Efflux of Glutathione and Glutathione Complexes from Human Erythrocytes in Response to Inorganic Arsenic Exposure

The objective of the present study was to investigate if arsenic exposure results in glutathione efflux from human erythrocytes. Arsenite significantly depleted intracellular nonprotein thiol level in a time- and concentration-dependent manner. The intracellular nonprotein thiol level was decreased to 0.767?±?0.0017???mol/ml erythrocyte following exposure to 10?mM of arsenite for 4?h. Extracellular nonprotein thiol level was increased concomitantly with the intracellular decrease and reached to 0.481?±?0.0005???mol/ml erythrocyte in 4?h. In parallel with the change in extracellular nonprotein thiol levels, significant increases in extracellular glutathione levels were detected. Extracellular glutathione levels reached to 0.122?±?0.0013, 0.226?±?0.003, and 0.274?±?0.004???mol/ml erythrocyte with 1, 5, and 10?mM of arsenite, respectively. Dimercaptosuccinic acid treatment of supernatants significantly increased the glutathione levels measured in the extracellular media. Utilization of MK571 and verapamil, multidrug resistance-associated protein 1 and Pgp inhibitors, decreased the rate of glutathione efflux from erythrocytes suggesting a role for these membrane transporters in the process. The results of the present study indicate that human erythrocytes efflux glutathione in reduced free form and in conjugated form or forms that can be recovered with dimercaptosuccinic acid when exposed to arsenite.     

Involvement of NO-guanylyl cyclase pathway for the depression of spinal monosynaptic reflex by Mesobuthus tamulus venom in neonatal rat in vitro

AimsThe present study was undertaken to evaluate the role of nitric oxide (NO) in Mesobuthus tamulus (MBT) venom-induced depression of spinal reflexes.Main methodsExperiments were performed on isolated hemisected spinal cords from 4 to 6 day old rats. Stimulation of a dorsal root with supramaximal strength evoked monosynaptic (MSR) and polysynaptic reflex (PSR) potentials in the corresponding segmental ventral root.Key findingsSuperfusion of MBT venom (0.3 μg/ml) depressed the spinal reflexes in a time-dependent manner and the maximum depression was seen at 10 min (MSR by 63%; PSR by 79%). The time to produce 50% depression (T-50) of MSR and PSR was 7.7 ± 1.3 and 5.7 ± 0.5 min, respectively. Pretreatment with bicuculline (1 μM; GABA_A receptor antagonist) or strychnine (1 μM; glycine_A receptor antagonist) did not block the venom-induced depression of spinal reflexes. However, Nω-nitro-L-arginine methyl ester (L-NAME, 100 or 300 μM; NO synthase inhibitor) or hemoglobin (Hb, 100 μM; NO scavenger) antagonized the venom-induced depression of MSR. Further, soluble guanylyl cylase inhibitors (1 H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one, ODQ; 1 μM or methylene blue, 100 μM) also antagonized the venom-induced depression of MSR but not PSR. Nitrite concentration (indicator of NO activity) of the cords exposed to venom (0.3 μg/ml) was not different from the control group.SignificanceThe results indicate that venom-induced depression of MSR is mediated via NO-guanylyl cyclase pathway without involving GABAergic or glycinergic system.     

Low serum zinc levels in patients undergoing coronary angiography correlate with immune activation and inflammation

