The role of extra- and intracellular calcium in pepsinogen extrusion by isolated gastric glands]

It was found that carbacholine stimulated pepsinogen extrusion by isolated guinea pig stomach glands which were incubated in Ca(2+)-free medium, containing EGTA (0.25 mM). This effect could be imitated by caffeine (10 mM), a specific activator of Ca2+ release from intracellular pools. Extracellular Ca2+ in the concentrations over 0.125 mM increased pepsinogen extrusion which was stimulated by carbacholine. The interdependence between the level of pepsinogen extrusion and Ca2+ concentration in the medium had S-shaped character. La3+ ions (10(-4) mM) inhibited pepsinogen extrusion already in the first minutes after its activation by carbacholine. When testing other cations (Sr2+, Mg2+, Ba2+) it was found that only Sr2+ had some influence on pepsinogen extrusion. Thus, it can be concluded that both intra- and extracellular Ca2+ take part in the activation of pepsinogen extrusion. Obviously the role of extracellular Ca2+ consists in the support of reactivity of stomach glands to the action of stimulators of secretion.     

Impact of single neonatal allylestrenol treatment on the estrus cycle of rats treated with FSH+LH or TSH.

Neonatal allylestrenol treatment administered to female rats significantly increases the duration of estrus phase in the sexual cycle. Treatment with follicle stimulating hormone (FSH) + luteinizing hormone (LH) in adulthood prolongs the duration of estrus even on its own; the effect, however, is more pronounced in those animals who were treated (imprinted) with allylestrenol neonatally. When administered to the control animals, the chemically related thyreotrop hormone (TSH) is either indifferent or it even decreases the estrus index. In animals having received neonatal allylestrenol treatment, however, TSH administration increases significantly the duration of the estrus phase. Either with or without FSH+LH treatment, the ratio of estrogenic to gestagenic phase increases following neonatal allylestrenol treatment. The experiments call attention to the potential functional risks inherent in neonatal allylestrenol treatment. The actual risks, however, seem to be smaller than the effects seen at the receptor level.     

VCP, the mammalian homolog of cdc48, is tyrosine phosphorylated in response to T cell antigen receptor activation.

Activation of T cells through the T cell antigen receptor (TCR) results in the rapid tyrosine phosphorylation of a number of cellular proteins, one of the earliest being a 100 kDa protein. We have sought to identify this 100 kDa substrate by partially purifying the protein by antiphosphotyrosine (APT) affinity purification, in order to obtain amino acid sequence data and, using this information, to isolate the cDNA clone encoding the molecule. We report here that the amino acid sequence data showed pp100 to be the murine equivalent of porcine valosin containing protein (VCP), a finding confirmed from the cloning and sequencing of the murine pp100 cDNA. Sequence analysis has shown VCP to be a member of a family of ATP binding, homo-oligomeric proteins, and the mammalian homolog of Saccharomyces cerevisiae cdc48p, a protein essential to the completion of mitosis in yeast. We also provide proof that both endogenous and expressed murine VCP are tyrosine phosphorylated in response to T cell activation. Thus we have identified a novel component of the TCR mediated tyrosine kinase activation pathway that may provide a link between TCR ligation and cell cycle control.     

Transposase-induced excision and circularization of the bacterial insertion sequence IS911.

We have investigated the role of three IS911-specified proteins in transposition in vivo: the products of the upstream (OrfA) and downstream (OrfB) open reading frames, and a transframe protein (OrfAB) produced by -1 translational frameshifting between orfA and orfB. The production of OrfAB alone is shown to lead both to excision and to circularization of the element and to be sufficient for intermolecular transposition into a plasmid target. Simultaneous and independent production of OrfA is shown to stimulate OrfAB-mediated intermolecular transposition while greatly reducing the appearance of transposon circles. We have not been able to detect a role for OrfB. Although under certain conditions, the vector plasmid undergoes precise resealing after IS911 excision, the data suggest that this is not normally the case and that the donor plasmid is not generally conserved. The use of IS911 derivatives carrying mutations in the terminal 2 bp suggested that circle formation represents a site-specific intramolecular transposition event. We present a model which explains both intra- and intermolecular transposition events in terms of a single reaction mechanism of the 'cut and paste' type.     

Interaction of Wnt-1 proteins with the binding protein BiP.

The mouse Wnt-1 gene, a target for insertional activation in mouse mammary tumor virus-induced mammary tumors, encodes poorly secreted, cysteine-rich glycoproteins required for proper central nervous system development. We have been analyzing the biosynthesis of Wnt-1 proteins in several cell lines that express Wnt-1 cDNA from heterologous promoters. A protein of 78 kDa was found to be associated with the intracellular forms of Wnt-1 proteins in mammalian and avian cells by using multiple antisera against Wnt-1 proteins. We have identified p78 as the binding protein BiP with anti-BiP antisera and by its release from Wnt-1 immunoprecipitates upon incubation with MgCl2 and ATP. Experiments with a Wnt-1 mutant that lacks the sequence encoding the signal peptide indicates that Wnt-1 proteins must enter the secretory pathway in order to interact with BiP. We demonstrate that Wnt-1 proteins are associated with BiP in cells in which active Wnt-1 proteins are produced, such as a cultured mammary epithelial cell line and Wnt-1 transgenic mouse mammary tumor cells. The association of Wnt-1 proteins with BiP may be a factor in determining the efficiency of secretion of Wnt-1 gene products.     

Nucleotide and deduced amino acid sequence of bovine adrenal medulla chromogranin B (secretogranin I)

1. A novel 1745-dalton pyroglutamyl peptide (BAM-1745)6 was recently isolated and characterized from bovine adrenal medulla chromaffin granules. Its amino acid sequence was found to be 93% identical to residues 580-593 of human chromogranin B (secretogranin I). 2. Based on this sequence a degenerate oligonucleotide probe was synthesized and used to identify a 2.4-kb bovine adrenal medulla chromogranin B cDNA. 3. The deduced polypeptide is 647 amino acids long and begins with a putative signal sequence of 20 residues as in the human, rat, and mouse proteins. Also conserved in the bovine protein is a tyrosine residue which may be sulfated, two N-terminal cysteines, and many paired basic amino acids which may serve as sites of posttranslational processing. The peptide BAM-1745 is flanked by paired basic amino acids and therefore is most likely a product of posttranslational processing. Bovine chromogranin B is 67, 58, and 58% identical to the human, rat, and mouse chromogranin B proteins, respectively. 4. The carboxyl terminus of bovine chromogranin B, including BAM-1745, was found to be the most conserved region of the polypeptide and may identify it as an important functional domain.