Ellman's Reagent Research Articles

Біохімічні показники життєдіяльності насіння гіркокаштанів, найпоширеніших у західному регіоні України

Визначено біохімічні показники життєдіяльності насіння гіркокаштанів для пошуку стійких проти каштанової мінуючої молі (Cameraria ohridella Desch. & Dem (Lepidoptera, Gracillariidaе)) видів і форм. Для експериментів у насінний рік в умовно чистих зонах і в місцях інтенсивного антропогенно-техногенного забруднення відібрано насіння чотирьох видів гіркокаштанів. Заготівлю насіння здійснено способом збирання із поверхні землі у період найінтенсивнішого його опадання. Досліджено вміст у насінні гіркокаштанів низки фізіолого-біологічних, морфологічних та основних біохімічних показників (білка, розчинних вуглеводнів і "сирого жиру"), золи, макро- і мікроелементів в умовно чистих зонах і в місцях інтенсивного антропогенно-техногенного забруднення. Встановлено рівень активності перекисного окислення ліпідів (ПОЛ) та вміст відновленого глутатіону. Вивчені зміни фізіолого-біологічних та біохімічних показників свідчать, що порівняно із трьома іншими досліджуваними видами дерева гіркокаштана кінського звичайного у стресових ґрунтово-кліматичних умовах великого міста з додатковим зниженням життєздатності каштановою мінуючою міллю характеризуються найбільшими негативними змінами у метаболізмі. Це свідчить, що за зростання в аналогічних ґрунтово-кліматичних умовах та за подібного навантаження чинників антропогенно-техногенного впливу, дерева і насадження жовтоквіткового (Aesculus octаndra Marsh.) і червоноквіткових видів гіркокаштанів (Aesculus pavia L. і Aesculus carnea Hayne) на сьогодні є життєво стійкішими, ніж дерева і насадження найпоширенішого у регіоні гіркокаштана кінського звичайного (Aesculus hippocastanum L.).

Determination of cysteamine in animal feeds by high performance liquid chromatography with diode-array detection (HPLC-DAD)

A HPLC-DAD method for the determination of cysteamine supplementation in commercial animal feeds was developed. Samples were extracted with a mixture of 0.5 % hydrochloric acid – acetonitrile (90:10, v/v), matrix interferences were removed with a C18 cartridge, cysteamine was derivatized using 5,5'-dithiobis-(2-nitrobenzoic) acid (DTNB) as Ellman's reagent targeting to the thiol group in the molecule. Quantification of cysteamine was performed on a c18 column with DAD at 323 nm. The developed method had LOD of 1.1 mg/l, good linearity of the calibration curve (R2 ≥ 0.9998), high recoveries (> 92 %), and high reproducibility (RSD < 2.0%).

Determination of Blood Plasma Aminothiols Using Derivatization-enhanced Capillary Transient Isotachophoresis.

A sensitive capillary electrophoresis method was developed for the determination of aminothiol (cysteine, homocysteine, and glutathione) total levels in human blood plasma. Analytes were derivatized with Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid)) after reduction with dithiothreitol. Liquid-liquid extraction was applied to purify the samples and concentrate the analytes. Total analysis time was 7.5 min using a silica capillary (50 μm i.d.; effective separation length 23.5 cm). Electrophoretic separation was performed using 50 mM citric acid with 20 mM triethanolamine (pH 3) containing 2% Ficoll 400. Detection limit was 0.8 μM for glutathione and 0.3 μM for both cysteine and homocysteine. Accuracy was 94 - 107%, repeatability and reproducibility were ca. 2.7 - 3.5 and 2.5 - 6.5%, respectively.

