Reduction Of Ferricytochrome Research Articles

Stability of the structure and redox state of ferricytochrome c in the desolvation process

Stability of the structure and redox state of ferricytochrome c during continuous changing of the hydration environment from dilute solution to condensed phase was studied based on real-time resonance Raman measurement. Our experimental results indicated that the structure and redox state of ferricytochrome c were sufficiently maintained during the entire desolvation process, which reflected that the stabilizing excluded-volume effect was stronger than the destabilizing crowder-protein interaction, at the case of globular mini-protein itself acting as the crowder. Rapid reduction of ferricytochrome c was observed at measurement sites at the time of solution to condensed phase transition. Similar phenomenon was observed on cytochrome c adsorbed on indium tin oxide surface. At present conditions, the rapid reduction was found to be derived mainly from thermal reduction under laser illumination and secondarily from photoreduction induced by the residual adsorbed ferrocyanide anions. After re-dissolved, the protein which had undergone sufficient desolvation recovered the structure and redox state in the initial solution phase, suggesting that the crowding effect in solution prevented large protein deformations observed in gas phase condition.

Prevention of Pulmonary Injury in Isolated Perfused Rat Lungs by Activated Human Neutrophils Preincubated With Anti-Mo1 Monoclonal Antibody

Neutrophil activation results in neutrophil adherence and may subsequently cause lung injury through the generation of oxidants, release of granule proteases, and generation of a variety of mediator substances. We hypothesized that inhibition of neutrophil adherence and subsequent lung sequestration would attenuate the lung injury caused by activated neutrophils. Using isolated perfused rat lungs, we determined if anti-Mo1 monoclonal antibody (binds to the alpha subunit of a neutrophil glycoprotein [gp 155.94] that facilitates adherence) would attenuate lung neutrophil sequestration and lung injury caused by human neutrophils stimulated by phorbol myristate acetate (PMA). PMA-stimulated neutrophils but not PMA or neutrophils alone caused lung injury as assessed by accumulation of 125I-bovine serum albumin into lung parenchyma and alveolar lavage fluid. Incubation of neutrophils with anti-Mo1 antibody prior to stimulation with PMA attenuated lung injury and neutrophil sequestration. Furthermore, a histological survey revealed that anti-Mo1 antibody inhibited neutrophils present in the lung from spreading following exposure to PMA. Anti-Mo1 antibody did not inhibit PMA-stimulated neutrophil release of granule constituents or toxic O2 metabolites as evidenced by lysozyme and lactoferrin release or the reduction of ferricytochrome c in the lung perfusate. The inhibition of lung injury caused by the anti-Mo1 antibody was not likely due to a nonspecific effect of the antibody, since another murine monoclonal antibody of the same class (anti-Mo5) did not inhibit lung neutrophil sequestration or lung injury. Thus, in this experimental model, interference with the close approximation of the neutrophil to its target site inhibited the ability of the activated human neutrophil to cause injury.

Protein Kinase C Beta II Peptide Modulation of Superoxide Release in Rat Polymorphonuclear Leukocytes

