Lateral clustering of platelet GP Ib-IX complexes leads to up-regulation of the adhesive function of integrin alpha IIbbeta 3.

Binding of von Willebrand factor (VWF) to GP Ib-IX mediates initial platelet adhesion and increases the subsequent adhesive function of alpha(IIb)beta(3). Because these responses are promoted most effectively by large VWF multimers, we hypothesized that receptor clustering modulates GP Ib-IX function. To test this, GP IX was fused at its cytoplasmic tail to tandem repeats of FKBP, and GP Ib-IX(FKBP)(2) and alpha(IIb)beta(3) were expressed in Chinese hamster ovary cells. Under flow conditions at wall shear rates of up to 2000 s(-1), GP Ib-IX(FKBP)(2) mediated cell tethering to immobilized VWF, just as in platelets. Conditional oligomerization of GP Ib-IX(FKBP)(2) by AP20187, a cell-permeable FKBP dimerizer, caused a decrease in cell translocation velocities on VWF (p < 0.001). Moreover, clustering of GP Ib-IX(FKBP)(2) by AP20187 led to an increase in alpha(IIb)beta(3) function, manifested under static conditions by increased cell adhesion to fibrinogen (p < 0.01) and under flow by increased stable cell adhesion to VWF (p < 0.04). Clustering of GP Ib-IX(FKBP)(2) also stimulated rapid tyrosine phosphorylation of ectopically expressed Syk, a putative downstream effector of GP Ib-IX in platelets. These studies establish that GP Ib-IX oligomerization, per se, affects the interaction of this receptor with VWF and its ability to influence the adhesive function of alpha(IIb)beta(3). By extrapolation, GP Ib-IX clustering in platelets may promote thrombus formation.     

Alternative entry receptors for herpes simplex virus and their roles in disease

Either herpesvirus entry mediator (HVEM, TNFRSF14) or nectin-1 (PVRL1) is sufficient for herpes simplex virus (HSV) infection of cultured cells. The contribution of individual receptors to infection in vivo and to disease is less clear. To assess this, Tnfrsf14(-/-) and/or Pvrl1(-/-) mice were challenged intravaginally with HSV-2. Infection of the vaginal epithelium occurred in the absence of either HVEM or nectin-1 but was virtually undetectable when both receptors were absent, indicating that either HVEM or nectin-1 was necessary. Absence of nectin-1 (but not HVEM) reduced efficiency of infection of the vaginal epithelium and viral spread to the nervous system, attenuating neurological disease and preventing external lesion development. While nectin-1 proved not to be essential for infection of the nervous system, it is required for the full manifestations of disease. This study illustrates the value of mutant mice for understanding receptor contributions to disease caused by a human virus.     

Peptidylarginine deiminase 2 suppresses inhibitory {kappa}B kinase activity in lipopolysaccharide-stimulated RAW 264.7 macrophages

Peptidylarginine deiminases (PADs) are enzymes that convert arginine to citrulline in proteins. In this study, we examined PAD-mediated citrullination and its effect on pro-inflammatory activity in the macrophage cell line RAW 264.7. Citrullination of 45-65-kDa proteins was induced when cells were treated with lipopolysaccharide (LPS; 1 μg/ml). Protein citrullination was suppressed by the intracellular calcium chelator BAPTA/AM (30 μM). LPS treatment up-regulated COX-2 levels in cells. Interestingly, overexpressing PAD2 reduced LPS-mediated COX-2 up-regulation by 50%. PAD2 overexpression also reduced NF-κB activity, determined by NF-κB-driven luciferase activity. The effect of PAD2 on NF-κB activity was further examined by using HEK 293 cells transfected with NF-κB luciferase, IκB β/γ kinase (IKKβ/γ) subunits, and PAD2. IKKβ increased NF-κB activity, but this increase was markedly suppressed when PAD2 was present in cells. IKKβ-mediated NF-κB activation was further enhanced by IKKγ in the presence of calcium ionophore A23187. However, this stimulatory effect of IKKβ/γ was abolished by PAD2. Coimmunoprecipitation of cell lysates showed that IKKγ and PAD2 can coimmunoprecipitate in the presence of the Ca(2+) ionophore. IKKγ coimmunoprecipitated truncation mutants, PAD2(1-385) and PAD2(355-672). The substitution of Gln-358 (a putative ligand for Ca(2+) binding) with an Ala abolished coimmunoprecipitation. Conversely, PAD2 coimmunoprecipitated truncation mutants IKKγ(1-196) and IKKγ(197-419). In other experiments, treating RAW 264.7 cells with LPS induced citrullination in the immunoprecipitates of IKKγ. In vitro citrullination assay showed that incubation of purified PAD2 and IKKγ proteins in the presence of Ca(2+) citrullinated IKKγ. These results demonstrate that PAD2 interacts with IKKγ and suppresses NF-κB activity.     

Chemiluminescent detection of sequential DNA hybridizations to high-density, filter-arrayed cDNA libraries: a subtraction method for novel gene discovery.

A chemiluminescent approach for sequential DNA hybridizations to high-density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently "deplexed" into individual components for specific probe localizations.     

