Regulation of Redox Signaling by Selenoproteins

The unique chemistry of oxygen has been both a resource and threat for life on Earth for at least the last 2.4 billion years. Reduction of oxygen to water allows extraction of more metabolic energy from organic fuels than is possible through anaerobic glycolysis. On the other hand, partially reduced oxygen can react indiscriminately with biomolecules to cause genetic damage, disease, and even death. Organisms in all three superkingdoms of life have developed elaborate mechanisms to protect against such oxidative damage and to exploit reactive oxygen species as sensors and signals in myriad processes. The sulfur amino acids, cysteine and methionine, are the main targets of reactive oxygen species in proteins. Oxidative modifications to cysteine and methionine can have profound effects on a protein’s activity, structure, stability, and subcellular localization. Non-reversible oxidative modifications (oxidative damage) may contribute to molecular, cellular, and organismal aging and serve as signals for repair, removal, or programmed cell death. Reversible oxidation events can function as transient signals of physiological status, extracellular environment, nutrient availability, metabolic state, cell cycle phase, immune function, or sensory stimuli. Because of its chemical similarity to sulfur and stronger nucleophilicity and acidity, selenium is an extremely efficient catalyst of reactions between sulfur and oxygen. Most of the biological activity of selenium is due to selenoproteins containing selenocysteine, the 21st genetically encoded protein amino acid. The most abundant selenoproteins in mammals are the glutathione peroxidases (five to six genes) that reduce hydrogen peroxide and lipid hydroperoxides at the expense of glutathione and serve to limit the strength and duration of reactive oxygen signals. Thioredoxin reductases (three genes) use nicotinamide adenine dinucleotide phosphate to reduce oxidized thioredoxin and its homologs, which regulate a plethora of redox signaling events. Methionine sulfoxide reductase B1 reduces methionine sulfoxide back to methionine using thioredoxin as a reductant. Several selenoproteins in the endoplasmic reticulum are involved in the regulation of protein disulfide formation and unfolded protein response signaling, although their precise biological activities have not been determined. The most widely distributed selenoprotein family in Nature is represented by the highly conserved thioredoxin-like selenoprotein W and its homologs that have not yet been assigned specific biological functions. Recent evidence suggests selenoprotein W and the six other small thioredoxin-like mammalian selenoproteins may serve to transduce hydrogen peroxide signals into regulatory disulfide bonds in specific target proteins.     

Analysis of the Relationship Between Hemorheologic Parameters,Aluminum, Manganese,and Selenium in Smokers

Smoking is a significant risk factor in fatal pathologies including cardio-cerebrovascular and respiratory diseases. Aluminum (Al) is a toxic element without known biological function, but with recognized toxic effects. Manganese (Mn) and selenium (Se) are essential trace elements involved in cellular antioxidant defense mechanisms. Al, Mn, and Se carry out their metabolic activities via blood flow and tissue oxygenation. The structure and number of red blood cells (RBC) play important role in tissue oxygenation throughout blood flow. Increased hematocrit (Hct) as a result of probable hypoxia induces disturbed blood flow, RBC aggregation (RBC Agg), RBC deformability index (Tk), and oxygen delivery index (ODI). Therefore, we aimed to investigate the effects of altered Al, Mn, and Se levels on number, structure, and function of RBCs (Hct, blood and plasma viscosity (BV and PV, respectively), RBC Agg, Tk, ODI) in smokers without diagnosis of chronic obstructive pulmonary disease (COPD) in a study group (n = 128) categorized as ex-smokers (ES), smokers (S), and healthy controls (HC). Elements were analyzed in serum using ICP-OES. BV and PV were measured via Brookfield and Harkness viscometers at 37 °C, respectively. Smokers had statistically higher serum Al and Mn levels, BV, RBC, Hgb, Hct, PV, fibrinogen, RBC Agg, Tk₄₅, and pulmonary blood flow rate, but lower serum Se levels and ODI₄₅ values versus HC. In conclusion, increased Al, Mn, and hemorheological parameters and decreased Se and ODI₄₅ might result from inflammatory response in defense mechanism in smokers without diagnosis of COPD. Our results point out that serum Al, Mn, and Se with hemorheological parameters may be beneficial markers of tissue oxygenation and defense mechanism before the clinic onset of COPD in smokers.

