Stably Transfected Human Cells Overexpressing Rat Brain Endopeptidase 3.4.24.16: Biochemical Characterization of the Activity and Expression of Soluble and Membrane-Associated Counterparts

Abstract: We recently cloned endopeptidase-24.16 (neurolysin; EC 3.4.24.16), a neurotensin-degrading peptidase likely involved in the physiological termination of the neurotensinergic signal in the central nervous system and in the gastrointestinal tract. We stably transfected human kidney cells with the pcDNA3-λ7aB1 construction bearing the whole open reading frame encoding the rat brain peptidase. Transfectants displayed endopeptidase-24.16 immunoreactivity and exhibited QFS- and neurotensin-hydrolyzing activities, the biochemical and specificity properties of which fully matched those observed with the purified murine enzyme. Cryoprotection experiments and substrate degradation by intact plated cells indicated that transfectants exhibited a membrane-associated form of endopeptidase-24.16, the catalytic site of which clearly faced the extracellular domain. Transfected cells were unable to secrete the enzyme. Overall, our experiments indicate that we have obtained stably transfectant cells that overexpress an enzymatic activity displaying biochemical properties identical to those of purified endopeptidase-24.16. The membrane-associated counterpart and lack of secretion of the enzyme were clearly reminiscent of what was observed with pure cultured neurons, but not with astrocytes. Therefore, the transfected cell model described here could prove useful for establishing, by a mutagenesis approach, the structural elements responsible for the "neuronal" phenotype exhibited by the enzyme in transfected cells.     

α-Secretase-Derived Product of β-Amyloid Precursor Protein Is Decreased by Presenilin 1 Mutations Linked to Familial Alzheimer's Disease

Abstract: Recent reports indicate that missense mutations on presenilin (PS) 1 are likely responsible for the main early-onset familial forms of Alzheimer's disease (FAD). Consensual data obtained through distinct histopathological, cell biology, and molecular biology approaches have led to the conclusion that these PS1 mutations clearly trigger an increased production of the 42-amino-acid-long species of β-amyloid peptide (Aβ). Here we show that overexpression of wild-type PS1 in HK293 cells increases Aβ40 secretion. By contrast, FAD-linked mutants of PS1 trigger increased secretion of both Aβ40 and Aβ42 but clearly favor the production of the latter species. We also demonstrate that overexpression of the wild-type PS1 augments the α-secretase-derived C-terminally truncated fragment of β-amyloid precursor protein (APPα) recovery, whereas transfectants expressing mutated PS1 secrete drastically lower amounts of APPα when compared with cells expressing wild-type PS1. This decrease was also observed when comparing double transfectants overexpressing wild-type β-amyloid precursor protein and either PS1 or its mutated congener M146V-PS1. Altogether, our data indicate that PS mutations linked to FAD not only trigger an increased ratio of Aβ42 over total Aβ secretion but concomitantly down-regulate the production of APPα.     

Study of numerical aberrations of chromosome 1 by fluorescent in situ hybridization and DNA content by densitometric analysis on (pre)-malignant cervical lesions

Summary In an attempt to determine whether the fluorescent in situ hybridization (FISH) can be used as a rapid approach for the identification of aneuploidy in premalignant cervical smears, a centromeric probe for chromosome 1 was used. The results from the FISH experiments were compared with measurements of the overall DNA content obtained by means of an image analysis system. With progression to neoplasia, a decrease of the frequency of cells with two spots was observed, due to an increasing polysomy of chromosome 1. As far as the DNA content was concerned, an increasing DNA index and 5C-exceeding ratio (fraction of cells with a DNA content higher than 5C) was observed. Classification of the FISH results by a linear discriminant analysis revealed that 67.6% of the cases were classified in agreement with the CIN classification. These data suggest that chromosome 1 may be considered as a marker chromosome for pre-malignant cervical lesions and that the DNA content measurements are complementary to the FISH results.     

