The K-Ras 4A isoform promotes apoptosis but does not affect either lifespan or spontaneous tumor incidence in aging mice

Ras proteins function as molecular switches in signal transduction pathways, and, here, we examined the effects of the K-ras4A and 4B splice variants on cell function by comparing wild-type embryonic stem (ES) cells with K-ras(tmDelta4A/tmDelta4A) (exon 4A knock-out) ES cells which express K-ras4B only and K-ras(-/-) (exons 1-3 knock-out) ES cells which express neither splice variant, and intestinal epithelium from wild-type and K-ras(tmDelta4A/tmDelta4A) mice. RT-qPCR analysis found that K-ras4B expression was reduced in K-ras(tmDelta4A/tmDelta4A) ES cells but unaffected in small intestine. K-Ras deficiency did not affect ES cell growth, and K-Ras4A deficiency did not affect intestinal epithelial proliferation. K-ras(tmDelta4A/tmDelta4A) and K-ras(-/-) ES cells showed a reduced capacity for differentiation following LIF withdrawal, and K-ras(-/-) cells were least differentiated. K-Ras4A deficiency inhibited etoposide-induced apoptosis in ES cells and intestinal epithelial cells. However, K-ras(tmDelta4A/tmDelta4A) ES cells were more resistant to etoposide-induced apoptosis than K-ras(-/-) cells. The results indicate that (1) K-Ras4A promotes apoptosis while K-Ras4B inhibits it, and (2) K-Ras4B, and possibly K-Ras4A, promotes differentiation. The findings raise the possibility that alteration of the K-Ras4A/4B isoform ratio modulates tumorigenesis by differentially affecting stem cell survival and/or differentiation. However, K-Ras4A deficiency did not affect life expectancy or spontaneous overall tumor incidence in aging mice.     

An immune response in the bumblebee, Bombus terrestris leads to increased food consumption

Background  

The concept of a costly immune system that must be traded off against other important physiological systems is fundamental to the burgeoning field of ecological immunity. Bumblebees have become one of the central models in this field. Although previous work has demonstrated costs of immunity in numerous life history traits, estimates of the more direct costs of bumblebee immunity have yet to be made.     

Characterization of lectin resistant cell populations derived from human colon carcinoma: correlation of K-Ras with beta1-6 branching of N-linked carbohydrate and CEA production.

Previous studies of cell lines derived from human colon carcinoma showed that the extent of beta1-6 branching on N-linked carbohydrate was associated with the presence of K-ras mutation and Ras-activation. We observed that the extent of Ras-activation in these cell lines depends not only upon the presence of an activating mutation in K-ras, but also on the amount of total K-Ras protein produced. Here we examined whether negative selective pressure by PHA-L against beta1-6 branching could select for cells having a lower level of K-Ras protein and Ras-activation. PHA-L binds specifically to the beta1-6 branch in N-linked carbohydrate. We utilized a K-ras mutant colon carcinoma cell line, HTB39, which had abundant beta1-6 branching and high levels of K-Ras mutant protein. Lectin resistant cell populations of HTB39 were generated and found to have less beta1-6 branching and less K-Ras protein than their parental counterpart. The lectin resistant cell populations produced lower levels of highly glycosylated CEA, which contributed to the lower level of beta1-6 branching in these cells. PHA-L resistant cell populations were two-fold less sensitive than the parental line to an inhibitor of farnesyl transferase (an enzyme essential for Ras processing and function). This suggested a decrease in dependence on K-ras mediated signaling. Collectively, the data indicated that beta1-6 branching of N-linked carbohydrate and CEA production were linked to K-Ras protein synthesis and activation of the Ras-signaling pathway.     

The behavioural responses of badgers (Meles meles) to exclusion from farm buildings using an electric fence

Behavioural investigations into the transmission of bovine tuberculosis (Mycobacterium bovis) between badgers and cattle suggest that badger activity in farm buildings may incur a significant risk of cross-infection. However, measures to exclude badgers from buildings have not been systematically field-tested. In the present study, remote surveillance and radio-tracking were used to monitor the effect of electric fencing manipulations on the frequency of badger incursions into feed stores and cattle housing, and on badger ranging behaviour. Electric fencing was effective in preventing access to the farm buildings where it was installed and also significantly reduced incursions into unfenced buildings. Badger home range and core activity areas tended to increase in size when the fencing was installed, although they did not extend beyond the boundaries of the relevant social group territories. We discuss the logistical constraints of using electric fencing in this context and conclude that it is a potentially useful method of reducing contact between badgers and cattle, within farm buildings and yards.     

