Differential vulnerability of immature murine neurons to oxygen-glucose deprivation

In vivo studies support selective neuronal vulnerability to hypoxia-ischemia (HI) in the developing brain. Since differences in intrinsic properties of neurons might be responsible, pure cultures containing immature neurons (6-8 days in vitro) isolated from mouse cortex and hippocampus, regions chosen for their marked vulnerability to oxidative stress, were studied under in vitro ischemic conditions-oxygen-glucose deprivation (OGD). Twenty-four hours of reoxygenation after 2.5 h of OGD induced significantly greater cell death in hippocampal than in cortical neurons (67.8% vs. 33.4%, P = 0.0068). The expression of neuronal nitric oxide synthase (nNOS) protein, production of nitric oxide (NO), and reactive oxygen species (ROS), as well as glutathione peroxidase (GPx) activity and intracellular levels of reduced glutathione (GSH), were measured as indicators of oxidative stress. Hippocampal neurons had markedly higher nNOS expression than cortical neurons by 24 h of reoxygenation, which coincided with an increase in NO production, and significantly greater ROS accumulation. GPx activity declined significantly in hippocampal but not in cortical neurons at 4 and 24 h after OGD. The decrease in GSH level in hippocampal neurons correlated with the decline of GPx activity. Our data suggest that developing hippocampal neurons are more sensitive to OGD than cortical neurons. This finding supports our in vivo studies showing that mouse hippocampus is more vulnerable than cortex after neonatal HI. An imbalance between excess prooxidant production (increased nNOS expression, and NO and ROS production) and insufficient antioxidant defenses created by reduced GPx activity and GSH levels may, in part, explain the higher susceptibility to OGD of immature hippocampal neurons.

Manipulation of antioxidant pathways in neonatal murine brain

To assess the role of brain antioxidant capacity in the pathogenesis of neonatal hypoxic-ischemic brain injury, we measured the activity of glutathione peroxidase (GPX) in both human-superoxide dismutase-1 (hSOD1) and human-GPX1 overexpressing transgenic (Tg) mice after neonatal hypoxia-ischemia (HI). We have previously shown that mice that overexpress the hSOD1 gene are more injured than their wild-type (WT) littermates after HI, and that H(2)O(2) accumulates in HI hSOD1-Tg hippocampus. We hypothesized that lower GPX activity is responsible for the accumulation of H(2)O(2). Therefore, increasing the activity of this enzyme through gene manipulation should be protective. We show that brains of hGPX1-Tg mice, in contrast to those of hSOD-Tg, have less injury after HI than WT littermates: hGPX1-Tg, median injury score = 8 (range, 0-24) versus WT, median injury score = 17 (range, 2-24), p < 0.01. GPX activity in hSOD1-Tg mice, 2 h and 24 h after HI, showed a delayed and bilateral decline in the cortex 24 h after HI (36.0 +/- 1.2 U/mg in naive hSOD1-Tg versus 29.1 +/- 1.7 U/mg in HI cortex and 29.2 +/- 2.0 for hypoxic cortex, p < 0.006). On the other hand, GPX activity in hGPX1-Tg after HI showed a significant increase by 24 h in the cortex ipsilateral to the injury (48.5 +/- 5.2 U/mg, compared with 37.2 +/- 1.5 U/mg in naive hGPX1-Tg cortex, p < 0.008). These findings support the hypothesis that the immature brain has limited GPX activity and is more susceptible to oxidative damage and may explain the paradoxical effect seen in ischemic neonatal brain when SOD1 is overexpressed.

Gamma-tocopherol supplementation inhibits protein nitration and ascorbate oxidation in rats with inflammation