Organisation de la phase minérale chez Vaceletia crypta (Vacelet)Démosponge,Sphinctozoaire actuelle. Comparaison avec des formes aragonitiques du Trias de Turquie

The fundamental microstructural and ultrastructuralcaracteristics of a non fibrous carbonate tissue has been revealed by the study of the only living Sphinctozoa: Vaceletia crypta (Vacelet), Demospongia.These observations yield important data for interpretation of homogeneous carbonate structure in the skeleton of some fossil sponges. Effectively, we notice that two triassic forms, morphologically very different (Sphinctozoa and Inozoa) show remarkable microstructural analogies with Vaceletia crypta. These considerations should not be leaved out to establish an only classification of Sponges, joining both living and fossil forms.Moreover, the elementary skeletal constituants ofthe two forms show dimensions which indicate a growth in size in comparison with the living species. This increase has probably a diagenetic origin (adjunction of aragonite). So it appears that the present aspect of the mineral components cannot result from a reduction in size (micritization), as has been sometimes asserted.     

Nuclear magnetic resonance monitoring of sodium in biological tissues

In recent year, the 23Na nuclear magnetic resonance (NMR) technique has been applied to the study of biological tissues. The advantages of this method are noninvasiveness and good sensitivity. The resonances of the intra- and extra-cellular sodium can be separated by the addition of shift reagents to the extracellular compartment. The method has been mostly applied to cell suspensions, kidney tubules, glands, and small organs. Owing to line-broadening effects, the NMR visibility of the intracellular sodium is reduced to 40% in most cases but can be lower or higher. Time-dependent measurements are possible with adequate life-supporting equipment, allowing the determination of transport parameters. 23Na relaxation times are short in tissues (below 50 ms) and highly dependent on the medium composition. The application of the 23Na NMR technique to intact organs can be hampered by the difficulty of getting a good distribution of the shift reagent in the extracellular milieu. A summary of the studies performed is presented with specific examples to illustrate typical applications.     

The phenology of fine root growth in a maple-dominated ecosystem: relationships with some soil properties

A two-year study was undertaken in a maple-dominated watershed of southern Québec, Canada, to examine relationships between trends in fine root growth, stem diameter growth, soil moisture, soil temperature, mineralized-N and extractable-P. Until September, soil temperature was consistently higher in 1995 than in 1994. Apart from the first sampling in mid-May, soil moisture was higher in 1994 than in 1995. In 1994, most fine roots were produced before leaf expansion, whereas in 1995, fine root production peaked in July. Annual fine root production was estimated to be 2.7 times higher in 1994 than in 1995. Stem growth was strongly associated with the seasonal and annual variation in soil temperature. Root and diameter growth were asynchronous in 1994 but not in 1995. Fine root production was associated with two groups of variables: a soil fertility (mineralized-N and extractable-P) group and a physical soil environment (moisture and temperature) group. Our results are consistent with the negative effect of high soil-N fertility on fine root production but are inconclusive as to the positive effect of high soil-P fertility. Soil conditions that are detrimental to root growth such as high N availability and anaerobiosis could modify the normal dynamics of fine root growth.     

Isolation of Bacillus thuringiensis from Stored Tobacco and Lasioderma serricorne (F.)

Bacillus thuringiensis was isolated from dried tobacco residues and dead tobacco beetles (Lasioderma serricorne (F.); Coleoptera: Anobiidae) collected in a large number of locations worldwide. Eighty-eight samples of stored tobacco were analyzed and yielded 78 B. thuringiensis strains which were characterized on the basis of parasporal crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles, and the results of an immunoblot analysis of the insecticidal crystal proteins. Flagellar antigen identification was used to differentiate selected isolates. Strains that produced rhomboidal crystals associated with the Coleoptera-specific pathotype (Cry III group) were the most abundant strains (59% of the isolates). Preliminary toxicity assays were performed with L. serricorne larvae, and the results suggested that activity is not restricted to isolates related to the Coleoptera-specific group. The results of our survey indicate that B. thuringiensis is part of the natural microflora in the stored-tobacco environment and that this special habitat represents a source of B. thuringiensis isolates that may be used to control stored-product pests.     