IntroductionLow serum zinc concentrations are associated with adverse outcomes. To explain this phenomenon we aimed to investigate whether low zinc levels are related to immune activation, renal function and coronary artery disease (CAD).MethodsSerum concentrations of zinc and the immune activation markers neopterin and C-reactive protein (CRP) were measured in 2048 patients derived from the LUdwigshafen RIsk and Cardiovascular Health (LURIC) study, a cohort study among patients referred for coronary angiography.ResultsZinc concentrations did not differ between patients with CAD (mean ± SD: 13.3 ± 2.4 μmol/L) and controls (13.3 ± 2.2 μmol/L; Welch's t test: p = n.s.) but CAD patients had higher neopterin (8.6 ± 7.4 nmol/L) and CRP (9.7 ± 19.6 mg/L) concentrations compared to controls (neopterin: 7.5 ± 4.8 nmol/L, p = 0.0005; CRP: 5.5 ± 10.0 mg/L, p < 0.0001). There was an inverse correlation between serum zinc concentrations and neopterin (Spearman's rank correlation: r_s = ?0.222) and CRP (r_s = ?0.166; both p < 0.0001) concentrations.ConclusionsOur results indicate increased inflammatory processes in patients with low zinc levels. Further studies should clarify whether inflammation related processes such as renal wasting contribute to zinc deficiency and underlie the adverse health consequences of low serum zinc levels.     

Propofol decreases the axonal excitability in rat primary sensory afferents

AimsThe aim of this present study was to investigate the changes of peripheral sensory nerve excitability produced by propofol.Main methodsIn a recently described in vitro model of rodent saphenous nerve we used the technique of threshold tracking (QTRAC®) to measure changes of axonal nerve excitability of Aβ-fibres caused by propofol. Concentrations of 10 μMol, 100 μMol and 1000 μMol were tested. Latency, peak response, strength-duration time constant (τSD) and recovery cycle of the sensory neuronal action potential (SNAP) were recorded.Key findingsOur results have shown that propofol decreases nerve excitability of rat primary sensory afferents in vitro. Latency increased with increasing concentrations (0 μMol: 0.96 ± 0.07 ms; 1000 μMol 1.10 ± 0.06 ms, P < 0.01). Also, propofol prolonged the relative refractory period (0 μMol: 1.79 ± 1.13 ms; 100 μMol: 2.53 ± 1.38 ms, P < 0.01), and reduced superexcitability (0 μMol: ? 14.0 ± 4.0%; 100 μMol: ? 9.5 ± 5.5%) and subexcitability (0 μMol: 7.5 ± 1.2%; 1000 μMol: 3.6 ± 1.2) significantly during the recovery cycle (P < 0.01).SignificanceOur results have shown that propofol decreases nerve excitability of primary sensory afferents. The technique of threshold tracking revealed that axonal voltage-gated ion channels are significantly affected by propofol and therefore might be at least partially responsible for earlier described analgesic effects.     

Aqueous humor nitric oxide in patients with central retinal vein occlusion

PurposeIt has been suggested that nitric oxide (NO) has a role in ischemic retinopathies. Since retinal ischemia may develop in retinal vein occlusion, we investigated the presence of nitric oxide in the pathogenesis of central retinal vein occlusion (CRVO).MethodsEighteen consecutive patients with CRVO were included in this study. Aqueous humor specimens were obtained within 21 days of diagnosis. Samples of aqueous humor were also collected from 20 control patients undergoing cataract surgery. For each sample after reduction of nitrate to nitrite with vanadium chloride (VCl₃), we used spectrophotometric method for simultaneous detection of nitrate and nitrite (NO_x).ResultsMean level of aqueous humor NO_x in CRVO and control group was 94.1 ± 23.2 μmol/l and 55.6 ± 11.0 μmol/l, respectively. The difference between two groups was statistically significant (p < 0.0001).ConclusionsOur results may support involvement of nitric oxide in the pathogenesis of CRVO.     

