Interactions of 12-lipoxygenase with phospholipase A(2) isoforms following platelet activation through the glycoprotein VI collagen receptor

Recent studies implicate the collagen receptor, glycoprotein VI (GPVI) in activation of platelet 12-lipoxygenase (p12-LOX). Herein, we show that GPVI-stimulated 12-hydro(peroxy)eicosatetraenoic acid (H(P)ETE) synthesis is inhibited by palmityl trifluromethyl ketone or oleyloxyethyl phosphocholine, but not bromoenol lactone, implicating secretory and cytosolic, but not calcium-independent phospholipase A(2) (PLA(2)) isoforms. Also, following GPVI activation, 12-LOX co-immunoprecipitates with both cytosolic and secretory PLA(2), (sPLA(2)). Finally, venoms containing sPLA(2) acutely activate p12-LOX in a dose-dependent manner. This study shows that platelet 12-H(P)ETE generation utilizes arachidonate substrate from both c- and sPLA(2) and that 12-LOX functionally associates with both PLA(2) isoforms. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Differential roles of integrins alpha(2)beta(1), and alpha(IIb)beta(3) in collagen and CRP-induced platelet activation

Collagen and collagen-related peptide (CRP) activate platelets by interacting with glycoprotein (GP)VI. In addition, collagen binds to integrin alpha(2)beta(1) and possibly to other receptors. In this study, we have compared the role of integrins alpha(2)beta(1) and alpha(IIb)beta(3) in platelet activation induced by collagen and CRP. Inhibitors of ADP and thromboxane A(2) (TxA(2)) substantially attenuated collagen-induced platelet aggregation and dense granule release, whereas CRP-induced responses were only partially inhibited. Under these conditions, a proportion of platelets adhered to the collagen fibres resulting in dense granule release and alpha(IIb)beta(3) activation. This adhesion was substantially mediated by alpha(2)beta(1). The alpha(IIb)beta(3) antagonist lotrafiban potentiated CRP-induced dense granule release, suggesting that alpha(IIb)beta(3) outside-in signalling may attenuate GPVI signals. By contrast, lotrafiban inhibited collagen-induced dense granule release. These results emphasise the differential roles of alpha(2)beta(1) and alpha(IIb)beta(3) in platelet activation induced by collagen and CRP. Further, they show that although ADP and TxA(2) greatly facilitate collagen-induced platelet activation, collagen can induce full activation of those platelets to which it binds in the absence of these mediators, via a mechanism that is dependent on adhesion to alpha(2)beta(1).

Differential cytopathogenesis of respiratory syncytial virus prototypic and clinical isolates in primary pediatric bronchial epithelial cells.

Background Human respiratory syncytial virus (RSV) causes severe respiratory disease in infants. Airway epithelial cells are the principle targets of RSV infection. However, the mechanisms by which it causes disease are poorly understood. Most RSV pathogenesis data are derived using laboratory-adapted prototypic strains. We hypothesized that such strains may be poorly representative of recent clinical isolates in terms of virus/host interactions in primary human bronchial epithelial cells (PBECs). Methods To address this hypothesis, we isolated three RSV strains from infants hospitalized with bronchiolitis and compared them with the prototypic RSV A2 in terms of cytopathology, virus growth kinetics and chemokine secretion in infected PBEC monolayers. Results RSV A2 rapidly obliterated the PBECs, whereas the clinical isolates caused much less cytopathology. Concomitantly, RSV A2 also grew faster and to higher titers in PBECs. Furthermore, dramatically increased secretion of IP-10 and RANTES was evident following A2 infection compared with the clinical isolates. Conclusions The prototypic RSV strain A2 is poorly representative of recent clinical isolates in terms of cytopathogenicity, viral growth kinetics and pro-inflammatory responses induced following infection of PBEC monolayers. Thus, the choice of RSV strain may have important implications for future RSV pathogenesis studies.