Previous studies have shown that protein kinase C (PKC) alpha, beta II (βII) and zeta inhibition attenuates polymorphonuclear leukocyte (PMN) superoxide (SO) release, whereas PKC delta inhibition augments this response via inhibiting phosphorylation of PMN NADPH oxidase (NOX‐2). However, to date, there is no information regarding the role of PKCβII peptide activator on regulating PMN SO release. PKCβII activator and inhibitor peptides work by augmenting or attenuating PKCβII translocation after stimulation by N‐formyl‐L‐methionyl‐Lleucyl‐L‐phenylalanine (fMLP; a PMN chemotactic receptor agonist) or phorbol 12‐myristate 13‐acetate (PMA; a diacylglycerol mimetic). It is known that myristic acid (myr‐) conjugation is an effective means to facilitate intracellular delivery of the cargo sequence to regulate PKCβII activity. We hypothesize that myr‐PKCβII peptide‐activator (N‐myr‐SVEIWD; myr‐PKCβ+) would increase PMA/fMLP‐induced rat PMN SO release, whereas, myr‐PKCβII peptideinhibitor (N‐myr‐SLNPEWNET; myr‐PKCβ−) would attenuate this response compared to non‐drug treated controls or unconjugated native PKCβ+/− peptide sequences. PMNs were isolated via peritoneal lavage from male Sprague‐Dawley rats (~500g) and were incubated (5x106) for 15min at 37°C in the presence/absence of native/myr‐PKCβ+/− (20 mM; n=8[fMLP]) or SO dismutase (SOD;10mg/mL; n=8) as positive control. fMLP or PMA induced PMN SO release was measured spectrophotometrically at 550nm via reduction of ferricytochrome c for 90 sec (fMLP;1mM; n=19) or 360 sec (PMA;100nM; n=17). fMLP‐induced PMN SO release increased absorbance to 0.155±0.018 in non‐drug controls, 0.162±0.037 in myr‐PKCβ+, 0.136±0.025 in native PKCβ+, 0.070±0.012 in myr‐PKCβ− and 0.146±0.018 in native PKCβ− treated PMNs at 30 sec. Myr‐PKCβ− significantly decreased fMLP‐induced PMN SO release compared to nondrug controls, myr‐PKCβ+ and native PKC β+/− peptides (p<0.05). PMA‐induced PMN SO release increased absorbance to 0.41±0.039 in non‐drug controls, 0.517±0.047(n=7) in myr‐PKCβ+, 0.345±0.071(n=5) in native PKCβ+, 0.241±0.055(n=6) in myr‐PKCβ−, and 0.308±0.052(n=6) in native PKC β− at 360 sec. Myr‐PKCβ‐significantly attenuated PMA‐SO release compared to non‐drug controls and myr‐PKCβ+ (p<0.05). SOD‐treated samples showed >90% reduction of fMLP or PMA‐induced SO release and native PKCβ+/− was not different from non‐drug controls. Cell viability ranged between 94±2% and 98±2% in all groups as determined by 0.2% trypan blue exclusion. Preliminary results suggest that myr‐PKCβ‐significantly attenuates fMLP/PMA‐induced SO release, whereas myr‐PKCβ+ only shows a trend to augment fMLP/PMA‐induced SO release. Additional studies will be conducted to determine if myr‐PKCβ+ will significantly augment PMN SO release.Support or Funding InformationThis research was supported by the Department of Bio‐Medical Sciences and the Division of Research at Philadelphia College of Osteopathic Medicine (PCOM) and by Young Therapeutics, LLC. 4170 City Avenue, Philadelphia, PA 19131This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Diagnostic Testing for Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a rare genetic immunodeficiency associated with recurrent bacterial infections, granulomas, and increased mortality. It is characterized by the inability of phagocytes (neutrophils, monocytes, etc.) to generate reactive oxygen species (ROS), a major component of the microbicidal repertoire of phagocytes. Diagnosis of patients with CGD is commonly based on the assessment of ROS production by neutrophils. Multiple assays to assess ROS production are described-a flow cytometric dihydrorhodamine assay and a histochemical nitroblue tetrazolium assay, both of which can be used to visualize ROS production in individual cells, and two quantitative assays-O2˙- reduction of ferricytochrome c and a ROS-dependent, luminol-enhanced chemiluminescence assay that will quantitate the response of a population of cells. In addition, two approaches to identify the defective phox protein defect are described-standard immunoblotting and flow cytometry of neutrophils stained with phox-specific antibodies. When determining the status of a patient, several assays should be used to assess ROS production and identify the protein defect. The results of these assays should agree and can be used to develop a comprehensive package, which includes confirmation of a diagnosis of CGD, identification of the specific protein target for genetic sequencing, and an indication of the prognosis for the patient.

Effect of uric acid on inflammatory COX-2 and ROS pathways in vascular smooth muscle cells

Hyperuricemia is thought to play a role in cardiovascular diseases (CVD), including hypertension, coronary artery disease and atherosclerosis. However, exactly how uric acid contributes to these pathologies is unknown. An underlying mechanism of inflammatory diseases, such as atherosclerosis, includes enhanced production of cyclooxygenase-2 (COX-2) and superoxide anion. Here, we aimed to examine the effect of uric acid on inflammatory COX-2 and superoxide anion production and to determine the role of losartan. Primarily cultured vascular smooth muscle cells (VSMCs) were time and dose-dependently induced by uric acid and COX-2 and superoxide anion levels were measured. COX-2 levels were determined by ELISA, and superoxide anion was measured by the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c method. Uric acid elevated COX-2 levels in a time-dependent manner. Angiotensin-II receptor blocker, losartan, diminished uric-acid-induced COX-2 elevation. Uric acid also increased superoxide anion level in VSMCs. Uric acid plays an important role in CVD pathogenesis by inducing inflammatory COX-2 and ROS pathways. This is the first study demonstrating losartan’s ability to reduce uric-acid-induced COX-2 elevation.

