Saccharomyces cerevisiae, key role of MIG1 gene in metabolic switching: putative fermentation/oxidation.

Saccharomyces cerevisiae can utilize a wide range of carbon sources; however, in the presence of glucose the use of alternate carbon sources would be repressed. Several genes involved in the metabolic pathways exert these effects. Among them, the zinc finger protein, Mig1 (multicopy inhibitor of GAL gene expression) plays important roles in glucose repression of Saccharomyces cerevisiae. To investigate whether the alleviation of glucose effect would result in a switch to oxidative production pathway, MIG1 were disrupted in a haploid laboratory strain (2805) of S. cerevisiae. The impact of this disruption was studied under fully aerobic conditions when glucose was the sole carbon source. Our results showed that glucose repression was partly alleviated; i.e., ethanol, as a significant fermentation marker, and acetate productions were respectively decreased by 14.13% and 43.71% compared to the wild type. In ΔMIG1 strain, the metabolic shifting on the aerobic pathway and a significant increase in pyruvate and glycerol production suggested it as an optimally productive industrial yeast strain. However, further studies are needed to confirm these findings.

EFFECTS OF MEDIAL SEPTAL LESION ON HIPPOCAMPAL EXTRACELLULAR GLUTAMATE AND GABA LEVELS DURING SPATIAL ALTERNATION TESTING.

The present study investigated spatial working memory assessed in spontaneous alternation (SA) task and hippocampal glutamate and GABA release prior to, during, and after SA test in sham-operated and electrolytic medial septal (MS) lesioned rats. Also, have been investigated the effects of MS lesion on KCl-stimulated release of glutamate and GABA in the hippocampus. Behavioral study showed that electrolytic lesion of MS significantly impaired SA performance. Although both groups of animals had an insignificant rise in their respective hippocampal glutamate efflux during the SA, the rise of MS lesioned animals was blunted when compared with control animals. Hippocampal GABA levels did not change during behavioral testing in both groups. Most of control animals showed increase in KCl-stimulated glutamate release. By contrast, only one MS lesioned rat showed increase in glutamate release in response to KCl stimulation. Most of control and MS lesioned rats were non-responders in GABA release in response to KCl stimulation. Decreased glutamate release (upon stimulation) in the MS lesioned rats may contribute to spatial working memory impairment in these animals. We propose that SA testing coupled with in vivo microdialysis sampling represents a suitable approach to revealing the neurochemical correlates of hippocampal-dependent memory function, and thus could be a useful tool for better understanding of the neurochemical basis of cognitive decline associated with various disorders and neurodegenerative diseases.

Knockdown of interleukin-10 induces the redistribution of sigma1-receptor and increases the glutamate-dependent NADPH-oxidase activity in mouse brain neurons.

BACKGROUND: In the central nervous system, interleukin-10 (IL-10) provides trophic and survival effects directly on neurons, modulates neurite plasticity, and has a pivotal importance in the neuronal regeneration in neurodegenerative and neuroinflammatory conditions. This cytokine is primarily produced by glial cells and has beneficial effects on the neuronal viability. However, the mechanisms of IL-10-elicited neuroprotection are not clear. RESULTS: Membrane preparations, isolated from wild-type (Wt) and IL-10 knockout (KO) mice brain were used in this study. It has been shown that compared to wild-type mice, in IL-10 KO mice brain, the amount of immunoglobulin binding protein (BiP) is greatly increased, whereas the content of sigma receptor-1 (SigR1) is not changed significantly. Co-immunoprecipitation experiments have shown that the association of SigR1 with small GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1), NR2B subunit of NMDA-receptor (NMDAR) and inositol-3-phosphate receptor (IP3R) is higher in the IL-10 KO mice brain than in the Wt mice brain. Besides, we have found that either glutamate or sigma ligands, separately or together, do not change glutamate-induced NADPH-oxidase (NOX) activity in Wt-type mice brain membrane preparations, whereas in IL-10 KO mice high concentration of glutamate markedly increases the NOX-dependent production of reactive oxygen species (ROS). Glutamate-dependent ROS production was decreased to the normal levels by the action of sigma-agonists. CONCLUSIONS: It has been concluded that IL-10 deprivation, at least in part, can lead to the induction of ER-stress, which causes BiP expression and SigR1 redistribution between components of endoplasmic reticulum (ER) and plasma membrane. Moreover, IL-10 deficiency can change the specific organization of NMDAR, increasing the surface expression of SigR1-sensitive NR2B-containing NMDAR. In these conditions, glutamate-dependent ROS production is greatly increased leading to the initiation of apoptosis. In this circumstances, sigma-ligands could play a preventive role against NMDA receptor-mediated excitotoxicity.

