer. EgGST2-3 might display novel properties able to influence the infection establishment. This is the first report of a stable heterodimeric GST built up by phylogenetically distant subunits. Copyright © 2016 Elsevier B.V. All rights reserved.
-
Chen, Junqin; Fontes, Ghislaine; Saxena, Geetu; Poitout, Vincent; Shalev, Anath
2010-02-01
We have previously shown that lack of thioredoxin-interacting protein (TXNIP) protects against diabetes and glucotoxicity-induced beta-cell apoptosis. Because the role of TXNIP in lipotoxicity is unknown, the goal of the present study was to determine whether TXNIP expression is regulated by fatty acids and whether TXNIP deficiency also protects beta-cells against lipoapoptosis. RESARCH DESIGN AND METHODS: To determine the effects of fatty acids on beta-cell TXNIP expression, INS-1 cells and isolated islets were incubated with/without palmitate and rats underwent cyclic infusions of glucose and/or Intralipid prior to islet isolation and analysis by quantitative real-time RT-PCR and immunoblotting. Using primary wild-type and TXNIP-deficient islets, we then assessed the effects of palmitate on apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]), mitochondrial death pathway (cytochrome c release), and endoplasmic reticulum (ER) stress (binding protein [BiP], C/EBP homologous protein [CHOP]). Effects of TXNIP deficiency were also tested in the context of staurosporine (mitochondrial damage) or thapsigargin (ER stress). Glucose elicited a dramatic increase in islet TXNIP expression both in vitro and in vivo, whereas fatty acids had no such effect and, when combined with glucose, even abolished the glucose effect. We also found that TXNIP deficiency does not effectively protect against palmitate or thapsigargin-induced beta-cell apoptosis, but specifically prevents staurosporine- or glucose-induced toxicity. Our results demonstrate that unlike glucose, fatty acids do not induce beta-cell expression of proapoptotic TXNIP. They further reveal that TXNIP deficiency specifically inhibits the mitochondrial death pathway underlying beta-cell glucotoxicity, whereas it has very few protective effects against ER stress-mediated lipoapoptosis.
-
DEFF Research Database (Denmark)
Piaggi, Simona; Marchi, Santino; Ciancia, Eugenio
2009-01-01
Barrett's esophagus (BE) represents a major risk factor for esophageal adenocarcinoma (AC). For this reason, patients with BE are subjected to a systematic endoscopic surveillance to detect initial evolution towards non-invasive neoplasia (NiN) and cancer, that eventually occurs only in a small......-S-transferase-omega 1 could be involved in the stress response of human cells playing a role in the cancer progression of Barrett's esophagus. Its immunohistochemical detection could represent a useful tool in the grading of Barrett's disease.......N in BE and to understand the mechanisms of the progression from BE to AC. We investigated the expression and subcellular localization of GSTO1 in biopsies from patients with BE and in human cancer cell lines subjected to heath shock treatment. A selective nuclear localisation of GSTO1 was found in 16/16 biopsies with low...
-
Senathilake, K S; Karunanayake, E H; Samarakoon, S R; Tennekoon, K H; de Silva, E D
2016-08-01
Human lymphatic filariasis (LF) is mainly caused by filarial parasite Wuchereria bancrofti and is the second leading cause of long term and permanent disability in tropical countries. To date, incapability to eliminate long lived adult parasites by current drugs remains the major challenge in the elimination of LF. Hence, in the current study, the efficacy of rhizome extracts of Curcuma zedoaria (a plant traditionally used in Sri Lanka in the management of LF) was evaluated as an effective filaricide in vitro. Sequential solvent extracts of C. zedoaria rhizomes were screened for in vitro antifilarial activity at 0.01-1 mg/mL concentrations by motility inhibition assay and 3-(4, 5 dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT) reduction assay using cattle parasite Setaria digitata as a model organism. Exposure of parasites to hexane and chloroform extracts of C. zedoaria caused a dose dependant reduction in motility and viability of microfilariae (IC50 = 72.42 μg/mL for hexane extract, 191.14 μg/mL for chloroform extract) and adult parasites (IC50 = 77.07 μg/mL for hexane extract, 259.87 μg/mL for chloroform extract). Both extracts were less toxic to human peripheral blood mononuclear cells when compared to filariae. A dose dependant increase in caspase 3/CED 3 and a decrease in total protein content, cyclooxygenase (COX) and protein tyrosine phosphatase (PTP) activities were observed in adult parasites treated with hexane or chloroform extract. A significant degree of chromatin condensation and apoptotic body formation were also observed in these worms by Hoechst 33342 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) staining respectively. Dose dependant chromosomal DNA laddering was observed in treated adult worms but not in microfilariae in response to both extracts. Oxidative stress parameters such as reduction in reduced glutathione (GSH) levels and increase in glutathione s transferase (GST
-
Motyka, Bruce; Fisicaro, Nella; Wang, Szu-I; Kratochvil, Annetta; Labonte, Katrina; Tao, Kesheng; Pearcey, Jean; Marshall, Thuraya; Mengel, Michael; Sis, Banu; Fan, Xiaohu; dʼApice, Anthony J F; Cowan, Peter J; West, Lori J
2016-06-01
ABO-incompatible (ABOi) organ transplantation is performed owing to unremitting donor shortages. Defining mechanisms of antibody-mediated rejection, accommodation, and tolerance of ABOi grafts is limited by lack of a suitable animal model. We report generation and characterization of a murine model to enable study of immunobiology in the setting of ABOi transplantation. Transgenesis of a construct containing human A1- and H-transferases under control of the ICAM-2 promoter was performed in C57BL/6 (B6) mice. A-transgenic (A-Tg) mice were assessed for A-antigen expression by histology and flow cytometry. B6 wild-type (WT) mice were sensitized with blood group A-human erythrocytes; others received passive anti-A monoclonal antibody and complement after heart transplant. Serum anti-A antibodies were assessed by hemagglutination. "A-into-O" transplantation (major histocompatibility complex syngeneic) was modeled by transplanting hearts from A-Tg mice into sensitized or nonsensitized WT mice. Antibody-mediated rejection was assessed by morphology/immunohistochemistry. A-Tg mice expressed A-antigen on vascular endothelium and other cells including erythrocytes. Antibody-mediated rejection was evident in 15/17 A-Tg grafts in sensitized WT recipients (median titer, 1:512), with 2 showing hyperacute rejection and rapid cessation of graft pulsation. Hyperacute rejection was observed in 8/8 A-Tg grafts after passive transfer of anti-A antibody and complement into nonsensitized recipients. Antibody-mediated rejection was not observed in A-Tg grafts transplanted into nonsensitized mice. A-Tg heart grafts transplanted into WT mice with abundant anti-A antibody manifests characteristic features of antibody-mediated rejection. These findings demonstrate an effective murine model to facilitate study of immunologic features of ABOi transplantation and to improve potential diagnostic and therapeutic strategies.
-
Mechanical sensitivity and psychological factors in patients with burning mouth syndrome.
Honda, Mika; Iida, Takashi; Kamiyama, Hirona; Masuda, Manabu; Kawara, Misao; Svensson, Peter; Komiyama, Osamu
2018-05-18
The aim of this study was to compare mechanical sensitivity on the tongue using quantitative sensory testing (QST) and psychological factors using the General Health Questionnaire (GHQ) between burning mouth syndrome (BMS) patients and healthy participants. Participants comprised 20 female BMS patients (68.1 ± 7.4 years) and 20 healthy females (65.4 ± 4.6 years). Psychological factors were evaluated with GHQ. Tactile detection thresholds (TDT) and filament-prick pain detection thresholds (FPT) were used to evaluate mechanical sensitivity on the tongue in all participants. TDT and FPT were measured on the tongue within both the painful area and the non-painful area in BMS patients, and on the tongue on both sides in healthy participants. As controls, TDT and FPT were measured with Semmes-Weinstein monofilaments on the skin of the mentum and palm in all participants. GHQ scores were significantly higher in BMS patients than in healthy participants (P = 0.024). No significant differences in TDT or FPT on the tongue, mentum, or palm were seen between BMS patients and healthy participants (P > 0.05). BMS patients showed no significant differences in TDT or FPT between the painful and non-painful areas on the tongue (P > 0.05). There were no significant correlations among TDT/FPT and GHQ score in BMS patients (P > 0.05). These findings could indicate a more important role for psychological factors than mechanical sensitivity in BMS pathophysiology. Pain on the tongue in elderly female patients with BMS may be more related to psychological factors.
-
Frequent video game players resist perceptual interference.
Directory of Open Access Journals (Sweden)
Aaron V Berard
Full Text Available Playing certain types of video games for a long time can improve a wide range of mental processes, from visual acuity to cognitive control. Frequent gamers have also displayed generalized improvements in perceptual learning. In the Texture Discrimination Task (TDT, a widely used perceptual learning paradigm, participants report the orientation of a target embedded in a field of lines and demonstrate robust over-night improvement. However, changing the orientation of the background lines midway through TDT training interferes with overnight improvements in overall performance on TDT. Interestingly, prior research has suggested that this effect will not occur if a one-hour break is allowed in between the changes. These results have suggested that after training is over, it may take some time for learning to become stabilized and resilient against interference. Here, we tested whether frequent gamers have faster stabilization of perceptual learning compared to non-gamers and examined the effect of daily video game playing on interference of training of TDT with one background orientation on perceptual learning of TDT with a different background orientation. As a result, we found that non-gamers showed overnight performance improvement only on one background orientation, replicating previous results with the interference in TDT. In contrast, frequent gamers demonstrated overnight improvements in performance with both background orientations, suggesting that they are better able to overcome interference in perceptual learning. This resistance to interference suggests that video game playing not only enhances the amplitude and speed of perceptual learning but also leads to faster and/or more robust stabilization of perceptual learning.
-
Frequent video game players resist perceptual interference.
Berard, Aaron V; Cain, Matthew S; Watanabe, Takeo; Sasaki, Yuka
2015-01-01
Playing certain types of video games for a long time can improve a wide range of mental processes, from visual acuity to cognitive control. Frequent gamers have also displayed generalized improvements in perceptual learning. In the Texture Discrimination Task (TDT), a widely used perceptual learning paradigm, participants report the orientation of a target embedded in a field of lines and demonstrate robust over-night improvement. However, changing the orientation of the background lines midway through TDT training interferes with overnight improvements in overall performance on TDT. Interestingly, prior research has suggested that this effect will not occur if a one-hour break is allowed in between the changes. These results have suggested that after training is over, it may take some time for learning to become stabilized and resilient against interference. Here, we tested whether frequent gamers have faster stabilization of perceptual learning compared to non-gamers and examined the effect of daily video game playing on interference of training of TDT with one background orientation on perceptual learning of TDT with a different background orientation. As a result, we found that non-gamers showed overnight performance improvement only on one background orientation, replicating previous results with the interference in TDT. In contrast, frequent gamers demonstrated overnight improvements in performance with both background orientations, suggesting that they are better able to overcome interference in perceptual learning. This resistance to interference suggests that video game playing not only enhances the amplitude and speed of perceptual learning but also leads to faster and/or more robust stabilization of perceptual learning.
-
Maize white seedling 3 (w3) has been used to study carotenoid deficiency for almost 100 years, although its genetic basis remained unknown. We show here that w3 phenotype is caused by disruption of homogentisate solanesyl transferase (HST), which catalyzes the first committed step in plastoquinone-9...
-
The broad utility of Trizac diamond tile
Gagliardi, John I.; Romero, Vincent D.; Sventek, Bruce; Zu, Lijun
2017-10-01
Sample finishing data from a broad range of materials — glasses, sapphire, silicon carbide, silicon, zirconium oxide, lithium tantalate, and flooring materials — are shown effectively processed with Trizact™ Diamond Tile (TDT). This data should provide the reader with an understanding of what to expect when using TDT on hard to grind or brittle materials. Keys to maintaining effective TDT pad wear rates, and therefore cost effect and stable processes, are described as managing 1) the proper lubricant flow rate for glasses and silicon-type materials and 2) the conditioning particle concentration for harder-to-grind materials
-
Response of Glutathione and Glutathione S-transferase in Rice Seedlings Exposed to Cadmium Stress
Directory of Open Access Journals (Sweden)
Chun-hua ZHANG
2008-03-01
Full Text Available A hydroponic culture experiment was done to investigate the effect of Cd stress on glutathione content (GSH and glutathione S-transferase (GST, EC 2.5.1.18 activity in rice seedlings. The rice growth was severely inhibited when Cd level in the solution was higher than 10 mg/L. In rice shoots, GSH content and GST activity increased with the increasing Cd level, while in roots, GST was obviously inhibited by Cd treatments. Compared with shoots, the rice roots had higher GSH content and GST activity, indicating the ability of Cd detoxification was much higher in roots than in shoots. There was a significant correlation between Cd level and GSH content or GST activity, suggesting that both parameters may be used as biomarkers of Cd stress in rice.
-
Immune- and Pollution-mediated DNA Damage in Two Wild Mya arenaria Clam Populations
Directory of Open Access Journals (Sweden)
François Gagné
2009-01-01
Full Text Available In aquatic environments, genotoxicity results from the effects of pollution combined with the inflammatory response triggered by the immune system. Indeed, the production of nitrosylated DNA and proteins are though to arise from the production of peroxinitrite during phagocytosis and inflammation. The purpose of this study was to examine new DNA biomarkers that differentiate between immune- and pollution-mediated genotoxicity in wild clam populations. Intertidal clam populations were sampled and analyzed for gonadal DNA strand breaks, DNA nitrosylation and xanthine oxidoreductase (XOR activity (purine salvage pathway. The clam weight-to-shell-length ratio, the gonado-somatic index (GSI, age status, lipid peroxidation, xenobiotic conjugation activity (glutathione S-transferase (GST and phagocytic activity were examined to shed light on their relationships with the observed genotoxic endpoints. XOR activity and DNA strand breaks were generally elevated at polluted sites and correlated significantly with clam weight-to-shell-length ratios and DNA nitrosylation. DNA nitrosylation was also higher at some sites and correlated significantly with phagocytic activity and with DNA strand breaks. This study showed that DNA strand breaks were associated with both immune- and pollution-mediated effects. This suggests that there is a loss of DNA repair capacity due to the combined effects of aging, pollution and immune response in wild clam populations that are impacted by anthropogenic activity.
-
Directory of Open Access Journals (Sweden)
Yong-Chao Ma
Full Text Available Both tyrosine kinase and topoisomerase II (TopII are important anticancer targets, and their respective inhibitors are widely used in cancer therapy. However, some combinations of anticancer drugs could exhibit mutually antagonistic actions and drug resistance, which further limit their therapeutic efficacy. Here, we report that HMNE3, a novel bis-fluoroquinolone chalcone-like derivative that targets both tyrosine kinase and TopII, induces tumor cell proliferation and growth inhibition. The viabilities of 6 different cancer cell lines treated with a range of HMNE3 doses were detected using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. Cellular apoptosis was determined using Hoechst 33258 fluorescence staining and the terminal deoxynucleotidyl transferase (TdT dUTP nick-end labeling (TUNEL assay. The expression of activated Caspase-3 was examined by immunocytochemistry. The tyrosine kinase activity was measured with a human receptor tyrosine kinase (RTK detection kit using a horseradish peroxidase (HRP-conjugated phosphotyrosine (pY20 antibody as the substrate. The topoisomerase II activity was measured using agarose gel electrophoresis with the DNA plasmid pBR322 as the substrate. The expression levels of the P53, Bax, Bcl-2, Caspase-3, -8, -9, p-cSrc, c-Src and topoisomerase II proteins were detected by western blot analysis. The proliferation of five of the six cancer cell lines was significantly inhibited by HMNE3 at 0.312 to 10 μmol/L in a time- and dose-dependent manner. Treatment of the Capan-1 and Panc-1 cells with 1.6 to 3.2 μM HMNE3 for 48 h significantly increased the percentage of apoptotic cells (P<0.05, and this effect was accompanied by a decrease in tyrosine kinase activity. HMNE3 potentially inhibited tyrosine kinase activity in vitro with an IC50 value of 0.64±0.34 μmol/L in Capan-1 cells and 3.1±0.86 μmol/L in Panc-1 cells. The activity of c-Src was significantly inhibited by HMNE3 in a dose
-
Guneidy, Rasha A; Shahein, Yasser E; Abouelella, Amira M K; Zaki, Eman R; Hamed, Ragaa R
2014-09-01
Rhipicephalus (Boophilus) annulatus is a bloodsucking ectoparasite that causes severe production losses in the cattle industry. This study aims to evaluate the in vitro effects of tannic acid, hematin (GST inhibitors) and different plant extracts (rich in tannic acid) on the activity of the recombinant glutathione S-transferase enzyme of the Egyptian cattle tick R. annulatus (rRaGST), in order to confirm their ability to inhibit the parasitic essential detoxification enzyme glutathione S-transferase. Extraction with 70% ethanol of Hibiscus cannabinus (kenaf flowers), Punica granatum (red and white pomegranate peel), Musa acuminata (banana peel) (Musaceae), Medicago sativa (alfalfa seeds), Tamarindus indicus (seed) and Cuminum cyminum (cumin seed) were used to assess: (i) inhibitory capacities of rRaGST and (ii) their phenolic and flavonoid contents. Ethanol extraction of red pomegranate peel contained the highest content of phenolic compounds (29.95mg gallic acid/g dry tissue) compared to the other studied plant extracts. The highest inhibition activities of rRaGST were obtained with kenaf and red pomegranate peel (P. granatum) extracts with IC50 values of 0.123 and 0.136mg dry tissue/ml, respectively. Tannic acid was the more effective inhibitor of rRaGST with an IC50 value equal to 4.57μM compared to delphinidine-HCl (IC50=14.9±3.1μM). Gossypol had a weak inhibitory effect (IC50=43.7μM), and caffeic acid had almost no effect on tick GST activity. The IC50 values qualify ethacrynic acid as a potent inhibitor of rRaGST activity (IC50=0.034μM). Cibacron blue and hematin showed a considerable inhibition effect on rRaGST activity, and their IC50 values were 0.13μM and 7.5μM, respectively. The activity of rRaGST was highest for CDNB (30.2μmol/min/mg protein). The enzyme had also a peroxidatic activity (the specific activity equals 26.5μmol/min/mg protein). Both tannic acid and hematin inhibited rRaGST activity non-competitively with respect to GSH and
-
Response transforms from comparative study of commercial pulsed-neutron-capture logging systems
International Nuclear Information System (INIS)
Salaita, G.N.
1992-01-01
This paper reports that three generations of Schlumberger's Thermal Decay Time (TDT SM ) logging devices - viz., TDT-K, TDT-M, and TDT-P - along with an Atlas Wireline PDK-100 SM system were run in a Saudi Aramco well. The wellbore (8 1/2 in. with 7-in. casing) penetrated a sequence of clean sand, shaly sand, and shale streaks as exhibited by the openhole natural gamma ray log. The initial wellbore fluid was diesel. The fluid was then changed to brines of 42,000 and 176,000 ppm NaCl, respectively. Three repeat passes at a logging speed of 900 ft/hr were obtained by each device for each borehole liquid. As a result of this extensive comparative study, a set of departure curves and mathematical transforms was developed primarily for standardizing the various Schlumberger tools to a common reference logging system and/or borehole environment. The transforms were used beneficially to determine residual oil saturation (ROS) from time-lapse logs in a Saudi Aramco reservoir
-
Directory of Open Access Journals (Sweden)
Feras I. Hawari
2014-10-01
Full Text Available Tobacco use negatively affects health and is a major risk factor for non-communicable diseases (NCDs. Today, tobacco use ranks third among risk factors in North Africa and the Middle East in terms of disease burden. Despite the established need for these services, tobacco dependence treatment (TDT services are still inadequate in the Eastern Mediterranean region (EMR. Among the main challenges hindering their expansion is the current lack of training opportunities. The provision of training and capacity-building—a key enabler of TDT—offers an excellent catalyst to launch TDT services in the region. This review discusses the need for TDT training in the EMR and describes a model for providing regional evidence-based training in line with international standards. The King Hussein Cancer Center in Amman, Jordan, is the regional host for Global Bridges, a worldwide TDT initiative. Using this model, they have trained 1,500 professionals and advocates from the EMR over the past three years.
-
DEFF Research Database (Denmark)
Revoredo, Leslie; Wang, Shengjun; Bennett, Eric Paul
2016-01-01
A large family of UDP-GalNAc:polypeptide GalNAc transferases (ppGalNAc-Ts) initiates and defines sites of mucin-type Ser/Thr-O-GalNAc glycosylation. Family members have been classified into peptide- and glycopeptide-preferring subfamilies, although both families possess variable activities agains...
-
Metabolic DDT resistance in Drosophila melanogaster has previously been associated with constitutive over-transcription of cytochrome P450s. Increased P450 activity has also been associated with increased oxidative stress. In contrast, over-transcription of glutathione S transferases (GSTs) has been...
