In order to explore the potential influences of the disulfide bridge on the physical and chemical properties of PrP protein, the expressed recombinant human wild-type PrP protein was purified for using in an established redox process for the reduction and oxidation of the ethanethiol group within PrP. Sedimentation tests illustrated that redox process remarkably promoted the aggregation of recombinant PrP. Thioflavin T binding assay revealed an enhanced fibrillization of the recombinant human PrP after redox process. Far-UV circular dichroism demonstrated that the PrP treated with redox process showed a significant p-sheet rich structure. Furthermore, PrP-specific Western blot identified that the recombinant PrP after redox possessed stronger proteinase K-resistance. Those data indicates that the formation of the disulfide bridge induces the alteration of the secondary structure and enhances the progresses of aggregation and fibrillization of PrP protein.
Amyloid ; chemistry ; Endopeptidase K ; metabolism ; Humans ; Oxidation-Reduction ; Prions ; chemistry ; metabolism ; Protein Multimerization ; Protein Structure, Secondary ; Proteolysis ; Sulfhydryl Compounds ; chemistry

The effect of selenium on the tissue sulfhydryl group content and lipid peroxide-destorying enzyme system in the liver, kidney and testis of rat treated with mercury was investigated. The male rats were injected s.c. with HgCl2 (10 micromoles/kg BW) and orally received Na2SeO3 (13 micromoles/kg BW) simultaneously. After 3 days, liver, kidney and testis were removed and analyzed. Mercury decreased the total sulfhydryl group content in the kidney by 25% and the total glutathione content in the kidney and testis by 50% and 36%, respectively, with no changes in other tissues. There was 12% increase in the total sulfhydryl group but not in the total glutathione content in kidney by a simul-taneous treatment of Se and Hg. Glutathione peroxidase (GSH-Px) activities were decreased by 63% in the liver and 69% in the kidney, and glutathione reductase (GSH-Rd) activity was increased in the tests by 16% by the Hg treatment with no changes in Other tissues. Hg had no effect upon glutathione-S-transferase activities in all organs examined. Simultaneous Se treatment increased GSH-Rd activity in the kidney by 23% and GSH-Px activities in liver and kidney by 24% and 21%, respectively, compared to the Hg-treated group. These data indicate that the alleviation of Hg toxicity by Se treatment is well correlated with the protein sulfhydryl group content and GSH-Px activity.
Animal ; Glutathione/metabolism* ; Glutathione Peroxidase/analysis ; Glutathione Reductase/analysis ; Male ; Mercury/toxicity* ; Rats ; Selenium/pharmacology* ; Sulfhydryl Compounds/analysis*

Redox signal transduction, especially the oxidative modification of proein thiols, correlates with many diseases and becomes an expanding research area. However, there was rare method for quick and specific detection of protein thiols and their oxidative modification in living cells. In this article, we review the current chemical strategies for the detection and quantification of protein thiols and related cysteine oxidation. We also look into the future of the development of fluorescent probes for protein thiols and their potential application in the research of reactive cysteine proteomes and early detection of redox-related diseases.
Animals ; Cysteine ; metabolism ; Fluorescent Dyes ; Humans ; Nitrosation ; Oxidation-Reduction ; Proteins ; chemistry ; metabolism ; Reactive Nitrogen Species ; metabolism ; Reactive Oxygen Species ; metabolism ; Sulfenic Acids ; analysis ; Sulfhydryl Compounds ; analysis ; chemistry ; metabolism

Genetic modification of barley variety can be an efficient way to improve beer quality. The objective of this study was to understand the effect of trxS gene on hydrolases activities in transgenic and non-transgenic barley seeds. The results showed that alpha-amylase, free beta-amylase and limit dextrinase activity were increased in transgenic seeds in comparison with non-transgenic seeds. Sulfhydryl content of protein in transgenic seeds was also higher than that in non-transgenic seeds, suggesting that trxS gene could express in barley seeds, which opens a new way for breeding new barley varieties to improve beer quality.
Germination ; genetics ; Glucosyltransferases ; metabolism ; Hordeum ; enzymology ; genetics ; Plants, Genetically Modified ; enzymology ; genetics ; Seeds ; enzymology ; genetics ; Sulfhydryl Compounds ; metabolism ; Thioredoxins ; genetics ; alpha-Amylases ; metabolism ; beta-Amylase ; metabolism

Holo glutaredoxin (Grx) is a homo-dimer that bridges a [2Fe-2S] cluster with two glutathione (GSH) ligands. In this study, both monothiol and dithiol holo Grxs are found capable of transferring their iron-sulfur (FeS) cluster to an apo ferredoxin (Fdx) through direct interaction, regardless of FeS cluster stability in holo Grxs. The ligand GSH molecules in holo Grxs are unstable and can be exchanged with free GSH, which inhibits the FeS cluster transfer from holo Grxs to apo Fdx. This phenomenon suggests a novel role of GSH in FeS cluster trafficking.
Circular Dichroism ; Dimerization ; Ferredoxins ; chemistry ; metabolism ; Glutaredoxins ; chemistry ; metabolism ; Glutathione ; metabolism ; Iron ; chemistry ; Ligands ; Magnetic Resonance Spectroscopy ; Sulfhydryl Compounds ; chemistry ; Sulfur ; chemistry ; Toluene ; analogs & derivatives ; chemistry

OBJECTIVETo study the relationship of renal aquaporin -1, -2, -3, and -4 (AQP1-4) expression with renal parenchymal thickness and glomerular filtration rate (GFR) in children with congenital hydronephrotis.

METHODSRenal tissue samples were obtained from 10 kidneys of 10 children (age: 62.3±18.3 months) with hydronephrosis and who underwent Anderson-Hynes pyeloplasty. Renal control samples were obtained from 6 children (age: 62.7±17.1 months) undergoing nephrectomy for nephroblastoma and were confirmed histologically as normal renal tissues. Renal parenchymal thickness of the hydronephrotic kidneys was measured by ultrasound preoperatively and was verified at operation. Renal GFR was assessed using 99mTc-DTPA scintigraphy preoperatively. Western blot was used to examine the expression of AQP1-4 in the renal tissues. The correlations of renal AQP1-4 expression with the renal parenchymal thickness and GFR were assessed by Pearson correlation analysis.

RESULTSThe expression of AQP1-4 in the hydronephrotis group was markedly reduced compared to that in the control group (P<0.05). The mean renal parenchymal thickness of the hydronephrotic kidney was 4.59±2.25 mm measured by ultrasound preoperatively. The mean GFR of the obstructed kidney was significantly lower than that of the contralateral kidney in the hydronephrosis group (40±12 mL/min vs 105±20 mL/min; P<0.05). The expression of AQP1, 2, 3 and 4 was positively correlated with preoperative renal GFR and renal parenchymal thickness in the hydronephrosis group (P<0.05). Renal parenchymal thickness was positively correlated with renal GFR (P<0.05).

CONCLUSIONSThe expression of renal AQP1-4 is reduced in children with congenital hydronephrosis. The expression levels of AQP1-4 are positively correlated with renal parenchymal thickness and GFR.

Aquaporins ; analysis ; Child ; Child, Preschool ; Female ; Glomerular Filtration Rate ; Humans ; Hydronephrosis ; congenital ; metabolism ; pathology ; Kidney ; pathology ; Male ; Sulfhydryl Compounds ; Technetium Tc 99m Aggregated Albumin ; Technetium Tc 99m Pentetate

Antirheumatic gold compounds have been shown to inhibit NF-kB activation by blocking IkB kinase (IKK) activity. To examine the possible inhibitory mechanism of gold compounds, we expressed wild type and mutant forms of IKk alpha and beta subunits in COS-7 cells and determined the effect of gold on the activity of these enzymes both in vivo and in vitro. Substitution of Cys-179 of IKK beta with alanine (C179A) rendered the enzyme to become resistant to inhibition by a gold compound auranofin, however, similar protective effect was not observed with an equivalent level of IKK alpha (C178A) mutant expressed in the cells. Auranofin inhibited constitutively active IKK alpha and beta and variants; IKK alpha (S176E, S180E) or IKK beta (S177E, S181E), suggesting that gold directly cause inhibition of activated enzyme. The different inhibitory effect of auranofin on IKK alpha (C178A) and IKK beta (C179A) mutants indicates that gold could inhibit the two subunits of IKK in a different mode, and the inhibition of NF- kB and IKK activation induced by inflammatory signals in gold-treated cells appears through its interaction with Cys-179 of IKK beta.
Amino Acid Substitution ; Animals ; Auranofin/*pharmacology ; COS Cells ; Cysteine/genetics/*metabolism ; Enzyme Activation/drug effects ; Gold Compounds/*pharmacology ; Protein Subunits/chemistry ; Protein-Serine-Threonine Kinases/*antagonists & inhibitors/chemistry/genetics/*metabolism ; Sulfhydryl Compounds/pharmacology

An experiment was designed to investigate the reaction mechanism of AP (apurinic or apyrimidinic) DNA endonucleases (APcI, APcII, APcIII) purified from rat liver chromatin. Sulfhydryl compounds (2-mercaptoethanol, dithiothreitol) brought about optimal activities of AP DNA endonucleases and N-ethylmaleimide or HgCl2 inhibited the enzyme activities, indicating the presence of sulfhydryl group at or near the active sites of the enzymes. Mg2+ was essential and 4mM of Mg2+ was sufficient for the optimal activities of AP DNA endonucleases. Km values of APcI, APcII and APcIII for the substrate (E. coli chromosomal AP DNA) were 0.53, 0.27 and 0.36 microM AP sites, respectively. AMP was the most potent inhibitor among adenine nucleotides tested and the inhibition was uncompetitive with respective to the substrate. The Ki values of APcI, APcII and APcIII were 0.35, 0.54 and 0.41mM, respectively. The degree of nick translation of AP DNAs nicked by APcI, APcII and APcIII with Klenow fragment in the presence and absence of T4 polynucleotide kinase or alkaline phosphatase were the same, suggesting that all 3 AP DNA endonucleases excise the phosphodiester bond of AP DNA strand to release 3-hydroxyl nucleotides and 5-phosphomonoester nucleotides.
Animals ; Binding Sites ; Chromatin/*enzymology ; DNA Damage/physiology ; DNA Repair/physiology ; DNA-(Apurinic or Apyrimidinic Site) Lyase ; Deoxyribonuclease IV (Phage T4-Induced) ; Endodeoxyribonucleases/antagonists & inhibitors/drug effects/*metabolism ; Kinetics ; Liver/drug effects/*enzymology ; Magnesium/pharmacology ; Rats ; Sulfhydryl Compounds/pharmacology

It has been reported that a change in the cellular redox state may be involved in the regulation of vascular tone, but the underlying mechanism is not fully understood. The present study was designed to investigate the cellular effect of sulfhydryl modifying agents in the coronary artery of rabbit using the tension measurement and whole cell clamping method. The application of diamide, a sulfhydryl oxidizing agent, relaxed the endothelium denuded coronary arteries in a dose dependent manner. The fact that this diamide-induced relaxation was significantly attenuated by a pretreatment of 4-AP, and the coronary arteries precontracted with 100 mM K+ instead of histamine, suggests the involvement of 4-AP sensitive K+ channels in the diamide-induced relaxation of coronary arteries. Whole cell patch clamp studies revealed that the 4-AP sensitive IdK was significantly enhanced by the membrane permeant oxidizing agents, diamide and DTDP, and were reversed by subsequent exposure to the reducing agent, DTT. Neither the membrane impermeant oxidizing or reducing agents, GSSG or GSH, had any effect on the activity of IdK, indicating that intracellular sulfhydryl modification is critical for modulating IdK activity. The Diamide failed to significantly alter the voltage dependence of the activation and inactivation parameters, and did not change the inactivation process, suggesting that diamide increases the number of functional channels without altering their gating properties. Since IdK has been believed to play an important role in regulating membrane potential and arterial tone, our results about the effect of sulfhydryl modifying agents on coronary arterial tone and IdK activity should help understand the pathophysiology of the diseases, where oxidative damage has been implicated.
Animal ; Arteries/physiology ; Arteries/drug effects ; Arteries/cytology ; Coronary Vessels/physiology ; Coronary Vessels/drug effects* ; Coronary Vessels/cytology ; Female ; Male ; Oxidants/pharmacology* ; Potassium Channels/physiology ; Rabbits ; Reducing Agents/pharmacology* ; Sulfhydryl Compounds/metabolism*

OBJECTIVEFlavonoids are present in foods such as fruits and vegetables. Several studies have demonstrated a relationship between the consumption of flavonoid-rich foods and prevention of human disease, including neurodegenerative disorders. We assessed the effect of rutin (quercetin-3-O-rutinoside) on oxidative stress in kainic acid (KA)-induced seizure.

METHODSThirty-six BALB/c mice were randomly divided into three groups. In the control group, saline (intra-peritoneal, i.p.) was administered for 7 d, and on the last day, KA (10 mg/kg, i.p.) was injected 30 min after administration of saline. In rutin groups, mice were pretreated with rutin (100 and 200 mg/kg, i.p.) for 7 d, and on the last day, KA (10 mg/kg, i.p.) was injected 30 min after administration of rutin. Subsequently, behavioural changes were observed in mice. Lipid peroxidation and oxidative stress were measured respectively in the early and late phases after KA-induced seizures.

RESULTSSeizure scores in the rutin groups were significantly lower than those in the control group (P < 0.01). Furthermore, rutin dose-dependently inhibited the number of wet-dog shakes (WDS) (P < 0.05). Malondialdehyde level in the hippocampus of the rutin groups was significantly lower than that in the hippocampus of the control group on days 1 and 21 after KA administration. In the rutin groups, the thiol levels observed on day 1 after KA administration were higher than that in the control group (P < 0.01).

CONCLUSIONThese results indicate that rutin has potential anticonvulsant and antioxidative activities against oxidative stress in KA-induced seizure in mice.

Animals ; Dose-Response Relationship, Drug ; Kainic Acid ; toxicity ; Lipid Peroxidation ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Oxidative Stress ; drug effects ; Rutin ; pharmacology ; Seizures ; chemically induced ; metabolism ; Sulfhydryl Compounds ; analysis