Prevotella bivia as a source of lipopolysaccharide in the vagina

ObjectivesTo compare vaginal lipopolysaccharides (LPS) concentrations between patients with and without bacterial vaginosis (BV), to evaluate the correlation between Prevotella bivia colonization density and LPS concentration, and to determine the impact of LPS on loss of dopamine neurons (DA).MethodsVaginal washes obtained from patients with (n = 43) and without (n = 59) BV were tested for quantity of P. bivia cells using quantitative PCR and for concentrations of LPS using the Limulus Amebocyte Lysate gel clot method. Prevotella bivia, Gardnerella vaginalis and Escherichia coli sonicated cell extracts were also tested for LPS production. DA neuron cells obtained from embryonic day (E) 14.5 pregnant rats were exposed to fluid from eight vaginal washes; tyrosine hydrolase immunoreactive staining was applied for visualization and cell counts.ResultsThe median LPS concentrations were dramatically higher among patients who had symptoms of BV compared to those who did not have symptoms (3235.0 vs 46.4 EU/ml, respectively, P < 0.001); patients who had BV also had much higher colonization densities of P. bivia (0.06 ± 0.36 vs 5.4 ± 2.2 log₁₀ CFU/ml, respectively, P < 0.001).Prevotella bivia cell lysates resulted in a higher LPS concentration (10,713.0 ± 306.6 EU/ml) than either E. coli (4679.0 ± 585.3 EU/ml) or G. vaginalis (0.07 ± 0.01 EU/ml of LPS).The loss of DA neuron was 20–27% in cultures treated with vaginal washes from BV-negative patients and 58–97% in cultures treated with vaginal washes from patients with BV.ConclusionP. bivia produces high LPS concentration, which may create a toxic vaginal environment that damages DA neurons.     

Intra-operative preparation of autologous bone marrow-derived CD34-enriched cellular products for cardiac therapy

Background and AimsWith the advent of regenerative therapy, there is renewed interest in the use of bone marrow as a source of adult stem and progenitor cells, including cell subsets prepared by immunomagnetic selection. Cell selection must be rapid, efficient and performed according to current good manufacturing practices. In this report we present a methodology for intra-operative preparation of CD34⁺ selected autologous bone marrow for autologous use in patients receiving coronary artery bypass grafts or left ventricular assist devices.Methods and ResultsWe developed a rapid erythrocyte depletion method using hydroxyethyl starch and low-speed centrifugation to prepare large-scale (mean 359 mL) bone marrow aspirates for separation on a Baxter Isolex 300i immunomagnetic cell separation device. CD34 recovery after erythrocyte depletion was 68.3 ± 20.2%, with an average depletion of 91.2 ± 2.8% and an average CD34 content of 0.58 ± 0.27%. After separation, CD34 purity was 64.1 ± 17.2%, with 44.3 ± 26.1% recovery and an average dose of 5.0 ± 2.7 × 10⁶ CD34⁺ cells/product. In uncomplicated cases CD34-enriched cellular products could be accessioned, prepared, tested for release and administered within 6 h. Further analysis of CD34⁺ bone marrow cells revealed a significant proportion of CD45^? CD34⁺ cells.ConclusionsIntra-operative immunomagnetic separation of CD34-enriched bone marrow is feasible using rapid low-speed Hetastarch sedimentation for erythrocyte depletion. The resulting CD34-enriched product contains CD45^? cells that may represent non-hematopoietic or very early hematopoietic stem cells that participate in tissue regeneration.     

Improved survival of mesenchymal stromal cell after hypoxia preconditioning: role of oxidative stress

AimsTo investigate the mechanisms underlying the beneficial effect of hypoxia preconditioning (HPC) on mesenchymal stromal cells (MSCs) and optimize novel non-invasive methods to assess the effect of biological interventions aimed to increased cell survival.Main methodsMSCs from rat femur, with or without HPC, were exposed to hypoxic conditions in cell culture (1% O₂ for 24 h) and cell survival (by the LDH release assay and Annexin-V staining) was measured. Oxidant status (conversion of dichloro-fluorescein-DCF- and dihydro-ethidium-DHE-, protein expression of oxidant enzymes) was characterized, together with the mobility pattern of cells under stress. Furthermore, cell survival was assessed non-invasively using state-of-the-art molecular imaging.Key findingsCompared to controls, Hypoxia resulted in increased expression of the oxidative stress enzyme NAD(P)H oxidase (subunit 67^phox: 0.05 ± 0.01 AU and 0.48 ± 0.02 AU, respectively, p < 0.05) and in the amount of ROS (DCF: 13 ± 1 and 42 ± 3 RFU/μg protein, respectively, p < 0.05) which led to a decrease in stem cell viability. Hypoxia preconditioning preserved cell biology, as evidenced by preservation of oxidant status (16 ± 1 RFU/μg protein, p < 0.05 vs. hypoxia), and cell viability. Most importantly, the beneficial effect of HPC can be assessed non-invasively using molecular imaging.SignificanceHPC preserves cell viability and function, in part through preservation of oxidant status, and its effects can be assessed using state-of-the-art molecular imaging. Understanding of the mechanisms underlying the fate of stem cells will be critical for the advancement of the field of stem cell therapy.     

