Modelling the postsynaptic location and magnitude of tonic conductance changes resulting from neurotransmitters or drugs

Tonic conductance changes, synaptically or drug-mediated, can occur near the soma, in one or more distal dendrites, or diffusely. Using simple analogue passive neuronal models, means of localizing tonic conductance changes from intrasomatic recordings were explored. The input resistance measured from the soma was more influenced by perisomatic than distal conductance changes. The membrane time constant, τ₀, was quite sensitive to shunt magnitude, but not to shunt location. Semilogarithmic plots of the voltage response to a short constant current pulse showed that proximal shunts caused a faster earlier decay than distal shunts. Combining these data it is often possible to estimate the location of a tonic conductance change in a neuron from intrasomatic recordings.     

The impact of vacuum freeze-drying on collagen sponges after gas plasma sterilization

The sterilization of porous collagen sponges remains a challenging procedure. Gamma irradiation denatures collagen, resulting in dramatic changes to its structure. Ethylene oxide leaves toxic residues requiring weeks to evaporate. This study investigated the impact on cell behavior of gas plasma treatment when combined with vacuum freeze-drying. The goal of this procedure is to eliminate the molecules of hydrogen peroxide remaining after the sterilization process, together with their decomposition products, from the scaffolds. These molecules hinder the immediate use of the porous designs. Collagen and EDC/NHS-heparinized collagen scaffolds were sterilized with gas plasma. H₂O₂ released by the collagen specimens was measured by peroxidase test both immediately and also 1 week after the plasma treatment. Further measurements were done 24, 36, 48 and 72 h after vacuum freeze-drying. The activity of these scaffolds was further evaluated in relation to the proliferation, migration and differentiation of human umbilical vein endothelial cells (HUVECs). Both immediately after exposure to gas plasma and also 1 week later, the collagen designs contained significantly higher concentrations of H₂O₂ than scaffolds having also undergone vacuum freeze-drying. This procedure achieved faster decontamination of the remaining H₂O₂. Following vacuum freeze-drying, sponges already allowed HUVEC proliferation after 48 h, but in non-lyophilized specimens after gas plasma treatment alone, cell death occurred as early as only 1 week later. These data highlight the advantages of carrying out vacuum freeze-drying following gas plasma sterilization. The results show the substantial impact of sterilization of porous materials made for tissue engineering.     

Mechanisms underlying the modulation of L-type Ca2+ channel by hydrogen peroxide in guinea pig ventricular myocytes

Although Cav1.2 Ca²⁺ channels are modulated by reactive oxygen species (ROS), the underlying mechanisms are not fully understood. In this study, we investigated effects of hydrogen peroxide (H₂O₂) on the Ca²⁺ channel using a patch-clamp technique in guinea pig ventricular myocytes. Externally applied H₂O₂ (1 mM) increased Ca²⁺ channel activity in the cell-attached mode. A specific inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) KN-93 (10 μM) partially attenuated the H₂O₂-mediated facilitation of the channel, suggesting both CaMKII-dependent and -independent pathways. However, in the inside-out mode, 1 mM H₂O₂ increased channel activity in a KN-93-resistant manner. Since H₂O₂-pretreated calmodulin did not reproduce the H₂O₂ effect, the target of H₂O₂ was presumably assigned to the Ca²⁺ channel itself. A thiol-specific oxidizing agent mimicked and occluded the H₂O₂ effect. These results suggest that H₂O₂ facilitates the Ca²⁺ channel through oxidation of cysteine residue(s) in the channel as well as the CaMKII-dependent pathway.     

