Quantitative detection of Legionella pneumophila in water samples by immunomagnetic purification and real-time PCR amplification of the dotA gene.

A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used.

Glucocorticoid activation of the neuronal nicotinic acetylcholine receptor alpha7 subunit gene: involvement of transcription factor Egr-1.

The alpha7 subunit is a component of nicotinic acetylcholine receptors expressed in bovine chromaffin cells. The peculiar localization of these receptors at adrenomedullary areas adjacent to the adrenal cortex suggests that factors, probably glucocorticoids, arising from the cortex might diffuse and regulate alpha7 receptor expression. In reporter gene transfection experiments, dexamethasone increased alpha7 promoter activity by up to fivefold in a concentration- and time-dependent manner despite the absence of consensus glucocorticoid receptor elements at the alpha7 promoter.

Modifications in the C terminus of the synaptosome-associated protein of 25 kDa (SNAP-25) and in the complementary region of synaptobrevin affect the final steps of exocytosis.

Fusion proteins made of green fluorescent protein coupled to SNAP-25 or synaptobrevin were overexpressed in bovine chromaffin cells in order to study the role of critical protein domains in exocytosis. Point mutations in the C-terminal domain of SNAP-25 (K201E and L203E) produced a marked inhibition of secretion, whereas single (Q174K, Q53K) and double mutants (Q174K/Q53K) of amino acids from the so-called zero layer only produced a moderate alteration in secretion. The importance of the SNAP-25 C-terminal domain in exocytosis was also confirmed by the similar effect on secretion of mutations in analogous residues of synaptobrevin (A82D, L84E).

Activity of the nicotinic acetylcholine receptor alpha5 and alpha7 subunit promoters in muscle cells.

The acetylcholine receptor alpha5 and alpha7 subunits are components of different nicotinic receptor subtypes expressed in the nervous system. However, they are also present in non-neuronal tissues. We have detected alpha5 and alpha7 transcripts in mouse C2C12 muscle cells.

Transcription factors NF-Y and Sp1 are important determinants of the promoter activity of the bovine and human neuronal nicotinic receptor beta 4 subunit genes.

The beta4 subunit is a component of the neuronal nicotinic acetylcholine receptors which control catecholamine secretion in bovine adrenomedullary chromaffin cells. The promoter of the gene coding for this subunit was characterized. A proximal region (from minus sign99 to minus sign64) was responsible for the transcriptional activity observed in chromaffin, C2C12, and COS cells.