Markers of vascular endothelial cell damage and P. falciparum malaria: association between levels of both sE-selectin and thrombomodulin, and cytokines, hemoglobin and clinical presentation

We investigated associations between markers of damage of vascular endothelial cells (MDVECs) and plasma cytokine levels, hemoglobin level and temperature in individuals with acute uncomplicated malaria, as well as healthy controls, using enzyme linked immunosorbent assay (ELISA) for the presence of soluble endothelial cell adhesion molecule-1 (sE-selectin), circulating granule membrane protein-140 (sP-selectin), circulating thrombomodulin (TM), circulating von Willebrand factor (VWf), interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). Significant differences were observed between falciparum malaria patients and the healthy people in term of levels of both sE-selectin and TM. The serum levels of sP-selectin and VWf were comparable between the two groups. The levels of both sE-Selectin and TM correlated positively with temperature, levels of IFN-gamma and levels of TNF-alpha; and negatively with hemoglobin levels. Trends of positive correlations were observed between level of sP-selectin or VWf and temperature. Furthermore, sE-selectin levels correlated with vomiting. These data suggest that sE-selectin and TM might be useful markers of endothelium activation in in vivo studies. Moreover, our results highlight the use of both sE-selctin and TM as markers of anemia.     

Recent advances in tRNA mitochondrial import

In many eukaryotes, tRNA import from the cytosol into mitochondria is essential for mitochondrial biogenesis and, consequently, for cell viability. Recent work has begun to unravel the molecular mechanisms involved in tRNA transport in yeast, trypanosomatids and plants. The mechanisms of tRNA targeting to, and translocation through, the double mitochondrial membrane in addition to how selectivity and regulation of these processes are achieved are the main questions that have been addressed. The characterization of both direct and co-import mechanisms involving distinct protein-import factors is in agreement with a polyphyletic origin of tRNA import. Moreover, our increased understanding of the tRNA-import pathway has been exploited recently to rescue dysfunctions associated with mitochondrial tRNA mutations.     

Replication of an association between 17q21 SNPs and asthma in a French-Canadian familial collection

Asthma is a complex trait which is influenced by environmental and genetic factors. In a recent genome-wide association study on asthma in European subjects, novel associations were reported between 17q21 single nucleotide polymorphisms (SNPs) and childhood asthma. We performed an association study with the ten SNPs that showed the strongest association in a French-Canadian asthmatic familial collection. Family-based association tests revealed significant associations for eight SNPs (0.005 < P < 0.017) and for two haplotypes (P = 0.0004 and 0.002), confirming the 17q21 as an asthma locus. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Apoptotic cell-derived sphingosine-1-phosphate promotes HuR-dependent cyclooxygenase-2 mRNA stabilization and protein expression

Removal of apoptotic cells by phagocytes is considered a pivotal immune regulatory process. Although considerable knowledge has been obtained on the postphagocytic macrophage phenotype, there is little information on molecular mechanisms, which provoke macrophage polarization. In this study, we show that human apoptotic Jurkat cells (AC) or AC-conditioned medium (CM) rapidly induces cyclooxygenase-2 (COX-2) expression in mouse RAW264.7 macrophages via sphingosine-1-phosphate (S1P). Pharmacological inhibition of S1P release from AC or using CM from cells with a knockdown of sphingosine kinase 2 in human MCF-7 cells abrogates this effect. Expression of COX-2 resulted from an increase in mRNA stability via its 3'-untranslated region (UTR), shown by COX-2-3'-UTR and AU-rich element-driven reporter assays. Western analysis corroborated increased nucleocytoplasmic shuttling of the RNA-binding protein HuR after CM treatment. RNA EMSA analysis revealed an S1P- and CM-mediated increase in HuR-RNA binding to a COX-2-specific UTR, whereas HuR knockdown pointed to its importance for S1P in CM-induced COX-2 expression. Immunofluorescence microscopy of phospholipase A2 (PLA2) and ELISA analysis of PGE2 revealed activation of PLA2 and production of PGE2 in response to CM but not S1P. S1P, released from AC, uses HuR to stabilize COX-2 mRNA and thus to increase COX-2 protein expression. However, only CM also activates PLA2 to provide the substrate for COX-2. Our data underscore the importance of S1P in AC-mediated immune regulation, by stabilizing COX-2 mRNA in macrophages, a prerequisite for PGE2 formation.     