-
Hechinger, Anne-Kathrin; Maas, Kristina; Dürr, Christoph; Leonhardt, Franziska; Prinz, Gabriele; Marks, Reinhard; Gerlach, Ulrike; Hofmann, Maike; Fisch, Paul; Finke, Jürgen; Pircher, Hanspeter; Zeiser, Robert
2013-01-01
Despite advances in immunosuppressive regimens, acute graft-versus-host disease remains a frequent complication of allogeneic hematopoietic cell transplantation. Pathogenic donor T cells are dependent on correct attachment of small GTPases to the cell membrane, mediated by farnesyl- or geranylgeranyl residues, which, therefore, constitute potential targets for graft-versus-host disease prophylaxis. A mouse model was used to study the impact of a farnesyl-transferase inhibitor and a geranylgeranyl-transferase inhibitor on acute graft-versus-host disease, anti-cytomegalovirus T-cell responses and graft-versus-leukemia activity. Treatment of mice undergoing allogeneic hematopoietic cell transplantation with farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor reduced the histological severity of graft-versus-host disease and prolonged survival significantly. Mechanistically, farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor treatment resulted in reduced alloantigen-driven expansion of CD4 T cells. In vivo treatment led to increased thymic cellularity and polyclonality of the T-cell receptor repertoire by reducing thymic graft-versus-host disease. These effects were absent when squalene production was blocked. The farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor did not compromise CD8 function against leukemia cells or reconstitution of T cells that were subsequently responsible for anti-murine cytomegalovirus responses. In summary, we observed an immunomodulatory effect of inhibitors of farnesyl-transferase and geranylgeranyl-transferase on graft-versus-host disease, with enhanced functional immune reconstitution. In the light of the modest toxicity of farnesyl-transferase inhibitors such as tipifarnib in patients and the potent reduction of graft-versus-host disease in mice, farnesyl-transferase and geranylgeranyl-transferase inhibitors could help to reduce graft-versus-host disease significantly without
-
Wang, Wei; Peng, Yizhi; Wang, Yuanyuan; Zhao, Xiaohui; Yuan, Zhiqiang
2009-09-01
1. Hypoxia-induced cardiomyocyte apoptosis contributes significantly to cardiac dysfunction following trauma, shock and burn injury. There is evidence that heat shock protein (HSP) 90 is anti-apoptotic in cardiomyocytes subjected to a variety of apoptotic stimuli. Because HSP90 acts as an upstream regulator of the serine/threonine protein kinase Akt survival pathway during cellular stress, we hypothesized that HSP90 exerts a cardioprotective effect via the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. 2. Neonatal rat cardiomyocytes were subjected to normoxia or hypoxia in the absence or presence of the HSP90 inhibitor geldanamycin (1 μg/mL). Cardiomyocyte apoptosis was assessed by release of lactate dehydrogenase (LDH), terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) staining and caspase 3 activity. Expression of HSP90, Akt, Bad and cytochrome c release was determined by western blot analysis. 3. Following exposure of cells to hypoxia, HSP90 was markedly elevated in a time-dependent manner, reaching a peak at 6 h (eightfold increase). Geldanamycin significantly increased hypoxia-induced release of LDH by 114%, the percentage of apoptotic cardiomyocytes by 102% and caspase 3 activity by 78%. Pretreatment of cells with geldanamycin also suppressed phosphorylation of both Akt and its downstream target Bad, but promoted the mitochondrial release of cytochrome c. 4. In conclusion, HSP90 activity is enhanced in cardiomyocytes following hypoxic insult. The anti-apoptotic effect of HSP90 on cardiomyocytes subjected to hypoxia is mediated, at least in part, by the PI3-K/Akt pathway. Key words: apoptosis, cardiomyocyte, heart failure, heat shock protein 90, hypoxia, phosphatidylinositol 3-kinase/Akt signalling pathway, serine/threonine protein kinase Akt.
-
Marita, Jane M; Hatfield, Ronald D; Rancour, David M; Frost, Kenneth E
2014-06-01
Grasses, such as Zea mays L. (maize), contain relatively high levels of p-coumarates (pCA) within their cell walls. Incorporation of pCA into cell walls is believed to be due to a hydroxycinnamyl transferase that couples pCA to monolignols. To understand the role of pCA in maize development, the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase (pCAT) was isolated and purified from maize stems. Purified pCAT was subjected to partial trypsin digestion, and peptides were sequenced by tandem mass spectrometry. TBLASTN analysis of the acquired peptide sequences identified a single full-length maize cDNA clone encoding all the peptide sequences obtained from the purified enzyme. The cDNA clone was obtained and used to generate an RNAi construct for suppressing pCAT expression in maize. Here we describe the effects of suppression of pCAT in maize. Primary screening of transgenic maize seedling leaves using a new rapid analytical platform was used to identify plants with decreased amounts of pCA. Using this screening method, mature leaves from fully developed plants were analyzed, confirming reduced pCA levels throughout plant development. Complete analysis of isolated cell walls from mature transgenic stems and leaves revealed that lignin levels did not change, but pCA levels decreased and the lignin composition was altered. Transgenic plants with the lowest levels of pCA had decreased levels of syringyl units in the lignin. Thus, altering the levels of pCAT expression in maize leads to altered lignin composition, but does not appear to alter the total amount of lignin present in the cell walls. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.
-
Oshodi, Y; Ojewunmi, O; Oshodi, T A; Ijarogbe, G T; Ogun, O C; Aina, O F; Lesi, Fea
2017-09-01
The role of oxidative stress has been identified in the development of autism spectrum disorder (ASD), and polymorphisms of glutathione S-transferase have been associated with some diseases linked to oxidative stress. Hence, we evaluated the serum levels of oxidative stress markers and investigated genetic polymorphisms of glutathione S-transferase associated with autism. Forty-two children clinically diagnosed with ASD using the Diagnostic and Statistical Manual for Mental Disorders (DSM-5) criteria and a clinical interview were included in the study. Twenty-three age-matched controls without any known genetic/developmental disorder were also recruited. Oxidative stress markers along with the genetic polymorphisms of glutathione S-transferase were determined. Reduced glutathione in ASD patients was significantly lower than the control (P = 0.008), whereas other oxidative stress markers measured were not significantly different in both the control and case populations. The frequencies of GSTT1 and GSTM1 null genotypes were lower among the controls compared with the cases, however, no association risk was observed. The observed risk of carrying Val/Val genotype among the cases was approximately six times that of the controls. Individuals with ASD showed a significant diminished level of reduced glutathione, however, the distribution of GSTT1, GSTM1, and GSTP1 polymorphisms was not found to be associated with autism in this study population.
-
The role of glutathione transferases in renal cell carcinoma
Directory of Open Access Journals (Sweden)
Ćorić Vesna
2016-01-01
Full Text Available Mounting evidence suggest that members of the subfamily of cytosolic glutathione S-transferases (GSTs possess roles far beyond the classical glutathione-dependent enzymatic conjugation of electrophilic metabolites and xenobiotics. Namely, monomeric forms of certain GSTs are capable of forming protein: protein interactions with protein kinases and regulate cell apoptotic pathways. Due to this dual functionality of cytosolic GSTs, they might be implicated in both the development and the progression of renal cell carcinoma (RCC. Prominent genetic heterogeneity, resulting from the gene deletions, as well as from SNPs in the coding and non-coding regions of GST genes, might affect GST isoenzyme profiles in renal parenchyma and therefore serve as a valuable indicator for predicting the risk of cancer development. Namely, GSTs are involved in the biotransformation of several compounds recognized as risk factors for RCC. The most potent carcinogen of polycyclic aromatic hydrocarbon diol epoxides, present in cigarette smoke, is of benzo(apyrene (BPDE, detoxified by GSTs. So far, the relationship between GST genotype and BPDE-DNA adduct formation, in determining the risk for RCC, has not been evaluated in patients with RCC. Although the association between certain individual and combined GST genotypes and RCC risk has been debated in a the literature, the data on the prognostic value of GST polymorphism in patients with RCC are scarce, probably due to the fact that the molecular mechanism supporting the role of GSTs in RCC progression has not been clarified as yet.
-
Bowman, B P; Snell, T W; Cochrane, B J
1990-01-01
1. The enzyme glutathione S-transferase (GST), a critical element in xenobiotic metabolism, was isolated from the marine rotifer Brachionus plicatilis and its freshwater congener B. calyciflorus. 2. In B. plicatilis, GST comprised 4.2% of cytosolic protein and was present as three separate isozymes with mol. wts 30,000, 31,400 and 33,700. Specific activity of crude homogenates was 56 nmol min-1 mg-1 protein, while that of affinity chromatography purified GST was 1850. 3. In B. calyciflorus, GST was present as two isozymes with mol. wts of 26,300 and 28,500, representing 1.0% of cytosolic protein. Crude GST specific activity was 1750 nmol min-1 mg-1 protein and purified was 72,400. 4. Rotifer GSTs are unusual because they are monomers whereas all other animals thus far investigated posses dimeric GSTs.
-
Mediation Analysis with Multiple Mediators.
VanderWeele, T J; Vansteelandt, S
2014-01-01
Recent advances in the causal inference literature on mediation have extended traditional approaches to direct and indirect effects to settings that allow for interactions and non-linearities. In this paper, these approaches from causal inference are further extended to settings in which multiple mediators may be of interest. Two analytic approaches, one based on regression and one based on weighting are proposed to estimate the effect mediated through multiple mediators and the effects through other pathways. The approaches proposed here accommodate exposure-mediator interactions and, to a certain extent, mediator-mediator interactions as well. The methods handle binary or continuous mediators and binary, continuous or count outcomes. When the mediators affect one another, the strategy of trying to assess direct and indirect effects one mediator at a time will in general fail; the approach given in this paper can still be used. A characterization is moreover given as to when the sum of the mediated effects for multiple mediators considered separately will be equal to the mediated effect of all of the mediators considered jointly. The approach proposed in this paper is robust to unmeasured common causes of two or more mediators.
-
O-GlcNAc regulates NEDD4-1 stability via caspase-mediated pathway
International Nuclear Information System (INIS)
Jiang, Kuan; Bai, Bingyang; Ta, Yajie; Zhang, Tingling; Xiao, Zikang; Wang, Peng George; Zhang, Lianwen
2016-01-01
O-GlcNAc modification of cytosolic and nuclear proteins regulates essential cellular processes such as stress responses, transcription, translation, and protein degradation. Emerging evidence indicates O-GlcNAcylation has a dynamic interplay with ubiquitination in cellular regulation. Here, we report that O-GlcNAc indirectly targets a vital E3 ubiquitin ligase enzyme of NEDD4-1. The protein level of NEDD4-1 is accordingly decreased following an increase of overall O-GlcNAc level upon PUGNAc or glucosamine stimulation. O-GlcNAc transferase (OGT) knockdown, overexpression and mutation results confirm that the stability of NEDD4-1 is negatively regulated by cellular O-GlcNAc. Moreover, the NEDD4-1 degradation induced by PUGNAc or GlcN is significantly inhibited by the caspase inhibitor. Our study reveals a regulation mechanism of NEDD4-1 stability by O-GlcNAcylation. - Highlights: • Reduced NEDD4-1 correlates with increased overall O-GlcNAc level. • OGT negatively regulates NEDD4-1 stability. • O-GlcNAc regulates NEDD4-1 through caspase-mediated pathway.
-
Collaborative Curriculum Design to Increase Science Teaching Self-Efficacy: A Case Study
Velthuis, C.H.; Fisser, Petra; Pieters, Julius Marie
2015-01-01
The purpose of this study was to establish whether participation in a teacher design team (TDT) is an effective way to increase the science teaching self-efficacy of primary school teachers who vary in their levels of experience and interest in science. A TDT is a group of at least 2 teachers from
-
Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway
Energy Technology Data Exchange (ETDEWEB)
Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing, E-mail: allenylq@hotmail.com; Liao, Er-Yuan, E-mail: eyliao@21cn.com
2013-11-01
Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. - Highlights: • We explored the effects of ghrelin on serum deprivation-induced MC3T3-E1 cells apoptosis. • Both ELISA and TUNEL were used to detect the apoptosis. • The receptor of ghrelin, GHSR, was expressed in MC3T3-E1
-
Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway
International Nuclear Information System (INIS)
Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing; Liao, Er-Yuan
2013-01-01
Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. - Highlights: • We explored the effects of ghrelin on serum deprivation-induced MC3T3-E1 cells apoptosis. • Both ELISA and TUNEL were used to detect the apoptosis. • The receptor of ghrelin, GHSR, was expressed in MC3T3-E1
-
Nakamura, Shoko; Horie, Masayuki; Daidoji, Tomo; Honda, Tomoyuki; Yasugi, Mayo; Kuno, Atsushi; Komori, Toshihisa; Okuzaki, Daisuke; Narimatsu, Hisashi; Nakaya, Takaaki; Tomonaga, Keizo
2016-02-15
mucin-type O-linked glycosylation is initiated by IAV infection and how mucin production affects viral replication have not yet been elucidated. In this study, we show that levels of two miRNAs that target the UDP-GalNAc transferase GALNT3 are markedly decreased during the early stage of IAV infection, resulting in the upregulation of GALNT3 mRNA. We also demonstrate that the expression of GALNT3 initiates mucin production and affects IAV replication in infected cells. This is the first report demonstrating the mechanism underlying the miRNA-mediated initiation of mucin-type O-glycosylation in IAV-infected cells and its role in viral replication. Our results have broad implications for understanding IAV replication and suggest a strategy for the development of novel anti-influenza approaches. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
-
Vontas, John G; Small, Graham J; Nikou, Dimitra C; Ranson, Hilary; Hemingway, Janet
2002-01-01
A novel glutathione S-transferase (GST)-based pyrethroid resistance mechanism was recently identified in Nilaparvata lugens [Vontas, Small and Hemingway (2001) Biochem. J. 357, 65-72]. To determine the nature of GSTs involved in conferring this resistance, the GSTs from resistant and susceptible strains of N. lugens were partially purified by anion exchange and affinity chromatography. The majority of peroxidase activity, previously correlated with resistance, was confined to the fraction tha...
-
Ikeguchi, M; Cai, J; Fukuda, K; Oka, S; Katano, K; Tsujitani, S; Maeta, M; Kaibara, N
2001-06-01
The aim of this study was to investigate whether angiogenic factors influence the occurrence of spontaneous apoptosis in advanced gastric cancer. The apoptotic indices (AIs) of 97 tumors from 97 patients with advanced gastric cancer (pT3, pN0, pM0, Stage II) were analyzed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) method. Intratumoral microvessel densities (IMVDs) of tumors stained with anti-CD34 monoclonal antibody were quantified under x 200 magnification using computer-assisted image analysis. The expressions of angiogenic factors, such as vascular endothelial growth factor (VEGF), thymidine phosphorylase (dThdPase), transforming growth factor-alpha (TGF-alpha), and p53 were analyzed immunohistochemically and compared with IMVDs and AIs. The mean IMVD of the 97 tumors was 365/mm2 (range 147-990/mm2). The mean AI of tumors was 2.1% (range 0-11.3%). A significant inverse correlation between the AIs and the IMVDs was shown (p = -0.278, P = 0.0064). The mean IMVDs of tumors with high expressions of dThdPase, TGF-alpha, or p53 were significantly higher than those of tumors with low expressions of these factors. The mean AI of tumors with high expressions of dThdPase was significantly lower than that of tumors with low expressions of dThdPase (P = 0.023). However, no significant correlations were detected between AIs and the expression levels of VEGF, TGF-alpha, or p53. In gastric cancer, dThdPase may play an important role in tumor progression by increasing microvessels and by suppressing apoptosis of cancer cells.
-
Classical swine fever virus induces pyroptosis in the peripheral lymphoid organs of infected pigs.
Yuan, Jin; Zhu, Mengjiao; Deng, Shaofeng; Fan, Shuangqi; Xu, Hailuan; Liao, Jiedan; Li, Peng; Zheng, Jingfang; Zhao, Mingqiu; Chen, Jinding
2018-05-02
Classical swine fever virus (CSFV) causes a highly lethal disease in pigs, which is characterized by immunosuppression. Leukopenia is known to be a possible mechanism of immunosuppression during CSFV infection. As a new and specialized form of cell death, pyroptosis is the key response of the innate immune system to pathogens, and is widely involved in the occurrence and development of infectious diseases. However, the relationship between CSFV and pyroptosis has not been explored. In this study, we investigated the occurrence of pyroptosis in pigs following CSFV infection. According to qRT-PCR assay results, the prevalence of this virus in peripheral lymphoid organs (tonsils, lymph nodes, and spleen) was much higher than that in other organs. Severe bleeding, necrosis, and a significant reduction in lymphocytes were found in the peripheral lymphoid organs of CSFV-infected pigs based on histological examination. In-depth studies showed that an increased ratio of deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells were present in the peripheral lymphoid organs of the CSFV-infected group according to immunohistochemistry. Meanwhile, the p10 subunit and activity of caspase-1, which is a regulator of pyroptosis, the N-terminal domain of gasdermin D, which is an executor of pyroptosis, and the cleavage and secretion of IL-1b, which is a product of pyroptosis were increased in the peripheral lymphoid organs of the CSFV-infected group. Together, these results demonstrated that pyroptosis is involved in CSFV-induced cell death in vivo, which provides a new understanding of the mechanism associated with lymphocyte depletion and immunosuppression in pigs infected with this virus. Copyright © 2018 Elsevier B.V. All rights reserved.
-
Srinivasan, Krishnamoorthy; Sharma, Shyam S
2011-11-20
Endoplasmic reticulum (ER) stress has been postulated to play a crucial role in the pathophysiology of cerebral ischemic/reperfusion (I/R) injury and diabetes. Diabetes is a major risk factor and also common amongst the people who suffer from stroke. In this study, we have investigated the neuroprotective potential of sodium 4-phenylbutyrate (SPB; 30-300mg/kg), a chemical chaperone by targeting ER stress in a rat model of transient focal cerebral ischemia associated with comorbid type 2 diabetes. Intraperitoneal treatment with SPB (100 and 300mg/kg) significantly ameliorated brain I/R damage as evidenced by reduction in cerebral infarct and edema volume. It also significantly improved the functional recovery of various neurobehavioral impairments (neurological deficit score, grip strength and rota rod) evoked by I/R compared with vehicle-treatment. Further, SPB (100mg/kg) significantly reduced the DNA fragmentation as shown by prominent reduction in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells. This effect was observed concomitantly with significant attenuation in upregulation of 78kDa glucose regulated protein (GRP78), CCAAT/enhancer binding protein homologous protein or growth arrest DNA damage-inducible gene 153 (CHOP/GADD153) and activation of caspase-12, specific markers of ER stress/apoptosis. The neuroprotection observed with SPB was independent of its effect on cerebral blood flow and blood glucose. In conclusion, this study demonstrates the neuroprotective effect of SPB owing to amelioration of ER stress and DNA fragmentation. It also suggest that targeting ER stress might offer a promising therapeutic approach and benefits against ischemic stroke associated with comorbid type 2 diabetes. Copyright © 2011 Elsevier B.V. All rights reserved.
-
Toker, M Berk; Alcay, Selim; Gokce, Elif; Ustuner, Burcu
2016-06-01
The scope of this study was investigation the affects of various antioxidants on 1% soybean lecithin-based semen extenders for ram semen cryopreservation. Ejaculates, collected via electrically stimulated ejaculation, that have a thick consistency, rapid wave motion (3-5 on a 0-5 scale) and >75% initial motility were pooled. The pooled samples were split into four equal aliquots as 5 mM Methionine, 5 mM Cysteamine, 1 mM Cysteine and a sample of antioxidant-free control group. Each sample group was diluted to a ratio of 1/5 (semen/extender, v/v) as final concentration and two step dilution method was used for cryopreservation. Extender groups were assessed for sperm motility, plasma membrane functional integrity using hypoosmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Semen samples also incubated for 6 h in humidified air with 5% CO2 at 39 °C to evaluate post-thaw incubation resilience of semen characteristics. The results showed that freezing and thawing procedures had negative effects on motility (P < 0.05), plasma membrane integrity (P < 0.05) and acrosomal integrity (P < 0.05). After 6 h of incubation time, the Cysteine supplemented extender group yielded significantly higher results than other extender groups in terms of spermatological parameters. Furthermore MDA levels in the antioxidant groups were lower than control group (P < 0.05). Nevertheless, there were no significant differences among antioxidant groups. Copyright © 2016 Elsevier Inc. All rights reserved.
-
Palmitate induces VSMC apoptosis via toll like receptor (TLR)4/ROS/p53 pathway.
