Chemoattractant receptors activate distinct pathways for chemotaxis and secretion. Role of G-protein usage

Human leukocyte chemoattractant receptors activate chemotactic and cytotoxic pathways to varying degrees and also activate different G-proteins depending on the receptor and the cell-type. To determine the relationship between G-protein usage and the biological and biochemical responses activated, receptors for the chemoattractants formyl peptides (FR), platelet-activating factor (PAFR), and leukotriene B(4) (BLTR) were transfected into RBL-2H3 cells. Pertussis toxin (Ptx) served as a Galpha(i) inhibitor. These receptors were chosen to represent the spectrum of G(i) usage as Ptx had differential effects on their ability to induce calcium mobilization, phosphoinositide hydrolysis, and exocytosis with complete inhibition of all responses by FR, intermediate effects on BLTR, and little effect on PAFR. Ptx did not affect ligand-induced phosphorylation of PAFR and BLTR but inhibited phosphorylation of FR. In contrast, chemotaxis to formylmethionylleucylphenylalanine, leukotriene B(4), and platelet-activating factor was completely blocked by Ptx. Wortmannin, a phosphotidylinositol 3-kinase inhibitor, also completely blocked ligand-induced chemotaxis by all receptors but did not affect calcium mobilization or phosphoinositide hydrolysis; however, it partially blocked the exocytosis response to formylmethionylleucylphenylalanine and the platelet-activating factor. Membrane ruffling and pseudopod extension via the BLTR was also completely inhibited by both Ptx and wortmannin. These data suggest that of the chemoattractant receptors studied, G-protein usage varies with FR being totally dependent on G(i), whereas BLTR and PAFR utilize both G(i) and a Ptx-insensitive G-protein. Both Ptx-sensitive and -insensitive G-protein usage can mediate the activation of phospholipase C, mobilization of intracellular calcium, and exocytosis by chemoattractant receptors. Chemotaxis, however, had an absolute requirement for a G(i)-mediated pathway.     

Genetic differentiation of the Western Capercaillie highlights the importance of South-eastern Europe for understanding the species phylogeography

The Western Capercaillie (Tetrao urogallus L.) is a grouse species of open boreal or high altitude forests of Eurasia. It is endangered throughout most mountain range habitat areas in Europe. Two major genetically identifiable lineages of Western Capercaillie have been described to date: the southern lineage at the species' southernmost range of distribution in Europe, and the boreal lineage. We address the question of genetic differentiation of capercaillie populations from the Rhodope and Rila Mountains in Bulgaria, across the Dinaric Mountains to the Slovenian Alps. The two lineages' contact zone and resulting conservation strategies in this so-far understudied area of distribution have not been previously determined. The results of analysis of mitochondrial DNA control region sequences of 319 samples from the studied populations show that Alpine populations were composed exclusively of boreal lineage; Dinaric populations of both, but predominantly (96%) of boreal lineage; and Rhodope-Rila populations predominantly (>90%) of southern lineage individuals. The Bulgarian mountains were identified as the core area of the southern lineage, and the Dinaric Mountains as the western contact zone between both lineages in the Balkans. Bulgarian populations appeared genetically distinct from Alpine and Dinaric populations and exhibited characteristics of a long-term stationary population, suggesting that they should be considered as a glacial relict and probably a distinct subspecies. Although all of the studied populations suffered a decline in the past, the significantly lower level of genetic diversity when compared with the neighbouring Alpine and Bulgarian populations suggests that the isolated Dinaric capercaillie is particularly vulnerable to continuing population decline. The results are discussed in the context of conservation of the species in the Balkans, its principal threats and legal protection status. Potential conservation strategies should consider the existence of the two lineages and their vulnerable Dinaric contact zone and support the specificities of the populations.     

Controlled pseudopod extension of human neutrophils stimulated with different chemoattractants

The formation of pseudopods and lamellae after ligation of chemoattractant sensitive G-protein coupled receptors (GPCRs) is essential for chemotaxis. Here, pseudopod extension was stimulated with chemoattractant delivered from a micropipet. The chemoattractant diffusion and convection mass transport were considered, and it is shown that when the delivery of chemoattractant was limited by diffusion there was a strong chemoattractant gradient along the cell surface. The diffusion-limited delivery of chemoattractant from a micropipet allowed for maintaining an almost constant chemoattractant concentration at the leading edge of single pseudopods during their growth. In these conditions, the rate of pseudopod extension was dependent on the concentration of chemoattractant in the pipet delivering chemoattractant. The pseudopod extension induced using micropipets was oscillatory even in the presence of a constant delivery of chemoattractant. This oscillatory pseudopod extension was controlled by activated RhoA and its downstream effector kinase ROCK and was abolished after the inhibition of RhoA activation with Clostridium botulinium C3 exoenzyme (C3) or the blocking of ROCK activation with Y-27632. The ability of the micropipet assay to establish a well-defined chemoattractant distribution around the activated cell over a wide range of molecular weights of the used chemoattractants allowed for comparison of the effect of chemoattractant stimulation on the dynamics of pseudopod growth. Pseudopod growth was stimulated using N-formylated peptide (N-formyl-methionyl-leucyl-phenylalanine (fMLP)), platelet activating factor (PAF), leukotriene B4 (LTB(4)), C5a anaphylotoxin (C5a), and interleukin-8 (IL-8), which represent the typical ligands for G-protein coupled chemotactic receptors. The dependence of the rate of pseudopod extension on the concentration of these chemoattractants and their equimolar mixture was measured and shown to be similar for all chemoattractants. The inhibition of the activity of phosphoinositide-3 kinase (PI3K) with wortmannin showed that 72%-80% of the rate of pseudopod extension induced with N-formyl-methionyl-leucyl-phenylalanine, platelet activating factor, and leukotriene B4 was phosphoinositide-3 kinase-dependent, in contrast to 55% of the rate of pseudopod extension induced with interleukin-8. The dependence of the rate of pseudopod extension on the concentration of individual chemoattractants and their equimolar mixture suggests that there is a common rate-limiting mechanism for the polymerization of cytoskeletal F-actin in the pseudopod region induced by G-protein coupled chemoattractant receptors.     

