Development of a homogeneous time-resolved fluorescence leukotriene B4 assay for determining the activity of leukotriene A4 hydrolase

Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotactic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4 hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA4 hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4 (LTB4 HTRF assay) and its application to establish an HTRF-based LTA4 hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4 HTRF assay to the LTA4 hydrolase reaction, an HTRF-based LTA4 hydrolase assay was established and validated. Using a test set of 16 LTA4 hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA4 hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase.     

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells.Biological membranes are conceptually simple structures that may be generated in vitro according to simple physicochemical principles. In vivo, however, membranes are highly complex and host a plethora of proteins that mediate the transfer of molecules and communication across the membrane. Proteins may be trapped in membrane by their transmembrane domains, anchored by lipid tails, or attach to membrane-integral proteins. A further level of complexity is seen when membrane proteins are not equally distributed but occupy only a limited fraction of the available surface (i.e. when they are polarly localized or when they form small membrane subdomains in the micrometer range). The question of how membrane proteins are retained locally and prevented from diffusing freely is of high importance to cell biology. Polarly localized proteins may be retained in their respective domains by membrane fences; in such a situation, polarly localized proteins are mobile in their domains but cannot diffuse through tightly packed scaffold proteins forming a molecular fence within the membrane. Membrane fences delimiting polar domains have been described in different organisms. For example, diffusion between membrane compartments is prevented in budding yeast (Saccharomyces cerevisiae) at the level of the bud neck (Barral et al., 2000; Takizawa et al., 2000); in ciliated vertebrate cells, between ciliary and periciliary membranes (Hu et al., 2010); in epithelial cells, between apical and basolateral membranes (van Meer and Simons, 1986); in neurons, between axon and soma (Kobayashi et al., 1992; Winckler et al., 1999; Nakada et al., 2003); and in spermatozoa, at the level of the annulus (Myles et al., 1984; Nehme et al., 1993). The existence of membrane scaffolds that prevent free protein diffusion has also been described in bacteria (Baldi and Barral, 2012; Schlimpert et al., 2012). In plants, we have shown the existence of a strict membrane fence in the root endodermis, where a median domain splits the cell in two lateral halves occupied by different sets of proteins (Alassimone et al., 2010). The situation in the plant endodermis is analogous to the separation of animal epithelia into apical and basolateral domains; indeed, a parallel between epithelia and endodermal cells has been drawn, despite the different origin of multicellularity in plants and animals (Grebe, 2011).The protein complexes responsible for the formation of membrane fences have been identified. Septins are a family of proteins able to oligomerize and form filaments (Saarikangas and Barral, 2011); their role in the formation of membrane fences has been demonstrated in several organisms and cellular situations, including the yeast bud neck (Barral et al., 2000; Takizawa et al., 2000), animal cilia (Hu et al., 2010), and mammalian spermatozoa (Ihara et al., 2005; Kissel et al., 2005; Kwitny et al., 2010). At the axonal initial segment of neurons, AnkyrinG is necessary to establish and maintain a membrane scaffold where different membrane proteins are immobilized and stabilized (Hedstrom et al., 2008; Sobotzik et al., 2009). In Caulobacter crescentus, the stalk protein Stp forms a complex that prevents diffusion between the cell body and stalk and between stalk compartments. Claudins and occludin are the main components of epithelial tight junctions (Furuse et al., 1993, 1998). Occludins are four-membrane-span proteins and belong to the MARVEL protein family (Sánchez-Pulido et al., 2002), as do Tricellulin and MARVELD3, which are also tight junction-associated proteins (Furuse et al., 1993; Ikenouchi et al., 2005; Steed et al., 2009).In Arabidopsis (Arabidopsis thaliana), our group identified a family of proteins that form a membrane fence in the endodermis (Roppolo et al., 2011). These CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASP1 to CASP5) are four-transmembrane proteins that form a median domain referred to as the Casparian strip membrane domain (CSD). CASPs are initially targeted to the whole plasma membrane, then they are quickly removed from lateral plasma membranes and remain localized exclusively at the CSD; there, they show an extremely low turnover, although they are eventually removed (Roppolo et al., 2011). The membrane proteins NOD26-LIKE INTRINSIC PROTEIN5;1 and BORON TRANSPORTER1 are restricted from diffusing through the CSD and remain polarly localized in the outer and inner lateral membranes, respectively; a fluorescent lipophilic molecule, when integrated in the outer endodermal membrane, was blocked at the level of the CSD and could not diffuse into the inner membrane (Roppolo et al., 2011). Besides making a plasma membrane diffusion barrier, CASPs have an important role in directing the modification of the cell wall juxtaposing their membrane domain: by interacting with secreted peroxidases, they mediate the deposition of lignin and the building up of the Casparian strips (Roppolo et al., 2011; Naseer et al., 2012; Lee et al., 2013). The two CASP activities, making membrane scaffolds and directing a modification of the cell wall, can be uncoupled: indeed, (1) formation of the CASP domain is independent from the deposition of lignin, and (2) interaction between CASPs and peroxidases can take place outside the CSD when CASPs are ectopically expressed (Lee et al., 2013).As CASPs are currently the only known proteins forming membrane fences in plants and because of their essential role in directing a local cell wall modification, we were interested in characterizing the repertoire of a large number of CASP-like (CASPL) proteins in the plant kingdom. Our aim was to provide the molecular basis for the discovery of additional membrane domains in plants and for the identification of proteins involved in local cell wall modifications. We extended our phylogenetic analysis outside of the plant kingdom and found conservation between CASPLs and the MARVEL protein family. Conserved residues are located in transmembrane domains, and we provide evidence suggesting that these domains are involved in CASP localization. We explored the potential use of the CASPL module in plants by investigating CASPL expression patterns and their ability to form membrane domains in the endodermis. Moreover, we related the appearance of the Casparian strips in the plant kingdom to the emergence of a CASP-specific signature that was not found in the genomes of plants lacking Casparian strips.     

Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1

Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP₃ receptors (IP₃Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP₃R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP₃R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP₃R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment.     

Decline of FoxP3+ Regulatory CD4 T Cells in Peripheral Blood of Children Heavily Exposed to Malaria

FoxP3+ regulatory CD4 T cells (T_regs) help to maintain the delicate balance between pathogen-specific immunity and immune-mediated pathology. Prior studies suggest that T_regs are induced by P. falciparum both in vivo and in vitro; however, the factors influencing T_reg homeostasis during acute and chronic infections, and their role in malaria immunopathogenesis, remain unclear. We assessed the frequency and phenotype of T_regs in well-characterized cohorts of children residing in a region of high malaria endemicity in Uganda. We found that both the frequency and absolute numbers of FoxP3+ T_regs in peripheral blood declined markedly with increasing prior malaria incidence. Longitudinal measurements confirmed that this decline occurred only among highly malaria-exposed children. The decline of T_regs from peripheral blood was accompanied by reduced in vitro induction of T_regs by parasite antigen and decreased expression of TNFR2 on T_regs among children who had intense prior exposure to malaria. While T_reg frequencies were not associated with protection from malaria, there was a trend toward reduced risk of symptomatic malaria once infected with P. falciparum among children with lower T_reg frequencies. These data demonstrate that chronic malaria exposure results in altered T_reg homeostasis, which may impact the development of antimalarial immunity in naturally exposed populations.     

Rural-urban differences on the rates and factors associated with early initiation of breastfeeding in Nigeria: further analysis of the Nigeria demographic and health survey, 2013

Background

This study investigates and compares the rates and factors associated with early initiation of breastfeeding (EIBF) within one hour of birth in rural and urban Nigeria.

Methods

Data from the 2013 Nigeria Demographic and Health Survey (NDHS) were analyzed. The rates of EIBF were reported using frequency tabulation. Associated factors were examined using Chi-Square test and further assessed on multivariable logistic regression analysis.