Zhang, Yuanjun; Xia, Guanghao; Zhang, Yaqiong; Liu, Juxiang; Liu, Xiaowei; Li, Weihua; Lv, Yaya; Wei, Suhong; Liu, Jing; Quan, Jinxing
2017-08-01
Toll-like receptor 4 (TLR4) has been implicated in vascular inflammation, as well as in the pathogenesis of atherosclerosis and diabetes. Vascular smooth muscle cell (VSMC) apoptosis has been shown to induce plaque vulnerability in atherosclerosis. Previous studies reported that palmitate induced apoptosis in VSMCs; however, the role of TLR4 in palmitate-induced apoptosis in VSMCs has not yet been defined. In this study, we investigated whether or not palmitate-induced apoptosis depended on the activation of the TLR4 pathway. VSMCs were treated with or without palmitate, CRISPR/Cas9z-mediated genome editing methods were used to deplete TLR4 expression, while NADPH oxidase inhibitors were used to inhibit reactive oxygen species (ROS) generation. Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, ROS was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) method, the mRNA and protein expression levels of caspase 3, caspase 9, BCL-2 and p53 were studied by real-time polymerase chain reaction (RT-PCR) and ELISA. Palmitate significantly promotes VSMC apoptosis, ROS generation, and expression of caspase 3, caspase 9 and p53; while NADPH oxidase inhibitor pretreatment markedly attenuated these effects. Moreover, knockdown of TLR4 significantly blocked palmitate-induced ROS generation and VSMC apoptosis accompanied by inhibition of caspase 3, caspase 9, p53 expression and restoration of BCL-2 expression. Our results suggest that palmitate-induced apoptosis depends on the activation of the TLR4/ROS/p53 signaling pathway, and that TLR4 may be a potential therapeutic target for the prevention and treatment of atherosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.
-
Yan, Lu-Lu; Zhang, Wei-Yang; Wei, Xiao-Hong; Yan, Li; Pan, Chun-Shui; Yu, Yang; Fan, Jing-Yu; Liu, Yu-Ying; Zhou, Hua; Han, Jing-Yan; Yao, Xin-Sheng
2018-01-01
Background: Gualou Xiebai Decoction (GLXB) is a classic prescription of Chinese medicine used for the treatment of cardiac problems. The present study was designed to explore the effect and mechanism of GLXB on ischemia/reperfusion (I/R) induced disorders in myocardial structure and function, focusing on the regulation of energy metabolism and the RhoA/ROCK pathway. Methods: After hyperlipidemic rat model was established by oral administration of high fat diet, the rats were treated with GLXB for 6 weeks and subjected to 30 min occlusion of the left anterior descending coronary artery (LADCA) followed by 90 min reperfusion to elicit I/R challenge. Myocardial infarct size was assessed by Evans blue-TTC staining. Myocardial blood flow (MBF) and cardiac function were evaluated. Enzyme-linked immunosorbent assay was performed to examine the content of ATP, ADP, AMP, CK, CK-MB, LDH, cTnT, cTnI, and IL-6. Double staining of F-actin and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was conducted to assess myocardial apoptosis. Expressions of ATP synthase subunit δ (ATP 5D), and RhoA and ROCK were determined by Western blotting. Results: Administration with GLXB at high dose for 6 weeks protected heart against I/R-induced MBF decrease, myocardial infarction and apoptosis, ameliorated I/R-caused impairment of cardiac function and myocardial structure, restored the decrease in the ratio of ADP/ATP and AMP/ATP, and the expression of ATP 5D with inhibiting the expression of RhoA and ROCK. Conclusions: Treatment with GLXB effectively protects myocardial structure and function from I/R challenge, possibly via regulating energy metabolism involving inactivation of RhoA/ROCK signaling pathway.
-
Epithelial apoptosis in mechanistically distinct methods of injury in the murine small intestine
Vyas, Dinesh; Robertson, Charles M; Stromberg, Paul E; Martin, James R.; Dunne, W. Michael; Houchen, Courtney W; Barrett, Terrence A; Ayala, Alfred; Perl, Mario; Buchman, Timothy G; Coopersmith, Craig M
2007-01-01
Gut epithelial apoptosis is involved in the pathophysiology of multiple diseases. This study characterized intestinal apoptosis in three mechanistically distinct injuries with different kinetics of cell death. FVB/N mice were subjected to gamma radiation, Pseudomonas aeruginosa pneumonia or injection of monoclonal anti-CD3 antibody and sacrificed 4, 12, or 24 hours post-injury (n=10/time point). Apoptosis was quantified in the jejunum by hematoxylin and eosin (H&E), active caspase-3, terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL), in situ oligoligation reaction (ISOL,) cytokeratin 18, and annexin V staining. Reproducible results were obtained only for H&E, active caspase-3, TUNEL and ISOL, which were quantified and compared against each other for each injury at each time point. Kinetics of injury were different with early apoptosis highest following radiation, late apoptosis highest following anti CD3, and more consistent levels following pneumonia. ISOL was the most consistent stain and was always statistically indistinguishable from at least 2 stains. In contrast, active caspase-3 demonstrated lower levels of apoptosis, while the TUNEL assay had higher levels of apoptosis in the most severely injured intestine regardless of mechanism of injury. H&E was a statistical outlier more commonly than any other stain. This suggests that regardless of mechanism or kinetics of injury, ISOL correlates to other quantification methods of detecting gut epithelial apoptosis more than any other method studied and compares favorably to other commonly accepted techniques of quantifying apoptosis in a large intestinal cross sectional by balancing sensitivity and specificity across a range of times and levels of death. PMID:17357092
-
Kim, Kyung-A; Hyun, Lee Chung; Jung, Sang Hoon; Yang, Sung Jae
2016-01-01
In this study, the beneficial effects of the oral administration of ethanol extract of Diospyros kaki (EEDK) were tested on a mouse dry eye model induced by benzalkonium chloride (BAC). A solution of 0.2% BAC was administered topically to mouse eyes for 14 days, twice daily, to induce dry eye. Various concentrations of EEDK were administrated daily by oral gavage for 14 days after BAC treatment. Preservative-free eye drops were instilled in the positive-control group. The tear secretion volume (Schirmer's test), tear break-up time (BUT), and fluorescein score were measured on the ocular surface. BAC-induced corneal damage was tested with hematoxylin-eosin staining. Moreover, apoptotic cell death in the corneal epithelial layer was investigated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The protein expression level of interleukin-1alpha (IL-1α), IL-1β, IL-6, tumor necrosis factor- alpha (TNF-α), and monocyte chemotactic protein-1 (MCP-1) was determined with western blot analysis. Furthermore, squamous metaplasia in the corneal epithelial layer was detected with immunofluorescent staining for cytokeratine-10. The cellular proliferation in the cornea was examined with immunohistochemical staining for Ki-67. EEDK treatment resulted in prolonged BUT, decreased fluorescein score, increased tear volume, and smoother epithelial cells compared with BAC treatment alone in the cornea. Moreover, EEDK treatment inhibited the inflammatory response and corneal epithelial cell death in a BAC-induced murine dry eye model, and changes in squamous cells were inhibited. Proliferative activity in the corneal epithelium cells was improved with EEDK. EEDK could be a potential therapeutic agent in the clinical treatment of dry eye.
-
A novel model for rapid induction of apoptosis in spiral ganglions of mice.
Lee, Ji Eun; Nakagawa, Takayuki; Kim, Tae Soo; Iguchi, Fukuichiro; Endo, Tsuyoshi; Dong, Youyi; Yuki, Kazuo; Naito, Yasushi; Lee, Sang Heun; Ito, Juichi
2003-06-01
The survival of the spiral ganglion (SG) is a critical issue in preservation of hearing. Research on topics related to this issue requires a mouse experimental model because such a model has advantages including use of genetic information and knockout or "knockin" mice. Thus, the aim of the study was to establish a mouse model for induction of apoptosis of SG neurons with a definite time course. Laboratory study using experimental animals. C57BL/6 mice were used as experimental animals and were subjected to direct application of cisplatin into the inner ear. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and immunostaining for Neurofilament 200-kD (NF) and peripherin were used for analysis of SG degeneration. In addition, generation of peroxynitrite in affected spiral ganglions was examined by immunostaining for nitrotyrosine. Cellular location of activated caspase-9 and cytochrome-c in dying SG neurons were examined for analysis of cell death pathway. The TUNEL assay and immunohistochemical analysis for NF and peripherin indicated that type I neurons in spiral ganglions were deleted through the apoptotic pathway over time. Spiral ganglion neurons treated with cisplatin exhibited expression of nitrotyrosine, indicating induction of peroxynitrite by cisplatin. In dying SG neurons, expression of activated caspase-9 and translocation of cytochrome-c from mitochondria to cytoplasm were observed, indicating the mitochondrial pathway of apoptosis. The predictable fashion of induction of apoptosis in SG neurons over a well-defined time course in the model in the study will aid studies of the molecular mechanism of cell death and elucidation of a strategy for prevention of SG degeneration.
-
Chen, Shuchen; Chen, Liangwan; Wu, Xiaonan; Lin, Jiangbo; Fang, Jun; Chen, Xiangqi; Wei, Shijin; Xu, Jianxin; Gao, Qin; Kang, Mingqiang
2012-11-01
It has been reported that ischemic postconditioning (IPO) or mesenchymal stem cell (MSC) engraftment could protect organs from ischemia/reperfusion (I/R) injury. We investigated the synergetic effects of combined treatment on lung injury induced by I/R. Adult Sprague-Dawley rats were randomly assigned to one of the following groups: sham-operated control, I/R, IPO, MSC engraftment, and IPO plus MSC engraftment. Lung injury was assessed by arterial blood gas analysis, the wet/dry lung weight ratio, superoxide dismutase level, malondialdehyde content, myeloperoxidase activity, and tissue histologic changes. Cytokine expression was detected using real-time polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. Cell apoptosis was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end assay and annexin V staining. MSC engraftment or IPO alone markedly attenuated the lung wet/dry weight ratio, malondialdehyde and myeloperoxidase production, and lung pathologic injury and enhanced arterial partial oxygen pressure, superoxide dismutase content, inhibited pro-inflammatory cytokine levels, and decreased cell apoptosis in lung tissue, compared with the I/R group. In contrast, IPO pretreatment enhanced the protective effects of MSC on I/R-induced lung injury compared with treatment alone. Moreover, in the combined treatment group, the number of MSC engraftments in the lung tissue was increased, associated with enhanced survival of MSCs compared with MSC treatment alone. Additional investigation showed that IPO treatment increased expression of vascular endothelial growth factor and stromal cell-derived factor-1 in I/R lung tissue. IPO might contribute to the homing and survival of transplanted MSCs and enhance their therapeutic effects through improvement of the microenvironment of I/R injury. Copyright © 2012 Elsevier Inc. All rights reserved.
-
Hughes, Amy; Mohanasundaram, Daisy; Kireta, Svjetlana; Jessup, Claire F; Drogemuller, Chris J; Coates, P Toby H
2013-03-15
The early loss of functional islet mass (50-70%) due to apoptosis after clinical transplantation contributes to islet allograft failure. Insulin-like growth factor (IGF)-II is an antiapoptotic protein that is highly expressed in β-cells during development but rapidly decreases in postnatal life. We used an adenoviral (Ad) vector to overexpress IGF-II in isolated rat islets and investigated its antiapoptotic action against exogenous cytokines interleukin-1β- and interferon-γ-induced islet cell death in vitro. Using an immunocompromised marginal mass islet transplant model, the ability of Ad-IGF-II-transduced rat islets to restore euglycemia in nonobese diabetic/severe combined immunodeficient diabetic recipients was assessed. Ad-IGF-II transduction did not affect islet viability or function. Ad-IGF-II cytokine-treated islets exhibited decreased cell death (40% ± 2.8%) versus Ad-GFP and untransduced control islets (63.2% ± 2.5% and 53.6% ± 2.3%, respectively). Ad-IGF-II overexpression during cytokine treatment resulted in a marked reduction in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells (8.3% ± 1.4%) versus Ad-GFP control (41% ± 4.2%) and untransduced control islets (46.5% ± 6.2%). Western blot analysis confirmed that IGF-II inhibits apoptosis via activation of the phosphatidylinositol 3-kinase/Akt signaling pathway. Transplantation of IGF-II overexpressing islets under the kidney capsule of diabetic mice restored euglycemia in 77.8% of recipients compared with 18.2% and 47.5% of Ad-GFP and untransduced control islet recipients, respectively (Pislet transplant outcomes in vivo. Antiapoptotic gene transfer is a potentially powerful tool to improve islet survival after transplantation.
-
Directory of Open Access Journals (Sweden)
Hajhosieni Laleh
2014-04-01
Full Text Available Objective: Ginger is a strong antioxidant and long-term treatment of streptozotocin (STZ-diabetic animals, and it has been shown to reduce oxidative stress. Prevalence oxidative stress among urban life and changes in antioxidant capacity are considered asplay an important role in the pathogenesis of chronic diabetes mellitus. Materials and Methods: Wistar male rat (n = 40 were divided into three groups, control group (n = 10 and Ginger Quercetin group that received 100 mg/kg (gavage, (n = 10, and diabetic group, which received 55 mg/kg intra peritoneal (IP STZ (n = 20, which was subdivided to two groups of 10; STZ group and treatment group. Treatment group received 55 mg/kg (IP STZ plus100 mg/kg ginger, daily for, 8 weeks, respectively; however, the control group just received an equal volume of distilled water daily (IP. Diabetes was induced by a single (IP injection of STZ (55 mg/kg. Animals were kept in standard condition. In 28 day after inducing diabetic 5 cc blood were collected for total antioxidant capacity, malondialdehyde and oxidized low density lipoprotein levels and kidney tissues of rat in whole groups were removed then prepared for apoptosis analysis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay (TUNEL method. Results: Apoptotic cells significantly decreased in group that has received 100 mg/kg ginger (P < 0.05 in comparison to experimental groups (P < 0.05. Conclusion: Since in our study 100 mg/kg ginger have significantly preventive effect on kidney cells damages by reducing number of apoptotic cells in kidney and hence it seems that using it can be effective for treatment in diabetic rat.
-
Expression of hPNAS-4 Radiosensitizes Lewis Lung Cancer
International Nuclear Information System (INIS)
Zeng Hui; Yuan Zhu; Zhu Hong; Li Lei; Shi Huashan; Wang Zi; Fan Yu; Deng Qian; Zeng Jianshuang; He Yinbo; Xiao Jianghong; Li Zhiping
2012-01-01
Purpose: This study aimed to transfer the hPNAS-4 gene, a novel apoptosis-related human gene, into Lewis lung cancer (LL2) and observe its radiosensitive effect on radiation therapy in vitro and in vivo. Methods and Materials: The hPNAS-4 gene was transfected into LL2 cells, and its expression was detected via western blot. Colony formation assay and flow cytometry were used to detect the growth and apoptosis of cells treated with irradiation/PNAS-4 in vitro. The hPNAS-4 gene was transferred into LL2-bearing mice through tail vein injection of the liposome/gene complex. The tumor volumes were recorded after radiation therapy. Proliferating cell nuclear antigen (PCNA) immunohistochemistry staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to detect the tumor cell growth and apoptosis in vivo. Results: The hPNAS-4 gene was successfully transferred into LL2 cells and tumor tissue, and its overexpressions were confirmed via western blot analysis. Compared with the control, empty plasmid, hPNAS-4, radiation, and empty plasmid plus radiation groups, the hPNAS-4 plus radiation group more significantly inhibited growth and enhanced apoptosis of LL2 cells in vitro and in vivo (P<.05). Conclusions: The hPNAS-4 gene was successfully transferred into LL2 cells and tumor tissue and was expressed in both LL2 cell and tumor tissue. The hPNAS-4 gene therapy significantly enhanced growth inhibition and apoptosis of LL2 tumor cells by radiation therapy in vitro and in vivo. Therefore, it may be a potential radiosensitive treatment of radiation therapy for lung cancer.
-
Fetoni, Anna Rita; Piacentini, Roberto; Fiorita, Antonella; Paludetti, Gaetano; Troiani, Diana
2009-02-27
The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS) also in noise induced hearing loss (NIHL) and anti-oxidants and free-radicals scavengers have been shown to attenuate the damage. Coenzyme Q(10) (CoQ(10)) or ubiquinone has a bioenergetic role as a component of the mithocondrial respiratory chain, it inhibits mitochondrial lipid peroxidation, inducing ATP production and it is involved in ROS removal and prevention of oxidative stress-induced apoptosis. However the therapeutic application of CoQ(10) is limited by the lack of solubility and poor bio- availability, therefore it is a challenge to improve its water solubility in order to ameliorate the efficacy in tissues and fluids. This study was conducted in a model of acoustic trauma in the guinea pig where the effectiveness of CoQ(10) was compared with a soluble formulation of CoQ(10) (multicomposite CoQ(10) Terclatrate, Q-ter) given intraperitoneally 1 h before and once daily for 3 days after pure tone noise exposure (6 kHz for 1 h at 120 dB SPL). Functional and morphological studies were carried out by measuring auditory brainstem responses, scanning electron microscopy for hair cell loss count, active caspase 3 staining and terminal deoxynucleotidyl transferase-mediated dUTP labelling assay in order to identify initial signs of apoptosis. Treatments decreased active caspase 3 expression and the number of apoptotic cells, but animals injected with Q-ter showed a greater degree of activity in preventing apoptosis and thus in improving hearing. These data confirm that solubility of Coenzyme Q(10) improves the ability of CoQ(10) in preventing oxidative injuries that result from mitochondrial dysfunction.
-
International Nuclear Information System (INIS)
Yasui, Hironobu; Inanami, Osamu; Asanuma, Taketoshi; Iizuka, Daisuke; Nakajima, Takayuki; Kon, Yasuhiro; Matsuda, Akira; Kuwabara, Mikinori
2007-01-01
Purpose: To examine the in vivo antitumor efficacy of X-irradiation combined with administration of a ribonucleoside anticancer drug, 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine (TAS106, ECyd), to tumor cell-transplanted mice. Methods and Materials: Colon26 murine rectum adenocarcinoma cells and MKN45 human gastric adenocarcinoma cells were inoculated into the footpad in BALB/c mice and severe combined immunodeficient mice, respectively. They were treated with a relatively low dose of X-irradiation (2 Gy) and low amounts of TAS106 (0.1 mg/kg and 0.5 mg/kg). The tumor growth was monitored by measuring the tumor volume from Day 5 to Day 16 for Colon26 and from Day 7 to Day 20 for MKN45. Histologic analyses for proliferative and apoptotic cells in the tumors were performed using Ki-67 immunohistochemical and terminal deoxynucleotidyl transferase-mediated nick end labeling staining. The expression of survivin, a key molecule related to tumor survival, was assessed by quantitative polymerase chain reaction and immunohistochemical analysis. Results: When X-irradiation and TAS106 treatment were combined, significant inhibition of tumor growth was observed in both types of tumors compared with mice treated with X-irradiation or TAS106 alone. Marked inhibition of tumor growth was observed in half of the mice that received the combined treatment three times at 2-day intervals. Parallel to these phenomena, the suppression of survivin expression and appearance of Ki-67-negative and apoptotic cells were observed. Conclusions: X-irradiation and TAS106 effectively suppress tumor growth in mice. The inhibition of survivin expression by TAS106 is thought to mainly contribute to the suppression of the tumor growth
-
Semaan, Maroun T; Zheng, Qing Y; Han, Fengchan; Zheng, Yuxi; Yu, Heping; Heaphy, John C; Megerian, Cliff A
2013-04-01
Spiral ganglion neurons (SGN) in the Phex male mouse, a murine model of postnatal endolymphatic hydrops (ELH) undergo progressive deterioration reminiscent of human and other animal models of ELH with features suggesting apoptosis as an important mechanism. Histologic analysis of the mutant's cochlea demonstrates ELH by postnatal Day (P) 21 and SGN loss by P90. The SGN loss seems to occur in a consistent topographic pattern beginning at the cochlear apex. SGN were counted at P60, P90, and P120. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative PCR, and immunohistochemical analyses of activated caspase-3, caspase-8, and caspase-9 were performed on cochlear sections obtained from mutants and controls. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay (TUNEL) was carried out on 2 mutants and 2 controls. Corrected SGN counts in control mice were greater in the apical turn of the cochleae at P90 and P120, respectively (p < 0.01). Increased expression of activated caspase-3, caspase-8, and caspase-9 was seen in the mutant. At later time points, activated caspase expression gradually declined in the apical turns and increased in basal turns of the cochlea. Quantitative and semiquantitative PCR analysis confirmed increased expression of caspase-3, caspase-8, and caspase-9 at P21 and P40. TUNEL staining demonstrated apoptosis at P90 in the apical and basal turns of the mutant cochleae. SGN degeneration in the Phex /Y mouse seems to mimic patterns observed in other animals with ELH. Apoptosis plays an important role in the degeneration of the SGN in the Phex male mouse.
