Paracrine repercussions of preconditioning on angiogenesis and apoptosis of endothelial cells

The mechanisms of cytoprotection conferred by stress preconditioning remain largely uncharacterized in endothelial cells (EC). We report that stress preconditioning of EC with serum starvation induces the release of soluble mediator(s) that confer resistance to apoptosis, increase proliferation, and enhance angiogenesis in a second set of "non-preconditioned" EC. Preconditioning was found to target specifically the mitochondrial control of apoptosis in EC with increased protein levels of Bcl-2, decreased protein levels of Bax, and decreased cytosolic release of cytochrome c. Regulators of apoptosis acting upstream and downstream of the mitochondria such as p53, cIAP-1, cIAP-2, and XIAP were not altered. Mediators classically associated with preconditioning in other cell types such as adenosine, opioids, and nitric oxide are not implicated in this cytoprotective loop. Blockade of protein kinase C-dependent signaling inhibited cytoprotection of EC. Further characterization of this paracrine pathway should provide insights into the molecular regulation of preconditioning in endothelial cells.     

Increased myocardial expression of RAMP1 and RAMP3 in rats with chronic heart failure

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) are potent vasodilators in humans and improved myocardial ischemia is observed after CGRP administration. Receptors for CGRP and ADM were already identified in heart. Receptor activity-modifying proteins (RAMPs) determine the ligand specificity of the calcitonin receptor-like receptor (CRLR); co-expression of RAMP1 and CRLR results in a CGRP receptor, whereas the association of RAMP2 or RAMP3 with CRLR gives an ADM receptor. As CGRP and ADM may play a beneficial role in heart failure, we investigated whether the CGRP and ADM receptors are upregulated in chronic heart failure. We have used semi-quantitative RT-PCR and Western-blot analysis to detect and quantify the mRNA and the protein of RAMP1 and RAMP3 in both atria and ventricles of failing hearts 6 months after aortic banding in rats. Our results showed for the first time an up-regulation of RAMP1 and RAMP3 mRNAs and proteins in this model of cardiac failure. No change was observed in mRNAs coding for CRLR, RAMP2, RDC1 (canine orphan receptor), and ADM. The present results suggested after congestive heart failure in adult rats, an up-regulation of the CGRP receptor (by an increase in RAMP1 that is associated with CRLR) in atria and ventricles and of ADM receptor (by increased RAMP3 expression that is associated with CRLR) in atria. These findings support a functional role for CGRP and ADM receptors to compensate the chronic heart failure in rats.     

NBT-II carcinoma behaviour is not dependent on cell-cell communication through gap junctions

To study the mechanism(s) underlying the proliferation of heterogeneous cell populations within a solid tumour, the NBT-II rat bladder carcinoma system was used. It has been first investigated whether the different cell populations are coupled through gap junctions (GJIC). Cells overexpressing the Cx43 were generated to test for any tumour suppressive activity in vivo. To determine whether GJIC is essential for tumour proliferation and the establishment of a cooperative community effect, NBT-II cells that are incompetent for cell coupling were generated. The data report that (i) carcinoma cells expressing or not FGF-1 are coupled through GJIC in vitro and in coculture and express the gap junction protein Cx43, (ii) overexpression of Cx43 in these cells does not affect their in vitro coupling capacities and in vivo tumourigenic growth properties, (iii) inhibition of GJIC through antisense strategy has no in vivo obvious consequence on the tumour growth properties of the carcinoma, and (iv) the community effect between two carcinoma cell populations does not critically involve cell coupling through gap junctions.     

Exploring the active site of Trypanosoma brucei phosphofructokinase by inhibition studies: specific irreversible inhibition

This work deals with the phosphofructokinase enzyme (PFK) of the parasite Trypanosoma brucei. Inhibitors which are analogues of fructose-6-phosphate (F6P) derived from 2,5-anhydromannitol and therefore blocked in a closed conformation, both nonphosphorylated and phosphorylated, were designed. They provided information on this class of ATP-dependent PFK (structurally more similar to PPi-dependent PFKs revealing (i) an ordered mechanism, ATP binding first, inducing an essential conformational change to increase the affinity for F6P, and (ii) a rather hydrophobic environment at the ATP binding site. Nonphosphorylated mannitol derivatives bind at both the ATP and F6P binding sites, whereas the phosphorylated derivatives only bind at the ATP binding site. The inhibitors bearing an aromatic ring substituted at the meta position indicate a polar interaction with lysine 227, which is specific to T. brucei PFK and is replaced by a glycine in human PFK. This lysine can be irreversibly bound, leading to inhibition when an electrophilic carbon atom is beta to the meta position on the ring. This lysine was identified by site-directed mutagenesis. This first example of a specific irreversible inactivation of T. brucei PFK offers an opportunity to develop biologically active compounds against the sleeping sickness, the causative agent of which is the trypanosome.     

