Heritability of glutathione and related metabolites in stored red blood cells

Red blood cells (RBCs) collected for transfusion deteriorate during storage. This deterioration is termed the “RBC storage lesion.” There is increasing concern over the safety, therapeutic efficacy, and toxicity of transfusing longer-stored units of blood. The severity of the RBC storage lesion is dependent on storage time and varies markedly between individuals. Oxidative damage is considered a significant factor in the development of the RBC storage lesion. In this study, the variability during storage and heritability of antioxidants and metabolites central to RBC integrity and function were investigated. In a classic twin study, we determined the heritability of glutathione (GSH), glutathione disulfide (GSSG), the status of the GSSG,2H⁺/2GSH couple (E_hc), and total glutathione (tGSH) in donated RBCs over 56 days of storage. Intracellular GSH and GSSG concentrations both decrease during storage (median net loss of 0.52±0.63 mM (median ± SD) and 0.032±0.107 mM, respectively, over 42 days). Taking into account the decline in pH, E_hc became more positive (oxidized) during storage (median net increase of 35±16 mV). In our study population heritability estimates for GSH, GSSG, tGSH, and E_hc measured over 56 days of storage are 79, 60, 67, and, 75%, respectively. We conclude that susceptibility of stored RBCs to oxidative injury due to variations in the GSH redox buffer is highly variable among individual donors and strongly heritable. Identifying the genes that regulate the storage-related changes in this redox buffer could lead to the development of new methods to minimize the RBC storage lesion.     

Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae

The reduction potential of a cell is related to its fate. Proliferating cells are more reduced than those that are differentiating, whereas apoptotic cells are generally the most oxidized. Glutathione is considered the most important cellular redox buffer and the average reduction potential (E_h) of a cell or organism can be calculated from the concentrations of glutathione (GSH) and glutathione disulfide (GSSG). In this study, triplicate groups of cod larvae at various stages of development (3 to 63 days post-hatch; dph) were sampled for analyses of GSSG/2GSH concentrations, together with activities of antioxidant enzymes and expression of genes encoding proteins involved in redox metabolism. The concentration of total GSH (GSH+GSSG) increased from 610±100 to 1260±150 μmol/kg between 7 and 14 dph and was then constant until 49 dph, after which it decreased to 810±100 μmol/kg by 63 dph. The 14- to 49-dph period, when total GSH concentrations were stable, coincides with the proposed period of metamorphosis in cod larvae. The concentration of GSSG comprised approximately 1% of the total GSH concentration and was stable throughout the sampling series. This resulted in a decreasing E_h from −239±1 to −262±7 mV between 7 and 14 dph, after which it remained constant until 63 dph. The changes in GSH and E_h were accompanied by changes in the expression of several genes involved in redox balance and signaling, as well as changes in activities of antioxidant enzymes, with the most dynamic responses occurring in the early phase of cod larval development. It is hypothesized that metamorphosis in cod larvae starts with the onset of mosaic hyperplasia in the skeletal muscle at approximately 20 dph (6.8 mm standard length (SL)) and ends with differentiation of the stomach and disappearance of the larval finfold at 40 to 50 dph (10–15 mm SL). Thus, metamorphosis in cod larvae seems to coincide with high and stable total concentrations of GSH.     

Glutathione disulfide liposomes – A research tool for the study of glutathione disulfide associated functions and dysfunctions

Glutathione disulfide (GSSG) is the oxidized form of glutathione (GSH). GSH is a tripeptide present in the biological system in mM concentration and is the major antioxidant in the body. An increase in GSSG reflects an increase in intracellular oxidative stress and is associated with disease sates. The increase has also been demonstrated to lead to an increase in protein S-glutathionylation that can affect the structure and function of proteins. Protein S-glutathionylation serves as a regulatory mechanism during cellular oxidative stress. Though GSSG is commercially available, its roles in various GSSG-associated normal/abnormal physiological functions have not been fully delineated due to the reason that GSSG is not cell membrane permeable and a lack of method to specifically increase GSSG in cells. We have developed cationic liposomes that can effectively deliver GSSG into cells. Various concentrations of GSSG liposomes can be conveniently prepared. At 1 mg/mL, the GSSG liposomes effectively increased intracellular GSSG by 27.1±6.9 folds (n=3) in 4 h and led to a significant increase in protein S-glutathionylation confirming that the increased GSSG is functionally effective. The Trypan blue assay demonstrated that GSSG liposomes were not cytotoxic; the cell viability was greater than 95% after cells were treated with the GSSG liposomes for 4 h. A stability study showed that the dry form of the GSSG liposomes were stable for at least 70 days when stored at ?80 °C. Our data demonstrate that the GSSG liposomes can be a valuable tool in studying GSSG-associated physiological/pathological functions.     