Vascular Immunotargeting to Endothelial Determinant ICAM-1 Enables Optimal Partnering of Recombinant scFv-Thrombomodulin Fusion with Endogenous Cofactor

The use of targeted therapeutics to replenish pathologically deficient proteins on the luminal endothelial membrane has the potential to revolutionize emergency and cardiovascular medicine. Untargeted recombinant proteins, like activated protein C (APC) and thrombomodulin (TM), have demonstrated beneficial effects in acute vascular disorders, but have failed to have a major impact on clinical care. We recently reported that TM fused with an scFv antibody fragment to platelet endothelial cell adhesion molecule-1 (PECAM-1) exerts therapeutic effects superior to untargeted TM. PECAM-1 is localized to cell-cell junctions, however, whereas the endothelial protein C receptor (EPCR), the key co-factor of TM/APC, is exposed in the apical membrane. Here we tested whether anchoring TM to the intercellular adhesion molecule (ICAM-1) favors scFv/TM collaboration with EPCR. Indeed: i) endothelial targeting scFv/TM to ICAM-1 provides ∼15-fold greater activation of protein C than its PECAM-targeted counterpart; ii) blocking EPCR reduces protein C activation by scFv/TM anchored to endothelial ICAM-1, but not PECAM-1; and iii) anti-ICAM scFv/TM fusion provides more profound anti-inflammatory effects than anti-PECAM scFv/TM in a mouse model of acute lung injury. These findings, obtained using new translational constructs, emphasize the importance of targeting protein therapeutics to the proper surface determinant, in order to optimize their microenvironment and beneficial effects.     

Ras-dependent apoptosis correlates with persistent activation of stress-activated protein kinases and induction of isoform(s) of Bcl-x

Ras proteins are signal transducers for many cellular responses. However, it is not well established whether Ras-signaling also contributes to apoptosis. We have constructed H-RasR12-transformed Rat1 fibroblasts using tetracycline operator/repressor (TetO/TetR)-based conditional vectors. Rat1/TetO-RasR12 (Rat1-Ras) cells produced high levels of H-RasR12 protein and exhibited oncogenic transformation. Treatment of Rat1-Ras cells with 0.1% serum triggered massive apoptosis. Rat1-Ras cells expressed increased basal activities of extracellular response kinase (ERK) and p46/p54 stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Interestingly, Ras-dependent apoptosis correlated with further persistent activation of both p46 and p54 SAPK/JNK and concurrent inhibition of ERK. Differential modulation of SAPK/JNK and ERK was not detected in tetracycline-treated cells that did not commit apoptosis. Furthermore, two Bcl-x related proteins of 15 kDa and 18 kDa were highly induced in apoptotic Rat1-Ras cells. Our results establish a direct role for Ras in apoptosis, and suggest a functional relationship between H-Ras, SAPK/JNK, ERK and Bcl-x in regulating apoptosis.     

A new model of forelimb ecomorphology for predicting the ancient habitats of fossil turtles

Various morphological proxies have been used to infer habitat preferences among fossil turtles and their early ancestors, but most are tightly linked to phylogeny, thereby minimizing their predictive power. One particularly widely used model incorporates linear measurements of the forelimb (humerus + ulna + manus), but in addition to the issue of phylogenetic correlation, it does not estimate the likelihood of habitat assignment. Here, we introduce a new model that uses intramanual measurements (digit III metacarpal + non‐ungual phalanges + ungual) to statistically estimate habitat likelihood and that has greater predictive strength than prior estimators. Application of the model supports the hypothesis that stem‐turtles were primarily terrestrial in nature and recovers the nanhsiungchelyid Basilemys (a fossil crown‐group turtle) as having lived primarily on land, despite some prior claims to the contrary.     

Non-hinge-binding pyrazolo[1,5-a]pyrimidines as potent B-Raf kinase inhibitors

As part of our research effort to discover B-Raf kinase inhibitors, we prepared a series of C-3 substituted N-(3-(pyrazolo[1,5-a]pyrimidin-7-yl)phenyl)-3-(trifluoromethyl)benzamides. X-ray crystallography studies revealed that one of the more potent inhibitors (10n) bound to B-Raf kinase without forming a hinge-binding hydrogen bond. With basic amine residues appended to C-3 aryl residues, cellular activity and solubility were enhanced over previously described compounds of this class.