     

Metal Pollution Assessment in Sediments of the Southeastern Black Sea Coast of Turkey

The main objective of this investigation is to determine the concentration and accumulation of heavy metals in the coastal sediments of the Southeastern Black Sea of Turkey. The selected sampling area has mainly been affected by anthropogenic activities, such as agricultural, untreated domestic and treated industrial wastewater, and mining. Metal enrichment factor (EF), geo-accumulation index (I_geo), and metal pollution index (MPI) have been calculated and relative contamination levels evaluated at all stations. In this study, the maximum EF values calculated for nickel (Ni), cobalt (Co), cadmium (Cd), lead (Pb), chromium (Cr), arsenic (As), zinc (Zn), and copper (Cu) were 1.4, 3.1, 5.2, 7.8, 7.8, 20, 26, and 42, respectively. Metal pollution has decreased in recent years, but the enrichment of heavy metals has been observed to be relatively high, especially in the uppermost 3-6 cm of the core sample from the Sürmene sampling station, which has been polluted by mining activities. In addition, factor analysis revealed that the coastal sediments from the Eastern Black Sea were influenced by several sources, namely lithogenic and anthropogenic activities (mining, wastewater discharging, agriculture).     

GLYCOALKALOID METABOLISM1 is required for steroidal alkaloid glycosylation and prevention of phytotoxicity in tomato

Steroidal alkaloids (SAs) are triterpene-derived specialized metabolites found in members of the Solanaceae family that provide plants with a chemical barrier against a broad range of pathogens. Their biosynthesis involves the action of glycosyltransferases to form steroidal glycoalkaloids (SGAs). To elucidate the metabolism of SGAs in the Solanaceae family, we examined the tomato (Solanum lycopersicum) GLYCOALKALOID METABOLISM1 (GAME1) gene. Our findings imply that GAME1 is a galactosyltransferase, largely performing glycosylation of the aglycone tomatidine, resulting in SGA production in green tissues. Downregulation of GAME1 resulted in an almost 50% reduction in α-tomatine levels (the major SGA in tomato) and a large increase in its precursors (i.e., tomatidenol and tomatidine). Surprisingly, GAME1-silenced plants displayed growth retardation and severe morphological phenotypes that we suggest occur as a result of altered membrane sterol levels caused by the accumulation of the aglycone tomatidine. Together, these findings highlight the role of GAME1 in the glycosylation of SAs and in reducing the toxicity of SA metabolites to the plant cell.     

Comet assay and analysis of micronucleus formation in patients with rheumatoid arthritis

Oxidants play a significant role in causing oxidative stress, which underlies the pathogenesis of rheumatoid arthritis (RA). Genetic factors that predispose individuals to RA are considered to play an important role in the development of the disease. The aim of this study was to determine, by use of the comet assay and the micronucleus (MN) test, whether DNA damage has an effect on the pathogenesis of RA. Furthermore, our aim was to show if there is an association between oxidative stress and DNA damage in RA. This study was conducted between January and June 2010 in the Erzurum Training and Research Hospital. We analyzed lymphocytes from patients with RA (12 in active and 31 in inactive periods) and 30 healthy controls for effects in the comet assay and the MN test. In addition, the levels of malondialdehyde (MDA) and superoxide dismutase (SOD), the activity of glutathione peroxidase (GSH-Px), the erythrocyte sedimentation rate (ESR) and the high-sensitivity C-reactive protein (hs-CRP) rate were determined in all the subjects. The comet-tail length, the MN frequencies and the MDA levels were significantly higher in patients--both in the active and the inactive period--than in the controls. In contrast, the SOD and GSH-Px levels were significantly lower in both patient groups than in the controls. Our results suggest that an increased plasma MDA level and decreased plasma GSH-Px and SOD levels reflect the higher degree of oxidative stress in RA patients, a situation that may impair genetic stability in those patients. Thus, the results suggest that increased DNA damage may play an important role in the pathogenesis of RA.     

Inter‐varietal structural variation in grapevine genomes

Grapevine (Vitis vinifera L.) is one of the world's most important crop plants, which is of large economic value for fruit and wine production. There is much interest in identifying genomic variations and their functional effects on inter‐varietal, phenotypic differences. Using an approach developed for the analysis of human and mammalian genomes, which combines high‐throughput sequencing, array comparative genomic hybridization, fluorescent in situ hybridization and quantitative PCR, we created an inter‐varietal atlas of structural variations and single nucleotide variants (SNVs) for the grapevine genome analyzing four economically and genetically relevant table grapevine varieties. We found 4.8 million SNVs and detected 8% of the grapevine genome to be affected by genomic variations. We identified more than 700 copy number variation (CNV) regions and more than 2000 genes subjected to CNV as potential candidates for phenotypic differences between varieties.     