Brassica napus subjected to copper excess: Phospholipases C and D and glutathione system in signalling

Brassica napus plants were subjected to an oxidative stress by incubating them with 100 μM CuSO₄ for different times. The early response to copper stress was evaluated studying changes at both root and leaf level in the putative lipid and antioxidative signals diacylglicerol (DAG), phosphatidic acid (PA) and glutathione, in order to achieve elucidation on how these two kind of signals are related to each other. Activation of phospholipases C (PLC) and D (PLD) was studied in roots and leaves whereas increases in the levels of total and reduced glutathione (GSH) and changes in its redox status were evaluated in roots, leaves and chloroplast stroma. PLC and PLD were measured by studying the production of DAG, PA and phosphatidylbutanol (PtdButOH). PA, PtdButOH as well as DAG increased in roots already after 1 min of the treatment whereas in leaves, where no translocation of the metal occurred, any increase in PA and DAG was observed and no PtdButOH was formed. Roots were affected by oxidative stress showing decreases in glutathione reductase (GR), in total glutathione (GSH + GSSG) and GSH, and increases in oxidised glutathione (GSSG). In leaves, GR was induced during the whole stress period and both GSH + GSSG and GSH showed a peak at 5 min of the treatment. In the stroma, the maximum presence in GSH + GSSG and GSH occurred with a time shift of 25 min compared with total leaf extract.     

Hemoglobin glutathionylation can occur through cysteine sulfenic acid intermediate: Electrospray ionization LTQ-Orbitrap hybrid mass spectrometry studies

Glutathionylated hemoglobin (Hb-SSG) is now recognized as a promising biomarker of systemic oxidative stress. Aim of this study is to gain a mechanistic insight into its formation. The ability of GSSG to form Hb-SSG through a thiol-disulfide exchange mechanism was firstly examined. For this purpose, GSSG (ranging from 0.23 to 230 μmol/g Hb, 15 μM–15 mM final concentrations) was incubated with 1 mM Hb and the relative content of Hb-SSG determined by direct infusion mass spectrometry (Orbitrap as analyzer). No detectable Hb-SSG was observed at a GSSG concentration range found in physiopathological conditions (0.13–0.23 μmol/g Hb). To reach a detectable Hb-SSG signal, the GSSG concentration was raised to 2.3 μmol/g Hb (0.5% relative abundance). The relative content of Hb-GSSG dose-dependently increased to 6% and 11% at 77 and 153 μmol/g Hb, respectively. The second step was to demonstrate whether Hb-SSG is formed through a sulfenic acid intermediate, a well-recognized mechanism of S-protein glutathionylation. Cys β93 sulfenic acid was found to be formed by oxidizing Hb with 1 mM H₂O₂, as demonstrated by direct infusion and LC–ESI-MS/MS experiments and using dimedone as derivatazing agent. When H₂O₂-treated Hb was incubated with physiological concentrations of GSH (9 μmol/g Hb), the corresponding Hb-SSG form was detected, reaching 15% of relative abundance. In summary, we here demonstrate that Hb glutathionylation can occur through a Cys sulfenic acid intermediate which is formed in oxidizing conditions. Hb glutathionylation is also mediated by a thiol-disulfide transfer mechanism, but this requires a concentration of GSSG which is far to be achieved in physiopathological conditions.     

