Free cholesterol accumulation impairs antioxidant activities and aggravates apoptotic cell death in menadione-induced oxidative injury.

Although the relationship between hypercholesterolemia and oxidative stress has been extensively investigated, direct evidence regarding to the roles of cholesterol accumulation in the generations of reactive oxygen species (ROS) and apoptotic cell death under oxidative stress is lack. In this study, we investigated productions of superoxide anions (O(2)(-)) and nitric oxide (NO), and apoptotic cell death in wild type Chinese hamster ovary (CHO) cells and cholesterol accumulated CHO cells genetically and chemically. Oxidative stress was induced by menadione challenge. The results revealed that abundance of free cholesterol (FC) promoted menadione-induced O(2)(-) and NO productions. FC accumulation down-regulated eNOS expression but up-regulated NADPH oxidases, and inhibited the activities of superoxide dismutase (SOD) and catalase. Treatment of menadione increased the expressions of iNOS and qp91 phox, enhanced the activities of SOD and catalase in the wild-type CHO cells but inhibited the activity of glutathione peroxidase in the cholesterol accumulated CHO cells. Moreover, FC abundance promoted apoptotic cell death in these cells. Taken together, those results suggest that free cholesterol accumulation aggravates menadione-induced oxidative stress and exacerbates apoptotic cell death.

Abnormal spatiotemporal processing of emotional facial expressions in childhood autism: dipole source analysis of event-related potentials.

Previous studies of face processing in autism suggest abnormalities in anatomical development, functioning and connectivity/coordination of distributed brain systems involved in social cognition, but the spatial sequence and time course of rapid (sub-second) neural responses to emotional facial expressions have not been examined in detail. Source analysis of high-density event-related potentials (ERPs) is an optimal means to examine both the precise temporal profile and spatial location of early electrical brain activity in response to emotionally salient stimuli. Therefore, we recorded 128-channel ERPs from high-functioning males with autism (aged 6-10 years), and age-, sex- and IQ-matched typically developing controls during explicit and implicit processing of emotion from pictures showing happy, angry, fearful, sad and neutral facial expressions. Children with autism showed normal patterns of behavioural and ERP (P1, N170 and P2) responses. However, dipole source analysis revealed that ERP responses relating to face detection (visual cortex) and configural processing of faces (fusiform gyrus), as well as mental state decoding (medial prefrontal lobe), were significantly weaker and/or slower in autism compared with controls during both explicit and implicit emotion-processing tasks. Slower- and larger-amplitude ERP source activity in the parietal somatosensory cortices possibly reflected more effortful compensatory analytical strategies used by the autism group to process facial gender and emotion. Such aberrant neurophysiological processing of facial emotion observed in children with autism within the first 300 ms of stimulus presentation suggests abnormal cortical specialization within social brain networks, which would likely disrupt the development of normal social-cognitive skills.

Cytoglobin overexpression protects against damage-induced fibrosis.

Cytoglobin (Cygb), a member of the hexacoordinate globin superfamily (hxHb), is expressed in fibroblasts from a broad range of tissues. The physiological functions of hxHb are still unclear, but biochemical studies reveal that they can scavenge toxic species, such as nitric oxide, peroxynitrite, and hydrogen peroxide. We demonstrate that the overexpression of Cygb in rat hepatic stellate cells, both in vitro and in vivo, protects against oxidative stress, inhibiting their differentiation into a myofibroblast-like phenotype. Accordingly, the overexpression of Cygb reduces extracellular matrix deposition in both toxic and cholestatic models of liver injury. The overexpression of Cygb also promotes recovery from previously initiated damage-induced fibrogenesis. By inhibiting free radical-induced activation of hepatic stellate cells, Cygb plays an important role in controlling tissue fibrosis. Therefore, the normal upregulation of Cygb during tissue injury has a homeostatic effect, inhibiting free radical-induced fibroblast activation and tissue fibrosis.

Nitric oxide down-regulates caveolin-1 expression in rat brains during focal cerebral ischemia and reperfusion injury.