Assessing Cysteine Residue Thiol Status of t‐DARPP, A Protein Involved in Chemoresistance

The gene PPP1R1B expresses t‐Darpp, a protein that activates protein kinase A and the AKT pathway in breast cancer cells. t‐Darpp, when overexpressed in breast cancer cells, also confers resistance to trastuzumab, a therapeutic that binds to the Her2 receptor. Although t‐Darpp expression is relatively high in gastric, breast and colon cancers, its structure is unknown. Human t‐Darpp has two cysteine residues that can be exploited to explore t‐Darpp structure, Cys36 and Cys119. Methoxypolyethylene glycol‐maleimide (Mal‐PEG) was used to determine whether recombinant t‐Darpp cysteine thiols are on the surface of the protein and to find out if they are reversibly oxidized. Later, a quantitative analysis was conducted using Ellman's test. 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (Ellman's reagent) was used to determine whether recombinant t‐Darpp cysteine thiols are free or not. Both studies suggest that the two cysteine thiols are either buried or oxidized. Further work will be conducted to distinguish between these two possibilities.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Somatostatin Decorated Quantum Dots for Targeting of Somatostatin Receptors.

Due to the unique optical properties like high brightness and narrow emission bands of Quantum dots, it is used as simple fluorescence materials in bio-imaging, immunoassays, microarrays, and other applications. To easy invistigate cell lines that overexpressed somtostatin receptors, somatostatin (SST) was conjugated with Quantum dots carrying PEG amine (Qdots-PEG-NH2). The conjugation of SST to Qdots-PEG-NH2 started with the thiolation of SST using Traut's reagent. Moreover, the Qdots-PEG-NH2 were subsequently activated by 500-fold molar excess of sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) dissolved in phosphate buffer. The Qdots-PEG-NH2-sulfo-SMCC was conjugated to the thiolated-SST to form Qdots-SST. The number of sulfhydryl groups can be controlled by the molar ratio of Traut´s reagent to SST. Thiolation was necessary for the conjugation of SST to Qdots-PEG-NH2. This was achieved by reacting the SST with Traut's reagent in a 1:1 molar ratio. Ellman's reagent was used to determine the number of sulfhydryle groups. Furthermore, cellular uptake study on triple negative breast cancer cells (HCC-1806) showed that the numbers of Qdots-SST per cell were significantly higher compared to unmodified Qdots-PEG-NH2 when quantified using inductively coupled plasma optical emission spectroscopy (ICP-OES). Moreover, the binding of Qdots-SST to cells can be suppressed by addition of free SST, indicating that the binding of Qdots-SST to cells is due to receptor-specific binding.

Sensitivity-Tunable Colorimetric Detection of Chloropicrin Vapor on Nylon-6 Nanofibrous Membrane Based on a Detoxification Reaction with Biological Thiols

Detoxification reaction of chloropicrin in the human body with biological thiols was selected for detection of chloropicrin in the air. The consumption of free sulfhydryl group in biological thiols by chloropicrin is colorimetrically detectable with the addition of the Ellman's reagent. In this study, glutathione, N-acetyl-l-cysteine, l-homocysteine, cysteamine, and thioglycolic acid were tested as sensing agents for chloropicrin vapor detection in ppb concentration range. The reactivity of the selected biological thiols was investigated based on both their redox properties and the nucleophilic strength of the sulfhydryl groups. Nylon-6 nanofibrous membrane and an organic solvent were used as a sensor matrix and a vapor sorbent, respectively, to provide solid supports with ultrahigh surface area and enhanced adsorption to chloropicrin vapor. The tunable sensitivity and detection range by using different biological thiols was achieved on the sensors due to the different reactivity of thiols toward chloropicrin.

Vitamin E Analogue Protects Red Blood Cells against Storage-Induced Oxidative Damage

Background: To investigate i) the effects of Trolox® or mannitol, which represent two different classes of antioxidants, on oxidative changes generated in manually isolated red blood cells (RBCs) from citrate-phosphate-dextrose (CPD) preserved whole blood, followed by up to 20 days refrigerated storage, and ii) whether Trolox supplemented to the blood bank-manufactured saline-adenine-glucose-mannitol (SAGM) preserved RBC units would offer better storage conditions compared with SAGM alone. Methods: The percentage of hemolysis and extracellular activity of lactate dehydrogenase (LDH) was measured to assess RBC membrane integrity. Lipid peroxidation, reduced glutathione (GSH) levels and total antioxidant capacity (TAC) were quantified by thiobarbituric acid-reactive substances (TBARS), Ellman's reagent and 2, 2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS^.+) based assay, respectively. Results: Trolox was little more effective than mannitol in protecting against progressive RBC hemolysis. Trolox (0.125-3.125 mmol/l) inhibited storage-induced leakage of LDH, lipid peroxidation, and to a lesser extent GSH depletion. Mannitol at these concentrations neither inhibited TBARS formation nor prevented GSH depletion. RBC units stored in SAGM-Trolox had significantly lower hemolysis, LDH leakage, and lipid peroxidation level compared to RBCs stored in SAGM. Conclusion: There is evidence of the beneficial effects of supplementing RBC-additive solutions with membrane-interacting antioxidants such as vitamin E analogues.