Thrombin specificity. Selective cleavage of antibody light chains at the joints of variable with joining regions and joining with constant regions

Selective cleavage of polypeptides by alpha-thrombin can be reasonably predicted [Chang, J.Y. (1985) Eur. J. Biochem. 151,217-224]. This knowledge was applied to the selective cleavage of antibody light chains with the aim of producing intact fragments of both variable region and constant region. (a) Mouse kappa light chains 10K26 and 10K44 from anti-(azobenzene arsonate) antibodies contain 20 Arg/Lys-Xaa bonds. Only two of them, one ProArg-Thr bond located at the joint of the variable region with the joining peptide and one ValLys-Ser bond located near the carboxyl-terminal end of the constant region, were selectively cleaved by alpha-thrombin. The ProArg-Thr bond has a 50% cleavage time of about 10 min under the designated conditions, whereas the ValLys-Ser has a 50% cleavage time approx. 9-10 h. A single selective cleavage at the joining position of the variable region and joining peptide can be achieved by short-time thrombin digestion. Fragments containing intact variable region (1-96) and intact joining peptide-constant region (97-214) obtained from both denatured and native light chains of 10K26 can be separated by gel filtration. (b) lambda light chains from both human and mouse all begin with the GlnProLys-(Ala/Ser) structure (positions 108-111) at their constant regions. This ProLys-Ala/Ser bond is also susceptible to specific thrombin cleavage. Four human lambda chain (KERN, NEI, NEW, VOR) and one mouse lambda chain (RPC20) were shown to be selectively cleaved by thrombin at these ProLys-Ala/Ser bonds. For human lambda chains, the 50% cleavage time at this ProLys-Ala bond was approx. 3-4 h under the designated conditions. Six additional thrombin specific cleavages were also detected within the variable regions of NEI, VOR and RPC-20. (c) Heparin inhibits thrombin cleavage of Arg/Lys-Xaa bonds located near the center of the antibody light chain, but slightly activates thrombin cleavage of those located near the amino or carboxyl-terminal ends of the protein. The significance of these findings is threefold. (a) It demonstrates that selective cleavage of large polypeptides by alpha-thrombin can also be reasonably predicted. (b) It provides a useful method for light chain fragmentation which can greatly facilitate amino acid sequencing of antibodies. (c) It serves to generate fragments containing intact variable regions and constant regions from antibody light chains of human and mouse. Such fragments may be useful for chemical semisynthesis of a human-mouse light chain chimeras.     

Biological activities of human recombinant interferon alpha/beta targeted by anti-Epstein-Barr virus monoclonal antibodies

The requirement of high doses of interferon (IFN) during therapy severely restrict its application. Thus a model using an Epstein-Barr virus (EBV) membrane antigen (MA) specific monoclonal antibody (MAb) was developed to assess the feasibility of coupling minimal amounts of IFN to a MAb and specifically delivering the IFN to the target cells. Coupled IFN was first shown to retain fully both its anti-viral and anti-proliferative properties when tested on human tumor cell lines QIMR-WIL (EBV-MA+) and the U-266 (EBV-MA-). A series of in vitro pulsing experiments demonstrated the specific targeting of both the anti-viral and anti-proliferative properties of IFN to the EBV-MA+ QIMR-WIL cells and not EBV-MA- cell lines.     

Linkage relationships and allelic associations of the cystic fibrosis locus and four marker loci

Summary The linkage relationships between the cystic fibrosis (CF) locus and four marker loci (MET-H, MET-D, D7S8 and D7S16), allelic associations between these loci and the extent of informativity at these marker loci were investigated in a sample of 206 families with at least one child affected by CF. The data were contributed by 11 laboratories from Europe and Israel. The maximum lod scores and recombination frequency estimates ( ) (and confidence limits of ) were: 18.3 at =0.007(0.001–0.038) for CF vs. MET, 11.0 at (0.001–0.068) for CF vs. D7S8, and 5.7 at =0.0(0.0–0.064) for CF vs. D7S16. A gene order of CF-MET-D7S8 was best supported by the data, but its preference to the order D7S8-CF-MET is mainly based on one single family. There are significant allelic associations between CF, MET, D7S8 and D7S16; these allelic associations affect the risk of random individuals to be carriers of CF.