Copper,zinc and iron levels in premature infants following red blood cell transfusion

This study aimed to investigate effect of erythrocyte suspension (ES) transfusion on Cu, Zn, and Fe levels. It was conducted on 53 premature infants who were admitted to Hacettepe Hospital and received EST for first time. Blood samples were drawn before and 96 h after ES transfusion to determine Cu, Zn, and Fe levels in plasma and/or erythrocytes. The mean plasma Cu levels were 99 ± 3 μg/dl and 113 ± 3 μg/dl; Zn levels were 105 ± 2 μg/dl and 115 ± 23 μg/dl; mean plasma Fe level was 58.1 ± 19.4 and 75.2 ± 25.4 μg/dl and mean erythrocyte Fe level was 4182 ± 2314 μg/ml and 7009 ± 5228 μg/ml, before and after ES transfusion. The differences between before and after ES transfusion in Cu, Zn and Fe levels were significant. Correlation between plasma and erythrocyte Fe levels was significant both before and after ES transfusion. Though Fe overload is a major cause of morbidity/mortality after ES transfusion, alterations in trace elements should also be considered when transfusing blood to infants and children.     

Resveratrol in combination with other dietary polyphenols concomitantly enhances antiproliferation and UGT1A1 induction in Caco-2 cells

AimsThe only FDA approved medication for colorectal cancer (CRC) prevention is celecoxib. Its adverse effects underline the need for safer drugs. Polyphenols like resveratrol are in clinical trials for this purpose. This study aimed at examining effects of resveratrol alone and in combination with curcumin or chrysin on UGT induction in Caco-2 cells. Phytochemical combinations were selected using drug combination analyses of various anti-proliferation ratios of resveratrol + curcumin and resveratrol + chrysin.Main methodsCell proliferation and UGT1A1 induction assays were carried out with individual polyphenols and combinations. Cell viability was determined with AlamarBlue assays. UGT1A1 mRNA was quantified via real time RT-PCR. UGT activity was determined with 4-methylumbelliferone (4MU) glucuronidation.Key findingsCell proliferation IC₅₀ estimates (± SE) for resveratrol, curcumin and chrysin were 20.8 ± 1.2, 20.1 ± 1.1 and 16.3 ± 1.3 μM respectively. Combination of anti-proliferative effects showed additivity for resveratrol + chrysin and resveratrol + curcumin. Resveratrol at its IC₅₀ mediated a four-fold induction of UGT1A1 mRNA in a concentration independent manner. Chrysin at its IC₅₀ induced UGT1A1 expression seven-fold while Curcumin at its IC₉₀ mediated a two-fold induction. The 20 μM:40 μM resveratrol + curcumin and 20 μM :32 μM resveratrol + chrysin combinations mediated the greatest increases in mRNA expression (12 and 22 folds respectively). Significant increase in 4-MU glucuronidation was observed with combinations exhibiting maximal mRNA induction.SignificancePhytochemical combinations can offer greater chemoprevention than single agents. These chemicals might offer safer options than present synthetic therapeutics for CRC prevention.     

