Replacement of histidine in position 105 in the alpha(5) subunit by cysteine stimulates zolpidem sensitivity of alpha(5)beta(2)gamma(2) GABA(A) receptors

Zolpidem is a positive allosteric modulator of GABA(A) receptors with sensitivity to subunit composition. While it acts with high affinity and efficacy at GABA(A) receptors containing the alpha(1) subunit, it has a lower affinity to GABA(A) receptors containing alpha(2), alpha(3), or alpha(5) subunits and has a very weak efficacy at receptors containing the alpha(5) subunit. Here, we show that replacing histidine in position 105 in the alpha(5) subunit by cysteine strongly stimulates the effect of zolpidem in receptors containing the alpha(5) subunit. The side chain volume of the amino acid residue in this position does not correlate with the modulation by zolpidem. Interestingly, serine is not able to promote the potentiation by zolpidem. The homologous residues to alpha(5)H105 in alpha(1), alpha(2), and alpha(3) are well-known determinants of the action of classical benzodiazepines. Other studies have shown that replacement of these histidines alpha(1)H101, alpha(2)H101, and alpha(3)H126 by arginine, as naturally present in alpha(4) and alpha(6), leads to benzodiazepine insensitivity of these receptors. Thus, the nature of the amino acid residue in this position is not only crucial for the action of classical benzodiazepines but in alpha(5) containing receptors also for the action of zolpidem.

Increased lipopolysaccharide-induced tumour necrosis factor-alpha, interferon-gamma and interleukin-10 production in atopic dermatitis

BACKGROUND: Atopic dermatitis (AD) is based on a genetic predisposition, but environmental factors may trigger skin inflammation. According to the hygiene hypothesis, decreased exposure to microbial products in early childhood does not allow sufficient maturation of the immune system that is associated with an increased risk of atopic sensitization. OBJECTIVES: The effect of lipopolysaccharide (LPS) on the cytokine production of peripheral blood mononuclear cells (PBMC) of AD patients and nonatopic controls was studied. PATIENTS AND METHODS: PBMC were isolated from heparinized blood of 10 patients with AD and 10 nonatopic individuals, suspended in culture medium and stimulated with LPS. Cytokine levels in the supernatants were measured by immunoassays. Results Upon stimulation with LPS, PBMC from AD patients produced significantly higher amounts of tumour necrosis factor-alpha, interferon-gamma and interleukin (IL)-10 compared with control PBMC. LPS stimulation blocked the increased spontaneous production of IL-4 and IL-5 by PBMC from AD patients, but had no effect on IL-13 production. CONCLUSIONS: These results demonstrate that the effects of LPS stimulation depend on both the type of cytokine and the origin of PBMC. Endotoxin exposure is suggested to modulate the disease course of AD.

[Interferon-gamma release assays in tuberculosis diagnostics]

Two years ago, CE certified interferon-gamma release assays (IGRA) were launched on the German market (QuantiFERON-TB Gold In-Tube and T-SPOT-TB). Since this time, a multitude of studies have analysed these assays. Guidelines have been elaborated by national expert committees of England, the USA and Switzerland. However, standards of tuberculosis diagnostics and management may vary from country to country. This statement provides practice relevant recommendations for indications, pre-analytics and the interpretation of IGRA test results under different clinical conditions. The IGRA are integrated into existing guidelines for the management of tuberculosis.

Anti-inflammatory properties of alpha- and gamma-tocopherol

Natural vitamin E consists of four different tocopherol and four different tocotrienol homologues (alpha,beta, gamma, delta) that all have antioxidant activity. However, recent data indicate that the different vitamin E homologues also have biological activity unrelated to their antioxidant activity. In this review, we discuss the anti-inflammatory properties of the two major forms of vitamin E, alpha-tocopherol (alphaT) and gamma-tocopherol (gammaT), and discuss the potential molecular mechanisms involved in these effects. While both tocopherols exhibit anti-inflammatory activity in vitro and in vivo, supplementation with mixed (gammaT-enriched) tocopherols seems to be more potent than supplementation with alphaT alone. This may explain the mostly negative outcomes of the recent large-scale interventional chronic disease prevention trials with alphaT only and thus warrants further investigation.