Unique mode of cell death in freshly isolated adult rat ventricular cardiomyocytes exposed to hydrogen peroxide

To address whether adult rat ventricular cardiomyocytes (ARVCs) exposed to oxidant stress die via apoptosis (secondarily by necrosis) or primarily by necrosis, we exposed ARVCs to hydrogen peroxide (H₂O₂; 0.1–100 μM) for up to 24 h and then compared them with isoproterenol-induced apoptotic and Triton X-induced necrotic controls. Cellular shrinkage preceded plasma membrane disruption, reflected by trypan blue uptake in ARVCs exposed to lower concentrations of H₂O₂ (<1 μM; an apoptotic pattern), but the order was reversed in cells exposed to higher concentrations of H₂O₂ (>1 μM; a necrotic pattern). DNA fragmentation, caspase-3 activation, mitochondrial membrane potential preservation, and ATP preservation were all apparent in ARVCs treated with low H₂O₂ (0.5 μM), but not in those treated with high H₂O₂ (10 μM). In addition, electron microscopy revealed unique morphology in H₂O₂-treated ARVCs; i.e., the nuclei had a homogeneous ground glass-like appearance that was never accompanied by chromatin condensation. Apparently, high concentrations of H₂O₂ caused primary necrosis in ARVCs, whereas low concentrations induced biochemically comparable apoptosis, although the latter did not satisfy the morphological criteria of apoptosis. These findings caution against the use of oxidant stress, H₂O₂ in particular, as an inducer of apoptosis in ARVCs.     

Effects of Low-Temperature Hydrogen Peroxide Gas Plasma Sterilization on In Vitro Cytotoxicity of Poly(ϵ-Caprolactone) (PCL)

Abstract

Our objective was to determine whether low-temperature hydrogen peroxide (H₂O₂) gas plasma sterilization of porous three-dimensional poly(?-caprolactone) (PCL) constructs significantly inhibits cellular metabolism of canine chondrocytes. Porous cylindrical constructs were fabricated using fused deposition modeling and divided into four sterilization groups. Two groups were sterilized with low-temperature H₂O₂ gas plasma (LTGP) and constructs from one of those groups were subsequently rinsed with Dulbecco’s Modified Essential Media (LTGPDM). Constructs in the other two groups were disinfected with either 70% isopropyl alcohol or exposure to UV light. Canine chondrocytes were seeded in 6-well tissue-culture plates and allowed to adhere prior to addition of PCL. Cellular metabolism was assessed by adding resazurin to the tissue-culture wells and assessing conversion of this substrate by viable cells to the fluorescent die resorufin. This process was performed at three times prior to addition of PCL and at four times after addition of PCL to the tissue-culture wells. Metabolism was not significantly different among the different tissue-culture wells at any of the 3 times prior to addition of PCL. Metabolism was significantly different among the treatment groups at 3 of 4 times after addition of PCL to the tissue culture wells. Metabolism was significantly lower with constructs sterilized by LTGP than all other treatment groups at all 3 of these times. We conclude that LTGP sterilization of PCL constructs resulted in significant cytotoxicity to canine chondrocytes when compared to PCL constructs disinfected with either UV light exposure or 70% isopropyl alcohol.     

Up-regulation of a hydrogen peroxide–responsive pre-mRNA binding protein in atherosclerosis and intimal hyperplasia

BackgroundMultiple lines of investigation have implicated hydrogen peroxide (H₂O₂) as an important endogenous mediator of cell proliferation in the vessel wall. Heterogeneous nuclear ribonucleoprotein C (hnRNP-C), a nuclear pre-mRNA binding protein that plays roles in vertebrate cell proliferation and differentiation, has been identified as a component of a vascular cell signaling pathway activated by low physiologic levels of H₂O₂. The expression of hnRNP-C in human arteries has not previously been assessed.MethodsSegments of human proximal internal carotid arteries were evaluated for the expression of hnRNP-C by immunohistochemistry.ResultsIn normal proximal internal carotid arteries, hnRNP-C is expressed predominantly by the endothelium, with significantly lower expression by medial smooth muscle. In preatherosclerotic intimal hyperplasia, hnRNP-C is up-regulated in the artery wall, due to the robust expression by the intimal smooth muscle cells, without up-regulation in the medial smooth muscle cells. In arteries with atherosclerotic lesions, there is strong expression of hnRNP-C not only by intimal cells but also by medial smooth muscle cells.ConclusionsThe H₂O₂ responsive pre-mRNA binding protein hnRNP-C is up-regulated in atherosclerosis and in preatherosclerotic intimal hyperplasia in humans, supporting the hypothesis that H₂O₂ is a regulator of vascular cell proliferation in these conditions. These data also suggest that hnRNP-C may be useful as a marker of vascular cell activation.     

