CD154 Is Released from T-cells by a Disintegrin and Metalloproteinase Domain-containing Protein 10 (ADAM10) and ADAM17 in a CD40 Protein-dependent Manner

CD154 (CD40 ligand) is a type II transmembrane protein that belongs to the tumor necrosis factor superfamily. The soluble form of CD154 (sCD154), which results from the shedding of membrane-bound CD154, plays a key role in the production of proinflammatory cytokines and has been linked to various autoimmune and vascular disorders. Therefore, elucidating the mechanisms by which CD154 is released from the cell surface following its interaction with its various receptors is of primordial importance. Using co-culture experiments, we show that CD154 is shed predominantly upon its engagement with CD40. Indeed, only CD40 (both membrane-bound and soluble) and not α5β1 or αMβ2 is involved in the cleavage and release of CD154 from Jurkat E6.1 T-cells. Interestingly, CD154 is cleaved independently of the formation of cell surface CD40 homodimers and independently of its association into lipid rafts. In contrast, we found that the protein kinase C (PKC) signaling family and the matrix metalloproteinases ADAM10 and ADAM17 are intimately involved in this process. In conclusion, our data indicate that CD154 is released from T-cells by ADAM10 and ADAM17 upon CD40 ligation. These findings add significant insights into the mechanisms by which CD154 is down-regulated and may lead to the generation of novel therapeutic targets for the treatment of CD154-associated disorders.     

Vitamin B12 regulates photosystem gene expression via the CrtJ antirepressor AerR in Rhodobacter capsulatus

The tetrapyrroles haem, bacteriochlorophyll and cobalamin (B₁₂) exhibit a complex interrelationship regarding their synthesis. In this study, we demonstrate that AerR functions as an antirepressor of the tetrapyrrole regulator CrtJ. We show that purified AerR contains B₁₂ that is bound to a conserved histidine (His145) in AerR. The interaction of AerR to CrtJ was further demonstrated in vitro by pull down experiments using AerR as bait and quantified using microscale thermophoresis. DNase I DNA footprint assays show that AerR containing B₁₂ inhibits CrtJ binding to the bchC promoter. We further show that bchC expression is greatly repressed in a B₁₂ auxotroph of Rhodobacter capsulatus and that B₁₂ regulation of gene expression is mediated by AerR's ability to function as an antirepressor of CrtJ. This study thus provides a mechanism for how the essential tetrapyrrole, cobalamin controls the synthesis of bacteriochlorophyll, an essential component of the photosystem.     

Purification and characterization of a fibrinogenolytic and hemorrhagic metalloproteinase isolated from Vipera lebetina venom

Serious clinical problems such as hemorrhage, edema and tissue necrosis are observed following viperid envenoming. A proteinase (VLH2) was isolated from Vipera lebetina by combination of two chromatographic steps of gel filtration on Sephadex G-75 followed by DEAE Sephadex A-50. This acidic proteinase, with a molecular mass of about 55 kDa and isoelectric point of 5.4, displayed a fibrinogenolytic and hemorrhagic activities. VLH2 hydrolyses rapidly the Aα-chain of fibrinogen, followed, more slowly, by the Bβ-chain, leaving the γ-chain unaffected. The proteolytic and hemorrhagic activities of VLH2 were inhibited by EDTA, EGTA and 1–10 phenanthroline. However, these activities were not affected by AEBSF, Aprotinine, and E64, suggesting that VLH2 is a metalloproteinase with an α-fibrinogenase activity, requiring calcium and zinc for its activity. The enzyme VLH2 did not have proteolytic activity towards extracellular components gelatin, laminin and fibronectin. The hemorrhagic metalloproteinase VLH2 has a myotoxic activity, as determined by serum CK level and histological observation of muscle tissue. Furthermore, VLH2 is able to induce apoptosis of C2C12 myotubes. These results indicate that VLH2 is implicated in the local and systemic bleeding, contributing thus in the toxicity of V. lebetina venom.     

Seedling and adult stage resistance to net form of net blotch (NFNB) in spring barley and stability of adult stage resistance to NFNB in Morocco

This study was conducted to identify stable resistance to net form of net blotch (NFNB) in spring barley in Moroccan environments. Seedling resistance to NFNB was evaluated by inoculating 336 barley genotypes with two NFNB isolates LDNH04Ptt-19 and TD-10 in the greenhouse. These genotypes were evaluated for adult plant resistance to NFNB under seven environments in Morocco in 2015 and 2016. The disease severity was estimated at GS 77–87 on barley leaves using a double-digit scale. To investigate stability of resistance, 149 barley genotypes were subjected to AMMI analysis. At the seedling stage, differential responses of barley genotypes to different NFNB isolates were identified, whereas genotypes had variable stability to NFNB resistance at the adult stages. Five genotypes, AM-68, AM-95, AM-250, AM-267 and AM-322, were resistant to both NFNB isolates at the seedling stage. There were significant (p < .001) effects of genotype (G) and G × E interaction on NFNB severity for barley genotypes at the adult stage. The principal components, IPCA1 and IPCA2, accounted for 48.4% and 18.7% variation for NFNB severity, respectively. The AMMI stability values (ASVs) ranged from 0.01 to 15.5, and fifty-nine barley genotypes had stable responses (ASV ≤ 0.05) across all seven environments. Specifically, two stable genotypes, AM-187 and AM-244, had lower mean NFNB severities across all environments, suggesting a quantitative resistance in these genotypes. Divergent environmental responses of NFNB severity were measured in Sidi El Ayedi 2015 and Sidi Allal Tazi 2016, suggesting that these environments may be suitable to capture resistance to diverse pathotypes. These stable genotypes are valuable resources for introgression of both qualitative resistance and quantitative resistance to NFNB in future.     

