The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells.

The adhesion protein E-cadherin plays a central part in the process of epithelial morphogenesis. Expression of this protein is downregulated during the acquisition of metastatic potential at late stages of epithelial tumour progression. There is evidence for a transcriptional blockage of E-cadherin gene expression in this process. Here we show that the transcription factor Snail, which is expressed by fibroblasts and some E-cadherin-negative epithelial tumour cell lines, binds to three E-boxes present in the human E-cadherin promoter and represses transcription of E-cadherin. Inhibition of Snail function in epithelial cancer cell lines lacking E-cadherin protein restores the expression of the E-cadherin gene.

Inhibition of p70/p85 S6 kinase activities in T cells by dexamethasone.

Glucocorticoids (GC) are potent immunosuppressive agents that interfere with interleukin-2 (IL-2)-dependent proliferation and IL-2 receptor signal transduction in T lymphocytes through complex mechanisms. Here we report that the basal activity, and IL-2- and phorbol ester-dependent activation of the p70/p85 S6 kinases (referred to collectively as pp70S6k) are inhibited by the glucocorticoid dexamethasone (Dex) in CTLL-20 cells. This Dex-induced inhibition is time- and dose-dependent, appears to be the consequence of pp70S6k dephosphorylation, and requires ongoing transcription. Attempts to establish a link between Dex action and those of known pp70S6k-regulating agents such as phosphatidylinositol 3-kinase, protein kinase A-stimulating agents, calyculin A-inhibited protein phosphatases, and rapamycin have been negative. Additional results with NIH3T3 cells suggest the existence of a T cell-specific blockade of pp70S6k by Dex. Implications are 2-fold: 1) pp70S6k inactivation may account for at least part of the immunosuppressive effects of GC in vivo, and 2) GC inactivation of pp70S6k is exerted through a novel, distinct mechanism that does not appear to be linked to any other known pp70S6k regulatory process.

Structural and functional analysis of pp70S6k.

The pp70/85-kDa S6 kinases, collectively referred to as pp70S6k, are thought to participate in transit through the G1 phase of the cell cycle. pp70S6k regulates the phosphorylation of the 40S ribosomal protein S6 and the transcription factor CREM tau. pp70S6k is regulated by serine/threonine phosphorylation, and although 1-phosphatidylinositol 3-kinase and phospholipase C have been implicated as upstream regulators, the mechanism of activation and identity of the upstream pp70S6k kinases remain unknown. To improve our understanding of how this mitogen-stimulated protein kinase is regulated by growth factors and the immunosuppressant rapamycin, we have initiated a structure/function analysis of pp70S6k. Our results indicate that both the N and C termini participate in the complex regulation of pp70S6k activity.

Activation of pp70/85 S6 kinases in interleukin-2-responsive lymphoid cells is mediated by phosphatidylinositol 3-kinase and inhibited by cyclic AMP.

Activation of phosphatidylinositol 3-kinase (PI3K) and activation of the 70/85-kDa S6 protein kinases (alpha II and alpha I isoforms, referred to collectively as pp70S6k) have been independently linked to the regulation of cell proliferation. We demonstrate that these kinases lie on the same signalling pathway and that PI3K mediates the activation of pp70 by the cytokine interleukin-2 (IL-2). We also show that the activation of pp70S6k can be blocked at different points along the signalling pathway by using specific inhibitors of T-cell proliferation. Inhibition of PI3K activity with structurally unrelated but highly specific PI3K inhibitors (wortmannin or LY294002) results in inhibition of IL-2-dependent but not phorbol ester (conventional protein kinase C [cPKC])-dependent pp70S6k activation. The T-cell immunosuppressant rapamycin potently antagonizes IL-2-(PI3K)- and phorbol ester (cPKC)-mediated activation of pp70S6k. Thus, wortmannin and rapamycin antagonize IL-2-mediated activation of pp70S6k at distinct points along the PI3K-regulated signalling pathway, or rapamycin antagonizes another pathway required for pp70S6k activity. Agents that raise the concentration of intracellular cyclic AMP (cAMP) and activate cAMP-dependent protein kinase (PKA) also inhibit IL-2-dependent activation of pp70S6k. In this case, inhibition appears to occur at least two points in this signalling path. Like rapamycin, PKA appears to act downstream of cPKC-mediated pp70S6k activation, and like wortmannin, PKA antagonizes IL-2-dependent activation of PI3K. The results with rapamycin and wortmannin are of added interest since the yeast and mammalian rapamycin targets resemble PI3K in the catalytic domain.