Knockdown of interleukin-10 induces the redistribution of sigma1-receptor and increases the glutamate-dependent NADPH-oxidase activity in mouse brain neurons

BACKGROUND: In the central nervous system, interleukin-10 (IL-10) provides trophic and survival effects directly on neurons, modulates neurite plasticity, and has a pivotal importance in the neuronal regeneration in neurodegenerative and neuroinflammatory conditions. This cytokine is primarily produced by glial cells and has beneficial effects on the neuronal viability. However, the mechanisms of IL-10-elicited neuroprotection are not clear. RESULTS: Membrane preparations, isolated from wild-type (Wt) and IL-10 knockout (KO) mice brain were used in this study. It has been shown that compared to wild-type mice, in IL-10 KO mice brain, the amount of immunoglobulin binding protein (BiP) is greatly increased, whereas the content of sigma receptor-1 (SigR1) is not changed significantly. Co-immunoprecipitation experiments have shown that the association of SigR1 with small GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1), NR2B subunit of NMDA-receptor (NMDAR) and inositol-3-phosphate receptor (IP3R) is higher in the IL-10 KO mice brain than in the Wt mice brain. Besides, we have found that either glutamate or sigma ligands, separately or together, do not change glutamate-induced NADPH-oxidase (NOX) activity in Wt-type mice brain membrane preparations, whereas in IL-10 KO mice high concentration of glutamate markedly increases the NOX-dependent production of reactive oxygen species (ROS). Glutamate-dependent ROS production was decreased to the normal levels by the action of sigma-agonists. CONCLUSIONS: It has been concluded that IL-10 deprivation, at least in part, can lead to the induction of ER-stress, which causes BiP expression and SigR1 redistribution between components of endoplasmic reticulum (ER) and plasma membrane. Moreover, IL-10 deficiency can change the specific organization of NMDAR, increasing the surface expression of SigR1-sensitive NR2B-containing NMDAR. In these conditions, glutamate-dependent ROS production is greatly increased leading to the initiation of apoptosis. In this circumstances, sigma-ligands could play a preventive role against NMDA receptor-mediated excitotoxicity.

Synaptic and non-synaptic mitochondria in hippocampus of adult rats differ in their sensitivity to hypothyroidism.

Hypothyroidism in humans provokes various neuropsychiatric disorders, movement, and cognitive abnormalities that may greatly depend on the mitochondrial energy metabolism. Brain cells contain at least two major populations of mitochondria that include the non-synaptic mitochondria, which originate from neuronal and glial cell bodies (CM), and the synaptic (SM) mitochondria, which primarily originate from the nerve terminals. Several parameters of oxidative stress and other parameters in SM and CM fractions of hippocampus of adult rats were compared among euthyroid (control), hypothyroid (methimazol-treated), and thyroxine (T4)-treated hypothyroid states. nNOS translocation to CM was observed with concomitant increase of mtNOS's activity in hypothyroid rats. In parallel, oxidation of cytochrome c oxidase and production of peroxides with substrates of complex I (glutamate + malate) were enhanced in CM, whereas the activity of aconitase and mitochondrial membrane potential (ΔΨm) were decreased. Furthermore, the elevation of mitochondrial hexokinase activity in CM was also found. No differences in these parameters between control and hypothyroid animals were observed in SM. However, in contrast to CM, hypothyroidism increases the level of pro-apoptotic K-Ras and Bad in SM. Our results suggest that hypothyroidism induces moderate and reversible oxidative/nitrosative stress in hippocampal CM, leading to the compensatory elevation of hexokinase activity and aerobic glycolysis. Such adaptive activation in glycolytic metabolism does not occur in SM, suggesting that synaptic mitochondria differ in their sensitivity to the energetic disturbance in hypothyroid conditions.

mGluR1 interacts with cystic fibrosis transmembrane conductance regulator and modulates the secretion of IL-10 in cystic fibrosis peripheral lymphocytes.