Treatment of whole blood (WB) and red blood cells (RBC) with S-303 inactivates pathogens and retains in vitro quality of stored RBC

A pathogen inactivation (PI) process has been developed using the frangible anchor linker effector (FRALE) compound S-303. A series of experiments were performed in whole blood (WB) to measure the level of viral and bacterial inactivation. The results showed that 0.2 mM S-303 and 2 mM glutathione (GSH) inactivated >6.5 logs of HIV, >5.7 logs of Bluetongue virus, >7.0 logs of Yersinia enterocolitica, 4.2 logs of Serratia marcescens, and 7.5 logs of Staphylococcus epidermidis. Recent development for S-303 is focused on optimization of the PI process for red blood cell concentrates (RBC). A series of studies in RBC showed that 0.2 mM S-303 and 20 mM GSH inactivated approximately 5 logs or greater of Y. enterocolitica, E. coli, S. marcescens, S. aureus, HIV, bovine viral diarrhoea virus, bluetongue virus and human adenovirus 5. In both applications of the S-303 process, in vitro parameters of RBC function and physiology were retained compared to conventional RBC. Results from these studies indicate that S-303 can be applicable for PI of RBC and WB.     

Glutathione,glutathione disulfide,and S-glutathionylated proteins in cell cultures

The analysis of the global thiol–disulfide redox status in tissues and cells is a challenging task since thiols and disulfides can undergo artificial oxido-reductions during sample manipulation. Because of this, the measured values, in particular for disulfides, can have a significant bias. Whereas this methodological problem has already been addressed in samples of red blood cells and solid tissues, a reliable method to measure thiols and disulfides in cell cultures has not been previously reported.Here, we demonstrate that the major artifact occurring during thiol and disulfide analysis in cultured cells is represented by glutathione disulfide (GSSG) and S-glutathionylated proteins (PSSG) overestimation, due to artificial oxidation of glutathione (GSH) during sample manipulation, and that this methodological problem can be solved by the addition of N-ethylmaleimide (NEM) immediately after culture medium removal. Basal levels of GSSG and PSSG in different lines of cultured cells were 3–5 and 10–20 folds higher, respectively, when the cells were processed without NEM. NEM pre-treatment also prevented the artificial reduction of disulfides that occurs during the pre-analytical phase when cells are exposed to an oxidant stimulus. In fact, in the absence of NEM, after medium removal, GSH, GSSG and PSSG levels restored their initial values within 15–30 min, due to the activity of reductases and the lack of the oxidant. The newly developed protocol was used to measure the thiol–disulfide redox status in 16 different line cells routinely used for biomedical research both under basal conditions and after treatment with disulfiram, a thiol-specific oxidant (0–200 μM concentration range).Our data indicate that, in most cell lines, treatment with disulfiram affected the levels of GSH and GSSG only at the highest concentration. On the other hand, PSSG levels increased significantly also at the lower concentrations of the drug, and the rise was remarkable (from 100 to 1000 folds at 200 μM concentration) and dose-dependent for almost all the cell lines. These data support the suitability of the analysis of PSSG in cultured cells as a biomarker of oxidative stress.     

GC–MS analysis of S-nitrosothiols after conversion to S-nitroso-N-acetyl cysteine ethyl ester and in-injector nitrosation of ethyl acetate