Protective Effect of Water Extracted Spirulina maxima on Glutamate-induced Neuronal Cell Death in Mouse Hippocampal HT22 Cell.

Introduction:Spirulina maxima was used as important nutritional source in the Aztec civilization because it is rich in proteins and vitamins. It contains various antioxidants such as phycocyanin and flavonoids. Based on abundant antioxidants, S. maxima is known to possess anti-inflammatory effect, especially on neuronal cells.Materials and Methods:S. maxima was extracted in water and contain of phycocyanin was identified by high-performance liquid chromatography. Cell viability test was performed with treatment of S. maxima extract. After, oxidative stress-related mechanisms were evaluated by detecting the accumulation of reactive oxygen species (ROS) and Ca2+ influx, and decrease of mitochondrial membrane potential (MMP) level. Then, the glutathione (GSH) related assays were conducted.Results:The water extracted S. maxima exerted the neuroprotective activity by attenuating the ROS and Ca2+ formation, maintaining the MMP level, and protecting the activity of the antioxidant enzymes by increasing reduced GSH against oxidative stress compared to control.Conclusion:The results suggested that water extracted S. maxima showed powerful neuroprotective effect through the mechanism related to antioxidant activity, able to preventing the radical-mediated cell death.SUMMARY Water extracted Spirulina maxima contains C-phycocyaninWater extracted Spirulina maxima exerts neuroprotective effect on HT22 cellTo investigate the protective mechanisms, reactive oxygen species, Ca2+, mitochondrial membrane potential, Glutathione-related assays were performed. Abbreviations used: ROS: Reactive oxygen species; MMP: Mitochondrial membrane potential; GSH: Glutathione; GSSG: Glutathione disulfide, oxidized glutathione; GPx: Glutathione peroxidase; GR: Glutathione reductase; DMEM: Dulbecco's modified Eagle's medium; FBS: Fetal bovine serum; DCF-DA: 2',7'-dichlorofluorescein diacetate; PBS: Phosphate buffered serum; Rho 123: Rhodamine 123; NADPH: Nicotinamide adenine dinucleotide phosphate; DTNB: 5,5'-dithiobis-2-nitrobenzoic acid, Ellman's reagent; GSSG-R: Glutathione disulfide reductase; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMSO: Dimethyl sulfoxide; HPLC: High-performance liquid chromatography.

Glycosylated Cell‐Penetrating Poly(disulfide)s: Multifunctional Cellular Uptake at High Solubility

The glycosylation of cell‐penetrating poly(disulfide)s (CPDs) is introduced to increase the solubility of classical CPDs and to achieve multifunctional cellular uptake. With the recently developed sidechain engineering, CPDs decorated with α‐d‐glucose (Glu), β‐d‐galactose (Gal), d‐trehalose (Tre), and triethyleneglycol (TEG) were readily accessible. Confocal laser scanning microscopy images of HeLa Kyoto cells incubated with the new CPDs at 2.5 μm revealed efficient uptake into cytosol and nucleoli of all glycosylated CPDs, whereas the original CPDs and TEGylated CPDs showed much precipitation into fluorescent aggregates at these high concentrations. Flow cytometry analysis identified Glu‐CPDs as most active, closely followed by Gal‐CPDs and Tre‐CPDs, and all clearly more active than non‐glycosylated CPDs. In the MTT assay, all glyco‐CPDs were non‐toxic at concentrations as high as 2.5 μm. Consistent with thiol‐mediated uptake, glycosylated CPDs remained dependent on thiols on the cell surface for dynamic covalent exchange, their removal with Ellman's reagent DTNB efficiently inhibited uptake. Multifunctionality was demonstrated by inhibition of Glu‐CPDs with d‐glucose (IC50 ca. 20 mm). Insensitivity toward l‐glucose and d‐galactose and insensitivity of conventional CPDs toward d‐glucose supported that glucose‐mediated uptake of the multifunctional Glu‐CPDs involves selective recognition by glucose receptors at the cell surface. Weaker but significant sensitivity of Gal‐CPDs toward d‐galactose but not d‐glucose was noted (IC50 ca. 110 mm). Biotinylation of Glu‐CPDs resulted in the efficient delivery of streptavidin together with a fluorescent model substrate. Protein delivery with Glu‐CPDs was more efficient than with conventional CPDs and remained sensitive to DTNB and d‐glucose, i.e., multifunctional.

Silver-Nanoparticle-Embedded Porous Silicon Disks Enabled SERS Signal Amplification for Selective Glutathione Detection.

As the major redox couple and nonprotein thiol source in human tissues, the level of glutathione (GSH) has been a concern for its relation with many diseases. However, the similar physical and chemical properties of interference molecules such as cysteine (Cys) and homocysteine (Hcy) make discriminative detection of GSH in complex biological fluids challenging. Here we report a novel surface-enhanced Raman scattering (SERS) platform, based on silver-nanoparticle-embedded porous silicon disks (PSDs/Ag) substrates for highly sensitive and selective detection of GSH in biofluids. Silver nanoparticles (AgNPs) were reductively synthesized and aggregated directly into pores of PSDs, achieving a SERS enhancement factor (EF) up to 2.59 × 107. Ellman's reagent 5,5'-ditho-bis (2-nitrobenzoic acid) (DTNB) was selected as the Raman reactive reporting agent, and the GSH quantification was determined using enzymatic recycling method, and allowed the detection limit of GSH to be down to 74.9 nM using a portable Raman spectrometer. Moreover, the significantly overwhelmed enhancement ratio of GSH over other substances enables the discrimination of GSH detection in complex biofluids.

Research of indicators of the endogenous antioxidant system in rats affected with sodium nitrite on the background of tobacco intoxication

Under conditions of progression of environmental pollution, the one of priority directions of toxicology and medicine is the study of features and mechanisms of the combined effect of xenobiotics – risk factors of many ecologically depending diseases. In last years there is widely studied the influence of tobacco smoking on the human organism. The numerous researches proved that the wide use of mineral fertilizers in agriculture resulted in environmental pollution, especially pollution of atmospheric air, drinking water and food and created the real threat for life of both human and other living beings. All these anthropogenic toxicants penetrate the human organism and lead to the oxidative stress and activation of free radical processes in the organism and imbalance in functioning of pro- and antioxidant systems.The aim of the research is to establish the level of the oxidative stress and activity of indicators of the system of antioxidant defense in rats, injured with sodium nitrite on the background of 45 days tobacco intoxication for choosing adequate schemes of correction of detected disorders.Experiments were realized on non-linear white male rats of different age groups, who underwent the effect of tobacco smoke during 45 days. Two groups of animals, injured by smoke, received sodium nitrite in dose 45 mg / kg of body mass for 24 hours and 72 hours before the end of experiment. Euthanasia was realized under thiopental anesthesia. Blood, blood serum liver and kidneys were taken for the study.For determining AOF, there was realized extraction of blood neutrophils by the method of gradient centrifugation. For measuring AOF level in neutrophils, dichlofluorescein diacetate was used. The level of AOF production was analyzed by the intensity of dye stuff lighting on the current cytometer. Catalase activity in the reaction with ammonium molybdate, superoxide dismutase activity with nitrotetrazolium blue, the content of restored glutathione at interaction of Ellman reagent with free SH-groups were determined in blood serum and tissues. The content of ceruloplasmin in blood serum was determined by the reaction of para-phenylendiamine oxidation.The studies were realized observing general principles of experiments on animals, coordinated with statements of the European convention about protection of vertebral animals, used for experimental and other scientific aims.Rats of all age groups, injured by tobacco smoke demonstrated the increase of AOF level in blood that resulted in the oxidative process development in the organism. The synchronous intoxication of rats with tobacco smoke and sodium nitrite deepened even more all detected disorders that testify to the reliable (p≤0,05) increase of AOF that acquired the maximal values in immature animals. The oxidative stress, observed in the organism of rats of all age groups, caused inhibition of antioxidant enzymes activity: superoxide dismutase, catalase and decrease of the non-enzyme component of the antioxidant system – restored glutathione. The synchronous injure of animals by sodium nitrite and tobacco smoke resulted in the decrease of catalase activity in lungs of mature rats in 2,4times compared with intact animals in the final term of the study. After sodium nitrite intoxication of rats, toxicated by smoke, the content of restored glutathione was maximally decreased in the liver of mature rats (in 4,5 times). Most expressed changes of indicators of the antioxidant system were observed in blood serum and organs of mature rats

