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.     

Evaluation of the mechanisms by which gamma-amino-butyric acid in association with phosphatidylserine exerts an antiepileptic effect in the rat

The i.p. injection in rats of GABA (740 mg/Kg) after sonication with an equal amount of phosphatidylserine (PS) has an antiepileptic effect. The injection of plain GABA has no such an effect. Blood, brain and synaptosomal accumulation of exogenous labeled GABA under the two circumstances are evaluated. In the case of GABA/PS injection there is a higher passage of the exogenous labeled neurotransmitter into the blood and brain nerve endings (synaptosomes). A higher synaptosomal accumulation of the exogenous labeled neurotransmitter is found even when GABA and PS are injected separately. Since these accumulation increases occur at a time when there is the antiepileptic effect, they seem relevant to it. Our interpretation of the chain of the events resulting in the antiepileptic action is that the phospholipid facilitates from the beginning the first passage of the exogenous neurotransmitter form the peritoneum to the blood. Then a higher passage to the brain tissue and eventually to the GABA-ergic nerve endings ensues. The brisker accumulation of the exogenous neurotransmitter in the nerve endings could be at the basis of a more efficient GABA-ergic inhibitory control in the brain.     

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.