Albumin decreases hydrogen peroxide and reperfusion injury in isolated rat hearts

Perfusion with human serum albumin decreased myocardial hydrogen peroxide (H₂O₂) levels (as assessed by inactivation of myocardial catalase activities following ammotriazole pretreatment) and increased myocardial ventricular developed pressures (DP), contractility (+dP/dt) but not relaxation rate (-dP/dt) in isolated crystalloid perfused rat hearts subjected to normothermic global ischemia (20 min) and then reperfusion (40 min). Albumin also decreased H₂O₂ concentrations in vitro. The findings support the possibility that albumin may act as a protective O₂ metabolite scavenger in vivo.     

Short-term response of brain tissue to cerebrospinal fluid shunts in vivo and in vitro.

The purpose of the studies was to determine how gross physical characteristics of cerebrospinal fluid (CSF) shunts and the cellular proliferative response to shunts contribute to shunt obstruction. Ventricular catheters with round holes, slots, and flanges were implanted into the lateral ventricles of rabbits for 4 weeks. All shunt designs were subject to ingrowth of tissue from the ventricle wall or choroid plexus. There were no qualitative or quantitative differences between normal and hydrocephalic rabbits. Astroglial cells from newborn mice were cultured on shunt catheters for 2 or 4 weeks. The growth of these cells was poor, probably because the cells cannot attach well to the silicone rubber substrate. Contact between the shunt catheter and vascularized brain tissue is the most important factor in the genesis of shunt obstruction.     

Modulatory effect of hydrogen peroxide on tumoral lymphocytes proliferation

BW 5147 (murine lymphoma cell line). We analyzed the effect of H₂O₂ in cell proliferation testing nitric oxide and apoptosis. Enzymes involved in the regulation of H₂O₂ levels as superoxide dismutase (SOD) and peroxidase (PER) were analyzed. H₂O₂ exerted a biphasic effect. The inhibitory effect of H₂O₂ was related to the activation of the ERK and P38 pathway, NO production and apoptosis. The high proliferation was associated with a low level of H₂O₂ related to a low SOD and a high PER activities. Drugs capable of producing an increase in H₂O₂ levels could be used in cancer.     

Mutation of alkyl hydroperoxide reductase gene ahpC of Xanthomonas oryzae pv. oryzae affects hydrogen peroxide accumulation during the rice–pathogen interaction

Hydrogen peroxide (H₂O₂) is usually generated by normal aerobic respiration of pathogens and by the host defense response during plant–pathogen interactions. In this study, histochemical localization of H₂O₂ accumulation in rice inoculated with the wild-type strain (PXO99^A) and the gene deletion mutant (ΔahpC) of alkyl hydroperoxide reductase subunit C (AhpC) of Xanthomonas oryzae pv. oryzae (Xoo), the bacterial blight pathogen of rice, was analyzed. The ΔahpC mutant displayed a significant decrease in endogenous H₂O₂ accumulation which was induced by the compensatory increase in H₂O₂ scavenging activity. The change in the bacterial endogenous H₂O₂ level affected the total amount of H₂O₂ accumulation during the interaction with rice plants. These results suggested that Xoo contributes to H₂O₂ accumulation in rice in a compatible interaction, and pathogen-driving H₂O₂ is in association with cell collapse of rice.     

Effect of Cold Exposure on Free Water Reabsorption in Hydropenic Man

With clearance technique, changes in tubular reabsorption of solute free water(T_H2_O^c), were studied in a group of 9 healthy hydropenic volunteers during a cold stress of 1 h at + 15°C. Increases were found in(T^c_H20)and in fractional excretion of sodium and osmoles during cold. Since(T^C_H2_O)was used as an index of sodium reabsorption in the ascending limb of Henle's loop, the findings indicated that under the conditions used in this study, the concentrating ability of the kidney was not negatively influenced by cold, despite a significant increase in arterial pressure. It is suggested that the reabsorption by the ascending limb of the loop was increased secondary to a reduced proximal tubular reabsorption of sodium.     

