Regulation of membrane functions and fatty acid composition in Escherichia coli by cyclic AMP receptor protein.

Adenosine 3′,5′-cyclic monophosphate (cAMP) has been shown to be involved in regulating a number of membrane functions in Escherichia coli cells, including various respiratory and transport activities. In this report we show that this regulation is mediated by the cAMP receptor protein (CRP). In addition, data are presented which show that the cAMP-CRP system is involved in regulating the E. coli fatty acid composition.     

Bcl-xL mediates a survival mechanism independent of the phosphoinositide 3-kinase/Akt pathway in prostate cancer cells

Among various molecular strategies by which prostate cancer cells evade apoptosis, phosphoinositide 3-kinase (PI3K)/Akt signaling represents a dominant survival pathway. However, different prostate cancer cell lines such as LNCaP and PC-3 display differential sensitivity to the apoptotic effect of PI3K inhibition in serum-free media, reflecting the heterogeneous nature of prostate cancer in apoptosis regulation. Whereas both cell lines are equally susceptible to LY294002-mediated Akt dephosphorylation, only LNCaP cells default to apoptosis, as evidenced by DNA fragmentation and cytochrome c release. In PC-3 cells, Akt deactivation does not lead to cytochrome c release, suggesting that the intermediary signaling pathway is short-circuited by an antiapoptotic factor. This study presents evidence that Bcl-xL overexpression provides a distinct survival mechanism that protects PC-3 cells from apoptotic signals emanating from PI3K inhibition. First, the Bcl-xL/BAD ratio in PC-3 cells is at least an order of magnitude greater than that of LNCaP cells. Second, ectopic expression of Bcl-xL protects LNCaP cells against LY294002-induced apoptosis. Third, antisense down-regulation of Bcl-xL sensitizes PC-3 cells to the apoptotic effect of LY294002. The physiological relevance of this Bcl-xL-mediated survival mechanism is further underscored by the protective effect of serum on LY294002-induced cell death in LNCaP cells, which is correlated with a multifold increase in Bcl-xL expression. In contrast to Bcl-xL, Bcl-2 expression levels are similar in both cells lines, and do not respond to serum stimulation, suggesting that Bcl-2 may not play a physiological role in antagonizing apoptosis signals pertinent to BAD activation in prostate cancer cells.     

Angiotensin II induces complex fractionated electrogram in a cultured atrial myocyte monolayer mediated by calcium and sodium-calcium exchanger

Angiotensin II (AngII) has been implicated in the mechanism of atrial fibrillation (AF). There may be calcium-dependent pro-fibrillatory effect of AngII on atrial myocytes. We used cultured confluent HL-1 atrial myocyte monolayer with spontaneously propagated depolarization to study direct pro-fibrillatory effect of AngII and its molecular mechanism. AngII stimulation induced fibrillatory-like complex electrogram and calcium wave propagation. AngII shortened action potential duration and augmented calcium transient, thus increasing electrochemical gradient of forward-mode sodium-calcium exchanger (NCX) current and induced frequent irregular afterdepolarizations. AngII increased expression of sodium-calcium exchanger (NCX), further increasing calcium-membrane voltage coupling gain. The fibrillatory effect of AngII was attenuated by NCX blocker SEA0400 and NCX siRNA knockdown. AngII increased expression of L-type calcium channel and augmented calcium transient through PKC and CREB. The fibrillatory effect of AngII was also attenuated by PKC inhibitor chelerythrine and dominant negative form of CREB. In conclusions, AngII itself may electrically contribute to the mechanism of AF through increasing NCX expression and augmenting calcium transient, which is PKC and CREB dependent. Specific genetic knockdown of NCX attenuated calcium mediated afterdepolarization and complex electrogram.     

Crosslinking CD81 results in activation of TCRgammadelta T cells

CD81 is expressed on most cells and is associated with other glycoproteins, including CD4 and CD8, to form multimolecular membrane complexes. Crosslinking of CD81 on TCRalphabeta(+) T cells results in costimulatory signals that have been proposed to be mediated via CD4 or CD8. In this study, we show that CD81 is also expressed on TCRgammadelta(+)CD4(-)CD8(-) T cells. CD81 crosslinking greatly enhanced anti-CD3 activation of both TCRalphabeta(+) (CD4+ and CD8+) and TCRgammadelta(+) T cells with regard to IFN-gamma production. However, crosslinking of CD81 molecules on TCRgammadelta(+) T cells, in the absence of anti-CD3 stimulation, resulted in cytokine production and enhanced IL-2-induced proliferation, demonstrating that physical association with CD4 or CD8 is not necessary for CD81 signaling. In contrast, crosslinking of CD81 on TCRalphabeta(+) T cells, in the absence of anti-CD3 stimulation, failed to activate these T cells. These results suggest that CD81 signaling may be mediated via a different mechanism(s) in TCRgammadelta(+) versus TCRalphabeta(+) T cells.     

DNA cloning without restriction enzyme and ligase

One common problem in using the traditional DNA cloning procedure is that suitable natural restriction sites are often unavailable for a given task. Creating new restriction sites is often time consuming. Here, I describe a simple technique of producing "customized cohesive ends" by a combination of PCR primer design and lambda exonuclease digestion. These complementary cohesive ends can form hybrids to link two sequences. Because the overhangs created by lambda exonuclease are slightly longer than the complementary sequence, after hybrid formation, a stretch of single-strand gap remains, which then is repaired by Klenow (3'-->5' exo-) enzyme. The repair process also stabilizes the linkage. Because of the independence from natural or artificial restriction sites, this method allows rapid and precise insertion of one DNA fragment into another at virtually any position. It also simplifies the planning of a cloning strategy, increases recombinant frequency and is suitable for automation.