A new family of highly variable proteins in the Chlamydophila pneumoniae genome

Chlamydiaceae are obligate intracellular bacterial pathogens characterized by a wide range of vertebrate host, tissue tropism and spectrum of diseases. To get insights into the biological mechanisms involved in these differences, we have put forward a computational and experimental procedure to identify the genome recombination hotspots, as frequent sequence variation allows rapid adaptation to environmental changes. We find a larger potential for recombination in Chlamydophila pneumoniae genomes as compared with Chlamydia trachomatis or Chlamydia muridarum. Such potential is mostly concentrated in a family of seven previously uncharacterized species-specific elements that we named ppp for C.pneumoniae polymorphic protein genes, which have the potential to vary by homologous recombination and slipped-mispair. Experimentally, we show that these sequences are indeed highly polymorphic among a collection of nine C.pneumoniae strains of very diverse geographical and pathological origins, mainly by slippage of a poly(C) tract. We also show that most elements are transcribed during infection. In silico analyses suggest that Ppps correspond to outer membrane proteins. Given their species specificity, their putative location in the outer membrane and their extreme polymorphism, Ppps are most likely to be important in the pathogenesis of C.pneumoniae and could represent targets for future vaccine development.     

M13 endopeptidases: New conserved motifs correlated with structure,and simultaneous phylogenetic occurrence of PHEX and the bony fish

M13 endopeptidase alignments have focused mainly on mammalian sequences and on the active site region defining the catalytic sequence signatures. Aligning all available M13 from bacteria to human on a full-length basis, we have performed a sequence analysis. This enabled us to highlight the origin and function of the M13 PHEX subtype family endopeptidase (phosphate regulating gene with homologies to endopeptidases on the X chromosome). New evolutionary conserved regions in both prokaryotes and eukaryotes have been detected and eukaryotic-specific regions clearly delineated. Using the recently solved neprilysin structure, we have observed that all new motifs, except one, localize in the spatial vicinity of the previously reported catalytic signatures. Interestingly, a highly hydrophobic pocket containing three newly reported motifs is centered by the C-terminal tryptophan residue. Extensive M13 searches in complete and in progress higher eukaryotic genomes have lead to the identification of Danio rerio as the simplest organism having PHEX. Finally, the human PHEX substrate, the parathyroid hormone-related peptide, PTHrP(107-139), is absent in bony fish: this suggests the existence of further PHEX substrates common to both bony fishes and higher vertebrates.     

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.     

Semiparametric regression modeling with mixtures of Berkson and classical error, with application to fallout from the Nevada test site

We construct Bayesian methods for semiparametric modeling of a monotonic regression function when the predictors are measured with classical error. Berkson error, or a mixture of the two. Such methods require a distribution for the unobserved (latent) predictor, a distribution we also model semiparametrically. Such combinations of semiparametric methods for the dose response as well as the latent variable distribution have not been considered in the measurement error literature for any form of measurement error. In addition, our methods represent a new approach to those problems where the measurement error combines Berkson and classical components. While the methods are general, we develop them around a specific application, namely, the study of thyroid disease in relation to radiation fallout from the Nevada test site. We use this data to illustrate our methods, which suggest a point estimate (posterior mean) of relative risk at high doses nearly double that of previous analyses but that also suggest much greater uncertainty in the relative risk.     

Asymmetrical membranes and surface tension

The (31)P-nuclear magnetic resonance chemical shift of phosphatidic acid in a membrane is sensitive to the lipid head group packing and can report qualitatively on membrane lateral compression near the aqueous interface. We have used high-resolution (31)P-nuclear magnetic resonance to evaluate the lateral compression on each side of asymmetrical lipid vesicles. When monooleoylphosphatidylcholine was added to the external monolayer of sonicated vesicles containing dioleoylphosphatidylcholine and dioleoylphosphatidic acid, the variation of (31)P chemical shift of phosphatidic acid indicated a lateral compression in the external monolayer. Simultaneously, a slight dilation was observed in the inner monolayer. In large unilamellar vesicles on the other hand the lateral pressure increased in both monolayers after asymmetrical insertion of monooleoylphosphatidylcholine. This can be explained by assuming that when monooleoylphosphatidylcholine is added to large unilamellar vesicles, the membrane bends until the strain is the same in both monolayers. In the case of sonicated vesicles, a change of curvature is not possible, and therefore differential packing in the two layers remains. We infer that a variation of lipid asymmetry by generating a lateral strain in the membrane can be a physiological way of modulating the conformation of membrane proteins.     