The actin-based motility of the facultative intracellular pathogen Listeria monocytogenes

Summary
The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular parasite that invades and multiplies within diverse eukaryotic cell types. An essential pathogenicity determinant is its ability to move in the host cell cytoplasm and to spread within tissues by directly passing from one cell to another. The propulsive force for intracellular movement is thought to be generated by continuous actin assembly at the rear end of the bacterium. Moving bacteria that reach the plasma membrane induce the formation of long membranous protrusions that are internalized by neighbouring cells, thus mediating the spread of infection. The unrelated pathogens Shigella and Rickettsia use a similar process of actin-based motility to disseminate in infected tissues. This review focuses on the bacterial and cellular factors involved in the actin-based motility of L monocytogenes.     

Constitutive activation of the Saccharomyces cerevislae mating response pathway by a MAP kinase kinase from Candida albicans

The HST7 gene of Candida albicans encodes a protein with structural similarity to MAP kinase kinases. Expression of this gene in Saccharomyces cerevisiae complements disruption of the Ste7 MAP kinase kinase required for both mating in haploid cells and pseudohyphal growth in diploids. However, Hst7 expression does not complement loss of either the Pbs2 (Hog4) MAP kinase kinase required for response to high osmolarity, or loss of the Mkk1 and Mkk2 MAP kinase kinases required for proper cell wall biosynthesis. Intriguingly, HST7 acts as a hyperactive allele of STE7; expression of Hst7 activates the mating pathway even in the absence of upstream signaling components including the Ste7 regulator Ste11, elevates the basal level of the pheromone-inducible FUS1 gene, and amplifies the pseudohyphal growth response in diploid cells. Thus Hst7 appears to be at least partially independent of upstream activators or regulators, but selective in its activity on downstream target MAP kinases. Creation of Hst7/Ste7 hybrid proteins revealed that the C-terminal two-thirds of Hst7, which contains the protein kinase domain, is sufficient to confer this partial independence of upstream activators.     

Comparison of the Lipolytic Effects of Norepinephrine and BRL 37344 in Rat Brown and White Adipocytes

The lipolytic effects of norepinephrine (a non-selective β-agonist) and BRL 37344 (a selective β₃-agonist) were compared in isolated rat brown and white adipocytes. Norepinephrine and BRL 37344 maximally stimulated lipolysis in brown and white adipocytes, approximately 10 times above basal values. However, adipocyte sensitivity for BRL 37344 was greater than that for norepinephrine, particularly in brown adipocytes [the EC₅₀ values (nM) for BRL 37344 and norepinephrine were 5 ± 1 and 103 ± 31 in brown adipocytes (P <0.01) versus 56 ± 9 and 124 ± 17 in white adipocytes (P <0.05), respectively]. On the other hand, the lipolytic effects of norepinephrine were totally blocked by 20–40 times superior concentrations of propranolol or bupranolol in brown as well as in white adipocytes. In contrast, the lipolytic effects of BRL 37344 were fully inhibited by concentrations of propranolol or bupranolol that were 200–1000 superior to the β₃ agonist concentration. The results demonstrate that: (1) the (β₃-agonist BRL 37344 is as effective as norepinephrine for maximally stimulating lipolysis in rat brown and white adipocytes, (2) both adipocyte types are more sensitive to the lipolytic effects of BRL 37344 than to those of norepinephrine, (3) although bupranolol is a better antagonist than propranolol on BRL 37344-stimulated lipolysis, it cannot be considered as a specific β₃-antagonist, (4) brown adipocytes are 10 times more sensitive than white adipocytes to the lipolytic effects of BRL 37344, suggesting an important role of β₃-receptors in brown adipose tissue.