As a signalling molecule of the integral membrane protein family, caveolin participates in cellular signal transduction via interaction with other signalling molecules. The nature of interaction between nitric oxide (NO) and caveolin in the brain, however, remains largely unknown. In this study we investigated the role(s) of NO in regulating caveolin-1 expression in rat ischemic brains with middle cerebral artery occlusion (MCAO). Exposure to 1 h ischemia induced the increases in neuronal nitric oxide synthase (nNOS) and NO concentration with concurrent down-regulation of caveolin-1 expression in the ischemic core of rat brains. Subsequent 24 h or more reperfusion time led to an increase in inducible NOS (iNOS) expression and NO production, as well as a decline of caveolin-1 protein at the core and penumbra of the ischemic brain. Afterwards, NOS inhibitors and an NO donor were utilized to clarify the link between NO production and caveolin-1 expression in the rats with 1 h ischemia plus 24 h reperfusion. N(G)-nitro-l-arginine methyl ester (L-NAME, a non-selective NOS inhibitor), N(6)-(1-iminoethyl)-lysine (NIL, an iNOS inhibitor), and 7-nitroindazole (7-NI, a nNOS inhibitor) prevented the loss of caveolin-1 in the core and penumbra of the ischemic brain, whereas l-N(5)-(1-iminoethyl)-ornithine (L-NIO, an endothelial NOS inhibitor) showed less effect than the other NOS inhibitors. S-Nitroso-N-acetylpenicillamine (SNAP, a NO donor) down-regulated the expression of caveolin-1 protein in normal and ischemic brains. These results, when taken together, suggest that NO modulates the expression of caveolin-1 in the brain and that the loss of caveolin-1 is associated with NO production in the ischemic brain.

Novel sparse component analysis approach to free radical EPR spectra decomposition.

Free radicals play important roles in many physiological and pathological pathways in biological systems. These free radicals can be detected and quantified by their EPR spectra. The measured EPR spectra are often mixtures of pure spectra of several different free radicals and other chemicals. Blind source separation can be applied to estimate the pure spectra of interested free radicals. However, since the pure EPR spectra are often not independent of each other, the approach based on independent component analysis (ICA) cannot accurately extract the required spectra. In this paper, a novel sparse component analysis method for blind source separation, which exploits the sparsity of the EPR spectra, is presented to reliably extract the pure source spectra from their mixtures with high accuracy. This method has been applied to the analysis of EPR spectra of superoxide, hydroxyl, and nitric oxide free radicals, for both simulated data and real world ex vivo experiment. Compared to the traditional self-modeling method and our previous ICA-based blind source separation method, the proposed sparse component analysis approach gives much better results and can give perfect separation for mixtures of superoxide spectrum and hydroxyl spectrum in the ideal noise-free case. This method can also be used in other similar applications of quantitative spectroscopy analysis.

Altered expression of aquaporin-2 in human explants with chronic renal allograft dysfunction.

OBJECTIVE: To investigate the distribution of aquaporins, a recently discovered family of transmembrane water channels, in human renal explants, with specific reference to chronic renal allograft dysfunction (CRAD). MATERIALS AND METHODS: Immunohistochemistry for aquaporin-1 and -2 was used in 11 explants, of which five had clinically and histologically confirmed CRAD. Controls were taken from the six explants unaffected by CRAD and from histologically normal areas of six kidneys excised for renal tumours. RESULTS: In the renal tumour control group, aquaporin-1 immunoreactivity was detected in the glomerular endothelium, Bowman's capsule, the proximal convoluted tubules and the thin limb of the loop of Henle, whereas immunoreactivity for aquaporin-2 was detected in the collecting ducts only. Of the explants without CRAD, where architecture was preserved, immunoreactivity for aquaporin-1 and -2 was the same as in the renal tumour controls. In the two explants with no CRAD and loss of collecting ducts, there was no aquaporin-2 immunoreactivity. In five explants with CRAD, immunoreactivity for aquaporin-2 was decreased or absent from the medulla to the cortex. The apparent decreased immunoreactivity of aquaporin-1 in this group was secondary to a decrease in the number of viable proximal tubules. CONCLUSION: There was less aquaporin-2 immunoreactivity in human renal explants diagnosed with CRAD, starting from the medullary region. In explants with no CRAD and viable collecting ducts, or in normal controls, aquaporin-2 immunoreactivity remained unchanged. Aquaporins might be useful as markers for CRAD.

Green tea polyphenols prevent toxin-induced hepatotoxicity in mice by down-regulating inducible nitric oxide-derived prooxidants.

