Rho kinase regulates the intracellular micromechanical response of adherent cells to rho activation

Local sol-gel transitions of the cytoskeleton modulate cell shape changes, which are required for essential cellular functions, including motility and adhesion. In vitro studies using purified cytoskeletal proteins have suggested molecular mechanisms of regulation of cytoskeleton mechanics; however, the mechanical behavior of living cells and the signaling pathways by which it is regulated remains largely unknown. To address this issue, we used a nanoscale sensing method, intracellular microrheology, to examine the mechanical response of the cell to activation of the small GTPase Rho. We observe that the cytoplasmic stiffness and viscosity of serum-starved Swiss 3T3 cells transiently and locally enhances upon treatment with lysophosphatidic acid, and this mechanical behavior follows a trend similar to Rho activity. Furthermore, the time-dependent activation of Rho decreases the degree of microheterogeneity of the cytoplasm. Our results reveal fundamental differences between intracellular elasticity and cellular tension and suggest a critical role for Rho kinase in the regulation of intracellular mechanics.     

<Emphasis Type="Italic">Sporotomaculum syntrophicum</Emphasis> sp. nov., a novel anaerobic,syntrophic benzoate-degrading bacterium isolated from methanogenic sludge treating wastewater from terephthalate manufacturing

An anaerobic, mesophilic, syntrophic benzoate-degrading bacterium, designated strain FB(T), was isolated from methanogenic sludge which had been used to treat wastewater from the manufacture of terephthalic acid. Cells were non-motile gram-positive rods that formed spores. The optimum temperature for growth was 35-40 degrees C, and the optimum pH was 7.0-7.2. A co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei converted benzoate to acetate, carbon dioxide, and methane. Butyrate transiently accumulated at a high concentration of 2.5 mM during degradation. Besides benzoate, no other compound tested supported growth of the co-culture. Crotonate supported growth of strain FB(T) in pure culture. Furthermore, the strain degraded benzoate in pure culture with crotonate as co-substrate to produce acetate and butyrate. The strain was not able to utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, or Fe(III) as electron acceptor. The G+C content of the DNA was 46.8 mol%. Strain FB(T) contained MK-7 as the major quinone and C(16:1) as the major fatty acid. 16S rDNA sequence analysis revealed that the strain was a member of the genus Sporotomaculum, even though it exhibited significant differences, such as the capacity for syntrophic growth, to the known member of the genus. Hence, we propose the name Sporotomaculum syntrophicum sp. nov. for strain FB(T). The type strain is strain FB(T) (DSM 14795, JCM 11475).     

Host-based divergence in populations of the pea aphid: insights from nuclear markers and the prevalence of facultative symbionts

In North America, the pea aphid Acyrthosiphon pisum encompasses ecologically and genetically distinct host races that offer an ideal biological system for studies on sympatric speciation. In addition to its obligate symbiont Buchnera, pea aphids harbour several facultative and phylogenetically distant symbionts. We explored the relationships between host races of A. pisum and their symbiotic microbiota to gain insights into the historical process of ecological specialization and symbiotic acquisition in this aphid. We used allozyme and microsatellite markers to analyse the extent of genetic differentiation between populations of A. pisum on pea, alfalfa and clover in France. In parallel, we examined: (i) the distribution of four facultative symbionts; and (ii) the genetic variation in the Buchnera genome across host-associated populations of A. pisum. Our study clearly demonstrates that populations of A. pisum on pea, clover and alfalfa in France are genetically divergent, which indicates that they constitute distinct host races. We also found a very strong association between host races of A. pisum and their symbiotic microbiota. We stress the need for phylogeographic studies to shed light on the process of host-race formation and acquisition of facultative symbionts in A. pisum. We also question the effects of these symbionts on aphid host fitness, including their role in adaptation to a host plant.     

Response of Penaeus indicus females at two different stages of ovarian development to a lethal infection with Vibrio penaeicida

An association between vitellogenesis and the immune system was suggested in crustaceans from studies on plasma lipoproteins. The present research studies the effect of an experimentally induced bacterial infection on vitellogenesis in females of the shrimp Penaeus indicus, as a model for penaeid species. Pre-vitellogenic and vitellogenic P. indicus females were experimentally infected with an extremely pathogenic bacterium, Vibrio penaeicida. The peak in mortality occurred earlier in pre-vitellogenic animals than in vitellogenic ones, although the final mortality level ( approximately 64-74%) 52h post-infection was nearly the same for the two groups. Twenty hours after infection, the total number of haemocytes was significantly reduced in vitellogenic females while there was no change in the pre-vitellogenic group. Protein synthesis in ovaries was not significantly affected by infection, at the two stages of ovarian development. No differences were found in mRNA levels of shrimp ovarian peritrophin protein (SOP), but preliminary results showed that mRNA expression of vitellin (VT) was reduced in a heavily infected vitellogenic female. The total amount of lipids in the haemolymph of vitellogenic females was almost twice higher than that of pre-vitellogenic ones. However, there was no change in the total content of lipids, lipid classes and fatty acid distribution in haemolymph or hepatopancreas following infection. Although vitellogenic and pre-vitellogenic females probably respond differently to a lethal bacterial infection, physiological differences may be concealed by the rapid onset of mortality.     

