Non-Selective Calcium Channel Blocker Bepridil Decreases Secondary Pathology in Mice after Photothrombotic Cortical Lesion

Experimental studies have identified a complex link between neurodegeneration, β-amyloid (Aβ) and calcium homeostasis. Here we asked whether early phase β-amyloid pathology in transgenic hAPP_SL mice exaggerates the ischemic lesion and remote secondary pathology in the thalamus, and whether a non-selective calcium channel blocker reduces these pathologies. Transgenic hAPP_SL (n = 33) and non-transgenic (n = 30) male mice (4–5 months) were subjected to unilateral cortical photothrombosis and treated with the non-selective calcium channel blocker bepridil (50 mg/kg, p.o., once a day) or vehicle for 28 days, starting administration 2 days after the operation. Animals were then perfused for histological analysis of infarct size, Aβ and calcium accumulation in the thalamus. Cortical photothrombosis resulted in a small infarct, which was associated with atypical Aβ and calcium accumulation in the ipsilateral thalamus. Transgenic mice had significantly smaller infarct volumes than non-transgenic littermates (P<0.05) and ischemia-induced rodent Aβ accumulation in the thalamus was lower in transgenic mice compared to non-transgenic mice (P<0.01). Bepridil decreased calcium load in the thalamus (P<0.01). The present data suggest less pronounced primary and secondary pathology in hAPP_SL transgenic mice after ischemic cortical injury. Bepridil particularly decreased calcium pathology in the thalamus following ischemia.     

Poly-N-Acetyllactosamine Glycans of Cellular Glycoproteins: Predominance of Linear Chains in Mouse Neuroblastoma and Rat Pheochromocytoma Cell Lines

To study the properties of protein-bound oligosaccharides in neuronally differentiating cells, two model systems were used: murine N1E-115 and N-18 neuroblastoma cells inducible by serum starvation and rat PC12 pheochromocytoma cells inducible by nerve growth factor. Glycopeptides were prepared from cells metabolically labeled with [3H]glucosamine and analyzed by gel filtration. The properties of the high-molecular-weight glycopeptides were studied using enzymatic digestion with neuraminidase and endo-beta-galactosidase. In contrast to other cell lines analyzed, the neuroblastoma and pheochromocytoma lines contained predominantly glycopeptides completely cleavable with endo-beta-galactosidase, which indicated that they were linear-type poly-N-acetyllactosamine glycans. The proportion of these linear chains in the high-molecular-weight fraction increased during neuronal differentiation in both cell systems. The linear nature of the glycans was also correlated with positive anti-i and negative anti-I reactivity of the cells in immunofluorescence microscopy. Specific cell surface labeling for poly-N-acetyllactosamine glycans and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed several glycoprotein components, some of which showed changes during neuronal differentiation. The high proportion of linear poly-N-acetyllactosamine chains in these neuronal cell lines and its increase during neuronal differentiation suggests that these glycans may be a characteristic feature of neuronal or neuronally differentiating cells.     

Comparative assessment of inorganic pyrophosphate and pyrophosphatase levels of Escherichia coli, Clostridium pasteurianum, and Clostridium thermoaceticum

Abstract Pyrophosphatase (PP_iase) specific activities were much higher in anaerobic cultures of Escherichia coli (0.54 units) than in Clostridium pasteurianum (0.067 units) and Clostridium thermoaceticum (0.017 units) (1 unit = 1 μ mole PP_i hydrolyzed/min per mg cell dry wt.), and were fairly constant throughout the growth of all three organisms. Conversely, intracellular levels of pyrophosphate (PP_i) were very low and constant in E. coli throughout growth (0.3 mM), while those of C. pasteurianum and C. thermoaceticum were higher (1.44 and 0.8 mM, respectively) and peaked sharply during mid log-phase of growth. PP_iase and intracellular PP_i remained relatively constant in E. coli when grown aerobically or anaerobically, and when growth was in medium containing PP_i as the sole source of supplemental phosphorus.     