Cystic fibrosis (CF) is caused by the mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. CFTR dysfunction in T cells could lead directly to aberrant immune responses. The action of glutamate on the secretion of IL-8 and IL-10 by lymphocytes derived from healthy subjects and cystic CF patients, as well as the expression of metabotropic glutamate receptor subtype 1 (mGluR1) in the membrane fractions of lymphocytes was investigated. Our results have shown that CF-derived T-cells in the presence of IL-2 produce more IL-8 and IL-10, than T-cell from healthy control. However, only in normal lymphocytes a significant increase (144%) in the IL-10 secretion during exposure to high concentration of glutamate (10(-4)M) was detected. Glutamate-dependent secretion of IL-10 was not inhibited either by NMDA-receptor (NMDAR), or by AMPA-receptor (AMPAR) antagonist. Only mGluR1 antagonist, LY367385, strongly decreases the production of IL-10. Furthermore, the content of mGluR1, as well as cystic fibrosis transmembrane conductance regulator-associated ligand (CAL), Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1), was analyzed in plasma membrane of lymphocytes after immunoprecipitation of CFTR. We have found that normal, non-mutated CFTR, as well as mutated forms of CFTR were associated with metabotropic mGluR1, but the level of surface exposed mGluR1 in CF-lymphocytes was much lower than in normal cells. Besides, our results have shown that normal, non-mutated CFTR, as well as mutated forms of CFTR were associated with NHERF-1 and CAL; however in lymphocytes with CFTR mutation the amount of cell-surface expressed CFTR-CAL complex was greatly decreased. We have concluded that CFTR and mGluR1 could compete for binding to CAL, which in turn downregulates the post-synthetic trafficking of mGluR1 and decreases the synthesis of IL-10.

S-nitrosylation decreases the adsorption of H-Ras in lipid bilayer and changes intrinsic catalytic activity.

Structural, chemical, and mutational studies have shown that C-terminal cysteine residues on H-Ras could potentially be oxidized by nitrosylation. For investigating the effect of nitrosylation of Ras molecule on the adsorption of farnesylated H-Ras into lipid layer, experiments with optical waveguide lightmode spectroscopy were used. The analysis of association/dissociation kinetics to planar phospholipids under controlled hydrodynamic conditions has shown that preliminary treatment of protein by S-nitroso-cysteine decreased the adsorption of farnesylated H-Ras. The authors have found that compared with nitrosylated forms, farnesylated H-Ras has more compact configuration, because of the smaller area occupied by protein upon absorption at the membrane. The association rate coefficient for unmodified H-Ras was lower than similar parameter for farnesylated and nitrosylated forms. However, the desorbability, i.e., parameter, which reflects the rate of dissociation of protein from lipids is higher for farnesylated H-Ras. In addition, it was have found that farnesylation of cytoplasmic H-Ras, in contrast to membrane-derived forms, inhibits intrinsic GTPase activity of protein, and preliminary treatment of H-Ras by S-nitroso-cysteine restores the activity to the control level. These data suggest that nitrosylation of H-Ras rearranges the adsorptive potential and intrinsic GTPase activity of H-Ras through modification of C-terminal cysteines of molecule.

Hypoinsulinemia alleviates the GRF1/Ras/Akt anti-apoptotic pathway and induces alterations of mitochondrial ras trafficking in neuronal cells.

Recent observations have established that interruption of insulin production causes deficits in learning and memory formation. We have studied the mechanism of insulin's neuroprotective effect on primary neuronal cells and in streptozotocin (STZ)-induced diabetic rat brain. We have found that in hippocampal neuronal cells insulin increases the content of farnesylated Ras and phosphorylated form of Akt. Besides, the treatment of cells by insulin leads to the activation of mitochondrial cytochrome oxidase, which is inhibited by manumycin, a farnesyltransferase inhibitor. During experimental diabetes, the content of membrane-bound GRF1 was decreased in rat hippocampus that was correlated with the reduction in mitochondrial Ras and phosphorylated forms of Akt. This redistribution in Ras-GRF system was accompanied by the alteration in the activities of CREB, NF-kB (p65) and c-Rel transcription factors. We have proposed that hypoinsulinemia induces the inhibition of Ras signalling in the neuronal cells additionally by abnormality of Ras trafficking into mitochondria.