Gamma-tocopherol (gammaT) complements alpha-tocopherol (alphaT) by trapping reactive nitrogen oxides to form a stable adduct, 5-nitro-gammaT [Christen et al., PNAS 94:3217-3222; 1997]. This observation led to the current investigation in which we studied the effects of gammaT supplementation on plasma and tissue vitamin C, vitamin E, and protein nitration before and after zymosan-induced acute peritonitis. Male Fischer 344 rats were fed for 4 weeks with either a normal chow diet with basal 32 mg alphaT/kg, or the same diet supplemented with approximately 90 mg d-gammaT/kg. Supplementation resulted in significantly higher levels of gammaT in plasma, liver, and kidney of control animals without affecting alphaT, total alphaT+gammaT or vitamin C. Intraperitoneal injection of zymosan caused a marked increase in 3-nitrotyrosine and a profound decline in vitamin C in all tissues examined. Supplementation with gammaT significantly inhibited protein nitration and ascorbate oxidation in the kidney, as indicated by the 29% and 56% reduction of kidney 3-nitrotyrosine and dehydroascorbate, respectively. Supplementation significantly attenuated inflammation-induced loss of vitamin C in the plasma (38%) and kidney (20%). Zymosan-treated animals had significantly higher plasma and tissue gammaT than nontreated pair-fed controls, and the elevation of gammaT was strongly accentuated by the supplementation. In contrast, alphaT did not significantly change in response to zymosan treatment. In untreated control animals, gammaT supplementation lowered basal levels of 3-nitrotyrosine in the kidney and buffered the starvation-induced changes in vitamin C in all tissues examined. Our study provides the first in vivo evidence that in rats with high basal amounts of alphaT, a moderate gammaT supplementation attenuates inflammation-mediated damage, and spares vitamin C during starvation-induced stress without affecting alphaT.

Analysis of plasma tocopherols alpha, gamma, and 5-nitro-gamma in rats with inflammation by HPLC coulometric detection

Reactive nitrogen oxide species (RNOS) have been implicated as effector molecules in inflammatory diseases. There is emerging evidence that gamma-tocopherol (gammaT), the major form of vitamin E in the North American diet, may play an important role in these diseases. GammaT scavenges RNOS such as peroxynitrite by forming a stable adduct, 5-nitro-gammaT (NGT). Here we describe a convenient HPLC method for the simultaneous determination of NGT, alphaT, and gammaT in blood plasma and other tissues. Coulometric detection of NGT separated on a deactivated reversed-phase column was linear over a wide range of concentrations and highly sensitive (approximately 10 fmol detection limit). NGT extracted from blood plasma of 15-week-old Fischer 344 rats was in the low nM range, representing approximately 4% of gammaT. Twenty-four h after intraperitoneal injection of zymosan, plasma NGT levels were 2-fold higher compared to fasted control animals when adjusted to gammaT or corrected for total neutral lipids, while alpha- and gammaT levels remained unchanged. These results demonstrate that nitration of gammaT is increased under inflammatory conditions and highlight the importance of RNOS reactions in the lipid phase. The present HPLC method should be helpful in clarifying the precise physiological role of gammaT.

gamma-tocopherol, the major form of vitamin E in the US diet, deserves more attention

gamma-tocopherol is the major form of vitamin E in many plant seeds and in the US diet, but has drawn little attention compared with alpha-tocopherol, the predominant form of vitamin E in tissues and the primary form in supplements. However, recent studies indicate that gamma-tocopherol may be important to human health and that it possesses unique features that distinguish it from alpha-tocopherol. gamma-Tocopherol appears to be a more effective trap for lipophilic electrophiles than is alpha-tocopherol. gamma-Tocopherol is well absorbed and accumulates to a significant degree in some human tissues; it is metabolized, however, largely to 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), which is mainly excreted in the urine. gamma-CEHC, but not the corresponding metabolite derived from alpha-tocopherol, has natriuretic activity that may be of physiologic importance. Both gamma-tocopherol and gamma-CEHC, but not alpha-tocopherol, inhibit cyclooxygenase activity and, thus, possess antiinflammatory properties. Some human and animal studies indicate that plasma concentrations of gamma-tocopherol are inversely associated with the incidence of cardiovascular disease and prostate cancer. These distinguishing features of gamma-tocopherol and its metabolite suggest that gamma-tocopherol may contribute significantly to human health in ways not recognized previously. This possibility should be further evaluated, especially considering that high doses of alpha-tocopherol deplete plasma and tissue gamma-tocopherol, in contrast with supplementation with gamma-tocopherol, which increases both. We review current information on the bioavailability, metabolism, chemistry, and nonantioxidant activities of gamma-tocopherol and epidemiologic data concerning the relation between gamma-tocopherol and cardiovascular disease and cancer.

