Effects of molecular crowding environment on the acquisition of toxic properties of wild-type SOD1.

BACKGROUND: Mutants of Cu,Zn-superoxide dismutase (SOD1) exhibit cytotoxicity such as aggregation and pro-oxidation after denaturation, which is thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). In the present study, we investigated the possibility of the acquisition of toxic properties for wild-type SOD1 (WT) in the demetalated (apo) form after denaturation. METHODS: Denaturation and subsequent pro-oxidant activity of SOD1 were confirmed by circular dichroism (CD) spectroscopy and fluorescence assay, respectively. The aggregation of SOD1 was investigated by native polyacrylamide gel electrophoresis (PAGE). Crowding environment was prepared by the addition of polyethylene glycol (PEG) into buffer solution. RESULTS: The structural stability of SOD1 is reduced by demetallation. Nevertheless, high temperatures around 45 °C are required to induce denaturation of apo-WT. The generated denaturated apo-WT exhibits pro-oxidant activity after the rebinding of Cu2+. In molecular crowding environment mimicked by PEG, apo-WT is found to exhibit denaturation even at physiological temperature. The denatured WT in molecular crowding environment has both the activities of pro-oxidation and aggregation. The acquisition of the pro-oxidant activity is accelerated for H43R, which is an ALS-related mutant, in molecular crowding environment. CONCLUSIONS: Apo-WT acquires the toxic properties at physiological temperature when subjected to molecular crowding environment. Molecular crowding environment also accelerates the induction of the toxicity for H43R. GENERAL SIGNIFICANCE: Molecular crowding environment in living cells becomes an instability factor inducing denaturation and subsequent toxicity for SOD1. Apo-WT also has the toxic properties in molecular crowding environment, which can be related to the pathogenesis of ALS.

The gene expression profile of human umbilical vein endothelial cells stimulated by tumor necrosis factor alpha using DNA microarray analysis.

Stimulation of vascular endothelial cells by tumor necrosis factor alpha (TNFalpha) plays a critical role in the pathogenesis of inflammation and vascular diseases. Changes in the gene expression profile in cultured human umbilical vein endothelial cells (HUVEC) treated with TNFalpha was analyzed with high-density oligonucleotide arrays comprised of 35,000 genes. TNFalpha stimulation profoundly induced genes involved in signal transduction, leukocyte adhesion and chemoattraction. ICAM-1 mRNA (fold change 111.9) was most profoundly induced followed by TNFalpha receptor-associated factor 1 (TRAF1) (95.5), Bcl3 (71.8), IL8 (65.4), fractalkaine (62.4), E-selectin (48.0), lymphotoxin beta (41.3) and VCAM-1 (31.7). In addition to these previously known genes, 18 poorly characterized or novel genes known as ESTs profoundly induced by TNFalpha. Initial sequencing analysis identified three of these the genes for squalene epoxydase, chromodomain helicase DNA binding protein 4, and CLP respectively. Further analysis of these genes will provide important information about TNFalpha signaling and function in vascular endothelial cells.

Observation of the nuCH Excited Vibrational Levels in the Ã1A" State of HCP by IR-UV Double Resonance Spectroscopy.

Rovibrational levels with one quantum of CH stretch mode, (1, v2, v3), in the Ã1A" electronic state of HCP were observed for the first time by IR-UV double resonance spectroscopy. Nine vibrational levels were identified in the energy region of 37 500-40 800 cm-1. Perturbations similar to those in the (0, v2, v3) levels were also observed in many of the vibrational levels. It was found that the CH stretch frequency in the Ã1A" state is reduced about 270 cm-1 relative to that of the electronic ground state. Copyright 1999 Academic Press.

IMMUNOLOCALIZATION OF AN ANTIGEN ASSOCIATED WITH THE INVERTEBRATE ELECTROGENIC 2Na+/1H+ ANTIPORTER

Epithelial plasma membranes from crustacean gut, kidney and gills have been shown recently to display an electrogenic 2Na+/1H+ antiporter that differs considerably in its physiological properties from the vertebrate electroneutral 1Na+/1H+ exchange paradigm. In this study, we describe the histological and cytological localization of an antigen associated with invertebrate electrogenic 2Na+/1H+ antiport in lobster (Homarus americanus) tissues using a monoclonal antibody (MAb 11) raised in mice against purified brush border membranes of the hepatopancreatic epithelium. Previous work showed that MAb 11 inhibited electrogenic 2Na+/1H+ and Ca2+/H+ exchange by hepatopancreatic brush border membrane vesicles, but was without effect on Na+-dependent d-glucose transport, suggesting a restricted inhibitory specificity to the cation exchanger. MAb 11 binding occurred at hepatopancreatic epithelial R-cell brush border membranes, at plasma membranes of the antennal gland and gill podocytes, and at vacuolar membranes of hepatopancreatic B- and R-cells, gill nephrocytes and epithelial cells of the antennal gland labyrinth and gill lamellae, as assessed by FITC-labelled secondary antibodies. Control FITC-labelled antibodies raised in mice against vertebrate keratin proteins displayed only weak non-specific binding to the tissues and cells responding intensely to MAb 11, supporting the specific nature of MAb 11 binding to its cognate antigen. The broad histological and cytological distribution of MAb 11 binding to plasma membranes and vacuolar membranes from several lobster organ systems suggests that the physiological activities regulated by its antigen, possibly an element of the invertebrate electrogenic cation exchanger, may be diverse.