Ribonuclease hydration and its heat stability in solutions of H2O and D2O

Ribonuclease hydration in wide concentration and temperature range using differential scanning microcalorimetry and NMR technique was studied. The temperature- concentration equilibrium diagram for H2O--ribonuclease system was suggested. Protein hydration in D2O was shown to be higher than in ordinary water. NMR measurements in dilute protein solutions showed that cooperative temperature denaturation was followed by solvation changes. Models of globular protein hydration are discussed.     

The effect of cooling rate and warming rate on the packing effect in human erythrocytes frozen and thawed in the presence of 2 M glycerol

The effect of hematocrit (2 versus 75%) has been studied on human red blood cells frozen and thawed in 2 M glycerol at a range of cooling rates (0.8-850 degrees C/min) and warming rates (0.1-200 degrees C/min). The data obtained at a hematocrit of 2% agree well with the data of R. H. Miller and P. Mazur (Cryobiology 13, 404-414, 1976). The results at a hematocrit of 75% show a decrease in recovery with increased cell packing, primarily dependent on warming rate at cooling rates less than 100 degrees C/min and on cooling rate at higher cooling rates. Rapid warming reduced the packing effect, whereas cooling faster than 100 degrees C/min accentuated it. It has been argued that these effects are unlikely to be due to modulation of the generally accepted mechanisms of freezing injury, that is, solution effects and intracellular freezing. It has been suggested that they may be explained by effects of cooling and warming rates on the dimensions of the liquid channels in which the cells are accommodated during freezing and thawing.     

The effect of cell concentration on the recovery of human erythrocytes after freezing and thawing in the presence of glycerol

Human erythrocytes, suspended in 2.5 M glycerol in phosphate-buffered saline, were frozen at 35 °C/min to between ?60 and ?65 °C and thawed at 5 °C/min. It was found that cell concentration had a marked effect on cell recovery. When the hematocrit was less than 20%, hemolysis was less than 1% but when the hematocrit exceeded 50%, hemolysis increased, reaching 16% at an hematocrit of 80%. Possible causes of this effect are discussed, and it is suggested that augmentation of solution effects and intracellular freezing may provide a sufficient explanation. The importance of this cell-packing effect for attempts to preserve whole organs is discussed.     

Permeability of erythrocytes to glycerol and its acylated derivatives in the camel and dog

• 1.1. Glycerol and glyeerol derivatives transport was compared in human, dog and camel erythrocytes.
• 2.2. The camel erythrocytes had the slowest glyeerol transport, that of the dog erythrocytes was also slow, while the quickest transport occurred in human erythrocytes.
• 3.3. In glyeerol mono-, di- and triacetates the transport in camel and dog erythrocytes increased until in the triacetate the quickest transport was found.
• 4.4. Addition of copper ions had no inhibiting effect on glyeerol transport in camel erythrocytes but a striking effect was seen in human erythrocytes.
• 5.5. Changes in pH greatly affected transport in camel erythrocytes, a decline in pH enhanced and an increase in pH slowed the transport. Storage of camel erythrocytes did not affect the glyeerol transport in camel erythrocytes.
• 6.6. It is concluded that the mechanism of glyeerol transport in camel erythrocytes is one of a non-facilitated diffusion.

     

Synthesis of Isozymes of Superoxide Dismutase in Maize Leaves in Response to O3, SO2 and Elevated O2

Matters, G. L. and Scandalios, J. G. 1987. Synthesis of isozymesof superoxide dismutase in maize leaves in response to O₃ SO₂and elevated O₂.—J. exp. Bot 38: 842–852. The activities of the enzymes superoxide dismutase (SOD) andcatalase were determined in maize leaves treated with O₃or SO₂for8 h, or with elevated levels of oxygen for up to 96 h. NeitherO₃nor SO₂significantly increased the levels of superoxide dismutaseor catalase activity. However, after 72 h in an atmosphere containing90% oxygen, superoxide dismutase activity was increased, butnot the activities of catalase, ascorbate pcroxidase, and malatedehydrogenase. Immunological analysis showed that amounts ofthe cytosolic superoxide dismutase isozymes, SOD-2 and SOD-4,were increased by the elevated oxygen but not the chroloplast(SOD-1) or mitochondrial (SOD-3) isozymes. Immunoprecipitationof translation products of leaf polysomes indicated that thehigher levels of SOD-2 and SOD-4 were due to increased amountsof polysome-bound mRNA coding for these proteins. The specificresponse of SOD-2 and SOD-4 to 90% oxygen treatments contrastswith the increase in all SOD isozymes in maize leaves treatedwith the herbicide paraquat. Key words: Air pollutants, maize, oxidative stress, oxygen, superoxide dismutase     

Enzymic Pathways Involved in Cell Response to H2O2

An influence of possible interaction of glutathione peroxidase and cyclooxygenase on the clonogenic survival of epithelial cells exposed in vitro to H₂O₂ was investigated. Indomethacin served as the inhibitor of cyclooxygenase, and the use of alkaline (7.5) or acidic (6.5) pH combined with controlled supply of glucose modified glutathione peroxidase activity. Indomethacin affected survival of cells exposed to H₂O₂ in a biphasic manner, enhancing cytotoxicity at lower hydrogen peroxide concentrations, and diminishing it at higher concentrations. The turning point moved gradually to higher concentrations of H₂O₂ corresponding to the augmented decomposition of hydrogen peroxide caused by increased activity of glutathione peroxidase. The data revealed that both enzymic pathways interact in the presence of H₂O₂, resulting in the overall cell survival different from that obtained after inhibition of either.     

Enzymic Pathways Involved in Cell Response to H2O2

An influence of possible interaction of glutathione peroxidase and cyclooxygenase on the clonogenic survival of epithelial cells exposed in vitro to H₂O₂ was investigated. Indomethacin served as the inhibitor of cyclooxygenase, and the use of alkaline (7.5) or acidic (6.5) pH combined with controlled supply of glucose modified glutathione peroxidase activity. Indomethacin affected survival of cells exposed to H₂O₂ in a biphasic manner, enhancing cytotoxicity at lower hydrogen peroxide concentrations, and diminishing it at higher concentrations. The turning point moved gradually to higher concentrations of H₂O₂ corresponding to the augmented decomposition of hydrogen peroxide caused by increased activity of glutathione peroxidase. The data revealed that both enzymic pathways interact in the presence of H₂O₂, resulting in the overall cell survival different from that obtained after inhibition of either.     

NMR studies of glycerol permeability in lipid vesicles, erythrocytes and the alga Dunaliella

Glycerol diffusional permeabilities through the cytoplasmic cell membrane of Dunaliella salina, the cell envelope of pig erythrocyte and egg phosphattidylcholine vesicles were measured by NMR spectroscopy employing the spin-echo method and nuclear T₁ relaxation. The following permeability coefficients (P) and corresponding enthalpies of activation (ΔH^‡) were determined for glycerol at 25°C: for phosphatidylcholine vesicles 5·10⁻⁶ cm/s and 11±2 kcal/mol; for pig erythrocytes 7·10⁻⁸ cm/s and 18±3 kcal/mol, respectively; for the cytoplasmic membrane of D. salina the permeability at 17°C was found to be exceptionally low and only a lower limit (P<5·10⁻¹¹cm/s) could be calculated. At temperatures above 50°C a change in membrane permeability occurred leading to rapid leakage of glycerol accompanied by cell death. The data reinforce the notion that the cytoplasmic membrane of Dunaliella represents a genuine anomaly in its exceptional low permeability to glycerol.     

Peroxiredoxin 2, glutathione peroxidase,and catalase in the cytosol and membrane of erythrocytes under H2O2-induced oxidative stress

Abstract

Erythrocytes are continuously exposed to risk of oxidative injury due to oxidant oxygen species. To prevent damage, they have antioxidant agents namely, catalase (Cat), glutathione peroxidase (GPx), and peroxiredoxin 2 (Prx2). Our aim was to contribute to a better understanding of the interplay between Prx2, Cat, and GPx under H₂O₂-induced oxidative stress, by studying their changes in the red blood cell cytosol and membrane, in different conditions. These three enzymes were quantified by immunoblotting. Malondialdehyde, that is, lipoperoxidation (LPO) in the erythrocyte membrane, and membrane-bound hemoglobin (MBH) were evaluated, as markers of oxidative stress. We also studied the erythrocyte membrane protein profile, to estimate how oxidative stress affects the membrane protein structure. We showed that under increasing H₂O₂ concentrations, inhibition of the three enzymes with or without metHb formation lead to the binding of Prx2 and GPx (but not Cat) to the erythrocyte membrane. Prx2 was detected mainly in its oxidized form and the linkage of metHb to the membrane seems to compete with the binding of Prx2. Catalase played a major role in protecting erythrocytes from high exogenous flux of H₂O₂, since whenever Cat was active there were no significant changes in any of the studied parameters. When only Cat was inhibited, Prx2 and GPx were unable to prevent H₂O₂-induced oxidative stress resulting in increasing MBH and membrane LPO. Additionally, the inhibition of one or more of these enzymes induced changes in the anchor/linker proteins of the junctional complexes of the membrane cytoskeleton–lipid bilayer, which might lead to membrane destabilization.     

Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol

When mouse epididymal spermatozoa were rapidly frozen in two steps (37 to -70 degrees C for solid CO2 and -70 to -196 degrees C for liquid nitrogen) as pellets, 18% raffinose provided the greatest protection to ICR mouse spermatozoa against cold-shock; sperm motility and fertilizing ability were 43% and 22.4%, respectively. A small proportion of spermatozoa frozen with 10% sucrose was motile but incapable of fertilizing ovulated oocytes. Glycerol and dimethylsulphoxide were less effective at any concentration examined. However, the fertilizing ability of frozen-thawed ICR spermatozoa was significantly improved (35.5%) by addition of glycerol (1.75% final concentration) to medium containing 18% raffinose. Spermatozoa from one outbred (ddY) and 5 inbred (C57BL/6N, C3H/HeN, DBA/2N, BALB/c and kk) strains of mice were successfully frozen in the presence of 18% raffinose and 1.75% glycerol, although the fertilization rates of frozen-thawed spermatozoa varied among strains (13% for C57BL/6N to 64% for DBA/2N). A small fraction of mouse eggs resulting from fertilization by frozen-thawed spermatozoa developed normally in vitro (37% in C57BL/6N to 71% in ICR) to the blastocyst stage and in vivo (19% for C57BL/6N spermatozoa and ddY oocytes) to Day 18 of gestation.     

Hemoglobin autoxidation and regulation of endogenous H2O2 levels in erythrocytes

Red cells from mice deficient in glutathione peroxidase-1 were used to estimate the hemoglobin autoxidation rate and the endogenous level of H2O2 and superoxide. Methemoglobin and the rate of catalase inactivation by 3-amino-2,4,5-triazole (3-AT) were determined. In contrast with iodoacetamide-treated red cells, catalase was not inactivated by 3-AT in glutathione peroxidase-deficient erythrocytes. Kinetic models incorporating reactions known to involve H2O2 and superoxide in the erythrocyte were used to estimate H2O2, superoxide, and methemoglobin levels. The experimental data could not be modeled unless the intraerythrocytic concentration of Compound I is very low. Two additional models were tested. In one, it was assumed that a rearranged Compound I, termed Compound II*, does not react with 3-AT. However, experiments with an NADPH-generating system provided evidence that this mechanism does not occur. A second model that explicitly includes peroxiredoxin II can fit the experimental findings. Insertion of the data into the model predicted a hemoglobin autoxidation rate constant of 4.5 x 10(-7) s(-1) and an endogenous H2O2 and superoxide concentrations of 5 x 10(-11) and 5 x 10(-13) M, respectively, lower than previous estimates.     

裂殖酵母Papl应答H2O2氧化胁迫的机制

转录因子Papl是裂殖酵母(Schizosaccharomyces pombe)应答H2O2氧化胁迫反应中的关键调控因子.高浓度的H2O2激活蛋白激酶Styl途径清除过量的H2O2,使H2O2降至较低浓度再活化Papl;低浓度的则直接氧化活化Papl,导致Papl快速向细胞核内运输从而激活Papl相关基因的表达.本文综述了裂殖酵母中转录因子Papl在不同浓度H2O2胁迫下的激活途径,以及蛋白激酶Styl对Papl激活的重要作用.     

Evidence of interspecies hydrogen transfer from glycerol in saline environments

Two halanaerobic bacteria--Halanaerobium saccharolytica subsp. senegalense and Halanaerobium sp. strain FR1H--produced acetate, H2, and CO2 from glycerol fermentation, but the glycerol consumption rate was low. In contrast, in the presence of the moderately halophilic hydrogenotrophic sulfate-reducing bacterium, Desulfohalobium retbaense, used as H2 scavenger in the coculture, glycerol oxidation by both halanaerobes significantly increased. Cocultures of both halanaerobes with D. retbaense on glycerol led to acetate, hydrogen sulfide, and CO2 production, whereas glycerol fermentation by the two strains led to the production of acetate, hydrogen, and CO2. The increased glycerol oxidation by H. saccharolytica and strain FRI H in coculture with D. retbaense resulted from low H2 partial pressure caused by the hydrogen-oxidizing activity of D. retbaense. These results provide the first evidence of interspecies hydrogen transfer in saline environments and indicate that this mechanism may play an important role in organic matter mineralization in hypersaline ecosystems.