The bovine seminal plasma protein PDC-109 extracts phosphorylcholine-containing lipids from the outer membrane leaflet

The bovine seminal plasma protein PDC-109 modulates the maturation of bull sperm cells by removing lipids, mainly phosphatidylcholine and cholesterol, from their cellular membrane. Here, we have characterized the process of extraction of endogenous phospholipids and of their respective analogues. By measuring the PDC-109-mediated release of fluorescent phospholipid analogues from lipid vesicles and from biological membranes (human erythrocytes, bovine epididymal sperm cells), we showed that PDC-109 extracts phospholipids with a phosphorylcholine headgroup mainly from the outer leaflet of these membranes. The ability of PDC-109 to extract endogenous phospholipids from epididymal sperm cells was followed by mass spectrometry, which allowed us to characterize the fatty acid pattern of the released lipids. From these cells, PDC-109 extracted phosphatidylcholine and sphingomyelin that contained an enrichment of mono- and di-unsaturated fatty acids as well as short-chain and lyso-phosphatidylcholine species. Based on the results, a model explaining the phospholipid specificity of PDC-109-mediated lipid release is presented. Astrid Tannert and Anke Kurz have contributed equally to this work. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Effects of High Fat Feeding and Diabetes on Regression of Atherosclerosis Induced by Low-Density Lipoprotein Receptor Gene Therapy in LDL Receptor-Deficient Mice

We tested whether a high fat diet (HFD) containing the inflammatory dietary fatty acid palmitate or insulin deficient diabetes altered the remodeling of atherosclerotic plaques in LDL receptor knockout (Ldlr^-/-) mice. Cholesterol reduction was achieved by using a helper-dependent adenovirus (HDAd) carrying the gene for the low-density lipoprotein receptor (Ldlr; HDAd-LDLR). After injection of the HDAd-LDLR, mice consuming either HFD, which led to insulin resistance but not hyperglycemia, or low fat diet (LFD), showed regression compared to baseline. However there was no difference between the two groups in terms of atherosclerotic lesion size, or CD68+ cell and lipid content. Because of the lack of effects of these two diets, we then tested whether viral-mediated cholesterol reduction would lead to defective regression in mice with greater hyperglycemia. In both normoglycemic and streptozotocin (STZ)-treated hyperglycemic mice, HDAd-LDLR significantly reduced plasma cholesterol levels, decreased atherosclerotic lesion size, reduced macrophage area and lipid content, and increased collagen content of plaque in the aortic sinus. However, reductions in anti-inflammatory and ER stress-related genes were less pronounced in STZ-diabetic mice compared to non-diabetic mice. In conclusion, HDAd-mediated Ldlr gene therapy is an effective and simple method to induce atherosclerosis regression in Ldlr^-/- mice in different metabolic states.     

Hypoxia-induced acute lung injury in murine models of sickle cell disease

Vaso-occlusive events are the major source of morbidity and mortality in sickle cell disease (SCD); however, the pathogenic mechanisms driving these events remain unclear. Using hypoxia to induce pulmonary injury, we investigated mechanisms by which sickle hemoglobin increases susceptibility to lung injury in a murine model of SCD, where mice either exclusively express the human alpha/sickle beta-globin (halphabetaS) transgene (SCD mice) or are heterozygous for the normal murine beta-globin gene and express the halphabetaS transgene (mbeta+/-, halphabetaS+/-; heterozygote SCD mice). Under normoxia, lungs from the SCD mice contained higher levels of xanthine oxidase (XO), nitrotyrosine, and cGMP than controls (C57BL/6 mice). Hypoxia increased XO and nitrotyrosine and decreased cGMP content in the lungs of all mice. After hypoxia, vascular congestion was increased in lungs with a greater content of XO and nitrotyrosine. Under normoxia, the association of heat shock protein 90 (HSP90) with endothelial nitric oxide synthase (eNOS) in lungs of SCD and heterozygote SCD mice was decreased compared with the levels of association in lungs of controls. Hypoxia further decreased association of HSP90 with eNOS in lungs of SCD and heterozygote SCD mice, but not in the control lungs. Pretreatment of rat pulmonary microvascular endothelial cells in vitro with xanthine/XO decreased A-23187-stimulated nitrite + nitrate production and HSP90 interactions with eNOS. These data support the hypotheses that hypoxia increases XO release from ischemic tissues and that the local increase in XO-induced oxidative stress can then inhibit HSP90 interactions with eNOS, decreasing *NO generation and predisposing the lung to vaso-occlusion.     

Validation of criteria for the selection of AFLP markers to assess the genetic variation of a breeders’ collection of evergreen azaleas

Fluorescent AFLP and automated data analysis were employed to assess the genetic conformity within a breeders’ collection of evergreen azaleas. The study included 75 genotypes of Belgian pot azaleas (Rhododendron simsii Planch. hybrids), Kurume and Hirado azaleas and wild ancestor species from the Tsutsusi subgenus. Fluorescent detection and addition of an internal size standard to each lane enabled the automated scoring of each fragment arising from a single AFLP primer combination (PC). The use of three PCs generated an initial data set with a total of 648 fragments ranging from 70 bp to 450 bp. Different marker selection thresholds for average fluorescent signal intensity and marker frequency were used to create eight extra restricted data subsets. Pairwise plant genetic similarity was calculated for the nine data sets using the Simple Matching coefficient (symmetrical, including double-zeros) and Jaccard coefficient (asymmetrical, excluding double zeros). The averages, the ranges and the correlation to one other (Mantel analysis) were compared for the obtained similarity matrices. This revealed the sensitivity of ordinations obtained by both similarity coefficients for the presence of weak or intensive markers or for the degree of polymorphism of the markers. For 34 cultivars, pedigree information (at maximum to the fifth ancestor generation) was available. Genetic similarity by descent (kinship coefficient) was turned into a genetic distance and correlated to the genetic conformity, as revealed by the different selections of AFLP markers (Mantel analysis). Use of a Simple Matching coefficient with no or moderate selection to signal intensity and excluding rare and abundant markers gave the best correlation with pedigree. Finally, the ordination of the studied genotypes by means of dendrograms and principal co-ordinate analysis was confronted with known or accepted relationships based on geographical origin, parentage and morphological characters. Genotypes could be assigned to three distinct groups: pot azaleas, Kurume azaleas and Hirado azaleas. Wild ancestor species appeared to be more related to the Japanese azaleas. Intermediate cultivars could be typified as crossings with Kurume or Hirado azaleas or with wild species. Received: 3 September 1998 / Accepted: 25 March 1999     

Structural basis for Myf and Psa fimbriae‐mediated tropism of pathogenic strains of Yersinia for host tissues

Three pathogenic species of the genus Yersinia assemble adhesive fimbriae via the FGL‐chaperone/usher pathway. Closely related Y. pestis and Y. pseudotuberculosis elaborate the pH6 antigen (Psa), which mediates bacterial attachment to alveolar cells of the lung. Y. enterocolitica, instead, assembles the homologous fimbriae Myf of unknown function. Here, we discovered that Myf, like Psa, specifically recognizes β1‐3– or β1‐4–linked galactose in glycosphingolipids, but completely lacks affinity for phosphatidylcholine, the main receptor for Psa in alveolar cells. The crystal structure of a subunit of Psa (PsaA) complexed with choline together with mutagenesis experiments revealed that PsaA has four phosphatidylcholine binding pockets that enable super‐high‐avidity binding of Psa‐fibres to cell membranes. The pockets are arranged as six tyrosine residues, which are all missing in the MyfA subunit of Myf. Conversely, the crystal structure of the MyfA‐galactose complex revealed that the galactose‐binding site is more extended in MyfA, enabling tighter binding to lactosyl moieties. Our results suggest that during evolution, Psa has acquired a tyrosine‐rich surface that enables it to bind to phosphatidylcholine and mediate adhesion of Y. pestis/pseudotuberculosis to alveolar cells, whereas Myf has specialized as a carbohydrate‐binding adhesin, facilitating the attachment of Y. enterocolitica to intestinal cells.     

4E-BP1 degradation and eIF4E truncation occur spatially distinctly in the porcine uterine epithelia and are features of noninvasive implantation in the pig

The implantation of the blastocyst into the endometrium is an indispensable premise for successful embryonic development. This process is regulated by maternal and embryonic signals that influence gene expression at the translational level, among other processes. Recently, we have shown that proteolytical cleavage of the prototypical 25‐kDa, mRNA cap‐binding protein eIF4E produces a stable variant with a molecular mass of approximately 23 kDa exclusively in the porcine endometrium during implantation. This is accompanied by dephosphorylation and reduction of the abundant repressor 4E‐BP1. Here, we investigate the distribution of the truncated eIF4E and of 4E‐BP1 in the porcine uterine tissue, their binding in native samples, and we analyzed eIF4E‐, eIF4G‐, and 4E‐BP1‐specific proteolytic activities. Our results show that in pigs, the truncated eIF4E is located in the endometrial luminal epithelium during implantation. Neither glandulary tissue nor stroma expressed any truncated eIF4E. The reduced abundance of 4E‐BP1 during implantation is mainly the result of decay in the glandular epithelia. Moreover, steroid replacements, in vitro protease assays, and cell lysate fractionation showed that eIF4E cleavage and 4E‐BP1 decay both depended on the ovarian steroid hormones estradiol and progestrone, but these effects are the result of different proteolytic activities. Although eIF4G cleavage also depends on calcium, stimulation by these steroids could not be established. We propose that the translation initiation process in the endometrium is differently regulated by the truncated eIF4E, utilizing different abundances of 4E‐BP1 and binding dynamic of eIF4E/4E‐BP1 in distinct forms of implantation. Mol. Reprod. Dev. 78:895–905, 2011. © 2011 Wiley Periodicals, Inc.