The human mas oncogene, which renders transfected NIH/3T3 cells tumorigenic, was identified as a subtype of angiotensin receptor by transient expression in Xenopus oocytes and stable expression in the mammalian neuronal cell line, NG115-401L. The mas receptor preferentially recognizes angiotensin III, and is expressed at high levels in brain. The mas/angiotensin receptor functions through the breakdown of inositol lipids and can drive DNA synthesis, unlike another inositol-linked peptide receptor, that for bradykinin. Comparative analysis of several early biochemical events elicited by either angiotensin or bradykinin stimulation of mas-transfected cells has not indicated a specific difference correlated with mitogenic activity. In particular, the inositol lipid kinase, phosphatidylinositol-3-kinase, thought to be involved in the mitogenic mechanism of platelet-derived growth factor receptors, is unaffected by activation of mas. These results have shown that a proto-oncogene encodes a neural peptide receptor, indicating that peptide receptors may be involved in differentiation and proliferation processes, as are other identified proto-oncogenes.

Amino Acid Sequence↗

PubMed1988

Villin, a specific marker for some epithelia specialized in transport, to study the differentiation of intestinal and kidney cells in vivo and in a human colon adenocarcinoma line HT29 in culture.

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Adenocarcinoma↗

PubMed1987

Changes in villin synthesis and subcellular distribution during intestinal differentiation of HT29-18 clones.

Brush border in enterocytes is a cell surface specialization intimately associated with terminal differentiation of these cells. HT29-18, a clone derived from the HT-29 human colonic adenocarcinoma cell line, and HT29-18-C1, a subclone from HT29-18 described in the companion paper (Huet, C., C. Sahuquillo-Merino, E. Coudrier, and D. Louvard, 1987, J. Cell Biol., 105:345-357), undergo terminal differentiation with brush borders in the absence of glucose or upon replacement of glucose by galactose in the medium. Taking advantage of this clone and its subclone which can be manipulated in vitro, we have studied the synthesis and subcellular distribution of villin, one major protein in the microvillus core of the brush border. For this study, a monoclonal antibody against villin (BDID2C3) has been isolated and characterized in detail. In addition an ELISA has been set up to measure villin accurately in total cell extracts. Villin content in differentiated HT29-18 cells is close to that seen in normal human colonic cells but 10 times lower in undifferentiated HT29-18 cells. The rate of villin synthesis is dramatically increased in the course of enterocytic differentiation, while villin is remarkably stable after synthesis. We have recently shown, using a cDNA probe for villin, that this change is controlled either at the transcription level or by RNA stabilization (Pringault, E., M. Arpin, A. Garcia, J. Finidori, and D. Louvard, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:3119-3124). As shown by immunofluorescence and immunogold labelings, villin is targeted to the brush border area of differentiated HT29-18 cells but remains diffusely distributed in undifferentiated ones.

Adenocarcinoma↗

PubMed1987

Correlation of cerebrospinal fluid endotoxinlike activity with clinical and laboratory variables in gram-negative bacterial meningitis in children.

Detection of endotoxinlike activity in cerebrospinal fluid by Limulus amebocyte lysate gelation has been suggested as a useful technique for the diagnosis of gram-negative bacterial meningitis. We prospectively screened 1,503 cerebrospinal fluid specimens with a Limulus amebocyte lysate microassay. The limit of sensitivity of the assay was 0.01 ng/ml. All specimens that were positive for endotoxinlike activity were subjected to confirmatory retesting, after which 38 (86%) remained positive. Comparison with available culture results revealed that 33 of 38 specimens (86%) were culture positive; 3 of the 5 culture-negative specimens were from patients on therapy for gram-negative bacterial meningitis, and 1 was from a neonate. The overall specificity of confirmed positive tests was 99.5%, with a positive predictive value of 97.3%. There was one false-negative specimen, giving an overall sensitivity of 97.3% and a negative predictive value of 99.9%. Endotoxinlike activities of greater than or equal to 150 ng/ml correlated with present illness of less than 2 days' duration (P = 0.024), elevated cerebrospinal fluid protein (P less than 0.05), and seizures (P = 0.004); levels of greater than or equal to 3,000 ng/ml correlated with neutropenia (P = 0.032), and levels of greater than or equal to 3.2 X 10(6) ng/ml correlated with death (P = 0.001). We conclude that the Limulus amebocyte lysate microslide gelation test has prognostic value as a sensitive, specific, simple, inexpensive screening test for gram-negative bacterial meningitis.

Bacterial Infections↗

PubMed1984

Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor.

S. cerevisiae kex2 mutants are defective for the production of two biologically active secreted peptides: killer toxin and the mating pheromone, alpha-factor. Both molecules are excised from larger precursor polypeptides. In normal cells, the alpha-factor precursor is core-glycosylated and proteolytically processed intracellularly. In kex2 mutants, however, prepro-alpha-factor is not proteolytically cleaved and is secreted in a highly glycosylated form. All kex2 mutants examined (three independent alleles) lack a Zn++-sensitive membrane-associated endopeptidase with specificity for cleaving on the carboxyl side of a pair of basic residues. Absence of this activity cosegregates with the other phenotypes of a kex2 lesion in genetic crosses. The normal KEX2 gene was isolated by complementation of three of the phenotypes conferred by the kex2-1 mutation. The cloned DNA, either on a multicopy plasmid or integrated into the genome, restores both enzymatic activity in vitro and the normal pattern of proteolytic processing and glycosylation of prepro-alpha-factor in vivo. Gene dosage effects suggest that KEX2 is the structural gene for the endopeptidase.

Arginine↗

PubMed1983

Yeast alpha factor is processed from a larger precursor polypeptide: the essential role of a membrane-bound dipeptidyl aminopeptidase.

Alpha factor mating pheromone is a peptide of 13 amino acids secreted by Saccharomyces cerevisiae alpha cells. Nonmating ("sterile," or ste) alpha-cell mutants bearing defects in the STE13 gene do not produce normal alpha factor, but release a collection of incompletely processed forms (alpha factor) that have a markedly reduced specific biological activity. The major alpha-factor peptides have the structures H2N-GluAlaGluAla-alpha factor and H2N-AspAlaGluAla-alpha factor. The ste13 mutants lack a membrane-bound heat-stable dipeptidyl aminopeptidase (DPAPase A) that specifically cleaves on the carboxyl side of repeating -X-Ala- sequences. Absence of DPAPase A and the other phenotypes of a ste13 lesion cosegregate in genetic crosses. The cloned STE13 gene on a plasmid causes yeast cells to overproduce DPAPase A severalfold. A different cloned DNA segment, which weakly suppresses the ste13 defects, causes overproduction of a heat-labile activity (DPAPase B) by about tenfold. Other experiments indicate that DPAPase A action may be rate-limiting for alpha-factor maturation in normal alpha cells.

Amino Acids↗