Identification of GR and TrxR systems in Setaria cervi: Purification and characterization of glutathione reductase

The glutathione reductase (GR) and thioredoxin reductase (TrxR) are important enzymes of the redox system that aid parasites to maintain an adequate intracellular redox environment. In the present study, the enzyme activity of GR and TrxR was investigated in Setaria cervi (S. cervi). Significant activity of both enzymes was detected in the somatic extract of adult and microfilariae stages of S. cervi. Both GR and TrxR were separated by partial purification using ammonium sulfate fractionation and DEAE ion exchange chromatography suggesting the presence of both glutathione and thioredoxin systems in S. cervi. The enzyme glutathione reductase (ScGR) was purified to homogeneity using affinity and ion exchange chromatography that resulted in 90 fold purification with a yield of 11.54%. The specific activity of the ScGR was 643 U/mg that migrated as a single band on SDS-PAGE. The subunit molecular mass was determined to be ~ 50 kDa while the optimum pH and temperature were found to be 7.0 and 35 °C respectively. The activation energy (Ea) was calculated from the slope of Arrhenius plot as 16.29 ± 1.40 kcal/mol. The Km and Vmax were determined to be 0.27 ± 0.045 mM; 30.30 ± 1.30 U/ml with NADPH and 0.59 ± 0.060 mM; 4.16 ± 0.095 U/ml with GSSG respectively. DHBA, a specific inhibitor for GR has completely inhibited the enzyme activity at 1 μM concentration. The inhibition of ScGR activity with NAI (IC₅₀ 0.71 mM), NEM (IC₅₀ 0.50 mM) and DEPC (IC₅₀ 0.27 mM) suggested the presence of tyrosine, cysteine and histidine residues at its active site. Further studies on characterization and understanding of these antioxidant enzymes may lead to designing of an effective drug against lymphatic filariasis.     

Zinc induced damage to kidney proximal tubular cells: Studies on chemical speciation leading to a mechanism of damage

This study was carried out to investigate whether zinc can potentiate renal toxicity using monolayer cultures of kidney proximal tubular cells and if so to establish the chemical species and the mechanism involved.MethodsZinc was prepared as the citrate complex at pH 7.4 in phosphate buffered saline. Monolayers of kidney proximal tubular cells under standard cell culture conditions were exposed to zinc concentrations of 0, 5 10, 20, 50 and 100 μmol/L. To assess cellular damage, thiazol blue (MTT) uptake, NAG and LDH release, DAPI staining and Tunel assay were used. Cytoprotective agents: trolox, cysteine, glutathione, ascorbic acid and sodium selenite were used to investigate if the damage was reversible.ResultsIncubation of kidney cells with zinc citrate showed a dose related reduction in cell viability (p < 0.005) associated with cellular uptake of zinc ions. After 24 h incubation with 100 μmol/L Zn citrate, NAG release was not significantly different compared to the control whereas LDH increased 3 fold. DAPI staining showed apoptotic bodies within the cells confirmed by Tunel assay using flow cytometry. Electron microscopy showed significant morphological changes including loss of brush border, vacuolated cytoplasm and condensed nuclei. Trolox almost completely (>85 ± 5%) and sodium selenite partially recovered (40 ± 4%) the viability of cells exposed to Zn but no protection was observed with other cytoprotectants, e.g. glutathione, cysteine or ascorbic acid.In conclusion zinc can induce damage to kidney cells by a mechanism dependent on zinc ions entering the cell, binding to the cell organelles and disrupting cellular processes rather than damage initiated by free radical and ROS production.     

The receptor for advanced glycation endproducts and its ligands in patients with myasthenia gravis