Neutrophils may directly synthesize both H2O2 and O2 − since surface stimuli induce their release in stimulus-specific ratios

Many stimuli induce neutrophils to undergo an oxidative burst and generate toxic oxygen metabolites. The major products are O₂ ^– and H₂O₂, the latter being presumed to arise by spontaneous dismutation of the former. If H₂O₂ were indeed derived exclusively from released O₂ ^– according to the equation 2O₂ ^– + 2H⁺ H₂O₂ + O₂, one would expect that relationship to be reflected in the ratio of the two metabolites detectable in the extracellular mileu of stimulated neutrophils. A second corollary is that H₂O₂ should not form when cytochromec is present to scavenge O₂ ^– before it can dismutate. Although H₂O₂ cannot be measured directly in the presence of cytochromec because it is consumed in reoxidizing reduced cytochromec, its presence can be detected indirectly by the ability of catalase to improve the apparent yield of reduced cytochromec. We found that the relative amounts of extracellular H₂O₂ and O₂ ^– that could be measured in the environment of stimulated neutrophils varied with the stimulus and that catalase protected reduced cytochromec from H₂O₂ oxidation when some stimuli were used but not with others. For example, the ratio of O₂ ^– to H₂O₂ produced by neutrophils exposed to PMA was about 2:1, the expected result if H₂O₂ were derived from O₂ ^–. However when cytochalasin B was added to the cells before the stimulus, the yield of H₂O₂ was reduced but not the yield of O₂ ^–. When cells were allowed to settle and spread on tissue culture plastic they produced equimolar amounts of O₂ ^– and H₂O₂. Coating the plastic with IgG doubled cytochromec reduction without effecting H₂O₂. In contrast, coating with albumin reduced H₂O₂ without effecting cytochromec reduction. Soluble IgG aggregates induced production of mostly O₂ ^– whereas immune complexes resulted in release of both metabolites.FMLP and A23187 were similar to the soluble IgG aggregates in their effects and induced release of proportionately more O₂ ^– than H₂O₂. The addition of catalase to the cytochromec solution improved the yield of reduced cytochromec when PMA or IgG was used to stimulate the cells but not when FMLP was used. These and other data suggest that H₂O₂ release is not a linear function of the amount of O₂ ^– generated and that either a variable fraction of O₂ ^– spontaneously dismutates to H₂O₂ or the neutrophil NADPH oxidase, in a manner analogous to xanthine oxidase, is capable, under some circumstances, of producing H₂O₂ as well as O₂ ^–. If the latter were true, the pathologic consequences of neutrophil activation would vary depending on whether O₂ ^– was the primary product (chemotactic activation) or whether H₂O₂ was released as well (immune complex stimulation).     

过氧化氢对血管平滑肌细胞增殖和明胶酶A及其抑制因子基因表达的影响

目的:探讨过氧化氢(H₂O₂)对体外培养大鼠血管平滑肌细胞(VSMC)增殖和明胶酶A(MMP-2)、及其抑制因子(TIMP-2)基因表达的影响。方法:先用四甲基偶氮唑(MTT)法筛选出H₂O₂对VSMC的毒性作用浓度和增殖作用浓度,在此基础上用逆转录聚合酶链反应(RT-PCR)法测定H₂O₂对MMP-2、TIMP-2mRNA表达的影响。结果:H₂O₂浓度大于300μmol/L后显示出对VSMC的致死毒性作用;H₂O₂浓度在0.01-5.0μmol/L内可以刺激VSMC的增殖,且呈时间依赖性;1μmol/LH₂O₂、10μmol/LH₂O₂对MMP-2基因转录有明显的促进作用;不同浓度的H₂O₂对TIMP-2基因转录无明显促进作用。结论:适当浓度的H₂O₂可以促进VSMC的增殖和MMP-2mRNA表达,但对TIMP-2mRNA表达无明显作用,提示H₂O₂参与了血管重塑的不同病理环节。     

