Protein interaction studies point to new functions for Escherichia coli glyceraldehyde-3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a multifunctional protein with defined functions in numerous mammalian cellular processes. GAPDH functional diversity depends on various factors such as covalent modifications, subcellular localization, oligomeric state and intracellular concentration of substrates or ligands, as well as protein-protein interactions. In bacteria, alternative GAPDH functions have been associated with its extracellular location in pathogens or probiotics. In this study, new intracellular functions of E. coli GAPDH were investigated following a proteomic approach aimed at identifying interacting partners using in vivo formaldehyde cross-linking followed by mass spectrometry. The identified proteins were involved in metabolic processes, protein synthesis and folding or DNA repair. Some interacting proteins were also identified in immunopurification experiments in the absence of cross-linking. Pull-down experiments and overlay immunoblotting were performed to further characterize the interaction with phosphoglycolate phosphatase (Gph). This enzyme is involved in the metabolism of 2-phosphoglycolate formed in the DNA repair of 3"-phosphoglycolate ends generated by bleomycin damage. We show that interaction between Gph and GAPDH increases in cells challenged with bleomycin, suggesting involvement of GAPDH in cellular processes linked to DNA repair mechanisms.

NADPH oxidase NOX4 mediates stellate cell activation and hepatocyte cell death during liver fibrosis development.

A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2−/−/p19ARF−/−, Stat3Δhc/Mdr2−/−) and a model of experimental induced fibrosis (CCl4) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-β pathway activation. In vitro TGF-β-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-β is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-β-induced epithelial-mesenchymal transition (EMT), but was required for TGF-β-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-β-induced death of hepatocytes.

BS-SEM evaluation of the tisular interactions between cortical bone and calcium-phosphate covered titanium implants

The improvement of the reliability of the contact between the osseous tissues and the implant materials has been tested by recovering the metallic implants with ceramic materials, usually calcium phosphates. In our study, the calcium phosphate recovering layers were deposited by means of a pulsed-laser deposition technique. Our aim was to to evaluate the tissue interactions established between cortical bone and titanium implants covered by five different layers, ranging from amorphous calcium phosphate to crystalline hydroxyapatite, obtained by altering the parameters of the laser ablation process. The surgical protocol of the study consisted in the simultaneous implantation of the five types of implants in both the tibial dyaphisis of three Beagle dogs, sacrificed respectively one, two and three months after the last surgical procedures. After the sacrifice, the samples were submitted to a scheduled procedure of embedding in plastic polymers without prior decalcification, in order to perform the ultrastructural studies: scanning microscopy with secondary and backscattered electrons (BS-SEM). Our observations show that both in terms of the calcified tissues appearing as a response to the presence of the different coatings and of time of recovering, the implants coated with crystalline calcium phosphate layers by laser ablation present a better result than the amorphous-calcium-phosphate-coated implants. Moreover, the constant presence of chondroid tissue, related with the mechanical induction by forces applied on the recovering area, strongly suggests that the mechanisms implied in osteointegration are related to endomembranous, rather than endochondral ossification processes

A radiochemical method for carbamoyl-phosphate synthetase I: application to rats fed a hyperproteic diet

A method for the measurement of carbamoyl-phosphate synthetase I activity in animal tissues has been developed using the livers of rats under normal and hyperproteic diets. The method is based on the incorporation of 14C-ammonium bicarbonate to carbamoyl-phosphate in the presence of ATP-Mg and N-acetyl-glutamate. The reaction is stopped by chilling, lowering the pH and adding ethanol. Excess bicarbonate is flushed out under a gentle stream of cold CO2. The only label remaining in the medium was that incorporated into carbamoyl-phosphate, since all 14C-CO2 from bicarbonate was eliminated. The method is rapid and requires only a low pressure supply of CO2 to remove the excess substrate. The reaction is linear up to 10 min using homogenate dilutions of 1:20 to 1:200 (w/v). Rat liver activity was in the range of 89±8 nkat/g. Hyperproteic diet resulted in a significant 1.4-fold increase. The design of the method allows for the processing of multiple samples at the same time, and incubation medium manipulation is unnecessary, since the plastic incubation vial and its contents are finally counted together.