The E3 Ubiquitin Ligase Triad3A Negatively Regulates the RIG-I/MAVS Signaling Pathway by Targeting TRAF3 for Degradation

The primary role of the innate immune response is to limit the spread of infectious pathogens, with activation of Toll-like receptor (TLR) and RIG-like receptor (RLR) pathways resulting in a pro-inflammatory response required to combat infection. Limiting the activation of these signaling pathways is likewise essential to prevent tissue injury in the host. Triad3A is an E3 ubiquitin ligase that interacts with several components of TLR signaling and modulates TLR activity. In the present study, we demonstrate that Triad3A negatively regulates the RIG-I RNA sensing pathway through Lys⁴⁸-linked, ubiquitin-mediated degradation of the tumor necrosis factor receptor-associated factor 3 (TRAF3) adapter. Triad3A was induced following dsRNA exposure or virus infection and decreased TRAF3 levels in a dose-dependent manner; moreover, Triad3A expression blocked IRF-3 activation by Ser-396 phosphorylation and inhibited the expression of type 1 interferon and antiviral genes. Lys⁴⁸-linked ubiquitination of TRAF3 by Triad3A increased TRAF3 turnover, whereas reduction of Triad3A expression by stable shRNA expression correlated with an increase in TRAF3 protein expression and enhancement of the antiviral response following VSV or Sendai virus infection. Triad3A and TRAF3 physically interacted together, and TRAF3 residues Y440 and Q442—previously shown to be important for association with the MAVS adapter—were also critical for Triad3A. Point mutation of the TRAF-Interacting-Motif (TIM) of Triad3A abrogated its ability to interact with TRAF3 and modulate RIG-I signaling. TRAF3 appears to undergo sequential ubiquitin “immuno-editing” following virus infection that is crucial for regulation of RIG-I-dependent signaling to the antiviral response. Thus, Triad3A represents a versatile E3 ubiquitin ligase that negatively regulates RIG-like receptor signaling by targeting TRAF3 for degradation following RNA virus infection.     

Proteomic analysis of pulmonary edema fluid and plasma in patients with acute lung injury

Proteomics is the large-scale analysis of protein profiles. This approach has not yet been reported in the study of acute lung injury (ALI). This study details protein profiles in plasma and pulmonary edema fluid (EF) from 16 ALI patients and plasma and bronchoalveolar lavage fluid (BALF) from 12 normal subjects. More than 300 distinct protein spots were evident in the EF and BALF of both normal subjects and ALI patients. Of these, 158 were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In the plasma and EF protein profile of ALI patients, there were multiple qualitative changes. For instance, in all normal subjects, but in only one of the ALI patients, seven distinct surfactant protein A isoforms were evident. Nearly all ALI patients also had protein spots that indicate truncation or other posttranslational modifications. Several of these novel changes could serve as new biomarkers of lung injury.     

Quantifying the dynamics of light tolerance in Arabidopsis plants during ontogenesis

The amount of light plants can tolerate during different phases of ontogenesis remains largely unknown. This was addressed here employing a novel methodology that uses the coefficient of photochemical quenching (qP) to assess the intactness of photosystem II reaction centres. Fluorescence quenching coefficients, total chlorophyll content and concentration of anthocyanins were determined weekly during the juvenile, adult, reproductive and senescent phases of plant ontogenesis. This enabled quantification of the protective effectiveness of non‐photochemical fluorescence quenching (NPQ) and determination of light tolerance. The light intensity that caused photoinhibition in 50% of leaf population increased from ～70 μmol m^?2 s^?1, for 1‐week‐old seedlings, to a maximum of 1385 μmol m^?2 s^?1 for 8‐week‐old plants. After 8 weeks, the tolerated light intensity started to gradually decline, becoming only 332 μmol m^?2 s^?1 for 13‐week‐old plants. The dependency of light tolerance on plant age was well‐related to the amplitude of protective NPQ (pNPQ) and the electron transport rates (ETRs). Light tolerance did not, however, show a similar trend to chlorophyll a/b ratios and content of anthocyanins. Our data suggest that pNPQ is crucial in defining the capability of high light tolerance by Arabidopsis plants during ontogenesis.     

Keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inflammation, and repair

A growing body of evidence indicates that the epithelial-specific growth factors keratinocyte growth factor (KGF), fibroblast growth factor (FGF)-10, and hepatocyte growth factor (HGF) play important roles in lung development, lung inflammation, and repair. The therapeutic potential of these growth factors in lung disease has yet to be fully explored. KGF has been best studied and has impressive protective effects against a wide variety of injurious stimuli when given as a pretreatment in animal models. Whether this protective effect could translate to a treatment effect in humans with acute lung injury needs to be investigated. FGF-10 and HGF may also have therapeutic potential, but more extensive studies in animal models are needed. Because HGF lacks true epithelial specificity, it may have less potential than KGF and FGF-10 as a targeted therapy to facilitate lung epithelial repair. Regardless of their therapeutic potential, studies of the unique roles played by these growth factors in the pathogenesis and the resolution of acute lung injury and other lung diseases will continue to enhance our understanding of the complex pathophysiology of inflammation and repair in the lung.     

Intraguild predation involving Harmonia axyridis: a review of current knowledge and future perspectives

As an effective generalist predator of aphids and other hemipteran pests H. axyridis has been a successful biological control agent. However, the very functional traits that have contributed to its success in this regard also implicate it as an intraguild predator that poses a significant risk not only to the diversity of other natural enemies of Hemiptera (and their associated ecosystem services), but to biodiversity more widely. In this paper we will specifically review the existing data on intraguild predation involving H. axyridis, and consider the strength and symmetry of such interactions both within its native guild and within exotic guilds where it has established as an invasive alien. We will use these studies to interpret the observed population declines in predator diversity in the field, predict species at risk in regions not yet invaded and consider implications for resulting ecosystem services. We will also indicate gaps in our knowledge that require further study in order to identify opportunities for mitigation.