An assay for dielectrophoresis: Applications to electromagnetically induced membrane adhesion and fusion

A new assay for dielectrophoresis, consisting of a platinum wire placed along the axis of symmetry of a hollow metal cylinder, was constructed. The main advantage of this device is that because of the simple axisymmetrical electric field the experimental data can be easily interpreted. Preliminary experimental data for adhesion and fusion of red blood cells are reported. The most interesting observation was that fusion of cell membranes can be induced without applying DC electrical pulses, merely because of the dielectrophoretic effect. A possible explanation of this phenomenon is suggested.     

The small Rho GTPase Rac1 controls normal human dermal fibroblasts proliferation with phosphorylation of the oncoprotein c-myc

Proliferation of dermal fibroblasts is crucial for the maintenance of skin. The small Rho GTPase, Rac1, has been identified as a key transducer of proliferative signals in various cell types, but in normal human dermal fibroblasts its significance to cell growth control has not been studied. In this study, we applied the method of RNA interference to suppress endogenous Rac1 expression and examined the consequences on human skin fibroblasts. Rac1 knock-down resulted in inhibition of DNA synthesis. This effect was not mediated by inhibition of the central transducer of proliferative stimuli, ERK1/2 or by activation of the pro-apoptotic p38. Rather, as a consequence of the suppressed Rac1 expression we observed a significant decrease in phosphorylation of c-myc, revealing for the first time that in human fibroblasts Rac1 exerts control on proliferation through c-myc phosphorylation. Thus Rac1 activates proliferation of normal fibroblasts through stimulation of c-myc phosphorylation without affecting ERK1/2 activity.     

Synthesis and biological activity of conjugates between paclitaxel and the cell delivery vector penetratin

Synthesis of paclitaxel-penetratin (pAntp) constructs, in which the 2'- or 7-position of paclitaxel was used as the attachment site for linker connecting the drug and peptide moieties, is described. Paclitaxel-2'-pAntp[43-58]-NH(2)3b and paclitaxel-2'-pAntp[52-58]-NH(2)3c showed excellent antitumour activity against human lung and breast cancer cell lines. These conjugates were highly soluble and stable with a half-life of >8h under cell culture conditions. The drug-peptide conjugates may be therapeutically useful due to improved pharmaceutical properties.     

Carbamoyl-PROXYL-enhanced MRI detects very small disruptions in brain vascular permeability induced by dietary cholesterol

Background

Gd-DTPA-enhanced magnetic resonance imaging (MRI) is a conventional method for non-invasive investigation of blood-brain-barrier (BBB) permeability in animal models. It allows the visualization of serious injury to the BBB. We developed a novel approach for detecting very small disruptions in BBB permeability induced by dietary cholesterol by using carbamoyl-PROXYL (CMP) as an MRI contrast probe.

Methods

Mice were separated into two groups: normal diet (ND-mice) and high cholesterol diet (CD-mice). MRI-signal dynamics, plasma cholesterol, matrix metalloproteinase (MMP-9, MMP-2), and the white blood cell profile were analyzed. For the MRI analysis, two regions-of-interest (ROI) were selected: brain (ROI-1) and surrounding area (ROI-2).

Results

In the ROI-2 of ND-mice, CMP- or Gd-enhanced MRI-signal followed typical kinetics with a half-life of signal decay (τ_1/2) ~ 8 or ~ 15 min, respectively. In CD-mice, the MRI-signal increased continuously without decay.In the ROI-1 of ND- and CD-mice, MRI-signal enhancement was not detected by Gd-DTPA. In the ROI-1 of ND-mice, CMP-induced MRI-signal enhancement was negligible, while in CD-mice, it was significant (τ_1/2 > 15 min).Hypercholesterolemia increased the plasma levels of MMP-9 and neutrophils.

Conclusions

Hypercholesterolemia increases vascular permeability, which is mediated by MMP-9 and neutrophils.

General significance

Even very small disruptions in brain vascular permeability could be detected by CMP-enhanced MRI but not by Gd-DTPA-enhanced MRI.     

Mechanically stimulated cytoskeleton rearrangement and cortical contraction in human neutrophils.

A mechanical test with micropipets is used to characterize cytoskeleton rearrangement and contraction induced by mechanical stresses in human neutrophils. The yield shear resultant of the cell cortex is on the order of 0.06 to 0.09 mN.m-1. The measured yield shear resultant suggests that the neutrophil cortex is a weakly cross-linked structure. When a tether is pulled out from the cell surface, a polymer structure starts to fill it and spreads out from the cell body. The rate of advancement of the polymerization front is almost constant and, therefore, is not diffusion limited. The measured rate is much smaller than the one of spontaneous actin polymerization, suggesting that the limiting process is either the dissociation of actin monomers from their dimers with the capping proteins or the rate of formation of new nucleation sites or both. Polymerization is also observed after applying sufficient mechanical stresses on a small portion of the cell surface. The polymerization is followed by mass transfer from the cell into the prestressed region and later on by contraction of the main cell body. The pressure generating the flow is located in the prestressed region and most probably is a result of its "swelling" and contraction. The contraction of the main cell body is very similar (in its time dependence and magnitude) to the contraction during phagocytosis. The measured maximum cortical tension is on the order of 0.5 mN.m-1, which for a 3.5-microns diameter pipet corresponds to a maximum contraction force of 11 nN.