Vaccine-associated varicella and rubella infections in severe combined immunodeficiency with isolated CD4 lymphocytopenia and mutations in IL7R detected by tandem whole exome sequencing and chromosomal microarray.

In areas without newborn screening for severe combined immunodeficiency (SCID), disease-defining infections may lead to diagnosis, and in some cases, may not be identified prior to the first year of life. We describe a female infant who presented with disseminated vaccine-acquired varicella (VZV) and vaccine-acquired rubella infections at 13 months of age. Immunological evaluations demonstrated neutropenia, isolated CD4 lymphocytopenia, the presence of CD8(+) T cells, poor lymphocyte proliferation, hypergammaglobulinaemia and poor specific antibody production to VZV infection and routine immunizations. A combination of whole exome sequencing and custom-designed chromosomal microarray with exon coverage of primary immunodeficiency genes detected compound heterozygous mutations (one single nucleotide variant and one intragenic copy number variant involving one exon) within the IL7R gene. Mosaicism for wild-type allele (20-30%) was detected in pretransplant blood and buccal DNA and maternal engraftment (5-10%) demonstrated in pretransplant blood DNA. This may be responsible for the patient's unusual immunological phenotype compared to classical interleukin (IL)-7Rα deficiency. Disseminated VZV was controlled with anti-viral and immune-based therapy, and umbilical cord blood stem cell transplantation was successful. Retrospectively performed T cell receptor excision circle (TREC) analyses completed on neonatal Guthrie cards identified absent TREC. This case emphasizes the danger of live viral vaccination in severe combined immunodeficiency (SCID) patients and the importance of newborn screening to identify patients prior to high-risk exposures. It also illustrates the value of aggressive pathogen identification and treatment, the influence newborn screening can have on morbidity and mortality and the significant impact of newer genomic diagnostic tools in identifying the underlying genetic aetiology for SCID patients.

Mechanisms of aluminum adjuvant toxicity and autoimmunity in pediatric populations.

Immune challenges during early development, including those vaccine-induced, can lead to permanent detrimental alterations of the brain and immune function. Experimental evidence also shows that simultaneous administration of as little as two to three immune adjuvants can overcome genetic resistance to autoimmunity. In some developed countries, by the time children are 4 to 6 years old, they will have received a total of 126 antigenic compounds along with high amounts of aluminum (Al) adjuvants through routine vaccinations. According to the US Food and Drug Administration, safety assessments for vaccines have often not included appropriate toxicity studies because vaccines have not been viewed as inherently toxic. Taken together, these observations raise plausible concerns about the overall safety of current childhood vaccination programs. When assessing adjuvant toxicity in children, several key points ought to be considered: (i) infants and children should not be viewed as "small adults" with regard to toxicological risk as their unique physiology makes them much more vulnerable to toxic insults; (ii) in adult humans Al vaccine adjuvants have been linked to a variety of serious autoimmune and inflammatory conditions (i.e., "ASIA"), yet children are regularly exposed to much higher amounts of Al from vaccines than adults; (iii) it is often assumed that peripheral immune responses do not affect brain function. However, it is now clearly established that there is a bidirectional neuro-immune cross-talk that plays crucial roles in immunoregulation as well as brain function. In turn, perturbations of the neuro-immune axis have been demonstrated in many autoimmune diseases encompassed in "ASIA" and are thought to be driven by a hyperactive immune response; and (iv) the same components of the neuro-immune axis that play key roles in brain development and immune function are heavily targeted by Al adjuvants. In summary, research evidence shows that increasing concerns about current vaccination practices may indeed be warranted. Because children may be most at risk of vaccine-induced complications, a rigorous evaluation of the vaccine-related adverse health impacts in the pediatric population is urgently needed.

Cytokines profile in neonatal and adult wild-type mice post-injection of U. S. pediatric vaccination schedule.