The determination of asialoglycoforms of serum glycoproteins by lectin blotting with Ricinus communis agglutinin

Serum proteins were fractionated by polyacrylamide gel electrophoresis under denaturing conditions and transferred to nitrocellulose membranes. The blotted polypeptides were probed with biotinylated Ricinus communis lectin (RCA120) followed by streptavidin/alkaline phosphatase. This procedure detected five asialoglycoproteins (a2-macroglobulin, transferrin, a1-antitrypsin, a1-antichymotrypsin and haptoglobin ß chain). The asialoform of the a1-trypsin inhibitor was found to be decreased in inflammation.

Lipoprotein-associated phospholipase A(2), inflammatory biomarkers, and risk of cardiovascular disease in the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER)

Objective: Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is an inflammatory biomarker that circulates mainly bound to LDL. We evaluated the association of Lp-PLA(2) with vascular events in the elderly where the importance of LDL is diminished as a risk factor for coronary disease.

In vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo

Respiratory syncytial virus (RSV) is the major viral cause of severe pulmonary disease in young infants worldwide. However, the mechanisms by which RSV causes disease in humans remain poorly understood. To help bridge this gap, we developed an ex vivo/in vitro model of RSV infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs), the primary targets of RSV infection in vivo. Our RSV/WD-PBEC model demonstrated remarkable similarities to hallmarks of RSV infection in infant lungs. These hallmarks included restriction of infection to noncontiguous or small clumps of apical ciliated and occasional nonciliated epithelial cells, apoptosis and sloughing of apical epithelial cells, occasional syncytium formation, goblet cell hyperplasia/metaplasia, and mucus hypersecretion. RSV was shed exclusively from the apical surface at titers consistent with those in airway aspirates from hospitalized infants. Furthermore, secretion of proinflammatory chemokines such as CXCL10, CCL5, IL-6, and CXCL8 reflected those chemokines present in airway aspirates. Interestingly, a recent RSV clinical isolate induced more cytopathogenesis than the prototypic A2 strain. Our findings indicate that this RSV/WD-PBEC model provides an authentic surrogate for RSV infection of airway epithelium in vivo. As such, this model may provide insights into RSV pathogenesis in humans that ultimately lead to successful RSV vaccines or therapeutics.

Primary motor cortex involvement in initial learning during visuomotor adaptation

Human motor behaviour is continually modified on the basis of errors between desired and actual movement outcomes. It is emerging that the role played by the primary motor cortex (M1) in this process is contingent upon a variety of factors, including the nature of the task being performed, and the stage of learning. Here we used repetitive TMS to test the hypothesis that M1 is intimately involved in the initial phase of sensorimotor adaptation. Inhibitory theta burst stimulation was applied to M1 prior to a task requiring modification of torques generated about the elbow/forearm complex in response to rotations of a visual feedback display. Participants were first exposed to a 30° clockwise (CW) rotation (Block A), then a 60° counterclockwise rotation (Block B), followed immediately by a second block of 30° CW rotation (A2). In the STIM condition, participants received 20s of continuous theta burst stimulation (cTBS) prior to the initial A Block. In the conventional (CON) condition, no stimulation was applied. The overt characteristics of performance in the two conditions were essentially equivalent with respect to the errors exhibited upon exposure to a new variant of the task. There were however, profound differences between the conditions in the latency of response preparation, and the excitability of corticospinal projections from M1, which accompanied phases of de-adaptation and re-adaptation (during Blocks B and A2). Upon subsequent exposure to the A rotation 24h later, the rate of re-adaptation was lower in the stimulation condition than that present in the conventional condition. These results support the assertion that primary motor cortex assumes a key role in a network that mediates adaptation to visuomotor perturbation, and emphasise that it is engaged functionally during the early phase of learning.