In vitro biosynthesis of 3-mercaptolactate by lactate dehydrogenases.

3-Mercaptolactate (3ML) is an interesting mercapto compound with special regard to the biosynthesis of new polythioesters (PTEs). Unfortunately, this thioester analog of lactic acid is currently not commercially available. For this reason, we developed an in vitro biosynthesis pathway to convert cysteine to 3-mercaptopyruvate (3MPy), which is then rapidly and efficiently converted to 3ML by suitable lactate dehydrogenases (LDHs). As liver LDH from Rattus norvegicus (LDHRn) was previously described to Exhibit 3MPy reduction activity, in silico studies based on homology to LDHRn were performed and led to the identification of four potentially suitable bacterial LDH candidates from Escherichia coli (LDHEc), Corynebacterium glutamicum (LDHCg), Bacillus cereus (LDHBc) and Gloeobacter violaceus (LDHGv). After heterologous expression in E. coli followed by purification, the enzymes were assessed for their potential to reduce 3MPy to 3ML in comparison to LDHRn. With 3MPy, LDHs from E. coli, C. glutamicum and B. cereus showed no or only very low specific activities of 0.23±0.1U/mg (LDHCg) and 0.08±0.2U/mg (LDHBc), respectively. In contrast, LDHGv exhibited a remarkable specific activity of 63.6±8.1U/mg, being even twice as active as the R. norvegicus LDH. To verify LDH-catalyzed biosynthesis of 3ML we developed and optimized a detection method allowing qualitative analysis and quantification of 3MPy and 3ML by derivatization with Ellman's reagent and liquid chromatography-mass spectrometry. This study shows once more the impressive versatility of LDHs and presents a rapid and efficient biosynthesis process for 3ML, a biotechnologically interesting, yet hard-to-obtain, compound.

Heavy metal ion removal by thiol functionalized aluminum oxide hydroxide nanowhiskers

In this study, we developed a cost effective method of using thiol functionalized γ-aluminum oxide hydroxide (γ-AlOOH) filters for removing three key heavy metals from water: mercury, lead, and cadmium under non-concomitant conditions. Compared to non-thiol treated γ-AlOOH filters, the introduction of thiol functional groups greatly improved the heavy metal removal efficiency under both static and dynamic filtration conditions. The adsorption kinetics of thiol functionalized γ-AlOOH were investigated using the Lagergren first order and pseudo-second order kinetics models; whereas the isothermal adsorption behavior of these membranes was revealed through the Langmuir and Freundlich models. Heavy metal concentration was quantified by Inductively Coupled Plasma-Mass Spectroscopy, and the thiol level on γ-AlOOH surface was measured by a colorimetric assay using Ellman's reagent. X-ray photoelectron spectroscopy was used to further address the surface sulfur state on the membranes after heavy metal exposure. Mechanisms for heavy metal adsorption were also discussed.