Glutamate decreases the secretion of IL-10 by peripheral blood lymphocytes in persons with autoimmune thyroiditis.

Human T lymphocytes expose ionotropic and metabotropic glutamate receptors, which control immune responses, cell activation, maturation, and death. Several cytokines release during inflammation which identification may have important physiological and clinical implications. Main biological function of IL-10 is limitation and termination of inflammatory responses and the regulation of differentiation and proliferation of several immune cells. Various inflammatory molecules regulated the secretion of IL-8 and IL-10, but the action of glutamate on the biosynthesis of cytokines is unknown. We have found that in peripheral blood lymphocytes glutamate at the concentrations within normal plasma levels (1 x 10(-5) M), as well as at lower concentration (0.3 x 10(-6) M) changes the secretion of immunosuppressive cytokine IL-10, whereas synthesis of proinflammatory chemokine, IL-8 did not changed significantly. Moreover, our results have shown that peripheral blood lymphocytes from patients with autoimmune thyroiditis release less IL-10 at both concentration of glutamate than peripheral blood lymphocytes from healthy persons. These data suggest that glutamate decrease the secretion of IL-10 by peripheral blood lymphocytes, especially in patients with autoimmune thyroiditis that may be responsible for prolongation of inflammation.

Ras proteins, nitrosylation and homocysteine metabolism.

Elevated levels of homocysteine and S-adenosylmethionine are causal risk factors for several neurological disorders. Homocysteine is a sulfur-containing, nonproteinogenic, neurotoxic amino acid biosynthesized during methyl cycles after demethylation of S-adenosylmethionine (SAM) to adenosylhomocysteine (SAH) and subsequent hydrolysis of SAH into homocysteine and adenosine. PC12 pheochromocytoma cells expressing a dominant inhibitory mutant of Ha-ras (M-M17-26, PC12 pheochromocytoma cells expressing a mutant v-ras gene (MVR) and PC12 cells transfected with normal c-rasH (M-CR3B) has been used to investigate the role of nitrosylation and farnesylation of Ras on the production of homocysteine and the activities of the redox-sensitive transcription factors NF-kB and c-Fos. We found that under serum and nerve growth factor withdrawal conditions undifferentiated apoptotic M-CR3B cells accumulated more homocysteine, than M-M17-26 cells and the production of homocysteine decreased under the action of manumycin (inhibitor of farnesyltransferase) and increased in the presence of L-NAME (inhibitor of nitric oxide synthase). Furthermore, we have shown that manumycin increased the activity of c-Fos in the M-CR3B cells and decreased the activity of NF-kB, while L-NAME reduced the activities of both transcription factors, and accelerated apoptosis of M-CR3B cells. In contrast to the M-CR3B cells, in M-M17-26 cells manumycin did not change the activity of c-Fos or the activity of NF-kB. Moreover, we have shown that L-NAME significantly changes the SAM/SAH ratio in both MCR and MVR cells. Moreover, these alterations have reciprocal character; in the MCR cells, the SAM/SAH ratio was raised, whereas in the MVR cells this ratio was reduced. We conclude that trophic factor withdrawal stimulates Ras, which apparently through the Rac/NADPH oxidase system induces permanent oxidative stress, modulates the activities of NF-kB and c-Fos, induces production of homocysteine and accelerates apoptosis. Nitrosylation of Ras is necessary for maintaining the survival of PC12 cells, while farnesylation of Ras stimulates apoptosis under withdrawal conditions. Besides, our results suggest that in conditions of a low level of nitric oxide PC12 cells with mutated oncogenic Ras produce more ROS than cells with wild type Ras and switch homocysteine metabolism toward to transsulfuration.

Inhibition of nitric oxide synthase and farnesyltransferase change the activities of several transcription factors.

