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.

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.

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.

[Isolation and study of some properties of new phosphopeptides from nerve tissue]. / Vydelenie i izuchenie nekotorykh svoistv novykh fosfopeptidov nervnoi tkani.

The phosphopeptides were extracted by organic solvents from the lipid-deleted homogenate of rat brain tissue. The latter was obtained by means of trichloroacetic acid from rats injected with 32P-NaH2PO4 into brain ventricles. Chromatography of the brain extract on DEAE-Sephadex A-25 and gel filtration through Sephadex G-25 and G-15 resulted in 3 homogenous fractions of the phosphopeptides characterized by the maximal rate of the label incorporation. These fractions appeared to be homogenous during polyacrylamide gel electrophoresis and thin-layer chromatography. The molecular weights of the fractions are equal to 1200, 1100 and 900, respectively; the isoelectric points lie at 3.2, 3.0 and 3.1, respectively. All the phosphopeptides tested contain large amounts of glycine, serine and glutamic acid and have alanine as N-terminal amino acid.

[Effect of thermostable protein kinase modulator on Mg, Ca-ATPase from nervous tissue]. / Deistvie termostabil'nogo moduliatora proteinkinazy na Mg2+, Ca2+-ATPazu nervnoi tkani.

Isoelectric focusing of a purified fraction of thermostable modulator of 3',5'-AMP-dependent protein kinase revealed five individual proteins, the main protein having an isoelectric point of 4,05. The molecular weight of this protein as determined by gel filtration is 8000--9000. The protein with a pI of 4,05 binds Ca2+ and in contrast to the original modulator inhibits the endogenous 3',5'-AMP-dependent phosphorylation of synaptic membranes. An addition of the original modulator fraction to the microsomes isolated from nervous tissue increases the Mg, Ca-ATPase activity and absorption of 45Ca. Neither the protein with a pI of 4,05 nor other individual proteins affect the activity of transport ATPase. The activating effect is partly restored after mixing of all the five subfractions. It is assumed that these proteins are aggregated by Ca2+ and change the activity of ATPase or membrane 3',5'-AMP-dependent protein kinase depending on the concentration of calcium ions.

[Reaction mechanism of succinyl CoA synthetase from pigeon thoracic muscle]. / Izuchenie mekhanizma reaktsii suktsinil-CoA-sintetazy grudnoí myshtsy golubia.

The ability of succinyl-CoA-synthetase from pigeon thoracic muscle to interact with ATP is investigated. gamma-32P-ATP and 8-14C-ATP were used in experiments. It is found that the enzyme, when reacting with ATP in the presence of Mg2+, forms a complex containing 2 moles of ATP residue and 2 moles of phosphoric acid residue (splitted from ATP) per 1 mole of protein. After 2 hours of incubation at 0-4 degrees C, the complex is converted into another one, containing 4 residues of phosphoric acid per 1 mole of @protein. Both complexes are active, and their incubation with succinate and CoA results in the formation of succinyl-CoA. The reaction capacity of these enzyme complexes with some reaction substrates is investigated. The enzyme complex containing 2 phosphoric acid residues and 2 nucleotide residues is found to interact neither with CoA, nor with succinate. The enzyme complex containing 4 phosphoric acid residues does not react with CoA, but it interacts with 14C-succinate, releasing inorganic phosphate in the amount equivalent to the equimolar amount of protein-binding succinic acid.

[Mechanism of the reaction of substrate phosphorylation in the tricarboxylic acid cycle]. / Mekhanizm reaktsii substratnogo fosforilirovaniia v tsikle trikarbonovykh kislot.

The suggested sequence of the reactions differs from those presented in literature and includes the following compounds as intermediate ones: the complex of enzyme having 2ATP and two residues of phosphoric acid; the complex of enzyme having four residues of phosphoric acid; the complex of enzyme containing two residues of phosphoric acid and two residues of succinic acid. Sequences of the reactions suggested in literature involve as intermediate compounds a phosphorylated derivative of the enzyme (E--P), phosphorylated derivative CoA linked with the enzyme (E.CoA--P), a high-ergic compound of the enzyme with CoA (E.CoA) and succinyl phosphate linked with the enzyme (E.succinyl--P).

[Participation of phosphopeptides in oxidative amino acid exchange in brain tissue]. / Ob uchastii fosfopeptidov v okislitel'nom obmene aminokislot v mozgovom tkani.

Specific proteins and phosphopeptides of nervous tissue soluble in acidified organic solvents have been shown to have an active participation in the metabolism of ammonia. Nitrogen phosphopeptides (amides) bound to phosphate residues which are non stable in acids. It was shown that acid-labil nitrogen (amide group) of phosphopeptides, bound to phosphate risidue, participates in the metabolism of ammonia formed during the oxidative deamination of amino acids. The data obtained indicate that 3'--5'-AMP dependent proteinkinase participates in the phosphorilation of phosphopeptides.