S-Nitrosothiols from low-molecular-mass and high-molecular-mass thiols, including glutathione, albumin and hemoglobin, are endogenous potent vasodilators and inhibitors of platelet aggregation. By utilizing the S-transnitrosation reaction and by using the lipophilic (pK_L 0.78) and strong nucleophilic synthetic thiol N-acetyl cysteine ethyl ester (NACET) we have developed a GC–MS method for the analysis of S-nitrosothiols and their ¹⁵N- or ²H–¹⁵N-labelled analogs as S-nitroso-N-acetyl cysteine ethyl ester (SNACET) and S¹⁵NACET or d₃-S¹⁵NACET derivatives, respectively, after their extraction with ethyl acetate. Injection of ethyl acetate solutions of S-nitrosothiols produced two main reaction products, compound X and compound Y, within the injector in dependence on its temperature. Quantification was performed by selected-ion monitoring of m/z 46 (i.e., [NO₂]^?) for SNACET and m/z 47 (i.e., [¹⁵NO₂]^?) for S¹⁵NACET/d₃-S¹⁵NACET for compound X, and m/z 157 for SNACET and m/z 160 for d₃-S¹⁵NACET for compound Y. In this article we describe the development, validation and in vitro and in vivo applications of the method to aqueous buffered solutions, human and rabbit plasma. Given the ester functionality of SNACET/S¹⁵NACET/d₃-S¹⁵NACET, stability studies were performed using metal chelators and esterase inhibitors. The method was found to be suitable for the quantitative determination of various S-nitrosothiols including SNACET externally added to human plasma (0–10 μM). Nitrite contamination in ethyl acetate was found to interfere. Our results suggest that the concentration of endogenous S-nitrosothiols in human plasma does not exceed about 200 nM in total. Oral administration of S¹⁵NACET to rabbits (40–63 μmol/kg body weight) resulted in formation of ALB-S¹⁵NO, [¹⁵N]nitrite and [¹⁵N]nitrate in plasma.     

Evaluation of insecticidal activity of the essential oil of Allium chinense G. Don and its major constituents against Liposcelis bostrychophila Badonnel

Water-distilled essential oil from the dried bulbs of Allium chinense (Liliaceae) was analyzed by gas chromatography–mass spectrometry (GC–MS). Eighteen compounds, accounting for 98.4% of the total oil, were identified and the main components of the essential oil of A. chinense were methyl allyl trisulfide (30.7%), dimethyl trisulfide (24.1%), methyl propyl disulfide (12.8%) and dimethyl disulfide (9.6%) followed by methyl allyl disulfide (3.4%) and methyl propyl trisulfide (3.6%). The essential oil exhibited contact toxicity against the booklice (Liposcelis bostrychophila) with an LC₅₀ value of 441.8 μg/cm² while the two major constituents, dimethyl trisulfide and methyl propyl disulfide had LC₅₀ values of 153.0 μg/cm² and 738.0 μg/cm² against the booklice, respectively. The essential oil of A. chinense possessed strong fumigant toxicity against the booklice with an LC₅₀ value of 186.5 μg/l while methyl allyl trisulfide (LC₅₀ = 90.4 μg/l) and dimethyl trisulfide (LC₅₀ = 114.2 μg/l) exhibited stronger fumigant toxicity than methyl propyl disulfide (LC₅₀ = 243.4 μg/l) and dimethyl disulfide (LC₅₀ = 340.8 μg/l) against the booklice. The results indicated that the essential oil and its major constituents have potential for development into natural insecticides or fumigants for control of insects in stored grains.     

Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze–thawing process

Oxidative damage to sperm resulting from reactive oxygen species generated by the cellular components of semen is one of the main causes for the decline in motility and fertility of sperm during the freeze–thawing process. The aim of this study was thus to determine the effects of anti-oxidants on standard semen parameters, lipid peroxidation (LPO) and anti-oxidant activities after the freeze–thawing of ram semen. Ejaculates collected from four Akkaraman rams, were pooled and evaluated at 33 °C. Semen samples were diluted in a Tris-based extender containing the anti-oxidants glutathione (GSH) (5 mM), oxidized glutathione (GSSG) (5 mM) or cysteine (5 mM) and an extender containing no anti-oxidants (control), cooled to 5 °C and frozen in 0.25 ml French straws. Frozen straws were thawed individually for 20 s in a water bath (37 °C) for microscopic evaluation. The use of an extender supplemented with cysteine led to the highest (P < 0.01) post-thaw motility (61.0 ± 1.9%), compared to the other treatment groups. No significant differences were observed in viability, acrosome damage and total abnormalities, and following the hypo-osmotic swelling test (HOST), following supplementation with anti-oxidants after the thawing of the semen. Following the thawing process, the levels of malondialdehyde (MDA) did not change with the addition of anti-oxidants, compared to the control. The GSH level and glutathione peroxidase (GSH-PX) activity remained significantly higher upon the addition of GSH (3.33 ± 0.14 nmol/ml and 22.02 ± 1.27 IU/g protein) and GSSG (3.24 ± 0.08 nmol/ml and 20.17 ± 3.38 IU/g protein) compared to the other treatment (P < 0.001) groups. Only cysteine significantly elevated the activity of catalase (CAT, 842.40 ± 90.42 kU/l) following the freeze–thawing process. The Vitamin E (VitE) level was significantly higher, when compared to GSSG, cysteine and the control, when GSH (4.21 ± 0.20 mg/dl) was added to the freezing extender (P < 0.001). It could be concluded that future efforts aimed on improving the efficiency of cryopreservation of ram sperm should concentrate on the use of anti-oxidant additives. The results obtained provide a new approach to the cryopreservation of ram semen, and could positively contribute to intensive sheep production.     