Proteomics characterization of cell model with renal fibrosis phenotype: osmotic stress as fibrosis triggering factor

Renal fibroblasts are thought to play a major role in the development of renal fibrosis (RF). The mechanisms leading to this renal alteration remain poorly understood. We performed differential proteomic analyses with two established fibroblast cell lines with RF phenotype to identify new molecular pathways associated with RF. Differential 2-DE combined with mass spectrometry analysis revealed the alteration of more than 30 proteins in fibrotic kidney fibroblasts (TK188) compared to normal kidney fibroblast (TK173). Among these proteins, markers of the endoplasmic reticulum (ER) stress- and the unfolded protein response (UPR) pathway (GRP78, GRP94, ERP57, ERP72, and CALR) and the oxidative stress pathway proteins (PRDX1, PRDX2, PRDX6, HSP70, HYOU1) were highly up-regulated in fibrotic cells. Activation of these stress pathways through long time exposition of TK173, to high NaCl or glucose concentrations resulted in TK188 like phenotype. Parallel to an increase in reactive oxygen species, the stressed cells showed significant alteration of fibrosis markers, ER-stress and oxidative stress proteins. Similar effects of osmotic stress could be also observed on renal proximal tubule cells. Our data suggest an important role of the ER-stress proteins in fibrosis and highlights the pro-fibrotic effect of osmotic stress through activation of oxidative stress and ER-stress pathways.     

Dosimetric study of Hounsfield number correction effect in areas influenced by contrast product in lungs case

BackgroundThe aim of the study was dosimetric effect quantification of exclusive computed tomography (CT) use with an intravenous (IV) contrast agent (CA ), on dose distribution of 3D-CRT treatment plans for lung cancer. Furthermore, dosimetric advantage investigation of manually contrast-enhanced region overriding, especially the heart.Materials and methodsTen patients with lung cancer were considered. For each patient two planning CT sets were initially taken with and without CA. Treatment planning were optimized based on CT scans without CA. All plans were copied and recomputed on scans with CA. In addition, scans with IV contrast were copied and density correction was performed for heart contrast enhanced. Same plans were copied and replaced to undo dose calculation errors that may be caused by CA. Eventually, dosimetric evaluations based on dose volume histograms (DVHs) of planning target volumes (PTV) and organs at-risk were studied and analyzed using the Wilcoxon’s signed rank test.ResultsThere is no statistically significant difference in dose calculation for the PTV maximum, mean, minimum doses, spinal cord maximum doses and lung volumes that received 20 and 30 Gy, between planes calculated with and without contrast scans (p > 0.05) and also for contrast scan, with manual regions overriding.ConclusionsDose difference caused by the contrast agent is negligible and not significant. Therefore, there is no justification to perform two scans, and using an IV contrast enhanced scan for dose calculation is sufficient.     

Characterization of the follicular dendritic cell reservoir of human immunodeficiency virus type 1

Throughout the natural course of human immunodeficiency virus (HIV) infection, follicular dendritic cells (FDCs) trap and retain large quantities of particle-associated HIV RNA in the follicles of secondary lymphoid tissue. We have previously found that murine FDCs in vivo could maintain trapped virus particles in an infectious state for at least 9 months. Here we sought to determine whether human FDCs serve as an HIV reservoir, based on the criteria that virus therein must be replication competent, genetically diverse, and archival in nature. We tested our hypothesis using postmortem cells and tissues obtained from three HIV-infected subjects and antemortem blood samples obtained from one of these subjects. Replication competence was determined using coculture, while genetic diversity and the archival nature of virus were established using phylogenetic and population genetics methods. We found that FDC-trapped virus was replication competent and demonstrated greater genetic diversity than that of virus found in most other tissues and cells. Antiretrovirus-resistant variants that were not present elsewhere were also detected on FDCs. Furthermore, genetic similarity was observed between FDC-trapped HIV and viral species recovered from peripheral blood mononuclear cells obtained 21 and 22 months antemortem, but was not present in samples obtained 4 and 18 months prior to the patient's death, indicating that FDCs can archive HIV. These data indicate that FDCs represent a significant reservoir of infectious and diverse HIV, thereby providing a mechanism for viral persistence for months to years.     

The role of free histidine in xylem loading of nickel in Alyssum lesbiacum and Brassica juncea

Exposure of the hyperaccumulator Alyssum lesbiacum to nickel (Ni) is known to result in a dose-dependent increase in xylem sap concentrations of Ni and the chelator free histidine (His). Addition of equimolar concentrations of exogenous L-His to an Ni-amended hydroponic rooting medium enhances Ni flux into the xylem in the nonaccumulator Alyssum montanum, and, as reported here, in Brassica juncea L. cv Vitasso. In B. juncea, reducing the entry of L-His into the root by supplying D-His instead of L-His, or L-His in the presence of a 10-fold excess of L-alanine, did not affect root Ni uptake, but reduced Ni release into the xylem. Compared with B. juncea, root His concentrations were constitutively about 4.4-fold higher in A. lesbiacum, and did not increase within 9 h of exposure to Ni. Cycloheximide did not affect root His or Ni concentrations, but strongly decreased the release of His and Ni from the root into the xylem of A. lesbiacum, whereas xylem sap concentrations of Ca and Mg remained unaffected. Near-quantitative chelation of Ni with nitrilotriacetate in the rooting medium did not enhance Ni flux into the xylem of A. lesbiacum and B. juncea, suggesting the absence of a significant apoplastic pathway for Ni entry into the xylem. The data suggest that in B. juncea roots, Ni(2+) uptake is independent of simultaneous uptake of His. In both species, enhanced release of Ni into the xylem is associated with concurrent release of His from an increased root free His pool.