-
Jung, Kyoung In; Kim, Jie Hyun; Park, Chan Kee
2015-10-15
Excitotoxicity, glutamate-induced toxic effects to retinal ganglion cells (RGCs), is one of several mechanisms of RGC loss suggested in glaucoma. In this study, we focused on the role of glutamate transporter of glial cells as well as N-methyl-d-aspartate (NMDA) receptor with regard to glutamate toxicity in glaucoma. We also investigated whether α2-adrenoceptor activation could modulate glutamate transporters and NMDA receptors in a chronic ocular hypertension model. Brimonidine 0.15% was administered topically to the eyes of experimental glaucoma and control animals twice daily. After 8 weeks of intraocular pressure (IOP) elevation, staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) revealed an increase in the ganglion cell layer, and the number of TUNEL-positive cells was reduced by brimonidine treatment (P<0.05). Animals with experimentally induced glaucoma exhibited an increase in retinal stress marker glial fibrillary acidic protein (GFAP) immunoreactivity; brimonidine treatment reduced GFAP. Excitatory amino acid transporter 1(EAAT1) expression remained stable throughout the period of chronic ocular hypertension. α2-Adrenergic treatment upregulated EAAT1 protein levels (P<0.05). NMDA receptor (GluN1) expression was stimulated by chronic elevation of IOP, and GluN1-positive cells in ganglion cell layer were co-localized with TUNEL staining. Brimonidine administration suppressed GluN1 levels (P<0.05). These results indicate that brimonidine decreased RGC apoptosis, upregulating EAAT1 and downregulating NMDA receptors. We suggest that topical brimonidine treatment may decrease the glutamate excitotoxicity through modulation of glutamate transporter and NMDA receptor in glaucoma. Copyright © 2015 Elsevier B.V. All rights reserved.
-
Directory of Open Access Journals (Sweden)
Susi Endrini
2015-01-01
Full Text Available Background: This study aimed to analyze the cytotoxicity effect of γ-sitosterol isolated from “Kejibeling” (Strobilanthes crispus, a medicinal plant, on several cancer cell lines. The mechanisms of the effects were studied through the expression of cancer-caused gene, c-myc and apoptotic pathways.Methods: This in vitro study was done using human colon cancer cell lines (Caco-2, liver cancer cell lines (HepG2, hormone-dependent breast cancer cell lines (MCF-7 and the normal liver cell lines (Chang Liver. The cytotoxic effect was measured through MTT assay and the potential cytotoxic value was calculated by determining the toxic concentration which may kill up to 50% of the total cell used (IC50. Meanwhile, the cytotoxic mechanism was studied by determining the effect of adding γ-sitosterol to the c-myc gene expression by reverse transciptase-polymerase chain reaction (RT-PCR. The effect of γ-sitosterol through apoptotic pathway was studied by using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay.Results: γ-sitosterol was cytotoxic against Caco-2, HepG2, and MCF-7 with IC50-values of 8.3, 21.8, and 28.8 μg/mL, respectively. There were no IC50-values obtained from this compound against Chang Liver cell line. This compound induced apotosis on Caco-2 and HepG2 cell lines and suppressed the c-myc genes expression in both cells.Conclusion: γ-sitosterol was cytotoxic against colon and liver cancer cell lines and the effect was mediated by down-regulation of c-myc expression and induction of the apoptotic pathways.
-
Sirtuin 1 participates in the process of age-related retinal degeneration.
Zeng, Ying; Yang, Ke
The process of aging involves retinal cell damage that leads to visual dysfunction. Sirtuin (Sirt) 1 can prevent oxidative stress, DNA damage, and apoptosis. In the present study, we measured the expression of Sirt1 as a functional regulator in the retina during the aging process. The visual function and Sirt1 expression in young (1 month) and old (19 months) Sprague-Dawley (SD) rats. Electroretinogram (ERG) and real-time polymerase chain reaction (PCR) or Western blotting were performed. Resveratrol, an activator of Sirt1, was orally administered to SD rats at a dose of 5 mg/kg/day for 19 months. The expression of Sirt1, brain-derived neurotrophic factor (BDNF), and tropomyosin receptor kinase B (TrkB) was evaluated in the retinas of mice that did and did not receive resveratrol treatment. Apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. With decreasing b-wave amplitude, the expression level of Sirt1 was significantly reduced in aged retinas compared to that in young retinas. After 19 months of treatment with resveratrol, the Sirt1 expression level and b-wave amplitude increased. In old rats treated with resveratrol, the expression levels of BDNF and TrkB were up-regulated. Compared to young retinas, the aged retinas exhibited higher apoptosis, but resveratrol delayed this process. Our data demonstrated a reduction of Sirt1 expression during the aging process of the retina, but enhancing Sirt1 expression reversed the degeneration of the retina. These results suggested that increasing Sirt1 expression may protect retinal neurons and visual function via regulating neurotrophin and its receptor. Copyright © 2015 Elsevier Inc. All rights reserved.
-
Badalzadeh, Reza; Azimi, Ako; Alihemmati, Alireza; Yousefi, Bahman
2017-02-01
It has been shown that diabetes modifies the myocardial responses to ischemia/reperfusion (I/R) and to cardioprotective agents. In this study, we aimed to investigate the effects of combined treatment with ischemic postconditioning (IPostC) and cyclosporine A (CsA) on inflammation and apoptosis of the diabetic myocardium injured by I/R. Eight weeks after induction of diabetes in Wistar rats, hearts were mounted on a Langendorff apparatus and were subsequently subjected to a 30-min regional ischemia followed by 45-min reperfusion. IPostC was induced at the onset of reperfusion, by 3 cycles of 30-s reperfusion/ischemia (R/I). The concentration of creatine kinase (CK), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were determined; the levels of total and phosphorylated glycogen synthase kinase 3 beta (p-GSK3β) and B-cell lymphoma 2 (Bcl-2) were quantified by western blotting, and the rate of apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. Administration of either IPostC or CsA alone in nondiabetic animals significantly reduced CK, TNF-α, IL-1β, and IL-6 concentrations, increased the p-GSK3β and Bcl-2, and decreased the level of apoptosis (P GSK3β and Bcl-2 and decreasing apoptosis and inflammation were restored in comparison with nonpostconditioned diabetic hearts. IPostC or CsA failed to affect apoptosis and inflammation and failed to protect the diabetic myocardium against I/R injury. However, combined administration of IPostC and CsA at reperfusion can protect the diabetic myocardium by decreasing the inflammatory response and apoptosis.
-
International Nuclear Information System (INIS)
Kiukkonen, Anu; Sahlberg, Carin; Partanen, Anna-Maija; Alaluusua, Satu; Pohjanvirta, Raimo; Tuomisto, Jouko; Lukinmaa, Pirjo-Liisa
2006-01-01
Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to mouse embryonic teeth, sharing features of early development with salivary glands in common, involves enhanced apoptosis and depends on the expression of epidermal growth factor (EGF) receptor. EGF receptor signaling, on the other hand, is essential for salivary gland branching morphogenesis. To see if TCDD impairs salivary gland morphogenesis and if the impairment is associated with EGF receptor signaling, we cultured mouse (NMRI) E13 submandibular glands with TCDD or TCDD in combination with EGF or fibronectin (FN), both previously found to enhance branching morphogenesis. Explants were examined stereomicroscopically and processed to paraffin sections. TCDD exposure impaired epithelial branching and cleft formation, resulting in enlarged buds. The glands were smaller than normal. EGF and FN alone concentration-dependently stimulated or inhibited branching morphogenesis but when co-administered with TCDD, failed to compensate for its effect. TCDD induced cytochrome P4501A1 expression in the glandular epithelium, indicating activation of the aryl hydrocarbon receptor. TCDD somewhat increased epithelial apoptosis as observed by terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling method but the increase could not be correlated with morphological changes. The frequency of proliferating cells was not altered. Corresponding to the reduced cleft sites in TCDD-exposed explants, FN immunoreactivity in the epithelium was reduced. The results show that TCDD, comparably with EGF and FN at morphogenesis-inhibiting concentrations, impaired salivary gland branching morphogenesis in vitro. Together with the failure of EGF and FN at morphogenesis-stimulating concentrations to compensate for the effect of TCDD this implies that TCDD toxicity to developing salivary gland involves reduced EGF receptor signaling
-
Directory of Open Access Journals (Sweden)
Zhang Kang-Jian
2012-02-01
Full Text Available Abstract Background Gene therapy and viral therapy are used for cancer therapy for many years, but the results are less than satisfactory. Our aim was to construct a new recombinant adenovirus which is more efficient to kill hepatocarcinoma cells but more safe to normal cells. Methods By using the Cancer Targeting Gene-Viro-Therapy strategy, Apoptin, a promising cancer therapeutic gene was inserted into the double-regulated oncolytic adenovirus AD55 in which E1A gene was driven by alpha fetoprotein promoter along with a 55 kDa deletion in E1B gene to form AD55-Apoptin. The anti-tumor effects and safety were examined by western blotting, virus yield assay, real time polymerase chain reaction, 3-(4,5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide assay, Hoechst33342 staining, Fluorescence-activated cell sorting, xenograft tumor model, Immunohistochemical assay, liver function analysis and Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling assay. Results The recombinant virus AD55-Apoptin has more significant antitumor effect for hepatocelluar carcinoma cell lines (in vitro than that of AD55 and even ONYX-015 but no or little impair on normal cell lines. Furthermore, it also shows an obvious in vivo antitumor effect on the Huh-7 liver carcinoma xenograft in nude mice with bigger beginning tumor volume till about 425 mm3 but has no any damage on the function of liver. The induction of apoptosis is involved in AD55-Apoptin induced antitumor effects. Conclusion The AD55-Apoptin can be a potential anti-hepatoma agent with remarkable antitumor efficacy as well as higher safety in cancer targeting gene-viro-therapy system.
-
International Nuclear Information System (INIS)
Libertin, C.R.; Woloschak, G.E.; Panozzo, J.; Groh, K.R.; Chang-Liu, Chin-Mei; Schreck, S.
1994-01-01
Previous work by our group and others has shown the modulation of human immunodeficiency virus (HIV) promoter or long terminal repeat (LTR) after exposure to neutrons and ultraviolet radiations. Using HeLa cells stably transfected with a construct containing the chloramphenicol acetyl transferase (CAT) gene, the transcription of which is mediated by the HIV-LTR, we designed experiments to examine the effects of exposure to different types of radiation (such as γ rays, ultraviolet and sunlight irradiations, electromagnetic fields and microwaves) in HIV-LTR-driven expression of CAT. These results demonstrated ultraviolet-light-induced transcription from the HIV promoter, as has been shown by others. Exposure to other DNA-damaging agents such as γ rays and sunlight (with limited exposures) had no significant effect on transcription mediated by HIV-LTR, suggesting that induction of HIV is not mediated by just any type of DNA damage but rather may require specific types of DNA damage. Microwaves did not cause cell killing when cells in culture were exposed in high volumes of medium, and the same cells showed no changes in expression. When microwave exposure was carried out in low volumes of medium (so that excessive heat was generated) induction of HIV-LTR transcription (as assayed by CAT activity) was evident. Electromagnetic field exposures had no effect on expression of HIV-LTR. These results demonstrate that not all types of radiation and not all DNA-damaging agents are capable of inducing HIV. We hypothesize that induction of HIV transcription may be mediated by several different signals exposure to radiation. 22 refs., 8 figs
-
Tilt testing results are influenced by tilt protocol.
Zyśko, Dorota; Fedorowski, Artur; Nilsson, David; Rudnicki, Jerzy; Gajek, Jacek; Melander, Olle; Sutton, Richard
2016-07-01
It is unknown how the return to supine position influences duration of loss of consciousness (LOC) and cardioinhibition during tilt test. Retrospective analysis of two datasets containing records of patients who underwent tilt testing for unexplained syncope in two centres was performed. Patients, totalling 1232, were included in the study: 262 in a Swedish centre and 970 patients in a Polish centre. In Sweden, tilt table with tilt-down time (TDT) of 18 s was used (Group II). In Poland, two different tilt tables were used, one of them with TDT of 10 s (Group I, n = 325), and the other with TDT of 47 s (Group III, n = 645). Cardioinhibitory reflex occurred most frequently in Group III, whereas number of pauses >3 s, frequency of very long asystole ≥30 s, and the total duration of pauses >3 s demonstrated a trend to increase from Group I to III. Duration of LOC in Groups II and III was significantly longer compared with Group I (32.0 and 33.7 s vs. 16.4 s). In the multivariate-adjusted regression model, cardioinhibitory reflex was predicted by tilt-table model (odds ratio per model with increasing TDT: 1.40; 95% confidence interval, 1.19-1.64; P < 0.0001), whereas LOC duration was longer with increasing TDT (P < 0.0001) and age (P < 0.0001). Longer TDT during induced vasovagal syncope increases the prevalence of cardioinhibitory reflex and prolongs the duration of LOC. Tilt-down time does not affect asystolic pause duration but delay may lead to occurrence of multiple pauses, higher frequency of very long asystole, and longer total asystole duration. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.
-
Matsumoto, M; Imagawa, M; Aoki, Y
1999-01-01
3,3',4,4',5-Pentachlorobiphenyl (PenCB), one of the most toxic co-planar polychlorinated biphenyl congeners, specifically induces class Pi glutathione S-transferase (GSTP1) as well as cytochrome P-450 1A1 in primary cultured rat liver parenchymal cells [Aoki, Matsumoto and Suzuki (1993) FEBS Lett. 333, 114-118]. However, the 5'-flanking sequence of the GSTP1 gene does not contain a xenobiotic responsive element, to which arylhydrocarbon receptor binds. Using a chloramphenicol acetyltransferas...
-
Itzhaki, H; Maxson, J M; Woodson, W R
1994-01-01
The increased production of ethylene during carnation petal senescence regulates the transcription of the GST1 gene encoding a subunit of glutathione-S-transferase. We have investigated the molecular basis for this ethylene-responsive transcription by examining the cis elements and trans-acting factors involved in the expression of the GST1 gene. Transient expression assays following delivery of GST1 5' flanking DNA fused to a beta-glucuronidase receptor gene were used to functionally define ...
-
Directory of Open Access Journals (Sweden)
Costa Sánchez, Carmen
2012-12-01
Full Text Available En español: La evolución del medio televisivo en nuestro país ha tenido como última etapa el apagón analógico y el consecuente nacimiento de múltiples programas que nutren la oferta de la Televisión Digital. En el presente artículo se analiza la reconfiguración de la oferta de Televisión Digital Terrestre (TDT en España después de la aprobación del Real-Decreto 365/2010 para asignar los múltiples de la TDT una vez finalizadas las emisiones analógicas In english: The evolution of television in our country has had like last stage the analogue switch off and the birth of multiple programs that nourish the offer of the Digital Television. In the present article we analyze the reconfiguration of the offer of Terrestrial Digital Television (TDT in our country, after the approval of the Real-Decree 365/2010 to assign the multiple of the TDT once finalized the analog broadcasts.
-
Directory of Open Access Journals (Sweden)
Kataria N.
2012-08-01
Full Text Available The investigation was carried out to determine serum gamma glutamyl transferase enzyme as a biomarker of stress and metabolic dysfunctions in Rathi cattle of arid tract in India. Blood samples were collected to harvest serum from healthy male and female, drought affected, ketotic cows, recently aborted cows, cows with diarrhoea, cows with traumatic pericarditis, calves with urinary calculi, cows affected with urea poisoning and cows affected with acidosis. The mean values of γ glutamyl transferase showed significant variations (p≤0.05 according to sex and age in the healthy group of animals. The normal range in healthy animals was from 12 to 34 UL-1. In affected group an average 23.69 times rise in the value was observed from that of healthy group. Cows affected with urea poisoning and acidosis were having highest mean values whereas drought affected animals were having least value. It was concluded that present study attempted to provide a new insight about an old enzyme. As the number of animals in the present study was statistically sufficient therefore the mean value of healthy group can be used as reference value for γ GT in Rathi cattle and other cattle breeds which can help to interpret the variations of serum γ GT in various metabolic diseases of cattle.
-
Association study of functional genetic variants of innate immunity related genes in celiac disease
Directory of Open Access Journals (Sweden)
Martín J
2005-08-01
Full Text Available Abstract Background Recent evidence suggest that the innate immune system is implicated in the early events of celiac disease (CD pathogenesis. In this work for the first time we have assessed the relevance of different proinflammatory mediators typically related to innate immunity in CD predisposition. Methods We performed a familial study in which 105 celiac families characterized by the presence of an affected child with CD were genotyped for functional polymorphisms located at regulatory regions of IL-1α, IL-1β, IL-1RN, IL-18, RANTES and MCP-1 genes. Familial data was analysed with a transmission disequilibrium test (TDT that revealed no statistically significant differences in the transmission pattern of the different genetic markers considered. Results The TDT analysis for IL-1α, IL-1β, IL-1RN, IL-18, and MCP-1 genes genetic variants did not reveal biased transmission to the affected offspring. Only a borderline association of RANTES promoter genetic variants with CD predisposition was observed. Conclusion Our results suggest that the analysed polymorphisms of IL-1α, IL-1β, IL-1RN, IL-18, RANTES and MCP-1 genes do not seem to play a major role in CD genetic predisposition in our population.
-
Bae, Young-An; Kim, Jeong-Geun; Kong, Yoon
2016-01-01
Glutathione transferase (GST) is one of the major antioxidant proteins with diverse supplemental activities including peroxidase, isomerase, and thiol transferase. GSTs are classified into multiple classes on the basis of their primary structures and substrate/inhibitor specificity. However, the evolutionary routes and physiological environments specific to each of the closely related bioactive enzymes remain elusive. The sigma-like GSTs exhibit amino acid conservation patterns similar to the prostaglandin D synthases (PGDSs). In this study, we analyzed the phylogenetic position of the GSTs of the biocarcinogenic liver fluke, Clonorchis sinensis. We also observed induction profile of the GSTs in association with the parasite's maturation and in response to exogenous oxidative stresses, with special attention to sigma-class GSTs and PGDSs. The C. sinensis genome encoded 12 GST protein species, which were separately assigned to cytosolic (two omega-, one zeta-, two mu-, and five sigma-class), mitochondrial (one kappa-class), and microsomal (one membrane-associated proteins in eicosanoid and glutathione metabolism-like protein) GST families. Multiple sigma GST (or PGDS) orthologs were also detected in Opisthorchis viverrini. Other trematode species possessed only a single sigma-like GST gene. A phylogenetic analysis demonstrated that one of the sigma GST lineages duplicated in the common ancestor of trematodes were specifically expanded in the opisthorchiids, but deleted in other trematodes. The induction profiles of these sigma GST genes along with the development and aging of C. sinensis, and against various exogenous chemical stimuli strongly suggest that the paralogous sigma GST genes might be undergone specialized evolution to cope with the diverse hostile biochemical environments within the mammalian hepatobiliary ductal system. Copyright © 2016 Elsevier B.V. All rights reserved.
-
Liu, Shiguo; Yu, Xiaoxia; Xu, Quanchen; Cui, Jiajia; Yi, Mingji; Zhang, Xinhua; Ge, Yinlin; Ma, Xu
2015-08-03
Recently, a genome-wide association study has indicated associations between single nucleotide polymorphisms in the Collagen Type XXVII Alpha 1 gene (COL27A1) and Tourette syndrome in several ethnic populations. To clarify the global relevance of the previously identified SNPs in the development of Tourette syndrome, the associations between polymorphisms in COL27A1 and Tourette syndrome were assessed in Chinese trios. PCR-directed sequencing was used to evaluate the genetic contributions of three SNPs in COL27A1(rs4979356, rs4979357 and rs7868992) using haplotype relative risk (HRR) and transmission disequilibrium tests (TDT) with a total of 260 Tourette syndrome trios. The family-based association was significant between Tourette syndrome and rs4979356 (TDT: χ2 = 4.804, P = 0.033; HRR = 1.75, P = 0.002; HHRR = 1.32, P = 0.027), and transmission disequilibrium was suspected for rs4979357 (TDT: χ2 = 3.969, P = 0.053; HRR = 1.84, P = 0.001; HHRR = 1.29, P = 0.044). No statistically significant allele transfer was found for rs7868992 (TDT: χ2 = 2.177, P = 0.158). Although the TDT results did not remain significant after applying the conservative Bonferroni correction (p = 0.005), the significant positive HRR analysis confirmed the possibility of showing transmission disequilibrium, which provides evidence for an involvement of COL27A1in the development of TS. However, these results need to be verified with larger datasets from different populations.
