A common atopy-associated variant in the Th2 cytokine locus control region impacts transcriptional regulation and alters SMAD3 and SP1 binding.

BACKGROUND: Type 2 immune responses directed by Th2 cells and characterized by the signature cytokines IL4, IL5, and IL13 play major pathogenic roles in atopic diseases. Single nucleotide polymorphisms in the human Th2 cytokine locus in particular in a locus control region within the DNA repair gene RAD50, containing several RAD50 DNase1-hypersensitive sites (RHS), have been robustly associated with atopic traits in genome-wide association studies (GWAS). Functional variants in IL13 have been intensely studied, whereas no causative variants for the IL13-independent RAD50 signal have been identified yet. This study aimed to characterize the functional impact of the atopy-associated polymorphism rs2240032 located in the human RHS7 on cis-regulatory activity and differential binding of transcription factors. METHODS: Differential transcription factor binding was analyzed by electrophoretic mobility shift assays (EMSAs) with Jurkat T-cell nuclear extracts. Identification of differentially binding factors was performed using mass spectrometry (LC-MS/MS). Reporter vector constructs carrying either the major or minor allele of rs2240032 were tested for regulating transcriptional activity in Jurkat and HeLa cells. RESULTS: The variant rs2240032 impacts transcriptional activity and allele-specific binding of SMAD3, SP1, and additional putative protein complex partners. We further demonstrate that rs2240032 is located in an RHS7 subunit which itself encompasses repressor activity and might be important for the fine-tuning of transcription regulation within this region. CONCLUSION: The human RHS7 critically contributes to the regulation of gene transcription, and the common atopy-associated polymorphism rs2240032 impacts transcriptional activity and transcription factor binding.

Asymmetric hemoglobins, their thiol content, and blood glutathione of the scalloped hammerhead shark, Sphyrna lewini.

Starch gel electrophoresis pH 8.6, or PAGE pH 8.9, of the scalloped hammerhead shark hemolysates showed three hemoglobins (Hb). An additional Hb between the two most mobile electrophoretic components was seen in starch gel electrophoresis, pH 8.1, and also in highly loaded PAGE gels. The relative concentration of these Hbs was variable among individuals, when accessed at pH 8.1. Dilution of hemolysates led to a redistribution of the Hb tetramer subunits. Under denaturing conditions, the unfractionated hemolysate was resolved in 3 Hb subunits. Isolated Hbs, named SL I-SL IV, showed unusual subunit compositions: SL I, the least mobile, is "b3c"; SL II is "a2bc"; SL III and SL IV are composed only by "a" subunits. Hemoglobins in the whole hemolysate have an average of two reactive cysteines per tetramer, which were not easily S-thiolated by glutathione, as is the case for related species. After hemoglobin denaturation, six additional -SH groups were titrated by Ellman's reagent. Methemoglobin content was low in the erythrocytes of nine examined specimens, 1.13 +/- 1.90%. High values for total erythrocyte glutathione (GSH) were found: 4.5 +/- 0.7 mM; n = 7. The ratio of 1.4 +/- 0.4 GSH/Hb is higher than usually reported for mammalians.

High sulfhydryl content in turtle erythrocytes: is there a relation with resistance to hypoxia?

Glutathione concentration in the erythrocytes of the fresh-water turtle Phrynops hilarii, as determined by the enzymic recycling method is 1.9 +/- 0.2 mM. The erythrocyte non-protein -SH, NPSH, content, as determined by the 5,5' dithiobis(2-nitrobenzoic acid), DTNB, reducing capacity is 4.6 +/- 0.8 mM. The total DTNB reducing capacity in the erythrocytes, including hemoglobin, is 26 +/- 5 mM. Incubation with oxidized glutathione, greatly increases the electrophoretic mobility of both hemoglobin components present in the erythrocytes of Phrynops hilarii, indicating the formation of mixed disulphides with glutathione. The high --SH content in the erythrocytes of P. hilarii might be part of a redox buffering principally an antioxidant mechanism involved in resistance to hypoxia, possibly along the hypoxic period as well as during reperfusion with oxygen.

High hemoglobin mixed disulfide content in hemolysates from stressed shark.

1. Hemolysate from heavily stressed smooth hammerhead shark, Sphyrna zygaena, shows three electrophoretic components, SZ I, SZ II and SZ III, whose relative concentrations are 36.4 +/- 6.8, 36.4 +/- 5.0 and 20.8 +/- 5.7%, respectively. After reduction with DTE only SZ I remained. 2. SZ I reacted with glutathione disulfide reconstitute SZ II and SZ III. 3. Non-reduced, DTE-reduced, and denatured hemoglobin were found to have 2.0 +/- 0.4, 3.7 +/- 0.6, and 9.4 +/- 0.7-SH groups, respectively. 4. Erythrocyte non-protein--SH (NPSH), including glutathione present as mixed disulfide with SZ II and SZ III, is 1.7 NPSH/Hb.

