Context-dependent monoclonal antibodies against protein carbamidomethyl-cysteine

Protein sulfhydryl residues participate in key structural and biochemical functions. Alterations in sulfhydryl status, regulated by either reversible redox reactions or by permanent covalent capping, may be challenging to identify. To advance the detection of protein sulfhydryl groups, we describe the production of new Rabbit monoclonal antibodies that react with carbamidomethyl-cysteine (CAM-cys), a product of iodoacetamide (IAM) labeling of protein sulfhydryl residues. These antibodies bind to proteins labeled with IAM (but not N-ethylmaleimide (NEM) or acrylamide) and identify multiple protein bands when applied to Western blots of cell lysates treated with IAM. The monoclonal antibodies label a subset of CAM-cys modified peptide sequences and purified proteins (human von Willebrand Factor (gene:vWF), Jagged 1 (gene:JAG1), Laminin subunit alpha 2 (gene:LAMA2), Thrombospondin-2 (gene:TSP2), and Collagen IV (gene:COL4)) but do not recognize specific proteins such as Bovine serum albumin (gene:BSA) and human Thrombospondin-1 (gene:TSP1), Biglycan (gene:BGN) and Decorin (gene:DCN). Scanning mutants of the peptide sequence used to generate the CAM-cys antibodies elucidated residues required for context dependent reactivity. In addition to recognition of in vitro labeled proteins, the antibodies were used to identify selected sulfhydryl-containing proteins from living cells that were pulse labeled with IAM. Further development of novel CAM-cys monoclonal antibodies in conjunction with other biochemical tools may complement current methods for sulfhydryl detection within specific proteins. Moreover, CAM-cys reactive reagents may be useful when there is a need to label subpopulations of proteins.

Effects of single and binary applications of bisphenol A and NaCl on Ceratopyllum demersum

Determining the physiological effects of single and binary applications of BPA and NaCl on the Ceratophyllum demersum L., which is a rootless submerged aquatic macrophyte, is the aim of the present study. For this purpose, the macrophyte was collected from a local pond that was not exposed to any pollution. Macrophytes were acclimatized in 10% nutrient solution for 14 days, than they were treated with 17.2 mg/L BPA; 10 and 100 mM NaCl; 17.2 mg/L BPA plus 10 mM NaCl and 17.2 mg/L BPA plus 100 mM NaCl. The macrophyte without added BPA or/and NaCl served as control (without treatment). Increasing concentrations of NaCl induced a progressive accumulation of Na in the macrophyte tissues. On the other hand, BPA application partially reduced Na uptake. Nutrient uptake was affected differently by the applications. The contents of some nutrients such as Cu, Zn, Mn, K and Mg were generally reduced, whereas the Fe and Ca contents were increased. In general, increases in contents of total carbohydrate, total phenolic and non-protein sulfhydryl groups were found, when compared to control. Protein and photosynthetic pigment contents, on the contrary to these, were decreased. According to findings, the increase in H2O2 and MDA levels showed that single and combined applications of BPA and NaCl in C. demersum tissues induced oxidative stress.

Effect of Berberine on Glycation, Aldose Reductase Activity, and Oxidative Stress in the Lenses of Streptozotocin-Induced Diabetic Rats In Vivo—A Preliminary Study

Diabetes mellitus affects the eye lens, leading to cataract formation by glycation, osmotic stress, and oxidative stress. Berberine, an isoquinoline alkaloid, is a natural compound that has been reported to counteract all these pathological processes in various tissues and organs. The goal of this study was to evaluate whether berberine administered at a dose of 50 mg/kg by oral gavage for 28 days to rats with streptozotocin-induced diabetes reveals such effects on the biochemical parameters in the lenses. For this purpose, the following lenticular parameters were studied: concentrations of soluble protein, non-protein sulfhydryl groups (NPSH), advanced oxidation protein products (AOPP), advanced glycation end-products (AGEs), thiobarbituric acid reactive substances (TBARS), and activities of aldose reductase (AR), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Diabetes induced unfavorable changes in the majority of the examined parameters. The administration of berberine resulted in an increased soluble protein level, decreased activity of AR, and lowered AOPP and AGEs levels. The results suggest that berberine administered orally positively affects the lenses of diabetic rats, and should be further examined with regard to its anticataract potential.

Comparison of Oxidative Stress Parameters in Heart Failure Patients Depending on Ischaemic or Nonischaemic Aetiology