Propofol protects against high glucose-induced endothelial apoptosis and dysfunction in human umbilical vein endothelial cells.

Perioperative hyperglycemia is a common clinical metabolic disorder. Hyperglycemia could induce endothelial apoptosis and dysfunction. Propofol is a widely used IV anesthetic drug in clinical settings. In the present study, we examined whether and how propofol reduced high glucose-induced endothelial apoptosis and dysfunction in human umbilical vein endothelial cells (HUVECs). HUVECs were cultured with different concentrations (5, 10, 15, and 25 mM) of glucose for different times (4, 8, 12, and 24 hours). To study the effect of propofol, cells were incubated with different concentrations (0.2, 1, 5, and 25 μM) of propofol for 2 hours. In parallel experiments, cells were incubated in 5 mM glucose as control. Nitric oxide (NO) production was measured with a nitrate reductase assay. Cell viability was determined with a Cell Counting Kit-8. Protein expression of active caspase 3, cytochrome c, endothelial NO synthase (eNOS), p-eNOS-Thr, p66, protein kinase C βII (PKCβII), and p-PKCβII-Ser was measured by Western blot analysis. Accumulation of superoxide anion (O2˙) was measured with the reduction of ferricytochrome c. Cell apoptosis was determined with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining. Compared with control, high glucose decreased NO production (P < 0.0001) and reduced cells viability (P < 0.0001) in HUVECs. Compared with high glucose treatment, pretreatment of cells with propofol (5 μM, 2 hours) reduced high glucose-induced inhibitory p-eNOS-Thr phosphorylation (P < 0.0001), increasing NO production (P = 0.0007), decreased high glucose-induced p66 expression (P < 0.0001) and p66 mitochondrial translocation (P < 0.0001), O2˙ accumulation (P < 0.0001), mitochondrial cytochrome c release (P < 0.0001), active caspase 3 expression (P < 0.0001), and enhancing endothelial viability (P < 0.0001). Furthermore, propofol inhibited high glucose-induced PKCβII expression (P = 0.0002) and p-PKCβII-Ser phosphorylation (P < 0.0001). Moreover, the observed protective effect of propofol was quite similar to that of PKCβII inhibitor. Propofol, by a mechanism of decreasing high glucose-induced PKCβII expression and p-PKCβII-Ser phosphorylation, inhibits high glucose-induced p66 mitochondrial translocation, therefore protecting HUVECs from high glucose-induced endothelial dysfunction and apoptosis.

FMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils.

N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8) release and nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP), diphenyleneiodonium (DPI), and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na+/H+ exchanger inhibitor) inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils.

Propofol protects against angiotensin II-induced mouse hippocampal HT22 cells apoptosis via inhibition of p66Shc mitochondrial translocation.

Hippocampal neuronal oxidative stress and apoptosis have been reported to be involved in cognitive impairment, and angiotensin II could induce hippocampal oxidative stress and apoptosis. Propofol is a widely used intravenous anesthetic agent in clinical practice, and it demonstrates significant neuroprotective activities. In this study, we investigated the mechanism how propofol protected mouse hippocampal HT22 cells against angiotensin II-induced oxidative stress and apoptosis. Cell viability was evaluated with CCK8 kit. Protein expressions of active caspase 3, cytochrome c, p66(Shc), p-p66(shc)-Ser(36), protein kinase C βII (PKCβII), Pin-1 and phosphatase A2 (PP2A) were measured by Western blot. Superoxide anion (O2(.-)) accumulation was measured with the reduction of ferricytochrome c. Compared with the control group, angiotensin II up-regulated expression of PKCβII, Pin-1 and PP2A, induced p66(Shc)-Ser(36) phosphorylation, and facilitated p66(Shc) mitochondrial translocation, resulting in O2(.-) accumulation, mitochondrial cytochrome c release, caspase 3 activation, and the inhibition of cell viability. Importantly, we found propofol inhibited angiotensin II-induced PKCβII and PP2A expression and improved p66(Shc) mitochondrial translocation, O2(.-) accumulation, mitochondrial cytochrome c release, caspase 3 activation, inhibition of cell viability. On the other hand, propofol had no effects on angiotensin II-induced Pin-1 expression and p66(Shc)-Ser(36) phosphorylation. Moreover, the protective effects of propofol on angiotensin II-induced HT22 apoptosis were similar with calyculin A, an inhibitor of PP2A and CGP53353, an inhibitor of PKCβII. However, the protective effect of propofol could be reversed by FTY720, an activator of PP2A, rather than PMA, an activator of PKCβII. Our data indicated that propofol down-regulated PP2A expression, inhibiting dephosphorylation of p66(Shc)-Ser(36) and p66(Shc) mitochondrial translocation, decreasing O2(.-) accumulation, reducing mitochondrial cytochrome c release, inhibiting caspase 3 activation. By these mechanisms, it protects mouse hippocampal HT22 cells against angiotensin II-induced apoptosis.