Affinity purification of bacterial luciferase and NAD(P)H:FMN oxidoreductases by FMN-sepharose for analytical applications

A modified purification method for bacterial luciferases and NAD(P)H:FMN oxidoreductases is described which uses FMN-Sepharose alone or coupled to DEAE ion exchange chromatography for the simultaneous purification of luciferase and the various oxidoreductases from Vibrio harveyi, a bright mutant of Vibrio harveyi, Vibrio fischeri, and Photobacterium phosphoreum. This purification method is compared with DEAE-Sepharose CI 6B fractionations from these organisms. Both methods allow the separation of oxidoreductases specific for either NADH or NADPH. The use of FMN-Sepharose coupled to DEAE-Sepharose fractionation allows the isolation of highly purified enzymes. Lacking interfering factors, these are very suitable for various analytical applications based on bacterial bioluminescence enzymes. The partially purified enzymes from the affinity column have higher specific activities than those obtained using DEAE-Sepharose.     

FGF-2 Inhibits Apoptosis in Human Teratocarcinoma Cells during Differentiation on Collagen Substratum

Tera-2 is a human teratocarcinoma cell line, which is induced to differentiate into neuronal direction by retinoic acid. Once differentiated, the cells form an almost nondividing population that can be maintained for weeks under conventional culture conditions. If differentiation by retinoic acid is induced while the cells are growing on type I collagen or if the already-differentiated cells are transferred onto collagen, they survive only a few days unless the cultures are repeatedly supplied with FGF-2. Lack of this growth factor induces programmed cell death (apoptosis) detectable after 24–48 h, as marked by DNA cleavage and nuclear fragmentation. The undifferentiated stem cells survive and proliferate readily on collagen without addition of FGF-2. Tera-2 cells express two members of the FGF family, FGF-2 and FGF-4. The expression of both FGFs is turned off during differentiation on collagen substratum, whereas when cultivated on plain tissue culture dish, the expression of only FGF-4 becomes undetectable. The results indicate that signaling through cell surface FGF receptors is vital for the cells, and differentiation on collagen substratum results in complete extinction of the autocrine stimulatory loop.In vivo,such induction of growth factor dependency upon differentiation would result in apoptotic death of those cells which fail to find adequate conditions for continuing FGF stimulation.     

An approach to mapping haplotype-specific recombination sites in human MHC class III

Studies of the major histocompatibility complex (MHC) in mouse indicate that the recombination sites are not randomly distributed and their occurrence is haplotype-dependent. No data concerning haplotype-specific recombination sites in human are available due to the low number of informative families. To investigate haplotype-specific recombination sites in human MHC, we here describe an approach based on identification of recombinant haplotypes derived from one conserved haplotype at the population level. The recombination sites were mapped by comparing polymorphic markers between the recombinant and assumed original haplotypes. We tested this approach on the extended haplotype HLA A3; B47; Bf ^* F; C4A ^* 1; C4B ^* Q0; DR7, which is most suitable for this analysis. First, it carries a number of rare markers, and second, the haplotype, albeit rare in the general population, is frequent in patients with 21-hydroxylase (21OH) defect. We observed recombinants derived from this haplotype in patients with 21OH defect. All these haplotypes had the centromeric part (from Bf to DR) identical to the original haplotype, but they differed in HLA A and B. We therefore assumed that they underwent recombinations in the segment that separates the Bf and HLA B genes. Polymorphic markers indicated that all break points mapped to two segments near the TNF locus. This approach makes possible the mapping of preferential recombination sites in different haplotypes.     

Acetate Degradation at 70°C in Upflow Anaerobic Sludge Blanket Reactors and Temperature Response of Granules Grown at 70°C

Anaerobic acetate degradation at 70°C and at 55°C (as a reference) was studied by running laboratory upflow anaerobic sludge blanket (UASB) reactors inoculated with mesophilic granular sludge. In UASB reactors fed with acetate-containing media (3 g of chemical oxygen demand [COD] per liter, corresponding to 47 mM acetate) approximately 50 days was needed at 70°C and less than 15 days was needed at 55°C to achieve an effluent COD of 500 to 700 mg/liter. In the UASB reactors at both 70 and 55°C up to 90% of the COD was removed. Batch assays showed that sludges from two 70°C UASB reactors, one run at a low effluent acetate concentration and the other run at a high effluent acetate concentration, exhibited slightly different responses to temperatures in the range from 37 to 70°C. Both 70°C sludges, as well as the 55°C sludge, produced methane at temperatures of 37 to 73°C. The 55°C sludge exhibited shorter lag phases than the 70°C sludges and higher specific methane production rates between 37 and 65°C.