Phosphatidylserine and phosphatidylethanolamine regulate the structure and function of FVIIa and its interaction with soluble tissue factor

Cell membranes have important functions in many steps of the blood coagulation cascade, including the activation of factor X (FX) by the factor VIIa (FVIIa)-tissue factor (TF) complex (extrinsic X_ase). FVIIa shares structural similarity with factor IXa (FIXa) and FXa. FIXa and FXa are regulated by binding to phosphatidylserine (PS)-containing membranes via their γ-carboxyglutamic acid-rich domain (Gla) and epidermal growth-factor (EGF) domains. Although FVIIa also has a Gla-rich region, its affinity for PS-containing membranes is much lower compared with that of FIXa and FXa. Research suggests that a more common endothelial cell lipid, phosphatidylethanolamine (PE), might augment the contribution of PS in FVIIa membrane-binding and proteolytic activity. We used soluble forms of PS and PE (1,2-dicaproyl-sn-glycero-3-phospho-l-serine (C6PS), 1,2-dicaproyl-sn-glycero-3-phospho-ethanolamine (C6PE)) to test the hypothesis that the two lipids bind to FVIIa jointly to promote FVIIa membrane binding and proteolytic activity. By equilibrium dialysis and tryptophan fluorescence, we found two sites on FVIIa that bound equally to C6PE and C6PS with K_d of ∼ 150–160 μM, however, deletion of Gla domain reduced the binding affinity. Binding of lipids occurred with greater affinity (K_d∼70–80 μM) when monitored by FVIIa proteolytic activity. Global fitting of all datasets indicated independent binding of two molecules of each lipid. The proteolytic activity of FVIIa increased by ∼50–100-fold in the presence of soluble TF (sTF) plus C6PS/C6PE. However, the proteolytic activity of Gla-deleted FVIIa in the presence of sTF was reduced drastically, suggesting the importance of Gla domain to maintain full proteolytic activity.     

Human Alanine-Glyoxylate Aminotransferase 2 Lowers Asymmetric Dimethylarginine and Protects from Inhibition of Nitric Oxide Production

Elevated blood concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric-oxide (NO) synthase, are found in association with diabetes, hypertension, congestive heart failure, and atherosclerosis. ADMA levels are controlled by dimethylarginine dimethylaminohydrolases (DDAHs), cytosolic enzymes that hydrolyze ADMA to citrulline and dimethylamine. ADMA also has been proposed to be regulated through an alternative pathway by alanine-glyoxylate aminotransferase 2 (AGXT2), a mitochondrial aminotransferase expressed primarily in the kidney. The goal of this study was to define the subcellular localization of human AGXT2 and test the hypothesis that overexpression of human AGXT2 protects from ADMA-induced inhibition in nitric oxide (NO) production. AGXT2 was cloned from human kidney cDNA and overexpressed in COS-7 cells and human umbilical vein endothelial cells with a C-terminal FLAG epitope tag. Mitochondrial localization of human AGXT2 was demonstrated by confocal microscopy and a 41-amino acid N-terminal mitochondrial cleavage sequence was delineated by N-terminal sequencing of the mature protein. Overexpression of human AGXT2 in the liver of C57BL/6 mice using an adenoviral expression vector produced significant decreases in ADMA levels in plasma and liver. Overexpression of human AGXT2 also protected endothelial cells from ADMA-mediated inhibition of NO production. We conclude that mitochondrially localized human AGXT2 is able to effectively metabolize ADMA in vivo resulting in decreased ADMA levels and improved endothelial NO production.     

The acetylcholine receptor as a cellular receptor for rabies virus

Characterization of specific host cell receptors for enveloped viruses is a difficult problem because many enveloped viruses bind to a variety of substrates which are not obviously related to tissue tropisms in the intact host. Viruses with a limited cellular tropism in infected animals present useful models for studying the mechanisms by which virus attachment regulates the disease process. Rabies virus is a rhabdovirus which exhibits a marked neuronotropism in infected animals. Limited data suggest that spread occurs by transsynaptic transfer of virus. The results of recent experiments at Yale suggest that viral antigen is localized very soon after injection at neuromuscular junctions, the motor nerve endings on muscle tissue. On cultured muscle cells, similar co-localization with the acetylcholine receptor is seen both before and after virus multiplication. Pretreatment of these cells with some ligands of the acetylcholine receptor results in reduced viral infection. These findings suggest that a neurotransmitter receptor or a closely associated molecule may serve as a specific host cell receptor for rabies virus and thus may be responsible for the tissue tropism exhibited by this virus. In addition to clarifying aspects of rabies virus pathogenesis, these studies have broad implications regarding the mechanism by which other viruses or viral immunizations might mediate autoimmune diseases such as myasthenia gravis.     

Assessment of methanogenie activity in anaerobic digestion: Apparatus and method

An apparatus is described for the rapid measurement and recording of methanogenic activity in anaerobic fermentations, and its application is demonstrated in the evaluation of the anaerobic contact process, using pear waste. The method is based on recording the rate of manometer liquid displacement in a Warburgtype vessel by means of optical sensors, appropriate electronic circuitry, and an event marking recorder or time-interval printer. Optimum conditions for measuring methanogenic activity included a pH of 6.7–6.9, a final phosphate buffer concentration of 0.07–015M, and formic and acetic acid contents of over 500 and 200 mg/liter, respectively. In comparisons of fermenter liquid and settled effluent, methanogenic activity can be assumed to be proportional to the number of methane formers present. The apparatus should be generally useful in recording rates of gas production or consumption.     

