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.     

Low virulence and immunogenicity in mice and in rabbits of variants ofHerpes simplex virus resistant to 5-iodo-2-deoxyuridine

Résumé Par passage en série sur des cellules en culture et en présence de 5-iodo-2-desoxyuridine (IUdR) on a obtenu une souche de virus herpétique résistant à l'IUdR. Cette souche est très peu virulente chez la souris et le lapin, mais est capable de protéger ces animaux contre le virus pathogène d'origine.

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This work has been supported by a Grant of Consiglio Nazionale delle Ricerche, Roma.     

Differences in cerebral morphology in 2 stocks of mutant mice heterozygous for the loop-tail (Lp)-gene

Summary Adult loop-tail heterozygotes (Lp/+) from a stock ofLp-mice which consistently fail to show head wobbling exhibit normal brain morphology with respect to size and shape of lateral ventricles and nearby nuclei. Loop-tail heterozygotes from a head wobbling stock ofLp-mice show enlargement and deformity of the lateral ventricles.Supported by research grant HD09562 from the National Institutes of Health.     

The age-dependent difference in the response of the vagina to the neonatal administration of testosterone propionate in Long-Evans female rats

Summary The ability of the vaginal epithelium to respond to testosterone propionate by producing keratin was established when female rats were 3 days of age, whereas the vaginal stroma from rats 2 days old or less was capable of responding to the androgen and might destroy the vaginal epithelium.     

Lack of mutagenicity of irradiated glucose in Salmonella typhimurium using host-mediated assay

Experiments were conducted to study the ability of irradiated glucose to induce reverse mutations in S. typhimurium by host-mediated assay. The results revealed no significant increase in the frequency of reverse mutations compared to controls.     

Injection of a soluble sperm fraction into sea-urchin eggs triggers the cortical reaction

Fertilization membranes form around unfertilized sea-urchin eggs after microinjection of a soluble spermatozoa fraction isosmotic with seawater. This demonstrates that the spermatozoon contains a chemical that triggers an increase in cytosolic calcium, leading to exocytosis of cortical granules. It also demonstrates that the triggering mechanism does not require an externally-activated egg-membrane process. Further experiments show that the chemical trigger is not calcium.