-
Ganapathy-Kanniappan, Shanmugasundaram; Geschwind, Jean-Francois H; Kunjithapatham, Rani; Buijs, Manon; Syed, Labiq H; Rao, Pramod P; Ota, Shinichi; Kwak, Byung Kook; Loffroy, Romaric; Vali, Mustafa
2010-03-01
Autophagy, a cellular response to stress, plays a role in resistance to chemotherapy in cancer cells. Resistance renders systemic chemotherapy generally ineffective against human hepatocellular carcinoma (HCC). Recently, we reported that the pyruvate analog 3-bromopyruvate (3-BrPA) promoted tumor cell death by targeting GAPDH. In continuance, we investigated the intracellular response of two human HCC cell lines (Hep3B and SK-Hep1) that differ in their status of key apoptotic regulators, p53 and Fas. 3-BrPA treatment induced endoplasmic reticulum (ER) stress, translation inhibition and apoptosis based on Western blot and qPCR, pulse labeling, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and active caspase-3 in both the cell lines. However, electron microscopy revealed that 3-BrPA treated SK-Hep1 cells underwent classical apoptotic cell death while Hep3B cells initially responded with the protective autophagy that failed to prevent eventual apoptosis. 3-BrPA treatment promotes apoptosis in human HCC cell lines, irrespective of the intracellular response.
-
The Root Extract of Gentiana macrophylla Pall. Alleviates Cardiac Apoptosis in Lupus Prone Mice.
Directory of Open Access Journals (Sweden)
Chih-Yang Huang
Full Text Available The roots of the perennial herb Gentiana macrophylla Pall. (GM are known as Qinjiao, which has been used for centuries to treat systemic lupus erythematosus (SLE. However, little is known about the effects of GM on cholesterol-aggravated cardiac abnormalities in SLE, and the mechanisms thereof. This study investigates whether GM exhibits anti-apoptotic effects, focusing on the left ventricle (LV of NZB/W F1 mice fed with high-cholesterol diet. The morphology and apoptotic status of ventricular tissues were determined by microscopy and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay. Levels of apoptotic biomarkers were determined by immunoblotting. The results thus obtained revealed that GM significantly reduced the cholesterol-aggravated apoptosis of LV in NZB/W F1 mice by suppressing both intrinsic and extrinsic apoptotic pathways. Additionally, GM significantly increased the cardiac insulin-like growth factors (IGF-1 survival signaling and anti-apoptotic proteins in LV tissues. Accordingly, GM is considered to be beneficial in alleviating cholesterol-aggravated cardiac damage in SLE, and therefore constitute an alternative treatment for SLE patients with cardiac abnormalities.
-
Protective effect of Urtica dioica on liver damage induced by biliary obstruction in rats.
Oguz, Serhat; Kanter, Mehmet; Erboga, Mustafa; Ibis, Cem
2013-10-01
The aim of this study was to evaluate the possible protective effects of Urtica dioica (UD) against liver damage in the common bile duct-ligated rats. A total of 24 male Sprague Dawley rats were divided into three groups, namely, control, bile duct ligation (BDL) and BDL + received UD groups, containing eight animals in each group. The rats in UD-treated groups were given UD oils (2 ml/kg) once a day intraperitoneally for 2 weeks starting 3 days prior to BDL operation. The change demonstrating the bile duct proliferation and fibrosis in expanded portal tracts includes the extension of proliferated bile ducts into the lobules; inflammatory cell infiltration into the widened portal areas were observed in BDL group. Treatment of BDL with UD attenuated alterations in liver histology. The α-smooth muscle actin, cytokeratin-positive ductular proliferation and the activity of terminal deoxynucleotidyl transferase dUTP nick end labeling in the BDL were observed to be reduced with the UD treatment. The data indicate that UD attenuates BDL-induced cholestatic liver injury, bile duct proliferation and fibrosis.
-
DEFF Research Database (Denmark)
Lira-Navarrete, Erandi; de Las Rivas, Matilde; Compañón, Ismael
2015-01-01
the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy......Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present...... and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity...
-
DEFF Research Database (Denmark)
Christensen, Camilla L; Gjetting, Torben; Poulsen, Thomas Tuxen
2010-01-01
Small cell lung cancer (SCLC) is a highly malignant cancer for which there is no curable treatment. Novel therapies are therefore in great demand. In the present study we investigated the therapeutic effect of transcriptionally targeted suicide gene therapy for SCLC based on the yeast cytosine...... deaminase (YCD) gene alone or fused with the yeast uracil phosphoribosyl transferase (YUPRT) gene followed by administration of 5-fluorocytosine (5-FC) prodrug. Experimental design: The YCD gene or the YCD-YUPRT gene was placed under regulation of the SCLC-specific promoter insulinoma-associated 1 (INSM1...
-
Phi Class of Glutathione S-transferase Gene Superfamily Widely Exists in Nonplant Taxonomic Groups.
Munyampundu, Jean-Pierre; Xu, You-Ping; Cai, Xin-Zhong
2016-01-01
Glutathione S-transferases (GSTs) constitute a superfamily of enzymes involved in detoxification of noxious compounds and protection against oxidative damage. GST class Phi (GSTF), one of the important classes of plant GSTs, has long been considered as plant specific but was recently found in basidiomycete fungi. However, the range of nonplant taxonomic groups containing GSTFs remains unknown. In this study, the distribution and phylogenetic relationships of nonplant GSTFs were investigated. We identified GSTFs in ascomycete fungi, myxobacteria, and protists Naegleria gruberi and Aureococcus anophagefferens. GSTF occurrence in these bacteria and protists correlated with their genome sizes and habitats. While this link was missing across ascomycetes, the distribution and abundance of GSTFs among ascomycete genomes could be associated with their lifestyles to some extent. Sequence comparison, gene structure, and phylogenetic analyses indicated divergence among nonplant GSTFs, suggesting polyphyletic origins during evolution. Furthermore, in silico prediction of functional partners suggested functional diversification among nonplant GSTFs.
-
Relation of serum γ-glutamyl transferase activity with copper in an adult population.
Peng, You-Fan; Wang, Chun-Fang; Pan, Guo-Gang
2017-10-26
The aim of this study was to evaluate the relationship between serum γ-glutamyl transferase (γ-GGT) activity and serum copper in an adult population. We analyzed 281 adult subjects who regularly attended the physical examination center at the Affiliated Hospital of Youjiang Medical University for Nationalities. The demographic and laboratory data of the participants were divided into two groups according to the median of serum γ-GGT activity. Serum copper concentrations in individuals with higher γ-GGT levels were significantly increased compared with those with lower γ-GGT concentrations (9.9±2.41 vs. 11.2±3.36 μmol/L, pcopper in all eligible subjects (r=0.198, p=0.001). Further, serum γ-GGT maintained a positive correlation with serum copper in both males and females (r=0.322, pcopper after adjusting for multiple potential confounders (b=0.464, p=0.001). This study suggests that serum γ-GGT activity is correlated with copper in the study population, indicating that serum γ-GGT may be a biomarker to evaluate serum copper levels in an adult population.
-
Collaborative Curriculum Design to Increase Science Teaching Self-Efficacy: A Case Study
Velthuis, Chantal; Fisser, Petra; Pieters, Jules
2015-01-01
The purpose of this study was to establish whether participation in a teacher design team (TDT) is an effective way to increase the science teaching self-efficacy of primary school teachers who vary in their levels of experience and interest in science. A TDT is a group of at least 2 teachers from the same or related subjects working together to…
-
DEFF Research Database (Denmark)
Kristiansen, Ole P; Nolsøe, Runa L; Larsen, Lykke
2003-01-01
The type 1 diabetes mellitus (T1DM) candidate gene SNP IL6-174G/C was genotyped in 253 Danish T1DM families (1129 individuals). TDT analysis demonstrated linkage in the presence of association between the IL6-174C allele and T1DM in the 416 T1DM offspring, P(tdt)=0.04. Gender conditioned TDT......DM versus non-T1DM females) excluded preferential meiotic segregation in females, P=4.6 x 10(-3), and demonstrated differences in the transmission patterns between female and male T1DM offspring, P=5.1 x 10(-3). The IL6-174 CC genotype was associated with younger age at onset of T1DM in females (P=0...
-
Regulation and control of nitric oxide (NO) in macrophages
DEFF Research Database (Denmark)
Kovacevic, Zaklina; Sahni, Sumit; Lok, K.H.
2017-01-01
We recently demonstrated that a novel storage and transport mechanism for nitric oxide (NO) mediated by glutathione-S-transferase P1 (GSTP1) and multidrug resistance protein 1 (MRP1/ABCC1), protects M1-macrophage (M1-MØ) models from large quantities of endogenous NO. This system stores and transp......We recently demonstrated that a novel storage and transport mechanism for nitric oxide (NO) mediated by glutathione-S-transferase P1 (GSTP1) and multidrug resistance protein 1 (MRP1/ABCC1), protects M1-macrophage (M1-MØ) models from large quantities of endogenous NO. This system stores...... be responsible for delivering cytotoxic NO as DNICs via MRP1 from M1-MØs, to tumor cell targets....
-
DEFF Research Database (Denmark)
Gerken, Thomas A; Revoredo, Leslie; Thome, Joseph J C
2013-01-01
and specificity that differ between transferase isoforms. For example, ppGalNAc T1, T2, and T14 prefer C-terminally placed GalNAc-O-Thr, whereas ppGalNAc T3 and T6 prefer N-terminally placed GalNAc-O-Thr. Several transferase isoforms, ppGalNAc T5, T13, and T16, display equally enhanced N- or C-terminal activities...... relative to the nonglycosylated control peptides. This N- and/or C-terminal selectivity is presumably due to weak glycopeptide binding to the lectin domain, whose orientation relative to the catalytic domain is dynamic and isoform-dependent. Such N- or C-terminal glycopeptide selectivity provides...
-
Directory of Open Access Journals (Sweden)
Muhammad Tariq Saeed
2016-09-01
Full Text Available The alteration of glucose metabolism, through increased uptake of glucose and glutamine addiction, is essential to cancer cell growth and invasion. Increased flux of glucose through the Hexosamine Biosynthetic Pathway (HBP drives increased cellular O-GlcNAcylation (hyper-O-GlcNAcylation and contributes to cancer progression by regulating key oncogenes. However, the association between hyper-O-GlcNAcylation and activation of these oncogenes remains poorly characterized. Here, we implement a qualitative modeling framework to analyze the role of the Biological Regulatory Network in HBP activation and its potential effects on key oncogenes. Experimental observations are encoded in a temporal language format and model checking is applied to infer the model parameters and qualitative model construction. Using this model, we discover step-wise genetic alterations that promote cancer development and invasion due to an increase in glycolytic flux, and reveal critical trajectories involved in cancer progression. We compute delay constraints to reveal important associations between the production and degradation rates of proteins. O-linked N-acetylglucosamine transferase (OGT, an enzyme used for addition of O-GlcNAc during O-GlcNAcylation, is identified as a key regulator to promote oncogenesis in a feedback mechanism through the stabilization of c-Myc. Silencing of the OGT and c-Myc loop decreases glycolytic flux and leads to programmed cell death. Results of network analyses also identify a significant cycle that highlights the role of p53-Mdm2 circuit oscillations in cancer recovery and homeostasis. Together, our findings suggest that the OGT and c-Myc feedback loop is critical in tumor progression, and targeting these mediators may provide a mechanism-based therapeutic approach to regulate hyper-O-GlcNAcylation in human cancer.
-
Creación de contenidos interactivos de deporte para televisión digital terrestre en Ecuador
Directory of Open Access Journals (Sweden)
Abel Suing
2016-08-01
Full Text Available Ecuador, al igual que otros países latinoamericanos, implementa el sistema ISDB-T para Televisión Digital Terrestre (TDT que permite la emisión de interactividad a través del middleware Ginga. La TDT puede implicar un cambio en la calidad de los contenidos pero sobre todo una oportunidad de acceso a la sociedad del conocimiento. La investigación describe una dinámica de trabajo y la integración de equipos multidisciplinarios en la producción de dos programas de deportes “Aventura-T”, empleando metodología cualitativa, los instrumentos usados son observación, entrevistas semiestructuradas y grupos focales. Se concluye que las aplicaciones en Ginga NCL generadas cumplen con estándares de calidad para emisiones de TDT.
-
Striatal FP-CIT uptake differs in the subtypes of early Parkinson's disease
International Nuclear Information System (INIS)
Spiegel, J.; Fassbender, K.; Dillmann, U.; Hellwig, D.; Samnick, S.; Moellers, M.-O.; Kirsch, C.-M.; Jost, W.
2007-01-01
In idiopathic Parkinson's disease (PD), a tremor-dominant type (TDT), an akinetic-rigid type (ART), and a mixed type (MT) are distinguished. We compared cerebral [I- 123 ]FP-CIT SPECT in the PD subtypes (67 patients Hoehn and Yahr stage 1:26 with ART, 19 with MT, 22 with TDT). We measured the ratios putamen/occipital lobe binding and caudate nucleus/occipital lobe binding. Parkinsonian motor symptoms were quantified by UPDRS motor scale. In both putamen and caudate nucleus contralateral to the clinically affected body side TDT patients showed a significantly higher FP-CIT uptake than ART or MT patients (ANOVA; p 0.05). The missing correlation between striatal FP-CIT uptake and tremor suggests, that further systems besides the nigrostriatal dopaminergic system may contribute to generation of parkinsonian tremor. (author)
-
Directory of Open Access Journals (Sweden)
Gisele Rodrigues Moreira
2004-05-01
Full Text Available O objetivo deste trabalho foi estudar a divergência genética e propor uma subcoleção representativa da traça-do-tomateiro, Tuta absoluta (TDT. O experimento foi conduzido com quatro populações do inseto procedentes de Uberlândia, MG, Viçosa, MG, Camocim de São Félix, PE, e Santa Teresa, ES, e cinco acessos de tomateiro, 'Santa Clara', 'Moneymaker', TOM-601, PI 126445 (Lycopersicon hirsutum f. typicum e PI 134417 (L. hirsutum f. glabratum. Foi realizada análise de agrupamento (método de Tocher, usando a distância de Mahalanobis como medida de dissimilaridade e verificada a importância relativa dos caracteres da TDT para a divergência genética entre populações por meio do método de Singh. As populações de cada grupo obtido pela análise de agrupamento foram combinadas e, para cada caráter, foi realizado o teste t de Student para uma média. Existe variabilidade genética entre populações da TDT provenientes de diferentes localidades do Brasil, quando estão infestando Lycopersicon spp. A mortalidade larval teve maior contribuição para a divergência genética entre as populações, com exceção de PI 134417, cujo caráter de maior contribuição foi o número de pupas fêmeas. Propõe-se uma subcoleção da TDT tomando-se por base a combinação das populações de Santa Teresa e Uberlândia.This work aimed to study the genetic diversity and to propose a representative tomato pinworm, Tuta absoluta (TDT, subcollection. Populations of insect originally from Uberlândia, MG, Viçosa (MG, Camocim de São Félix, PE, and Santa Teresa, ES, Brazil, and five tomato accessions, 'Santa Clara', 'Moneymaker', TOM-601, PI 126445 (Lycopersicon hirsutum f. typicum and PI 134417 (L. hirsutum f. glabratum were used. Grouping analysis (Tocher method, using the Mahalanobis distance as dissimilarity measurement was performed and the relative importance of TDT characters to the genetic divergence among populations was evaluated by Singh
-
Wei, Pengcheng; Song, Guangji; Shi, Mengyang; Zhou, Yafei; Liu, Yang; Lei, Jun; Chen, Peng; Yin, Lei
2018-02-01
Colistin is considered a last-resort antibiotic against most gram-negative bacteria. Recent discoveries of a plasmid-mediated, transferable mobilized colistin-resistance gene ( mcr-1) on all continents have heralded the imminent emergence of pan-drug-resistant superbacteria. The inner-membrane protein MCR-1 can catalyze the transfer of phosphoethanolamine (PEA) to lipid A, resulting in colistin resistance. However, little is known about the mechanism, and few drugs exist to address this issue. We present crystal structures revealing the MCR-1 catalytic domain (cMCR-1) as a monozinc metalloprotein with ethanolamine (ETA) and d-glucose, respectively, thus highlighting 2 possible substrate-binding pockets in the MCR-1-catalyzed PEA transfer reaction. Mutation of the residues involved in ETA and d-glucose binding impairs colistin resistance in recombinant Escherichia coli containing full-length MCR-1. Partial analogs of the substrate are used for cocrystallization with cMCR-1, providing valuable information about the family of PEA transferases. One of the analogs, ETA, causes clear inhibition of polymyxin B resistance, highlighting its potential for drug development. These data demonstrate the crucial role of the PEA- and lipid A-binding pockets and provide novel insights into the structure-based mechanisms, important drug-target hot spots, and a drug template for further drug development to combat the urgent, rising threat of MCR-1-mediated antibiotic resistance.-Wei, P., Song, G., Shi, M., Zhou, Y., Liu, Y., Lei, J., Chen, P., Yin, L. Substrate analog interaction with MCR-1 offers insight into the rising threat of the plasmid-mediated transferable colistin resistance.
-
Xue, Di; Li, Yanan; Jiang, Zhongjia; Deng, Guangcun; Li, Min; Liu, Xiaoming; Wang, Yujiong
2017-05-01
Mycoplasma Ovipneumoniae (M. ovipneumoniae) is a primary etiological agent of enzootic pneumonia in sheep and goats. It can enter and colonize ovine respiratory epithelial cells to establish an infection, which leads a serious cell death of epithelial cells. However, the nature of the interaction between pathogen of M. ovipneumoniae and host cells in the cell injury is currently not well understood. In this study, we investigated the epithelial cell apoptosis caused by an infection of M. ovipneumoniae in sheep primary air-liquid interface (ALI) epithelial cultures. The results showed that M. ovipneumoniae could specifically bind to ciliated cells at early stage of infection. Flow cytometric analysis demonstrated that an infection of M. ovipneumoniae induced a time-dependent cell apoptotic cell death, accompanied with an increased production of extracellular nitric oxide (NO), intracellular reactive oxygen species (ROS) production and activation of caspase-3 signaling in sheep bronchial epithelial cells. The induced cell apoptosis was further confirmed by a transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay. Interestingly, the M. ovipneumoniae-induced apoptosis and activation of caspase-3 were correlated with the production of ROS but not NO. Mechanistically, M. ovipneumoniae-induced cell apoptosis was mediated by a mechanism by increasing the expression of phosphorylation of p38 and pro-apoptotic proteins, and activating caspase-3, caspase-8 and poly ADP-ribose polymerase (PARP) cleavage. These results suggest a ROS-dependent and caspase-3-mediated cell apoptosis in sheep bronchial epithelial cells in response to M. ovipneumoniae infections. Copyright © 2017. Published by Elsevier B.V.
-
Successful adaptation to ketosis by mice with tissue-specific deficiency of ketone body oxidation.
Cotter, David G; Schugar, Rebecca C; Wentz, Anna E; d'Avignon, D André; Crawford, Peter A
2013-02-15
During states of low carbohydrate intake, mammalian ketone body metabolism transfers energy substrates originally derived from fatty acyl chains within the liver to extrahepatic organs. We previously demonstrated that the mitochondrial enzyme coenzyme A (CoA) transferase [succinyl-CoA:3-oxoacid CoA transferase (SCOT), encoded by nuclear Oxct1] is required for oxidation of ketone bodies and that germline SCOT-knockout (KO) mice die within 48 h of birth because of hyperketonemic hypoglycemia. Here, we use novel transgenic and tissue-specific SCOT-KO mice to demonstrate that ketone bodies do not serve an obligate energetic role within highly ketolytic tissues during the ketogenic neonatal period or during starvation in the adult. Although transgene-mediated restoration of myocardial CoA transferase in germline SCOT-KO mice is insufficient to prevent lethal hyperketonemic hypoglycemia in the neonatal period, mice lacking CoA transferase selectively within neurons, cardiomyocytes, or skeletal myocytes are all viable as neonates. Like germline SCOT-KO neonatal mice, neonatal mice with neuronal CoA transferase deficiency exhibit increased cerebral glycolysis and glucose oxidation, and, while these neonatal mice exhibit modest hyperketonemia, they do not develop hypoglycemia. As adults, tissue-specific SCOT-KO mice tolerate starvation, exhibiting only modestly increased hyperketonemia. Finally, metabolic analysis of adult germline Oxct1(+/-) mice demonstrates that global diminution of ketone body oxidation yields hyperketonemia, but hypoglycemia emerges only during a protracted state of low carbohydrate intake. Together, these data suggest that, at the tissue level, ketone bodies are not a required energy substrate in the newborn period or during starvation, but rather that integrated ketone body metabolism mediates adaptation to ketogenic nutrient states.
-
The Biochemistry of O-GlcNAc Transferase: Which Functions Make It Essential in Mammalian Cells?
Levine, Zebulon G; Walker, Suzanne
2016-06-02
O-linked N-acetylglucosamine transferase (OGT) is found in all metazoans and plays an important role in development but at the single-cell level is only essential in dividing mammalian cells. Postmitotic mammalian cells and cells of invertebrates such as Caenorhabditis elegans and Drosophila can survive without copies of OGT. Why OGT is required in dividing mammalian cells but not in other cells remains unknown. OGT has multiple biochemical activities. Beyond its well-known role in adding β-O-GlcNAc to serine and threonine residues of nuclear and cytoplasmic proteins, OGT also acts as a protease in the maturation of the cell cycle regulator host cell factor 1 (HCF-1) and serves as an integral member of several protein complexes, many of them linked to gene expression. In this review, we summarize current understanding of the mechanisms underlying OGT's biochemical activities and address whether known functions of OGT could be related to its essential role in dividing mammalian cells.