Vascular dysfunction and reduced circulating endothelial progenitor cells in young healthy UK South Asian men

OBJECTIVES: The objective of this study was to examine determinants of excess coronary artery disease risk in UK South Asians, more prevalent in this population than UK Caucasians, by examining differences in risk factors, vascular function, and endothelial progenitor cells (EPCs). METHODS AND RESULTS: 24 South Asian and 25 Caucasian healthy age-matched nonsmoking men were studied. Vascular function was assessed by flow-mediated and GTN brachial artery dilatation and blood flow responses to infusion of ACh, SNP, and L-NMMA. EPC number and function were measured by flow cytometry (CD34, CD133, and KDR positive cells), and CFU/migration assays. Traditional risk factors and anthropometric measurements were similar in the groups. South Asians had higher fasting insulin levels (6.01 versus 3.62 microU/mL; P = 0.02). South Asians had lower FMD (6.9 versus 8.5%; P = 0.003), L-NMMA response (0.8 versus 1.3 mL/min/100 mL; P = 0.03), mean SNP response (9.5+/-0.6 versus 11.6+/-0.6; P = 0.02), EPC number (0.046+/-0.005% versus 0.085+/-0.009%; P = < 0.001), and CFU ability (CFU 4.29+/-1.57 versus 18.86+/-4.00; P = 0.005). EPC number was the strongest predictor of FMD. Ethnicity was the strongest predictor of EPC number. CONCLUSIONS: Healthy South Asian men are more insulin resistant, and demonstrate endothelial dysfunction and reduced EPC number and function compared with Caucasians. These abnormalities may contribute to their increased CAD risk.

Recurrent rearrangements of chromosome 1q21.1 and variable pediatric phenotypes

BACKGROUND: Duplications and deletions in the human genome can cause disease or predispose persons to disease. Advances in technologies to detect these changes allow for the routine identification of submicroscopic imbalances in large numbers of patients. METHODS: We tested for the presence of microdeletions and microduplications at a specific region of chromosome 1q21.1 in two groups of patients with unexplained mental retardation, autism, or congenital anomalies and in unaffected persons. RESULTS: We identified 25 persons with a recurrent 1.35-Mb deletion within 1q21.1 from screening 5218 patients. The microdeletions had arisen de novo in eight patients, were inherited from a mildly affected parent in three patients, were inherited from an apparently unaffected parent in six patients, and were of unknown inheritance in eight patients. The deletion was absent in a series of 4737 control persons (P=1.1x10(-7)). We found considerable variability in the level of phenotypic expression of the microdeletion; phenotypes included mild-to-moderate mental retardation, microcephaly, cardiac abnormalities, and cataracts. The reciprocal duplication was enriched in nine children with mental retardation or autism spectrum disorder and other variable features (P=0.02). We identified three deletions and three duplications of the 1q21.1 region in an independent sample of 788 patients with mental retardation and congenital anomalies. CONCLUSIONS: We have identified recurrent molecular lesions that elude syndromic classification and whose disease manifestations must be considered in a broader context of development as opposed to being assigned to a specific disease. Clinical diagnosis in patients with these lesions may be most readily achieved on the basis of genotype rather than phenotype.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k

To study the role of the epithelial calcium channel transient receptor potential vanilloid type 6 (TRPV6) and the calcium-binding protein calbindin-D9k in intestinal calcium absorption, TRPV6 knockout (KO), calbindin-D9k KO, and TRPV6/calbindin-D(9k) double-KO (DKO) mice were generated. TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice have serum calcium levels similar to those of wild-type (WT) mice ( approximately 10 mg Ca2+/dl). In the TRPV6 KO and the DKO mice, however, there is a 1.8-fold increase in serum PTH levels (P < 0.05 compared with WT). Active intestinal calcium transport was measured using the everted gut sac method. Under low dietary calcium conditions there was a 4.1-, 2.9-, and 3.9-fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D9k KO mice, respectively (n = 8-22 per group; P > 0.1, WT vs. calbindin-D9k KO, and P < 0.05, WT vs. TRPV6 KO on the low-calcium diet). Duodenal calcium transport was increased 2.1-fold in the TRPV6/calbindin-D9k DKO mice fed the low-calcium diet (P < 0.05, WT vs. DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or KO mice. 1,25-Dihydroxyvitamin D3 administration to vitamin D-deficient null mutant and WT mice also resulted in a significant increase in duodenal calcium transport (1.4- to 2.0-fold, P < 0.05 compared with vitamin D-deficient mice). This study provides evidence for the first time using null mutant mice that significant active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D9k, thus challenging the dogma that TRPV6 and calbindin-D9k are essential for vitamin D-induced active intestinal calcium transport.