INTRODUCTION: A recent study from our laboratory demonstrated a number of neurobehavioral abnormalities in mice colony injected with a mouse-weight equivalent dose of all vaccines that are administered to infants in their first 18 months of life according to the U. S. pediatric vaccination schedule.Cytokines have been studied extensively as blood immune and inflammatory biomarkers, and their association with neurodevelopmental disorders. Given the importance of cytokines in early neurodevelopment, we aimed to investigate the potential post-administration effects of the U. S. pediatric vaccines on circulatory cytokines in a mouse model.In the current study, cytokines have been assayed at early and late time points in mice vaccinated early in postnatal life and compared with placebo controls. MATERIALS AND METHODS: Newborn mouse pups were divided into three groups: i) vaccine (V1), ii) vaccine â€‹× â€‹3 (V3) and iii) placebo control. V1 group was injected with mouse weight-equivalent of the current U. S. pediatric vaccine schedule. V3 group was injected with same vaccines but at triple the dose and the placebo control was injected with saline. Pups were also divided according to the sampling age into two main groups: acute- and chronic-phase group. Blood samples were collected at postnatal day (PND) 23, two days following vaccine schedule for the acute-phase group or at 67 weeks post-vaccination for the chronic-phase groups. Fifteen cytokines were analyzed: GM-CSF, IFN-γ, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17A, MCP-1, TNF-α, and VEGF-A. Wilcoxon Rank Sum test or unpaired Student's t-test was performed where applicable. RESULTS: IL-5 levels in plasma were significantly elevated in the V1 and V3 group compared with the control only in the acute-phase group. The elevation of IL-5 levels in the two vaccine groups were significant irrespective of whether the sexes were combined or analyzed separately. Other cytokines (VEGF-A, TNF-α, IL-10, MCP-1, GM-CSF, IL-6, and IL-13) were also impacted, although to a lesser extent and in a sex-dependent manner. In the acute-phase group, females showed a significant increase in IL-10 and MCP-1 levels and a decrease in VEGF-A levels in both V1 and V3 group compared to controls. In the acute-phase, a significant increase in MCP-1 levels in V3 group and CM-CSF levels in V1 and V3 group and decrease in TNF-α levels in V1 group were observed in treated males as compared with controls. In chronic-phase females, levels of VEGF-A in V1 and V3 group, TNF-α in V3 group, and IL-13 in V1 group were significantly decreased in contrast with controls. In chronic-phase males, TNF-α levels were significantly increased in V1 group and IL-6 levels decreased in V3 group in comparison to controls. The changes in levels of most tested cytokines were altered between the early and the late postnatal assays. CONCLUSIONS: IL-5 levels significantly increased in the acute-phase of the treatment in the plasma of both sexes that were subjected to V1 and V3 injections. These increases had diminished by the second test assayed at week 67. These results suggest that a profound, albeit transient, effect on cytokine levels may be induced by the whole vaccine administration supporting our recently published observations regarding the behavioral abnormalities in the same mice. These observations support the view that the administration of whole pediatric vaccines in a neonatal period may impact at least short-term CNS functions in mice.

Dominant versus recessive traits conveyed by allelic mutations - to what extent is nonsense-mediated decay involved?

Mutations in ROR2, encoding a receptor tyrosine kinase, can cause autosomal recessive Robinow syndrome (RRS), a severe skeletal dysplasia with limb shortening, brachydactyly, and a dysmorphic facial appearance. Other mutations in ROR2 result in the autosomal dominant disease, brachydactyly type B (BDB1). No functional mechanisms have been delineated to effectively explain the association between mutations and different modes of inheritance causing different phenotypes. BDB1-causing mutations in ROR2 result from heterozygous premature termination codons (PTCs) in downstream exons and the conveyed phenotype segregates as an autosomal dominant trait, whereas heterozygous missense mutations and PTCs in upstream exons result in carrier status for RRS. Given that the distribution of PTC mutations revealed a correlation between the phenotype and the mode of inheritance conveyed, we investigated the potential role for the nonsense-mediated decay (NMD) pathway in the abrogation of possible aberrant effects of selected mutant alleles. Our experiments show that triggering or escaping NMD may cause different phenotypes with a distinct mode of inheritance. We generalize these findings to other disease-associated genes by examining PTC mutation distribution correlation with conveyed phenotype and inheritance patterns. Indeed, NMD may explain distinct phenotypes and different inheritance patterns conveyed by allelic truncating mutations enabling better genotype-phenotype correlations in several other disorders.

