Expression of NADPH Oxidase (NOX) 5 in Rabbit Corneal Stromal Cells

To determine whether NOX 5 is expressed in rabbit corneal stromal cells (RCSC). NADPH oxidases (NOXes) are enzymes that preferentially use NADPH as a substrate and generate superoxide. Several isoforms of NOXes function as multi-protein complexes while NOX5 and DUOXs do not require the accessory proteins for their activity and possess calcium binding EF hands.Human NOX5 primers were used to amplify the rabbit NOX5 by RT-PCR. Amplified product was sequenced to confirm its identity. The protein encoded by the NOX5 was identified by western blot analysis. NOX5 siRNA was used to reduce transcript, protein, and calcium stimulated activity. In silico analyses were performed to establish the putative structure, functions, and evolution of rabbit NOX5.NOX activity was measured in RCSC with NADPH rather than NADH as a substrate. RT-PCR with NOX5 primers amplified 288 bp product using RCSC cDNA, which, when sequenced, confirmed its identity to human NOX5 mRNA. This sequence was used to predict the rabbit (Oryctolagus cuniculus) NOX5 gene. NOX5 siRNA reduced amounts of NOX5 mRNA in RCSC and reduced ionomycin stimulated superoxide production. A protein of about 65 to 70 kDa encoded by the NOX5 was detected by western blot analysis. In silico analysis predicted a putative rabbit NOX5 protein containing 801 amino acids. Motif searches predicted the presence of at least 3 putative EF-hands in N-terminus and a NOX domain in C terminal region.The data document that the NOX5 gene was expressed in cells of lagomorphs unlike rodents, making the rabbit an interesting model to study NOX5 functions. The activity of the rabbit NOX5 was calcium stimulated, a trait of NOX5 in general. NOX5 may also prove to be a useful genetic marker for studying the taxonomic position of lagomorphs and the Glires classification

The NOX toolbox: validating the role of NADPH oxidases in physiology and disease

Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis

Changes in gut bacterial populations and their translocation into liver and ascites in alcoholic liver cirrhotics

Background The liver is the first line of defence against continuously occurring influx of microbial-derived products and bacteria from the gut. Intestinal bacteria have been implicated in the pathogenesis of alcoholic liver cirrhosis. Escape of intestinal bacteria into the ascites is involved in the pathogenesis of spontaneous bacterial peritonitis, which is a common complication of liver cirrhosis. The association between faecal bacterial populations and alcoholic liver cirrhosis has not been resolved. Methods Relative ratios of major commensal bacterial communities (Bacteroides spp., Bifidobacterium spp., Clostridium leptum group, Enterobactericaea and Lactobacillus spp.) were determined in faecal samples from post mortem examinations performed on 42 males, including cirrhotic alcoholics (n = 13), non-cirrhotic alcoholics (n = 15), non-alcoholic controls (n = 14) and in 7 healthy male volunteers using real-time quantitative PCR (RT-qPCR). Translocation of bacteria into liver in the autopsy cases and into the ascites of 12 volunteers with liver cirrhosis was also studied with RT-qPCR. CD14 immunostaining was performed for the autopsy liver samples. Results Relative ratios of faecal bacteria in autopsy controls were comparable to those of healthy volunteers. Cirrhotics had in median 27 times more bacterial DNA of Enterobactericaea in faeces compared to the healthy volunteers (p = 0.011). Enterobactericaea were also the most common bacteria translocated into cirrhotic liver, although there were no statistically significant differences between the study groups. Of the ascites samples from the volunteers with liver cirrhosis, 50% contained bacterial DNA from Enterobactericaea, Clostridium leptum group or Lactobacillus spp.. The total bacterial DNA in autopsy liver was associated with the percentage of CD14 expression (p = 0.045). CD14 expression percentage in cirrhotics was significantly higher than in the autopsy controls (p = 0.004). Conclusions Our results suggest that translocation of intestinal bacteria into liver may be involved as a one factor in the pathogenesis of alcoholic liver cirrhosis.BioMed Central open acces

Innovation Sequences over Iterated Offerings: A Relative Innovation, Comfort, and Stimulation Framework of Consumer Responses

Innovations commonly involve changes to iterated market offerings (e.g., new games, car models, film sequels). To better understand consumer iteration responses, the authors develop and test a theoretical framework grounded in (1) prior innovations serving as reference states (comparators) for later innovations and (2) consumer desires for both comfort and stimulation. In Study 1's online game, prior innovations and loss aversion (greater loss than gain impact) moderate evaluations of current innovations, whereby an introduction-weaker-stronger innovation sequence (Periods 1-3 of four periods) generates more entertainment than an introduction-stronger-weaker sequence because the former's weak-opening-then-rise does less harm than the latter's strong-opening-then-drop. Study 2 replicates Study 1 and shows that an introduction-weaker-weaker sequence produces enough habituation and diminishing negative returns to outperform an introduction-stronger-weaker sequence at Period 4. Study 3 offers marketplace corroboration with a film industry test in which minor (fewer) innovations perform better (e.g., sales, return on investment) earlier in franchises, whereas major (many) innovations perform better later, thereby reconciling prior research's opposing prescriptions for the use of major versus minor sequel innovations. The framework and results implicate carefully sequenced innovations for managing consumer iteration responses, including the possibility of interspersing weaker/minor innovations among stronger/major innovations

Bromodomain and extra-terminal domain (BET) proteins regulate melanocyte differentiation.

Background Pharmacologic inhibition of bromodomain and extra-terminal (BET) proteins is currently being explored as a new therapeutic approach in cancer. Some studies have also implicated BET proteins as regulators of cell identity and differentiation through their interactions with lineage-specific factors. However, the role of BET proteins has not yet been investigated in melanocyte differentiation. Melanocyte inducing transcription factor (MITF) is the master regulator of melanocyte differentiation, essential for pigmentation and melanocyte survival. In this study, we tested the hypothesis that BET proteins regulate melanocyte differentiation through interactions with MITF. Results Here we show that chemical inhibition of BET proteins prevents differentiation of unpigmented melanoblasts into pigmented melanocytes and results in de-pigmentation of differentiated melanocytes. BET inhibition also slowed cell growth, without causing cell death, increasing the number of cells in G1. Transcriptional profiling revealed that BET inhibition resulted in decreased expression of pigment-specific genes, including many MITF targets. The expression of pigment-specific genes was also down-regulated in melanoma cells, but to a lesser extent. We found that RNAi depletion of the BET family members, bromodomain-containing protein 4 (BRD4) and bromodomain-containing protein 2 (BRD2) inhibited expression of two melanin synthesis enzymes, TYR and TYRP1. Both BRD4 and BRD2 were detected on melanocyte promoters surrounding MITF-binding sites, were associated with open chromatin structure, and promoted MITF binding to these sites. Furthermore, BRD4 and BRD2 physically interacted with MITF. Conclusion These findings indicate a requirement for BET proteins in the regulation of pigmentation and melanocyte differentiation. We identified changes in pigmentation specific gene expression that occur upon BET inhibition in melanoblasts, melanocytes, and melanoma cells