Angiotensin II stimulates the production of NO and peroxynitrite in endothelial cells

Angiotensin II(ANG II) produces vasoconstriction by a direct action on smooth musclecells via AT₁ receptors. Thesereceptors are also present in the endothelium, but their function ispoorly understood. This study was therefore undertaken to determinewhether ANG II elicits the release of nitric oxide (NO) from cultured rat aortic endothelial cells. NO production, measured by theaccumulation of nitrite and nitrate, was enhanced by10⁷ M ANG II. Thebiological activity of the NO released by ANG II action was evaluatedby measuring its guanylate cyclase-stimulating activity in smoothmuscle cells. The guanosine 3',5'-cyclic monophosphate (cGMP) content of smooth muscle cells was significantly increased byexposure of supernatant from ANG II-stimulated endothelial cells. Theseeffects resulted from the activation of NO synthase, as they wereinhibited by the L-arginineanalogs. These ANG II actions were mediated by theAT₁ receptor, as shown by theirinhibition by the AT₁ antagonistlosartan. The cGMP production by reporter cells was inhibited by thecalmodulin antagonist W-7, suggesting that ANG II activates endothelialcalmodulin-dependent NO synthase. This hypothesis is also supported bythe increase of intracellular free calcium induced by ANG II inendothelial cells. ANG II also stimulated luminol-enhancedchemiluminescence in endothelial cells. This effect was inhibited byN-monomethyl-L-arginine andsuperoxide dismutase, suggesting that this luminol-enhancedchemiluminescence reflected an increase in peroxynitrite production.Thus ANG II stimulates NO release from macrovascular endothelium, whichmay modulate the direct vasoconstrictor effect of ANG II on smoothmuscle cells. However, this beneficial effect may be counteracted bythe simultaneous production of peroxynitrite, which could contribute toseveral pathological processes in the vascular wall.

     

Proteome wide evaluation of the separation ability of hydrophobic interaction chromatography by fluorescent dye binding analysis

Hydrophobic interaction chromatography (HIC) is an important tool in the industrial purification of proteins from various sources. The HIC separation behavior of individual (or model) proteins has been widely researched by others. On the contrary, this study focused on the fractionation ability of HIC when it is challenged with whole proteomes. The impact of the nature of three different proteomes, that is, yeast, soybean, and Chinese hamster ovary cells, on HIC separation was investigated. In doing so, chromatography fractions obtained under standardized conditions were evaluated in terms of their overall hydrophobicity—as measured by fluorescence dye binding. This technique allowed for the calculation of an average protein surface hydrophobicity (S₀) for each fraction; a unique correlation between S₀ and the observed chromatographic behavior was established in each case. Following a similar strategy, the effect of three different ligands (polypropylene glycol, phenyl, and butyl) and two adsorbent particle sizes (65 and 100 µm) on the chromatographic behavior of the yeast proteome was evaluated. As expected, the superficial hydrophobicity of the proteins eluted is correlated with the salt concentration of its corresponding elution step. The findings reveled how—and in which extent—the type of ligand and the size of the beads actually influenced the fractionation of the complex biological mixture. Summarizing, the approach presented here can be instrumental to the study of the performance of chromatography adsorbents under conditions close to industrial practice and to the development of downstream processing strategies. Copyright © 2013 John Wiley & Sons, Ltd.     

Scrutiny of electrochemically-driven electrocatalysis of C-19 steroid 1α-hydroxylase (CYP260A1) from Sorangium cellulosum So ce56

Direct electrochemistry and bioelectrocatalysis of a newly discovered C-19 steroid 1α-hydroxylase (CYP260A1) from the myxobacterium Sorangium cellulosum So ce56 were investigated. CYP260A1 was immobilized on screen-printed graphite electrodes (SPE) modified with gold nanoparticles, stabilized by didodecyldimethylammonium bromide (SPE/DDAB/Au). Cyclic voltammograms in argon-saturated substrate free 0.1 M potassium phosphate buffer, pH 7.4, and in enzyme-substrate complex with androstenedione demonstrated a redox processes with a single redox couple of E^0′ of −299 ± 16 mV and −297.5 ± 21 mV (vs. Ag/AgCl), respectively. CYP260A1 exhibited an electrocatalytic activity detected by an increase of the reduction current in the presence of dissolved oxygen and upon addition of the substrate (androstenedione) in the air-saturated buffer. The catalytic current of the enzyme correlated with substrate concentration in the electrochemical system and this dependence can be described by electrochemical Michaelis-Menten model. The products of CYP260A1-depended electrolysis at controlled working electrode potential of androstenedione were analyzed by mass-spectrometry. MS analysis revealed a mono-hydroxylated product of CYP260A1-dependent electrocatalytic reaction towards androstenedione.     

XPO1 Inhibition Preferentially Disrupts the 3D Nuclear Organization of Telomeres in Tumor Cells

Previous work has shown that the three‐dimensional (3D) nuclear organization of telomeres is altered in cancer cells and the degree of alterations coincides with aggressiveness of disease. Nuclear pores are essential for spatial genome organization and gene regulation and XPO1 (exportin 1/CRM1) is the key nuclear export protein. The Selective Inhibitor of Nuclear Export (SINE) compounds developed by Karyopharm Therapeutics (KPT‐185, KPT‐330/selinexor, and KPT‐8602) inhibit XPO1 nuclear export function. In this study, we investigated whether XPO1 inhibition has downstream effects on the 3D nuclear organization of the genome. This was assessed by measuring the 3D telomeric architecture of normal and tumor cells in vitro and ex vivo. Our data demonstrate for the first time a rapid and preferential disruption of the 3D nuclear organization of telomeres in tumor cell lines and in primary cells ex vivo derived from treatment‐naïve newly diagnosed multiple myeloma patients. Normal primary cells in culture as well as healthy lymphocyte control cells from the same patients were minimally affected. Using both lymphoid and non‐lymphoid tumor cell lines, we found that the downstream effects on the 3D nuclear telomere structure are independent of tumor type. We conclude that the 3D nuclear organization of telomeres is a sensitive indicator of cellular response when treated with XPO1 inhibitors. J. Cell. Physiol. 231: 2711–2719, 2016. © 2016 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.     