Several types of cellular proteins can be modified by farnesylation and nitrosylation, of which the most significant is Ras. We used manumycin, a farnesyltransferase inhibitor, and L-NAME (Nomega-nitro-L-arginine methyl ester), a nitric oxide synthase (NOS) inhibitor, for characterization of Ras-dependent downstream targets activities. Our results suggest that change of the steady-state levels of nitric oxide and inhibition of farnesylation modified the activities of several transcription factors. We have found that the inhibition of farnesylation by manumycin decreased the DNA-binding activity of nuclear factor (NF)-kappaB, did not change the DNA-binding activities of STAT, Sp1, ATF-2, and CREB, and increased the activities of c-Fos, JunD, and c-Jun. Under such conditions, phosphorylation of Akt was decreased, whereas phosphorylation of extracellular signal-regulated kinase (ERK) was increased and phosphorylation of JNK did not change. Furthermore, our results show that reduction of intracellular concentration of nitric oxides by L-NAME increases the activities of c-Fos, ATF-2 and JunD and decreases the activities of CREB, STAT, Sp1, and c-Jun. The activities of all of these transcription factors are restored to normal levels in the presence of manumycin, suggesting that simultaneous modifications of proteins by farnesylation and nitrosylation change the direction of Ras-controlled downstream pathways. Our results provide further evidence of the significance of posttranslational modifications of Ras for the specificity of transducing cascade networks and physiological outcome.

Haloperidol induces neurotoxicity by the NMDA receptor downstream signaling pathway, alternative from glutamate excitotoxicity.

The NMDA receptor is believed to be important in a wide range of nervous system functions including neuronal migration, synapse formation, learning and memory. In addition, it is involved in excitotoxic neuronal cell death that occurs in a variety of acute and chronic neurological disorders. Besides of agonist/coagonist sites, other modulator sites, including butyrophenone site may regulate the N-methyl-D-aspartate receptor. It has been shown that haloperidol, an antipsychotic neuroleptic drug, interacts with the NR2B subunit of NMDA receptor and inhibits NMDA response in neuronal cells. We found that NMDA receptor was co-immunoprecipitated by anti-Ras antibody and this complex, beside NR2 subunit of NMDA receptor contained haloperidol-binding proteins, nNOS and Ras-GRF. Furthermore, we have shown that haloperidol induces neurotoxicity of neuronal cells via NMDA receptor complex, accompanied by dissociation of Ras-GRF from membranes and activation of c-Jun-kinase. Inclusion of insulin prevented relocalization of Ras-GRF and subsequent neuronal death. Haloperidol-induced dissociation of Ras-GRF leads to inhibition of membrane-bound form of Ras protein and changes downstream regulators activity that results in the initiation of the apoptotic processes via the mitochondrial way. Our results suggest that haloperidol induces neuronal cell death by the interaction with NMDA receptor, but through the alternative from glutamate excitotoxicity signaling pathway.

[Concentrational changes of mannose-binding proteins in blood plasma during noncomplicated pregnancy and in blood plasma of pregnant women with some infections].

Value of mannose-binding proteins was determined in plasma of pregnant women with no complications and also in pregnant women with some infections using affinitive chromatography. It was found that the concentration of mannose-binding proteins in blood plasma of women equals 0.152+/-0.025 mg/ml. The concentration of mannose-binding proteins in blood plasma increase during the consecutive trimesters of noncomplicated pregnancy. In the first trimester it equals 0.198+/-0.032 mg/ml, in the second 0.257+/-0.027 mg/ml and in the third trimester 0.345+/-0.034 mg/ml. We also found that the concentration of mannose-binding proteins in blood plasma of pregnant woman suffering with chlamydial infection equals 1.025+/-0.115 mg/ml, and in blood plasma of pregnant woman suffering with cytomegalovirus infection 1.278+/-0.144 mg/ml. The obtained data confirm a hypothesis of increased activity of innate immune system during pregnancy. Also according to this results complications accompanied by chlamydial and cytomegalovirus infections during pregnancy is the result of increasing activity of mannose-binding proteins.

[The action of dextrorphan and sigma ligands on the spontaneous secretion of interleukins by jurkat cell line].