Ultracold fluorine production via Doppler cooled BeF

Large parts of the periodic table cannot be cooled by current laser-based methods. We investigate whether zero energy fragmentation of laser cooled fluorides is a potential source of ultracold fluorine atoms. We report new ab initio calculations on the lowest electronic states of the BeF diatomic molecule including spin-orbit coupling, the calculated minima for the valence electronic states being within 1 pm of the spectroscopic values. A four colour cooling scheme based on the A2? ? X2S+ transition is shown to be feasible for this molecule. Multi-Reference Configuration Interaction (MRCI) potentials of the lowest energy Rydberg states are reported for the first time and found to be in good agreement with experimental data. A series of multi-pulse excitation schemes from a single rovibrational level of the cooled molecule are proposed to produce cold fluorine atoms.

Tyrosine nitration of prostacyclin synthase is associated with enhanced retinal cell apoptosis in diabetes

The risk of diabetic retinopathy is associated with the presence of both oxidative stress and toxic eicosanoids. Whether oxidative stress actually causes diabetic retinopathy via the generation of toxic eicosanoids, however, remains unknown. The aim of the present study was to determine whether tyrosine nitration of prostacyclin synthase (PGIS) contributes to retinal cell death in vitro and in vivo. Exposure of human retinal pericytes to heavily oxidized and glycated LDL (HOG-LDL), but not native forms of LDL (N-LDL), for 24 hours significantly increased pericyte apoptosis, accompanied by increased tyrosine nitration of PGIS and decreased PGIS activity. Inhibition of the thromboxane receptor or cyclooxygenase-2 dramatically attenuated HOG-LDL-induced apoptosis without restoring PGIS activity. Administration of superoxide dismutase (to scavenge superoxide anions) or L-N(G)-nitroarginine methyl ester (L-NAME, a nonselective nitric oxide synthase inhibitor) restored PGIS activity and attenuated pericyte apoptosis. In Akita mouse retinas, diabetes increased intraretinal levels of oxidized LDL and glycated LDL, induced PGIS nitration, enhanced apoptotic cell death, and impaired blood-retinal barrier function. Chronic administration of tempol, a superoxide scavenger, reduced intraretinal oxidized LDL and glycated LDL levels, PGIS nitration, and retina cell apoptosis, thereby preserving the integrity of blood-retinal barriers. In conclusion, oxidized LDL-mediated PGIS nitration and associated thromboxane receptor stimulation might be important in the initiation and progression of diabetic retinopathy.

How well do the public understand palliative care? A mixed methods study

Background: International research suggests that the general public appear to be confused about what palliative care is and who provides it.1 2 An understanding of public views is needed in order to target education and policy campaigns and to manage future needs, expectations and resourcing of care.

Aim: The aim of this study was to establish the current levels of awareness and perceptions of palliative care among the general public in Northern Ireland.

Methods: A mixed methods study comprising two phases was undertaken. A community-based cross-sectional survey with a population of 3,557 individuals aged over 17 years was performed. Information was collected using a structured questionnaire consisting of 17 items. Open questions were subject to content analysis; closed questions were subject to descriptive statistics with inferential testing as appropriate. This was followed by semi structured telephone interviews (n=50).

Results: Responses indicated limited knowledge about palliative care. Respondents who worked in healthcare themselves or who had a close relative or friend who had used a palliative care service were more aware of palliative care and the availability of different palliative care services. The main barriers to raising awareness were fear, lack of interaction with health services and perception of lack of resources. A key aspect identified for promoting palliative care was the development of understanding and use of the term itself and targeted educational strategies.

Conclusions: Public awareness of the concept of palliative care and of service availability remains insufficient. An increased awareness of palliative care is needed, in order to improve knowledge of and access to services when required, empower individuals, involve communities and ultimately to improve the delivery of palliative and end-of-life care.

