Changes in cellular gene expression in rat thyroid epithelial cells transformed by the Kirsten murine sarcoma virus

We have exploited a recently characterized system of rat thyroid epithelial cells transformed by the wild-type (wt) and a temperature-sensitive (ts) mutant strain of the Kirsten murine sarcoma virus (Ki-MSV) in order to study the effects of the K-ras oncogene on the gene expression of differentiated thyroid epithelial cells. By using cDNAs isolated from normal thyroid glands as probes, we were able to identify three sets of cellular sequences whose expression is influenced by the v-K-ras oncogene. The first set of genes is irreversibly repressed by transformation with both the wt and the ts viruses. The second set of genes is repressed in the ts-Ki-MSV-transformed cells but not in the same cells grown at the nonpermissive temperature. A third set of genes is present at higher levels at the nonpermissive temperature than at the permissive temperature. This system has allowed us to isolate and characterize a number of cDNA clones belonging to each of these three sets of genes. These specific cDNAs are suitable probes to study phenotypical changes during transformation of epithelial cells.     

Role for the Vacuolar H+-ATPase in Regulating the Cytoplasmic pH: An in Vivo Study Carried out in chl1, an Arabidopsis thaliana Mutant Impaired in NO-3 Transport

The effects of the growth in a medium containing NH₄NO₃ as nitrogensource were studied on cell sap pH, cytoplasmic pH and malatecontent in chl1, an Arabidopsis thaliana mutant impaired inchlorate and nitrate transport. In all the conditions testedthe pH of the cytoplasm in chl1 was more alkaline, and thatof the vacuole was more acidic as compared with those measuredin wt. Treatment with bafilomycin A1, a specific inhibitor ofthe vacuolar H⁺-ATPase, induced a small alkalinization of thevacuole, and a significant acidification of the cytoplasm, theseeffects being greater in chl1 than in wt. The greater responseof the mutant to bafilomycin Al suggests that, in the absenceof the inhibitor, the activity of the tonoplast H⁺-ATPase inchl1 is higher than in wt, this diversity being a possible reasonfor the differences in intracellular pH detected between thetwo strains. A possible role for the vacuolar H⁺-ATPase in regulatingthe cytoplasmic pH is discussed. (Received August 2, 1995; Accepted February 1, 1996)     

Molecular Cloning, Expression Pattern, and Chromosomal Localization of the Human Na–Cl Thiazide-Sensitive Cotransporter (SLC12A3)

Electrolyte homeostasis is maintained by several ion transport systems. Na–(K)–Cl cotransporters promote the electrically silent movement of chloride across the membrane in absorptive and secretory epithelia. Two kidney-specific Na–(K)–Cl cotransporter isoforms are known, so far, according to their sensitivity to specific inhibitors. We have cloned the human cDNA coding for the renal Na–Cl cotransporter selectively inhibited by the thiazide class of diuretic agents. The predicted protein sequence of 1021 amino acids (112 kDa) shows a structure common to the other members of the Na–(K)–Cl cotransporter family: a central region harboring 12 transmembrane domains and the 2 intracellular hydrophilic amino and carboxyl termini. The ex- pression pattern of the human Na–Cl thiazide-sensitive cotransporter (hTSC, HGMW-approved symbol SLC12A3) confirms the kidney specificity. hTSC has been mapped to human chromosome 16q13 by fluorescencein situhybridization. The cloning and characterization of hTSC now render it possible to study the involvement of this cotransport system in the pathogenesis of tubulopathies such as Gitelman syndrome.     

Polygalacturonase-inhibiting protein accumulates in Phaseolus vulgaris L. in response to wounding, elicitors and fungal infection

Polygalacturonase-inhibiting protein (PGIP) is a cell wall-associated protein that specifically binds to and inhibits the activity of fungal endopolygalacturonases. The Phaseolus vulgaris gene encoding PGIP has been cloned and characterized. Using a fragment of the cloned pgip gene as a probe in Northern blot experiments, it is demonstrated that the pgip mRNA accumulates in suspension-cultured bean cells following addition of elicitor-active oligogalacturonides or fungal glucan to the medium. Rabbit polyclonal antibodies specific for PGIP were generated against a synthetic peptide designed from the N-terminal region of PGIP; the antigenicity of the peptide was enhanced by coupling to KLH. Using the antibodies and the cloned pgip gene fragment as probes in Western and Northern blot experiments, respectively, it is shown that the levels of PGIP and its mRNA are increased in P. vulgaris hypocotyls in response to wounding or treatment with salicylic acid. Using gold-labeled goat-anti-rabbit secondary antibodies in EM studies, it has also been demonstrated that, in bean hypocotyls infected with Colletotrichum lindemuthianum, the level of PGIP preferentially increases in those cells immediately surrounding the infection site. The data support the hypothesis that synthesis of PGIP constitutes an active defense mechanism of plants that is elicited by signal molecules known to induce plant defense genes.     

A histochemical study of the binding of 125I-HCG to the rat ovary throughout the estrous cycle

Summary Changes in the distribution of the in vitro uptake of ¹²⁵I-HCG by the ovaries of adult rats were examined histochemically throughout the estrous cycle.Only in follicles wider than 500 m, occurring mainly at diestrus and proestrus, could granulosa cells bind the labelled hormone. The labelling increased with follicular size and decreased in intensity from the peripheral granulosa cells inwards. No uptake occurred in the oocytes, in the cells of the cumulus oophorus nor in the granulosa cells of the atretic follicles.The binding capacity of the newly-formed corpora lutea of estrus was less than that of preovulatory follicles. The uptake of ¹²⁵I-HCG by corpora lutea during the first cycle reached its maximum at diestrus but fell sharply by proestrus. The uptake was patchy in the corpora lutea of the second cycle and not significant in the older ones.The uptake of ¹²⁵I-HCG by thecae increased with follicular size and was greater in the thecae of atretic follicles than in the thecae of growing follicles of like size. There was a greater uptake in the last formed interstitial tissue than there was in older tissue.At proestrus, the uptake of ¹²⁵I-HCG was unaffected by the LH surge at 18.00h but had decreased slightly at 24.00 h.The implications of these data in relation to the regulation of receptor sites, is discussed.