A role for hydrogen peroxide in inflammatory hyperalgesia of the mouse hindpaw

Reactive oxygen species, including hydrogen peroxide (H₂O₂), are released from activated leucocytes and resident cells in the joint during inflammation. We hypothesize that H₂O₂ is an important mediator of inflammatory pain and, thus, the aim of our experiments was to investigate the effect of H₂O₂ on inflammation of the mouse hindpaw. We performed behavioural studies in CD1 mice to determine the effect of H₂O₂ on both thermal and mechanical hyperalgesia and oedema formation 20 minutes after intraplantar injection into the mouse hindpaw. The Hargreaves technique was used to test thermal hyperalgesia and a dynamic plantar aesthesiometer was used for mechanical hyperalgesia studies. Oedema was determined by measuring paw mass. Our experiments showed intraplantar injection of H₂O₂ (2200–8800 nmoles) causes significant thermal and mechanical hyperalgesia of the mouse hindpaw. H₂O₂ also causes significant oedema formation. Thus, we propose that H₂O₂ is a potential target for the treatment of inflammatory pain.     

Cell based metabolic barriers to glucose diffusion: Macrophages and continuous glucose monitoring

It is assumed that MQ are central to glucose sensor bio-fouling and therefore have a major negative impact on continuous glucose monitoring (CGM) performance in vivo. However to our knowledge there is no data in the literature to directly support or refute this assumption. Since glucose and oxygen (O₂) are key to glucose sensor function in vivo, understanding and controlling glucose and O₂ metabolic activity of MQ is likely key to successful glucose sensor performance. We hypothesized that the accumulation of MQ at the glucose sensor-tissue interface will act as “Cell Based Metabolic Barriers” (CBMB) to glucose diffusing from the interstitial tissue compartment to the implanted glucose sensor and as such creating an artificially low sensor output, thereby compromising sensor function and CGM. Our studies demonstrated that 1) direct injections of MQ at in vivo sensor implantation sites dramatically decreased sensor output (measured in nA), 2) addition of MQ to glucose sensors in vitro resulted in a rapid and dramatic fall in sensor output and 3) lymphocytes did not affect sensor function in vitro or in vivo. These data support our hypothesis that MQ can act as metabolic barriers to glucose and O₂ diffusion in vivo and in vitro.     

Local flow velocities in the cat carotid body tissue

With the aid of a new three-dimensional mathematical model local flow velocities in the specific carotid body tissue of the cat measured by hydrogen clearances were calculated to have a mean value of 0.006 cm/s at a perfusion pressure of 50 mm Hg, 0.014 cm/s at a perfusion pressure of 120 mm Hg, and 0.018 cm/s at a perfusion pressure of 170 mm Hg. These results indicate that the carotid body specific tissue possesses a distinct flow heterogeneity with normal capillary flow velocities and a high shunt flow. During hypoxia, the smallest decrease in tissuePO₂ was significantly correlated with the highest decrease in flow velocity. This suggests that the carotid body capillary network itself exhibits aPO₂ sensor mechanisms amplifying the chemoreceptive process in the specific cell elements.     

Effects of hydroxyl radicals on KATP channels in guinea-pig ventricular myocytes

 We studied the effects of oxygen free radicals on the ATP-sensitive potassium channel (K_ATP channel) of guinea-pig ventricular myocytes. Single K_ATP channel currents were recorded from inside-out patches in the presence of symmetrical K⁺ concentrations (140 mM in both bath and pipette solutions). Reaction of xanthine oxidase (0.1 U/ml) on hypoxanthine (0.5 mM) produced superoxide anions (·O₂ ^-) and hydrogen peroxide (H₂O₂). Exposure of the patch membrane to ·O₂ ^- and H₂O₂ increased the opening of K_ATP channels, but this activation was prevented by adding 1 μM glibenclamide to the bath solution. In the presence of ferric iron (Fe³⁺: 0.1 mM), the same procedure produced hydroxyl radicals (·OH) via the iron-catalysed Haber-Weiss reaction. ·OH also activated K_ATP channels; however, this activation could not be prevented by, even very high concentrations of glibenclamide (10 μM). These different effects of glibenclamide suggest that the mode of action of these oxygen free radicals on K_ATP channels is different and that ·OH is more potent than ·O₂ ^-/H₂O₂ in activating K_ATP channels in the heart. Received: 14 July 1998 / Received after revision: 5 September 1998 / Accepted: 14 September 1998     

