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[en] CD40L was found to reduce doxorubicin-induced apoptosis in non Hodgkin's lymphoma cell lines through caspase-3 dependent mechanism. Whether this represents a general mechanism for other tumor types is unknown. The resistance induced by CD40L against apoptosis induced by a panel of cytotoxic chemotherapeutic drugs in non Hodgkin's lymphoma and breast carcinoma cell lines was investigated. Doxorubicin, cisplatyl, etoposide, vinblastin and paclitaxel increased apoptosis in a dose-dependent manner in breast carcinoma as well as in non Hodgkin's lymphoma cell lines. Co-culture with irradiated L cells expressing CD40L significantly reduced the percentage of apoptotic cells in breast carcinoma and non Hodgkin's lymphoma cell lines treated with these drugs. In breast carcinoma cell lines, these 5 drugs induced an inconsistent increase of caspase-3/7 activity, while in non Hodgkin's lymphoma cell lines all 5 drugs increased caspase-3/7 activity up to 28-fold above baseline. Co-culture with CD40L L cells reduced (-39% to -89%) the activation of caspase-3/7 induced by these agents in all 5 non Hodgkin's lymphoma cell lines, but in none of the 2 breast carcinoma cell lines. Co culture with CD40L L cells also blocked the apoptosis induced by exogenous ceramides in breast carcinoma and non Hodgkin's lymphoma cell lines through a caspase-3-like, 8-like and 9-like dependent pathways. These results indicate that CD40L expressed on adjacent non tumoral cells induces multidrug resistance to cytotoxic agents and ceramides in both breast carcinoma and non Hodgkin's lymphoma cell lines, albeit through a caspase independent and dependent pathway respectively
[en] Campylobacter jejuni contains a post-translational N-glycosylation system in which a STT3 homologue, PglB, functions as the oligosaccharyltransferase. Herein, we established a method for obtaining relatively large quantities of homogenous PglB proteins. PglB was overexpressed in Escherichia coli C43(DE3) at a level of 1 mg/L cell cultures. The activity of purified PglB was verified using a chemically synthesized sugar donor: N-acetylgalactosamine-diphospho-undecaprenyl (GalNAc-PP-Und) and a synthesized peptide acceptor. The result confirms that PglB is solely responsible for the oligosaccharyltransferase activity and complements the finding that PglB exhibits relaxed sugar substrate specificity. In addition, we performed the topology mapping of PglB using the PhoA/LacZ fusion method. The topological model shows that PglB possesses 11 transmembrane segments and two relatively large periplasmic regions other than the C-terminal domain, which is consistent with the proposal of the common Ncyt-Cperi topology with 11 transmembrane segments for the STT3 family proteins.
[en] Protein A coated magnetic particles are used for the preparative purification of antibodies from up to 100 L cell culture supernatant. The comparison with conventional column and expanded bed chromatography results in similar yield and purity of the product but much faster separation times
[en] The radiation modifying effect and toxicity of tetraethylthiuram disulfide (disulfiram) have been studied. Disulfiram (DSM) inhibits aldehyde dehydrogenase, dopamine-beta-oxygenase, microsomal mixed-function oxidases and cytochrome P-450 enzymes. It is widely used for aversion therapy in alcoholism. Disulfiram also inhibits tumor formation by several known carcinogens. A biphasic toxicity pattern of DSM is reported in the L-929 mouse fibroblast culture system. Disulfiram is 100 percent toxic at 2 X 10(-7) M (0.05 micrograms per ml), 23 percent toxic at 3 X 10(-7) M (0.1 microgram per ml), and 100 percent toxic again at 3.4 X 10(-6) M (1.0 microgram per ml). The pattern is similar to the biphasic toxicity pattern of DMS's major metabolite, sodium diethyldithiocarbamate (DTC). Reports of both radiation protection and radiation enhancement by DTC exist. Previously, a radioprotective effect by 2 X 10(-6) M DTC (dose modifying factor = 1.26) has been demonstrated in the L-929 cell system. To date, no radiation modifying properties of DSM have been reported. Our investigation of DSM as a radiation modifier at 3 X 10(-7) M (0.1 microgram per ml) did not show significant improvement in survival of irradiated cells treated with DSM relative to the irradiated control group, as determined by absence of a difference in the Do of the two groups. Considering DSM's close structural relationship to DTC, it is possible that DSM may exhibit a radioprotective effect when applied in a different concentration than what was used in our research
[en] Mouse L-929 fibroblasts, an established line of cells, are very sensitive to lysis by human lymphotoxin (hTNF-beta). Specific binding of a highly purified preparation of hTNF-beta to these cells was examined. Recombinant DNA-derived hTNF-beta was radiolabeled with [3H]propionyl succinimidate at the lysine residues of the molecule to a specific activity of 200 microCi/nmol of protein. [3H]hTNF-beta was purified by high performance gel permeation chromatography and the major fraction was found to be monomeric by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeled hTNF-beta was fully active in causing lysis of L-929 fibroblasts and bound specifically to high affinity binding sites on these cells. Scatchard analysis of the binding data revealed the presence of a single class of high affinity receptors with an apparent Kd of 6.7 X 10(-11) M and a capacity of 3200 binding sites/cell. Unlabeled recombinant DNA-derived hTNF-beta was found to be approximately 5-fold more effective competitive inhibitor of binding than the natural hTNF-beta. The binding of hTNF-beta to these mouse fibroblasts was also correlated with the ultimate cell lysis. Neutralizing polyclonal antibodies to hTNF-beta efficiently inhibited the binding of [3H]hTNF-beta to the cells. The authors conclude that the specific high affinity binding site is the receptor for hTNF-beta and may be involved in lysis of cells