Proteasome inhibition improves diaphragm function in congestive heart failure rats

In congestive heart failure (CHF), diaphragm weakness is known to occur and is associated with myosin loss and activation of the ubiquitin-proteasome pathway. The effect of modulating proteasome activity on myosin loss and diaphragm function is unknown. The present study investigated the effect of in vivo proteasome inhibition on myosin loss and diaphragm function in CHF rats. Coronary artery ligation was used as an animal model for CHF. Sham-operated rats served as controls. Animals were treated with the proteasome inhibitor bortezomib (intravenously) or received saline (0.9%) injections. Force generating capacity, cross-bridge cycling kinetics, and myosin content were measured in diaphragm single fibers. Proteasome activity, caspase-3 activity, and MuRF-1 and MAFbx mRNA levels were determined in diaphragm homogenates. Proteasome activities in the diaphragm were significantly reduced by bortezomib. Bortezomib treatment significantly improved diaphragm single fiber force generating capacity (approximately 30-40%) and cross-bridge cycling kinetics (approximately 20%) in CHF. Myosin content was approximately 30% higher in diaphragm fibers from bortezomib-treated CHF rats than saline. Caspase-3 activity was decreased in diaphragm homogenates from bortezomib-treated rats. CHF increased MuRF-1 and MAFbx mRNA expression in the diaphragm, and bortezomib treatment diminished this rise. The present study demonstrates that treatment with a clinically used proteasome inhibitor improves diaphragm function by restoring myosin content in CHF.     

Tyrosine kinase-deficient Wv c-kit induces mast cell adhesion and chemotaxis

W/W^v mice are deficient intissue mast cells, and mast cells cultured from these mice do notproliferate in response to the c-kit ligand, stem cell factor (SCF). Inthis paper, we report that mouse bone marrow cultured mast cellsderived from W/W^v mice do adhereto fibronectin in the presence of SCF and exhibit chemotaxis to SCF,and we explore this model for the understanding of c-kit-mediatedsignaling pathways. Both in vitro and in vivo (in intact cells)phosphorylation experiments demonstrated a low residual level ofW/W^v c-kit proteinphosphorylation. SCF-induced responses inW/W^v mast cells were abolished bythe tyrosine kinase inhibitor herbimycin A and by thephospatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin butwere not affected by protein kinase C inhibitors. These observationsare consistent with the conclusions thatW^v c-kit initiates a signalingprocess that is PI 3-kinase dependent and that mutatedW^v c-kit retains the ability toinitiate mast cell adhesion and migration.

     

Paraoxonase Genotype,LDL-oxidation and Carotid Atherosclerosis in Male Life-long Smokers

Paraoxonase (PON-1) is a high-density lipoprotein (HDL) associated enzyme that hydrolyzes lipid peroxides in vitro, which may therefore protect against the onset of atherosclerosis. Heavy smokers are more exposed to oxidative stress and hence at high-risk for oxidative modification of LDL.

Our hypothesis is that the anti-oxidative properties of PON-1 inhibit LDL oxidation, especially in populations exposed to high oxidative stress.

We have studied the effects of PON-1 genotype and smoking to variation in oxidative status parameters and intima-media thickness (IMT), a surrogate marker of atherosclerosis. The contribution of two common polymorphisms in the PON-1 gene (Q192R and L55M) to LDL oxidizability, autoantibodies directed against oxLDL and IMT were studied in 207 male life-long smokers. Smokers were classified into average, heavy and excessive smokers based on pack years of cigarettes smoked.

PON-1 genotype was not associated with autoantibodies to oxLDL, LDL oxidizability or IMT. Smoking was associated with IMT in subgroups with the high levels of LDL, but not in the population at large.

The lack of association of PON-1 genotype with oxidative status parameters and IMT suggests that PON-1 is not a major inhibitor of LDL oxidation in a population of life-long smokers.     

Maternal TLR4 and NOD2 gene variants, pro-inflammatory phenotype and susceptibility to early-onset preeclampsia and HELLP syndrome

Background

Altered maternal inflammatory responses play a role in the development of preeclampsia and the hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome. We examined whether allelic variants of the innate immune receptors Toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain 2 (NOD2), that impair the inflammatory response to endotoxin, are related to preeclampsia and HELLP syndrome.

Methods and Findings

We determined five common mutations in TLR4 (D299G and T399I) and NOD2 (R702W, G908R and L1007fs) in 340 primiparous women with a history of early-onset preeclampsia, of whom 177 women developed HELLP syndrome and in 113 women with a history of only uneventful pregnancies as controls. In addition, we assessed plasma levels of pro-inflammatory biomarkers C-reactive protein, interleukin-6, soluble intercellular adhesion molecule-1, fibrinogen and von Willebrand factor in a subset of 214 women included at least six months after delivery. After adjustment for maternal age and chronic hypertension, attenuating allelic variants of TLR4 were more common in women with a history of early-onset preeclampsia than in controls (OR 2.9 [95% CI 1.2–6.7]). Highest frequencies for TLR4 variants were observed in women who developed HELLP syndrome (adjusted OR 4.1 [95% CI 1.7–9.8]). In addition, high levels of interleukin-6 and fibrinogen were associated with a history of early-onset preeclampsia. Combined positivity for any of the TLR4 and NOD2 allelic variants and high levels of interleukin-6 was 6.9-fold more common in women with a history of early-onset preeclampsia (95% CI 2.1–23.2) compared to controls.

Conclusions

We observed an association of common TLR4 and NOD2 gene variants, and pro-inflammatory phenotype with a history of early-onset preeclampsia and HELLP syndrome. These findings suggest involvement of the maternal innate immune system in severe hypertensive disorders of pregnancy.     

A public genome-scale lentiviral expression library of human ORFs

Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.