Modulation of antioxidant enzyme activities and metabolites ratios by nitric oxide in short-term salt stressed soybean root nodules

Several abiotic factors cause molecular damage to plants either directly or through the accumulation of reactive oxygen species such as hydrogen peroxide (H₂O₂). We investigated if application of nitric oxide (NO) donor 2,2′-(hydroxynitrosohydrazono) bis-ethanimine (DETA/NO) could reduce the toxic effect resulting from short-term salt stress. Salt treatment (150 mM NaCl) alone and in combination with 10 μM DETA/NO or 10 μM DETA were given to matured soybean root nodules for 24 h. Salt stress resulted in high H₂O₂ level and lipid peroxidation while application of DETA/NO effectively reduced H₂O₂ level and prevented lipid peroxidation in the soybean root nodules. NO treatment increased the activities of ascorbate peroxidase and dehydroascorbate reductase under salt stress. Whereas short-term salt stress reduced AsA/DHAsA and GSH/GSSG ratios, application of the NO donor resulted in an increase of the reduced form of the antioxidant metabolites thus increasing the AsA/DHAsA and GSH/GSSG ratios. Our data suggests a protective role of NO against salt stress.     

Parietin in the tolerant lichen Xanthoria parietina (L.) Th. Fr. increases protection of Trebouxia photobionts from cadmium excess

Secondary metabolites of lichens can be involved in production of chelates with heavy metals. We hypothesized that parietin plays important role in protection of photobiont cells in Xanthoria parietina from an excess of cadmium ions. Two types of X. parietina lichen thalli, natural with presence of secondary metabolite parietin (p+) as well as without parietin (p−) were exposed to different doses of cadmium (up to 300 μmol g⁻¹ dw). Based on determination of the total and intracellular Cd-accumulation, ergosterol and thiobarbituric acid reactive substances (TBARS) content did not show statistically significant differences in the response of both types of thalli (p+ and p−). However, a stronger toxic effect of the highest Cd-dose on photosynthetic pigment content and chlorophyll a fluorescence was observed in the parietin-depleted thalli. The protective role of parietin against Cd excess was better supported and concluded from the differences observed in the production of non-protein thiol compounds (cysteine, glutathione and phytochelatins) involved in Cd detoxification. In the p+ thalli Cys content was stable but GSH content slightly decreased in the studied Cd range, while in the p− thalli these compounds were completely absent at high Cd doses. At Cd doses higher than 37.5 μmol Cd g⁻¹ dw, toxic to both types of X. parietina thalli, Cys and GSH contents were significantly higher in p+ than in p− thalli. Also, the photobiont partner in the p+ thalli was better protected of the metal exposition, and able to produce phytochelatins (PCs) over the whole range of metal, while in the p− thalli the production was completely inhibited at 75 μmol Cd g⁻¹ dw and higher concentrations, together with the inhibition of cysteine (Cys) and reduced glutathione (GSH) production. The obtained results indicate that the parietin layer is a natural barrier decreasing Cd access to algal cells in X. parietina. Comparison of PCs production appeared to be the most sensitive marker for estimation of Cd availability to photobiont in the symbiotic system.     