First record of giant anteater (xenarthra, myrmecophagidae) in north america.

A right metacarpal III represents the first North American record of the giant anteater (Myrmecophaga tridactyla). Recovered in northwestern Sonora, Mexico, with a rich vertebrate fauna of early Pleistocene (Irvingtonian) age, it belongs to a cohort of large mammals that dispersed from South America to North America along a savanna corridor. Presumably habitat and climatic changes have subsequently driven this mammalian family more than 3000 kilometers back into Central America from its former expansion into temperate North America.

Sildenafil reduces alcohol-induced gastric damage: just say 'NO'.

Although sildenafil (Viagra) and other phosphodiesterase V (PDE V) inhibitors are increasingly recognized for their use in the treatment of male erectile dysfunction and perhaps more recently pulmonary artery hypertension, less is known of their potential beneficial effects in other situations. Medeiros et al., in the current issue of the British Journal of Pharmacology, report that sildenafil dramatically reduces alcohol-induced gastric damage in rats. The authors provide convincing evidence that such protection not only occurs via the nitric oxide (NO)/cGMP pathway, but also involves regulation of ATP-sensitive potassium channels. Therefore, in addition to exerting anti-impotence efficacy, PDE V inhibitors may provide significant beneficial effects from mucosal injury induced by alcohol.

Taurine release in developing mouse hippocampus is modulated by glutathione and glutathione derivatives.

Glutathione (reduced form GSH and oxidized form GSSG) constitutes an important defense against oxidative stress in the brain, and taurine is an inhibitory neuromodulator particularly in the developing brain. The effects of GSH and GSSG and glycylglycine, gamma-glutamylcysteine, cysteinylglycine, glycine and cysteine on the release of [(3)H]taurine evoked by K+-depolarization or the ionotropic glutamate receptor agonists glutamate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) were now studied in slices from the hippocampi from 7-day-old mouse pups in a perfusion system. All stimulatory agents (50 mM K(+), 1 mM glutamate, 0.1 mM kainate, 0.1 mM AMPA and 0.1 mM NMDA) evoked taurine release in a receptor-mediated manner. Both GSH and GSSG significantly inhibited the release evoked by 50 mM K+. The release induced by AMPA and glutamate was also inhibited, while the kainate-evoked release was significantly activated by both GSH and GSSG. The NMDA-evoked release proved the most sensitive to modulation: L-Cysteine and glycine enhanced the release in a concentration-dependent manner, whereas GSH and GSSG were inhibitory at low (0.1 mM) but not at higher (1 or 10 mM) concentrations. The release evoked by 0.1 mM AMPA was inhibited by gamma-glutamylcysteine and cysteinylglycine, whereas glycylglycine had no effect. The 0.1 mM NMDA-evoked release was inhibited by glycylglycine and gamma-glutamylcysteine. In turn, cysteinylglycine inhibited the NMDA-evoked release at 0.1 mM, but was inactive at 1 mM. Glutathione exhibited both enhancing and attenuating effects on taurine release, depending on the glutathione concentration and on the agonist used. Both glutathione and taurine act as endogenous neuroprotective effectors during early postnatal life.

The protease-activated receptor 4 Ala120Thr variant alters platelet responsiveness to low-dose thrombin and protease-activated receptor 4 desensitization, and is blocked by non-competitive P2Y12 inhibition.