Effect of temperature and parasite density on three species ofAphytis (Hymenoptera: Aphelinidae), parasitising California red scale

Summary Three species ofAphytis parasites of California red scale,Aonidiella aurantii (Maskell) were each confined at different densities with approximately equal numbers of scale insects at several constant temperatures to determine the effect of these factors on progeny production and distribution and the search rate. Egg location on the host body by the parasites was unaffected by temperature or parasite density.A. melinus laid its eggs both dorsally and ventrally in equal proportions whileA. lingnanensis andA. chrysomphali laid their eggs ventrally. Progeny production byA. melinus andA. lingnanensis increased at the higher temperature; that ofA. chrysomphali did not. Unlike the other species,A. chrysomphali failed to oviposit at 32°C. Although increasing parasite density reduced progeny production in bothA. melinus andA. lingnanensis, they were able to maintain an almonst constant search rate. This was due to their ability to distribute their eggs among the hosts more regularly at the higher parasite densities. While the mechanism of this process is easy to understand forA. melinus which behaved as a gregarious parasite, it is unclear forA. lingnanensis, which is almost a solitary parasite. The progeny production and the search rate ofA. chrysomphali dropped significantly with increasing parasite density.     

Hyphal growth ofHypomyces chlorinus Tul.: An autoradiographic study

Summary The incorporation of the chitin precursor N-acetyl-D-(1-³H) glucosamine byH. chlorinus has been studied by light and electron microscopic autoradiography. Light microscopic autoradiography showed that the incorporation occured preferentially at the hyphal apex. Autoradiograms from electron microscopy were quantitatively evaluated to determine the relative radioactivity incorporation between the cell wall and cytoplasm: this showed that (³H) incorporation took place mainly in the plasmalemma-wall complex. However a small amount of N-acetyl glucosamine can enter into the cytoplasmic space and is then transported by endomembranes (Golgi apparatus-vesicles) to the plasmalemma-cell wall interface before polymerization.Abbreviations PATAg Periodic acid-thiocarbohydrazide-silver proteinate - PTA phosphotungstic acid     

WW-domain-containing oxidoreductase is associated with low plasma HDL-C levels

Low serum HDL-cholesterol (HDL-C) is a major risk factor for coronary artery disease. We performed targeted genotyping of a 12.4 Mb linked region on 16q to test for association with low HDL-C by using a regional-tag SNP strategy. We identified one SNP, rs2548861, in the WW-domain-containing oxidoreductase (WWOX) gene with region-wide significance for low HDL-C in dyslipidemic families of Mexican and European descent and in low-HDL-C cases and controls of European descent (p = 6.9 × 10⁻⁷). We extended our investigation to the population level by using two independent unascertained population-based Finnish cohorts, the cross-sectional METSIM cohort of 4,463 males and the prospective Young Finns cohort of 2,265 subjects. The combined analysis provided p = 4 × 10⁻⁴ to 2 × 10⁻⁵. Importantly, in the prospective cohort, we observed a significant longitudinal association of rs2548861 with HDL-C levels obtained at four different time points over 21 years (p = 0.003), and the T risk allele explained 1.5% of the variance in HDL-C levels. The rs2548861 resides in a highly conserved region in intron 8 of WWOX. Results from our in vitro reporter assay and electrophoretic mobility-shift assay demonstrate that this region functions as a cis-regulatory element whose associated rs2548861 SNP has a specific allelic effect and that the region forms an allele-specific DNA-nuclear-factor complex. In conclusion, analyses of 9,798 subjects show significant association between HDL-C and a WWOX variant with an allele-specific cis-regulatory function.     

Translocation of the Serine Protease Omi/HtrA2 from Mitochondria into the Cytosol Upon Seizure-Induced Hippocampal Injury in the Neonatal Rat Brain

Omi/HtrA2 is a pro-apoptotic mitochondrial serine protease involved in caspase-dependent as well as caspase-independent cell death upon various brain injuries. However, the role of Omi/HtrA2 in neuronal death induced by status epilepticus (SE) in the immature brain has not been reported. In this study, we analyzed the contribution of serine protease Omi/HtrA2, its substrate X-linked inhibitor of apoptosis protein (XIAP) and the caspase-3 activation to damage of hippocamplal CA1 cells following lithium-pilocarpine SE in P14 rat pups. Status epilepticus in the immature brain significantly induced translocation of Omi/HtrA2 from mitochondria into the cytosol, increased cytosolic accumulation of Omi/HtrA2, induced appearance of XIAP-breakdown products and enhanced caspase-3 activity in the selectively vulnerable hippocampal CA1-subfield. Taken together, these results demonstrate for the first time that SE in the immature brain results in Omi/HtrA2 accumulation in the cytosol, where it probably promotes neuronal death by neutralizing and cleaving XIAP, one of the most potent endogenous inhibitors of apoptosis.