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.

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.