ObjectiveMyasthenia gravis (MG) is a T- and B-cell mediated autoimmune disorder affecting the neuromuscular junction. The receptor for advanced glycation endproducts (RAGE) plays a role in the amplification of chronic inflammatory disorders and autoimmune diseases. We sought to investigate the role of RAGE and its ligands in the pathophysiology of MG.MethodsIn this cross-sectional study we enrolled 42 patients with MG and 36 volunteers. We employed enzyme-linked immunosorbent assays to determine the concentration of soluble RAGE (sRAGE) and high mobility group box 1 (HMGB1) in serum of patients and volunteers. In a subpopulation of patients we measured the serum levels of endogenous secretory (es) RAGE and various RAGE ligands, such as S100B, S100A8 and advanced glycation endproducts (AGE-CML). Reported are means and standard error mean.ResultsWe found significantly reduced levels of the soluble receptors sRAGE and esRAGE in patients with MG compared to volunteers without MG (sRAGE [pg/ml] 927.2 ± 80.8 vs. 1400.1 ± 92.4; p < 0.001; esRAGE [pg/ml] 273.5 ± 24.6 vs. 449.0 ± 22.4; p < 0.001). Further categorization of patients with MG according to the distribution of muscle involvement revealed the following sRAGE concentrations: generalized MG 999.4 ± 90.8 and ocular MG 696.1 ± 161.8 (vs. control; One-way ANOVA: p < 0.001; Post hoc analysis: generalized vs. ocular MG: p = 0.264, generalized MG vs. control: p = 0.008, ocular MG vs. control: p = 0.001). In patients with detectable antibodies specific for acetylcholine receptors (Anti-AChR positive) the sRAGE concentration was 970.0 ± 90.2 compared to those without (seronegative) 670.6 ± 133.1 (vs. control; One-way ANOVA: p < 0.001; Post hoc analysis: Pos vs. Neg.: p = 0.418, Pos vs. control: p = 0.003, Neg. vs. control: p = 0.008). We next investigated the role of RAGE ligands in MG. The concentrations of RAGE ligands in patients with MG and controls were as follows: (HMGB1 [ng/ml] 1.7 ± 0.1 vs. 2.1 ± 0.2; p = 0.058; S100B [pg/ml] 22.5 ± 22.5 vs. 14.4 ± 9.2; p = 0.698; S100A8 [pg/ml] 107.0 ± 59.3 vs. 242.5 ± 103.6; p = 0.347; and AGE-CML [ng/ml] 1100.8 ± 175.1 vs. 1399.8 ± 132.8; p = 0.179).ConclusionsOur data suggest a role for the RAGE pathway in the pathophysiology of MG. Further studies are warranted to elucidate more about this immunological axis in patients with MG.     

Characterization of A-935142, a hERG enhancer, in the presence and absence of standard hERG blockers

AimsIn a previous study we found that A-935142 enhanced hERG current in a concentration-dependent manner by facilitating activation, reducing inactivation, and slowing deactivation (Su et al., 2009). A-935142 also shortened action potential duration (APD₉₀) in canine Purkinje fibers and guinea pig atrial tissue. This study focused on the combined effects of the prototypical hERG enhancer, A-935142 and two hERG current blockers (sotalol and terfenadine).Main methodsThe whole-cell voltage clamp method with HEK 293 cells heterologously expressing the hERG channel (Kv 11.1) was used.Key findingsA-935142 did not compete with ³H-dofetilide binding, suggesting that A-935142 does not overlap the binding site of typical hERG blockers. In whole-cell voltage clamp studies we found: 1) 60 μM A-935142 enhanced hERG current in the presence of 150 μM sotalol (57.5 ± 5.8%) to a similar extent as seen with A-935142 alone (55.6 ± 5.1%); 2) 150 μM sotalol blocked hERG current in the presence of 60 μM A-935142 (43.5 ± 1.5%) to a similar extent as that seen with sotalol alone (42.0 ± 3.2%) and 3) during co-application, hERG current enhancement was followed by current blockade. Similar results were obtained with 60 nM terfenadine combined with A-935142.SignificanceThese results suggest that the hERG enhancer, A-935142 does not compete with these two known hERG blockers at their binding site within the hERG channel. This selective hERG current enhancement may be useful as a treatment for inherited or acquired LQTS (Casis et al., 2006).     