Characterization, crystallization and preliminary X—ray diffraction analysis of acutohaemolysin, a haemolytic toxin from Agkistrodon acutus venom

Acutohaemolysin, a phospholipase A2 (PLA2) from the venom of the snake Agkistrodon acutus, has been isolated and purified to homogeneity by anion-exchange chromatography on a DEAE-Sepharose column followed by cation-exchange chromatography on a CM-Sepharose column. It is an alkaline protein with an isoelectric point of 10.5 and is comprised of a single polypeptide chain of 13 938 Da. Its N-terminal amino-acid sequence shows very high similarity to Lys49-type PLA2 proteins from other snake venoms. Although its PLA2 enzymatic activity is very low, acutohaemolysin has a strong indirect haemolytic activity and anticoagulant activity. Acutohaemolysin crystals with a diffraction limit of 1.60 Å were obtained by the hanging-drop vapour-diffusion method. The crystals belong to the space group C2, with unit-cell parameters a = 45.30, b = 59.55, c = 46.13 Å, [beta] = 117.69°. The asymmetric unit contains one molecule

Characterization of the Kepler-101 planetary system with HARPS-N. A hot super-Neptune with an Earth-sized low-mass companion

We characterize the planetary system Kepler-101 by performing a combined differential evolution Markov chain Monte Carlo analysisof Kepler data and forty radial velocities obtained with the HARPS-N spectrograph. This system was previously validated and iscomposed of a hot super-Neptune, Kepler-101b, and an Earth-sized planet, Kepler-101c. These two planets orbit the slightly evolvedand metal-rich G-type star in 3.49 and 6.03 days, respectively. With mass Mp = 51.1+5.1−4.7 M⊕, radius Rp = 5.77+0.85−0.79 R⊕, and density ρp = 1.45+0.83 −0.48 g cm−3, Kepler-101b is the first fully characterized super-Neptune, and its density suggests that heavy elements makeup a significant fraction of its interior; more than 60% of its total mass. Kepler-101c has a radius of 1.25+0.19−0.17 R⊕, which implies theabsence of any H/He envelope, but its mass could not be determined because of the relative faintness of the parent star for highly precise radial-velocity measurements (Kp = 13.8) and the limited number of radial velocities. The 1σ upper limit, Mp < 3.8 M⊕, excludes a pure iron composition with a probability of 68.3%. The architecture of the planetary system Kepler-101 − containing aclose-in giant planet and an outer Earth-sized planet with a period ratio slightly larger than the 3:2 resonance − is certainly of interest for scenarios of planet formation and evolution. This system does not follow the previously reported trend that the larger planet has the longer period in the majority of Kepler systems of planet pairs with at least one Neptune-sized or larger planet.

CaV3.1 T-Type Ca2+ Channels Contribute to Myogenic Signalling in Rat Retinal Arterioles

Purpose: Although L-type Ca2+ channels are known to play a key role in the myogenic reactivity of retinal arterial vessels, the involvement of other types of voltage-gated Ca2+ channels in this process remains unknown. In the present study we have investigated the contribution of T-type Ca2+ channels to myogenic signalling in arterioles of the rat retinal microcirculation.

Methods: Confocal immunolabelling of wholemount preparations was used to investigate the localisation of CaV3.1-3 channels in retinal arteriolar smooth muscle cells. T-type currents and the contribution of T-type channels to myogenic signalling were assessed by whole-cell patch-clamp recording and pressure myography of isolated retinal arteriole segments.

Results: Strong immunolabelling for CaV3.1 was observed on the plasma membrane of retinal arteriolar smooth muscle cells. In contrast, no expression of CaV3.2 or CaV3.3 could be detected in retinal arterioles, although these channels were present on glial cell end feet surrounding the vessels and retinal ganglion cells, respectively. TTA-A2 sensitive T-type currents were recorded in retinal arteriolar myocytes with biophysical properties distinct from those of the L-type currents present in these cells. Inhibition of T-type channels using TTA-A2 or ML-218 dilated isolated, myogenically active, retinal arterioles.

Conclusions: CaV3.1 T-type Ca2+ channels are functionally expressed on arteriolar smooth muscle cells of retinal arterioles and play an important role in myogenic signalling in these vessels. The work has important implications concerning our understanding of the mechanisms controlling blood flow autoregulation in the retina and its disruption during ocular disease.