Lysophosphatidic acid can activate platelets without increasing 32P-labelling of phosphatidic acid

Stimulation of platelets by thrombin produced a rise in [32P]phosphatidic acid labelling of platelets which was greater in medium without added calcium than in medium with 2.5 mM calcium. A rise in [32P]lysophosphatidic acid was also seen in platelets stimulated by thrombin in the presence of 2.5 mM extracellular calcium, though it was of lesser magnitude (average 35%) than the rise in phosphatidic acid. In platelets resuspended without added calcium no change in [32P]lysophosphatidic acid was seen in response to thrombin. Lysophosphatidic acid can itself induce platelet aggregation. Similarly to the calcium ionophore A23187, lysophosphatidic acid produced minimal change (in medium with no added calcium) to no change (in medium with 2.5 mM external calcium) in [32P]lysophosphatidic acid. The endoperoxide analog U46619 produced changes in 32P-labelling of platelet phosphatidic and lysophosphatidic acid similar to those produced by thrombin but of lesser magnitude. The results of these studies show that the action of lysophosphatidic acid on platelets differs from the action of thrombin, U46619 and platelet-activating factor, which produce a rapid rise in [32P]phosphatidic acid, and suggests that lysophosphatidic acid, like A23187, largely bypasses the initial receptor-coupled breakdown of phosphoinositides leading to formation of diacylglycerols and phosphatidic acid.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Stimulation of platelet protein phosphorylation by arachidonic acid and endoperoxide analogs

The present study has investigated the influence of arachidonate, endoperoxide analogs, and the calcium ionophore A23187 on platelet aggregation and on the phosphorylation of platelet proteins. Following stimulation of platelets by these agents a rapid increase in phosphorylation of three proteins was observed which began at the same time as the initial formation of platelet aggregates. These three proteins were the 260,000 dalton actin-binding protein, a 40,000 dalton protein of unknown function, and the 20,000 dalton myosin light chain. When extensive aggregation was reached, the extent of phosphorylation returned toward baseline. Pretreatment of platelets with aspirin completely inhibited both aggregation and protein phosphorylations induced by arachidonate, but had only partial inhibitory effects on endoperoxide analogs or A23187. Since endoperoxide analogs and A23187 may trigger endogenous production of prostaglandin endoperoxides and thromboxane A₂, in addition to having a direct effect of their own, it is probable that the partial inhibition seen was due to inhibition of that component of their effect due to this endogenous production, through other effects of aspirin can not be entirely ruled out. Since recent evidence shows that phosphorylation of myosin light chain results from calcium stimulation of a protein kinase in the presence of calmodulin, the results are consistent with mobilization of calcium as the primary role of the arachidonate-endoperoxide-thromboxane pathway.     

Two frameshift mutations in the cystic fibrosis gene

Cystic fibrosis (CF) is a recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have identified in exon 7 two frameshift mutations, one caused by a two-nucleotide insertion and the other caused by a one-nucleotide deletion; these mutations--CF1154insTC and CF1213delT, respectively, are predicted to shift the reading frame of the protein and to introduce UAA(ochre) termination codons at residues 369 and 368.     

Zinc inhibition of electron transfer: mechanism of beta receptor inhibition?

Zinc has been shown to inhibit β-receptor activation of adenylate cyclase at a post receptor site. We have postulated that the β-receptor is one of several receptors activated by reduction, followed by transmembrane elector transfer accelerated by GTP. GTP accelerates electron transfer in a model system and this accelerated electron transfer is inhibited by zinc. This could explain the mechanism of the post receptor inhibition by zinc of the adenylate cyclase stimulation which follows β-receptor activation.     

Programmatic Cost Evaluation of Nontargeted Opt-Out Rapid HIV Screening in the Emergency Department

Background

The Centers for Disease Control and Prevention recommends nontargeted opt-out HIV screening in healthcare settings. Cost effectiveness is critical when considering potential screening methods. Our goal was to compare programmatic costs of nontargeted opt-out rapid HIV screening with physician-directed diagnostic rapid HIV testing in an urban emergency department (ED) as part of the Denver ED HIV Opt-Out Trial.

Methods

This was a prospective cohort study nested in a larger quasi-experiment. Over 16 months, nontargeted rapid HIV screening (intervention) and diagnostic rapid HIV testing (control) were alternated in 4-month time blocks. During the intervention phase, patients were offered HIV testing using an opt-out approach during registration; during the control phase, physicians used a diagnostic approach to offer HIV testing to patients. Each method was fully integrated into ED operations. Direct program costs were determined using the perspective of the ED. Time-motion methodology was used to estimate personnel activity costs. Costs per patient newly-diagnosed with HIV infection by intervention phase, and incremental cost effectiveness ratios were calculated.

Results

During the intervention phase, 28,043 eligible patients were included, 6,933 (25%) completed testing, and 15 (0.2%, 95% CI: 0.1%–0.4%) were newly-diagnosed with HIV infection. During the control phase, 29,925 eligible patients were included, 243 (0.8%) completed testing, and 4 (1.7%, 95% CI: 0.4%–4.2%) were newly-diagnosed with HIV infection. Total annualized costs for nontargeted screening were $148,997, whereas total annualized costs for diagnostic HIV testing were $31,355. The average costs per HIV diagnosis were $9,932 and $7,839, respectively. Nontargeted HIV screening identified 11 more HIV infections at an incremental cost of $10,693 per additional infection.

Conclusions

Compared to diagnostic testing, nontargeted HIV screening was more costly but identified more HIV infections. More effective and less costly testing strategies may be required to improve the identification of patients with undiagnosed HIV infection in the ED.     

Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition

Lysine is one of the most limiting amino acids in plants and its biosynthesis is carefully regulated through inhibition of the first committed step in the pathway catalyzed by dihydrodipicolinate synthase (DHDPS). This is mediated via a feedback mechanism involving the binding of lysine to the allosteric cleft of DHDPS. However, the precise allosteric mechanism is yet to be defined. We present a thorough enzyme kinetic and thermodynamic analysis of lysine inhibition of DHDPS from the common grapevine, Vitis vinifera (Vv). Our studies demonstrate that lysine binding is both tight (relative to bacterial DHDPS orthologs) and cooperative. The crystal structure of the enzyme bound to lysine (2.4 Å) identifies the allosteric binding site and clearly shows a conformational change of several residues within the allosteric and active sites. Molecular dynamics simulations comparing the lysine-bound (PDB ID 4HNN) and lysine free (PDB ID 3TUU) structures show that Tyr132, a key catalytic site residue, undergoes significant rotational motion upon lysine binding. This suggests proton relay through the catalytic triad is attenuated in the presence of lysine. Our study reveals for the first time the structural mechanism for allosteric inhibition of DHDPS from the common grapevine.     

Reducing infectivity of HIV upon exposure to surfaces coated with N,N‐dodecyl,methyl‐polyethylenimine

The infectivity of high‐titer, cell‐free HIV in culture media and human milk is rapidly reduced upon exposure to polyethylene slides painted with the linear hydrophobic polycation N,N‐dodecyl,methyl‐polyethylenimine (DMPEI). Accompanying viral p24 protein and free viral RNA analysis of solutions exposed to DMPEI‐coated surfaces suggests that virion attachment to the polycationic surface and its subsequent inactivation are the likely mechanism of this phenomenon. Biotechnol. Bioeng. 2013; 110: 2058–2062. © 2013 Wiley Periodicals, Inc.