The effect of cysteine and glutathione on sperm and oxidative stress parameters of post-thawed bull semen

The aim of this study was to determine the effects of antioxidants such as reduced glutathione (GSH) and cysteine in Laiciphose® extender on semen parameters, fertilizing ability, lipid peroxidation (LPO) level and glutathione peroxidise (GPx) activity of post-thawed bull semen. Totally 54 ejaculates of three bulls were used in the study. Five groups, namely; GSH (0.5 and 2 mM), cysteine (5 and 10 mM) and control group, were conducted to test the antioxidants in Laiciphose®. Insemination doses were processed that each 0.25-mL straw contained 15 × 10⁶ sperm. The addition of antioxidants did not present any significant effect on the percentages of post-thaw sperm morphology (acrosome and total abnormalities), subjective, CASA and progressive motilities, as well as sperm motility characteristics (VAP, VSL, VCL, LIN and ALH), compared to the control groups (P > 0.05). GSH 0.5 mM (55.5 ± 7.38%) and cysteine 10 mM (48 ± 5.65%) led to lower rates of DNA damage, compared to control (P < 0.05). As regards to MDA level, cysteine at 10 mM dose gave the highest level (4.99 ± 0.44 nmol/L) (P < 0.001). GPx activity was demonstrated to be higher level upon the addition of 5 mM cysteine when compared to the other groups (P < 0.05). With respect to fertility results based on 60-day non-returns, the supplementation of antioxidants did not present significant differences (P > 0.05). The results of this study may provide an useful information for the future studies in this area. So, further studies could be suggested to achieve better information in terms of the DNA damage and fertilizing capacity of bull sperm frozen with effective antioxidants.     

Right ventricular pacing and sensing function in high posterior septal and apical lead placement in cardiac resynchronization therapy

BackgroundThe conventional right ventricular (RV) lead position in cardiac resynchronization therapy pacemakers (CRT-P) is the RV apex (RV-A). Little is known about electrophysiological stability and associated complications of pacing leads in RV high posterior septal (RV-HS) position in CRT-P.MethodsTwo hundred and thirty-five consecutive CRT-P patients were included from 1999-2010. Pacing thresholds at 0.5 ms and 2.5 V, sensing electrograms and lead impedances were measured at implant and repeated 1,3,6,12,18 and 24 months after CRT-P. Electrophysiological measurements of leads located in RV-A and RV-HS were analyzed retrospectively. Bipolar RV leads were used, including high impedance leads, passive fixation and active fixation.ResultsRV pacing leads were implanted in RV-A (n = 79) and RV-HS (n = 156). Average RV pacing thresholds from CRT implant procedure to 24-month follow-up at 0.5 ms were 0.77 ± 0.69 V in RV-A and 0.71 ± 0.35 V in RV-HS (P = 0.31), and at 2.5 V were 0.06 ± 0.08 ms in RV-A and 0.07 ± 0.05 ms in RV-HS (P = 0.12). Average RV electrogram amplitudes from baseline to 24 months after CRT were 15.3 ± 6.9 mV in RV-A and 12.1 ± 6.0 mV in RV-HS (P = 0.55). Average RV impedances during follow-up were 850 ± 286Ω in RV-A and 618 ± 147Ω in RV-HS (P = 0.57). Similar RV lead revisions between RV-A and RV-HS were observed after 2-year follow-up (P = 0.55).ConclusionsThe RV-HS lead position demonstrated stable and acceptable long-term pacing and sensing function, with rates of complications comparable to conventional RV-A lead position in CRT. The RV-HS lead position is feasible in CRT-P.     

Uptake and toxicity of arsenite and arsenate in cultured brain astrocytes

Inorganic arsenicals are environmental toxins that have been connected with neuropathies and impaired cognitive functions. To investigate whether such substances accumulate in brain astrocytes and affect their viability and glutathione metabolism, we have exposed cultured primary astrocytes to arsenite or arsenate. Both arsenicals compromised the cell viability of astrocytes in a time- and concentration-dependent manner. However, the early onset of cell toxicity in arsenite-treated astrocytes revealed the higher toxic potential of arsenite compared with arsenate. The concentrations of arsenite and arsenate that caused within 24 h half-maximal release of the cytosolic enzyme lactate dehydrogenase were around 0.3 mM and 10 mM, respectively. The cellular arsenic contents of astrocytes increased rapidly upon exposure to arsenite or arsenate and reached after 4 h of incubation almost constant steady state levels. These levels were about 3-times higher in astrocytes that had been exposed to a given concentration of arsenite compared with the respective arsenate condition. Analysis of the intracellular arsenic species revealed that almost exclusively arsenite was present in viable astrocytes that had been exposed to either arsenate or arsenite. The emerging toxicity of arsenite 4 h after exposure was accompanied by a loss in cellular total glutathione and by an increase in the cellular glutathione disulfide content. These data suggest that the high arsenite content of astrocytes that had been exposed to inorganic arsenicals causes an increase in the ratio of glutathione disulfide to glutathione which contributes to the toxic potential of these substances.     