BS-SEM evaluation of the tisular interactions between cortical bone and calcium-phosphate covered titanium implants

The improvement of the reliability of the contact between the osseous tissues and the implant materials has been tested by recovering the metallic implants with ceramic materials, usually calcium phosphates. In our study, the calcium phosphate recovering layers were deposited by means of a pulsed-laser deposition technique. Our aim was to to evaluate the tissue interactions established between cortical bone and titanium implants covered by five different layers, ranging from amorphous calcium phosphate to crystalline hydroxyapatite, obtained by altering the parameters of the laser ablation process. The surgical protocol of the study consisted in the simultaneous implantation of the five types of implants in both the tibial dyaphisis of three Beagle dogs, sacrificed respectively one, two and three months after the last surgical procedures. After the sacrifice, the samples were submitted to a scheduled procedure of embedding in plastic polymers without prior decalcification, in order to perform the ultrastructural studies: scanning microscopy with secondary and backscattered electrons (BS-SEM). Our observations show that both in terms of the calcified tissues appearing as a response to the presence of the different coatings and of time of recovering, the implants coated with crystalline calcium phosphate layers by laser ablation present a better result than the amorphous-calcium-phosphate-coated implants. Moreover, the constant presence of chondroid tissue, related with the mechanical induction by forces applied on the recovering area, strongly suggests that the mechanisms implied in osteointegration are related to endomembranous, rather than endochondral ossification processes

BS-SEM evaluation of the tisular interactions between cortical bone and calcium-phosphate covered titanium implants

The improvement of the reliability of the contact between the osseous tissues and the implant materials has been tested by recovering the metallic implants with ceramic materials, usually calcium phosphates. In our study, the calcium phosphate recovering layers were deposited by means of a pulsed-laser deposition technique. Our aim was to to evaluate the tissue interactions established between cortical bone and titanium implants covered by five different layers, ranging from amorphous calcium phosphate to crystalline hydroxyapatite, obtained by altering the parameters of the laser ablation process. The surgical protocol of the study consisted in the simultaneous implantation of the five types of implants in both the tibial dyaphisis of three Beagle dogs, sacrificed respectively one, two and three months after the last surgical procedures. After the sacrifice, the samples were submitted to a scheduled procedure of embedding in plastic polymers without prior decalcification, in order to perform the ultrastructural studies: scanning microscopy with secondary and backscattered electrons (BS-SEM). Our observations show that both in terms of the calcified tissues appearing as a response to the presence of the different coatings and of time of recovering, the implants coated with crystalline calcium phosphate layers by laser ablation present a better result than the amorphous-calcium-phosphate-coated implants. Moreover, the constant presence of chondroid tissue, related with the mechanical induction by forces applied on the recovering area, strongly suggests that the mechanisms implied in osteointegration are related to endomembranous, rather than endochondral ossification processes

GROWTH, YIELD AND POSTHARVEST QUALITY OF WAX APPLE AS AFFECTED BY NAPHTHALENE ACETIC ACID APPLICATION

Results of this study represent the first report of the effect of Naphthalene Acetic Acid (NAA) on the pre and post harvest quality of wax apple fruit. The wax apple trees were spray treated with 0, 5, 10 and 20 mg L-1 NAA under field conditions during 2008 to 2011. The experiments were carried out in Completely Randomized Design (CRD) with six replications. Leaf chlorophyll content, chlorophyll fluorescence, photosynthetic yield, net photosynthetic rate, drymatter content of leaves and total soluble solids and K+content of wax apple fruits were significantly increased after treatments with 10 mg L-1. Polygalacturonase activity significantly decreased with NAA treatments. The application of 5 mg L-1 NAA increased 27% more bud and reduced 42% less fruit drop compared to the control. In addition, higher protein and phosphate synthase activity of leaves, fruit set, fruit growth, larger fruit size and yield were recorded in NAA treated plants. In storage, treated fruits exhibited higher TSS and firmness and less weight loss, browning, titratable acidity, respiration and ethylene production than the control. It is concluded that spraying with 5 and 10 mg L-1 NAA once a week under field conditions produced better fruit growth and yield of the wax apple and maintained better fruit quality in postharvest storage.