Essentials The rs773902 SNP results in differences in platelet protease-activated receptor (PAR4) function. The functional consequences of rs773902 were analyzed in human platelets and stroke patients. rs773902 affects thrombin-induced platelet function, PAR4 desensitization, stroke association. Enhanced PAR4 Thr120 effects on platelet function are blocked by ticagrelor. SUMMARY: Background F2RL3 encodes protease-activated receptor (PAR) 4 and harbors an A/G single-nucleotide polymorphism (SNP) (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP) AYPGKF. Objectives To determine the functional effects of rs773902 on stimulation by a physiological agonist, thrombin, and on antiplatelet antagonist activity. Methods Healthy human donors were screened and genotyped for rs773902. Platelet function in response to thrombin was assessed without and with antiplatelet antagonists. The association of rs773902 alleles with stroke was assessed in the Stroke Genetics Network study. Results As compared with rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation in response to subnanomolar concentrations of thrombin, increased granule secretion, and decreased sensitivity to PAR4 desensitization. In the presence of PAR1 blockade, this genotype effect was abolished by higher concentrations of or longer exposure to thrombin. We were unable to detect a genotype effect on thrombin-induced PAR4 cleavage, dimerization, and lipid raft localization; however, rs773902 AA platelets required a three-fold higher level of PAR4-AP for receptor desensitization. Ticagrelor, but not vorapaxar, abolished the PAR4 variant effect on thrombin-induced platelet aggregation. A significant association of modest effect was detected between the rs773902 A allele and stroke. Conclusion The F2RL3 rs773902 SNP alters platelet reactivity to thrombin; the allelic effect requires P2Y12 , and is not affected by gender. Ticagrelor blocks the enhanced reactivity of rs773902 A platelets. PAR4 encoded by the rs773902 A allele is relatively resistant to desensitization and may contribute to stroke risk.

Magnetic resonance microscopy and immunohistochemistry of the CNS of the mutant SOD murine model of ALS reveals widespread neural deficits.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that primarily affects motor neurons and descending motor tracts of the CNS. We have evaluated the CNS of a murine model of familial ALS based on the over-expression of mutant human superoxide dismutase (mSOD; G93A) using magnetic resonance microscopy (MRM) and immunohistochemistry (IHC). Three-dimensional volumetric analysis was performed from 3D T2*-weighted images acquired at 17.6 T at isotropic resolutions of 40 mum. Compared to controls, mSOD mice had significant reductions in the volumes of total brain, substantia nigra, striatum, hippocampus, and internal capsule, with decreased cortical thickness in primary motor and somatosensory cortices. In the spinal cord, mSOD mice had significantly decreased volume of both the total grey and white matter; in the latter case, the volume change was confined to the dorsal white matter. Increased apoptosis, GFAP positive astrocytes, and/or activated microglia were observed in all those CNS regions that showed volume loss except for the hippocampus. The MRM findings in mSOD over-expressing mice are similar to data previously obtained from a model of ALS-parkinsonism dementia complex (ALS-PDC), in which neural damage occurred following a diet of washed cycad flour containing various neurotoxins. The primary difference between the two models involves a significantly greater decrease in spinal cord white matter volume in mSOD mice, perhaps reflecting variations in degeneration of the descending motor tracts. The extent to which several CNS structures are impacted in both murine models of ALS argues for a reevaluation of the nature of the pathogenesis of ALS since CNS structures involved in Parkinson's and Alzheimer's diseases appear to be affected as well.

Late appearance of glutamate transporter defects in a murine model of ALS-parkinsonism dementia complex.

