Changes in membrane phospholipid distribution during platelet activation

(1) Exposure of phospholipids at the outer surface of activated and control platelets was studied by incubation with a mixture of phospholipase A₂ from Naja naja and bee venom, solely or in combination with sphingomyelinase from Staphylococcus aureus, using conditions under which cell lysis remained below 10%. (2) Incubation with phospholipase A₂ alone revealed a markedly increased susceptibility of the phospholipids in platelets activated by a mixture of collagen plus thrombin, by the SH-oxydizing compound diamide, or by calcium ionophore A23187, as compared to control platelets or platelets activated separately by collagen or thrombin. (3) Collagen plus thrombin, diamide, and ionophore treated platelets revealed an increased exposure of phosphatidylserine at the outer surface accompanied by a decreased exposure of sphingomyelin, as could be concluded from incubations with a combination of phospholipase A₂ and sphingomyelinase. These alterations were much less apparent in platelets activated either by thrombin or by collagen alone. (4) The increased exposure of phosphatidylserine in activated platelets is accompanied by an increased ability of the platelets to enhance the conversion of prothrombin to thrombin by coagulation factor Xa, in the presence of factor Va and calcium. (5) It is concluded that the altered orientation of the phospholipids in the plasma membrane of platelets activated by collagen plus thrombin, by diamide, or by calcium ionophore, is the result of a transbilayer movement. Moreover, the increased exposure of phosphatidylserine in platelets stimulated by the combined action of collagen and thrombin might be of considerable importance for the hemostatic process.     

Barium- and calcium-permeable channels open at negative membrane potentials in rat ventricular myocytes

Summary Ca²⁺- and Ba²⁺-permeable channel activity from adult rat ventricular myocytes, spontaneously appeared in the three single-channel recording configurations: cell-attached, and excised inside-out or outside-out membrane patches. Single-channel activity was recorded at steady-state applied membrane potentials including the entire range of physiologic values, and displayed no rundown in excised patches. This activity occurred in irregular bursts separated by quiescent periods of 5 to 20 min in cell-attached membrane patches, whereas in excised patch experiments, this period was reduced to 2 to 10 min. During activity, a variety of kinetic behaviors could be observed with more or less complex gating patterns. Three conductance levels: 22, 45 and 78 pS were routinely observed in the same excised membrane patch, sometimes combining to give a larger level. These channels were significantly permeable to divalent cations and showed little or no permeability to potassium or sodium ions. The inorganic blockers of voltage-gated Ca channels, cobalt (2mm), cadmium (0.5mm) or nickel (3mm), had no apparent effect on these spontaneous unitary currents carried by barium ions. Under 10^–5 m bay K 8644 or nitrendipine, the activity was clearly increased in about half of the tested excised inside-out membrane patches. Both dihydropyridines enhanced openings of the larger conductance level, which was only very occasionally seen under control conditions. When the single-channel activity became sustained under 5×10^–6 m Bay K 8644, it was possible to calculate the mean unitary current at different membrane potentials and show that the mean current value increased with membrane potential.     

Strategies for the identification and purification of ligands for orphan biomolecules

The isolation of related genes with evolutionary conserved motifs by the application ofpolymerase chain reaction-based molecular biology techniques, or from database searchingstrategies, has facilitated the identification of new members of protein families. Many of theseprotein molecules will be involved in protein–protein interactions (e.g. growth factors,receptors, adhesion molecules), since such interactions are intrinsic to virtually every cellularprocess. However, the precise biological function and specific binding partners of these novelproteins are frequently unknown, hence they are known as orphan molecules.Complementary technologies are required for the identification of the specific ligands orreceptors for these and other orphan proteins (e.g., antibodies raised against crude biologicalextracts or whole cells). We describe herein several alternative strategies for the identification,purification and characterisation of orphan peptide and protein molecules, specifically thesynergistic use of micropreparative HPLC and biosensor techniques.     

Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation

Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways.     

Comparative assessment of the intracellular survival of the Burkholderia pseudomallei bopC mutant

Burkholderia pseudomallei, the causative agent of melioidosis, is a Gram-negative saprophytic bacterium capable of surviving within phagocytic cells. To assess the role of BopC (a type III secreted effector protein) in the pathogenesis of B. pseudomallei, a B. pseudomallei bopC mutant was used to infect J774A.1 macrophage-like cells. The bopC mutant showed significantly reduced intracellular survival in infected macrophages compared to wild-type B. pseudomallei. In addition, the bopC mutant displayed delayed escape from endocytic vesicles compared with the wild-type strain. This indicates that BopC is important, and at least in part, needed for intracellular survival of B. pseudomallei.     