Phosphate transporters

Acquisition of phosphate from the soil and its distribution across plant tissues, as well as between the cytosol and organelles, is dependent on an array of transporters, which include proton-phosphate cotransporters belonging to the family of PHT proteins, the PHO1 phosphate exporter, as well as organellar phosphate exchangers. The expression of these transporters is regulated both at the transcriptional and post-transcriptional levels, and their activity and localisation is controlled by modifications such as phosphorylation and ubiquitination. Proteins including the PHR1 and WRKY6 transcription factors, PHO2 and NLA involved in ubiquitination, as well as SPX proteins, form a network which enables plants to regulate phosphate transport activity under both nutrient-sufficient and -deficient conditions, allowing them to survive, grow and produce seeds under adverse conditions.

JNK Inhibition Reduced Retinal Ganglion Cell Death after Ischemia/Reperfusion In Vivo and after Hypoxia In Vitro.

Mitogen-activated protein kinases (MAPKs) are key regulators that have been linked to cell survival and death. Among the main classes of MAPKs, c-jun N-terminal kinase (JNK) has been shown to mediate cell stress responses associated with apoptosis. In Vitro, hypoxia induced a significant increase in 661W cell death that paralleled increased activity of JNK and c-jun. 661W cells cultured in presence of the inhibitor of JNK (D-JNKi) were less sensitive to hypoxia-induced cell death. In vivo, elevation in intraocular pressure (IOP) in the rat promoted cell death that correlated with modulation of JNK activation. In vivo inhibition of JNK activation with D-JNKi resulted in a significant and sustained decrease in apoptosis in the ganglion cell layer, the inner nuclear layer and the photoreceptor layer. These results highlight the protective effect of D-JNKi in ischemia/reperfusion induced cell death of the retina.

Characterization of hepatic fatty acids in mice with reduced liver fat by ultra-short echo time (1)H-MRS at 14.1 T in vivo.

Alterations in the hepatic lipid content (HLC) and fatty acid composition are associated with disruptions in whole body metabolism, both in humans and in rodent models, and can be non-invasively assessed by (1)H-MRS in vivo. We used (1)H-MRS to characterize the hepatic fatty-acyl chains of healthy mice and to follow changes caused by streptozotocin (STZ) injection. Using STEAM at 14.1 T with an ultra-short TE of 2.8 ms, confounding effects from T2 relaxation and J-coupling were avoided, allowing for accurate estimations of the contribution of unsaturated (UFA), saturated (SFA), mono-unsaturated (MUFA) and poly-unsaturated (PUFA) fatty-acyl chains, number of double bonds, PU bonds and mean chain length. Compared with in vivo (1) H-MRS, high resolution NMR performed in vitro in hepatic lipid extracts reported longer fatty-acyl chains (18 versus 15 carbons) with a lower contribution from UFA (61 ± 1% versus 80 ± 5%) but a higher number of PU bonds per UFA (1.39 ± 0.03 versus 0.58 ± 0.08), driven by the presence of membrane species in the extracts. STZ injection caused a decrease of HLC (from 1.7 ± 0.3% to 0.7 ± 0.1%), an increase in the contribution of SFA (from 21 ± 2% to 45 ± 6%) and a reduction of the mean length (from 15 to 13 carbons) of cytosolic fatty-acyl chains. In addition, SFAs were also likely to have increased in membrane lipids of STZ-induced diabetic mice, along with a decrease of the mean chain length. These studies show the applicability of (1)H-MRS in vivo to monitor changes in the composition of the hepatic fatty-acyl chains in mice even when they exhibit reduced HLC, pointing to the value of this methodology to evaluate lipid-lowering interventions in the scope of metabolic disorders.

A frequent hypofunctional IRAK2 variant is associated with reduced spontaneous hepatitis C virus clearance.