Effects of EDP-420 on penicillin-resistant and quinolone- and penicillin-resistant pneumococci in the rabbit meningitis model

OBJECTIVES: To test the efficacy of EDP-420, a new ketolide, in experimental pneumococcal meningitis and to determine its penetration into the CSF. METHODS: The experimental rabbit model was used in this study and EDP-420 was tested against a penicillin-resistant and a penicillin- and quinolone-resistant mutant. EDP-420 was also tested against both strains in time-killing assays over 8 h in vitro. RESULTS: In experimental meningitis, EDP-420 produced a bactericidal activity comparable to the standard regimen based on a combination of vancomycin with ceftriaxone against a penicillin-resistant Streptococcus pneumoniae and a penicillin- and quinolone-resistant S. pneumoniae isolate. The penetration of EDP-420 into inflamed meninges was 38% after an i.v. injection of 10 mg/kg. The bactericidal activity of EDP-420 was also confirmed in in vitro time-killing assays. CONCLUSIONS: EDP-420 is an efficacious alternative treatment in pneumococcal meningitis, especially when resistant strains are suspected.

Dynamics of settling charged particles in turbulence : theory and experiments

It has been proposed that inertial clustering may lead to an increased collision rate of water droplets in clouds. Atmospheric clouds and electrosprays contain electrically charged particles embedded in turbulent flows, often under the influence of an externally imposed, approximately uniform gravitational or electric force. In this thesis, we present the investigation of charged inertial particles embedded in turbulence. We have developed a theoretical description for the dynamics of such systems of charged, sedimenting particles in turbulence, allowing radial distribution functions to be predicted for both monodisperse and bidisperse particle size distributions. The governing parameters are the particle Stokes number (particle inertial time scale relative to turbulence dissipation time scale), the Coulomb-turbulence parameter (ratio of Coulomb ’terminalar speed to turbulence dissipation velocity scale), and the settling parameter (the ratio of the gravitational terminal speed to turbulence dissipation velocity scale). For the monodispersion particles, The peak in the radial distribution function is well predicted by the balance between the particle terminal velocity under Coulomb repulsion and a time-averaged ’drift’ velocity obtained from the nonuniform sampling of fluid strain and rotation due to finite particle inertia. The theory is compared to measured radial distribution functions for water particles in homogeneous, isotropic air turbulence. The radial distribution functions are obtained from particle positions measured in three dimensions using digital holography. The measurements support the general theoretical expression, consisting of a power law increase in particle clustering due to particle response to dissipative turbulent eddies, modulated by an exponential electrostatic interaction term. Both terms are modified as a result of the gravitational diffusion-like term, and the role of ’gravity’ is explored by imposing a macroscopic uniform electric field to create an enhanced, effective gravity. The relation between the radial distribution functions and inward mean radial relative velocity is established for charged particles.

Performance of a new, 3D-monitor based random-dot stereotest for children under 4 years of age

BACKGROUND: The aim of this study was to determine the performance of a new, 3D-monitor based, objective stereotest in children under the age of four. METHODS: Random-dot circles (diameter 10 cm, crossed, disparity of 0.34 degrees) randomly changing their position were presented on an 3D-monitor while eye movements were monitored by infrared photo-oculography. If > or = 3 consecutive stimuli were seen, a positive response was assumed. One hundred thirty-four normal children aged 2 months to 4 years (average 17+/-15.3 months) were examined. RESULTS: Below the age of 12 months, we were not able to obtain a response to the 3D stimulus. For older children the following rates of positive responses were found: 12-18 months 25%, 18-24 months 10%, 24-30 months 16%, 30-36 months 57%, 36-42 months 100%, and 42-48 months 91%. Multiple linear logistic regression showed a significant influence on stimulus recognition of the explanatory variables age (p<0.00001) and child cooperation (p<0.001), but not of gender (p>0.1). CONCLUSIONS: This 3D-monitor based stereotest allows an objective measurement of random-dot stereopsis in younger children. It might open new ways to screen children for visual abnormalities and to study the development of stereovision. However, the current experimental setting does not allow determining random-dot stereopsis in children younger than 12 months.