BACKGROUND: Recently, considerable attention has been focused on dietary and medicinal phytochemicals that inhibit, reverse, or retard diseases caused by oxidative and inflammatory processes. Green tea polyphenols have both antioxidant and antiinflammatory properties. OBJECTIVE: We examined the effects of green tea polyphenols in carbon tetrachloride-treated mice, a model of liver injury in which oxidant stress and cytokine production are intimately linked. We tested the effect of a pure form of epigallocatechin gallate (EGCG), the major polyphenol in green tea, in mice treated with carbon tetrachloride. DESIGN: Eight-week-old ICR mice were administered 20 microL/CCl(4) kg dissolved in olive oil. Two different doses of EGCG, 50 and 75 mg/kg, were tested. Control mice were treated with saline and olive oil. We analyzed liver histopathology, lipid peroxidation, and messenger RNA and protein concentrations of inducible nitric oxide synthase. Additionally, nitric oxide-generated radicals were assessed by electron paramagnetic resonance spectroscopy, and protein concentrations were measured by immunohistochemistry and Western blot analysis. RESULTS: Carbon tetrachloride administration caused an intense degree of liver necrosis associated with increases in lipid peroxidation, inducible nitric oxide synthase messenger RNA and protein, nitrotyrosine, and nitric oxide radicals. EGCG administration led to a dose-dependent decrease in all of the histologic and biochemical variables of liver injury observed in the carbon tetrachloride-treated mice. CONCLUSIONS: Green tea polyphenols reduce the severity of liver injury in association with lower concentrations of lipid peroxidation and proinflammatory nitric oxide-generated mediators. Green tea polyphenols can be a useful supplement in the treatment of liver disease and should be considered for liver conditions in which proinflammatory and oxidant stress responses are dominant.

COX-2-deficient mice are less prone to MPTP-neurotoxicity than wild-type mice.

The primary lesion in Parkinson's disease is the death of dopaminergic neurons in the substantia nigra. The role of cyclooxygenase (COX)-2 in the etiology of Parkinson's disease was explored using COX-2 gene knockout mice. Mortality after injection of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP, a chemical known to cause parkinsonism in humans) in heterozygous COX-2-deficient mice was lower than that in wild-type mice. The number of tyrosine hydroxylase immunoreactive neurons in the substantia nigra pars compacta of MPTP-treated wild-type mice declined to a greater extent than in heterozygous mice. Inhibition of COX-2 protein expression decreased the lesion caused by MPTP and protected the dopaminergic neurons in substantia nigra pars compacta. This result suggested that inhibition of COX-2 has potential therapeutic implications.

Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism.

The effect of the cholesterol content of the plasma membrane on the intracellular concentration of oxygen in Chinese hamster ovary (CHO) cells and their mutants was investigated by EPR oximetry. Total and free cholesterol content was significantly higher in 25 RA CHO cells as compared to wild-type and M 19 CHO cells, with most of the free cholesterol in normal and mutant CHO cells located in the plasma membrane. The plasma membrane cholesterol content also was altered by various biochemical means, and the effect on the oxygen gradient was studied. Comparing the three cell lines, the gradient was larger with increased content of cholesterol in the plasma cell membrane. This result also is supported by an additional increase in the oxygen gradients with the incorporation of additional cholesterol in the plasma membrane and a decrease in the oxygen gradient when the cholesterol was depleted from the plasma membrane. The results indicate that the concentration of cholesterol in the plasma membrane can be an important factor for the magnitude of the oxygen gradient observed across the cell membrane.

Nitric oxide and oxygen radicals induced apoptosis via bcl-2 and p53 pathway in hypoxia-reoxygenated cardiomyocytes.

Neonatal rat cardiomyocytes were subjected to 24 h of hypoxia 95%N2/5%CO2 and 24 h of hypoxia plus 4 h of reoxygenation 95%O2/5%CO2. 24 h of hypoxia increased the levels of NO, NO2-/NO3-, TBARS and LDH. 24 h of hypoxia plus 4 h of reoxygenation decreased the levels of NO, NO2-/NO3-, but further increased TBARS and LDH. The hypoxia up-regulated the expression of bcl-2, p53 and p21/waf1/cip1 but the reoxygenation down-regulated the expression of bcl-2, and further up-regulated p53 and p21/waf1/cip1. The hypoxia increased cell apoptosis and reoxygenation further increased both apoptotic and necrotic cell death. NO, NO2-/NO3- TBARS, DNA fragmentation and cell apoptosis were enhanced by SNP and inhibited by L-NAME respectively. In addition, SOD/catalase down-regulated the expression of p53, p21/wafl/cipl and TBARS but up-regulated bcl-2 and increased indirectly the level of NO, NO2-/NO3-, and inhibited DNA fragmentation. The results suggest that hypoxia-induced cell death is associated with the activation of NO, bcl-2 and p53 pathway, while hypoxia-reoxygenation induced cell death via the generation of reactive oxygen species and activation of p53 pathway. The present study clarified that NO may be an initiative signal to apoptotic cell death and the activation of bcl-2, p53 and p21/waf1/cip1 pathway in hypoxic and hypoxia-reoxygenated cardiomyocytes.

