Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin

Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na(+) and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl(-) efflux through chloride channels and Na(+) influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.     

Lateral clustering of TLR3:dsRNA signaling units revealed by TLR3ecd:3Fabs quaternary structure

Toll-like receptor 3 (TLR3) recognizes dsRNA and initiates an innate immune response through the formation of a signaling unit (SU) composed of one double-stranded RNA (dsRNA) and two TLR3 molecules. We report the crystal structure of human TLR3 ectodomain (TLR3ecd) in a quaternary complex with three neutralizing Fab fragments. Fab15 binds an epitope that overlaps the C-terminal dsRNA binding site and, in biochemical assays, blocks the interaction of TLR3ecd with dsRNA, thus directly antagonizing TLR3 signaling through inhibition of SU formation. In contrast, Fab12 and Fab1068 bind TLR3ecd at sites distinct from the N- and C-terminal regions that interact with dsRNA and do not inhibit minimal SU formation with short dsRNA. Molecular modeling based on the co-structure rationalizes these observations by showing that both Fab12 and Fab1068 prevent lateral clustering of SUs along the length of the dsRNA ligand. This model is further supported by cell-based assay results using dsRNA ligands of lengths that support single and multiple SUs. Thus, their antagonism of TLR3 signaling indicates that lateral clustering of SUs is required for TLR3 signal transduction.     

Increased expression of the anti-apoptotic protein Bcl-xL in the brain is associated with resilience to stress-induced depression-like behavior

Clinical observations and the results of animal studies have implicated changes in neuronal survival and plasticity in both the etiology of mood disorders, especially stress-induced depression, and anti-depressant drug action. Stress may predispose individuals toward depression through down-regulation of neurogenesis and an increase in apoptosis in the brain. Substantial individual differences in vulnerability to stress are evident in humans and were found in experimental animals. Recent studies revealed an association between the brain anti-apoptotic protein B cell lymphoma like X, long variant (Bcl-xL) expression and individual differences in behavioral vulnerability to stress. The ability to increase Bcl-xL gene expression in the hippocampus in response to stress may be an important factor for determining the resistance to the development of stress-induced depression. Treatment with anti-depressant drugs may change Bcl-xL response properties. In the rat brainstem, expression of this anti-apoptotic gene becomes sensitive to swim stress during the long-term fluoxetine treatment, an effect that appeared concomitantly with the anti-depressant-like action of the drug in the forced swim test, suggesting that Bcl-xL may be a new target for depression therapy. The processes and pathways linking stress stimuli to behavior via intracellular anti-apoptotic protein are discussed here in the context of Bcl-xL functions in the mechanisms of individual differences in behavioral resilience to stress and anti-depressant-induced effects on the behavioral despair.     

Accelerated leaf senescence takes part in enhanced resistance in cucumber mosaic virus inoculated pepper leaves

Altered photosynthetic reactions in cucumber mosaic virus (CMV) inoculated leaves of virus resistant lines L113 and L57 and susceptible pepper (Capsicum annuum L.) plants cv. Albena grown in controlled environment and in the field were investigated. The CMV inoculated leaves of virus resistant lines developed different symptoms—necrotic local lesions on L113 and chlorotic spots on L57 while the same leaves of susceptible cv. Albena were symptomless. The changes in Photosystem II (PSII) and PSI electron transport were evaluated by chlorophyll fluorescence, and far-red (FR) light induced leaf absorbance A _810–860. CMV infection caused a decrease in maximal PSII quantum yield, F _v/F _m, in susceptible leaves. Increased non-photochemical fluorescence quenching in CMV-inoculated leaves of both resistant lines were observed. In CMV-inoculated leaves of all tested plants FR light induced P700 oxidation was decreased. In the present study, the viral-infected pepper plants grown in controlled environment to avoid the effects of abiotic factors were used as model system that allow us to investigate the differences in leaf senescence in CMV-inoculated leaves of susceptible and resistant pepper lines expressing different symptoms. Earlier leaf falls of inoculated leaves as a result of accelerated leaf senescence is important for building successful secondary virus resistance strategy following fast responses such as hypersensitive reaction.     

On the mechanism of αC polymer formation in fibrin

Our previous studies revealed that the fibrinogen αC-domains undergo conformational changes and adopt a physiologically active conformation upon their self-association into αC polymers in fibrin. In the present study, we analyzed the mechanism of αC polymer formation and tested our hypothesis that self-association of the αC-domains occurs through the interaction between their N-terminal subdomains and may include β-hairpin swapping. Our binding experiments performed by size-exclusion chromatography and optical trap-based force spectroscopy revealed that the αC-domains self-associate exclusively through their N-terminal subdomains, while their C-terminal subdomains were found to interact with the αC-connectors that tether the αC-domains to the bulk of the molecule. This interaction should reinforce the structure of αC polymers and provide the proper orientation of their reactive residues for efficient cross-linking by factor XIIIa. Molecular modeling of self-association of the N-terminal subdomains confirmed that the hypothesized β-hairpin swapping does not impose any steric hindrance. To "freeze" the conformation of the N-terminal subdomain and prevent the hypothesized β-hairpin swapping, we introduced by site-directed mutagenesis an extra disulfide bond between two β-hairpins of the bovine Aα406-483 fragment corresponding to this subdomain. The experiments performed by circular dichroism revealed that Aα406-483 mutant containing Lys429Cys/Thr463Cys mutations preserved its β-sheet structure. However, in contrast to wild-type Aα406-483, this mutant had lower tendency for oligomerization, and its structure was not stabilized upon oligomerization, in agreement with the above hypothesis. On the basis of the results obtained and our previous findings, we propose a model of fibrin αC polymer structure and molecular mechanism of assembly.     

Bi-enzyme L-arginine-selective amperometric biosensor based on ammonium-sensing polyaniline-modified electrode

A novel L-arginine-selective amperometric bi-enzyme biosensor based on recombinant human arginase I isolated from the gene-engineered strain of methylotrophic yeast Hansenula polymorpha and commercial urease is described. The biosensing layer was placed onto a polyaniline-Nafion composite platinum electrode and covered with a calcium alginate gel. The developed sensor revealed a good selectivity to L-arginine. The sensitivity of the biosensor was 110 ± 1.3 nA/(mM mm(2)) with the apparent Michaelis-Menten constant (K(M)(app)) derived from an L-arginine (L-Arg) calibration curve of 1.27 ± 0.29 mM. A linear concentration range was observed from 0.07 to 0.6mM, a limit of detection being 0.038 mM and a response time - 10s. The developed biosensor demonstrated good storage stability. A laboratory prototype of the proposed amperometric biosensor was applied to the samples of three commercial pharmaceuticals ("Tivortin", "Cytrarginine", "Aminoplazmal 10% E") for L-Arg testing. The obtained L-Arg-content values correlated well with those declared by producers.