Age-related NADH oxidase (arNOX)-catalyzed oxidative damage to skin proteins.

Age-related NADH oxidase (arNOX), a cell surface-located hydroquinone oxidase capable of superoxide generation, appears at age 30 and increases with age thereafter. The ectodomain of arNOX is shed from the cell surface into body fluids including sera and saliva where its activity was measured spectrophotometrically using a reduction of ferricytochrome c as a measure of superoxide generation. The autofluorescence of advanced glycation end products correlates with epidermal arNOX activity as well. To demonstrate protein cross-linking, a fluorescence-labeled analog of tyrosine, tyramine, that would react with proteins carrying arNOX-generated tyrosyl radicals was used. The assay demonstrated the potential for arNOX-induced oxidative damage (dityrosine formation) to human collagen and elastin and to other surface proteins of intact human embryo fibroblasts and frozen sections from epidermal punch biopsies. The findings support a role for arNOX as a major source of oxidative damage leading to cross-linking of skin proteins.

Mitoquinone exerts antioxidant effects independent of mitochondrial targeted effects in phorbol‐12‐myristate‐13‐acetate stimulated polymorphonuclear leukocyte superoxide release (667.3)

MitoQ is a mitochondrial‐targeted coenzyme Q antioxidant analog that dose‐dependently restored cardiac function and reduced infarct size in isolated perfused rat hearts subjected to ischemia reperfusion (I/R). Moreover, mitoQ also dose‐dependently attenuated PMA stimulated PMN superoxide (SO) release at the same concentration (10uM) as the cardioprotective dose. NADPH oxidase is the principle source of PMN SO release. We speculate that mitoQ may exert antioxidant effects independent of the mitochondria. Therefore, we hypothesized that inhibition of mitoQ on PMN‐SO release will be similar as other coenzyme Q analogs: coenzyme Q1 and decylubiquinone without affecting cell viability. SO release was measured spectrophotometrically from isolated rat PMNs measured by the reduction of ferricytochrome c and were stimulated with 100nM PMA. The absorbance was measured at 550 nm up to 360sec. Positive control samples were given SO dismutase (SOD; 10ug/ml) which inhibited PMA induced SO release by &gt;90%. MitoQ significantly inhibited SO release by 56 + 3% (10uM, n=10 , P&lt;0.05); coenzyme Q1 by 49 + 9% (40uM, n=6) 68 + 7% (80uM, n=5),( both P&lt;0.05); and decylubiquinone by 61 + 9% (80uM, n=8, P&lt;0.05). Additionally, there was no significant change in cell viability. These preliminary results suggest that mitoQ may exert its antioxidant effects independent of the mitochondria. .

Dissociations in the effects of β2-adrenergic receptor agonists on cAMP formation and superoxide production in human neutrophils: support for the concept of functional selectivity.