Response to copper stress in aposymbiotically grown lichen mycobiont Cladonia cristatella: uptake,viability, ergosterol and production of non-protein thiols

The mycobiont of lichens usually determines the morphology of the symbiotic organism and is also dominates in terms of biomass. However, its role for sensitivity or tolerance of lichens to heavy metals is almost unknown. In the present study, the influence of copper (Cu) on the aposymbiotically-grown mycobiont of Cladonia cristatella was assessed. Intracellular Cu uptake was correlated with increasing Cu concentrations over a 24-h exposure time. Viability, measured as the degree of reduction of triphenyltetrazolium chloride to triphenyl formazan, as well as to ergosterol levels, decreased with growing Cu concentrations tested. Reduced glutathione (GSH) was found to be the most abundant low-molecular-weight thiol in the hyphae of C. cristatella and its intracellular content increased at concentrations of 10 μm Cu. Higher Cu concentrations caused a significant decrease in GSH, possibly due to heavy metal-induced oxidation of GSH to glutathione disulphide (GSSG). Free cysteine levels were relatively constant. As expected, we did not observe the production of phytochelatins in the mycobiont, contrary to what is found in intact lichens and axenic cultures of their photobionts.     

Multiparametric protocol for the determination of thiol redox state in living matter

Thiol redox state (TRS) evaluation is mostly restricted to the estimation of GSH and GSSG. However, these TRS parameters can estimate the GSSG/GSH potential, which might be useful for indicating abnormalities in redox metabolism. Nonetheless, evaluation of the multiparameric nature of TRS is required for a more accurate assessment of its physiological role. The present protocol extends the partial assessment of TRS by current methodologies. It measures 15 key parameters of TRS by two modular subprotocols: one for the glutathione (GSH)- and cysteine (CSH)-based nonprotein (NP) thiols/mixed disulfides (i.e., GSH, GSSG, GSSNP, CSH, CSSNP, NPSH, NPSSNP, NP_xSH_NPSSNP, NP_xSH_NPSH), and the other for their protein (P) thiols/mixed disulfides (i.e., PSH, PSSG, PSSC, PSSNP, PSSP, NP_xSH_PSSNP). The protocol eliminates autoxidation of GSH and CSH (and thus overestimation of GSSG and CSSNP). Its modularity allows the determination GSH and GSSG also by other published specific assays. The protocol uses three assays; two are based on the photometric reagents 4,4′-dithiopyridine (DTP) and ninhydrin (NHD), and the third on the fluorometric reagent o-phthaldialdehyde (OPT). The initial assays employing these reagents have been extensively modified and redesigned for increased specificity, sensitivity, and simplicity. TRS parameter values and their standard errors are estimated automatically by sets of Excel-adapted algebraic equations. Protocol sensitivity for NPSH, PSH, NPSSNP, PSSP, PSSNP, CSH, CSSNP, PSSC, NP_xSH_NPSSNP, and NP_xSH_NPSH is 1 nmol –SH/CSH, for GSSNP 0.2 nmol, for GSH and GSSG 0.4 nmol, and for PSSG 0.6 nmol. The protocol was applied on human plasma, a sample of high clinical value, and can be also applied in any organism.     

Determination of 2,3-dimercaptosuccinic acid in mice blood and tissues by HPLC with fluorescence detection

2,3-Dimercaptosuccinic acid (DMSA) is an orally effective chelating agent for the treatment of heavy metal poisoning. The increasing therapeutic use of DMSA has stimulated the need for sensitive and selective methods for its determination in biological samples, as well as study on pharmacokinetics and tissue distribution. According to the previously reported method, an improved method was established for the determination of DMSA in mice blood and tissues, in which oxidized DMSA was reduced by the disulfide-reducing agent, dithiothreitol (DTT), and DMSA was converted to a highly fluorescent and stable derivative by reaction with monobromobimane (mBBr) in alkaline solution. Acetonitrile was used for deproteinization and dichloromethane was used for condensation and purification, which significantly shortened the amount of time used to process the sample. Meanwhile isocratical elution was performed and excellent separation of the DMSA derivative was obtained, this enabled a run finish within 20 min. The limits of quantitation were 0.025 μg/ml in brain and 0.1 μg/ml in blood, lung, heart, intestine, liver, spleen and kidney, respectively. The calibration curves were linear in all samples (r² > 0.992) with a range of 0.025–1.6 μg/ml for brain homogenate and 0.1–6.4 μg/ml for blood and homogenates of lung, heart, intestine, liver, spleen and kidney, respectively. Therefore, the method is simple, rapid and sensitive, and it could be applicable to the studies in an animal model to evaluate the distribution of DMSA in blood and tissues.     