Background. Abnormalities in the oxidative and antioxidant states causing oxidative stress were both found in heart failure (HF) of various aetiologies and atherosclerosis. Aim of Study. The goals of the study were as follows: comparison of oxidative stress parameters (OSP) in ischaemic cardiomyopathy (ICM) (n=479) and nonischaemic cardiomyopathy (nICM) (n=295) patients; assessment of the relationships of OSP with functional capacity (NYHA class), maximal oxygen consumption (max.O2), left ventricle ejection fraction (LVEF), and NT-proBNP concentration; and determination of the mutual relations of OSP in subgroups of patients with ICM and n-ICM. Methods. Serum concentrations of total antioxidant capacity (TAC), total oxidant status (TOS), uric acid (UA), bilirubin, albumin, protein sulfhydryl groups (PSH), and malondialdehyde (MDA) were measured. The oxidative stress index (OSI) and MDA/PSH ratio were calculated. Results. Higher concentrations of TAC (1.14 vs 1.11 mmol/l; p<0.001) and MDA (1.80 vs 1.70 μmol/l; p<0.05) and higher MDA/PSH ratios (0.435 vs 0.358; p<0,001) were observed in ICM than in nICM patients. Simultaneously, lower values of the OSI index (4.27 vs 4.6; p<0,05), PSH (4.10 vs 4.75 μmol/g of protein; p<0,001), and bilirubin (12.70 vs 15.40 μmol/l; p<0,001) concentrations were indicated in ICM patients. There were no differences in TOS, UA, and albumin between the examined groups. The NYHA class and VO2max correlate with MDA, bilirubin, and albumin in both groups, while with UA only in the ICM group. Correlations between the NYHA class, VO2max, and PSH were indicated in nICM. The association of LVEF with UA, bilirubin, and albumin has been demonstrated in the ICM group. The study showed negative correlations between TAC, MDA, and PSH and positive between TAC and MDA in both groups. In ICM patients, MDA positively correlated with UA. A negative correlation between PSH and concentrations of UA and bilirubin was expressed only in the nICM group. Conclusion. The obtained results confirm the relationship between the severity of HF and oxidative stress. The mechanisms of oxidative stress and antioxidant defence are partially different in the ICM and the nICM patients.

Depletion of protein thiols and the accumulation of oxidized thioredoxin in Parkinsonism disclosed by a red-emitting and environment-sensitive probe

Protein sulfhydryl groups play a vital role in maintaining cellular redox homeostasis and protein functions and have attracted increasing interests for the selective detection of protein thiols over low-molecular-weight thiols (LMWTs).

Phytic acid, inorganic phosphorus, antioxidants in bread and durum wheat and their associations with agronomic traits

The phytic acid (PA), inorganic phosphorus (Pi), yellow pigment (YP), water soluble phenolics (WSPH) and free protein sulfhydryl groups (PSH) of 15 Triticum aestivum and 15 Triticum durum wheats grown at six different environments were evaluated for variability, intra- and inter- relations with agronomic traits. The most variable properties in bread wheat were YP and WSPH with coefficients of variation (CV) amounting to 14.59% and 14.10%, respectively, while in durum wheat those were PSH and YP with CV of 17.59% and 15.02%, respectively. PA in durum wheat showed the least variability (CV 3.61%). Among others, positive associations were obtained between WSPH and YP in bread and durum wheat, YP and PSH, WSPH and PSH, yield (YLD) and WSPH, YLD and PSH in durum wheat, while negative associations were recorded for PA and YP, PA and WSPH, YLD and antioxidants (YP and WSPH) in bread wheat, PA and antioxidants, PA and YLD in durum wheat.

Carbonyl and sulfhydryl groups of chicken meat proteins after dietary modulation with selenium

The objective of the study was to investigate the effects of selenium modulation on the alteration of carbonyl and sulfhydryl groups in proteins in chicken breast and leg meat. Selenium, in the form of sodium selenite and selenized yeast, was applied to broiler chicken at 0.26, 0.38 and 0.50 mg Se kg-1. The alteration of protein carbonyl and sulfhydryl groups, as well as total antioxidative potential of breast and leg meat were analyzed in fresh, chilled and frozen material. Protein reactive groups were effectively protected against oxidatively induced changes (carbonyl groups formation) by dietary selenium supplementation, during frozen storage of both types of chicken muscles, either with white fibre domination (breast) or with red fibre domination (leg). The inorganic form of selenium was effective in decreasing the loss of protein sulfhydryl groups in muscles with red fibre domination during frozen storage. In conclusion, selenium compounds can be used in broiler nutrition as a protein antioxidizing agent, especially in perspective of the long storage of meat under freezing conditions.

Genotype dependent tolerance to herbicides of maize (Zea mays L.) inbred lines

Herbicide application in seed maize requires special attention due to their larger sensitivity than hybrid maize. The aim of study was to examine and define the sensitivity/tolerance of the five maize inbred lines with different susceptibility to herbicides (belonging to different heterotic groups), based on alterations of secondary metabolites (phenolics, protein sulfhydryl groups, phytic and inorganic phosphorus). Two groups of herbicides: triketons (mesotrione and topramezone) and sulfonylureas (rimsulfuron and foramsulfuron) were tested. Lines from independent heterotic group, which were sensitive to herbicides expressed visible damages together with significant reduce in grain yield, mainly induced by sulfonylurea herbicides. Parallel with that, significant increase in phenolics, phytic and inorganic phosphorus, as well as drop in protein sulfhydryl groups were observed in their leaves. Tolerant lines (belonging to Lancaster group) had mainly insignificant grain yield reduce, also with lesser variations in sulfhydryl groups, content of phytic and inorganic phosphorus, as well as increase in phenolics content. Among examined secondary metabolites, phytate is the main factor, contributing to herbicide tolerance in maize lines. Owing to lesser yield decrease and variation in content of examined secondary metabolites, expressed in treatments with triketone herbicides, they usage could be safe in maize lines.