UNLABELLED: Patients carrying very rare loss-of-function mutations in interleukin-1 receptor-associated kinase 4 (IRAK4), a critical signaling mediator in Toll-like receptor signaling, are severely immunodeficient, highlighting the paramount role of IRAK kinases in innate immunity. We discovered a comparatively frequent coding variant of the enigmatic human IRAK2, L392V (rs3844283), which is found homozygously in ∼15% of Caucasians, to be associated with a reduced ability to induce interferon-alpha in primary human plasmacytoid dendritic cells in response to hepatitis C virus (HCV). Cytokine production in response to purified Toll-like receptor agonists was also impaired. Additionally, rs3844283 was epidemiologically associated with a chronic course of HCV infection in two independent HCV cohorts and emerged as an independent predictor of chronic HCV disease. Mechanistically, IRAK2 L392V showed intact binding to, but impaired ubiquitination of, tumor necrosis factor receptor-associated factor 6, a vital step in signal transduction. CONCLUSION: Our study highlights IRAK2 and its genetic variants as critical factors and potentially novel biomarkers for human antiviral innate immunity. (Hepatology 2015;62:1375-1387).

Proteinuria Increases Plasma Phosphate by Altering Its Tubular Handling.

Proteinuria and hyperphosphatemia are cardiovascular risk factors independent of GFR. We hypothesized that proteinuria induces relative phosphate retention via increased proximal tubule phosphate reabsorption. To test the clinical relevance of this hypothesis, we studied phosphate handling in nephrotic children and patients with CKD. Plasma fibroblast growth factor 23 (FGF-23) concentration, plasma phosphate concentration, and tubular reabsorption of phosphate increased during the proteinuric phase compared with the remission phase in nephrotic children. Cross-sectional analysis of a cohort of 1738 patients with CKD showed that albuminuria≥300 mg/24 hours is predictive of higher phosphate levels, independent of GFR and other confounding factors. Albuminuric patients also displayed higher plasma FGF-23 and parathyroid hormone levels. To understand the molecular mechanisms underlying these observations, we induced glomerular proteinuria in two animal models. Rats with puromycin-aminonucleoside-induced nephrotic proteinuria displayed higher renal protein expression of the sodium-phosphate co-transporter NaPi-IIa, lower renal Klotho protein expression, and decreased phosphorylation of FGF receptor substrate 2α, a major FGF-23 receptor substrate. These findings were confirmed in transgenic mice that develop nephrotic-range proteinuria resulting from podocyte depletion. In vitro, albumin did not directly alter phosphate uptake in cultured proximal tubule OK cells. In conclusion, we show that proteinuria increases plasma phosphate concentration independent of GFR. This effect relies on increased proximal tubule NaPi-IIa expression secondary to decreased FGF-23 biologic activity. Proteinuria induces elevation of both plasma phosphate and FGF-23 concentrations, potentially contributing to cardiovascular disease.

Randomized study of reduced-intensity chemotherapy combined with imatinib in adults with Ph-positive acute lymphoblastic leukemia.

In this study, we randomly compared high doses of the tyrosine kinase inhibitor imatinib combined with reduced-intensity chemotherapy (arm A) to standard imatinib/hyperCVAD (cyclophosphamide/vincristine/doxorubicin/dexamethasone) therapy (arm B) in 268 adults (median age, 47 years) with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). The primary objective was the major molecular response (MMolR) rate after cycle 2, patients being then eligible for allogeneic stem cell transplantation (SCT) if they had a donor, or autologous SCT if in MMolR and no donor. With fewer induction deaths, the complete remission (CR) rate was higher in arm A than in arm B (98% vs 91%; P = .006), whereas the MMolR rate was similar in both arms (66% vs 64%). With a median follow-up of 4.8 years, 5-year event-free survival and overall survival (OS) rates were estimated at 37.1% and 45.6%, respectively, without difference between the arms. Allogeneic transplantation was associated with a significant benefit in relapse-free survival (hazard ratio [HR], 0.69; P = .036) and OS (HR, 0.64; P = .02), with initial white blood cell count being the only factor significantly interacting with this SCT effect. In patients achieving MMolR, outcome was similar after autologous and allogeneic transplantation. This study validates an induction regimen combining reduced-intensity chemotherapy and imatinib in Ph+ ALL adult patients and suggests that SCT in first CR is still a good option for Ph+ ALL adult patients. This trial was registered at www.clinicaltrials.gov as #NCT00327678.