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.     

Sperm viability in the reproductive tract of females in a population of Sceloporus mucronatus exhibiting asynchronous reproduction

Asynchronous reproduction is a common phenomenon in high-elevation populations of lizards from Central México. Sperm storage in the reproductive tract of females is the mechanism for making oocyte fertilization possible. Our study addresses questions related to functional oviductal sperm storage of females mating on different dates throughout the reproductive season. A population of Sceloporus mucronatus with copulation in the summer and ovulation in the fall was chosen for this experiment. Eleven females that copulated in the field during June and 13 females that copulated in captivity during August were maintained in the laboratory until parturition. The number of pregnant females and the litter sizes produced in each experimental group were indicative of the viability of the stored sperm. Sperm stored in the reproductive tract of females were able to fertilize eggs after 4 months. No significant differences were found in the number of pregnant females between the 2 experimental groups nor in the litter sizes that they produced. We found that the amount of time sperm were held in the female reproductive tract (ca. 3 months) had no effect on the capacity of sperm to fertilize eggs. Histological examination of 8 oviducts collected before the mating season eliminated the possibility of sperm storage from one year to the next. In this system, sperm retention could have evolved as a response mechanism to deal with the asynchrony between sexes in the reproductive cycles. However, we cannot rule out alternative hypotheses.     

Attributes associated with probability of infestation by the pi?on ips, Ips confusus (Coleoptera: Scolytidae), in pi?on pine, Pinus edulis

We examined attributes of pi?on pine ( Pinus edulis ) associated with the probability of infestation by pi?on ips ( Ips confuses ) in an outbreak in the Coconino National Forest, Arizona. We used data collected from 87 plots, 59 infested and 28 uninfested, and a logistic regression approach to estimate the probability of infestation based on plot and tree-level attributes. Pi?on pine stand density index was a good predictor of the likelihood of infestation by pi?on ips at the plot level, and a cross-validation analysis confirmed that the model correctly classified 82% of the cases. Diameter at root collar and pi?on dwarf mistletoe infestation level were good predictors of individual tree infestation, and a cross-validation analysis indicated that the model correctly classified 72% of the cases. Results suggest that the occurrence of pi?on ips infestations may be related to stress factors associated with increased stocking and pi?on dwarf mistletoe infestations.     

Non-caspase proteases: triggers or amplifiers of apoptosis?

Caspases are the most important effectors of apoptosis, the major form of programmed cell death (PCD) in multicellular organisms. This is best reflected by the appearance of serious development defects in mice deficient for caspase-8, -9, and -3. Meanwhile, caspase-independent PCD, mediated by other proteases or signaling components has been described in numerous publications. Although we do not doubt that such cell death exists, we propose that it has evolved later during evolution and is most likely not designed to execute, but to amplify and speed-up caspase-dependent cell death. This review shall provide evidence for such a concept.     

Visualization of subcellular NAD pools and intra-organellar protein localization by poly-ADP-ribose formation

Poly-ADP-ribose polymerases (PARPs) use NAD⁺ as substrate to generate polymers of ADP-ribose. We targeted the catalytic domain of human PARP1 as molecular NAD⁺ detector into cellular organelles. Immunochemical detection of polymers demonstrated distinct subcellular NAD⁺ pools in mitochondria, peroxisomes and, surprisingly, in the endoplasmic reticulum and the Golgi complex. Polymers did not accumulate within the mitochondrial intermembrane space or the cytosol. We demonstrate the suitability of this compartment-specific NAD⁺ and poly-ADP-ribose turnover to establish intra-organellar protein localization. For overexpressed proteins, genetically endowed with PARP activity, detection of polymers indicates segregation from the cytosol and consequently intra-organellar residence. In mitochondria, polymer build-up reveals matrix localization of the PARP fusion protein. Compared to presently used fusion tags for subcellular protein localization, these are substantial improvements in resolution. We thus established a novel molecular tool applicable for studies of subcellular NAD metabolism and protein localization.     

Interaction and interrelation of P2X7 and P2X4 receptor complexes in mouse lung epithelial cells

P2X4 and P2X7 receptors are ATP-gated ion channels that are co-expressed in alveolar epithelial type I cells. Both receptors are localized to the plasma membrane and partly associated with lipid rafts. Here we report on our study in an alveolar epithelial cell line of the molecular organization of P2X7R and P2X4R receptors and the effect of their knockdown. Native gel electrophoresis reveals three P2X7R complexes of ~430, ~580 and ~760 kDa. The latter two correspond exactly in size to signals of Cav-1, the structural protein of caveolae. Interestingly knockdown of P2rx7 affects protein levels, the intracellular distribution and the supramolecular organization of Cav-1 as well as of P2X4R, which is mainly detected in a complex of ~430 kDa. Our data suggest upregulation of P2X4R as a compensatory mechanism of P2X7R depletion.     

Endocrine and exocrine pancreatic function after camostate-induced growth of the organ

It is well known that oral administration of camostate induces hyperplasia and hypertrophy of the rat pancreas. It is not clear, however, whether pancreatic hormone and enzyme secretion are affected by camostate treatment.In rats, daily administration of 200 mg camostate/kg b. wt for 14 days significantly increased pancreatic weight and pancreatic content of DNA, protein, amylase, lipase, trypsin and chymotrypsin, as well as the amount of insulin, glucagon and somatostatin. In the intact animal, blood glucose levels and serum concentrations of insulin and glucagon in response to an oral glucose load were not impaired after camostate treatment. In the isolated perfused pancreas, however, insulin and glucagon secretions were reduced, whereas somatostatin release was not affected. The volume of pancreatic juice produced by the unstimulated isolated perfused organ, as well as protein and enzyme secretion, were increased after camostate treatment. Likewise, the isolated perfused pancreas from camostate-treated rats secreted a larger volume of pancreatic juice and more protein in response to cholecystokinin (CCK), while enzyme secretion was affected in a non-parallel manner: amylase release was markedly reduced, lipase release was unchanged, and release of trypsin and chymotrypsin was increased.