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.     

Adhesion of blood platelets to subendothelial surface: Distinct from adhesion to collagen

Zusammenfassung Das subendotheliale Gewebe von Kaninchenaorten besteht zur Hauptsache aus Mikrofibrillen, Elastin und amorphem Material. Die Adhesion von Blutplättchen an subendothelialem Gewebe ist von der Präsenz von Erythrozyten und chelierbaren Ionen abhängig. Dies steht im Gegensatz zur Adhesion von Plättchen an Kollagen.     

Circadian rhythm in adrenal adenyl cyclase and corticosterone abolished by medial forebrain bundle transection in the rat

Zusammenfassung Nachweis, dass die Nebennierenrinde von Ratten nicht nur eine rhythmische Tagesschwankung in ihrem Gehalt an Corticosteron, sondern auch eine Schwankung im Gehalt an Adenyl-Cyclase aufweist. Diese Rhythmen können durch eine stereotaktisch ausgeführte Trennung des medianen Vorderhirnbündels, welches zum Hypothalamus führt, aufgehoben werden.

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This work was supported by research grants No. NS-05002 and No. HD-04581 from the National Institutes of Health, USPHS.     

The influence of reserpine on incorporation of3H-thymidine into mouse liver DNA

Zusammenfassung Der Einbau von³H-Thymidin in die DNA der Leber von weiblichen weissen Mäusen wurde 60 h nach der Verabreichung von 2 mg/kg Reserpin und ausserdem 60 h nach Futterentzug an nicht reserpinisierten Tieren gemessen. Reserpin führt unabhängig von der unter dieser Substanz auftretenden Hypothermie und Anorexie zu einer Hemmung des³H-Thymidin-Einbaus in die DNS der Leber um etwa 60%.     

Sialidase of transitional epithelium of sheep urinary tract

Resumen El epitelio de transición de cordero contiene actividad de sialidasa. Se demuestra que esta enzima se encuentra en lisosomas de este tejido y posee el pH óptimo ácido característico de enzimas lisosomales.

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The technical assistance of MissE. Prado and Mr.L. Iwakawa is acknowledged. This investigation was supported by a grant of the Consejo Nacional de Investigaciones Cientificas y Técnicas of Argentina.