Sigma receptor was demonstrated to have at least two subtypes, mediating pharmacological effects of various preparations including psychoactive, neuroleptic, cardioprotector, anti-inflammatory, immunosuppressive compounds and several steroid hormones. The stimulation of sigma receptor induces transient increase of intracellular calcium and amplifies signals from different stimuli. Pentazocine, SKF 10 047, dextrorphan, and other sigma ligands including phencyclidine and haloperidol were investigated for their potential immunoregulatory properties. We have found, that pentazocine, SKF 10 047, dextrorphan reduce spontaneous secretion of IL-8, IL-6 and IL-10 and selectively changes synthesis of IL-4 by Jurkat human T lymphocyte cells lines. Dextrorphan significantly enhanced, pentazocine, haloperidol and phencyclidine had no effect, while SKF 10 047 suppressed production of IL-4. Spontaneous secretion of IL-4 and IL-8 correlates with synthesis of nitric oxide, suggesting that NO and transitory S-nitrosylation of up-stream proteins participate in the sigma ligand dependent expression of IL-4 and IL-8 genes.

Production of homocysteine in serum-starved apoptotic PC12 cells depends on the activation and modification of Ras.

PC12 pheochromocytoma cells expressing a dominant inhibitory mutant of Ha-Ras (M-M17-26) and PC12 cells transfected with normal c-RasH (M-CR3B) have been used to investigate the role of nitrosylation and farnesylation of Ras on the production of homocysteine and the activities of the redox-sensitive transcription factors NF-kappaB and c-Fos. We found that under serum and nerve growth factor withdrawal conditions undifferentiated apoptotic M-CR3B cells accumulated more homocysteine than M-M17-26 cells, and the production of homocysteine decreased in the presence of manumycin and increased in the presence of l-NAME. Furthermore, we have shown that manumycin increased the activity of c-Fos in the M-CR3B cells and decreased the activity of NF-kappaB, while l-NAME decreased the activities of both transcription factors, and accelerated apoptosis of M-CR3B cells. In contrast, in M-M17-26 cells manumycin did not change the activity of c-Fos, nor the activity of NF-kappaB. We conclude that trophic factor withdrawal stimulates Ras, which apparently through the Rac/NADPH oxidase system induces permanent oxidative stress, modulates the activities of NF-kappaB and c-Fos, induces production of homocysteine and accelerates apoptosis. Nitrosylation of Ras is necessary for maintaining the survival of PC12 cells, while farnesylation of Ras stimulates apoptosis under withdrawal conditions.

Adhesion and clustering of charge isomers of myelin basic protein at model myelin membranes.

The association of myelin basic protein charge isomers with the lipid part of the myelin membrane was investigated at the microscopic (molecular) level in a model membrane system, using optical waveguide lightmode spectrometry to determine with high precision the kinetics of association and dissociation to planar phospholipid membranes under controlled hydrodynamic conditions and over a range of protein concentrations. Detailed analysis of the data revealed a rich and intricate behaviour and clearly showed that the membrane protein affinity is characterized by at least four independent parameters: (i) the association rate coefficient characterizing the protein-membrane interaction energy as the protein approaches the fluid-membrane interface; (ii) the protein-membrane adhesion, i.e., the probability that it will remain at the membrane after arrival; (iii) the protein conformation at the membrane; and (iv) the protein's tendency to cluster at the membrane. Some of these parameters varied in characteristic ways as the bulk solution concentration of the protein was varied, giving further clues to the detailed molecular comportment of the protein. The parameters and their characteristic variations with bulk concentration were markedly different for the different isomers. Implications of these results for neurological disorders involving demyelination, such as multiple sclerosis, are discussed.

Different effects of lectins on the ligand binding of the NMDA receptors and sigma sites in rat brain hippocampus synaptic membranes.

The effects of the lectins concanavalin A, WGA, ricin, abrin, and the mistletoe lectins from Viscum album MLI, MLII, and MLIII on the binding of ligands of the NMDA and sigma receptors in rat hippocampus synaptic plasma membranes were investigated. Binding of [3H]MK-801, [3H]glutamate, [3H]5,7-DCKA, and [3H]glycine to the membranes was decreased by 40-60% after addition of galactose-specific lectins (mistletoe lectins MLI, MLII, ricin, abrin) at concentrations of 0.01 mg/ml, but was not affected by the glucose- and mannose-specific lectin Con A, an acetylglucosamine-specific lectin WGA, or an acetylgalactosamine-specific lectin MLIII. The binding of [3H]SKF 10047 was decreased only in the presence of MLIII and did not change after addition of the other lectins. It is suggested that lectin-sensitive ligand binding sites of sigma- and NMDA receptors are located separately, and that the carbohydrate side chains of the sigma receptor do not participate in the modulation of the NMDA-receptor.