Excitotoxicity has been widely hypothesized to play a major role in various neurodegenerative diseases. We have used a mouse model of ALS-parkinsonism dementia complex (ALS-PDC) of the Western Pacific to explore this hypothesis. Mice fed washed cycad flour, the major epidemiological link to ALS-PDC, showed significant and progressive motor, cognitive, and sensory behavioural deficits [Wilson, J.M., Khabazian, I., Wong, M.C., Seyedalikhani, A., Bains, J.S., Pasqualotto, B.A., Williams, D.E., Andersen, R.J., Simpson, R.J., Smith, R., Craig, U.K., Kurland, L.T., Shaw, C.A., 2002. Behavioral and neurological correlates of ALS-parkinsonism dementia complex in adult mice fed washed cycad flour. Neuromol. Med. 1 (3), 207-221]. In addition, glutamate transporter (GLT-1/EAAT2) levels measured by immunohistochemistry with antibodies specific for two glial glutamate transporter splice variants (GLT-1alpha and GLT-1B) were significantly down-regulated showing a 'patchy' loss of antibody label centered on blood vessels [Wilson, J.M., Khabazian, I., Pow, D.V., Craig, U.K., Shaw, C.A., 2003. Decrease in glial glutamate transporter variants and excitatory amino acid receptor down-regulation in a murine model of ALS-PDC. Neuromol. Med. 3 (2), 105-118]. Receptor binding assays showed decreased NMDA and AMPA receptor levels combined with increased GABA(A) receptor levels in various CNS regions. The alterations in GLT-1 variants and the ionotropic receptors are consistent with an increased level of extracellular glutamate. The interaction between environmental toxicity and genetic susceptibility was also tested using mice expressing various Apolipoprotein E (ApoE) genotypes. Mice lacking the ApoE gene showed relative resistance to cycad-induced toxicity as measured by GLT-1B labeling, but all mice expressing the human ApoE isoforms showed a similar loss of GLT-1B. We have further shown that an isolated cycad toxin (beta-sitosterol-beta-d-glucoside, BSSG), previously shown to release glutamate in vitro [Wilson, J.M., Khabazian, I., Wong, M.C., Seyedalikhani, A., Bains, J.S., Pasqualotto, B.A., Williams, D.E., Andersen, R.J., Simpson, R.J., Smith, R., Craig, U.K., Kurland, L.T., Shaw, C.A., 2002. Behavioral and neurological correlates of ALS-parkinsonism dementia complex in adult mice fed washed cycad flour. Neuromol. Med. 1 (3), 207-221], can be directly toxic to motor neurons in vivo [Wilson, J.M., Petrik, M.S., Moghadasian, M.H., Shaw, C.A., 2005. Examining the interaction of apo E and neurotoxicity on a murine model of ALS-PDC. Can. J. Physiol. Pharmacol. 83 (2), 131-141]. However, BSSG-fed mice did not show altered GLT-1B labeling in the spinal cord suggesting that an initial excitotoxic mechanism may not be responsible for the final neuronal loss observed. While glutamate-mediated excitotoxicity is likely involved in the outcomes following cycad/BSSG exposure, the precise location in the cascade of events ultimately leading to neuronal death remains to be determined.

Cycad toxins, Helicobacter pylori and parkinsonism: cholesterol glucosides as the common denomenator.

Understanding sporadic cases of age-dependent neurodegenerative diseases such as parkinsonism requires the evaluation of potential environmental factors. Amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC), a neurological disorder in which features of parkinsonism are present and for which no consistent genetic explanation has been found, has been linked to the consumption of cycad (Cycas micronesica). Similarly, epidemiological evidence suggests an association between parkinsonism and gastric ulcer caused by Helicobacter pylori infection. While common immunological and inflammatory changes have been proposed to account for the link between parkinsonism and H. pylori infection, we propose an alternate explanation based on our work on the "cycad theory" of ALS-PDC. Recent experiments in our laboratory have identified several sterol glucosides in cycad that have neurotoxic properties in vitro and that appear to be linked to the development of neurodegenerative disease in vivo. Specifically, mice fed cycad display behavioural symptoms of parkinsonism such as reduced gait length, as well as neuropathological signs such as a loss of striatal dopaminergic (DAergic) terminals and an upregulation of the dopamine D2 receptor. These cycad-derived sterol glucosides are structurally similar to cholesterol glucosides that account for a significant part pf the lipid profile of H. pylori. We hypothesize that cholesterol glucosides arising from H. pylori infection may act as neurotoxins, promoting the degeneration of the DAergic neurons affected in parkinsonism, in a similar reaction to that which is thought to link cycad consumption and ALS-PDC. This hypothesis will be tested in future studies that will include exposing mice to purified sterol or cholestorol glucosides derived from cycad and comparing these mice behaviourally and neuropathologically to ones chronically infected with H. pylori.

