Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

Induction of apoptosis by a cachectic-factor in murine myotubes and inhibition by eicosapentaenoic acid

Treatment of C₂C₁₂ myotubes with a tumour-derived proteolysis-inducing factor (PIF) at concentrations between 1 and 10 nM was shown to stimulate the activity of the apoptotic initiator caspases-8 and -9 and the apoptotic effector caspases-2, -3 and -6. This increased caspase activity was attenuated in myotubes pretreated with 50 M eicosapentaenoic acid (EPA). At least part of the increase in caspase activity may be related to the increased proteasome proteolytic activity, since a caspase-3 inhibitor completely attenuated the PIF-induced increase in chymotrypsin-like enzyme activity, the predominant proteolytic activity of the proteasome. However, Western blot analysis showed that PIF induced an increase in expression of the active form of caspase-3, which was also attenuated by EPA.Further Western blot analysis showed PIF increased the cytosolic content of cytochrome c, as well as expression of the pro-apoptotic protein bax but not the anti-apoptotic protein bcl-2, which were both attenuated by 50 M EPA. Induction of apoptosis by PIF in murine myotubes was confirmed by an increase in free nucleasomes formation and increased DNA fragmentation evidenced by a nucleasomal ladder typical of apoptotic cells. This process was again inhibited by pre-incubation with EPA. These results suggest that in addition to activating the proteasome, PIF induces apoptosis in C₂C₁₂ myotubes, possibly through the common intermediate arachidonic acid. Both of these processes would contribute to the loss of skeletal muscle in cancer cachexia.     

Structural and kinetic analyses of the protease from an amprenavir-resistant human immunodeficiency virus type 1 mutant rendered resistant to saquinavir and resensitized to amprenavir

Recent drug regimens have had much success in the treatment of human immunodeficiency virus (HIV)-infected individuals; however, the incidence of resistance to such drugs has become a problem that is likely to increase in importance with long-term therapy of this chronic illness. An analysis and understanding of the molecular interactions between the drug(s) and the mutated viral target(s) is crucial for further progress in the field of AIDS therapy. The protease inhibitor amprenavir (APV) generates a signature set of HIV type 1 (HIV-1) protease mutations associated with in vitro resistance (M46I/L, I47V, and I50V [triple mutant]). Passage of the triple-mutant APV-resistant HIV-1 strain in MT4 cells, in the presence of increasing concentrations of saquinavir (SQV), gave rise to a new variant containing M46I, G48V, I50V, and I84L mutations in the protease and a resulting phenotype that was resistant to SQV and, unexpectedly, resensitized to APV. This phenotype was consistent with a subsequent kinetic analysis of the mutant protease, together with X-ray crystallographic analysis and computational modeling which elucidated the structural basis of these observations. The switch in protease inhibitor sensitivities resulted from (i) the I50V mutation, which reduced the area of contact with APV and SQV; (ii) the compensating I84L mutation, which improved hydrophobic packing with APV; and (iii) the G-to-V mutation at residue 48, which introduced steric repulsion with the P3 group of SQV. This analysis establishes the fine detail necessary for understanding the loss of protease binding for SQV in the quadruple mutant and gain in binding for APV, demonstrating the powerful combination of virology, molecular biology, enzymology, and protein structural and modeling studies in the elucidation and understanding of viral drug resistance.     

1-Thio-beta-D-galactofuranosides: synthesis and evaluation as beta-D- galactofuranosidase inhibitors

Beta-D-galactofuranosidase is a good chemotherapeutic target for the design of inhibitors, since beta-D-galactofuranose is a constituent of important parasite glycoconjugates but is not present in the host mammals. With this aim, we have synthesized for the first time alkyl, benzyl and aryl 1-thio-beta-D-galactofuranosides by condensation of penta-O-benzoyl-alpha,beta-D-galactofuranose with the corresponding thiols, in the presence of SnCl4as catalyst. The complete chemical and spectroscopical characterization of these compounds showed that the reaction was stereoselective. Debenzoylation with sodium methoxide afforded the beta-S-galactofuranosides in high yield. The thioglycosides were tested as inhibitors of the beta-D- galactofuranosidase of Penicillium fellutanum, using for the first time 4-nitrophenyl-beta-D-galactofuranoside as chromogenic substrate. The 4- aminophenyl-1-thio-beta-D-galactofuranoside, obtained by catalytic hydrogenation of the nitrophenyl derivative, was the best inhibitor being then an adequate ligand for the preparation of an affinity phase aimed at the isolation of beta-d-galactofuranosidases from different sources. Also the inhibitory activity of d-galactono-1, 4-lactone was shown.      

Characterization of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Beijing isolates from the Mediterranean area

Background  

The Beijing lineage of Mycobacterium tuberculosis is causing concern due to its global distribution and its involvement in severe outbreaks. Studies focused on this lineage are mainly restricted to geographical settings where its prevalence is high, whereas those in other areas are scarce. In this study, we analyze Beijing isolates in the Mediterranean area, where this lineage is not prevalent and is mainly associated with immigrant cases.     