In neutrophils, activation of the β2-adrenergic receptor (β2AR), a Gs-coupled receptor, inhibits inflammatory responses, which could be therapeutically exploited. The aim of this study was to evaluate the effects of various β2AR ligands on adenosine-3′,5′-cyclic monophosphate (cAMP) accumulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion (O2 •−) production in human neutrophils and to probe the concept of ligand-specific receptor conformations (also referred to as functional selectivity or biased signaling) in a native cell system. This is an important question because so far, evidence for functional selectivity has been predominantly obtained with recombinant systems, due to the inherent difficulties to genetically manipulate human native cells. cAMP concentration was determined by HPLC/tandem mass spectrometry, and O2 •− formation was assessed by superoxide dismutase-inhibitable reduction of ferricytochrome c. β2AR agonists were generally more potent in inhibiting fMLP-induced O2 •− production than in stimulating cAMP accumulation. (−)-Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but partially inhibited fMLP-induced O2 •− production. Moreover, (−)-adrenaline was equi-efficacious in both assays whereas the efficacy of salbutamol was more than two-fold higher in the O2 •− assay. Functional selectivity was visualized by deviations of ligand potencies and efficacies from linear correlations for various parameters. We obtained no evidence for involvement of protein kinase A in the inhibition of fMLP-induced O2 •− production after β2AR-stimulation although cAMP-increasing substances inhibited O2 •− production. Taken together, our data corroborate the concept of ligand-specific receptor conformations with unique signaling capabilities in native human cells and suggest that the β2AR inhibits O2 •− production in a cAMP-independent manner.

Ability of different isolates of Staphlococcal aureus to induce reactive metabolites in human polymorphovuclear leucocytes (P4429)

Abstract Staphylococcus aureus is a gram positive bacterium that causes a number of diseases such as abscesses, infective endocarditis, septic arthritis, etc. It is acquiring resistance against many antibiotics like methicillin due to which its control is becoming difficult. Phagocytes interact with bacteria and phagocytose them. The phagocytosis leads to killing of bacteria by activation of respiratory burst during which different bactericidal metabolites like reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) are produced. For this one hundred different isolates of S. aureus were obtained from various hospitals of Lahore in which fifty isolates were methicillin resistant while rest of the fifty isolates were methicillin sensitive. Peripheral blood from healthy individuals was used to obtain polymorphonuclear leucocytes by dextran density gradient centrifugation. Superoxide concentration was determined by the reduction of ferricytochrome c and nitric oxide concentration was measured by Griess method.Methicillin sensitive S. aureus (MSSA) has showed more phagocytosis by human polymorphonuclear leucocytes as compare to methicillin resistant S. aures (MRSA). Similarly, a significant difference was observed between two isolates of S. aures to induce ROI and RNI by human PMNs. MRSA produced more mean value of superoxide and nitric oxide as compare to MSSA.

Assessment of the cytotoxicity of chlorhexidine by employing an in vitro mammalian test system

Background/purposeChlorhexidine (CHX), a chlorophenyl biguanide with broad antibacterial action, has been widely used in dentistry. The initial uses of CHX in dentistry were to wash operation site and to disinfect root canals. Recently, the addition of CHX into many dental materials has improved the overall therapeutic efficacy. The aim of this study was to evaluate the potential toxicological implications of CHX employing an in vitro mammalian test system. Materials and methodsCytotoxicity, mode of cell death, and generation of superoxide anion were performed to elucidate the toxic effects of CHX on Chinese hamster ovary cells. Cytotoxicity was judged using tetrazolium bromide reduction assay. The mode of cell death was determined by flow cytometry. Superoxide anion generation was determined by the superoxide dismutase-inhibitable reduction of ferricytochrome c. ResultsCHX demonstrated a cytotoxic effect on Chinese hamster ovary cells in a dose-dependent and time-dependent manner (P < 0.05). The mode of cell death changed from apoptosis to necrosis as the concentrations of CHX elevated. CHX demonstrated a significant superoxide anion generation in a dose-dependent manner (P < 0.05). The addition of superoxide dismutase decreased the cytotoxicity induced by CHX (P < 0.05). ConclusionCHX was demonstrated to exhibit cytotoxicity that could disrupt the stable cellular redox balance, resulting in increasing levels of free radical generation and subsequent cell death. CHX has significant potential for cytotoxicity.