Amyotrophic lateral sclerosis: the involvement of intracellular Ca2+ and protein kinase C.

The neurodegenerative disease, amyotrophic lateral sclerosis (ALS), is characterized by the selective death of motoneurones and corticospinal tract neurones. Abnormalities in excitatory amino acids and their receptors, as well as disordered function of voltage-dependent Ca2+ channels and superoxide dismutase have been reported in ALS patients. Furthermore, the activity of protein kinase C (PKC), a Ca2+, phospholipid-dependent enzyme, is also substantially increased in tissue from ALS patients, suggesting that alterations in intracellular free Ca2+ may be central to many of the diverse pathogenic mechanisms potentially responsible for ALS as discussed here by Charles Krieger and colleagues. Increased PKC activity, in turn, may have direct or indirect effects on neuronal viability and influence the pathogenic process in ALS by modifying the phosphorylation of voltage-dependent Ca2+ channels, neurotransmitter receptors and structural proteins.

Age-dependent expression, phosphorylation and function of neurotransmitter receptors: pharmacological implications.

In recent years, a number of experimental controversies have arisen in the pharmacological sciences literature. Two possibly related examples concern the role of phosphorylation in receptor regulation and the occurrence of paradoxical effects of neurally active drugs in patients. In this article, Ruth Lanius and colleagues review these two issues and suggest that some of the recently reported age-dependent aspects of neurotransmitter receptor regulation and function may explain drug action. An appreciation of the dynamic interactions involved in receptor regulation, especially during development, may offer novel perspectives on neural function.

Localization of coxsackie virus and adenovirus receptor (CAR) in normal and regenerating human muscle.

The primary receptor for Adenovirus and Coxsackie virus (CAR) serves as main port of entry of the adenovirus vector mediating gene transfer into skeletal muscle. Information about CAR expression in normal and diseased human skeletal muscle is lacking. C'- or N'-terminally directed polyclonal antibodies against CAR were generated and immunohistochemical analysis of CAR on morphologically normal and regenerating human skeletal muscle of children and adults was performed. In morphologically normal human muscle fibers, CAR immunoreactivity was limited to the neuromuscular junction. In regenerating muscle fibers, CAR was abundantly co-expressed with markers of regeneration. The function of CAR at the neuromuscular junction is currently unknown. Co-expression of CAR with markers of regeneration suggests that CAR is developmentally regulated, and may serve as a marker of skeletal muscle fiber regeneration.

Comparative genomic hybridisation using a proximal 17p BAC/PAC array detects rearrangements responsible for four genomic disorders.

BACKGROUND: Proximal chromosome 17p is a region rich in low copy repeats (LCRs) and prone to chromosomal rearrangements. Four genomic disorders map within the interval 17p11-p12: Charcot-Marie-Tooth disease type 1A, hereditary neuropathy with liability to pressure palsies, Smith-Magenis syndrome, and dup(17)(p11.2p11.2) syndrome. While 80-90% or more of the rearrangements resulting in each disorder are recurrent, several non-recurrent deletions or duplications of varying sizes within proximal 17p also have been characterised using fluorescence in situ hybridisation (FISH). METHODS: A BAC/PAC array based comparative genomic hybridisation (array-CGH) method was tested for its ability to detect these genomic dosage differences and map breakpoints in 25 patients with recurrent and non-recurrent rearrangements. RESULTS: Array-CGH detected the dosage imbalances resulting from either deletion or duplication in all the samples examined. The array-CGH approach, in combination with a dependent statistical inference method, mapped 45/46 (97.8%) of the analysed breakpoints to within one overlapping BAC/PAC clone, compared with determinations done independently by FISH. Several clones within the array that contained large LCRs did not have an adverse effect on the interpretation of the array-CGH data. CONCLUSIONS: Array-CGH is an accurate and sensitive method for detecting genomic dosage differences and identifying rearrangement breakpoints, even in LCR-rich regions of the genome.