Revisiting ischemia and reperfusion injury as a possible cause of necrotizing enterocolitis: Role of nitric oxide and superoxide dismutase.

BACKGROUND/PURPOSE: The pathogenesis of necrotizing enterocolitis (NEC) is unknown. Ischemia and reperfusion (I/R) injury has been considered a major contributing factor. Nitric oxide (NO) and superoxide dismutases (SODs) have been shown to protect bowel from I/R injury. This study aims to assess (1) the ability of premature intestine to resist I/R injury compared with mature intestine and (2) the possible role of NO and SODs in modulating such response. METHODS: Intestines from 5 groups of rats (n = 6 for each study group) were studied: (1) premature, gestational age 20 days; (2) premature, gestational age 22 days; (3) full-term, newborn; (4) infant, day 15; (5) infant, day 30. EXPERIMENTS: (1) The degrees of I/R injury after 0, 30, 60, 90 and 120 minutes, respectively, of ischemia and 25 minutes of I/R were assessed histologically by a pathologist who was unaware of the operative details. (2) Tissue NO and copper levels were measured by electroparamagnetic resonance (EPR) study; and nitric oxide synthases, copper zinc (CuZn) SODs and manganese (Mn) SODs were examined immunohistochemically. (3) and (4) I/R injury was assessed in rats that had received intraperitoneal injections of L-arginine (NO donor) and L-NAME (NO antagonist), respectively. RESULTS: For premature (1,2), newborn (3) and mature (4,5) intestines, grades of injury at maximum I/R period studied (120 minutes of ischemia, 25 minutes of reperfusion) were 0, 0, and 3, respectively (P <.05); NO levels were 1 u +/- 1.5, 3 +/- 2.5, and 22 u +/- 3.5, respectively (P <.05); Cu levels were 150 u +/- 15, 200 u +/- 41 and 12 u +/- 2, respectively (P <.05); NOS in intestines were +, +, +++ and CuZnSODs were ++, +++, +, respectively; and MnSODs were +++, ++, -, respectively. No change in NO levels was detected in groups (1), (2), or (3) after L-arginine and L-NAME injections. CONCLUSIONS: Premature rat intestine is highly resistant to I/R injury, which may indicate that I/R alone, in the absence of other predisposing factors (eg, bacterial colonization) may not be sufficient in causing NEC. Nitric oxide does not have a protective role for immature and newborn intestines in I/R as in mature intestine. The high level of SODs of the immature and newborn intestine may play an important role in its high resistance to I/R injury.

Isopestacin, an isobenzofuranone from Pestalotiopsis microspora, possessing antifungal and antioxidant activities.

Isopestacin is an isobenzofuranone obtained from the endophytic fungus Pestalotiopsis microspora. While a few other isobenzofuranones are known from natural sources, isopestacin is the only one having a substituted benzene ring attached at the C-3 position of the furanone ring. The compound was isolated from culture broths of the fungus and crystallized and its structure was determined by X-ray crystallography. Both proton and carbon NMR spectral assignments are also reported for isopestacin. This compound possesses antifungal activity and, as measured by electron spin resonance specroscopy, it also behaves as an antioxidant scavenging both superoxide and hydroxy free radicals.

Neuropeptide Y-Y1 receptor modulates nitric oxide level during stroke in the rat.

