PubMed · 6934342
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L W Henderson. 1980. Technical considerations in hemofiltration.. https://pubmed.ncbi.nlm.nih.gov/6934342/
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Phosphorylation can both positively and negatively regulate activity of the Raf kinases. Akt has been shown to phosphorylate and inhibit C-Raf activity. We have recently reported that Akt negatively regulates B-Raf kinase activation by phosphorylating multiple residues within its amino-terminal regulatory domain. Here we investigated the regulation of B-Raf by serum and glucocorticoid-inducible kinase, SGK, which shares close sequence identity with the catalytic domain of Akt but lacks the pleckstrin homology domain. We observed that SGK inhibits B-Raf activity. A comparison of substrate specificity between SGK and Akt indicates that SGK is a potent negative regulator of B-Raf. In contrast to Akt, SGK negatively regulates B-Raf kinase activity by phosphorylating only a single Akt consensus site, Ser(364). Under similar experimental conditions, SGK displays a measurably stronger inhibitory effect on B-Raf kinase activity than Akt, whereas Akt exhibits a more inhibitory effect on the forkhead transcription factor, FKHR. The selective substrate specificity is correlated with an enhanced association between Akt or SGK and their preferred substrates, FKHR and B-Raf, respectively. These results indicate that B-Raf kinase activity is negatively regulated by Akt and SGK, suggesting that the cross-talk between the B-Raf and other signaling pathways can be mediated by both Akt and SGK.
BACKGROUND: Most myocardial infarctions are caused by the rupture of small rather than large plaques in the arteries of the heart that are beyond the detection limit of current technologies. METHODS AND RESULTS: Recently, optical coherence tomography (OCT) has demonstrated considerable potential as a method for high-resolution assessment of vulnerable plaque. However, intravascular OCT imaging is complicated by the need to remove blood from the imaging field because blood results in substantial signal attenuation. This work examines index matching as a method for increasing penetration. Index matching is based on the hypothesis that the predominant source of scattering in blood is the difference in refractive index between the cytoplasm of erythrocytes and serum. By increasing the refractive index of serum to a value near that of the cytoplasm, or index matching, scattering can be substantially reduced. The concept was tested with a system that pumped blood in vitro through transparent tubing. The test compounds, dextran and intravenous contrast agent, both led to significant improvements in penetration (69+/-12% and 45+/-4%). No significant effect was seen with the saline control. For dextran, the effect could not be attributed to reductions of red cell number or volume because changes in these parameters were not different from the control. In the case of intravenous contrast, a small but significant relative reduction in red cell volume was seen. CONCLUSIONS: This study demonstrates the feasibility of index matching for improving OCT imaging through blood. Future studies are required to identify compounds for effective index matching in vivo.
We investigated the mode of interaction of lipoplexes (DOTAP:DOPE/DNA) with HeLa cells, focusing on the analysis of the initial steps involved in the process of gene delivery. We evaluated the effect of different factors, namely the stoichiometry of cationic lipids and DNA, the presence of serum in the cell culture medium, and the incorporation of the ligand transferrin into the lipoplexes, on the extent of binding, association and fusion (lipid mixing) of the lipoplexes with the cells. Parallel experiments were performed upon cell treatment with inhibitors of endocytosis. Our results indicate that a decrease of the net charge of the complexes (upon addition of DNA) generally leads to a decrease in the extent of binding, cell association and fusion, except for the neutral complexes. Association of transferrin to the lipoplexes resulted in a significant enhancement of the interaction processes referred to above, which correlates well with the promotion of transfection observed under the same conditions. Besides triggering internalization of the complexes, transferrin was also shown to mediate fusion with the endosomal membrane. The extent of fusion of this type of complexes was reduced upon their incubation with cells in the presence of serum, suggesting that serum components limit the transferrin fusogenic properties. Results were analyzed by using a theoretical model which allowed to estimate the kinetic parameters involved in lipoplex--cell interactions. The deduced fusion and endocytosis rate constants are discussed and compared with those obtained for other biological systems. From the kinetic studies we found a twofold enhancement of the fusion rate constant (f) for the ternary lipoplexes. We also concluded that HeLa cells yield a relatively low rate of endocytosis. Overall, our results estimate the relative contribution of fusion of lipoplexes with the plasma membrane, endocytosis and fusion with the endosomal membrane to their interactions with cells, this information being of crucial importance for the development of gene therapy strategies.