Freeze tolerance in the frog,Rana sylvatica

Summary Wood frogs survive extracellular freezing at moderate subzero temperatures (–4°C) for at least 11 days. Freezing survival is aided by the accumulation of high concentrations of glucose as a cryoprotectant in blood and tissues. Glucose production was accompanied by a rapid decline in liver, but not muscle, glycogen levels suggesting that liver is the organ controlling cryoprotectant synthesis.Acknowledgments. This study was supported by grants to L.M.G. from the Kroc Foundation (Santa Ynez, California) and from the National Institute of Dental Research (Grant No. DE-03987). The authors wish to thank K. Yorko, M. Shakin, J. Finan and Mrs N. Manivannan for their technical and secretarial assistance.Acknowledgments. I thank Dr J. Ballantyne, Dr F. Schueler and I. McMurray for help with frog collections and Dr. W. Schmid, Dr J. Bogart and Dr F. Cook for helpful discussions. Supported by an N.S.E.R.C. operating grant and by a grant from the Atkinson Charitable Foundation.     

Novel oscillations in cell suspensions ofDictyostelium discoideum

Summary With a light-scattering technique, two novel rhythms were discovered in cell suspensions ofDictyostelium discoideum. One is a damped oscillation with a period of 2 to 2.5 min (at 23°C) induced by folate in EDTA-dissociated undifferentiated cells. The other is a sinusoidal oscillation with a period of about 12 min occasionally observed with late differentiated cells. Obviously, the repertoire of rhythms of this simple eukaryotic organism is larger than previously assumed.     

Paradoxical inhibition of the effects of bradykinin by some sulfhydryl reagents

Résumé Plusieurs dérivés thiol et deux antioxidants inhibent la bronchoconstriction chez le cobaye, une partie de l'hypotension chez le lapin et la libération de «rabbit aorta contracting substance» (RCS) dues à la bradykinine Il est proposé que les effets communs à la bradykinine, à la SRS-A, SRS-C et à l'acide arachidonique, qui sont bloqués par des antiinflammatoires, acides, et par les dérivés thiol, sont dus à la formation de RCS, constituée par des péroxydes cicliques analogues aux précurseurs instables des prostaglandines E 2 et F 2 alpha.

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We thank MrsB. Charpentier andC. Bellevrat for technical help, Prof.H. Clauser andJ. R. Vane and DrH. O. J. Collier for comments and DrS. H. Ferreira for advice concerning the superfusion technique and for BPP9. Squibb provided BPP5. This work is part of a thesis leading to the degree of Docteur-ès-Sciences, Orsay (B.B.V.).     

Differential basal synthesis of Hsp70/Hsc70 contributes to interindividual variation in Hsp70/Hsc70 inducibility

The source of intraspecies variation in the expression of heat shock proteins (HSPs) remains unresolved but could shed light on differential stress tolerance and disease susceptibility. This study investigated the influence of variable basal HSP synthesis on differential inducibility of HSP synthesis. Basal and heat-induced synthesis of the major HSP families in peripheral blood monocytes from healthy donors (n=42) were analysed using biometabolic labelling and densitometry. Basal Hsp70/Hsc70 synthesis and percentage induction of Hsp70/Hsc70 synthesis were significantly correlated (r=−0.57, p<0.0001), and described most accurately by an exponential decay equation (R=0.68, R²=0.46). This regression equation suggests that increasing levels of basal Hsp70/Hsc70 synthesis are accompanied byan exponential decrease in the percentage induction of Hsp70/Hsc70 synthesis. The model fits data from European and non-European population groups independently, although both coefficients in the regression equation were larger for non-Europeans. This implies population group as an additional factor influencing differential HSP expression. The differential inducibility of Hsp70/Hsc70 due to variable basal synthesis of Hsp70/Hsc70 and based upon population group may contribute to differential stress tolerance or disease susceptibility. Received 27 March 2000; received after revision 19 June 2000; accepted 20 June 2000     

Cellular internalization of proinsulin C-peptide

Proinsulin C-peptide is known to bind specifically to cell membranes and to exert intracellular effects, but whether it is internalized in target cells is unknown. In this study, using confocal microscopy and immunostained or rhodamine-labeled peptide, we show that C-peptide is internalized and localized to the cytosol of Swiss 3T3 and HEK-293 cells. In addition, transport into nuclei was found using the labeled peptide. The internalization was followed at 37°C for up to 1 h, and was reduced at 4°C and after preincubation with pertussis toxin. Hence, it is concluded to occur via an energy-dependent, pertussis toxin-sensitive mechanism and without detectable degradation within the experimental time course. Surface plasmon resonance measurements demonstrated binding of HEK-293 cell extract components to C-peptide, and subsequent elution of bound material revealed the components to be intracellular proteins. The identification of C-peptide cellular internalization, intracellular binding proteins, absence of rapid subsequent C-peptide degradation and apparent nuclear internalization support a maintained activity similar to that of an intracrine peptide hormone. Hence, the data suggest the possibility of one further C-peptide site of action. Received 31 October 2006; received after revision 27 December 2006; accepted 30 December 2006     

Beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation

The Wnt/beta-catenin/TCF4 pathway plays critical roles in the maintenance of small intestinal epithelium; however, downstream targets of the beta-catenin/TCF4 complex are not extensively characterized. We identified miR-30e as an immediate target activated by the beta-catenin/TCF4 complex. miR-30e was detected in the peri-nuclear region of the intestinal crypt IEC-6 cells. Bioinformatics analysis revealed clustered beta-catenin/TCF4 binding sites within the miR-30e promoter region. This promoter region was cloned into pGL3-control luciferase reporter vector, with the enhancer region removed. Transfection of pCMV-SPORT6-beta-catenin expression vector dose-dependently increased luciferase activity, and co-transfection of pCMV-SPORT6-TCF4 expression vector further enhanced the promoter activity. Dexamethasone-induced IEC-6 cells differentiation caused a 2.5-fold increase in miR-30e expression, and upon beta-catenin siRNA transfection, miR-30e increased 1.3-fold. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed the binding between beta-catenin/TCF4 complexes from IEC-6 nuclear extracts and the putative sequences in the miR-30e promoter. These results demonstrate that beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation.