Thiols are main determinants of total antioxidant capacity of cellular homogenates

While the total antioxidant capacity (TAC) of blood plasma is mainly accounted for by urate, TAC of cell interior can be expected to depend more on other antioxidants, especially glutathione and protein -SH groups. We studied TAC of homogenates of several lines of cultured cells subjected to the action of thiol-modifying agents. Comparison of changes of TAC of the homogenates and of the level of total thiols (determined with a biradical spin label) demonstrates that alterations in cellular thiol content is the main determinant of changes of TAC of cell homogenates. These results show that estimation of TAC of cell extracts may be a useful parameter of assessment of oxidative stress, primarily of oxidation of thiol groups, yielding information different than TAC of body fluids.     

Myricetin ameliorates high glucose-induced endothelial dysfunction in human umbilical vein endothelial cells

Endothelial dysfunction is recognized as the initial detectable stage of cardiovascular disease, a serious complication of diabetes. In this study, we evaluated effects of myricetin on high glucose (HG)-elicited oxidative damage in human umbilical vein endothelial cells (HUVECs). The cells were pre-incubated with myricetin and then treated with HG to induce apoptosis. The effect of myricetin on viability was investigated by MTT assay. The levels of lipid peroxidation (LPO) were determined by thiobarbituric acid (TBA) method. The protein expression of Bax, Bcl-2 and caspase-3 was measured by western blot analysis. Moreover, the effect of myricetin on total antioxidant capacity (TAC) and total thiol molecules was also determined. Our results showed that myricetin was able to markedly restore the viability of endothelial cells under oxidative stress. Myricetin reduced HG-caused increase in LPO levels. Also, TAC and total thiol molecules were notably elevated by myricetin. Incubation with myricetin decreased the protein expression levels of Bax, whereas it increased the expression levels of the Bcl-2, compared with HG treatment alone. Furthermore, myricetin significantly decreased cleaved caspase-3 protein expression. It is concluded that myricetin may protect HUVECs from oxidative stress induced by HG via increasing cell TAC and reducing Bax/Bcl-2 protein ratio, and caspase-3 expression.     

Antioxidative and prooxidative effects of quercetin on A549 cells

Quercetin, a common plant polyphenol, has been reported to show both antioxidant and prooxidant properties. We studied the effects of quercetin on A549 cells in in vitro culture. We found that low concentrations of the flavonoid stimulated cell proliferation and increased total antioxidant capacity (TAC) of the cells; while higher concentrations of the flavonoid decreased cell survival and viability, thiol content, TAC and activities of superoxide dismutase, catalase and glutathione S-transferase. Quercetin decreased production of reactive oxygen species in the cells but produced peroxides in the medium. The cellular effects of quercetin are therefore complex and include both antioxidant effects and induction of oxidative stress due to formation of reactive oxygen species in the extracellular medium.     

Studies on the binding of mercury in tissue homogenates

1. This paper describes an attempt to learn more about the binding of Hg(2+) to tissues at pharmacological concentrations of this metal. Other methods were not applicable to such low concentrations of mercury. 2. The method involved equilibrium dialysis of Hg(2+) against 1% homogenates of rat kidney or liver in the presence of penicillamine. Two classes of mercury-binding sites were observed, one class having a chemical affinity for mercury 100-fold greater than the other class. The binding capacities of the class of higher and lower affinity were respectively 1.0x10(-7) and 30x10(-7)mole of mercury/g. wet wt. of tissue. The same classes of binding sites were found in both liver and kidney homogenates. 3. The binding sites of both classes reacted with only one valency of Hg(2+), the other valency forming a bond with penicillamine. Thus the total binding capacities of both classes are equivalent to 50% of the total reactive protein-bound thiol groups in the homogenate. 4. The results eliminate three possible mechanisms for the preferential accumulation of mercury by kidney. They support the idea that the permeability changes in kidney cells resulting in diuresis are similar to the permeability changes produced on the membranes of other mammalian cell species by mercury.     

Total synthesis of largazole and analogues: HDAC inhibition, antiproliferative activity and metabolic stability

The total synthesis of largazole and four analogues is reported. All analogues were nanomolar HDAC inhibitors. The antiproliferative activity is driven by lipophilicity and cell permeability. In murine liver homogenates, largazole is rapidly metabolized (half-life ≤5 min) to the thiol which has a half-life of 51 min.     

Metabolism of cysteine in Ehrlich ascites tumour, mouse skeletal muscles and liver

The cell membrane of intact Ehrlich ascites tumour (EAT) cells limits the penetration and thus the utilization of exogenous cysteine in vitro. In homogenates the uptake of cysteine is the greatest in skeletal muscle, smaller in EAT and the smallest in liver. 2-Oxoglutarate, initiating the pyruvate pathway of cysteine catabolism, raises cysteine utilization only in liver homogenates. Although it also raises taurine formation, it shifts the equilibrium from the oxidative toward the anaerobic pyruvate pathway of cysteine metabolism. 2-Oxoglutarate has no effect on cysteine metabolism in EAT and only a small effect on this process in muscle homogenates. Limitation of cysteine metabolism in the pyruvate pathway in tumour cells is not compensated by increased cysteine oxidation. The greatest increase in protein thiol groups was observed in EAT homogenates; it was markedly reduced in the presence of 2-oxoglutarate.     

Inhibition of creatine kinase activity from rat cerebral cortex by D-2-hydroxyglutaric acid in vitro

D-2-Hydroxyglutaric acid (DGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as D-2-hydroxyglutaric aciduria (DHGA). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of DGA on total, cytosolic, and mitochondrial creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas cytosolic and mitochondrial activities were measured in the cytosolic and mitochondrial preparations from cerebral cortex. We verified that CK activities were significantly inhibited by DGA (11-34% inhibition) at concentrations as low as 0.25 mM, being the mitochondrial fraction the most affected activity. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of DGA on tCK activity is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK activity for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA may be related to the neurodegeneration of patients affected by DHGA.     

Dynamic changes of red cell membrane thiol groups followed by bimane fluorescent labeling

Monobromobimane labels red cell membrane protein thiol groups; bands exhibit fluorescence after sodium dodecyl sulfate acrylamide gel electrophoresis and correspond to almost all of those staining with Coomassie blue. The response of membrane protein thiol groups to oxidative challenge and the dynamics of recovery of the thiol groups may be followed. Diminished labeling is found after oxidation with diamide, with both intrachain and interchain disulfide bond formation demonstrated by sodium dodecyl sulfate acrylamide gel electrophoresis. Regeneration of thiol groups under physiological conditions (incubation with glucose) after a moderate degree of diamide oxidation is shown to be complete (with respect to thiol group content and degree and distribution of bimane label) in normal human red blood cell membranes. Even after oxidation of almost half of the membrane protein thiol groups (maximum degree of oxidation achieved), regeneration of thiol groups is almost complete; a minor fraction resides in the form of disulfide-linked high molecular weight proteins (demonstrated by the electrophoretic profile) which may be reduced completely with dithiothreitol.Bimane fluorescent labeling provides a convenient and sensitive method for following membrane thiol group status under physiological conditions.     

Investigation of albumin properties in patients with chronic renal failure

The aim of this study was the investigation of HSA properties and its structural changes after modification induced in vivo among patients with CRF who underwent haemodialysis. Application of different fluorescent dyes allowed the investigation of different regions of albumin molecule using ANS, bis-ANS, piren, piren maleimide and fluorescein isothiocyanate. As markers of oxidative modification, the total protein thiol, carbonyls, glycosylated plasma proteins and hydroperoxide were estimated in plasma. Additionally, this study investigated plasma viscosity and total antioxidant capacity (TAC) of the plasma. Results show that haemodialysis provoked significant changes in conformational properties of plasma albumin, which resulted in the loss of its biological functions. These findings suggest that oxidative stress and glycation of proteins in plasma are developed during haemodialysis. The results depict that one of the features of uraemia is the presence of signs of oxidative stress before haemodialysis. Nevertheless, oxidative stress and glycation of proteins in plasma are exacerbated during haemodialysis and are a complex process.     

Improving the endothelial dysfunction in type 2 diabetes with chromium and vitamin D3 byreducing homocysteine and oxidative stress: A randomized placebo-controlled trial

BackgroundChromium picolinate (CrPic) and vitamin D3 are known as two antioxidant micronutrients. Through inducing endothelial dysfunction, oxidants such as homocysteine (Hct) and malondialdehyde (MDA) lead to cardiovascular disease in type 2 diabetes mellitus (T2DM). No published data has directly examined the effects of these two antioxidants on improving the endothelial dysfunction in T2DM throughreducing homocysteine and oxidative stress.MethodsSubjects (n = 92) in this randomized, double blind, placebo-control study were randomly assigned to receive oral placebo (group I), D₃ (group II: 50,000 IU/ week), chromium picolinate (CrPic) (group III: 500 μg/day), and both vitamin D₃ and CrPic (group IV) for four months. Fasting blood samples were drawn at study baseline and following intervention to determine Hct, MDA, total antioxidant capacity (TAC), total thiol groups (SHs), vascular cell adhesion molecule- 1 (VCAM-1), and plasminogen activator inhibitor-1 (PAI-1).ResultsAfter intervention, MDA significantly decreased in groups II and IV; TAC significantly increased in group IV, and SHs significantly augmented in group III; Hct was significantly reduced in groups II, III, and IV; and VCAM-1 significantly decreased in groups III and IV and PAI-1 was significantly reduced in groups II, III, and IV.ConclusionOur findings suggest that through reducing homocysteine and oxidative stress and improving endothelial dysfunction, chromium and vitamin D₃ co-supplementation might be predictive and preventive of cardiovascular diseasesassociated with T2DM.IRCT, IRCT20190610043852N1, registered 21 October 2019, https://fa.irct.ir/user/trial/42293/view     

Purification and characterization of a cytosol protease from dormant cysts of the brine shrimp Artemia

A thiol protease has been isolated and purified from the postribosomal fraction of encysted embryos of the brine shrimp Artemia using a six-step procedure. The purified enzyme has a molecular weight of 55,000 +/- 4,200 and is composed of subunits of Mr 31,500 +/- 559 and 25,867 +/- 1,087. Isoelectric focusing revealed two discrete bands, one at pH 4.6 and the other at pH 5.1. The protease appears to be a member of the thiol group of proteases based on its inhibition by leupeptin, antipain, chymostatin, Ep-475, and several other thiol protease inhibitors. The enzyme was stimulated by heavy metal chelators and thiol reagents. At pH 3.5-4.0 the thiol protease hydrolyzed a wide range of proteins including bovine serum albumin, hemoglobin, Artemia embryo soluble proteins, Artemia lipovitelline, and protamine, whereas at pH 6.0-6.5 the enzyme showed a high degree of specificity for Artemia elongation factor 2 and lipovitelline alpha 1. The total amount of protease activity in crude homogenates of Artemia embryos decreased by about 50% during the first 24 h of development, while the amount of free, active enzyme decreased proportionally for 9 h of development then remained constant during the next 26-27 h of development. These changes in protease activity appear to reflect changing levels of an endogenous protease inhibitor during development.     

Reactive thiol groups in rat liver acid phosphatase

Dimeric rat liver acid phosphatase P1 of Mr 92,000 is inactivated by p-chloromercuribenzoate and fluorescein mercuriacetate (FMA). The enzyme is protected against the mercurials by the substrate analogue Pi. The reaction with FMA is accompanied by changes in absorbance at 495 nm and in fluorescence emission at 520 nm that are characteristic of reaction of this compound with thiol groups. Titration of P1 with FMA monitored by spectrophotometry or by fluorimetry indicated that equivalence is reached at an FMA/P1 ratio of 3. Since FMA can act as a bifunctional reagent, it is likely that P1 contains either 3 or 6 reactive thiol groups per molecule. Analysis of FMA inactivation/modification data by a statistical method suggests that of 6 reactive thiol groups, 2 are essential so that there are probably 3 thiol groups per subunit, one of which is located at the active site. If the total thiol number is 3, analysis suggests 1 essential thiol per subunit.     

Effects of sex differences on constitutive nitric oxide synthase expression and activity in response to pressure overload in rats

Clinical studies have documented sex differences in left ventricular (LV) hypertrophy patterns, but the mechanisms are so far poorly defined. This study aimed to determine whether 1) severe pressure overload altered expression and/or activity of cardiac constitutive nitric oxide synthase (NOS1 and NOS3) and 2) these changes were modulated according to sex. Analyses were performed 0.4-20 wk after thoracic aortic constriction (TAC) in male and female Wistar rats. Male rats with TAC exhibited early signs of cardiac dysfunction, as shown by echocardiographic and LV end-diastolic pressure measurements, whereas females with TAC exhibited higher LV hypertrophy (+96% vs. males at 20 wk; P < 0.05). After TAC, cardiac NOS1 expression was rapidly induced (0.4 wk) and stable afterward in males (P < 0.05 vs. sham groups), whereas it was delayed in females. Accordingly, specific NOS1 activity was increased by 2 wk in male rats with TAC (+122%; P < 0.001 vs. sham groups) and only by 20 wk in females (+220%; P < 0.001 vs. sham groups). NOS1 activity was correlated with NOS1 level. Regarding cardiac NOS3, expression was unaffected by TAC, and the decrease in activity observed at early and late times in male and female rats with TAC, respectively, is shown to be related to NOS3 allosteric regulator caveolin-1 level. The data demonstrated a unique sex-dependent regulation of the constitutive NOSs in response to TAC in rats; such a difference might play a role in the sex-dependent adaptability of the heart in response to pressure overload.     

Inhibition of mitochondrial creatine kinase activity from rat cerebral cortex by methylmalonic acid

Accumulation of methylmalonic acid (MMA) in tissues and biological fluids is the biochemical hallmark of patients affected by the neurometabolic disorder known as methylmalonic acidemia (MMAemia). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are not totally established. In the present study, we investigated the effect of MMA, as well as propionic (PA) and tiglic (TA) acids, whose concentrations are also increased but to a lesser extend in MMAemia, on total (tCK), cytosolic (Cy-CK) and mitochondrial (Mi-CK) creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas Cy-CK and Mi-CK were determined, respectively, in cytosolic and mitochondrial preparations from rat cerebral cortex. We verified that tCK and Mi-CK activities were significantly inhibited by MMA at concentrations as low as 1 mM, in contrast to Cy-CK which was not affected by the presence of the acid in the incubation medium. Furthermore, PA and TA, at concentrations as high as 5 mM, did not alter CK activity. We also observed that the inhibitions provoked by MMA were fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of MMA was possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of MMA may contribute to the neurodegeneration of patients affected by MMAemia and explain previous reports showing an impairment of brain energy metabolism and a reduction of brain phosphocreatine levels caused by MMA.     

Effect of heat-stable and heat-labile enterotoxins of Escherichia coli on intestinal brush border membrane enzymes of mice

The activities of intestinal brush border membrane (BBM) enzymes alkaline phosphatase, maltase, lactase, sucrase, gamma-glutamyl transpeptidase and leucine aminopeptidase were determined in intestinal homogenates and purified BBMs from control, heat-stable and heat-labile enterotoxin treated mice. The activities of all the enzymes except lactase were decreased significantly (p less than 0.01) in homogenates while increased significantly (p less than 0.001) in BBMs of experimental groups as compared to controls. Calmodulin activities were increased significantly (p less than 0.01) as compared to control in heat-stable enterotoxin treated mice but remained unaltered in heat-labile enterotoxin treated mice. DNA contents of intestinal homogenates were decreased in experimental groups demonstrating the decrease in cell number in these groups. The altered BBM enzyme activities could not be attributed to changes in calmodulin activities. The increase in enzyme activities in BBMs may reflect a compensatory phenomenon in the remaining cells.     

Expression and Changes of Hyperoxidized Peroxiredoxins in Non-Pyramidal and Polymorphic Cells in the Gerbil Hippocampus During Normal Aging

Oxidative stress is one of predisposing factors to age-related neurodegeneration in the brain. In particular, thiol-containing groups are susceptible to oxidative stress, which induces the formation of the disulfide bond and/or hyperoxidized form of thiol-containing proteins. We observed the protein thiol levels in the hippocampal homogenates and also investigated changes in hyperoxidized form of peroxiredoxin (Prx–SO₃) immunoreactivity and proteins levels in the gerbil hippocampal subregions during normal aging. Levels of total thiol, non-protein thiol, and protein thiol were decreased in the hippocampal homogenates with age. At post-natal month 1 (PM 1), pyramidal and non-pyramidal cells in the hippocampal CA1 region (CA1) showed Prx–SO₃ immunoreactivity. Prx–SO₃ immunoreactivity in the cells was decreased by PM 12, thereafter, Prx–SO₃ immunoreactivity in the cells increased again with age. In the CA2/3, Prx–SO₃ immunoreactivity in pyramidal cells was not significantly changed; however, the immunoreactivity in pyramidal cells was very low at PM 12. Prx–SO₃ immunoreactivity in the dentate gyrus (DG) was distinctly changed during aging. At PM 1, Prx–SO₃ immunoreactivity in granule and polymorphic cells was weak and strong, respectively. The immunoreactivity in the neurons was decreased with age, not shown in any neurons at PM 12. Thereafter, Prx–SO₃ immunoreactivity increased again with age. In addition, Prx–SO₃ protein level in the hippocampus was lowest at PM 12. These results suggest that thiol-containing proteins are changed during aging and Prx–SO₃ immunoreactivity was different according to cells in the hippocampal subregion during aging.     

Hypochlorous acid damages erythrocyte membrane proteins and alters lipid bilayer structure and fluidity

Treatment of human erythrocyte membranes with active forms of chlorine (hypochlorous acid and chloramine T) resulted in a concentration-dependent inhibition of the membrane Na(+), K(+)- and Mg(2+)-ATPases. Membrane protein thiol group oxidation was consistent with inactivation of enzymes and preceded oxidation of tryptophan residues and chloramine formation. Erythrocyte exposure to hypochlorous acid led to complex changes of cell membrane rigidity and cell morphological transformations: cell swelling, echinocyte formation, and haemolysis. The inhibition of ion pump ATPases of human erythrocyte membranes may be due to direct oxidation of essential residues of enzyme (thiol groups) and structural rearrangement of the membrane.     

Activity and thermal stability of acid phosphatase in homogenates of Amoeba proteus, acclimated to various temperatures

Activity and thermoresistance of acid phosphatase were determined in supernatant of Amoeba proteus homogenates using 1-naphthyl phosphate (pH 4.0) and p-nitrophenyl phosphate (pH 5.5). Although tartrate-resistant and tartrate-sensitive acid phosphatases hydrolyse both substrates, the former mainly hydrolyses p-nitrophenyl phosphate and the latter 1-naphthyl phosphate. A decrease in the activity of the total and tartrate-sensitive acid phosphatases, when using 1-naphthyl phosphate, and of the total and tartrate-resistant acid phosphatases, when using p-nitrophenyl phosphate, was found in amoebae acclimated to 10 degrees C (10 degrees-amoebae) compared to those acclimated to 25 degrees C (25 degrees-amoebae). Using 1-naphthyl phosphate, the thermoresistance of the total acid phosphatase was lower in 10 degrees-amoebae than in 25 degrees-amoebae, but the thermostability of tartrate-resistant enzyme was the same in both groups of amoebae. Using p-nitrophenyl phosphate, the thermoresistance of the total and tartrate-resistant acid phosphatases was lower (the latter only slightly) in 10 degrees-amoebae than in 25 degrees-amoebae. It is suggested that at least with the use of 1-naphthyl phosphate a decrease in thermostability of the total acid phosphatase may be due to a decrease in thermoresistance of tartrate-sensitive enzyme. The results obtained confirm the author's previous data on the activity and thermostability of electrophoretic forms of acid phosphatase using 2-naphthyl phosphate in 10- and 25 degrees-amoebae (Sopina, 2001). It is the first case of discovering a correlation between changes in primary cell thermoresistance of amoebae cultured at different temperatures and changes in the activity and thermostability of acid phosphatase in their homogenates, with the number of electrophoretic forms of this enzyme and their mobility being permanent.     

Modulatory Role of Glutathione on μ-Opioid, Substance P/Neurokinin-1, and Kainic Acid Receptor Binding Sites

Reduced glutathione (L-gamma-glutamyl-L-cysteinylglycine; GSH) is an endogenous tripeptide involved in the formation and maintenance of protein thiol groups as well as in various detoxification reactions. Because multiple receptor types contain thiol groups or disulfide bridges, effects of GSH treatments on mu-opioid, neurokinin-1/substance P, and kainic acid receptor binding sites were investigated and compared with those produced by dithiothreitol (DTT), a potent synthetic reducing agent. GSH inhibited binding more potently than did DTT at all three receptor types in porcine striatal membrane homogenates as well as in CHAPS-solubilized preparations of the mu and neurokinin-1 sites. GSH-induced inhibitory effects were associated with decreases in maximal binding capacity (Bmax) without significant alteration in apparent affinity (KD). Cysteine, the functional moiety of GSH, mimicked GSH effects albeit with lower potencies, whereas oxidized glutathione had no effects at similar concentrations. In CHAPS-solubilized preparations, the combination of low concentrations of GSH and guanylylimidodiphosphate markedly decreased the Bmax values of the binding of [3H][D-Ala2,Gly-ol5]enkephalin and [3H]substance P. This GSH-mediated mechanism may be important to prevent cell overstimulation by accelerating receptor uncoupling, desensitization, and/or internalization. This is in keeping with purported roles of GSH related to the maintenance of cellular integrity.     

Proteolytic and transhydrogenolytic activities in isolated pancreatic islets of rats.

In homogenates and subcellular fractions of pancreatic islets of Wistar rats we could demonstrate three groups of protein degrading enzymes. The proteinases of group 1 are characterized by both trypsin-like and carboxypeptidase B-like specificities with slightly acid pH optima (pH 5.5-6.5) and seem to play important roles in the conversion of proinsulin into insulin. The properties suggest that these enzymes localized in the secretion granule/mitochondria fraction are related to the tissue cathepsins. Group 2 enzymes are thiol-depending proteinases with a pH optimum at 7.0 occuring mainly in the cytosol and to a lesser extent in the fraction of nuclei and cell debris. Group 3 represents the thiol protein oxidoreductase with a pH optimum of 7.0. This enzyme degrading disulfide bonds could also be important in the formation of the disulfide bonds during protein folding after synthesis.