Glutathione mixed disulfides and heterogeneity of chicken hemoglobins.

1. Adult chicken hemoglobins Hb A and Hb D interact with glutathione disulfide, GSSG. The major hemoglobin, Hb A, forms at least two new components, termed GHb AI and GHb AII, and Hb D forms at least one, GHb DI. 2. At pH 8.0 and 5 degrees C, glutathione disulfide (GSSG) in a molar excess of 50 x took 6 days to complete the reaction, although at pH 8.6 and 41 degrees C only 1 hr was needed, where the hemoglobins Hb A and Hb D were converted to their most mobile forms GHb AII and GHb DI. 3. Slight molar excess (2.7 GSSG/Hb, pH 7.4, 41 degrees C), reacting for 1 hr, showed extensive formation of GHb AI and some GHb AII. 4. Electrophoretic patterns, from the reaction products of 54 GSSG/Hb excess at different times, showed a marked pH dependence. 5. Titration with pCMB (p-chloromercuribezoic acid) of DTE (dithioerythrytol)-reduced samples showed 8.0 +/- 0.4 (N = 5) -SH (sulfhydryl) per tetramer. In hemolysates not reacted with DTE, 6.0 +/- 0.4 (N = 3) -SH were detected. 6. DTE-reduced and GSSG-reacted hemoglobins showed 4.6 +/- 0.5 (N = 7) -SH and 1.5 +/- 0.4 (N = 6) -SH, respectively, as titrated by DTNB, pH 8.0. DTE-reduced hemoglobins showed four fast-reacting -SH groups, no longer present in GSSG-reacted hemoglobins. 7. Our data indicate that chicken GHb AI and GHb DI probably have two glutathionyl residues per tetramer whereas GHb AII has four.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidative stress causes intracellular reversible S-thiolation of chicken hemoglobin under diamide and xanthine oxidase treatment.

Time courses of total (GSH-t), disulfide (GSSG), and mixed disulfide (PSSG) forms of glutathione were studied in chicken blood submitted to oxidative stress induced by diamide or by the reactive oxygen species (ROS)-producing system xanthine/xanthine oxidase (X/XO). Diamide-treated blood induced an immediate increase in GSSG and PSSG, while X/XO produced a slow and sustained stress with increased values of GSSG and PSSG only after 30 and/or 60 min of incubation. Both total protein S-thiolation (mixed disulfide with glutathione) and dethiolation and hemoglobin A S-thiolation and dethiolation were clearly observed. Hemoglobin A (Hb A) was the major S-thiolated protein. We further characterized chicken Hb S-thiolation through the reaction of Hb with GSSG or the GSH/GSSG redox couple. Methemoglobin levels did not change with diamide or with X/XO treatment. Present results suggest that the most reactive cysteine pair of Hb A, the major chicken Hb, might function as an antioxidant under in vivo oxidative stress conditions.

[Hemoglobins of reptiles. Expression of alpha-D-genes in the turtles, Chrysemys picta bellii and Phrynops hilarii (Testudines)]. / Hämoglobine der Reptilien. Expression von alpha D-Genen bei Schildkröten, Chrysemys picta bellii und Phrynops hilarii (Testudines).

The hemoglobins of two turtles (Testudines)--Chrysemys picta bellii (suborder Cryptodira) and Phrynops hilarii (suborder Pleurodira)--were investigated. In both specimens we found two hemoglobin components with two distinct alpha-chains. The alpha-chains of the component HbD of Chrysemys picta bellii and of the component CII of Phyrynops hilarii belong to the alpha D-type, which has so far been reported to occur only in birds. The complete amino-acid sequences of both alpha D-chains are presented. Our further investigations on hemoglobins of other reptiles (Crocodilia, Lacertilia, Serpentes) did not give any evidence for the expression of alpha D-globin genes in the species examined. These findings are discussed with especial reference to the physiology of respiration. It is supposed that alpha D-genes were of certain significance in earlier times. There are findings suggesting that alpha D-genes are embryonic genes with persistent expression in many adult birds and turtles.

Kinetic study of hemoglobin Porto Alegre [beta 9(A6)Ser----Cys] disulfide polymer reduction.