-
Sun, Ying; Wang, Yu; Huang, Xing; Gu, Xiaobing; Lai, Weimin; Peng, Xuerong; Yang, Guangyou
2017-08-15
Taenia multiceps is a widespread zoonotic tapeworm parasite which infects cloven-hoofed animals around the world. Animal infection with Coenurus cerebralis, the coenurus larvae of T. multiceps (Tm), is often fatal, which is a major cause of economic losses in stockbreeding. This study amplified the glutathione S-transferase (GST) gene from the total RNA of C. cerebralis. The resulting protein, Tm-GST, consisted of 201 amino acids, and had a predicted molecular mass of 23.1kDa. Its amino acid sequence shares 77.61% similarity with Echinococcus granulosus GST. Recombinant Tm-GST (rTm-GST) was expressed in Escherichia coli. The protein reacted with serum from goats infected with T. multiceps. Immunofluorescence signals indicated that Tm-GST was largely localized in the parenchymatous area of adult T. multiceps; in addition, it was also apparent in the coenurus. An enzyme-linked immunosorbent assay based on rTm-GST showed specificity of 92.8% (13/14) and sensitivity of 90% (18/20) in detecting anti-GST antibodies in serum from naturally infected animals. This study suggests that Tm-GST has the potential to be used as a diagnostic antigen for Coenurosis. Copyright © 2017. Published by Elsevier B.V.
-
In-vitro effect of flavonoids from Solidago canadensis extract on glutathione S-transferase.
Apáti, Pál; Houghton, Peter J; Kite, Geoffrey; Steventon, Glyn B; Kéry, Agnes
2006-02-01
Solidago canadensis is typical of a flavonoid-rich herb and the effect of an aqueous ethanol extract on glutathione-S-transferase (GST) activity using HepG2 cells was compared with those of the flavonol quercetin and its glycosides quercitrin and rutin, found as major constituents. The composition of the extract was determined by HPLC and rutin was found to be the major flavonoidal component of the extract. Total GST activity was assessed using 1-chloro-2,4-dinitrobenzene as a substrate. The glycosides rutin and quercitrin gave dose-dependent increases in GST activity, with a 50% and 24.5% increase at 250 mM, respectively, while the aglycone quercetin inhibited the enzyme by 30% at 250 mM. The total extract of the herb gave an overall dose-dependent increase, the fractions corresponding to the flavonoids showed activating effects while those containing caffeic acid derivatives were inhibitory. The activity observed corresponds to that reported for similar compounds in-vivo using rats, thus the HepG2 cell line could serve as a more satisfactory method of assessing the effects of extracts and compounds on GST.
-
Mediation Analysis with Multiple Mediators
VanderWeele, T.J.; Vansteelandt, S.
2014-01-01
Recent advances in the causal inference literature on mediation have extended traditional approaches to direct and indirect effects to settings that allow for interactions and non-linearities. In this paper, these approaches from causal inference are further extended to settings in which multiple mediators may be of interest. Two analytic approaches, one based on regression and one based on weighting are proposed to estimate the effect mediated through multiple mediators and the effects throu...
-
Cao, Zhixin; Yang, Qianqian; Yin, Haiyan; Qi, Qi; Li, Hongrui; Sun, Gaoying; Wang, Hongliang; Liu, Wenwen; Li, Jianfeng
2017-11-01
Peroxynitrite (ONOO - ) is a potent and versatile oxidant implicated in a number of pathophysiological processes. The present study was designed to investigate the effect of ONOO - on the cultured cochlear hair cells (HCs) of C57BL/6 mice in vitro as well as the possible mechanism underlying the action of such an oxidative stress. The in vitro primary cultured cochlear HCs were subjected to different concentrations of ONOO - , then, the cell survival and morphological changes were examined by immunofluorescence and transmission electron microscopy (TEM), the apoptosis was determined by Terminal deoxynucleotidyl transferase dUNT nick end labeling (TUNEL) assay, the mRNA expressions of Caspase-3, Caspase-8, Caspase-9, Apaf1, Bcl-2, and Bax were analyzed by RT-PCR, and the protein expressions of Caspase-3 and AIF were assessed by immunofluorescence. This work demonstrated that direct exposure of primary cultured cochlear HCs to ONOO - could result in a base-to-apex gradient injury of HCs in a concentration-dependent manner. Furthermore, ONOO - led to much more losses of outer hair cells than inner hair cells mainly through the induction of apoptosis of HCs as evidenced by TEM and TUNEL assays. The mRNA expressions of Caspase-8, Caspase-9, Apaf1, and Bax were increased and, meanwhile, the mRNA expression of Bcl-2 was decreased in response to ONOO - treatment. Of interesting, the expression of Caspase-3 had no significant change, whereas, the expression alteration of AIF was observed. These results suggested that ONOO - can effectively damage the survival of cochlear HCs via triggering the apoptotic pathway. The findings from this work suggest that ONOO - -induced apoptosis is mediated, at least in part, via a Caspase-independent pathway in cochlear HCs.
-
Directory of Open Access Journals (Sweden)
Yu Ah Hong
2017-06-01
Full Text Available Background: Vitamin D is considered to exert a protective effect on various renal diseases but its underlying molecular mechanism remains poorly understood. This study aimed to determine whether paricalcitol attenuates inflammation and apoptosis during lipopolysaccharide (LPS-induced renal proximal tubular cell injury through the prostaglandin E₂ (PGE₂ receptor EP4. Methods: Human renal tubular epithelial (HK-2 cells were pretreated with paricalcitol (2 ng/mL for 1 hour and exposed to LPS (1 μg/mL. The effects of paricalcitol pretreatment in relation to an EP4 blockade using AH-23848 or EP4 small interfering RNA (siRNA were investigated. Results: The expression of cyclooxygenase-2, PGE₂, and EP4 were significantly increased in LPS-exposed HK-2 cells treated with paricalcitol compared with cells exposed to LPS only. Paricalcitol prevented cell death induced by LPS exposure, and the cotreatment of AH-23848 or EP4 siRNA offset these cell-protective effects. The phosphorylation and nuclear translocation of p65 nuclear factor-kappaB (NF-κB were decreased and the phosphorylation of Akt was increased in LPS-exposed cells with paricalcitol treatment. AH-23848 or EP4 siRNA inhibited the suppressive effects of paricalcitol on p65 NF-κB nuclear translocation and the activation of Akt. The production of proinflammatory cytokines and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells were attenuated by paricalcitol in LPS exposed HK-2 cells. The cotreatment with an EP4 antagonist abolished these anti-inflammatory and antiapoptotic effects. Conclusion: EP4 plays a pivotal role in anti-inflammatory and antiapoptotic effects through Akt and NF-κB signaling after paricalcitol pretreatment in LPS-induced renal proximal tubule cell injury.
-
Shibata, Naomi; Watari, Jiro; Fujiya, Mikihiro; Tanno, Satoshi; Saitoh, Yusuke; Kohgo, Yutaka
2003-01-01
To clarify the biological impact and molecular pathogenesis of cellular phenotype in differentiated-type gastric cancers (DGCs), we investigated cell kinetics and genetic instabilities in early stage of DGCs. A total of 43 early gastric cancers (EGCs) were studied. EGCs were divided into 3 phenotypic categories: gastric (G type, n = 11), ordinary (O type, n = 20), and complete intestinal (CI type, n = 12) based on the combination of HGM, ConA, MUC2, and CD10. Proliferative index (PI), apoptotic index (AI), and p53 overexpression were investigated by immunohistochemical staining with anti-Ki-67, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method, and p53 antibody, respectively. Using a high-resolution fluorescent microsatellite analysis system, microsatellite instability (MSI) and loss of heterozygosity (LOH) were examined. Frameshift mutation analysis of transforming growth factor-beta type II receptor (TGF-betaRII) and bcl-2-associated X (BAX) in cancers with MSI was also performed. The mean AI/PI ratio values were 0.04 for G-type, 0.10 for O-type, and 0.13 for CI-type cancers--significantly lower in G type than in O and CI types (P = 0.02 and P = 0.001, respectively). No difference in the incidence of MSI and LOH was seen among the 3 cellular phenotypes. However, the major pattern of MSI, which showed drastic and widely dispersed changes and is related to an increased risk for cancer, was significantly higher in G and O types than in CI type (P cancers. These results indicate that G-type cancers are likely to show more aggressive behaviors than CI-type cancers, and that O-type cancers show the intermediate characteristics of both types. However, the molecular pathogenesis of each phenotypic cancer is not associated with microsatellite alterations. Copyright 2003, Elsevier Science (USA). All rights reserved.
-
Zinc finger protein 139 expression in gastric cancer and its clinical significance.
Li, Yong; Zhao, Qun; Fan, Li-Qiao; Wang, Li-Li; Tan, Bi-Bo; Leng, Yan-Li; Liu, Yu; Wang, Dong
2014-12-28
To investigate the expression of zinc finger protein 139 (ZNF139) in gastric cancer (GC), and to analyze its clinical significance. A total of 108 patients who were diagnosed with GC and underwent surgery between January 2005 and March 2007 were enrolled in this study. Gastric tumor specimens and paired tumor-adjacent tissues were collected and paraffin-embedded, and the clinicopathologic characteristics and prognosis were recorded. The expression of ZNF139, Bcl-2, Bax, and caspase-3 were determined by immunohistochemistry, and apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. SPSS 13.0 software was used for data processing and analyses, and significance was determined at P stage, lymphatic metastasis, and blood vessel invasion (all Ps < 0.05). Patients with overexpression of ZNF139 had a poorer prognosis (P < 0.01), and overexpression of ZNF139 was an independent factor for the prognosis of GC patients by a Cox survival analysis (P = 0.02). A negative relationship between ZNF139 and the apoptosis index was observed (r = -0.686; P < 0.01). The expression of Bcl-2 in GC was stronger than in tumor-adjacent tissues (66.67% vs 41.67%), whereas the expression levels of Bax and caspase-3 were lower in primary tumors (54.63% and 47.22%, respectively) than in tumor-adjacent tissues (73.15% and 73.15%, respectively) (all Ps < 0.05). The expression of ZNF139 negatively correlated with caspase-3 (r = -0.370; P < 0.01). The expressions of Bcl-2 and Bax were also negatively correlated (r = -0.231; P = 0.02). The expressions of caspase-3 and Bax protein were positively correlated (r = 0.217; P = 0.024). ZNF139 is related to clinicopathologic characteristics and prognosis of GC. Furthermore, it is overexpressed and involved in apoptosis in GC tissues by regulating caspase-3.
-
International Nuclear Information System (INIS)
Gao, Feng-Hou; Liu, Feng; Zhao, Ying-Zheng; Fang, Yong; Chen, Fang-Yuan; Wu, Ying-Li; Hu, Xiao-Hui; Li, Wei; Liu, Hua; Zhang, Yan-Jie; Guo, Zhu-Ying; Xu, Mang-Hua; Wang, Shi-Ting; Jiang, Bin
2010-01-01
Oridonin, a tetracycline diterpenoid compound, has the potential antitumor activities. Here, we evaluate the antitumor activity and action mechanisms of oridonin in colorectal cancer. Effects of oridonin on cell proliferation were determined by using a CCK-8 Kit. Cell cycle distribution was determined by flow cytometry. Apoptosis was examined by analyzing subdiploid population and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Senescent cells were determined by senescence-associated β-galactosidase activity analysis. Semi-quantitative RT-PCR was used to examine the changes of mRNA of p16, p21, p27 and c-myc. The concomitant changes of protein expression were analyzed with Western blot. Expression of AcH3 and AcH4 were examined by immunofluorescence staining and Western blots. Effects of oridonin on colony formation of SW1116 were examined by Soft Agar assay. The in vivo efficacy of oridonin was detected using a xenograft colorectal cancer model in nude mice. Oridonin induced potent growth inhibition, cell cycle arrest, apoptosis, senescence and colony-forming inhibition in three colorectal cancer cell lines in a dose-dependent manner in vitro. Daily i.p. injection of oridonin (6.25, 12.5 or 25 mg/kg) for 28 days significantly inhibited the growth of SW1116 s.c. xenografts in BABL/C nude mice. With western blot and reverse transcription-PCR, we further showed that the antitumor activities of oridonin correlated with induction of histone (H3 and H4) hyperacetylation, activation of p21, p27 and p16, and suppression of c-myc expression. Oridonin possesses potent in vitro and in vivo anti-colorectal cancer activities that correlated with induction of histone hyperacetylation and regulation of pathways critical for maintaining growth inhibition and cell cycle arrest. Therefore, oridonin may represent a novel therapeutic option in colorectal cancer treatment
-
Işık, S; Karaman, M; Çilaker Micili, S; Çağlayan-Sözmen, Ş; Bağrıyanık, H Alper; Arıkan-Ayyıldız, Z; Uzuner, N; Karaman, Ö
In previous studies, anti-inflammatory, anti-apoptotic and immunomodulatory effects of ursodeoxycholic acid (UDCA) on liver diseases have been shown. In this study, we aimed to investigate the effects of UDCA on airway remodelling, epithelial apoptosis, and T Helper (Th)-2 derived cytokine levels in a murine model of chronic asthma. Twenty-seven BALB/c mice were divided into five groups; PBS-Control, OVA-Placebo, OVA-50mg/kg UDCA, OVA-150mg/kg UDCA, OVA-Dexamethasone. Mice in groups OVA-50mg/kg UDCA, OVA-150mg/kg UDCA, OVA-Dexamethasone received the UDCA (50mg/kg), UDCA (150mg/kg), and dexamethasone, respectively. Epithelium thickness, sub-epithelial smooth muscle thickness, number of mast and goblet cells of samples isolated from the lung were measured. Immunohistochemical scorings of the lung tissue for matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEG-F), transforming growth factor-beta (TGF-β), terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL) and cysteine-dependent aspartate-specific proteases (caspase)-3 were determined. IL-4, IL-5, IL-13, Nitric oxide, ovalbumin-specific immunoglobulin (Ig) E levels were quantified. The dose of 150mg/kg UDCA treatment led to lower epithelial thickness, sub-epithelial smooth muscle thickness, goblet and mast cell numbers compared to placebo. Except for MMP-9 and TUNEL all immunohistochemical scores were similar in both UDCA treated groups and the placebo. All cytokine levels were significantly lower in group IV compared to the placebo. These findings suggested that the dose of 150mg/kg UDCA improved all histopathological changes of airway remodelling and its beneficial effects might be related to modulating Th-2 derived cytokines and the inhibition of apoptosis of airway epithelial cells. Copyright © 2017 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.
-
Ladenheim, B; Krasnova, I N; Deng, X; Oyler, J M; Polettini, A; Moran, T H; Huestis, M A; Cadet, J L
2000-12-01
Increasing evidence implicates apoptosis as a major mechanism of cell death in methamphetamine (METH) neurotoxicity. The involvement of a neuroimmune component in apoptotic cell death after injury or chemical damage suggests that cytokines may play a role in METH effects. In the present study, we examined if the absence of IL-6 in knockout (IL-6-/-) mice could provide protection against METH-induced neurotoxicity. Administration of METH resulted in a significant reduction of [(125)I]RTI-121-labeled dopamine transporters in the caudate-putamen (CPu) and cortex as well as depletion of dopamine in the CPu and frontal cortex of wild-type mice. However, these METH-induced effects were significantly attenuated in IL-6-/- animals. METH also caused a decrease in serotonin levels in the CPu and hippocampus of wild-type mice, but no reduction was observed in IL-6-/- animals. Moreover, METH induced decreases in [(125)I]RTI-55-labeled serotonin transporters in the hippocampal CA3 region and in the substantia nigra-reticulata but increases in serotonin transporters in the CPu and cingulate cortex in wild-type animals, all of which were attenuated in IL-6-/- mice. Additionally, METH caused increased gliosis in the CPu and cortices of wild-type mice as measured by [(3)H]PK-11195 binding; this gliotic response was almost completely inhibited in IL-6-/- animals. There was also significant protection against METH-induced DNA fragmentation, measured by the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeled (TUNEL) cells in the cortices. The protective effects against METH toxicity observed in the IL-6-/- mice were not caused by differences in temperature elevation or in METH accumulation in wild-type and mutant animals. Therefore, these observations support the proposition that IL-6 may play an important role in the neurotoxicity of METH.
-
Effects of hepatitis B virus S protein exposure on sperm membrane integrity and functions.
Directory of Open Access Journals (Sweden)
XiangJin Kang
Full Text Available BACKGROUND: Hepatitis B is a public health problem worldwide. Viral infection can affect a man's fertility, but only scant information about the influence of hepatitis B virus (HBV infection on sperm quality is available. The purpose of this study was to investigate the effect of hepatitis B virus S protein (HBs on human sperm membrane integrity and functions. METHODS/PRINCIPAL FINDINGS: Reactive oxygen species (ROS, lipid peroxidation (LP, total antioxidant capacity (TAC and phosphatidylserine (PS externalization were determined. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL assays and flow cytometric analyses were performed. (1 After 3 h incubation with 25 µg/ml of HBs, the average rates of ROS positive cells, annexin V-positive/propidium iodide (PI-negative cells, Caspases-3,-8,-9 positive cells and TUNEL-positive cells were significantly increased in the test groups as compared to those in the control groups, while TAC level was decreased when compared with the control. The level of malondialdehyde (MDA in the sperm cells exposed to 50 µg/ml of HBs for 3 h was significantly higher than that in the control (P<0.05-0.01. (2 HBs increased the MDA levels and the numbers of ROS positive cells, annexin V-positive/PI-negative cells, caspases-3, -8, -9 positive cells and TUNEL-positive cells in a dose-dependent manner. (3 HBs monoclonal antibody (MAb and N-Acetylcysteine (NAC reduced the number of ROS-positive sperm cells. (4 HBs decreased the TAC levels in sperm cells in a dose-dependent manner. CONCLUSION: HBs exposure could lead to ROS generation, lipid peroxidation, TAC reduction, PS externalization, activation of caspases, and DNA fragmentation, resulting in increased apoptosis of sperm cells and loss of sperm membrane integrity and causing sperm dysfunctions.
-
Behavioral and anatomical correlates of chronic episodic hypoxia during sleep in the rat.
Gozal, D; Daniel, J M; Dohanich, G P
2001-04-01
The role played by chronic episodic hypoxia (EHYP) in the neurocognitive morbidity of obstructive sleep apnea (OSA) is unknown. Sleep recordings, Morris water maze experiments, and immunohistochemistry for NMDA NR1 glutamate receptor, c-fos protein, and apoptosis [nuclear immunoreactivity for single-stranded DNA and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assay] were conducted in EHYP-exposed Sprague Dawley male rats. Exposures consisted of up to14 d in an environmental chamber in which O(2) concentrations were cycled between 10 and 21% every 90 sec or 30 min during 12 hr of daylight. For the remaining 12 hr, EHYP rats breathed room air, while controls spent 14 d in room air. Although EHYP induced significant disruption of sleep architecture during the initial day of exposure, sleep patterns normalized thereafter. Marked increases in apoptosis occurred in the CA1 hippocampal region (sevenfold) and cortex (Cx; eightfold) after 1-2 d of EHYP but not in CA3 and were followed by decreases toward normoxic levels by 14 d. Double labeling for NMDA NR1 and c-fos revealed marked architectural disorganization in CA1 and Cx with increases in c-fos over time. Rats exposed to EHYP displayed significantly longer escape latencies and swim path lengths to escape a hidden platform during 12 training trials given over 2 d. Differences in the performances of EHYP and control rats, although reduced, persisted after 14 d of recovery. We conclude that EHYP is associated with marked cellular changes over time within neural regions associated with cognitive functions. Furthermore, EHYP impaired performance during acquisition of a cognitive spatial task without affecting sensorimotor function. Such changes may underlie components of the learning and memory impairments found in OSA.