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.

gamma-tocopherol traps mutagenic electrophiles such as NO(X) and complements alpha-tocopherol: physiological implications

Peroxynitrite, a powerful mutagenic oxidant and nitrating species, is formed by the near diffusion-limited reaction of .NO and O2.- during activation of phagocytes. Chronic inflammation induced by phagocytes is a major contributor to cancer and other degenerative diseases. We examined how gamma-tocopherol (gammaT), the principal form of vitamin E in the United States diet, and alpha-tocopherol (alphaT), the major form in supplements, protect against peroxynitrite-induced lipid oxidation. Lipid hydroperoxide formation in liposomes (but not isolated low-density lipoprotein) exposed to peroxynitrite or the .NO and O2.- generator SIN-1 (3-morpholinosydnonimine) was inhibited more effectively by gammaT than alphaT. More importantly, nitration of gammaT at the nucleophilic 5-position, which proceeded in both liposomes and human low density lipoprotein at yields of approximately 50% and approximately 75%, respectively, was not affected by the presence of alphaT. These results suggest that despite alphaT's action as an antioxidant gammaT is required to effectively remove the peroxynitrite-derived nitrating species. We postulate that gammaT acts in vivo as a trap for membrane-soluble electrophilic nitrogen oxides and other electrophilic mutagens, forming stable carbon-centered adducts through the nucleophilic 5-position, which is blocked in alphaT. Because large doses of dietary alphaT displace gammaT in plasma and other tissues, the current wisdom of vitamin E supplementation with primarily alphaT should be reconsidered.

Inhibition by interferon-gamma of human mononuclear cell-mediated low density lipoprotein oxidation. Participation of tryptophan metabolism along the kynurenine pathway

In this study we examined the potential inhibition by interferon-gamma (IFN gamma) of the early stages of low density lipoprotein (LDL) oxidation mediated by human peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM) in Ham's F-10 medium supplemented with physiological amounts of L-tryptophan (Trp). We assessed LDL oxidation by measuring the consumption of LDL's major antioxidant (i.e., alpha-tocopherol) and targets for oxidation (cholesteryllinoleate and cholesterylarachidonate), together with the accumulation of cholesterylester hydroperoxides and the increase in relative electrophoretic mobility of the lipoprotein particle. Exposure of PBMC or MDM to IFN gamma induced the degradation of extracellular Trp with concomitant accumulation of kynurenine, anthranilic and 3-hydroxyanthranilic acid (3HAA) in the culture medium. Formation of 3HAA, but neither Trp degradation nor formation of kynurenine and anthranilic acid, was inhibited by low amounts of diphenylene iodonium (DPI) in a concentration-dependent manner. In contrast to oxidative Trp metabolism, exposure of human PBMC or MDM to IFN gamma failed to induce degradation of arginine, and nitrite was not detected in the cell supernatant, indicating that nitric oxide synthase was not induced under these conditions. Incubation of LDL in Trp-supplemented F-10 medium resulted in a time-dependent oxidation of the lipoprotein that was accelerated in the presence of PBMC or MDM but inhibited strongly in the presence of both cells and IFN gamma, i.e., when Trp degradation and formation of 3HAA were induced. In contrast, when IFN gamma was added to PBMC or MDM in F-10 medium that was virtually devoid of Trp, inhibition of cell-accelerated LDL oxidation was not observed. Exogenous 3HAA added to PBMC or purified monocytes in the absence of IFN gamma also strongly and in a concentration-dependent manner inhibited LDL oxidation. Selective inhibition of IFN gamma-induced formation of 3HAA by DPI caused reversion of the inhibitory action of this cytokine on both PBMC- and MDM-mediated LDL oxidation. These results show that IFN gamma treatment of human PBMC or MDM in vitro attenuates the extent of LDL oxidation caused by these cells, and indicate that Trp degradation with formation of 3HAA is a major contributing factor to this inhibitory activity.

Measuring energy spectra of TeV gamma-ray emission from the Cygnus region of our galaxy with Milagro

High energy gamma rays can provide fundamental clues to the origins of cosmic rays. In this thesis, TeV gamma-ray emission from the Cygnus region is studied. Previously the Milagro experiment detected five TeV gamma-ray sources in this region and a significant excess of TeV gamma rays whose origin is still unclear. To better understand the diffuse excess the separation of sources and diffuse emission is studied using the latest and most sensitive data set of the Milagro experiment. In addition, a newly developed technique is applied that allows the energy spectrum of the TeV gamma rays to be reconstructed using Milagro data. No conclusive statement can be made about the spectrum of the diffuse emission from the Cygnus region because of its low significance of 2.2 σ above the background in the studied data sample. The entire Cygnus region emission is best fit with a power law with a spectral index of α=2.40 (68% confidence interval: 1.35-2.92) and a exponential cutoff energy of 31.6 TeV (10.0-251.2 TeV). In the case of a simple power law assumption without a cutoff energy the best fit yields a spectral index of α=2.97 (68% confidence interval: 2.83-3.10). Neither of these best fits are in good agreement with the data. The best spectral fit to the TeV emission from MGRO J2019+37, the brightest source in the Cygnus region, yields a spectral index of α=2.30 (68% confidence interval: 1.40-2.70) with a cutoff energy of 50.1 TeV (68% confidence interval: 17.8-251.2 TeV) and a spectral index of α=2.75 (68% confidence interval: 2.65-2.85) when no exponential cutoff energy is assumed. According to the present analysis, MGRO J2019+37 contributes 25% to the differential flux from the entire Cygnus at 15 TeV.