In a rat endovascular middle cerebral artery occlusion (MCAO) stroke model, we previously showed that intracerebroventricular (ICV) injection of neuropeptide Y (NPY) or an Y1 receptor agonist, [Leu(31),Pro(34)]-NPY, increased the infarct volume, that an Y1 receptor antagonist, BIBP3226, reduced the infarct volume, and that an Y2 receptor agonist, NPY3-36, had no effect. In this study, we used electron paramagnetic resonance (EPR) spectroscopy to measure nitric oxide (NO) and examined how ICV administration of NPY or its receptor analogs would modulate the brain NO level between the bregma levels +2 and -4 mm during MCAO, since excessive NO mediates ischemic damage. The relative brain NO concentration was increased to 131.94 +/- 7.99% (mean +/- SEM; n = 8) at 15 min of MCAO. NPY treatment further increased the relative brain NO concentration to 250.94 +/- 50.48% (n = 8), whereas BIBP3226 significantly reduced the brain NO concentration to 69.63 +/- 8.84% (n = 8). [Leu(31),Pro(34)]-NPY (137.61 +/- 14.54%; n = 7) or NPY3-36 (129.23 +/- 21.77%; n = 8) did not affect the brain NO concentration at 15 min of MCAO. Our results suggest that the NPY-Y1 receptor activation mediates ischemic injury via NO overproduction and that inhibition of the Y1 receptor may confer protection via suppression of excessive NO production during ischemia.

Augmentation of hypoxia-induced nitric oxide generation in the rat carotid body adapted to chronic hypoxia: an involvement of constitutive and inducible nitric oxide synthases.

Acute hypoxia increases the endogenous release of nitric oxide (NO) in rat carotid body and the expression of nitric oxide synthases is modulated by chronic hypoxia. The aim of the study was to examine hypoxia-induced NO generation in rat carotid body adapted to chronic hypoxia with inspired oxygen at 10% for 4 weeks. The concentration of NO was measured electrochemically with a Pt/Nafion/Pd-IrOx/POAP modified electrode inserted into the isolated carotid body superfused with bicarbonate-buffer saline at 35 degrees C. Acute hypoxia increased the concentration of NO by 471.3+/-71.4 nM in the carotid body of chronically hypoxic (CH) rats. The amount of NO release induced by hypoxia was significantly augmented when compared with that of the normoxic control (87.6+/-15.9 nM). The hypoxia-induced NO generation was markedly attenuated by pretreatment with L- NG-nitroarginine methylester (L-NAME; 500 microM), a non-selective nitric oxide synthase (NOS) inhibitor and also by removal of extracellular calcium with the calcium chelator EGTA (5 mM). Additionally, NO generation during hypoxia was reduced by 30% in the CH carotid body treated with S-methylisothiourea (SMT; 50 microM), a specific blocker of inducible NOS (iNOS). Immunohistochemical study revealed that positive iNOS protein immunoreactivity was detected in clusters of glomus cells in the carotid bodies of CH rats, but not in the normoxic group. Thus, chronic hypoxia enhances hypoxia-induced NO generation mediated by calcium-dependent NOSs and iNOS in the carotid body. Extracellular recording of sinus nerve activity of CH carotid bodies showed that L-NAME treatment enhanced the afferent discharge in response to hypoxia, confirming that the generation of NO suppresses the activities of carotid chemoreceptors. Taken together, our results suggest that hypoxia-induced NO production increases in the rat carotid body adapted to chronic hypoxia and that constitutive and inducible NOSs are involved in the NO generation. The enhancement of NO generation may play a physiological role in blunting the hypoxic chemosensitivity during chronic hypoxia.

Physical effects of reuse and repeated ethylene oxide sterilization on transscleral cyclophotocoagulation laser G-probes.

PURPOSE: The authors documented the physical effects of reuse and repeated ethylene oxide sterilization on transscleral cyclophotocoagulation laser G-probes. METHODS: Transscleral cyclophotocoagulation was performed using G-probes on fresh porcine eyes. Each of two G-probes was used for four transscleral cyclophotocoagulation procedures, with three cycles of ethylene oxide sterilization in between. The power output from the G-probes was measured by a laser output meter before and after each transscleral cyclophotocoagulation procedure. The G-probes were also examined under a slit lamp for signs of physical damage. RESULTS: Repeated use of the G-probe in transscleral cyclophotocoagulation, with ethylene oxide sterilization in between, resulted in an average decrease of 3% in laser energy delivered per repeated cycle of use up to the fourth cycle. No signs of physical damage were found. CONCLUSIONS: Laser G-probes remain functional after repeated use and ethylene oxide resterilization for up to four cycles. No visible physical damage to the probes was identified. It is safe and cost-effective to reuse G-probe for transscleral cyclophotocoagulation with ethylene oxide sterilization, provided the surgeon stays alert for signs of probe damage. This alertness should be retained regardless of whether new or old G-probes are used.