Ferricytochrome c Directly Oxidizes Aminoacetone to Methylglyoxal, a Catabolite Accumulated in Carbonyl Stress

Age-related diseases are associated with increased production of reactive oxygen and carbonyl species such as methylglyoxal. Aminoacetone, a putative threonine catabolite, is reportedly known to undergo metal-catalyzed oxidation to methylglyoxal, NH4 + ion, and H2O2 coupled with (i) permeabilization of rat liver mitochondria, and (ii) apoptosis of insulin-producing cells. Oxidation of aminoacetone to methylglyoxal is now shown to be accelerated by ferricytochrome c, a reaction initiated by one-electron reduction of ferricytochrome c by aminoacetone without amino acid modifications. The participation of O2 •− and HO• radical intermediates is demonstrated by the inhibitory effect of added superoxide dismutase and Electron Paramagnetic Resonance spin-trapping experiments with 5,5′-dimethyl-1-pyrroline-N-oxide. We hypothesize that two consecutive one-electron transfers from aminoacetone (E0 values = −0.51 and −1.0 V) to ferricytochrome c (E0 = 0.26 V) may lead to aminoacetone enoyl radical and, subsequently, imine aminoacetone, whose hydrolysis yields methylglyoxal and NH4 + ion. In the presence of oxygen, aminoacetone enoyl and O2 •− radicals propagate aminoacetone oxidation to methylglyoxal and H2O2. These data endorse the hypothesis that aminoacetone, putatively accumulated in diabetes, may directly reduce ferricyt c yielding methylglyoxal and free radicals, thereby triggering redox imbalance and adverse mitochondrial responses.

Vaspin attenuates high glucose-induced vascular smooth muscle cells proliferation and chemokinesis by inhibiting the MAPK, PI3K/Akt, and NF-κB signaling pathways

BackgroundVaspin has insulin-sensitizing effects, as well as additional beneficial effects on metabolic diseases. However, little is known about the direct effects of vaspin on vascular complications mediated by diabetes. The objective of this study is to determine the efficacy and mechanism of vaspin on hyperglycemia-induced vascular smooth muscle cells (VSMCs) proliferation, chemokinesis and cell signaling. MethodsRat VSMCs proliferation was determined with 5-ethynyl-2′-deoxyuridine cell proliferation assays, chemokinesis was monitored with scratch assays, and reactive oxygen species (ROS) production was assessed using H2DCFDA and SOD-inhibited reduction of ferricytochrome c assay. Luciferase activity is assayed using a Dual Luciferase Reporter Assay System. Cell signaling is assessed by immunblotting. ResultsVaspin significantly inhibited VSMCs proliferation and chemokinesis, as well as ROS generation and NADPH oxidase activity, induced by high glucose (HG) treatment. Compared with HG, vaspin significantly decreased VSMCs proliferation by 40 ± 8% at 100 ng/ml. Vaspin also decreased ROS production by 16 ± 8% at 100 ng/ml and 30 ± 8% at 300 ng/ml (all P < 0.01). Vaspin significantly abolished HG-induced phosphorylation of oxidase subunits p47phox, Akt, p38, and JNK1/2 without affecting their total levels, and attenuated HG-induced phosphorylation of insulin receptor and its downstream IRS-1 and IRS-2. For downstream targets, NF-κB activity and IκBα phosphorylation were both enhanced significantly after HG stimulation, and these effects were inhibited by vaspin. Vaspin also significantly abolished HG-induced PCNA and cyclin D1 expression. ConclusionsVaspin inhibits HG-induced VSMCs proliferation and chemokinesis by preventing ROS activation and MAPK, PI3K/Akt, and NF-κB signaling.

Genetically Determined Severity of Anti-Myeloperoxidase Glomerulonephritis

Myeloperoxidase (MPO) is a target antigen for antineutrophil cytoplasmic autoantibodies (ANCA). There is evidence that MPO-ANCA cause necrotizing and crescentic glomerulonephritis (NCGN) and vasculitis. NCGN severity varies among patients with ANCA disease, and genetic factors influence disease severity. The role of genetics in MPO-ANCA NCGN severity was investigated using 13 inbred mouse strains, F1 and F2 hybrids, bone marrow chimeras, and neutrophil function assays. Mouse strains include founders of the Collaborative Cross. Intravenous injection of anti-MPO IgG induced glomerular crescents in >60% of glomeruli in 129S6/SvEv and CAST/EiJ mice, but <1% in A/J, DBA/1J, DBA/2J, NOD/LtJ, and PWK/PhJ mice. C57BL6J, 129S1/SvImJ, LP/J, WSB/EiJ, NZO/HILtJ, and C3H mice had intermediate severity. High-density genotypes at 542,190 single nucleotide polymorphisms were used to identify candidate loci for disease severity by identifying genomic regions that are different between 129S6/SvEv and 129S1/SvImJ mice, which are genetically similar but phenotypically distinct. C57BL/6 × 129S6 F2 mice were genotyped at 76 SNPs to capture quantitative trait loci for disease severity. The absence of a dominant quantitative trait locus suggests that differences in severity are the result of multiple gene interactions. Invivo studies using bone marrow chimeric mice and invitro studies of neutrophil activation by anti-MPO IgG indicated that severity of NCGN is mediated by genetically determined differences in the function of neutrophils.