Sidechain Engineering in Cell‐Penetrating Poly(disulfide)s

Cell‐penetrating poly(disulfide)s (CPDs) have been introduced recently to explore new ways to enter into cells. In this report, we disclose a general method to covalently modify the sidechains of CPDs. Compatibility of copper‐catalyzed alkyne‐azide cycloaddition (CuAAC) with the addition of either strained cyclic disulfides of varied ring tension or increasing numbers of guanidinium and phosphonium cations is demonstrated. Reloading CPDs with disulfide ring tension results in an at least 20‐fold increase in activity with preserved sensitivity toward inhibition with the Ellman's reagent. The cumulation of permanent positive charges by sidechain engineering affords Ellman‐insensitive CPDs with similarly increased activity. Co‐localization experiments indicate that the CPDs reach endosomes, cytosol and nucleus, depending on their nature and their concentration. Supported by pertinent controls, these trends confirm that CPDs operate with combination of counterion‐ and thiol‐mediated uptake, and that the balance between the two can be rationally controlled. For the most active CPDs, uptake can be observed at substrate (fluorophore) concentrations as low as 5 nm.

Strain-Promoted Thiol-Mediated Cellular Uptake of Giant Substrates: Liposomes and Polymersomes.

Simple cyclic disulfides under high tension mediate the uptake of giant substrates, that is, liposomes and polymersomes with diameters of up to 400 nm, into HeLa Kyoto cells. To place them at the surface of the vesicles, the strained disulfides were attached to the head-group of cationic amphiphiles. Bell-shaped dose response curves revealed self-activation of the strained amphiphiles by self-assembly into microdomains at low concentrations and self-inhibition by micelle formation at high concentrations. Poor colocalization of internalized vesicles with endosomes, lysosomes, and mitochondria indicate substantial release into the cytosol. The increasing activity with disulfide ring tension, inhibition with Ellman's reagent, and inactivity of maleimide and guanidinium controls outline a distinct mode of action that deserves further investigation and is promising for practical applications.

Strain‐Promoted Thiol‐Mediated Cellular Uptake of Giant Substrates: Liposomes and Polymersomes

AbstractSimple cyclic disulfides under high tension mediate the uptake of giant substrates, that is, liposomes and polymersomes with diameters of up to 400 nm, into HeLa Kyoto cells. To place them at the surface of the vesicles, the strained disulfides were attached to the head‐group of cationic amphiphiles. Bell‐shaped dose response curves revealed self‐activation of the strained amphiphiles by self‐assembly into microdomains at low concentrations and self‐inhibition by micelle formation at high concentrations. Poor colocalization of internalized vesicles with endosomes, lysosomes, and mitochondria indicate substantial release into the cytosol. The increasing activity with disulfide ring tension, inhibition with Ellman's reagent, and inactivity of maleimide and guanidinium controls outline a distinct mode of action that deserves further investigation and is promising for practical applications.

Investigation of the Low Molecular Weight Thiol Composition in a Metastatic Prostate Cancer Cell Line (LNCaP) by LC-UV-MS and NMR after Labelling with the Ellman Reagent

The low molecular weight thiols present in the deproteinized extract of a prostate cancer cell line (LNCaP-FGC) were analysed after derivatization with the Ellman reagent (ESSE). The mixed disulphides formed (RSSE) were fractionated, characterized and quantified by liquid chromatography on a C-18 column using UV detection. This revealed the presence, in femtomoles per cell, of glutathione (8.30 ± 0.73), cysteine (2.71 ± 0.04) and cysteinylglycine (0.83 ± 0.10), accounting for the bulk of the thiol present. Further analysis of the cell extracts using a novel and sensitive mass spectrometry technique allowed the detection of low level of an additional derivative which was identified as cysteinylglycerate using NMRspectroscopy.

Protein glutathionylation protects wheat (Triticum aestivum Var. Sonalika) against Fusarium induced oxidative stress