Cytotoxic activity of butane type of 1,7-seco-2,7′-cyclolignanes and apoptosis induction by Caspase 9 and 3

All stereoisomers of methoxybutane and fluorobutane type of 1,7-seco-2,7′-cyclolignane were synthesized and cytotoxic activities of these compounds were compared with those of all stereoisomers of butane and butanol type compounds. Both enantiomers of butane type secocyclolignane showed higher cytotoxic activity (IC₅₀ = 16–20 μM) than methoxy type compounds, whereas none was observed for all the stereoisomers of butanol type secocyclolignane, however, (−)-Kadangustin J showed stereospecific cytotoxic activity (IC₅₀ = 47–67 μM). Since (R)-9′-fluoro derivative 23 was most potent (IC₅₀ = 19 μM) among the corresponding fluoro stereoisomers, (R)-9′-alkyl derivatives were synthesized, hydrophobic 9′-heptyl derivative 27 showing highest activity (IC₅₀ = 3.7 μM against HL-60, IC₅₀ = 3.1 μM against HeLa) in this experiment. Apoptosis induction caused by Caspase 3 and 9 for (R)-9′-heptyl derivative 27 was observed in the research on the mechanism. A degradation of DNA into small fragments was also shown by DNA ladder assay.     

Statistical optimization and multiple objective programming of lysozyme refolding catalyzed by recombinant DsbA in vitro

DsbA (disulfide bond formation protein A) is essential for disulfide bond formation directly affecting the nascent peptides folding to the correct conformation in vivo. In this paper, recombinant DsbA protein was employed to catalyze denatured lysozyme refolding and inhibit the aggregation of folding intermediates in vitro. Statistical methods, i.e., Plackett–Burman design and small central composite design, were adopted to screen out important factors affecting the refolding process and correlating these parameters with the refolding efficiency including both protein recovery and specific activity of refolded lysozyme. Four important parameters: initial lysozyme concentration, urea concentration, KCl concentration and GSSG (glutathione disulfide) concentration were picked out and operating conditions were optimized by introducing the effectiveness coefficient method and transforming the multiple objective programming into an ordinary constrained optimization issue. Finally, 99.7% protein recovery and 25,600 U/mg specific activity of lysozyme were achieved when 281.35 μg/mL denatured lysozyme refolding was catalyzed by an equivalent molar of DsbA at the optimal settings. The results indicated that recombinant DsbA protein could effectively catalyze the oxidized formation and reduced isomerization of intramolecular disulfide bonds in the refolding of lysozyme in vitro.     

Light-induced disulfide dimerization of recoverin under ex vivo and in vivo conditions

Despite vast knowledge of the molecular mechanisms underlying photochemical damage of photoreceptors, linked to progression of age-related macular degeneration, information on specific protein targets of the light-induced oxidative stress is scarce. Here, we demonstrate that prolonged intense illumination (halogen bulb, 1500 lx, 1–5 h) of mammalian eyes under ex vivo (cow) or in vivo (rabbit) conditions induces disulfide dimerization of recoverin, a Ca²⁺-dependent inhibitor of rhodopsin kinase. Western blotting and mass spectrometry analysis of retinal extracts reveals illumination time-dependent accumulation of disulfide homodimers of recoverin and its higher order disulfide cross-linked species, including a minor fraction of mixed disulfides with intracellular proteins (tubulins, etc.). Meanwhile, monomeric bovine recoverin remains mostly reduced. These effects are accompanied by accumulation of disulfide homodimers of visual arrestin. Histological studies demonstrate that the light-induced oxidation of recoverin and arrestin occurs in intact retina (illumination for 2 h), while illumination for 5 h is associated with damage of the photoreceptor layer. A comparison of ex vivo levels of disulfide homodimers of bovine recoverin with redox dependence of its in vitro thiol–disulfide equilibrium (glutathione redox pair) gives the lowest estimate of redox potential in rod outer segments under illumination from −160 to −155  mV. Chemical crosslinking and dynamic light scattering data demonstrate an increased propensity of disulfide dimer of bovine recoverin to multimerization/aggregation. Overall, the oxidative stress caused by the prolonged intense illumination of retina might affect rhodopsin desensitization via concerted disulfide dimerization of recoverin and arrestin. The developed herein models of eye illumination are useful for studies of the light-induced thiol oxidation of visual proteins.     