Analysis of neurological disease in four dimensions: insight from ALS-PDC epidemiology and animal models.

The causal factor(s) responsible for sporadic neurological diseases are unknown and the stages of disease progression remain undefined and poorly understood. We have developed an animal model of amyotrophic lateral sclerosis-parkinsonism dementia complex which mimics all the essential features of the disease with the initial neurological insult arising from neurotoxins contained in washed cycad seeds. Animals fed washed cycad develop deficits in motor, cognitive, and sensory behaviors that correlate with the loss of neurons in specific regions of the central nervous system. The ability to recreate the disease by exposure to cycad allows us to extend the model in multiple dimensions by analyzing behavioral, cellular, and biochemical changes over time. In addition, the ability to induce toxin-based neurodegeneration allows us to probe the interactions between genetic and epigenetic factors. Our results show that the impact of both genetic causal and susceptibility factors with the cycad neurotoxins are complex. The article describes the features of the model and suggests ways that our understanding of cycad-induced neurodegeneration can be used to decipher and identify the early events in various human neurological diseases.

Specific glutathione binding sites in pig cerebral cortical synaptic membranes.

Glutathione (gamma-glutamylcysteinylglycine) is a neuromodulator at glutamate receptors, but may also act as a neurotransmitter at sites of its own. The Na+-independent binding of [3H]glutathione to pig cortical synaptic membranes was characterized here using glycine, cysteine analogs, dipeptides and glutathione derivatives, and ligands selective for known glutamate receptors. L-Glutamate, pyroglutamate, quinolinate, (S)-5-fluorowillardiine and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione were weak inhibitors at concentrations of 0.5 or 1 mM. D-Glutamate, L- and D-aspartate, glutamine, quisqualate, kynurenate, other N-methyl-D-aspartate receptor ligands and non-N-methyl-D-aspartate receptor ligands failed to displace [3H]glutathione. Except for weak inhibition by D-serine (0.5 mM), glycine and other ligands of the glycine co-activatory site in the N-methyl-D-aspartate receptors had no displacing effect. Similarly, metabotropic glutamate group I, II and III receptor agonists and antagonists and compounds acting at the glutamate uptake sites were generally inactive. Glutathione, oxidized glutathione, S-nitrosoglutathione, gamma-L-glutamylcysteine, cysteinylglycine, cysteine, cysteamine and cystamine were the most potent displacers (IC50 values in the micromolar range), followed by dithiothreitol, glutathione sulfonate and the S-alkyl derivatives of glutathione (S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione). L-Homocysteinate and aminomethanesulfonate exhibited a moderate efficacy. Thiokynurenate, a cysteine analog and an antagonist at the N-methyl-D-aspartate receptor glycine co-activatory site, was a potent activator of glutathione binding. At 1 mM, some dipeptides also slightly activated the binding, gamma-L-glutamylleucine and gamma-L-glutamyl-GABA being the most effective. The specific binding sites for glutathione in brain synaptic membranes are not identical to any known excitatory amino acid receptor. The cysteinyl moiety is crucial in the binding of glutathione. The oxidation or alkylation of the cysteine thiol group reduces the binding affinity. The strong activation by thiokynurenate may indicate that the glutathione receptor protein contains a modulatory site to which co-agonists may bind and allosterically activate glutathione binding. The novel population of specific binding sites of glutathione gives rise to the possibility that they may have profound effects on synaptic functions in the mammalian central nervous system. The glutathione binding sites may be an important, and for the most part unrecognized, component in signal transduction in the brain.