Alteration in the expression of GLUT-1 and GLUT-4 protein and messenger RNA levels in denervated rat muscles.

Denervation induces insulin resistance of the glucose transport process in skeletal muscle. To determine whether this is due to alterations in the expression of muscle glucose transporters (GLUT) in different fiber types, we evaluated the amount of GLUT-1 and GLUT-4 protein and messenger RNA (mRNA) in extensor digitorum longus (EDL) and soleus at 1, 2, and 3 days after sciatotomy. Denervation elevated the basal rate of 2-[1,2-3H]deoxy-D-glucose (2-DOG) uptake in the EDL and decreased the insulin-stimulated DOG uptake in both muscles. Denervation after 1 day did not modify the GLUT-1 or the GLUT-4 protein level in either muscle. However, it increased GLUT-1 mRNA by 66% and decreased GLUT-4 mRNA by 70% in the EDL, but not in the soleus (P < 0.05). After 2 days of denervation, by which time GLUT-1 mRNA was increased 2-fold and GLUT-4 mRNA was reduced by 70%, we observed a 2-fold increase in GLUT-1 protein (P < 0.01) in the EDL and a 40-45% decrease in GLUT-4 protein in both muscles (P < 0.01). These results indicate that modifications in the expression of GLUT-1 and GLUT-4 protein cannot explain the insulin resistance of the glucose transport process in the EDL or soleus 1 day after denervation. After 2 days of denervation, however, alterations in GLUT-1 and GLUT-4 protein levels may contribute to the change in basal and insulin-stimulated DOG uptake in both the EDL and the soleus muscles.

Effect of diabetes and fasting on GLUT-4 (muscle/fat) glucose-transporter expression in insulin-sensitive tissues. Heterogeneous response in heart, red and white muscle.

1. GLUT-4 glucose-transporter protein and mRNA levels were assessed in heart, red muscle and white muscle, as well as in brown and white adipose tissue from 7-day streptozotocin-induced diabetic and 48 h-fasted rats. 2. In agreement with previous data, white adipose tissue showed a substantial decrease in GLUT-4 mRNA and protein levels in response to both diabetes and fasting. Similarly, GLUT-4 mRNA and protein markedly decreased in brown adipose tissue in both insulinopenic conditions. 3. Under control conditions, the level of expression of GLUT-4 protein content differed substantially in heart, red and white skeletal muscle. Thus GLUT-4 protein was maximal in heart, and red muscle had a greater GLUT-4 content compared with white muscle. In spite of the large differences in GLUT-4 protein content, GLUT-4 mRNA levels were equivalent in heart and red skeletal muscle. 4. In heart, GLUT-4 mRNA decreased to a greater extent than GLUT-4 protein in response to diabetes and fasting. In contrast, red muscle showed a greater decrease in GLUT-4 protein than in mRNA in response to diabetes or fasting, and in fact no decrease in GLUT-4 mRNA content was detectable in fasting. On the other hand, preparations of white skeletal muscle showed a substantial increase in GLUT-4 mRNA under both insulinopenic conditions, and that was concomitant to either a modest decrease in GLUT-4 protein in diabetes or to no change in fasting. 5. These results indicate that (a) the effects of diabetes and fasting are almost identical and lead to changes in GLUT-4 expression that are tissue-specific, (b) white adipose tissue, brown adipose tissue and heart respond similarly to insulin deficiency by decreasing GLUT-4 mRNA to a larger extent than GLUT-4 protein, and (c) red and white skeletal muscle respond to insulinopenic conditions in a heterogeneous manner which is characterized by enhanced GLUT-4 mRNA/protein ratios.