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.     

APP processing is regulated by cytoplasmic phosphorylation

Amyloid-beta peptide (Abeta) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers and the beta-secretase, BACE1. Western blot analysis reveals increased levels of T668-phosphorylated APP COOH-terminal fragments in hippocampal lysates from many AD but not control subjects. Importantly, P-APP cofractionates with endosome markers and BACE1 in an iodixanol gradient and displays extensive colocalization with BACE1 in rat primary cortical neurons. Furthermore, APP COOH-terminal fragments generated by BACE1 are preferentially phosphorylated on T668 verses those produced by alpha-secretase. The production of Abeta is significantly reduced when phosphorylation of T668 is either abolished by mutation or inhibited by T668 kinase inhibitors. Together, these results suggest that T668 phosphorylation may facilitate the BACE1 cleavage of APP to increase Abeta generation.     

Ha-ras overexpression mediated cell apoptosis in the presence of 5-fluorouracil

By using a mouse NIH3T3 derivate designed 7-4 harboring the inducible Ha-ras oncogene, we demonstrated the close relationship between Ha-ras expression level and sensitization of 5-flurouracil (5-FU)-treated cells. Further studies revealed that the cells susceptible to 5-FU treatment died of apoptosis, which was demonstrated by caspase-3 activation, loss of mitochondria membrane potential (MMP), and DNA fragmentation. The 7-4 cells coexpressing dominant negative Ras (Ras(Asn17)), dominant negative Raf-1 (Raf-1(CB4)), Bcl-2, or active form of phosphatidylinositol 3-kinase (PI3K) became resistant to 5-FU, and apoptosis was prevented. In contrast, the cells coexpressing dominant negative Rac 1 (Rac1(Asn17)) or dominant negative Rho A (RhoA(Asn19)) showed no change of sensitivity to 5-FU. These results indicate that Ras, Bcl-2, as well as Raf-1 and PI3K pathways play pivotal roles in 5-FU-induced apoptosis under Ha-ras-overexpressed condition. Aberrant levels of cyclin E and p21(Cip/WAF-1) expression as well as Cdc 2 phosphorylation at Tyrosine 15 suggest that perturbation of G1/S and G2/M transitions in cell cycle might be responsible for 5-FU triggered apoptosis. Sensitization of Ha-ras-related cells to 5-FU was also demonstrated in human bladder cancer cells. Through understanding the mechanism of 5-FU induced apoptosis in tumor cells, a new direction toward the treatment of Ha-ras oncogene-related cancers with 5-FU at more optimal dosages is possible and combinational therapy with other drugs that suppress PI3K and Bcl-2 activities can also be considered.     

Single molecule characterization of P-selectin/ligand binding

P-selectin expressed on activated platelets and vascular endothelium mediates adhesive interactions to polymorphonuclear leukocytes (PMNs) and colon carcinomas critical to the processes of inflammation and blood-borne metastasis, respectively. How the overall adhesiveness (i.e. the avidity) of receptor/ligand interactions is controlled by the affinity of the individual receptors to single ligands is not well understood. Using single molecule force spectroscopy, we probed in situ both the tensile strength and off-rate of single P-selectin molecules binding to single ligands on intact human PMNs and metastatic colon carcinomas and compared them to the overall avidity of these cells for P-selectin substrates. The use of intact cells rather than purified proteins ensures the proper orientation and preserves post-translational modifications of the P-selectin ligands. The P-selectin/PSGL-1 interaction on PMNs was able to withstand forces up to 175 pN and had an unstressed off-rate of 0.20 s(-1). The tensile strength of P-selectin binding to a novel O-linked, sialylated protease-sensitive ligand on LS174T colon carcinomas approached 125 pN, whereas the unstressed off-rate was 2.78 s(-1). Monte Carlo simulations of receptor/ligand bond rupture under constant loading rate for both P-selectin/PSGL-1 and P-selectin/LS174T ligand binding give distributions and mean rupture forces that are in accord with experimental data. The pronounced differences in the affinity for P-selectin/ligand binding provide a mechanistic basis for the differential abilities of PMNs and carcinomas to roll on P-selectin substrates under blood flow conditions and underline the requirement for single molecule affinity measurements.