Fusarium induced oxidative stress could be recovered by reversible protein oxidative modification through the process of glutathionylation in co-stressed (low-dose (50 μM) Cd2+ pre-treatment followed by Fusarium inoculation) wheat seedlings. Co-stressed seedlings showed low disease severity index as compared to Fusarium infected seedlings. A reduced level of hydrogen peroxide (H2O2) and carbonyl contents due to irreversible protein oxidation were observed in co-stressed seedlings as compared to Fusarium infected seedlings. Further, a comparative biochemical assay showed an enhanced glutathione content in co-stressed tissues as compared to Fusarium infected tissues. In an investigation, reduced glutathione pre-coated agarose gel beads were used to pull down proteins having affinity with GSH. Fructose-1, 6-bisphosphate aldolase and 3-Phosphoglycerate kinase were observed to be co-existed in co-stressed seedlings when analysed by LC-MS/MS after being processed through protein-pull assay. Co-stressed tissues showed an enhanced free protein thiol content as compared to Fusarium infected tissues. The ratio of free thiol to thiol disulfides was also observed to be increased in co-stressed tissues as compared to Fusarium infected tissues. In contrast, the quantitative assay by Ellman's reagent and qualitative analysis by diagonal gel electrophoresis showed enhanced protein thiol disulfides in Fusarium infected tissues as compared to co-stressed tissues. Further, glutaredoxin, responsible for the reverse reduction of proteins was observed to be enhanced in co-stressed tissues as compared to Fusarium infected tissues. Thus, a low dose Cd2+ triggered glutathionylation is suggestive of offering tolerance against Fusarium induced oxidative stress and protects target proteins from irreversible modification and permanent damage in wheat.

Serum free sulfhydryl status is associated with patient and graft survival in renal transplant recipients

Oxidative stress contributes significantly to graft failure, morbidity and mortality in renal transplant recipients (RTR). In cells, free sulfhydryl groups (reduced thiols, R-SH) are the transducers of redox-regulated events; their oxidation status is modulated by interaction with reactive oxygen and nitrogen oxide species and thought to be in equilibrium with the circulating pool. We hypothesized that high levels of serum free thiols are a reflection of a favorable redox status and therefore positively associated with cardiovascular risk parameters, patient and graft survival in RTR.To test this, reactive free thiol groups (R-SH; corrected for total protein) were quantified in serum of 695 RTR (57% male, 53±13yr, functioning graft ≥1yr) using Ellman's Reagent, and R-SH determinants were evaluated with multivariable linear regression models. Associations between R-SH and mortality or graft failure were assessed using multivariable Cox regression analyses.In multivariable models, male gender, estimated glomerular filtration rate and serum thiosulfate positively associated with R-SH while BMI, HbA1c, corrected calcium and NT-pro-BNP inversely associated with R-SH (model R2=0.26). During follow-up (3.1 [2.7–3.9] yrs), 79 (11%) patients died and 45 (7%) patients developed graft failure. R-SH correlated inversely with all-cause mortality (HR 0.58 [95% CI 0.45–0.75] per SD increase) and graft failure (HR 0.42 [0.30–0.59]; both P<0.001), independent of parameters with which R-SH significantly associated in the multivariable regression analyses, except for NT-pro-BNP.Serum R-SH are associated with a beneficial cardiovascular risk profile and better patient and graft survival in RTR, suggesting potential usefulness as low-cost, high-throughput screening tool for whole-body redox status in translational studies. Whether R-SH modification improves long-term outcome of RTR warrants further exploration.

Thiolated α-Cyclodextrin: The Invisible Choice to Prolong Ocular Drug Residence Time

It was the aim of this study to develop cysteamine-conjugated α-cyclodextrin (α-CD) enabled to form disulfide bonds with cysteine-rich substructures of the ocular mucus layer to provide a prolonged residence time of incorporated drugs at the site of action. Cysteamine was covalently attached to oxidized α-CD via reductive amination. The resulting α-CD-cysteamine conjugates (α-CD-Cys) were characterized regarding the amount of free thiol groups attached to the oligomer backbone via Ellman's reagent; resazurin assay was conducted for cytotoxicity, and mucoadhesive properties were evaluated on porcine intestinal and ocular mucosal tissues. Furthermore, albino rabbits were used for assessing the irritation-masking effects of α-CD-Cys. Free thiol groups attached to the backbone were in the range of 558 ± 24-1143 ± 92 μmol/g. None of these α-CD-Cys unduly affected the viability of Caco-2 cells in a concentration of 0.5%. Mucoadhesive properties of α-CD-Cys were up to 32-fold improved compared to unmodified α-CD. Encapsulation of cetirizine into α-CD-Cys resulted in significantly reduced local ocular mucosal irritation of this model drug. According to these results, α-CD-Cys is a promising new tool to prolong drug residence time on the ocular mucosal surface.