Rapid fluorescent visualization of multiple NAD(P)H oxidoreductases in homogenate,permeabilized cells,and tissue slices

Intracellular NAD(P)H oxidoreductases are a class of diverse enzymes that are the key players in a number of vital processes. The method we present and validate here is based on the ability of many NAD(P)H oxidoreductases to reduce the superoxide probe lucigenin, which is structurally similar to flavins, to its highly fluorescent water-insoluble derivative dimethylbiacridene. Two modifications of the method are proposed: (i) an express method for tissue homogenate and permeabilized cells in suspensions and (ii) a standard procedure for cells in culture and acute thin tissue slices. The method allows one to assess, visualize, and localize, using fluorescent markers of cellular compartments, multiple NADH and NADPH oxidoreductase activities. The application of selective inhibitors (e.g., VAS2870, a NOX2 inhibitor; plumbagin, a NOX4 inhibitor) allows one to distinguish and compare specific NAD(P)H oxidoreductase activities in cells and tissues and to attribute them to known enzymes. The method is simple, rapid, and flexible. It can be easily adapted to a variety of tasks. It will be useful for investigations of the role of various NAD(P)H oxidoreductases in a number of physiological and pathophysiological processes.     

P2-P1' macrocyclization of P2 phenylglycine based HCV NS3 protease inhibitors using ring-closing metathesis

Macrocyclization is a commonly used strategy to preorganize HCV NS3 protease inhibitors in their bioactive conformation. Moreover, macrocyclization generally leads to greater stability and improved pharmacokinetic properties. In HCV NS3 protease inhibitors, it has been shown to be beneficial to include a vinylated phenylglycine in the P2 position in combination with alkenylic P1' substituents. A series of 14-, 15- and 16-membered macrocyclic HCV NS3 protease inhibitors with the linker connecting the P2 phenylglycine and the alkenylic P1' were synthesized by ring-closing metathesis, using both microwave and conventional heating. Besides formation of the expected macrocycles in cis and trans configuration as major products, both ring-contracted and double-bond migrated isomers were obtained, in particular during formation of the smaller rings (14- and 15-membered rings). All inhibitors had K(i)-values in the nanomolar range, but only one inhibitor type was improved by rigidification. The loss in inhibitory effect can be attributed to a disruption of the beneficial π-π interaction between the P2 fragment and H57, which proved to be especially deleterious for the d-phenylglycine epimers.     

Peroxisomal cholesterol biosynthesis and Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS), caused by 7-dehydrocholesterol-reductase (DHCR7) deficiency, shows variable severity independent of DHCR7 genotype. To test whether peroxisomes are involved in alternative cholesterol synthesis, we used [1-(14)C]C24:0 for peroxisomal beta-oxidation to generate [1-(14)C]acetyl-CoA as cholesterol precursor inside peroxisomes. The HMG-CoA reductase inhibitor lovastatin suppressed cholesterol synthesis from [2-(14)C]acetate and [1-(14)C]C8:0 but not from [1-(14)C]C24:0, implicating a peroxisomal, lovastatin-resistant HMG-CoA reductase. In SLOS fibroblasts lacking DHCR7 activity, no cholesterol was formed from [1-(14)C]C24:0-derived [1-(14)C]acetyl-CoA, indicating that the alternative peroxisomal pathway also requires this enzyme. Our results implicate peroxisomes in cholesterol biosynthesis but provide no link to phenotypic variation in SLOS.     

Novel nuclear-encoded subunits of the chloroplast NAD(P)H dehydrogenase complex

The NAD(P)H dehydrogenase (NDH) complex functions in photosystem I cyclic electron transfer in higher plant chloroplasts and is crucial for plant responses to environmental stress. Chloroplast NDH complex is a close relative to cyanobacterial NDH-1L complex, and all fifteen subunits so far identified in NDH-1L have homologs in the chloroplast NDH complex. Here we report on the identification of two nuclear-encoded proteins NDH48 and NDH45 in higher plant chloroplasts and show their intimate association with the NDH complex. These two membrane proteins are shown to interact with each other and with the NDH complex enriched in stroma thylakoids. Moreover, the deficiency of either the NDH45 protein or the NDH48 protein in respective mutant plants leads to severe defects in both the accumulation and the function of the NDH complex. The NDH48 and NDH45 proteins are not components of the hydrophilic connecting domain of the NDH complex but are strongly attached to the hydrophobic membrane domain. We conclude that NDH48 and NDH45 are novel nuclear-encoded subunits of the chloroplast NDH complex and crucial both for the stable structure and function of the NDH complex.     