Comparison of N-acetyl-L-cysteine and L-cysteine in respect to their transmembrane fluxes

The objective of the present study was to compare cysteine and N-acetyl-L-cysteine in respect to their transmembrane fluxes and find out which one is a better available precursor for the cells and thus better supports the intracellular glutathione synthesis. Cysteine can directly participate in glutathione synthesis, whereas N-acetyl-L-cysteine must be first deacetylated before its incorporation to glutathione. In the present study we investigated and compared the efficiencies of cysteine and N-acetyl-L-cysteine influx and efflux through the erythrocyte membrane. Erythrocytes transported both cysteine and N-acetyl-L-cysteine in a concentration-dependent manner. However, our results demonstrated that cysteine crosses the erythrocyte membranes more efficiently as compared to N-acetyl-L-cysteine. Treatment of erythrocytes with 5 mM of cysteine or N-acetyl-L-cysteine for 1 hr raised the intracellular free sulfhydryl group (free-SH) levels to 3.37 ± 0.006 or 2.23 ± 0.08 μ mol/ml erythrocyte, respectively. Cysteine more effectively than N-acetyl-L-cysteine restored the intracellular free-SH level depleted beforehand. In erythrocytes previously depleted of free-SH, 5 mM cysteine raised the free-SH level to 1.45 ± 0.075 μ mol/ml within 1 hr, whereas N-acetyl-L-cysteine at the same concentration raised this level to 0.377 ± 0.034 μmol/ml only. The results of our study also revealed that both cysteine and N-acetyl-L-cysteine influx and efflux processes are temperature dependent indicating that their transport requires biological activity. Our results demonstrate that cysteine is a better thiol precursor for the erythrocytes. Availability of cysteine for the cells is higher than that of N-acetyl-L-cysteine. The article is published in the original.     

A targeted high-efficiency angiogenesis strategy as therapy for myocardial infarction

AimsThe aim of this study was to prove that an intramyocardial injection of a mixture of low-dose human growth factor (HGF) plasmid and microbubbles (MB) in combination with insonation was an effective therapy for myocardial infarction.Main methodsTwenty dogs with myocardial infarction were divided into 4 groups: (1) HGF, MB and ultrasound (HGF-US/MB), (2) HGF and US (HGF-US), (3) HGF alone and (4) surgery alone (control). In the HGF-US/MB group, HGF plasmid DNA (500 μg) mixed with 0.5 ml of MB solution was injected 5 min after coronary occlusion followed by insonation. With the exception of the control group, the other dogs were divided into two groups, one treated with the HGF gene and insonation and the other with the HGF gene only.Key findingsCompared to the HGF group, infarct size decreased from 32% ± 7% (control) to 23% ± 5% in the HGF-US/MB group 28 d later (P < 0.05). Capillary density increased from 21.7 ± 4.2/mm² (control) to 114.3 ± 28.9/mm² in the HGF-US/MB group (P < 0.01). Compared to the HGF group, there was a 14% decrease in the ratio of left ventricle weight/body weight and a 25% decrease in hydroxyproline content. We also observed a 29% and 20% decrease in collagen volume fraction of type I and type III collagen, respectively in the HGF-US/MB group.SignificanceIntramyocardial injection of HGF and MB in combination with insonation enhances neovascularization and reduces ventricular remodeling and infarct size.