Ripples and the formation of anisotropic lipid domains: imaging two-component supported double bilayers by atomic force microscopy

Direct visualization of the fluid-phase/ordered-phase domain structure in mica-supported bilayers composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphocholine mixtures is performed with atomic force microscopy. The system studied is a double bilayer supported on a mica surface in which the top bilayer (which is not in direct contact with the mica) is visualized as a function of temperature. Because the top bilayer is not as restricted by the interactions with the surface as single supported bilayers, its behavior is more similar to a free-standing bilayer. Intriguing straight-edged anisotropic fluid-phase domains were observed in the fluid-phase/ordered-phase coexistence temperature range, which resemble the fluid-phase/ordered-phase domain patterns observed in giant unilamellar vesicles composed of such phospholipid mixtures. With the high resolution provided by atomic force microscopy, we investigated the origin of these anisotropic lipid domain patterns, and found that ripple phase formation is directly responsible for the anisotropic nature of these domains. The nucleation and growth of fluid-phase domains are found to be directed by the presence of ripples. In particular, the fluid-phase domains elongate parallel to the ripples. The results show that ripple phase formation may have implications for domain formation in biological systems.     

The motion of a single molecule,the lambda-receptor,in the bacterial outer membrane

Using optical tweezers and single particle tracking, we have revealed the motion of a single protein, the lambda-receptor, in the outer membrane of living Escherichia coli bacteria. We genetically modified the lambda-receptor placing a biotin on an extracellular site of the receptor in vivo. The efficiency of this in vivo biotinylation is very low, thus enabling the attachment of a streptavidin-coated bead binding specifically to a single biotinylated lambda-receptor. The bead was used as a handle for the optical tweezers and as a marker for the single particle tracking routine. We propose a model that allows extraction of the motion of the protein from measurements of the mobility of the bead-molecule complex; these results are equally applicable to analyze bead-protein complexes in other membrane systems. Within a domain of radius approximately 25 nm, the receptor diffuses with a diffusion constant of (1.5 +/- 1.0) x 10(-9) cm(2)/s and sits in a harmonic potential as if it were tethered by an elastic spring of spring constant of ~1.0 x 10(-2) pN/nm to the bacterial membrane. The purpose of the protein motion might be to facilitate transport of maltodextrins through the outer bacterial membrane.     

Methylpurine DNA glycosylase of the hyperthermophilic archaeon Archaeoglobus fulgidus

Base excision repair of DNA alkylation damage is initiated by a methylpurine DNA glycosylase (MPG) function. Such enzymes have previously been characterized from bacteria and eukarya, but not from archaea. We identified activity for the release of methylated bases from DNA in cell-free extracts of Archaeoglobus fulgidus, an archaeon growing optimally at 83 degrees C. An open reading frame homologous to the alkA gene of Escherichia coli was overexpressed and identified as a gene encoding an MPG enzyme (M(r) = 34 251), hereafter designated afalkA. The purified AfalkA protein differs from E. coli AlkA by excising alkylated bases only, from DNA, in the following order of efficiency: 3-methyladenine (m(3)A) > 3-methylguanine approximately 7-methyladenine > 7-methylguanine. Although the rate of enzymatic release of m(3)A is highest in the temperature range of 65-75 degrees C, it is only reduced by 50% at 45 degrees C, a temperature that does not support growth of A. fulgidus. At temperatures above 75 degrees C, nonenzymatic release of methylpurines predominates. The results suggest that the biological function of AfalkA is to excise m(3)A from DNA at suboptimal and maybe even mesophilic temperatures. This hypothesis is further supported by the observation that the afalkA gene function suppresses the alkylation sensitivity of the E. coli tag alkA double mutant. The amino acid sequence similarity and evolutionary relationship of AfalkA with other MPG enzymes from the three domains of life are described and discussed.