-
Pressly, Jeffrey D; Mustafa, Suni M; Adibi, Ammaar H; Alghamdi, Sahar; Pandey, Pankaj; Roy, Kuldeep K; Doerksen, Robert J; Moore, Bob M; Park, Frank
2018-02-01
Ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI), which is an increasing problem in the clinic and has been associated with elevated rates of mortality. Therapies to treat AKI are currently not available, so identification of new targets that can be modulated to ameliorate renal damage upon diagnosis of AKI is essential. In this study, a novel cannabinoid receptor 2 (CB2) agonist, SMM-295 [3'-methyl-4-(2-(thiophen-2-yl)propan-2-yl)biphenyl-2,6-diol], was designed, synthesized, and tested in vitro and in silico. Molecular docking of SMM-295 into a CB2 active-state homology model showed that SMM-295 interacts well with key amino acids to stabilize the active state. In human embryonic kidney 293 cells, SMM-295 was capable of reducing cAMP production with 66-fold selectivity for CB2 versus cannabinoid receptor 1 and dose-dependently increased mitogen-activated protein kinase and Akt phosphorylation. In vivo testing of the CB2 agonist was performed using a mouse model of bilateral IRI, which is a common model to mimic human AKI, where SMM-295 was immediately administered upon reperfusion of the kidneys after the ischemia episode. Histologic damage assessment 48 hours after reperfusion demonstrated reduced tubular damage in the presence of SMM-295. This was consistent with reduced plasma markers of renal dysfunction (i.e., creatinine and neutrophil gelatinase-associated lipocalin) in SMM-295-treated mice. Mechanistically, kidneys treated with SMM-295 were shown to have elevated activation of Akt with reduced terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL)-positive cells compared with vehicle-treated kidneys after IRI. These data suggest that selective CB2 receptor activation could be a potential therapeutic target in the treatment of AKI. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.
-
Puntoni, Matteo; Branchi, Daniela; Argusti, Alessandra; Zanardi, Silvia; Crosta, Cristiano; Meroni, Emanuele; Munizzi, Francesco; Michetti, Paolo; Coccia, Gianni; De Roberto, Giuseppe; Bandelloni, Roberto; Turbino, Laura; Minetti, Egle; Mori, Marco; Salvi, Sandra; Boccardo, Simona; Gatteschi, Beatrice; Benelli, Roberto; Sonzogni, Angelica; DeCensi, Andrea
2013-02-01
Inflammation and oxidative stress play a crucial role in the development of colorectal cancer (CRC) and interference with these mechanisms represents a strategy in CRC chemoprevention. Allopurinol, a safe molecular scavenger largely used as antigout agent, has been shown to increase survival of patients with advanced CRC and to reduce CRC incidence in long-term gout users in epidemiologic studies. We conducted a randomized, double-blind, placebo-controlled preoperative trial in subjects with colorectal adenomatous polyps to assess the activity of allopurinol on biomarkers of colorectal carcinogenesis. After complete colonoscopy and biopsy of the index polyp, 73 subjects with colorectal adenomas were assigned to either placebo or one of two doses of allopurinol (100 mg or 300 mg) and treated for four weeks before polyp removal. Change of Ki-67 labeling index in adenomatous tissue was the primary endpoint. Secondary endpoints were the immunohistochemical (IHC) expression of NF-κB, β-catenin, topoisomerase-II-α, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in adenomatous polyps and normal adjacent colonic tissue. Compared with placebo, Ki-67 levels were not significantly modulated by allopurinol, whereas β-catenin and NF-κB expression levels decreased significantly in adenomatous tissue, with a mean change from baseline of -10.6%, 95% confidence interval (CI), -20.5 to -0.7, and -8.1%, 95% CI, -22.7 to 6.5, respectively. NF-κB also decreased significantly in normal adjacent tissue (-16.4%; 95% CI, -29.0 to -3.8). No dose-response relationship was noted, except for NF-κB expression in normal tissue. Allopurinol can inhibit biomarkers of oxidative activation in colon adenomatous polyps and normal adjacent tissue. Further studies should define its potential chemopreventive activity.
-
Chen, Wenxia; Yan, Yongbin; Song, Chundong; Ding, Ying; Du, Tao
2017-12-14
Studies have demonstrated that microvesicles (MVs) derived from human Wharton's Jelly mesenchymal stromal cells (hWJMSCs) could ameliorate renal ischemia/reperfusion injury (IRI); however, the underlying mechanisms were not clear yet. Here, MVs were isolated and injected intravenously into rats immediately after ischemia of the left kidney, and Erk1/2 activator hepatocyte growth factor (HGF) or inhibitor U0126 was administrated. Tubular cell proliferation and apoptosis were identified by Ki67 or terminal-deoxynucleotidyl transferase-mediated nick end labeling immunostaining. Masson's tri-chrome straining and alpha-smooth muscle actin staining were used for assessing renal fibrosis. The mRNA or protein expression in the kidney was measured by quantitative reverse transcription-PCR or Western blot, respectively. The total collagen concentration was also determined. In vitro , NRK-52E cells that treated with MVs under hypoxia injury and with HGF or U0126 administration were used, and cell cycle analysis was performed. The effects of hWJMSC-MVs on enhancing the proliferation and mitigating the apoptosis of renal cells, abrogating IRI-induced fibrosis, improving renal function, decreasing collagen deposition, and altering the expression levels of epithelial-mesenchymal transition and cell cycle-related proteins in IRI rats were found. In vitro experiment showed that hWJMSC-MVs could induce G2/M cell cycle arrest and decrease the expression of collagen deposition-related proteins in NRK-52E cells after 24 or 48 h. However, U0126 treatment reversed these effects. In conclusion, MVs derived from hWJMSCs ameliorate IR-induced renal fibrosis by inducing G2/M cell cycle arrest via Erk1/2 signaling. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.
-
International Nuclear Information System (INIS)
Kijima, Kazuyasu; Toyosawa, Kaoru; Yasuba, Masashi; Matsuoka, Nobuo; Adachi, Tetsuya; Komiyama, Masatoshi; Mori, Chisato
2004-01-01
To investigate the effects of di(2-ethylhexyl) phthalate (DEHP) on gene expression in rat testis, 6-week-old male Sprague-Dawley rats were given a single oral dose of 20 or 2000 mg/kg and euthanized 3, 6, 24, or 72 h thereafter. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly increased in the testis at 24 and 72 h after the exposure to 2000 mg/kg of DEHP. On cDNA microarray analysis, in addition to apoptosis-related genes, genes associated with atrophy, APEX nuclease, MutS homologue (E. coli), testosterone-repressed-prostatic-message-2 (TRPM-2), connective tissue growth factor, collagen alpha 2 type V, and cell adhesion kinase were differentially expressed. To investigate the relationship between histopathological alteration and gene expression, we selected genes associated with apoptosis and analyzed their expression by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). With 20 mg/kg of DEHP treatment, bcl-2, key gene related to apoptosis, was increased. Up-regulation of bcl-2, inhibitor of Apaf-1/caspase-9/caspase-2 cascade of apoptosis, may be related to the fact that no morphological apoptotic change was induced after dosing of 20 mg/kg DEHP. With 2000 mg/kg of DEHP treatment, the apoptotic activator cascade, Fas/FasL, FADD/caspase-8/caspase-3 cascade, and Apaf-1/caspase-9/caspase-2 cascade were increased and bcl-2 was decreased. Thus, these gene regulations might lead the cells into apoptosis in the case of high exposure to DEHP. In contrast, FADD/caspase-10/caspase-6 cascade and caspase-11/caspase-3 cascade were not increased. These results indicate that the cascades of FADD/caspase-10/caspase-6 and caspase-11/caspase-3 are not related to apoptosis with DEHP treatment
-
Energy Technology Data Exchange (ETDEWEB)
Kim, Won; Yoon, Jung Hwan; Kim, Chung Yang [Seoul National University College of Medicine, Seoul (Korea, Republic of); Cheon, Gi Jeoog; Lee, Tae Sup; Woo, Kwang Sun; Chung, Wee Sup [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)
2005-07-01
We have recently demonstrated that hypoxia stimulates hepatocellular carcinoma (HCC) cell growth through hexokinase II induction, and its inhibition induces apoptotic cell death through activating mitochondrial apoptotic signaling cascades. In this study, we were apt to evaluate the antitumoral effect of 3-bromopyruvate (3-BP) on in vivo model of HCC by apoptotic imaging using Tc-99m labeled annexin V. In vivo model of HCC was established in C3H mice intradermally implanted with MH134 cells, a mouse HCC cell line, and 3-BP (0, 5, 10 mg/kg) was subsequently administered intraperitoneally. Tc-99m-HYNIC-annexin V (185 KBq) was injected via tail vein at one and three days after the 3-BP treatment, planar scan was acquired at a hour after the injection using gamma camera. The anti-tumor effect was evaluated by measuring tumor volumes and quantification of apoptotic cells using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Tumor volume was significantly reduced in mice treated with 3-BP in a dose-dependent manner (mean tumor volume 1.07 vs. 0.58 vs. 0.39 cm{sup 3} in 3-BP 0, 5, 10 mg/kg, respectively: p=0.047). The percentage of TUNEL staining-positive cells was significantly increased in 3-BP-treated mice (0.53 vs. 1.40 vs. 1.84% in 3-BP 0, 5, 10 mg/kg, respectively; p=0.018). On Tc-99m-HYNIC annexin V imaging, tumor-to-background uptake ratio (UR) was 1.92 at one day and 4.23 at three days after 3-BP treatment of 5 mg/kg (non-treated tumor showed UR of 2.93). Apoptosis-inducing anti-tumor effect of 3-BP was able to be demonstrated in in vivo model of HCC by apoptotic in vivo imaging using Tc-99m-HYNIC annexin V.
-
International Nuclear Information System (INIS)
Kim, Won; Yoon, Jung Hwan; Kim, Chung Yang; Cheon, Gi Jeoog; Lee, Tae Sup; Woo, Kwang Sun; Chung, Wee Sup
2005-01-01
We have recently demonstrated that hypoxia stimulates hepatocellular carcinoma (HCC) cell growth through hexokinase II induction, and its inhibition induces apoptotic cell death through activating mitochondrial apoptotic signaling cascades. In this study, we were apt to evaluate the antitumoral effect of 3-bromopyruvate (3-BP) on in vivo model of HCC by apoptotic imaging using Tc-99m labeled annexin V. In vivo model of HCC was established in C3H mice intradermally implanted with MH134 cells, a mouse HCC cell line, and 3-BP (0, 5, 10 mg/kg) was subsequently administered intraperitoneally. Tc-99m-HYNIC-annexin V (185 KBq) was injected via tail vein at one and three days after the 3-BP treatment, planar scan was acquired at a hour after the injection using gamma camera. The anti-tumor effect was evaluated by measuring tumor volumes and quantification of apoptotic cells using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Tumor volume was significantly reduced in mice treated with 3-BP in a dose-dependent manner (mean tumor volume 1.07 vs. 0.58 vs. 0.39 cm 3 in 3-BP 0, 5, 10 mg/kg, respectively: p=0.047). The percentage of TUNEL staining-positive cells was significantly increased in 3-BP-treated mice (0.53 vs. 1.40 vs. 1.84% in 3-BP 0, 5, 10 mg/kg, respectively; p=0.018). On Tc-99m-HYNIC annexin V imaging, tumor-to-background uptake ratio (UR) was 1.92 at one day and 4.23 at three days after 3-BP treatment of 5 mg/kg (non-treated tumor showed UR of 2.93). Apoptosis-inducing anti-tumor effect of 3-BP was able to be demonstrated in in vivo model of HCC by apoptotic in vivo imaging using Tc-99m-HYNIC annexin V
-
McClusky, L M
2005-01-01
To understand the processes involved in the spatial and temporal maturation of testicular cells in Squalus acanthias, we used standard morphometry, proliferating-cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) immunohistochemistry. Except for immature spermatocysts (germinal zone, GZ; early-stage pre-meiotic, E-PrM), the number of cysts in all subsequent stages and the total number of cysts in the spermatogenic progression varied seasonally. The spermatogenic cycle spans about 2 years and is interrupted by germcell clone deletion via apoptosis at the mitosis-meiosis transition in April/May, manifesting as a zone of degeneration (ZD). Rate of displacement of the ZD across the testis diameter indicates that late-stage premeiotic (L-PrM) generations 12-13 require 9-10 months to reach the mature-spermatid stage. Also, the number of cysts completing spermatogenesis is approximately 4-5-fold less than the number that entered spermatogenesis proper 2 years earlier. Pronounced gonocytogenesis in the germinal ridge was coincident with ZD formation in April/May, but it was absent in the fall when mature spermatogonial and meiotic activities had resumed. Whereas strong Sertoli cell PCNA immunoreactivity dominated the GZ cyst cell-cycle activities throughout the year, except during the spring/summer months, the spermatogonial- and Sertoli-cell PCNA indices in E-PrM cysts were inversely related. PCNA immunoreactivity in spermatocytes was seasonal and dependent on the stage of meiosis. TUNEL labelling was limited to spermatogonia and increased stage-dependently in the PrM region (L-PrM = mid-stage PrM >E-PrM >GZ), correlating with ZD formation, in a season-dependent manner. Results imply that effects of normal regulatory factors in Squalus are stage- and process-specific.
-
International Nuclear Information System (INIS)
Navarrete, Maria Alicia H; Maier, Carolina M; Falzoni, Roberto; Quadros, Luiz Gerk de Azevedo; Lima, Geraldo R; Baracat, Edmund C; Nazário, Afonso CP
2005-01-01
During the menstrual cycle, the mammary gland goes through sequential waves of proliferation and apoptosis. In mammary epithelial cells, hormonal and non-hormonal factors regulate apoptosis. To determine the cyclical effects of gonadal steroids on breast homeostasis, we evaluated the apoptotic index (AI) determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in human mammary epithelial cells during the spontaneous menstrual cycle and correlated it with cellular proliferation as determined by the expression of Ki-67 during the same period. Normal breast tissue samples were obtained from 42 randomly selected patients in the proliferative (n = 21) and luteal (n = 21) phases. Menstrual cycle phase characterization was based on the date of the last and subsequent menses, and on progesterone serum levels obtained at the time of biopsy. The proliferation index (PI), defined as the number of Ki-67-positive nuclei per 1,000 epithelial cells, was significantly larger in the luteal phase (30.46) than in the follicular phase (13.45; P = 0.0033). The AI was defined as the number of TUNEL-positive cells per 1,000 epithelial cells. The average AI values in both phases of the menstrual cycle were not statistically significant (P = 0.21). However, the cell renewal index (CRI = PI/AI) was significantly higher in the luteal phase (P = 0.033). A significant cyclical variation of PI, AI and CRI was observed. PI and AI peaks occurred on about the 24th day of the menstrual cycle, whereas the CRI reached higher values on the 28th day. We conclude that proliferative activity is dependent mainly on hormonal fluctuations, whereas apoptotic activity is probably regulated by hormonal and non-hormonal factors
-
Wang, Yayun; Chen, Manhua
2017-07-06
BACKGROUND Acute pancreatitis (AP) is a sudden inflammation of the pancreas. It results in multiple, severe complications, and 15-20% of patients develop severe acute pancreatitis (SAP) with mortality as high as 30%. Consequently, it is imperative to develop an effective therapy for SAP. MATERIAL AND METHODS We used 30 adult male Sprague Dawley (SD) rats. Rats were randomly divided into 3 groups - sham, SAP, and fentanyl+SAP - with 10 rats in each group. An automatic biochemical analyzer was used to analyze the concentration of creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH). Terminal-deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay was applied to assess the cell apoptosis rate. Pathological changes in pancreas/heart were detected with hematoxylin and eosin (HE) staining. Western immunoblot assay was used to analyze protein levels of interleukin (IL)-1β, IL-6, and IκB. RESULTS Fentanyl pre-treatment inhibits SAP-induced elevation of CK-MB/LDH concentrations in serum. Compared with the sham group, SAP generates a higher brown/yellow staining rate, which is abated by fentanyl. In the pancreas, SAP generated more serious interstitial edema/hemorrhage and fat necrosis than in the sham group, which are attenuated by fentanyl. Likewise, compared to the sham group, SAP generates swelled/disordered myocardial fibers and congested blood vessels in myocardium, which are ameliorated by fentanyl. In the sham group, there was little IL-1β/IL-6, and fentanyl significantly inhibited SAP-induced up-regulation of IL-1β/IL-6 levels. Compared with the sham group, SAP significantly reduced IκB level, which was rescued by fentanyl. CONCLUSIONS Fentanyl effectively alleviates SAP-induced pancreas and heart injuries through regulating the nuclear factor-κB (NF-κB) signaling pathway.
-
Directory of Open Access Journals (Sweden)
Wanchao Yang
Full Text Available Therapeutic hypercapnia has the potential for neuroprotection after global cerebral ischemia. Here we further investigated the effects of different degrees of acute systemic hypoxia in combination with hypercapnia on brain damage in a rat model of hypoxia and ischemia. Adult wistar rats underwent unilateral common carotid artery (CCA ligation for 60 min followed by ventilation with normoxic or systemic hypoxic gas containing 11%O2,13%O2,15%O2 and 18%O2 (targeted to PaO2 30-39 mmHg, 40-49 mmHg, 50-59 mmHg, and 60-69 mmHg, respectively or systemic hypoxic gas containing 8% carbon dioxide (targeted to PaCO2 60-80 mmHg for 180 min. The mean artery pressure (MAP, blood gas, and cerebral blood flow (CBF were evaluated. The cortical vascular permeability and brain edema were examined. The ipsilateral cortex damage and the percentage of hippocampal apoptotic neurons were evaluated by Nissl staining and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL assay as well as flow cytometry, respectively. Immunofluorescence and western blotting were performed to determine aquaporin-4 (AQP4 expression. In rats treated with severe hypoxia (PaO2 50 mmHg, hypercapnia protected against these pathophysiological changes. Moreover, hypercapnia treatment significantly reduced brain damage in the ischemic ipsilateral cortex and decreased the percentage of apoptotic neurons in the hippocampus after the CCA ligated rats were exposed to mild or moderate hypoxemia (PaO2 > 50 mmHg; especially under mild hypoxemia (PaO2 > 60 mmHg, hypercapnia significantly attenuated the expression of AQP4 protein with brain edema (p < 0.05. Hypercapnia exerts beneficial effects under mild to moderate hypoxemia and augments detrimental effects under severe hypoxemia on brain damage in a rat model of hypoxia-ischemia.
-
Zhou, Tian; Dong, Qinglei; Shen, Yang; Wu, Wei; Wu, Haide; Luo, Xianglin; Liao, Xiaoling; Wang, Guixue
Micro/nanoparticles could cause adverse effects on cardiovascular system and increase the risk for cardiovascular disease-related events. Nanoparticles prepared from poly(ethylene glycol) (PEG)- b -poly( ε -caprolactone) (PCL), namely PEG- b -PCL, a widely studied biodegradable copolymer, are promising carriers for the drug delivery systems. However, it is unknown whether polymeric PEG- b -PCL nano-micelles give rise to potential complications of the cardiovascular system. Zebrafish were used as an in vivo model to evaluate the effects of PEG- b -PCL nano-micelle on cardiovascular development. The results showed that PEG- b -PCL nano-micelle caused embryo mortality as well as embryonic and larval malformations in a dose-dependent manner. To determine PEG- b -PCL nano-micelle effects on embryonic angiogenesis, a critical process in zebrafish cardiovascular development, growth of intersegmental vessels (ISVs) and caudal vessels (CVs) in flk1-GFP transgenic zebrafish embryos using fluorescent stereomicroscopy were examined. The expression of fetal liver kinase 1 (flk1), an angiogenic factor, by real-time quantitative polymerase chain reaction (qPCR) and in situ whole-mount hybridization were also analyzed. PEG- b -PCL nano-micelle decreased growth of ISVs and CVs, as well as reduced flk1 expression in a concentration-dependent manner. Parallel to the inhibitory effects on angiogenesis, PEG- b -PCL nano-micelle exposure upregulated p53 pro-apoptotic pathway and induced cellular apoptosis in angiogenic regions by qPCR and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay. This study further showed that inhibiting p53 activity, either by pharmacological inhibitor or RNA interference, could abrogate the apoptosis and angiogenic defects caused by PEG- b -PCL nano-micelles, indicating that PEG- b -PCL nano-micelle inhibits angiogenesis by activating p53-mediated apoptosis. This study indicates that polymeric PEG- b -PCL nano-micelle could
-
Directory of Open Access Journals (Sweden)
Sayo Koike
2016-09-01
Full Text Available Vascular calcification, especially medial artery calcification, is associated with cardiovascular death in patients with diabetes mellitus and chronic kidney disease (CKD. To determine the underlying mechanism of vascular calcification, we have demonstrated in our previous report that advanced glycation end-products (AGEs stimulated calcium deposition in vascular smooth muscle cells (VSMCs through excessive oxidative stress and phenotypic transition into osteoblastic cells. Since AGEs can induce apoptosis, in this study we investigated its role on VSMC apoptosis, focusing mainly on the underlying mechanisms. A rat VSMC line (A7r5 was cultured, and treated with glycolaldehyde-derived AGE-bovine serum albumin (AGE3-BSA. Apoptotic cells were identified by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL staining. To quantify apoptosis, an enzyme-linked immunosorbent assay (ELISA for histone-complexed DNA fragments was employed. Real-time PCR was performed to determine the mRNA levels. Treatment of A7r5 cells with AGE3-BSA from 100 µg/mL concentration markedly increased apoptosis, which was suppressed by Nox inhibitors. AGE3-BSA significantly increased the mRNA expression of NAD(PH oxidase components including Nox4 and p22phox, and these findings were confirmed by protein levels using immunofluorescence. Dihydroethidisum assay showed that compared with cBSA, AGE3-BSA increased reactive oxygen species level in A7r5 cells. Furthermore, AGE3-induced apoptosis was significantly inhibited by siRNA-mediated knockdown of Nox4 or p22phox. Double knockdown of Nox4 and p22phox showed a similar inhibitory effect on apoptosis as single gene silencing. Thus, our results demonstrated that NAD(PH oxidase-derived oxidative stress are involved in AGEs-induced apoptosis of VSMCs. These findings might be important to understand the pathogenesis of vascular calcification in diabetes and CKD.
