The role of c-FLIP splice variants in urothelial tumours

Deregulation of apoptosis is common in cancer and is often caused by overexpression of anti-apoptotic proteins in tumour cells. One important regulator of apoptosis is the cellular FLICE-inhibitory protein (c-FLIP), which is overexpressed, for example, in melanoma and Hodgkin's lymphoma cells. Here, we addressed the question whether deregulated c-FLIP expression in urothelial carcinoma impinges on the ability of death ligands to induce apoptosis. In particular, we investigated the role of the c-FLIP splice variants c-FLIPlong (c-FLIPL) and c-FLIPshort (c-FLIPS), which can have opposing functions. We observed diminished expression of the c-FLIPL isoform in urothelial carcinoma tissues as well as in established carcinoma cell lines compared with normal urothelial tissues and cells, whereas c-FLIPS was unchanged. Overexpression and RNA interference studies in urothelial cell lines nevertheless demonstrated that c-FLIP remained a crucial factor conferring resistance towards induction of apoptosis by death ligands CD95L and TRAIL. Isoform-specific RNA interference showed c-FLIPL to be of particular importance. Thus, urothelial carcinoma cells appear to fine-tune c-FLIP expression to a level sufficient for protection against activation of apoptosis by the extrinsic pathway. Therefore, targeting c-FLIP, and especially the c-FLIPL isoform, may facilitate apoptosis-based therapies of bladder cancer in otherwise resistant tumours

Evaluation of Brain Nuclear Medicine Imaging Tracers in a Murine Model of Sepsis-Associated Encephalopathy

PURPOSE: The purpose of this study was to evaluate a set of widely used nuclear medicine imaging agents as possible methods to study the early effects of systemic inflammation on the living brain in a mouse model of sepsis-associated encephalopathy (SAE). The lipopolysaccharide (LPS)-induced murine systemic inflammation model was selected as a model of SAE. PROCEDURES: C57BL/6 mice were used. A multimodal imaging protocol was carried out on each animal 4 h following the intravenous administration of LPS using the following tracers: [(99m)Tc][2,2-dimethyl-3-[(3E)-3-oxidoiminobutan-2-yl]azanidylpropyl]-[(3E)-3-hyd roxyiminobutan-2-yl]azanide ([(99m)Tc]HMPAO) and ethyl-7-[(125)I]iodo-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carbox ylate ([(125)I]iomazenil) to measure brain perfusion and neuronal damage, respectively; 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) to measure cerebral glucose uptake. We assessed microglia activity on another group of mice using 2-[6-chloro-2-(4-[(125)I]iodophenyl)-imidazo[1,2-a]pyridin-3-yl]-N-ethyl-N-methyl -acetamide ([(125)I]CLINME). Radiotracer uptakes were measured in different brain regions and correlated. Microglia activity was also assessed using immunohistochemistry. Brain glutathione levels were measured to investigate oxidative stress. RESULTS: Significantly reduced perfusion values and significantly enhanced [(18)F]FDG and [(125)I]CLINME uptake was measured in the LPS-treated group. Following perfusion compensation, enhanced [(125)I]iomazenil uptake was measured in the LPS-treated group's hippocampus and cerebellum. In this group, both [(18)F]FDG and [(125)I]iomazenil uptake showed highly negative correlation to perfusion measured with ([(99m)Tc]HMPAO uptake in all brain regions. No significant differences were detected in brain glutathione levels between the groups. The CD45 and P2Y12 double-labeling immunohistochemistry showed widespread microglia activation in the LPS-treated group. CONCLUSIONS: Our results suggest that [(125)I]CLINME and [(99m)Tc]HMPAO SPECT can be used to detect microglia activation and brain hypoperfusion, respectively, in the early phase (4 h post injection) of systemic inflammation. We suspect that the enhancement of [(18)F]FDG and [(125)I]iomazenil uptake in the LPS-treated group does not necessarily reflect neural hypermetabolism and the lack of neuronal damage. They are most likely caused by processes emerging during neuroinflammation, e.g., microglia activation and/or immune cell infiltration

Purification and characterization of a microbial dehydrogenase - A vanillin : NAD(P)(+) oxidoreductase

Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native M, value estimated by gel-filtration chromatography gave a value of 210,000. The enzyme made use of NAD(+) less effectively than NADP(+). Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD(+) or NADP(+). The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl2. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD(+) and NADP(+) protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD(+) or NADP(+). We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases