Urokinase receptor is necessary for adequate host defense against pneumococcal pneumonia

Cell recruitment is a multistep process regulated by cytokines, chemokines, and growth factors. Previous work has indicated that the urokinase plasminogen activator receptor (uPAR) may also play a role in this mechanism, presumably by an interaction with the beta(2) integrin CD11b/CD18. Indeed, an essential role of uPAR in neutrophil recruitment during pulmonary infection has been demonstrated for beta(2) integrin-dependent respiratory pathogens. We investigated the role of uPAR and urokinase plasminogen activator (uPA) during pneumonia caused by a beta(2) integrin-independent respiratory pathogen, Streptococcus pneumoniae. uPAR-deficient (uPAR(-/-)), uPA-deficient (uPA(-/-)), and wild-type (Wt) mice were intranasally inoculated with 10(5) CFU S. pneumoniae. uPAR(-/-) mice showed reduced granulocyte accumulation in alveoli and lungs when compared with Wt mice, which was associated with more S. pneumoniae CFU in lungs, enhanced dissemination of the infection, and a reduced survival. In contrast, uPA(-/-) mice showed enhanced host defense, with more neutrophil influx and less pneumococci in the lungs compared with Wt mice. These data suggest that uPAR is necessary for adequate recruitment of neutrophils into the alveoli and lungs during pneumonia caused by S. pneumoniae, a pathogen eliciting a beta(2) integrin-independent inflammatory response. This function is even more pronounced when uPAR is unoccupied by uPA.     

Effects of phosphatidylethanolamine glycation on lipid-protein interactions and membrane protein thermal stability

Non-enzymatic glycation of biomolecules has been implicated in the pathophysiology of aging and diabetes. Among the potential targets for glycation are biological membranes, characterized by a complex organization of lipids and proteins interacting and forming domains of different size and stability. In the present study, we analyse the effects of glycation on the interactions between membrane proteins and lipids. The phospholipid affinity for the transmembrane surface of the PMCA (plasma-membrane Ca(2+)-ATPase) was determined after incubating the protein or the phospholipids with glucose. Results show that the affinity between PMCA and the surrounding phospholipids decreases significantly after phosphospholipid glycation, but remains unmodified after glycation of the protein. Furthermore, phosphatidylethanolamine glycation decreases by approximately 30% the stability of PMCA against thermal denaturation, suggesting that glycated aminophospholipids induce a structural rearrangement in the protein that makes it more sensitive to thermal unfolding. We also verified that lipid glycation decreases the affinity of lipids for two other membrane proteins, suggesting that this effect might be common to membrane proteins. Extending these results to the in vivo situation, we can hypothesize that, under hyperglycaemic conditions, glycation of membrane lipids may cause a significant change in the structure and stability of membrane proteins, which may affect the normal functioning of membranes and therefore of cells.     

Somatic embryogenesis in asparagus: the role of explants and growth regulators

Summary The explant used to initiate embryogenic callus and the growth regulators used in subsequent induction (IM) and embryo development media (EDM) both influenced rate of somatic embryo development and conversion to plantlets in asparagus. Embryogenic callus derived from spear-cross sections (SS), in vitro crowns (IVC) and lateral buds (LB) was cultured on IM of MS salts and vitamins with 2, 4-D or NAA at 0, 0.01, 0.1, 1.0 or 10 mg/l and kinetin at 0, 0.1, 1.0 or 10 mg/l. The auxin 2,4-D at 1–10 mg/l, in combination with kinetin at 0–1 mg/l, in IM induced the highest frequency of embryos after four weeks; callus derived from SS, IVC and LB had means of 394, 382, and 344 small globular embryos, and 4, 11 and 9 bipolar embryos per gram of callus, respectively. After 6 weeks on EDM, 128, 116 and 51 bipolar embryos (4–7 mm in length) occurred per gram callus and 4.5, 1.4 and 2.1 embryos converted for IVC, SS and LB, respectively. NAA at 1–10 mg/l, in combinations with kinetin 0–1 mg/l, yielded means of 64, 175 and 225 small globular embryos per gram callus on IM for SS, IVC and LB, respectively. NAA promoted a higher rate of embryo development: means of 27, 54 and 91 bipolar embryos per gram callus for SS, LB and IVC, respectively, on EDM. There were 0.5, 9.4 and 11.9 plantlets from these respective callus sources. There was no difference between kinetin levels of 0–1 mg/l on callus growth and embryogenesis, whereas, 10 mg/l in IM was inhibitory.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - EDM embryo development medium - IAA indole-3-acetic acid - IM induction media - IVC in vitro crowns - LB lateral bud - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962) - NAA naphthaleneacetic acid - SS spear-cross section     

Timing of precipitation in an arid environment: Effects on population performance of a large herbivore

Climate models predict that shifts in temperature and precipitation patterns are likely to occur across the globe. Changing climate will likely have strong effects on arid environments as a result of increased temperatures, increasing frequency and intensity of droughts, and less consistent pulses of rainfall. Therefore, understanding the link between patterns of precipitation, temperature, and population performance of species occupying these environments will continue to increase in importance as climatic shifts occur within these natural ecosystems. We sought to evaluate how individual, maternal, population, and environmental, particularly temperature and precipitation, level factors influence population performance of a large herbivore in an arid environment. We used mule deer (Odocoileus hemionus) as a representative species and quantified juvenile survival to test hypotheses about effects of environmental factors on population performance. Precipitation events occurring in mid‐ to late‐pregnancy (January–April) leading to spring green‐up, as indexed by normalized difference in vegetation index, had the strongest positive effect on juvenile survival and recruitment. In addition, larger neonates had an increased probability of survival. Our findings indicate that timing and amount of precipitation prior to parturition have strong influences on maternal nutritional condition, which was passed on to young. These results have important implications for understanding how animal populations may benefit from timing of precipitation during spring and prior to parturition, especially in arid environments.     

Diaminobutyric acid: A tool for discriminating between carrier-mediated and non-carrier-mediated release of GABA from synaptosomes?

The carrier-mediated transport of GABA in rat brain synaptosomes was strongly and permanently inhibited byl-2,4-diaminobutyric acid (DAB). In order to discriminate between carrier-mediated and non-carrier-mediated release of [³H]GABA, synaptosomes prelabeled with 0.5 M [³H]GABA in the presence of 100 M DAB, or with 0.2 M [³H]GABA without DAB, were superfused in conditions stimulating the release of [³H]GABA. Only the release elicited by unlabeled GABA or DAB (by homo- and heteroexchange, respectively) was strongly inhibited in DAB-pretreated synaptosomes. The spontaneous release and the release induced by 56 mM KCl in the presence of CaCl₂, by the ionophore A23187, by ouabain, by lack of K⁺, or by purified black widow spider toxin were unaffected or only barely decreased in DAB-treated synaptosomes, and therefore do not seem to be mediated by the DAB-blocked GABA carrier.     

Fluorescent fructose derivatives for imaging breast cancer cells

Breast cancer cells are known to overexpress Glut5, a sugar transporter responsible for the transfer of fructose across the cell membrane. Since Glut5 transporter is not significantly expressed in normal breast cells, fructose uptake can potentially be used to differentiate between normal and cancerous cells. Fructose was labeled with two fluorophores at the C-1 position: 7-nitro-1,2,3-benzadiazole (NBD) and Cy5.5. The labeling site was chosen on the basis of the presence and substrate specificity of the key proteins involved in the first steps of fructose metabolism. Using fluorescence microscopy, the uptake of the probes was studied in three breast cancer cell lines: MCF 7, MDA-MB-435, and MDA-MB-231. Both fluorescent fructose derivatives showed a very good uptake in all tested cell lines. The level of uptake was comparable to that of the corresponding glucose analogs, 2-NBDG and Cy5.5-DG. Significant uptake of 1-NBDF derivative was not observed in cells lacking Glut5 transporter, while the uptake of the 1-Cy5.5-DF derivative was independent of the presence of a fructose-specific transporter. While 1-NBDF showed Glut5-specific accumulation, the coupling of a large fluorophore such as Cy5.5 likely introduces big structural and electronic changes, leading to a fructose derivative that does not accurately describe the uptake of fructose in cells.     

Effects of CRP infusion on endothelial function and coagulation in normocholesterolemic and hypercholesterolemic subjects

C-reactive protein (CRP) has been suggested to exert direct adverse effects on the vasculature in experimental setups, including endothelial dysfunction and proinflammatory changes. Here, we assessed the consequences of 1.25 mg/kg highly purified recombinant human CRP, administered as an intravenous bolus, in six patients with familial hypercholesterolemia (FH) and six normocholesterolemic subjects. Endothelium-dependent and -independent vasoreactivity to serotonin and nitroprusside, respectively, were assessed using venous occlusion plethysmography before and after CRP infusion. For biochemical analyses, blood was drawn at different time points. At baseline, FH patients showed blunted endothelium-dependent vasodilation (maximum, 89.2 +/- 30.0% vs. 117.7 +/- 13.1% in normolipidemic subjects; P = 0.037). Procoagulant activity was also higher in FH patients, illustrated by increased prothrombin fragment 1+2 (F(1+2)) levels (P = 0.030) and plasminogen activator inhibitor type-1 (PAI-1) activity (P = 0.016). Upon CRP challenge, endothelium-dependent vasodilator capacity further deteriorated in FH patients (P = 0.029), whereas no change in vascular reactivity was observed in normolipidemic subjects. Additionally, coagulation activation was augmented in FH patients compared with normolipidemic subjects (P = 0.009 for F(1+2) levels; P = 0.018 and P = 0.003 for PAI-1 antigen and activity, respectively). No difference in inflammatory responses was observed between groups. In hypercholesterolemic patients, CRP aggravates endothelial dysfunction and also evokes augmented procoagulant responses. These findings suggest that particularly in hypercholesterolemia, CRP-lowering strategies should be considered in addition to LDL reduction.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.