Propofol protects against high glucose-induced endothelial adhesion molecules expression in human umbilical vein endothelial cells

BackgroundHyperglycemia could induce oxidative stress, activate transcription factor nuclear factor kappa B (NF-κB), up-regulate expression of endothelial adhesion molecules, and lead to endothelial injury. Studies have indicated that propofol could attenuate oxidative stress and suppress NF-κB activation in some situations. In the present study, we examined whether and how propofol improved high glucose-induced up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells (HUVECs).MethodsProtein expression of endothelial adhesion molecules, NF-κB, inhibitory subunit of NF-κBα (IκBα), protein kinase Cβ2 (PKCβ2), and phosphorylation of PKCβ2 (Ser660) were measured by Western blot. NF-κB activity was measured by electrophoretic mobility shift assay. PKC activity was measured with SignaTECT PKC assay system. Superoxide anion (O2.-) accumulation was measured with the reduction of ferricytochrome c assay. Human peripheral mononuclear cells were prepared with Histopaque-1077 solution.ResultsHigh glucose induced the expression of endothelial selectin (E-selectin), intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and increased mononuclear-endothelial adhesion. High glucose induced O2.- accumulation, PKCβ2 phosphorylation and PKC activation. Further, high glucose decreased IκBα expression in cytoplasm, increased the translocation of NF-κB from cytoplasm to nuclear, and induced NF-κB activation. Importantly, we found these high glucose-mediated effects were attenuated by propofol pretreatment. Moreover, CGP53353, a selective PKCβ2 inhibitor, decreased high glucose-induced NF-κB activation, adhesion molecules expression, and mononuclear-endothelial adhesion.ConclusionPropofol, via decreasing O2.- accumulation, down-regulating PKCβ2 Ser660 phosphorylation and PKC as well as NF-κB activity, attenuated high glucose-induced endothelial adhesion molecules expression and mononuclear-endothelial adhesion.

Cytotoxicity and genotoxicity of chlorhexidine on macrophages in vitro

Chlorhexidine (CHX) is the most widely used antiseptic for wound, skin disinfection, and dental hygiene. The aim of this study is to investigate the possible correlation between CHX-induced cytogenotoxicity and alterations in normal cell cycle on RAW264.7 macrophages. The cytotoxicity, mechanism of cell death, mitotic activity, and reactive oxygen species (ROS) generation were determined by tetrazolium bromide reduction assay, flow cytometry, cytokinesis-block proliferation index, and superoxide dismutase-inhibitable reduction of ferricytochrome c, respectively. The genotoxicity was measured using comet assay and cytokinesis-block micronucleus assay. The cytotoxicity of CHX in RAW264.7 cells presented a dose- and time-dependent manner (p < 0.05). The mode of cell death shifted from apoptosis to necrosis when the dosage of CHX increased. The genotoxicity of CHX in RAW264.7 cells had shown DNA damage in a dose-dependent manner (p < 0.05). Prolongation of cell cycle and the increase of ROS generation also expressed in a dose-dependent manner (p < 0.05). Taken together, the data suggested that CHX-induced cytotoxicity and genotoxicity on macrophages may be via ROS generation.

