The Cu/Zn superoxide dismutase +35A/C (rs2234694) variant correlates with altered levels of protein carbonyls and glutathione and associates with severity of COPD in a Tunisian population

Chronic obstructive pulmonary disease (COPD) is a major cause of mortality that has been associated with inflammation and oxidative stress. The purpose of the present case–control study was to determine the relationships between oxidative stress-related genetic variants and the risk and severity of COPD, as well as, the influence of these variants on inflammatory and oxidative stress parameters. Genotyping of superoxide dismutase 1 (SOD1) + 35 A/C (rs2234694), catalase [A-21T (rs7943316), C-262T (rs1001179)] and glutathione peroxidase 1 (reduced glutathione (GSH)-Px1) 198Pro/Leu (rs1050450) was carried out in 143 patients with COPD and 216 healthy controls using PCR-RFLP. Serum levels of IL-6 and TNF-α were determined by enzyme-linked immunosorbent assays (ELISA), while the levels of reduced GSH, total antioxidant status (TAS), H₂O₂, lipid peroxides (TBARS) and protein carbonyls (PCs) were determined using spectrophotometric methods. We also evaluated the activities of GSH-Px, catalase, and superoxide dismutase (SOD) in both plasma and erythrocytes. We did not observe significant differences in the genotype and allele frequencies of chosen variants between COPD patients and healthy controls. A significant correlation was retrieved between the SOD1?+?35A/C variant and disease severity (odds ratios (OR) = 0.15, p?=?0.04). In addition, patients having the +35AC genotype presented increased plasma levels of GSH and a reduced level of PCs (p?=?0.03, p?=?0.04, respectively). The present data highlighted the important role of antioxidant enzymes and their genetic variants in the oxidative stress-mediated pathogenesis and progression of COPD.     

Differential effects of unaggregated and aggregated amyloid β protein (1–40) on K+ channel currents in primary cultures of rat cerebellar granule and cortical neurones

The effects of amyloid beta protein on voltage-gated K(+) channel currents were studied using the whole-cell patch-clamp technique. The 1-40 amino acid form of amyloid beta protein was applied to primary cultures of rat cerebellar granule and cortical neurones for 24 h. Both the unaggregated and aggregated forms of the peptide, which have differing biological activities, were used. In cerebellar granule neurones, 24-h pre-incubation with 1 microM unaggregated amyloid beta protein resulted in a 60% increase in the 'A'-type component of K(+) current. Increased delayed rectifier activity was Cd(2+)-sensitive and was presumed to be secondary to an increase in voltage-gated Ca(2+) channel current activity. Unaggregated amyloid beta protein had no effect on any component of the K(+) channel current in cortical neurones. One micromolar of aggregated amyloid beta protein had no effect on K(+) channel current in either cell type but reduced cell survival within 24 h as measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assays. The unaggregated form of amyloid beta protein had no neurotoxic effects when applied to either neurone type for up to 72 h. These data indicate that the unaggregated, non-pathological form of amyloid beta protein causes changes in the ion channel function of neurones, possibly reflecting a physiological role for the peptide.     

Membrane targeting, shedding and protein interactions of brain acetylcholinesterase

The early stages of Alzheimer's disease are characterized by cholinergic deficits and the preservation of cholinergic function through the use of acetylcholinesterase inhibitors is the basis for current treatments of the disease. Understanding the causes for the loss of basal forebrain cholinergic neurons in neurodegeneration is therefore a key to developing new therapeutics. In this study, we review novel aspects of cholinesterase membrane localization in brain and propose mechanisms for its lipid domain targeting, secretion and protein-protein interactions. In erythrocytes, acetylcholinesterase (AChE) is localized to lipid rafts through a GPI anchor. However, the main splice form of AChE in brain lacks a transmembrane peptide anchor region and is bound to the 'proline-rich membrane anchor', PRiMA, in lipid rafts. Furthermore, AChE is secreted ('shed') from membranes and this shedding is stimulated by cholinergic agonists. Immunocytochemical studies on rat brain have shown that membrane-associated PRiMA immunofluorescence is located selectively at cholinergic neurons of the basal forebrain and striatum. A strong association of AChE with the membrane via PRiMA seems therefore to be a specific requirement of forebrain cholinergic neurons. α7 nicotinic acetylcholine receptors are also associated with lipid rafts where they undergo rapid internalisation on stimulation. We are currently probing the mechanism(s) of AChE shedding, and whether this process and its apparent association with α7 nicotinic acetylcholine receptors and metabolism of the Alzheimer's amyloid precursor protein is determined by its association with lipid raft domains either in normal or pathological situations.     