Sumoylation of peroxisome proliferator-activated receptor gamma by apoptotic cells prevents lipopolysaccharide-induced NCoR removal from kappaB binding sites mediating transrepression of proinflammatory cytokines

Efficient clearance of apoptotic cells (AC) by professional phagocytes is crucial for tissue homeostasis and resolution of inflammation. Macrophages respond to AC with an increase in antiinflammatory cytokine production but a diminished release of proinflammatory mediators. Mechanisms to explain attenuated proinflammatory cytokine formation remain elusive. We provide evidence that peroxisome proliferator-activated receptor gamma (PPARgamma) coordinates antiinflammatory responses following its activation by AC. Exposing murine RAW264.7 macrophages to AC before LPS stimulation reduced NF-kappaB transactivation and lowered target gene expression of, that is, TNF-alpha and IL-6 compared with controls. In macrophages overexpressing a dominant negative mutant of PPARgamma, NF-kappaB transactivation in response to LPS was restored, while macrophages from myeloid lineage-specific conditional PPARgamma knockout mice proved that PPARgamma transmitted an antiinflammatory response, which was delivered by AC. Expressing a PPARgamma-Delta aa32-250 deletion mutant, we observed no inhibition of NF-kappaB. Analyzing the PPARgamma domain structures within aa 32-250, we anticipated PPARgamma sumoylation in mediating the antiinflammatory effect in response to AC. Interfering with sumoylation of PPARgamma by mutating the predicted sumoylation site (K77R), or knockdown of the small ubiquitin-like modifier (SUMO) E3 ligase PIAS1 (protein inhibitor of activated STAT1), eliminated the ability of AC to suppress NF-kappaB. Chromatin immunoprecipitation analysis demonstrated that AC prevented the LPS-induced removal of nuclear receptor corepressor (NCoR) from the kappaB site within the TNF-alpha promoter. We conclude that AC induce PPARgamma sumoylation to attenuate the removal of NCoR, thereby blocking transactivation of NF-kappaB. This contributes to an antiinflammatory phenotype shift in macrophages responding to AC by lowering proinflammatory cytokine production.     

Amplification of Adipogenic Commitment by VSTM2A

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Synthesis and biological evaluation of new donepezil-like Thiaindanones as AChE inhibitors

Pharmacomodulations of previously reported thiaindanones related to donepezil were achieved with the aim to enhance their AChE inhibitory activity. Condensation of the cyclopentane carbonyl group into hydrazone or cyanolefine derivatives, as well as its hydrogenation and the subsequent substitution of the resulting hydroxyl group led to new 2-(4-benzylpiperazin-1-yl)-N-(1,3-dibromo-6-hydroxy-5,6-dihydro-4H-cyclopenta[c]thien-4-yl) acetamides. The in vitro evaluation of this new series, according to the method of Ellman, shows however that it conserved only partially the biological activity. The best compound remains the alcohol 11 (IC(50) = 0.40 microM, against 0.02 microM for donepezil).     

Characterization of organic cation/carnitine transporter family in human sperm

Spermatozoan maturation, motility, and fertility are, in part, dependent upon the progressive increase in epididymal and spermatozoal carnitine, critical for mitochondrial fatty acid oxidation, as sperm pass from the caput to the cauda of the epididymis. We demonstrate that the organic cation/carnitine transporters, OCTN1, OCTN2, and OCTN3, are expressed in sperm as three distinct proteins with an expected molecular mass of 63 kDa, using Western blot analysis and our transporter-specific antibodies. Carnitine uptake studies in normal control human sperm samples further support the presence of high-affinity (OCTN2) carnitine uptake (K(m) of 3.39+/-1.16 microM; V(max) of 0.23+/-0.14 pmol/min/mg sperm protein; and mean+/-SD; n=12), intermediate-affinity (OCTN3) carnitine uptake (K(m) of 25.9+/-14.7 microM; V(max) of 1.49+/-1.03 pmol/min/mg protein; n=26), and low-affinity (OCTN1) carnitine uptake (K(m) of 412.6+/-191 microM; V(max) of 32.7+/-20.5 pmol/min/mg protein; n=18). Identification of individuals with defective sperm carnitine transport may provide potentially treatable etiologies of male infertility, responsive to L-carnitine supplementation.     

Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin

Background

Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin.

Methods

To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (F_unb) in the absence and presence of calponin or phosphorylated calponin.

Results

F_unb from F-actin alone (0.12 ± 0.01 pN; mean ± SE) was significantly increased in the presence of calponin (0.20 ± 0.02 pN). This enhancement was lost when calponin was phosphorylated (0.12 ± 0.01 pN). To further verify that this enhancement of F_unb was due to the cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the F_unb obtained at a [KCl] of 25 mM (0.21 ± 0.02 pN; mean ± SE) was significantly decreased at a [KCl] of 150 mM, (0.13 ± 0.01 pN).

Conclusions

This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state.

General significance

This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.