-
The 1-Tosylpentan-3-one Protects against 6-Hydroxydopamine-Induced Neurotoxicity
Directory of Open Access Journals (Sweden)
Chien-Jen Kao
2017-05-01
Full Text Available Previous studies have demonstrated that the marine compound austrasulfone, isolated from the soft coral Cladiella australis, exerts a neuroprotective effect. The intermediate product in the synthesis of austrasulfone, dihydroaustrasulfone alcohol, attenuates several inflammatory responses. The present study uses in vitro and in vivo methods to investigate the neuroprotective effect of dihydroaustrasulfone alcohol-modified 1-tosylpentan-3-one (1T3O. Results from in vitro experiments show that 1T3O effectively inhibits 6-hydroxydopamine-induced (6-OHDA-induced activation of both p38 mitogen-activated protein kinase (MAPK and caspase-3 in SH-SY5Y cells; and enhances nuclear factor erythroid 2–related factor 2 (Nrf2 and heme oxygenase-1 (HO-1 expression via phosphoinositide 3-kinase (PI3K/protein kinase B (Akt signaling. Hoechst staining and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL staining results reveal that 1T3O significantly inhibits 6-OHDA-induced apoptosis. In addition, the addition of an Akt or HO-1 inhibitor decreases the protective effect of 1T3O. Thus, we hypothesize that the anti-apoptotic activity of 1T3O in neuronal cells is mediated through the regulation of the Akt and HO-1 signaling pathways. In vivo experiments show that 1T3O can reverse 6-OHDA-induced reduction in locomotor behavior ability in zebrafish larvae, and inhibit 6-OHDA-induced tumor necrosis factor-alpha (TNF-α increase at the same time. According to our in vitro and in vivo results, we consider that 1T3O exerts its anti-apoptotic activities at SH-SY5Y cells after 6-OHDA challenges, probably via the regulation of anti-oxidative signaling pathways. Therefore, this compound may be a promising therapeutic agent for neurodegenerations.
-
Chen, Lili; He, Zhengxiang; Qin, Li; Li, Qinyan; Shi, Xibao; Zhao, Siting; Chen, Ling; Zhong, Nanshan; Chen, Xiaoping
2011-01-01
Lung cancer is the most common malignancy in humans and its high fatality means that no effective treatment is available. Developing new therapeutic strategies for lung cancer is urgently needed. Malaria has been reported to stimulate host immune responses, which are believed to be efficacious for combating some clinical cancers. This study is aimed to provide evidence that malaria parasite infection is therapeutic for lung cancer. Antitumor effect of malaria infection was examined in both subcutaneously and intravenously implanted murine Lewis lung cancer (LLC) model. The results showed that malaria infection inhibited LLC growth and metastasis and prolonged the survival of tumor-bearing mice. Histological analysis of tumors from mice infected with malaria revealed that angiogenesis was inhibited, which correlated with increased terminal deoxynucleotidyl transferase-mediated (TUNEL) staining and decreased Ki-67 expression in tumors. Through natural killer (NK) cell cytotoxicity activity, cytokine assays, enzyme-linked immunospot assay, lymphocyte proliferation, and flow cytometry, we demonstrated that malaria infection provided anti-tumor effects by inducing both a potent anti-tumor innate immune response, including the secretion of IFN-γ and TNF-α and the activation of NK cells as well as adaptive anti-tumor immunity with increasing tumor-specific T-cell proliferation and cytolytic activity of CD8(+) T cells. Notably, tumor-bearing mice infected with the parasite developed long-lasting and effective tumor-specific immunity. Consequently, we found that malaria parasite infection could enhance the immune response of lung cancer DNA vaccine pcDNA3.1-hMUC1 and the combination produced a synergistic antitumor effect. Malaria infection significantly suppresses LLC growth via induction of innate and adaptive antitumor responses in a mouse model. These data suggest that the malaria parasite may provide a novel strategy or therapeutic vaccine vector for anti-lung cancer
-
Directory of Open Access Journals (Sweden)
Lili Chen
Full Text Available BACKGROUND: Lung cancer is the most common malignancy in humans and its high fatality means that no effective treatment is available. Developing new therapeutic strategies for lung cancer is urgently needed. Malaria has been reported to stimulate host immune responses, which are believed to be efficacious for combating some clinical cancers. This study is aimed to provide evidence that malaria parasite infection is therapeutic for lung cancer. METHODOLOGY/PRINCIPAL FINDINGS: Antitumor effect of malaria infection was examined in both subcutaneously and intravenously implanted murine Lewis lung cancer (LLC model. The results showed that malaria infection inhibited LLC growth and metastasis and prolonged the survival of tumor-bearing mice. Histological analysis of tumors from mice infected with malaria revealed that angiogenesis was inhibited, which correlated with increased terminal deoxynucleotidyl transferase-mediated (TUNEL staining and decreased Ki-67 expression in tumors. Through natural killer (NK cell cytotoxicity activity, cytokine assays, enzyme-linked immunospot assay, lymphocyte proliferation, and flow cytometry, we demonstrated that malaria infection provided anti-tumor effects by inducing both a potent anti-tumor innate immune response, including the secretion of IFN-γ and TNF-α and the activation of NK cells as well as adaptive anti-tumor immunity with increasing tumor-specific T-cell proliferation and cytolytic activity of CD8(+ T cells. Notably, tumor-bearing mice infected with the parasite developed long-lasting and effective tumor-specific immunity. Consequently, we found that malaria parasite infection could enhance the immune response of lung cancer DNA vaccine pcDNA3.1-hMUC1 and the combination produced a synergistic antitumor effect. CONCLUSIONS/SIGNIFICANCE: Malaria infection significantly suppresses LLC growth via induction of innate and adaptive antitumor responses in a mouse model. These data suggest that the malaria
-
Directory of Open Access Journals (Sweden)
Zhao Ying-Zheng
2010-11-01
Full Text Available Abstract Background Oridonin, a tetracycline diterpenoid compound, has the potential antitumor activities. Here, we evaluate the antitumor activity and action mechanisms of oridonin in colorectal cancer. Methods Effects of oridonin on cell proliferation were determined by using a CCK-8 Kit. Cell cycle distribution was determined by flow cytometry. Apoptosis was examined by analyzing subdiploid population and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Senescent cells were determined by senescence-associated β-galactosidase activity analysis. Semi-quantitative RT-PCR was used to examine the changes of mRNA of p16, p21, p27 and c-myc. The concomitant changes of protein expression were analyzed with Western blot. Expression of AcH3 and AcH4 were examined by immunofluorescence staining and Western blots. Effects of oridonin on colony formation of SW1116 were examined by Soft Agar assay. The in vivo efficacy of oridonin was detected using a xenograft colorectal cancer model in nude mice. Results Oridonin induced potent growth inhibition, cell cycle arrest, apoptosis, senescence and colony-forming inhibition in three colorectal cancer cell lines in a dose-dependent manner in vitro. Daily i.p. injection of oridonin (6.25, 12.5 or 25 mg/kg for 28 days significantly inhibited the growth of SW1116 s.c. xenografts in BABL/C nude mice. With western blot and reverse transcription-PCR, we further showed that the antitumor activities of oridonin correlated with induction of histone (H3 and H4 hyperacetylation, activation of p21, p27 and p16, and suppression of c-myc expression. Conclusion Oridonin possesses potent in vitro and in vivo anti-colorectal cancer activities that correlated with induction of histone hyperacetylation and regulation of pathways critical for maintaining growth inhibition and cell cycle arrest. Therefore, oridonin may represent a novel therapeutic option in colorectal cancer treatment.
-
Isoflurane produces sustained cardiac protection after ischemia-reperfusion injury in mice.
Tsutsumi, Yasuo M; Patel, Hemal H; Lai, N Chin; Takahashi, Toshiyuki; Head, Brian P; Roth, David M
2006-03-01
Isoflurane reduces myocardial ischemia-reperfusion injury within hours to days of reperfusion. Whether isoflurane produces sustained cardiac protection has never been examined. The authors studied isoflurane-induced cardiac protection in the intact mouse after 2 h and 2 weeks of reperfusion and determined the dependence of this protection on adenosine triphosphate-dependent potassium channels and the relevance of this protection to myocardial function and apoptosis. Mice were randomly assigned to receive oxygen or isoflurane for 30 min with 15 min of washout. Some mice received mitochondrial (5-hydroxydecanoic acid) or sarcolemmal (HMR-1098) adenosine triphosphate-dependent potassium channel blockers with or without isoflurane. Mice were then subjected to a 30-min coronary artery occlusion followed by 2 h or 2 weeks of reperfusion. Infarct size was determined at 2 h and 2 weeks of reperfusion. Cardiac function and apoptosis were determined 2 weeks after reperfusion. Isoflurane did not change hemodynamics. Isoflurane reduced infarct size after reperfusion when compared with the control groups (27.7 +/- 6.3 vs. 41.7 +/- 6.4% at 2 h and 19.6 +/- 5.9 vs. 28.8 +/- 9.0% at 2 weeks). Previous administration of 5-hydroxydecanoic acid, but not HMR-1098, abolished isoflurane-induced cardiac protection. At 2 weeks, left ventricular end-diastolic diameter was decreased significantly and end-systolic pressure and maximum and minimum dP/dt were improved by isoflurane. Isoflurane-treated mice subjected to ischemia and 2 weeks of reperfusion showed less expression of proapoptotic genes, significantly decreased expression of cleaved caspase-3, and significantly decreased deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling-positive nuclei compared with the control group. Cardiac protection induced by isoflurane against necrotic and apoptotic cell death is associated with an acute memory period that is sustained and functionally relevant 2 weeks after
-
International Nuclear Information System (INIS)
Saradha, B.; Vaithinathan, S.; Mathur, P.P.
2009-01-01
Lindane, an organochlorine pesticide, is known to impair testicular functions and fertility. To elucidate the mechanism(s) underpinning the gonadal effects of lindane, we sought to investigate the levels of apoptosis-related proteins, namely cytochrome c, caspase-3 and-9, Fas and FasL in the testis of adult rats. Furthermore, the study aims to delineate whether nuclear factor kappa B (NF-κB) is involved in meditating the testicular effects of lindane. Animals were administered with a single dose of lindane (5 mg/kg body weight) and sacrificed at specific post-treatment intervals (0, 3, 6, 12, 24 and 72 h). Significant elevations in the levels of cytosolic cytochrome c with a parallel increase in pro-caspase-9 were observed as early as 6 h following exposure. Time-dependent elevations in the levels of Fas, FasL and caspase-3 were observed. Immunofluorescence studies revealed increased colocalization of Fas and caspase-3 in peritubular germ cells. FasL levels were increased in Sertoli and peritubular germ cells. The cytoplasmic levels of NF-κB p65 decreased from 3 h following exposure with a maximal decline at 12 and 24 h. Changes in the localization of NF-κB were observed with maximal nuclear translocation in germ cells at 12 and 24 h. Terminal deoxynucleotidyl transferase-mediated dUTP nickend-labeling (TUNEL) assay revealed a time-dependent increase in the number of apoptotic cells. Taken together, the data illustrate induction of testicular apoptosis in adult rats following exposure to a single dose of lindane. Early activation of NF-κB in contrast to late increase in Fas expression suggests a pro-apoptotic role of NF-κB in testicular response to lindane
-
Human lactoferrin stimulates skin keratinocyte function and wound re-epithelialization.
Tang, L; Wu, J J; Ma, Q; Cui, T; Andreopoulos, F M; Gil, J; Valdes, J; Davis, S C; Li, J
2010-07-01
Human lactoferrin (hLF), a member of the transferrin family, is known for its antimicrobial and anti-inflammatory effects. Recent studies on various nonskin cell lines indicate that hLF may have a stimulatory effect on cell proliferation. To study the potential role of hLF in wound re-epithelialization. The effects of hLF on cell growth, migration, attachment and survival were assessed, with a rice-derived recombinant hLF (holo-rhLF), using proliferation analysis, scratch migration assay, calcein-AM/propidium iodide staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) method, respectively. The mechanisms of hLF on cell proliferation and migration were explored using specific pathway inhibitors. The involvement of lactoferrin receptor low-density lipoprotein receptor-related protein 1 (LRP1) was examined with RNA interference technique. An in vivo swine second-degree burn wound model was also used to assess wound re-epithelialization. Studies revealed that holo-rhLF significantly stimulated keratinocyte proliferation which could be blocked by mitogen-activated protein kinase (MAPK) kinase 1 inhibitor. Holo-rhLF also showed strong promoting effects on keratinocyte migration, which could be blocked by either inhibition of the MAPK, Src and Rho/ROCK pathways, or downregulation of the LRP1 receptor. With cells under starving or 12-O-tetradecanoylphorbol-13-acetate exposure, the addition of holo-rhLF was found greatly to increase cell viability and inhibit cell apoptosis. Additionally, holo-rhLF significantly increased the rate of wound re-epithelialization in swine second-degree burn wounds. Our studies demonstrate the direct effects of holo-rhLF on wound re-epithelialization including the enhancement of keratinocyte proliferation and migration as well as the protection of cells from apoptosis. The data strongly indicate its potential therapeutic applications in wound healing.
-
Li, Huixian; Dong, Xiuhua; Jin, Mu; Cheng, Weiping
2018-01-18
Delayed paraplegia due to spinal cord ischemia/reperfusion injury (IRI) remains one of the most severe complications of thoracoabdominal aneurysm surgery, for which effective prevention and treatment is still lacking. The current study investigates whether spinal cord stimulation (SCS) postconditioning has neuroprotective effects against spinal cord IRI. Ninety-six New Zealand white male rabbits were randomly divided into four groups as follows: a sham group and three experimental groups (C group, 2 Hz group, and 50 Hz group) n = 24/group. Spinal cord ischemia was induced by transient infrarenal aortic balloon occlusion for 28 min, after which rabbits in group C underwent no additional intervention, while rabbits in the other two experimental groups underwent 2 Hz or 50 Hz epidural SCS for 30 min at the onset of reperfusion and then daily until sacrifice. Hind limb neurologic function of rabbits was assessed using Jacob scale. Lumbar spinal cords were harvested immediately after sacrifice for histological examination and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The number of viable α-motor neurons in ventral horn was counted and TUNEL-positive rate of α-motor neurons was calculated. Spinal cord IRI was caused by transient infrarenal aorta occlusion for 28 min. Both 2 Hz and 50 Hz SCS postconditioning had neuroprotective effects, particularly the 2 Hz SCS postconditioning. Comparing to C group and 50 Hz group, rabbits in the 2 Hz group demonstrated better hind limb motor function and a lower rate of TUNEL-positive α-motor neuron after eight hours, one day, three days, and seven days of spinal cord reperfusion. More viable α-motor neurons were preserved after one and three days of spinal cord reperfusion in 2 Hz group rabbits than in C group and 50 Hz group rabbits. SCS postconditioning at 2 Hz protected the spinal cord from IRI. © 2018 International Neuromodulation Society.
-
Li, Long-Gang; Xu, Hui-Mian
2005-01-01
AIM: To detect the presence of inducible nitric oxide synthase (iNOS), nitrotyrosine (NT) and apoptosis in gastric adenocarcinomas and their possible correlations with the clinicopathological characteristics and prognosis of gastric adenocarcinoma. METHODS: Sixty-six specimens of gastric adenocarcinoma and corresponding adjacent normal gastric tissues were studied. Immunohistochemistry was employed to localize iNOS and NT protein and an immunohistochemical scoring system was used. The occurrence of apoptotic cell death (apoptotic index [AI]) was analyzed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) method. RESULTS: Results showed that iNOS expression was detected at an intermediate or high level in 41 of 66 (62%) specimens of gastric adenocarcinoma. NT expression was 58%. Neither of them was found in the normal gastric tissues; there were significant positive correlations among iNOS expression, NT expression and AI. Many clinicopathologic characteristics of gastric adenocarcinoma, such as tumor size, depth of invasion, lymph node metastasis and TNM staging, were related to iNOS and NT expressions (P<0.05). In 66 surviving patients, the 5-year survival rate of 41 patients who had tumors with intermediate or high iNOS expressions and high AIs (4.09%; 19.96%) was significantly lower than that of 25 patients who had tumors with negative or low iNOS expressions and low AIs (0.79%; 47.14%) (P = 0.001). COX’s multivariate analysis revealed that the iNOS expression was identified as one of the significant independent prognostic factors predictive of a poor survival (relative risk [RR] = 2.69). CONCLUSION: NO produced by iNOS may play a stronger role in promoting gastric adenocarcinoma growth than in suppressing its growth. iNOS and NT expressions by gastric adenocarcinoma may correlate with a poor survival. PMID:15849807
-
Effects of ghrelin on the apoptosis of human neutrophils in vitro
Li, Bin; Zeng, Mian; Zheng, Haichong; Huang, Chunrong; He, Wanmei; Lu, Guifang; Li, Xia; Chen, Yanzhu; Xie, Ruijie
2016-01-01
Acute respiratory distress syndrome (ARDS) is characterized by lung inflammation and the diffuse infiltration of neutrophils into the alveolar space. Neutrophils are abundant, short-lived leukocytes that play a key role in immune defense against microbial infections. These cells die via apoptosis following the activation and uptake of microbes, and will also enter apoptosis spontaneously at the end of their lifespan if they do not encounter pathogens. Apoptosis is essential for the removal of neutrophils from inflamed tissues and for the timely resolution of neutrophilic inflammation. Ghrelin is an endogenous ligand for the growth hormone (GH) secretagogue receptor, produced and secreted mainly from the stomach. Previous studies have reported that ghrelin exerts anti-inflammatory effects in lung injury through the regulation of the apoptosis of different cell types; however, the ability of ghrelin to regulate alveolar neutrophil apoptosis remains largely undefined. We hypothesized that ghrelin may have the ability to modulate neutrophil apoptosis. In this study, to examine this hypothesis, we investigated the effects of ghrelin on freshly isolated neutrophils in vitro. Our findings demonstrated a decrease in the apoptotic ratio (as shown by flow cytometry), as well as in the percentage of cells with decreased mitochondrial membrane potential (ΔΨm) and in the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling-positive rate, accompanied by an increased B-cell lymphoma 2/Bax ratio and the downregulation of cleaved caspase-3 in neutrophils following exposure to lipopolysaccharide (100 ng/ml). However, pre-treatment with ghrelin at a physiological level (100 nM) did not have a notable influence on the neutrophils in all the aforementioned tests. Our findings suggest that ghrelin may not possess the ability to modulate the neutrophil lifespan in vitro. PMID:27431014
-
Flores-Rentería, Lluvia; Orozco-Arroyo, Gregorio; Cruz-García, Felipe; García-Campusano, Florencia; Alfaro, Isabel; Vázquez-Santana, Sonia
2013-09-01
The sexual separation in dioecious species has interested biologists for decades; however, the cellular mechanism leading to unisexuality has been poorly understood. In this study, the cellular changes that lead to male sterility in the functionally dioecious cactus, Opuntia stenopetala, are described. The spatial and temporal patterns of programmed cell death (PCD) were determined in the anthers of male and female flowers using scanning electron microscopy analysis and histological observations, focusing attention on the transition from bisexual to unisexual development. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assays were used as an indicator of DNA fragmentation to corroborate PCD. PCD was detected in anthers of both female and male flowers, but their patterns differed in time and space. Functionally male individuals developed viable pollen, and normal development involved PCD on each layer of the anther wall, which occurred progressively from the inner (tapetum) to the outer layer (epidermis). Conversely, functional female individuals aborted anthers by premature and displaced PCD. In anthers of female flowers, the first signs of PCD, such as a nucleus with irregular shape, fragmented and condensed chromatin, high vacuolization and condensed cytoplasm, occurred at the microspore mother cell stage. Later these features were observed simultaneously in all anther wall layers, connective tissue and filament. Neither pollen formation nor anther dehiscence was detected in female flowers of O. stenopetala due to total anther disruption. Temporal and spatial changes in the patterns of PCD are responsible for male sterility of female flowers in O. stenopetala. Male fertility requires the co-ordination of different events, which, when altered, can lead to male sterility and to functionally unisexual individuals. PCD could be a widespread mechanism in the determination of functionally dioecious species.