Nucleolar retention of a translational C/EBPα isoform stimulates rDNA transcription and cell size

The messenger RNA of the intronless CEBPA gene is translated into distinct protein isoforms through the usage of consecutive translation initiation sites. These translational isoforms have distinct functions in the regulation of differentiation and proliferation due to the presence of different N‐terminal sequences. Here, we describe the function of an N‐terminally extended protein isoform of CCAAT enhancer‐binding protein α (C/EBPα) that is translated from an alternative non‐AUG initiation codon. We show that a basic amino‐acid motif within its N‐terminus is required for nucleolar retention and for interaction with nucleophosmin (NPM). In the nucleoli, extended‐C/EBPα occupies the ribosomal DNA (rDNA) promoter and associates with the Pol I‐specific factors u pstream‐b inding f actor 1 (UBF‐1) and SL1 to stimulate rRNA synthesis. Furthermore, during differentiation of HL‐60 cells, endogenous expression of extended‐C/EBPα is lost concomitantly with nucleolar C/EBPα immunostaining probably reflecting the reduced requirement for ribosome biogenesis in differentiated cells. Finally, overexpression of extended‐C/EBPα induces an increase in cell size. Altogether, our results suggest that control of rRNA synthesis is a novel function of C/EBPα adding to its role as key regulator of cell growth and proliferation.     

Apricot Melanoidins Prevent Oxidative Endothelial Cell Death by Counteracting Mitochondrial Oxidation and Membrane Depolarization

The cardiovascular benefits associated with diets rich in fruit and vegetables are thought to be due to phytochemicals contained in fresh plant material. However, whether processed plant foods provide the same benefits as unprocessed ones is an open question. Melanoidins from heat-processed apricots were isolated and their presence confirmed by colorimetric analysis and browning index. Oxidative injury of endothelial cells (ECs) is the key step for the onset and progression of cardiovascular diseases (CVD), therefore the potential protective effect of apricot melanoidins on hydrogen peroxide-induced oxidative mitochondrial damage and cell death was explored in human ECs. The redox state of cytoplasmic and mitochondrial compartments was detected by using the redox-sensitive, fluorescent protein (roGFP), while the mitochondrial membrane potential (MMP) was assessed with the fluorescent dye, JC-1. ECs exposure to hydrogen peroxide, dose-dependently induced mitochondrial and cytoplasmic oxidation. Additionally detected hydrogen peroxide-induced phenomena were MMP dissipation and ECs death. Pretreatment of ECs with apricot melanoidins, significantly counteracted and ultimately abolished hydrogen peroxide-induced intracellular oxidation, mitochondrial depolarization and cell death. In this regard, our current results clearly indicate that melanoidins derived from heat-processed apricots, protect human ECs against oxidative stress.     

Virtually Naked: Virtual Environment Reveals Sex-Dependent Nature of Skin Disclosure

The human tendency to reveal or cover naked skin reflects a competition between the individual propensity for social interactions related to sexual appeal and interpersonal touch versus climatic, environmental, physical, and cultural constraints. However, due to the ubiquitous nature of these constraints, isolating on a large scale the spontaneous human tendency to reveal naked skin has remained impossible. Using the online 3-dimensional virtual world of Second Life, we examined spontaneous human skin-covering behavior unhindered by real-world climatic, environmental, and physical variables. Analysis of hundreds of avatars revealed that virtual females disclose substantially more naked skin than virtual males. This phenomenon was not related to avatar hypersexualization as evaluated by measurement of sexually dimorphic body proportions. Furthermore, analysis of skin-covering behavior of a population of culturally homogeneous avatars indicated that the propensity of female avatars to reveal naked skin persisted despite explicit cultural norms promoting less revealing attire. These findings have implications for further understanding how sex-specific aspects of skin disclosure influence human social interactions in both virtual and real settings.