Adding glutathione to parenteral nutrition prevents alveolar loss in newborn Guinea pig

Bronchopulmonary dysplasia, a main complication of prematurity, is characterized by an alveolar hypoplasia. Oxidative stress is suspected to be a trigger event in this population who has a low level of glutathione, a main endogenous antioxidant, and who receives high oxidative load, particularly ascorbylperoxide from their parenteral nutrition. Hypothesis: the addition of glutathione (GSSG) in parenteral nutrition improves detoxification of ascorbylperoxide by glutathione peroxidase and therefore prevents exaggerated apoptosis and loss of alveoli.Methods: Ascorbylperoxide is assessed as substrate for glutathione peroxidase in Michaelis-Menten kinetics. Three-days old guinea pig pups were divided in 6 groups to receive, through a catheter in jugular vein, the following solutions: 1) Sham (no infusion); 2) PN(-L): parenteral nutrition protected against light (low ascorbylperoxide); 3) PN(+L): PN without photo-protection (high ascorbylperoxide); 4) 180 μM ascorbylperoxide; 5) PN(+L)+10 μM GSSG; 6) ascorbylperoxyde+10 μM GSSG. After 4 days, lungs were sampled and prepared for histology and biochemical determinations. Data were analysed by ANOVA, p<0.05Results: The Km of ascorbylperoxide for glutathione peroxidase was 126±6 μM and Vmax was 38.4±2.5 nmol/min/ U. The presence of GSSG in intravenous solution has prevented the high GSSG, oxidized redox potential of glutathione, activation of caspase-3 (apoptosis marker) and loss of alveoli induced by PN(+L) or ascorbylperoxide.Conclusion: A correction of the low glutathione levels observed in newborn animal on parenteral nutrition, protects lungs from toxic effect of ascorbylperoxide. Premature infants having a low level of glutathione, this finding is of high importance because it provides hope in a possible prevention of bronchopulmonary dysplasia.     

Effect of Zinc nanoparticles on oxidative stress-related genes and antioxidant enzymes activity in the brain of Oreochromis niloticus and Tilapia zillii

This study was carried out to determine the median lethal concentrations (LC₅₀) of Zinc nanoparticles (ZnNPs) on Oreochromis niloticus and Tilapia zillii. The biochemical and molecular potential effects of ZnNPs (500 and 2000 μg L⁻¹) on the antioxidant system in the brain tissue of O. niloticus and T. zillii were investigated. Four hundred fish were used for acute and sub-acute studies. ZnNP LC₅₀ concentrations were investigated in O. niloticus and T. zillii. The effect of 500 and 2000 μg L⁻¹ ZnNPs on brain antioxidants of O. niloticus and T. zillii was investigated. The result indicated that 69 h LC₅₀ was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. Fish exposed to 500 μg L⁻¹ ZnNPs showed a significant increase in reduced glutathione (GSH), total glutathione (tGSH) levels, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activity and gene expression. On the contrary, malondialdehyde (MDA) levels significantly decreased. Meanwhile, fish exposed to 2000 μg L⁻¹ ZnNPs showed a significant decrease of GSH, tGSH levels, SOD, CAT, GR, GPx and GST activity and gene expression. On the contrary, MDA levels significantly increased. It was concluded that, the 96 h LC₅₀ of ZnNPs was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. ZnNPs in exposure concentrations of 2000 μg/L induced a deleterious effect on the brain antioxidant system of O. nilotica and T. zillii. In contrast, ZnNPs in exposure concentrations of 500 μg L⁻¹ produced an inductive effect on the brain antioxidant system of O. nilotica and T. zillii.