[Diagnosis by DNA analysis in familial isolated growth hormone deficiency type I-A]. / Diagnóstico mediante análisis del DNA de la deficiencia aislada y familiar de hormona de crecimiento tipo I-A.

We show the autoradiograms of DNA from one child affected of familial isolated growth hormone deficiency type I-A. Restriction endonuclease analysis of DNA isolated from leukocytes was done using 32P-labeled human GH cDNA clone as a probe. DNA analysis using the restriction endonuclease Bam HI revealed that the 3.8 kb restriction fragment, which contain the normal hGH-N gene, was absent. Since these deletions preclude production of any GH-N protein, affected individuals tend to be immunologically intolerant to exogenous GH. The child was homozygote and after treatment with exogenous GH developed a high titre of antibodies to GH and growth arrest. This is the first case of this genetic disorder studied in Spain.

Energy intake of rats fed a cafeteria diet.

The proportion of lipid, carbohydrate and protein energy self-selected by male and female rats from a cafeteria diet has been studied for a 48-day period (36-day in female rats). The diet consisted in 12 different items and was offered daily, in excess and under otherwise standard conditions, to rats--caged in groups of three--from weaning to adulthood. Groups of control animals were studied in parallel and compared with the cafeteria groups. Cafeteria diet fed groups of rats ingested more energy and lowered their metabolic efficiency with age. Male rats ate more than females and increased their body weight even after female practically stopped growing. There was a wide variation in the aliments consumed each day by the cafeteria-fed rats. However, the proportion of lipid, protein and carbohydrate the rats ate remained constant. Male rats ingested more lipid than females. Carbohydrate consumption was constant in control and cafeteria fed groups of rats independently of sex. Protein consumption was higher in cafeteria rats than in controls, but the differences were not so important as with liquid. Fiber content of the cafeteria diet was lower than that of the control diet. The cafeteria diet selected by the rats was, thus, hypercaloric and hyperlipidic, with practically the same amount of carbohydrate than the control diet, slightly hyperproteic and, nevertheless, remarkably constant in its composition with respect to time. Cafeteria rats had a higher water intake than controls. All these trends were maintained despite the observed changes in the animals' tastes and their differential consumption of the ailments of the diet.

Effects of a cafeteria diet and starvation of global 14C(U)-glucose disposal.

The label distribution in control and cafeteria-diet fed rats, either in basal conditions or after 24 hours of food deprivation, 10 minutes after the i.v. injection of carrier-free D-14C-(U)-glucose, has been studied. The radioactivity recovered in the different fractions of liver, kidney, heart, striated muscle and white adipose tissue showed comparable patterns of change with starvation in both dietary groups. Most of the radioactivity was found in the free amino acid fraction as well as in proteins, with significant proportions also in lipid and liver glycogen. However, most of the label was lost due to its oxidation, remaining in the combined indicated tissues 10-20% of the injected label. On the whole, cafeteria rats consumed more glucose than controls, the lowest oxidation corresponding to the starved-control group. The amount of glucose oxidized by cafeteria rats was actually comparable to that of fed controls. The availability of other energetic sources--i.e. lipid--allows for an increased glucose utilization in cafeteria rats, even in the starved state.

Effects of starvation and a high-energy diet on rat blood compartmentation of injected radioactive alanine and glucose.

The effects of starvation upon blood compartmentation and differential handling of glucose and alanine have been studied in control and cafeteria-fed rats. The injection of radioactive glucose resulted in higher specific radioactivities in intracellular glucose but lower intracellular amino acid specific radioactivities when compared with the plasma values. The cells/plasma specific radioactivity ratios increased dramatically in cafeteria rats with starvation. The injection of radioactive alanine resulted in higher cell than plasma glucose specific activities, and lower cell amino acid specific activities. All these parameters increased after a 24-hours starvation period. It is concluded that glucose synthetised by the liver is released mainly into the blood intracellular pool, being later liberated into the plasma or directly into the tissues.