Thermal unfolding used as a probe to characterize the intra- and intersubunit stabilizing interactions in phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus

Tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus can be described as a dimer of dimers with three nonequivalent interfaces. To investigate the contribution of intra- and intersubunit interactions to GAPDH thermostability, 10 residues located either at the cofactor domain (amino acids 1-148 and 313-333) or at the catalytic domain (amino acids 149-312) were mutated and the thermal unfolding of the mutants was studied by differential scanning calorimetry in the absence and presence of saturating concentrations of NAD. Disruptions of intrasubunit interactions lead to a drastic decrease in thermostability of the N313T, Y283V, and W310F mutants. Moreover, for the N313T mutant, a weakening of cooperative interactions between the catalytic and the cofactor domains and an inefficient binding of NAD are observed. This is likely the consequences of modification or loss of the hydrogen bonding network associating N313 and residues 236-238 and N313 and the nicotinamide carboxyamide of NAD, respectively. For the residues Y283 and W310, which are involved in stacking hydrophobic interactions, mutating both positions does not affect the efficiency of NAD binding. This shows that the factors involved in the thermostability of the tetrameric apo GAPDH are then different from those induced by NAD binding. Disruption of intersubunit hydrogen bonds between the catalytic domain and the NAD-binding domain of a neighboring subunit also leads to a significant destabilization of the apo tetrameric form as observed for the D282G mutant. Moreover, no efficient binding of NAD is observed. Both results are likely the consequence of a loss of hydrogen bonds across the P-axis and the Q-axis between D282 and R197 and between D282 and R52, respectively. Similar results, i.e., a destabilizing effect and inefficient NAD binding, are observed with the T34Q/T39S/L43Q mutant in which steric hindrance is introduced at the S-loop of the R-axis-related subunit via mutations at the adenosine subsite. The dimeric form of the D282G mutant exhibits a single partial heat absorption peak, whereas the Y46G/R52G mutant which exists only as a dimer shows two peaks. Taking into account the recent small-angle X-ray scattering studies which suggested that the dimeric form of the D282G mutant and of the dimeric Y46G/R52G mutant are of the O-R and O-P types, respectively (Vachette, unpublished results), we propose that the presence of one or two peaks in thermal unfolding of dimers is a signature of the dimer type.     

Early-glycation of apolipoprotein E: effect on its binding to LDL receptor,scavenger receptor A and heparan sulfates

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) causes substantial morbidity afflicting approximately 10% of adult males. Treatment is often empirical and ineffective since the etiology is unknown. Other prostate and genitourinary diseases have genetic components suggesting that CP/CPPS may also be influenced by genetic predisposition. We recently reported a highly polymorphic short tandem repeat (STR) locus near the phosphoglycerate kinase gene within Xq11-13. Because this STR is in a region known to predispose towards other prostate diseases, we compared STR polymorphisms in 120 CP/CPPS patients and 300 control blood donors. Nine distinct allele sizes were detected, ranging from 8 to 15 repeats of the tetrameric STR plus a mutant allele (9.5) with a six base deletion in the flanking DNA sequence. The overall allele size distribution in the CP/CPPS patients differed from controls (Chi-square=19.252, df=8, P=0.0231). Frequencies of two specific alleles, 9.5 and 15, differed significantly in CP/CPPS vs. control subjects and allele 10 differed with marginal significance. Alleles 9.5 and 10 were both more common in CP/CPPS patients than controls while allele 15 was less common. These observations suggest that Xq11-13 may contain one or more genetic loci that predispose toward CP/CPPS. Further investigations involving family studies, larger patient populations, and other control groups may help elucidate this potential genetic predisposition in CP/CPPS.     

Biodistribution of long-circulating PEG-liposomes in a murine model of established subcutaneous abscesses

The biodistribution of long-circulating PEG-liposomes in a subcutaneous mouse model of established mixed infection abscesses was investigated to assess their possible role as drug carriers in the treatment of small, undrainable intra-abdominal abscesses. There was a 10-30-fold greater localisation of (67)Ga-labelled PEG-liposomes in abscesses compared to uninfected normal skin samples. Over 3% of the injected dose (ID) of liposomes was present in the abscesses 24 h after liposome administration in contrast to 0.1% in normal skin sections. The percentage ID present in the liver, spleen and kidneys was 17%, 4% and 2% per organ respectively. Five days after liposome injection, 2% ID could still be recovered from the abscesses. Using colloidal gold-labelled PEG-liposomes, it was shown that there was a 4-fold greater density of liposome clusters in the subcutaneous tissue surrounding the capsule than in the core of the abscesses. The clusters within the abscesses were distributed evenly. We conclude that PEG-liposomes localise to a significant degree at the infection focus in our mouse model and may provide a new approach to the antimicrobial treatment of intra-abdominal abscesses.