Heterogeneous expression of Ca(2+) handling proteins in rabbit sinoatrial node.

We investigated the densities of the L-type Ca(2+) current, i(Ca,L), and various Ca(2+) handling proteins in rabbit sinoatrial (SA) node. The density of i(Ca,L), recorded with the whole-cell patch-clamp technique, varied widely in sinoatrial node cells. The density of i(Ca,L) was significantly (p<0.001) correlated with cell capacitance (measure of cell size) and the density was greater in larger cells (likely to be from the periphery of the SA node) than in smaller cells (likely to be from the center of the SA node). Immunocytochemical labeling of the L-type Ca(2+) channel, Na(+)-Ca(2+) exchanger, sarcoplasmic reticulum Ca(2+) release channel (RYR2), and sarcoplasmic reticulum Ca(2+) pump (SERCA2) also varied widely in SA node cells. In all cases there was significantly (p<0.05) denser labeling of cells from the periphery of the SA node than of cells from the center. In contrast, immunocytochemical labeling of the Na(+)-K(+) pump was similar in peripheral and central cells. We conclude that Ca(2+) handling proteins are sparse and poorly organized in the center of the SA node (normally the leading pacemaker site), whereas they are more abundant in the periphery (at the border of the SA node with the surrounding atrial muscle).     

ACE Inhibitory and Antioxidant Activities of Novel Peptides from <Emphasis Type="Italic">Scorpaena notata</Emphasis> By-product Protein Hydrolysate

This study describes the isolation of angiotensin I converting enzyme and antioxidative peptides from head protein hydrolysate of red scorpionfish (Scorpaena notata) prepared by treatment with a protease from the fungus Penicillium digitatum. After ultrafiltration, three peptides were isolated by a two-step procedure: size exclusion chromatography on a Toyopearl HW-40 followed by reversed-phase high performance liquid chromatography (RP-HPLC) with a high purification yield of 2.22 mg of peptide/g of initial protein. Active peptides were then identified by nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS–MS), corresponding to the following sequences: Gln–Gln–Pro–His–Ser–Arg–Ser–Lys–Gly–Phe–Pro–Gly–Pro (1424.724 Da), Gly–Gln–Lys–Ser–Val–Pro–Glu–Val–Arg (1000.565 Da) and Val–Glu–Gly–Lys–Ser–Pro–Asn–Val (830.448 Da). Peptides D-I, E-I and F-I showed high angiotensin-I converting enzyme inhibitory activity with an IC₅₀ values of 0.98, 1.69 and 1.44 µM, respectively as well as a synergistic antioxidant activity between the different fractions. Thus, we have demonstrated that underutilized wastes can be valorized by production of peptides that can be used as potential therapeutic compounds active against oxidative stress and hypertension.     

p53 differentially inhibits cell growth depending on the mechanism of telomere maintenance

Telomere stabilization is critical for tumorigenesis. A number of tumors and cell lines use a recombination-based mechanism, alternative lengthening of telomeres (ALT), to maintain telomere repeat arrays. Current data suggest that the mutation of p53 facilitates the activation of this pathway. In addition to its functions in response to DNA damage, p53 also acts to suppress recombination, independent of transactivation activity, raising the possibility that p53 might regulate the ALT mechanism via its role as a regulator of recombination. To test the role of p53 in ALT we utilized inducible alleles of human p53. We show that expression of transactivation-incompetent p53 inhibits DNA synthesis in ALT cell lines but does not affect telomerase-positive cell lines. The expression of temperature-sensitive p53 in clonal cell lines results in ALT-specific, transactivation-independent growth inhibition, due in part to the perturbation of S phase. Utilizing chromatin immunoprecipitation assays, we demonstrate that p53 is associated with the telomeric complex in ALT cells. Furthermore, the inhibition of DNA synthesis in ALT cells by p53 requires intact specific DNA binding and suppression of recombination functions. We propose that p53 causes transactivation-independent growth inhibition of ALT cells by perturbing telomeric recombination.