Sclerotial formation in Corticium olivascens and other hymenomycetes

Sclerotia ofCorticium olivascens are reported and described for the first time. Examples include sclerotia collected from a stump ofPinus virginiana in Greenbelt, Maryland, and those formed in several cultures originally developed from spores produced by basidiocarps. Among outstanding characteristics of basidiocarps ofC. olivascens are the greenish or olivaceous color of the hymenial surface, constant presence of clamp-connections, development of septate cystidia, and production of nonamyloid but dextrinoid basidiospores. Cultural characteristics are described, and the negative oxidase reaction is noted.C. olivascens is a highly distinctive fungus which requires further taxonomic attention.

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Zusammenfassung Sclerotia vonCorticium olivascens sind das erste Mal mitgeteilt und beschrieben. Beispiele betrifft Sclerotia, die vom Baumstumpf vonPinus virginiana in Greenbelt, Maryland gesammelt worden sind und diejenigen, die sich in mehreren Kulturen, ursprünglich von Sporen der Basidiocarpen entwickelt haben. Unter den besonderen Kennzeichen der Basidiocarpen vonC. olivascens sind die grünliche oder olive Farbe der Hymenialoberfläche, ständiges Vorkommen der Klammerverbindungen, Entwicklung septierter Cystidien und die Produktion von nicht-amyloiden, sondern dextrinoiden Basidiosporen. Kulturkennzeichen sind beschrieben und die negative Oxidasenreaktion ist erwähnt.C. olivascens ist ein hoch distinguierter Pilz, der eine weitere, taxonomische Aufmerksamkeit verlangt.

     

Proteolytic processing of human preproapolipoprotein A-I. A proposed defect in the conversion of pro A-I to A-I in Tangier's disease

The primary translation product of human intestinal apolipoprotein A-I mRNA was isolated from wheat germ and ascites cell-free translation systems. Comparison of its NH2-terminal sequence with that of plasma high density lipoprotein-associated A-I showed that it is initially synthesized as a preproprotein. Like rat preproapolipoprotein A-I, it contains an 18-amino acid prepeptide and a 6-amino acid propeptide. The highly unusual COOH-terminal Gln-Gln dipeptide present in the rat pro-segment is also represented at the same position in the human sequence. The functional division of the 24-amino acid NH2-terminal extention into pro- and presegments was verified by finding that the stable intracellular form of A-I in a human hepatoma cell line was the proprotein. Edman degradation of radiolabeled intracellular and extracellular A-I indicated that this apolipoprotein was secreted without proteolytic cleavage of its hexapeptide prosegment. Therefore, it appears that apolipoprotein A-I undergoes an additional proteolytic processing step before it is fully integrated into plasma high density lipoprotein. Two-dimensional gel electrophoresis of purified proapolipoprotein A-I isolated from the hepatocyte cell culture media indicated that it corresponds to isoforms 2 and 3, the basic A-I isoproteins which are the precursors of plasma A-I and the predominant plasma A-I isoforms found in patients with Tangier's disease (Zannis, V. I., Lees, A. M., Lees, R. S., and Breslow, J. L. (1982) J. Biol. Chem., 257, 4978-4986). Therefore this pathologic state probably arises from a defect in the conversion of proapolipoprotein A-I to apolipoprotein A-I.     

Fluorescence and calorimetric studies of phase transitions in phosphatidylcholine multilayers: kinetics of the pretransition

Discrepancies between calorimetric and fluorescence depolarization monitoring of the pretransition in multilamellar vesicles of synthetic phosphatidylcholines are shown to result primarily from the slow rate of this transition. The depolarization of fluorescence of the membrane-associated dye 1,6-diphenyl-1,3,5-hexatriene was used to determine the temperature of the pretransition for a series of heating and cooling scan rates. These temperatures, when plotted vs. scan rate, extrapolated linearly to the transition temperature at zero-scan rate, Tm = 29.8 +/- 0.8 degrees C. The slopes obtained from these plots yielded characteristic times for the transition of 8 to 30 min. In addition, analysis of temperature-jump experiments, assuming first-order kinetics, gave characteristic times in the range 4--8 min. The data are taken to suggest a most likely value for the pretransition characteristic time of 5 +/- 2 min, with larger values possibly explainable by supercooling effects. Slight differences between the calorimetrically and fluorimetrically determined main transition temperatures appear to result from perturbation of the phosphatidylcholine bilayer by the fluorescent probe.     

Assessment of qualitative and quantitative features in coronary artery MRA

In this paper, an analysis of the coronary trees using magnetic resonance angiography (MRA) is performed. The objective is to estimate how much MRA is capable to provide insights into the vascular network. A qualitative exploration of the MRA volumes with anatomical labelling by experts is first performed, Quantitative vessel features are then manually extracted providing a ground truth which is further compared to a semi-automatic extraction. This evaluation is carried out on 10 datasets of the SSFP MRA sequence and allows getting a more precise view on the current state-of-the- art as well as on future achievements to be done.