The cleavage of HbPA disulfide polymer by GSH and its indirect cleavage by yeast glutathione reductase, via reduced glutathione is obtained. Decreasing the initial proportion of GSH in the hemolysate increases the formation of HbPA disulfide polymer. In the experimental conditions used, yeast glutathione reductase is unable to perform the direct cleavage of the mixed disulfide of HbPA and GSH, using it as substrate. The reduction of HbPA polymer to tetramers by DTE is analyzed by a pseudo-first-order kinetic and two rate constants are obtained. That of 265 X 10(-3) min-1 should be concerned with one disulfide of the closed ring and one of the open ring structure of dodecamer, while that of 38 X 10(-3) min-1 is related to disulfide reduction of the octamer. The enthalpy of activation values of 8.0 kcal.mol-1 an 17.4 kcal.mol-1 obtained, from the Arrhenius plot, for the "fast" and "slow" rate disulfide reduction, respectively, are indicative that a strong conformational strain of S--S bonds in the closed ring structure is maintained. The entropy of activation values of 24 e.u. and 52 e.u. are found for the activation of disulfides from dodecamers and octamers, respectively.

Bohr effect, electron spin resonance spectroscopy and subunit dissociation of the hemoglobin components from the turtle Phrynops hilarii.

Isoelectric points of the two haemoglobin components from the fresh-water turtle, Phrynops hilarii, were estimated by isoelectric focusing electrophoresis (IEF) to be 7.6 and 6.3, respectively for component I (CI) and component II (CII). For further studies, CI and CII were isolated by ion exchange chromatography. At pH 7.1, the estimated intraerythrocytic pH, at 25 degrees C, the values of P50, for the stripped haemoglobins, are: 19 torr for CI; 40 torr for CII and 28 torr for the whole haemolysate (pht). Oxygen binding with the stripped isolated components (CI and CII) and with the whole haemolysate (pht) show that delta log P50/delta pH, in the pH interval from 6.8 to 8.5, to be: CI = -0.35; CII = -0.80 and pht = -0.47. The Bohr curves for CI and CII intercept at pH 7.8. eta 50 for CI was 3 and invariant with pH. CII, at the more acid pH range showed biphasic Hill plots. pht shows a eta 50 that varies with pH. CI is affected by ATP, ADP, GTP and slightly by CO2, whereas CII is totally insensitive to CO2 and ATP. Additionally both components are not affected by inositol phosphates. Electron spin resonance (ESR) spectra of both components at pH 8.4 and 6.4 show that CI fails to undergo a R----T transition, while CII, with the higher Bohr effect, undergoes the R----T transition driven by pH. Molecular sieving studies of the liquid nitrogen stored CI, CII and pht show them to be disulphide polymers, CI with an apparent mol. wt of 150 X 10(3) and CII and pht of 100 X 10(3).(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen binding and acid base status of the blood from the freshwater turtle, Phrynops hilarii.

Oxygenation studies with the whole blood of Phrynops hilarii show a P50 of 38 torr at extracellular pH (pHe) of 7.4 which corresponds to an intracellular pH (pHi) of 7.05 at 25 degrees C. The blood CO2 Bohr effect was -0.56 when related to pHi. pHi is related to pHe by the following equation: pHi = 0.75.pHe + 1.54 (r = 0.99); pHi = 0.72. pHe + 1.72 (r = 0.96) at 10 and 25 degrees C respectively. Blood pHe, for 25 degrees C, was 7.519 +/- 0.254 (n = 6). Blood gas partial pressures were: pCO2 = 25.8 +/- 3.8 torr (n = 6); pO2 = 61.7 +/- 21.2 torr (n = 6). The major red cell phosphates, in mmole/l erythrocytes, n = 6, were: ATP (3.66 +/- 0.86); GTP (0.53 +/- 0.28); 2.3-DPG (0.32 +/- 0.12) and inorganic phosphates (2.00 +/- 0.35). The plasma inorganic ion composition, n = 6, was, in mEq/l: K+ (3.04 +/- 0.40); Na+ (148.4 +/- 12.6); Ca2+ (4.75 +/- 1.32); Cl- (106.6 +/- 5.0). Additional blood parameters of interest (n = 6) were: lactate (2.07 +/- 1.72 mM in plasma); erythrocytes/mm3 (416 X 10(3) +/- 4.6 X 10(3)); leucocytes/mm3 (44636 +/- 2618); haematocrit (%) (14.5 +/- 3.6); haemoglobin, g/dl (3.2 +/- 0.5); plasma protein g/dl (4.4 +/- 0.4); osmolarity (293 +/- 10 mOsm/l). The non-bicarbonate buffer value was -22.6 mmol/kg H2O/pH. For a constant CO2 content, delta pHe/delta t = 0.0141 +/- 0.002 (n = 18) and delta pHi/delta t = 0.0157 +/- 0.003 (n = 18).