Genetics of VEGF serum variation in human isolated populations of cilento: importance of VEGF polymorphisms

Vascular Endothelial Growth Factor (VEGF) is the main player in angiogenesis. Because of its crucial role in this process, the study of the genetic factors controlling VEGF variability may be of particular interest for many angiogenesis-associated diseases. Although some polymorphisms in the VEGF gene have been associated with a susceptibility to several disorders, no genome-wide search on VEGF serum levels has been reported so far. We carried out a genome-wide linkage analysis in three isolated populations and we detected a strong linkage between VEGF serum levels and the 6p21.1 VEGF region in all samples. A new locus on chromosome 3p26.3 significantly linked to VEGF serum levels was also detected in a combined population sample. A sequencing of the gene followed by an association study identified three common single nucleotide polymorphisms (SNPs) influencing VEGF serum levels in one population (Campora), two already reported in the literature (rs3025039, rs25648) and one new signal (rs3025020). A fourth SNP (rs41282644) was found to affect VEGF serum levels in another population (Cardile). All the identified SNPs contribute to the related population linkages (35% of the linkage explained in Campora and 15% in Cardile). Interestingly, none of the SNPs influencing VEGF serum levels in one population was found to be associated in the two other populations. These results allow us to exclude the hypothesis that the common variants located in the exons, intron-exon junctions, promoter and regulative regions of the VEGF gene may have a causal effect on the VEGF variation. The data support the alternative hypothesis of a multiple rare variant model, possibly consisting in distinct variants in different populations, influencing VEGF serum levels.     

Subplasmalemmal mitochondria modulate the activity of plasma membrane Ca2+-ATPases

Mitochondria are dynamic organelles that modulate cellular Ca2+ signals by interacting with Ca2+ transporters on the plasma membrane or the endoplasmic reticulum (ER). To study how mitochondria dynamics affects cell Ca2+ homeostasis, we overexpressed two mitochondrial fission proteins, hFis1 and Drp1, and measured Ca2+ changes within the cytosol and the ER in HeLa cells. Both proteins fragmented mitochondria, decreased their total volume by 25-40%, and reduced the fraction of subplasmalemmal mitochondria by 4-fold. The cytosolic Ca2+ signals elicited by histamine were unaltered in cells lacking subplasmalemmal mitochondria as long as Ca2+ was present in the medium, but the signals were significantly blunted when Ca2+ was removed. Upon Ca2+ withdrawal, the free ER Ca2+ concentration decreased rapidly, and hFis1 cells were unable to respond to repetitive histamine stimulations. The loss of stored Ca2+ was due to an increased activity of plasma membrane Ca2+-ATPase (PMCA) pumps and was associated with an increased influx of Ca2+ and Mn2+ across store-operated Ca2+ channels. The increased Ca2+ influx compensated for the loss of stored Ca2+, and brief Ca2+ additions between successive agonist stimulations fully corrected subsequent histamine responses. We propose that the lack of subplasmalemmal mitochondria disrupts the transfer of Ca2+ from plasma membrane channels to the ER and that the resulting increase in subplasmalemmal [Ca2+] up-regulates the activity of PMCA. The increased Ca2+ extrusion promotes ER depletion and the subsequent activation of store-operated Ca2+ channels. Cells thus adapt to the lack of subplasmalemmal mitochondria by relying on external rather than on internal Ca2+ for signaling.     

Annexin in mineralization process

Annexins are necessary for mineralization process. They seem to play a major role in regulation of cells competent in mineralization as well as in direct formation of mineral phase in the extracellular matrix. Their ability to accommodate to different functions in different cellular compartments is associated with their property to bind to biological membranes in a lipid- and Ca2+-dependent and independent manners. The aim of this review is to describe potential functions of the annexin family of proteins in a mineralization process with special emphasis to structure-function relationships of annexins.     

The role of RuvA octamerization for RuvAB function in vitro and in vivo

RuvA plays an essential role in branch migration of the Holliday junction by RuvAB as part of the RuvABC pathway for processing Holliday junctions in Escherichia coli. Two types of RuvA-Holliday junction complexes have been characterized: 1) complex I containing a single RuvA tetramer and 2) complex II in which the junction is sandwiched between two RuvA tetramers. The functional differences between the two forms are still not clear. To investigate the role of RuvA octamerization, we introduced three amino acid substitutions designed to disrupt the E. coli RuvA tetramer-tetramer interface as identified by structural studies. The mutant RuvA was tetrameric and interacted with both RuvB and junction DNA but, as predicted, formed complex I only at protein concentrations up to 500 nm. We present biochemical and surface plasmon resonance evidence for functional and physical interactions of the mutant RuvA with RuvB and RuvC on synthetic junctions. The mutant RuvA with RuvB showed DNA helicase activity and could support branch migration of synthetic four-way and three-way junctions. However, junction binding and the efficiency of branch migration of four-way junctions were affected. The activity of the RuvA mutant was consistent with a RuvAB complex driven by one RuvB hexamer only and lead us to propose that one RuvA tetramer can only support the activity of one RuvB hexamer. Significantly, the mutant failed to complement the UV sensitivity of E. coli DeltaruvA cells. These results indicate strongly that RuvA octamerization is essential for the full biological activity of RuvABC.