Predictive Factors of Herpes Zoster HIV-Infected Patients: Another Adverse Effect of Crack Cocaine

A retrospective cohort study was conducted on 1541 HIV-infected patients to determine variables associated with the incidence of herpes zoster. A single failure Cox model showed that herpes zoster incidence increased following the first 6 months of antiretroviral treatment adjusted hazard ratio (AHR)=5 (95%CI=2.6-9.2), P<0.001; in the >60 years age group AHR=2 (95%CI=1-4), P=0.04; in patients in the top CD8 quartile AHR=2.1 (95%CI=1.3-3.6), P<0.001; and in patients previously reported to use crack cocaine AHR=5.9, (95%CI=1.4-25), P=0.02. Herpes zoster incidence increased in patients with CD4 counts<500 per mm³ and gradually declined since 1992-1996, with AHR=0.3 (95%CI=0.2-0.5), P<0.001 for the 1997-2002 period and AHR=0.24 (95%CI=0.14-0.4), P<0.001 for the 2002-2008 period. Contrary to what has been described elsewhere, there was no specific effect of protease inhibitors on herpes zoster incidence. The present study is the first to suggest that crack cocaine is associated with an increased incidence of herpes zoster. The neurological or immunological effects of crack are discussed.     

Overview of the Oldest Existing Set of Substrate-optimized Anaerobic Processes: Digestive Tracts

Over millions of years, living organisms have explored and optimized the digestion of a wide variety of substrates. Engineers who develop anaerobic digestion processes for waste treatment and energy production can learn much from this accumulated ‘experience’. The aim of this work is a survey based on the comparison of 190 digestive tracts (vertebrate and insect) considered as ‘reactors’ and their anaerobic processes. Within a digestive tract, each organ is modeled as a type of reactor (continuous stirred-tank, such reactors in series, plug-flow or batch) associated with chemical aspects such as pH or enzymes. Based on this analysis, each complete digestion process has been rebuilt and classified in accordance with basic structures which take into account the relative size of the different reactors. The results show that all animal digestive structures can be grouped within four basic types. Size and/or position in the structure of the different reactors (pre/post treatment and anaerobic microbial digestion) are closely correlated to the degradability of the feed (substrate). Major common features are: (i) grinding, (ii) an extreme pH compartment, and (iii) correlation between the size of the microbial compartment and the degradability of the feed. Thus, shared answers found by animals during their evolution can be a source of inspiration for engineers in designing optimal anaerobic processes.     

Derivation of endothelial cells from human embryonic stem cells in fully defined medium enables identification of lysophosphatidic acid and platelet activating factor as regulators of eNOS localization

The limited availability of human vascular endothelial cells (ECs) hampers research into EC function whilst the lack of precisely defined culture conditions for this cell type presents problems for addressing basic questions surrounding EC physiology. We aimed to generate endothelial progenitors from human pluripotent stem cells to facilitate the study of human EC physiology, using a defined serum-free protocol. Human embryonic stem cells (hESC-ECs) differentiated under serum-free conditions generated CD34⁺KDR⁺ endothelial progenitor cells after 6 days that could be further expanded in the presence of vascular endothelial growth factor (VEGF). The resultant EC population expressed CD31 and TIE2/TEK, took up acetylated low-density lipoprotein (LDL) and up-regulated expression of ICAM-1, PAI-1 and ET-1 following treatment with TNFα. Immunofluorescence studies indicated that a key mediator of vascular tone, endothelial nitric oxide synthase (eNOS), was localised to a perinuclear compartment of hESC-ECs, in contrast with the pan-cellular distribution of this enzyme within human umbilical vein ECs (HUVECs). Further investigation revealed that that the serum-associated lipids, lysophosphatidic acid (LPA) and platelet activating factor (PAF), were the key molecules that affected eNOS localisation in hESC-ECs cultures. These studies illustrate the feasibility of EC generation from hESCs and the utility of these cells for investigating environmental cues that impact on EC phenotype. We have demonstrated a hitherto unrecognized role for LPA and PAF in the regulation of eNOS subcellular localization.