A conserved mechanism for steroid receptor translocation to the plasma membrane

Multiple steroid receptors (SR) have been proposed to localize to the plasma membrane. Some structural elements for membrane translocation of the estrogen receptor alpha (ER alpha) have been described, but the mechanisms relevant to other steroid receptors are entirely unknown. Here, we identify a highly conserved 9 amino acid motif in the ligand binding domains (E domains) of human/mouse ER alpha and ER beta, progesterone receptors A and B, and the androgen receptor. Mutation of the phenylalanine or tyrosine at position-2, cysteine at position 0, and hydrophobic isoleucine/leucine or leucine/leucine combinations at positions +5/6, relative to cysteine, significantly reduced membrane localization, MAP and PI 3-kinase activation, thymidine incorporation into DNA, and cell viability, stimulated by specific SR ligands. The localization sequence mediated palmitoylation of each SR, which facilitated caveolin-1 association, subsequent membrane localization, and steroid signaling. Palmitoylation within the E domain is therefore a crucial modification for membrane translocation and function of classical sex steroid receptors.     

Mitochondrial nitric-oxide synthase stimulation causes cytochrome c release from isolated mitochondria. Evidence for intramitochondrial peroxynitrite formation.

Nitric oxide (NO) is synthesized by members of the NO synthase (NOS) family. Recently the existence of a mitochondrial NOS (mtNOS), its Ca(2+) dependence, and its relevance for mitochondrial bioenergetics was reported (Ghafourifar, P., and Richter, C. (1997) FEBS Lett. 418, 291-296; Giulivi, C., Poderoso, J. J., and Boveris, A. (1998) J. Biol. Chem. 273, 11038-11043). Here we report on the possible involvement of mtNOS in apoptosis. We show that uptake of Ca(2+) by mitochondria triggers mtNOS activity and causes the release of cytochrome c from isolated mitochondria in a Bcl-2-sensitive manner. mtNOS-induced cytochrome c release was paralleled by increased lipid peroxidation. The release of cytochrome c as well as increase in lipid peroxidation were prevented by NOS inhibitors, a superoxide dismutase mimic, and a peroxynitrite scavenger. We show that mtNOS-induced cytochrome c release is not mediated via the mitochondrial permeability transition pore because the release was aggravated by cyclosporin A and abolished by blockade of mitochondrial calcium uptake by ruthenium red. We conclude that, upon Ca(2+)-induced mtNOS activation, peroxynitrite is formed within mitochondria, which causes the release of cytochrome c from isolated mitochondria, and we propose a mechanism by which elevated Ca(2+) levels induce apoptosis.     

Association of IL-15 and IP-10 Serum Levels with Cytomegalovirus Infection,CMV Viral Load and Cyclosporine Level after Kidney Transplantation

Background:Cytomegalovirus (CMV) infection is the most common complications following kidney transplantation. Natural killer (NK) cells demonstrated critical anti-viral role in controlling and elimination of CMV after transplantation. Interleukin-15 (IL-15) is a pleiotropic cytokine that promotes the activity of NK cells and strengthens the acquired immune system. Also, IP10 (CXCL10) is a chemotactic factor which regulates NK cell recruitment and antiviral immune response. We aimed to determine the correlation between the serum levels of IL-15 and IP-10 cytokines with CMV infection, CMV viral load, and cyclosporine as a major immunosuppressive treatment after transplantation.Methods:Fifty-eight kidney transplant recipient patients without evidence of CMV virus disease before transplantation surgery were included in the study. From the day of transplant surgery, the patients were evaluated based on the presence of CMV Ag pp65, CMV viral load, serum levels of IL-15 & IP-10, Cyclosporine levels (C0 & C2), Glomerular Filtration Rate (GFR), and hematological & biochemical Index, up to 75 days.Results:Comparison analysis of serum levels of IL-15 and IP-10 showed no significant association with CMV infection in kidney transplant recipients. In addition, CMV viral load and cyclosporine levels at C0 and C2 did not affect patients'' IL-15 and IP-10 levels.Conclusion:The levels of IP-10 and IL-15 cytokines are not affected with CMV infection, even if a viral infection occurs in the early days after transplantation or long afterwards. In addition, taking the different levels of cyclosporine did not affect the cytokines levels. Other mechanisms may play a role in maintaining the levels of these cytokines.Key Words: Cytokine, Cytomegalovirus, IP-10, Interleukin-15, Transplantation     

Effect of surface modification of rat erythrocytes of different ages on their partitioning behavior in charge-sensitive two-polymer aqueous phases

Partitioning differences between cells in two-polymer aqueous phase systems originate from subtle differences between the surface properties of cells. Because of the exponential relation between the parameters affecting the partition ratio (P) and the P itself, differences in membrane components suspected of effecting the differential partitioning of closely related cell populations cannot be directly established by conventional chemical assay techniques. In order to study the chemical nature of the components responsible for the age-related changes in surface properties of rat red cells we have devised an approach which uses a combination of isotopic labeling of erythrocyte subpopulations of distinct cell age with different enzyme and/or chemical treatments followed by countercurrent distribution in charge-sensitive two-polymer aqueous phase systems. These studies show that: neuraminidase-susceptible sialic acid is not responsible for the cell age-related surface differences detected by partitioning; the component(s) responsible for the cell age-related surface differences can be extracted (from aldehyde-fixed red cells) with ethanol or cleaved with dilute sulfuric acid. Our data are consistent with the hypothesis that ganglioside-linked sialic acid is the chemical moiety responsible for the cell charge-associated surface differences among rat red blood cells of different ages.     