Results

The rates of EIBF were 30.8% (95% confidence interval [CI] 29.0, 32.6) and 41.9% (95% CI 39.6, 44.3) in rural and urban residences, respectively (p?<?0.001). The North-Central region had the highest EIBF rates both in rural (43.5%) and urban (63.5%) residences. Greater odds of EIBF in rural residence were significantly associated with higher birth order (Adjusted Odds Ratio [AOR] 1.29, 95% CI 1.10, 1.60), large birth size (AOR 1.33, 95% CI 1.10, 1.60), and health facility delivery (AOR 1.46, 95% CI 1.23, 1.72). Rural mothers in the rich wealth index, not working and whose husbands obtained at least a secondary school education had significantly higher odds of early initiation of breastfeeding. Regardless of residence, greater odds of EIBF were significantly associated with non-cesarean delivery (Rural AOR 3.50, 95% CI 1.84, 6.62; Urban AOR 2.48, 95% CI 1.60, 3.80) and living in North-Central (Rural AOR 1.84, 95% CI 1.34, 2.52; Urban AOR 4.40, 95% CI 3.15, 6.15) region. Also, higher odds of EIBF were significantly associated with living in North-East (Rural AOR 1.48, 95% CI 1.05, 2.08; Urban AOR 3.50, 95% CI 2.55, 4.83), South-South (Rural AOR 1.51, 95% CI 1.11, 2.10; Urban AOR 2.84, 95% CI 2.03, 3.97) and North-West (Urban residence only AOR 2.08, 95% CI 1.54, 2.80) regions.

Conclusions

Rural-urban differences in the rates and factors associated with EIBF exist in Nigeria with rural residence having significantly lower rates. Intervention efforts which address the risk factors identified in this study may contribute to improved EIBF rates. Efforts need to prioritize rural mothers generally, (particularly, those in rural North-West region) as well as mothers in urban South-West region of Nigeria.

     

The xipotl mutant of Arabidopsis reveals a critical role for phospholipid metabolism in root system development and epidermal cell integrity

Phosphocholine (PCho) is an essential metabolite for plant development because it is the precursor for the biosynthesis of phosphatidylcholine, which is the major lipid component in plant cell membranes. The main step in PCho biosynthesis in Arabidopsis thaliana is the triple, sequential N-methylation of phosphoethanolamine, catalyzed by S-adenosyl-l-methionine:phosphoethanolamine N-methyltransferase (PEAMT). In screenings performed to isolate Arabidopsis mutants with altered root system architecture, a T-DNA mutagenized line showing remarkable alterations in root development was isolated. At the seedling stage, the mutant phenotype is characterized by a short primary root, a high number of lateral roots, and short epidermal cells with aberrant morphology. Genetic and biochemical characterization of this mutant showed that the T-DNA was inserted at the At3g18000 locus (XIPOTL1), which encodes PEAMT (XIPOTL1). Further analyses revealed that inhibition of PCho biosynthesis in xpl1 mutants not only alters several root developmental traits but also induces cell death in root epidermal cells. Epidermal cell death could be reversed by phosphatidic acid treatment. Taken together, our results suggest that molecules produced downstream of the PCho biosynthesis pathway play key roles in root development and act as signals for cell integrity.     

Interleukin-1-induced neurotoxicity is mediated by glia and requires caspase activation and free radical release

Interleukin (IL)-1 expression is induced rapidly in response to diverse CNS insults and is a key mediator of experimentally induced neuronal injury. However, the mechanisms of IL-1-induced neurotoxicity are unknown. The aim of the present study was to examine the toxic effects of IL-1 on rat cortical cell cultures. Treatment with IL-1beta did not affect the viability of pure cortical neurones. However, IL-1 treatment of cocultures of neurones with glia or purified astrocytes induced caspase activation resulting in neuronal death. Neuronal cell death induced by IL-1 was prevented by pre-treatment with the IL-1 receptor antagonist, the broad spectrum caspase inhibitor Boc-Asp-(OMe)-CH(2)F or the antioxidant alpha-tocopherol. The NMDA receptor antagonist dizolcipine (MK-801) attenuated cell death induced by low doses of IL-1beta but the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) had no effect. Inhibition of inducible nitric oxide synthase with N(omega)-nitro-l-arginine methyl ester had no effect on neuronal cell death induced by IL-1beta. Thus, IL-1 activates the IL-1 type 1 receptor in astrocytes to induce caspase-dependent neuronal death, which is dependent on the release of free radicals and may contribute to neuronal cell death in CNS diseases.