Aliphatic alcohols of illegally produced spirits can act synergistically on superoxide-anion production by human granulocytes

Context: Aliphatic alcohols present in illegally produced spirits in a large number of low and middle income countries have been implicated in the etiology of chronic liver disease and cirrhosis. Previous studies have confirmed that chronic alcoholism can lead to increased susceptibility to infectious diseases. Reduced superoxide-anion (O2·−) production by granulocytes could provide a mechanism by which antimicrobial defense is impaired in alcoholics. In vitro experiments have also demonstrated that ethanol can inhibit granulocyte O2·− generation. Aliphatic alcohols consumed as contaminants of illicit spirits may also influence O2·− production thereby contributing to a decrease in microbicidal activity.Objective: The aim of this study was to investigate this possibility. It measured the O2·− production by human granulocytes following treatment of the cells with aliphatic alcohol contaminants found in illicit spirits.Materials and methods: Granulocytes were isolated from human buffy coats with centrifugal elutriation and then treated with individual aliphatic alcohols and their mixture. The O2·− production was stimulated with phorbol-12-13-dibutyrate and N-formyl-methionyl-leucyl-phenylalanine (FMLP) and measured by superoxide dismutase inhibitable reduction of ferricytochrome c.Results: Aliphatic alcohols of illegally produced spirits inhibited the FMLP-induced O2·− production in a concentration dependent manner. They suppressed O2·− generation at 2.5–40 times lower concentrations when combined than when tested individually.Discussion and conclusion: Aliphatic alcohols found in illegally produced spirits can inhibit FMLP-induced O2·− production by granulocytes in a concentration-dependent manner. Due to their synergistic effects, it is possible that, in combination with ethanol, they may inhibit O2·− formation in heavy episodic drinkers.

Propofol Protects Against High Glucose–Induced Endothelial Dysfunction in Human Umbilical Vein Endothelial Cells

Hyperglycemia, via peroxynitrite-mediated endothelial nitric oxide synthase (eNOS) enzymatic uncoupling, induced endothelial dysfunction. Propofol has been reported to improve high glucose-induced endothelial dysfunction. However, its mechanisms of action remain unclear. We hypothesized that propofol could improve hyperglycemia-induced endothelial dysfunction by decreasing the peroxynitrite level and thus restoring eNOS coupling. At the end of 3 days of incubation in medium with 30 mM glucose, human umbilical vein endothelial cells were treated with different concentrations (0.2, 1, 5, and 25 μM) of propofol for different times (0.5, 1, 2, and 4 hours). In parallel experiments, cells were cultured in 5 mM glucose for 3 days as a control. Nitric oxide (NO) production was measured with a nitrate reductase assay. Superoxide anion (O(2)(·-)) accumulation was measured with the reduction of ferricytochrome c and dihydroethidine fluorescence assay. The treatment that had maximal effect on 30 mM glucose-induced NO production and O(2)(·-) accumulation was applied in the following studies to examine the underlying signaling pathways. eNOS total protein, eNOS dimer and monomer expression, eNOS phosphorylation at Ser(1177), inducible NO synthase total protein, inducible NO synthase dimer and monomer expression, peroxynitrite, and guanosine triphosphate cyclohydrolase I expression were measured by Western blot. Tetrahydrobiopterin (BH(4)) level was measured with liquid chromatography-mass spectrometry. Compared with 5 mM glucose treatment, 30 mM glucose significantly decreased NO production by 60% (P < 0.001) and increased O(2)(·-) accumulation by 175% (P = 0.0026), which were both attenuated by propofol in a concentration- and time-dependent manner. Compared with 5 mM glucose treatment, total eNOS protein expression was increased by 30 mM glucose (P < 0.001), whereas the ratio of eNOS dimer/monomer (P = 0.0001) and eNOS phosphorylation (P < 0.001) were decreased by 30 mM glucose. Propofol did not affect 30 mM glucose-induced total eNOS protein expression, but restored the ratio of eNOS dimer/monomer (P = 0.0005) and increased eNOS phosphorylation (P < 0.001). 30 mM glucose-induced O(2)(·-) accumulation was inhibited by the eNOS inhibitor hydrochloride. Furthermore, compared with 5 mM glucose treatment, 30 mM glucose decreased the BH(4) level (P = 0.0001) and guanosine triphosphate cyclohydrolase I expression (P < 0.001), whereas it increased peroxynitrite level (P = 0.0003), which could all be reversed by propofol (P = 0.0045, P < 0.001, P = 0.0001 vs 30 mM glucose treatment, respectively). Propofol has beneficial effects on 30 mM glucose-induced NO reduction and O(2)(·-) accumulation in human umbilical vein endothelial cells. This may be mediated through inhibiting peroxynitrite-mediated BH(4) reduction, and restoring eNOS coupling.