Endothelin Receptors on Cultured Fetal Rat Diencephalic Glia

The recently described family of proteins, the endothelins, are produced in neurons and bind to extravascular sites in the CNS. To characterize these receptors, we carried out studies on cultures of fetal rat diencephalic glia. Scatchard analysis of saturation binding studies was done for astrocytes (greater than 95% glial fibrillary acidic protein positive). For endothelin 3 (ET-3) and ET-1, respectively, a single receptor class of KD 0.41 +/- 0.05 and 0.62 +/- 0.04 nM and a receptor density of 42 +/- 0.8 and 58 +/- 1.1 fmol/mg of glial protein was found. Bound and cross-linked 125I-ET-3 or ET-1 showed a single predominant receptor band at Mr 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis; a minor band at 50,000 was also seen. At concentrations equal to the receptor KD, the major brain form of ET, ET-3, stimulated a nearly 200% increase in the incorporation of tritiated thymidine into glia. ET-3 and ET-1 significantly impaired the ability of atrial natriuretic peptide (ANP) to generate cyclic GMP, and isoproterenol to generate cyclic AMP. The ability of ET to inhibit ANP-induced cyclic GMP generation was reversed by cycloheximide and actinomycin-D, whereas the inhibition of isoproterenol-induced cyclic AMP generation was partially and significantly blocked by inhibitors of calcium influx, protein kinase C action, or G protein activation, as well. Astrocytes from this part of the brain are a potential target cell for endothelin, assuming these findings are present in vivo. This neuropeptide may serve as a growth stimulator for astrocytes and modulator of the actions of catecholamines or ANP on glia by inhibiting second messenger generation.     

Novel spiro[indene-1,2′-quinazolin]-4′(3′H)-one derivatives as potent anticonvulsant agents: One-pot synthesis,in vivo biological evaluation,and molecular docking studies

A new series of spiro[indene-1,2′-quinazolin]-4′(3′H)-one derivatives 4a–m were synthesized via a one-pot method and evaluated for anticonvulsant activities using pentylenetetrazole (PTZ) and maximal electroshock (MES)-induced seizures. Obtained results demonstrated that these compounds have not anticonvulsant activity in PTZ test while are active in the MES test. Among the synthesized compounds, the best anticonvulsant activity was obtained with compound 4h . This compound also was not neurotoxic. Given that the title new compounds have the pharmacophore requirement for benzodiazepine (BZD) receptor agonist, the most potent compound was assayed in vivo and in silico as BZD receptor agonist. After treatment with flumazenil as a standard BZD receptor antagonist, anticonvulsant activity of compound 4h decreased. Therefore, the involvement of BZD receptors in anticonvulsant activity of this compound confirmed. Furthermore, docking study of compound 4h in the BZD-binding site of GABA_A receptor confirmed that this compound interacted with the important residues.     

Evidence that the plastid translocon Tic40 components possess modulating capabilities

The transport of proteins into the plastid is a process that faces changing cellular needs such as the situation found in different plant organs or developing tissues. The plastid translocon must therefore be responsive to the changing cell environment to deliver efficiently different arrays of structurally diverse proteins. Although the Tic40-related envelope proteins appear to be translocon components designed to address the varying needs of protein translocation, details of their involvement remain elusive. This study was thus designed to combine plant-based experiments and yeast mitochondrion-based approaches for unveiling clues related to how the Tic40 components may behave during the protein translocation process. The main findings related to how Tic40 proteins may work are: 1) natural fluctuations are apparent in developing tissues, in different organs of the same plant, and in different species; 2) transgenic Arabidopsis seedlings can tolerate functionally a wide range of variations in Tic40 levels, from partial suppression to excessive production; 3) the Tic40 proteins themselves exhibit configurational changes in their association with yeast mitochondria in response to different carbon sources; 4) the presence of Tic40 proteins in yeast mitochondria influences regulatory aspects of the mitochondrial translocon; and 5) the Tic40 proteins associate with mitochondrial translocon components involved in regulatory-like events. The combined data provide evidence that Tic40 proteins possess modulating capabilities.     

Dextran causes aggregation of mitochondria and influences their oxidoreductase activities and light scattering

It has been reported that dextrans diminish the intermembrane space of mitochondria, increase the number of contact sites between the inner and the outer mitochondrial membranes, decrease the outer membrane permeability to adenosine 5(')-diphosphate, and change the kinetic properties of mitochondrial kinases. In the present work the influence of dextran M40 (5% w/v) on the oxidoreductase activities of the inner and outer membranes of mitochondria, the interaction of cytochrome c with mitochondrial membranes, and the light scattering by rat liver mitochondria were studied. No influence of dextran on the release of cytochrome c from mitochondria or its interaction with mitochondrial membranes was observed. Decreases in the NADH-oxidase (to 80+/-2% of the control), NADH-cytochrome c reductase (to 26+/-2%), succinate-cytochrome c reductase (to 70+/-5%), and NADH-ferricyanide reductase (to 75+/-3%) activities induced by dextran, which may be due to the mitochondrial aggregation, were observed. The formation of aggregates was registered by light scattering, confirmed by light microscopy, and explained within the framework of the Gouy-Chapman theory of the electrical double layer. The observed mitochondrial aggregation seems to be useful also for understanding the mechanisms of mitochondrial condensation and perinuclear clustering during apoptosis.