Signaling pathways initiated by beta-hydroxy-beta-methylbutyrate to attenuate the depression of protein synthesis in skeletal muscle in response to cachectic stimuli

To investigate the mechanism by which beta-hydroxy-beta-methylbutyrate (HMB) attenuates the depression of protein synthesis in the skeletal muscle of cachectic mice, a study has been carried out in murine myotubes in the presence of proteolysis-inducing factor (PIF). PIF inhibited protein synthesis by 50% within 4 h, and this was effectively attenuated by HMB (25-50 muM). HMB (50 muM) alone stimulated protein synthesis, and this was attenuated by rapamycin (27 nM), an inhibitor of mammalian target of rapamycin (mTOR). Further evidence for an involvement of this pathway was shown by an increased phosphorylation of mTOR, the 70-kDa ribosomal S6 kinase (p70(S6k)), and initiation factor 4E-binding protein (4E-BP1) and an increased association of eukaryotic initiation factor 2 (eIF4E) with eIF4G. PIF alone induced a transient (1-2 h) stimulation of phosphorylation of mTOR and p70(S6k). However, in the presence of HMB, phosphorylation of mTOR, p70(S6k), and 4E-BP1 was increased, and inactive 4E-BP1-eIF4E complex was reduced, whereas the active eIF4G.eIF4E complex was increased, suggesting continual stimulation of protein synthesis. HMB alone reduced phosphorylation of elongation factor 2, but this effect was not seen in the presence of PIF. PIF induced autophosphorylation of the double-strand RNA-dependent protein kinase (PKR), leading to phosphorylation of eIF2 on the alpha-subunit, which would inhibit protein synthesis. However, in the presence of HMB, phosphorylation of PKR and eIF2alpha was attenuated, and this was also observed in skeletal muscle of cachectic mice administered HMB (0.25 g/kg). These results suggest that HMB attenuates the depression of protein synthesis by PIF in myotubes through multiple mechanisms.     

Mechanism of attenuation of angiotensin-II-induced protein degradation by insulin-like growth factor-I (IGF-I)

Insulin-like growth factor-I (IGF-I) has been shown to attenuate protein degradation in murine myotubes induced by angiotensin II through downregulation of the ubiquitin-proteasome pathway, although the mechanism is not known. Angiotensin II is known to upregulate this pathway through a cellular signalling mechanism involving release of arachidonic acid, activation of protein kinase Calpha (PKCalpha), degradation of inhibitor-kappaB (I-kappaB) and nuclear migration of nuclear factor-kappaB (NF-kappaB), and all of these events were attenuated by IGF-I (13.2 nM). Induction of the ubiquitin-proteasome pathway has been linked to activation of the RNA-activated protein kinase (PKR), since an inhibitor of PKR attenuated proteasome expression and activity in response to angiotensin II and prevented the decrease in the myofibrillar protein myosin. Angiotensin II induced phosphorylation of PKR and of the eukaryotic initiation factor-2 (eIF2) on the alpha-subunit, and this was attenuated by IGF-I, by induction of the expression of protein phosphatase 1, which dephosphorylates PKR. Release of arachidonic acid and activation of PKCalpha by angiotensin II were attenuated by an inhibitor of PKR and IGF-I, and the effect was reversed by Salubrinal (15 muM), an inhibitor of eIF2alpha dephosphorylation, as was activation of PKCalpha. In addition myotubes transfected with a dominant-negative PKR (PKRDelta6) showed no release of arachidonate in response to Ang II, and no activation of PKCalpha. These results suggest that phosphorylation of PKR by angiotensin II was responsible for the activation of the PLA(2)/PKC pathway leading to activation of NF-kappaB and that IGF-I attenuates protein degradation due to an inhibitory effect on activation of PKR.     

Antibiotic dosing in the 'at risk' critically ill patient: Linking pathophysiology with pharmacokinetics/pharmacodynamics in sepsis and trauma patients

Background

Critical illness, mediated by trauma or sepsis, can lead to physiological changes that alter the pharmacokinetics of antibiotics and may result in sub-therapeutic concentrations at the sites of infection. The first aim of this project is to identify the clinical characteristics of critically ill patients with significant trauma that have been recently admitted to ICU that may predict the dosing requirements for the antibiotic, cefazolin. The second aim of this is to identify the clinical characteristics of critically ill patients with sepsis that may predict the dosing requirements for the combination antibiotic, piperacillin-tazobactam.

Methods/Design

This is an observational pharmacokinetic study of patients with trauma (cefazolin) or with sepsis (piperacillin-tazobactam). Participants will have samples from blood and urine, collected at different intervals. Patients will also have a microdialysis catheter inserted into subcutaneous tissue to measure interstitial fluid penetration of the antibiotic. Participants will be administered sinistrin, indocyanine green and sodium bromide as well as have cardiac output monitoring performed and tetrapolar bioimpedance to determine physiological changes resulting from pathology. Analysis of samples will be performed using validated liquid chromatography tandem mass-spectrometry. Pharmacokinetic analysis will be performed using non-linear mixed effects modeling to determine individual and population pharmacokinetic parameters of antibiotics.

Discussion

The study will describe cefazolin and piperacillin-tazobactam concentrations in plasma and the interstitial fluid of tissues in trauma and sepsis patients respectively. The results of this study will guide clinicians to effectively dose these antibiotics in order to maximize the concentration of antibiotics in the interstitial fluid of tissues.     

The relation between cartilage biomarkers (C2C,C1,2C,CS846, and CPII) and the long-term outcome of rheumatoid arthritis patients within the CAMERA trial

Introduction  

The aim of this study was to investigate whether serum biomarker levels of C2C, C1,2C, CS846, and CPII can predict the long-term course of disease activity and radiographic progression early in the disease course of rheumatoid arthritis (RA).