Effect of insulin on the rate of hydrogen peroxide generation in mitochondria

We studied the effect of insulin on H₂O₂ generation by mitochondria in rat liver and heart. Insulin markedly increased the rate of H₂O₂ generation, which was realized via short-term activation of mitochondrial succinate dehydrogenase. In terms of the Michaelis-Menten equation describing the dependence of H₂O₂ generation by mitochondria on succinate concentration (succinate dehydrogenase substrate), insulin decreased the Michaelis-Menten constant and increased the maximum rate of H₂O₂ generation compared to the control.     

Anti-Oxidant Effects of Pomegranate Juice on Saccharomyces Cerevisiae Cell Growth

Background

Pomegranate juice has a number of positive effects on both human and animal subjects.

Material and methods

Four groups were used in this study. i: Control group, ii: H₂O₂ group, iii: Pomegranate juice (PJ) group and iv: PJ + H₂O₂ group. Following the sterilization method for pomegranate juice (10%) and H₂O₂ (6% v/v), Saccharomyces cerevisiae cultures were added and the cultivation incubated at 35°C for 72 hours. Fatty acids and vitamin concentrations were measured using HPLC and GC and the total protein bands profile were determined by SDS-PAGE.

Results

According to our results statistically significant differences have been determined among the study groups in terms of fatty acids and vitamin (p<0,05). Fatty acid synthesis, vitamin control and cell density increased in groups to which PJ was given in comparison with the control group (p<0,05). Pomegranate juice increased vitamins, fatty acids and total protein expression in Saccharomyces cerevisiae in comparison with the control.

Conclusion

Pomegranate juice has a positive effect on fatty acid, vitamin and protein synthesis by Saccharomyces cerevisiae. Accordingly, we believe that it has significantly decreased oxidative damage thereby making a positive impact on yeast development.     

Opposing role of JNK-p38 kinase and ERK1/2 in hydrogen peroxide-induced oxidative damage of human trophoblast-like JEG-3 cells

Trophoblasts play a crucial role in embryo implantation and maintenance of normal pregnancy. Recently, oxidative stress has been considered as one important factor in the pathogenesis of spontaneous abortion and preeclampsia. Many studies have reported that the plasma levels of hydrogen peroxide (H₂O₂) are significantly increased in women with preeclampsia, but the mechanisms involved in H₂O₂-induced cell cytotoxicity in trophoblasts are still not completely explained. Our present study was undertaken to provide a united understanding of the role of oxidative stress generated by H₂O₂ on human trophoblasts and the underlying intracellular signaling pathways. Exposure to H₂O₂ resulted in a concentration-dependent growth decrease and apoptosis in human trophoblast-like JEG-3 cells. H₂O₂ treatment also caused intracellular reactive oxygen species (ROS) production and concomitant dissipation of the mitochondrial membrane potential. The three MAPK subfamilies, ERK1/2, JNK and p38 kinase, were all activated under H₂O₂-induced oxidative stress. Blocking the activation of JNK and p38 kinase increased cell viability and decreased apoptosis induced by H₂O₂ with their respective inhibitors, SP600125 and SB203580. However, preventing ERK1/2 activation further increased H₂O₂-induced cell death with U0126, an inhibitor of ERK upstream kinase MEK1/2. Taken together, these findings suggest that the mitochondria-dependent pathways and JNK-p38 kinase pathways are involved in H₂O₂-induced oxidative damage of human trophoblast-like JEG-3 cells, while ERK1/2 pathway may play an active role in cell survival following oxidant injury.