Neuroblastoma Differentiation Involves the Expression of Two Isoforms of the α-Subunit of Go

The regulation of GTP-binding proteins (G proteins) was examined during the course of differentiation of neuroblastoma N1E-115 cells. N1E-115 cell membranes possess three Bordetella pertussis toxin (PTX) substrates assigned to alpha-subunits (G alpha) of Go (a G protein of unknown function) and "Gi (a G protein inhibitory to adenylate cyclase)-like" proteins and one substrate of Vibrio cholerae toxin corresponding to an alpha-subunit of Gs (a G protein stimulatory to adenylate cyclase). In undifferentiated cells, only one form of Go alpha was found, having a pI of 5.8 Go alpha content increased by approximately twofold from the undifferentiated state to 96 h of cell differentiation. This is mainly due to the appearance of another Go alpha form having a pI of 5.55. Both Go alpha isoforms have similar sizes on sodium dodecyl sulfate-polyacrylamide gels, are recognized by polyclonal antibodies to bovine brain Go alpha, are ADP-ribosylated by PTX, and are covalently myristylated in whole N1E-115 cells. In addition, immunofluorescent staining of N1E-115 cells with Go alpha antibodies revealed that association of Go alpha with the plasma membrane appears to coincide with the expression of the most acidic isoform and morphological cell differentiation. In contrast, the levels of both Gi alpha and Gs alpha did not significantly change, whereas that of the common beta-subunit increased by approximately 30% over the same period. These results demonstrate specific regulation of the expression of Go alpha during neuronal differentiation.     

Inherited haemolytic anaemia created by insertional inactivation of the α-spectrin gene

In the process of generating transgenic mice, inserted foreign DNA can cause insertional inactivation of the flanking genetic locus and simultaneously provide a molecular tag for localizing and cloning the inactivated gene. We describe the case of an insertional mutation leading, in animals homozygous for the insertion, to severe anaemia that was lethal within a few days after birth. The haemolytic anaemia and microspherocytosis of the red cells strongly suggested membrane abnormalities of the erythrocytes. Byin situ localization of the integration site, protein analysis of the red cell membranes, northern and Southern blot analyses, we were able to demonstrate that the integrated transgene had affected the α-spectrin gene locus.     

p-Chlorophenylalanine-Induced Alteration of Somatodendritic Levels of Tryptophan Hydroxylase Within the Rat Mesencephalic Raphe Nuclei

Abstract: Tryptophan hydroxylase distribution was examined across the nuclei raphe dorsalis, medianus, and pontis of the adult rat, under basal conditions and 2 days after a single injection of p -chlorophenylalanine, an irreversible tryptophan hydroxylase inhibitor. Tryptophan hydroxylase-expressing cells were numbered in transverse sections processed for immunohistochemistry, and the area of tryptophan hydroxylase distribution was delineated in adjacent sections transferred onto nitrocellulose and processed for immunoautoradiography. Two distinct areas were visualized: an inner zone, corresponding to the area displaying tryptophan hydroxylase-immunoreactive cells (so-called somatic area), and an outer zone, here called perisomatic, devoid of perikarya yet rich in tryptophan hydroxylase-positive neuropil in the histological sections. After treatment with p -chlorophenylalanine, a significant decrease in the number of tryptophan hydroxylase-immunoreactive cells could be observed only in the rostral raphe dorsalis, particularly within its ventromedian and dorsomedian subdivisions. In all raphe nuclei, the topological reconstruction of the somatic area was not modified. Based on the densitometric measurements in the immunoautoradiographs, however, a dramatic decrease in the content, concentration, and volume of expression of tryptophan hydroxylase could be documented in the three raphe nuclei. Detailed analysis of these results led to the conclusion that (a) tryptophan hydroxylase expression is differentially regulated in different serotoninergic cell body subpopulations of the raphe, some of which are more sensitive to p -chlorophenylalanine, and (b) distribution of tryptophan hydroxylase protein is modified also in the somatodendritic area in all raphe nuclei.     

Population dynamics of a motile and a non-motile Azospirillum lipoferum strain during rice root colonization and motility variation in the rhizosphere

Abstract: Azospirillum lipoferum 4B and non-motile A. lipoferum 4T have been simultaneously isolated from rice rhizosphere at the same frequency. A. lipoferum 4T showed stable morphological and metabolic traits which are atypical for A. lipoferum species such as lack of motility, carbohydrate metabolism and laccase activity. Inoculation experiments showed that A. lipoferum 4T, but not A. lipoferum 4B, needed rice roots to stabilize in sterile soil. Both strains were able to colonize efficiently rice roots (10⁸ cfu g⁻¹ fresh roots) but motile form 4B remained dominant. In spite of their phenotypical differences, A. lipoferum 4B and 4T co-existed without exclusion in sterile soil (planted or not) and rice rhizosphere. Inoculation of rice roots with A. lipoferum 4B showed that rice rhizosphere enhanced the frequency of appearance of stable non-motile forms (40%). This percentage was weaker in plantlet growth medium (4%). However, these non-motile bacteria kept the same biochemical traits than the motile parental strain 4B (carbohydrates metabolism, laccase activity).     

Virulence and vaccine potential of Salmonella typhlmurium mutants deficient in the expression of the RpoS (σs) regulon

The alternative sigma factor RpoS (σ^s) is required for Salmonella virulence in mice. We report the immunizing capacity of Salmonella typhimurlum rpoS and rpoS aroA mutants to protect susceptible BALB/c mice against subsequent oral challenge with virulent S. typhimurium. When administered orally or intraperitoneally, rpoS derivatives of the mouse-virulent S. typhimurium strains, C52 and SL1344, were highly attenuated and were efficient single-dose live vaccines. rpoS aroA mutants were more attenuated than corresponding single aroA or rpoS mutants, as assessed after oral or intraperitoneal administration, but retained significant ability to protect mice against salmonellosis. Salmonella rpoS and rpoS aroA mutants therefore deserve serious consideration for rational vaccine design. Consistent with this, Salmonella typhi Ty2, a ‘wild-type’ strain used widely for the development of human live-vaccine candidates against typhoid fever, was shown to be defective for rpoS. In addition, our results demonstrate that rpoS not only controls the growth and persistence of S. typhimurium in deep lymphoid organs, but also plays a role during the initial stages of oral infection.     

Glucose and Galactose Transport in Bifidobacterium bifidum DSM 20082

Sugar uptake was measured with ³H-galactose and ¹⁴C-glucose. Galactose transport system was not modified by inhibitors of known translocases and did not present a saturation kinetic with high concentration of galactose. Glucose incorporation was inhibited by lasalocid (cation symport inhibitor) and increased by KCl. The kinetic parameters K _M and V _max were respectively 9.16 mM and 26.56 nmol/min/mg cell protein. On the basis of this study, galactose crossed through the membrane by diffusion, and glucose was incorporated by a cation symport which is regulated by K⁺ ions. Received: 19 February 1997 / Accepted: 20 March 1997