Sulfhydryl Residues Research Articles

Powdered Medical Adhesive with Long Lasting Adhesion in Water Environment.

Medical adhesives have been used under surgical conditions. However, it is always a big challenge to maintain long-term adhesion in a water environment. Besides, it usually takes a long time to complete the adhesion, and the operation might be complicated. In this study, tannic acid and gelatin solution under acidic conditions were mixed, flocculated, lyophilized, and crushed; thus, a powdered medical adhesive (POWDER) was prepared with long-lasting adhesion in a water environment, convenience, and low price. Tannic acid bound gelatin and maintained adhesive force primarily through hydrogen bonding and reacted with amino sulfhydryl and other amino acid residues after oxidation into aldehyde, exhibiting excellent underwater adhesion. Oxidized dextran (ODex) powder rich in an aldehyde group was introduced to provide covalent binding in the adhesive. In vitro and in vivo studies showed that POWDER could quickly adhere to various tissues in the water environment. In vitro skin adhesion experiments demonstrated that it could achieve effective adhesion in a water environment for up to 60 days. Its blood compatibility, low cytotoxicity, and biodegradability were also verified. The POWDER developed in this study is of great significance for patients who need rapid wound treatments.

Carbon Free Radical (R⋅) Inactivates NF‐κB for Radical Capping Therapy

AbstractInactivating hyperactivated transcription factors can overcome tumor therapy resistance, but their undruggable features limit the development of conventional inhibitors. Here, we report that carbon‐centered free radicals (R⋅) can inactivate NF‐κB transcription by capping the active sites in both NF‐κB and DNA. We construct a type of thermosensitive R⋅ initiator loaded amphiphilic nano‐micelles to facilitate intracellular delivery of R⋅. At a temperature of 43 °C, the generated R⋅ engage in electrophilic radical addition towards double bonds in nucleotide bases, and simultaneously cap the sulfhydryl residues in NF‐κB through radical chain reaction. As a result, both NF‐κB nuclear translocation and NF‐κB‐DNA binding are suppressed, leading to a remarkable NF‐κB inhibition of up to 94.1 %. We have further applied R⋅ micelles in a clinical radiofrequency ablation tumor therapy model, showing remarkable NF‐κB inactivation and consequently tumor metastasis inhibition. Radical capping strategy not only provides a method to solve the heat‐sink effect in clinic tumor hyperthermia, but also suggests a new perspective for controllable modification of biomacromolecules in cancer therapy.

Carbon Free Radical (R⋅) Inactivates NF-κB for Radical Capping Therapy.

Inactivating hyperactivated transcription factors can overcome tumor therapy resistance, but their undruggable features limit the development of conventional inhibitors. Here, we report that carbon-centered free radicals (R⋅) can inactivate NF-κB transcription by capping the active sites in both NF-κB and DNA. We construct a type of thermosensitive R⋅ initiator loaded amphiphilic nano-micelles to facilitate intracellular delivery of R⋅. At a temperature of 43 °C, the generated R⋅ engage in electrophilic radical addition towards double bonds in nucleotide bases, and simultaneously cap the sulfhydryl residues in NF-κB through radical chain reaction. As a result, both NF-κB nuclear translocation and NF-κB-DNA binding are suppressed, leading to a remarkable NF-κB inhibition of up to 94.1 %. We have further applied R⋅ micelles in a clinical radiofrequency ablation tumor therapy model, showing remarkable NF-κB inactivation and consequently tumor metastasis inhibition. Radical capping strategy not only provides a method to solve the heat-sink effect in clinic tumor hyperthermia, but also suggests a new perspective for controllable modification of biomacromolecules in cancer therapy.

Mass spectrometric investigations of the action of hypochlorous acid on monomeric and oligomeric components of glycosaminoglycans

Hypochlorous acid (HOCl) is a strong non-radical oxidant, which is generated during inflammatory processes under the catalysis of the enzyme myeloperoxidase (MPO). HOCl reacts particularly with sulfhydryl and amino acid residues but affects also many other biomolecules. For instance, the glycosaminoglycans of articular cartilage and synovial fluids (such as hyaluronan) undergo degradation in the presence of HOCl at which the native polysaccharide is fragmented into oligosaccharides in a complex reaction.This is an initial mass spectrometry (MS)-based investigation dealing with the HOCl-induced degradation of glycosaminoglycans and the conversion of the related monosaccharides into chlorinated products. In particular, it will be shown that the reaction between HOCl and hyaluronan is slower than originally assumed and results in the generation of different products (particularly the hyaluronan monosaccharides) by the cleavage of the β-1,3/1,4-glycosidic linkages. The MS detection of chlorinated products is, however, only possible in the case of the monosaccharides. Potential reasons will be discussed.

S-Nitrosylation of OTUB1 Alters Its Stability and Ubc13 Binding.

S-Nitrosylation is a reversible post-translational modification that regulates protein function involving the covalent attachment of the nitric oxide (NO) moiety to sulfhydryl residues of the protein. It is an important regulator in the cell signaling process under physiological conditions. However, the release of an excess amount of NO due to dysregulated NOS machinery causes aberrant S-nitrosylation of proteins, which affects protein folding, localization, and activity. Here, we have shown that OTUB1, a deubiquitinating enzyme, undergoes S-nitrosylation under redox stress conditions in vivo and in vitro. Previously, we have shown that OTUB1 forms an amyloid-like structure that promotes phosphorylation of α-synuclein and neuronal toxicity. However, the mechanistic insight into OTUB1 aggregation remains elusive. Here, we identified that OTUB1 undergoes S-nitrosylation in SH-SY5Y neuroblastoma cells under rotenone-induced stress, as well as excitotoxic conditions, and in rotenone-treated mouse brains. The in vitro S-nitrosylation of OTUB1 followed by mass-spectrometry analysis has identified cysteine-23 and cysteine-91 as S-nitrosylation sites. S-Nitrosylated OTUB1 (SNO-OTUB1) diminished its catalytic activity, impaired its native structure, promoted amyloid-like aggregation, and compromised its binding with Ubc13. Thus, our results demonstrated that nitrosylation of OTUB1 might play a crucial role in regulating the ubiquitin signaling and Parkinson's disease pathology.

Stability of a Novel PEGylation Site on a Putative Haemoglobin-Based Oxygen Carrier.

PEGylation of protein sulfhydryl residues is a common method used to create a stable drug conjugate to enhance vascular retention times. We recently created a putative haemoglobin-based oxygen carrier using maleimide-PEG to selectively modify a single engineered cysteine residue in the α subunit (αAla19Cys). However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions, with consequent cross-conjugation to endogenous plasma thiols such as those found on human serum albumin or glutathione. In previous studies mono-sulfone-PEG adducts have been shown to be less susceptible to deconjugation. We therefore compared the stability of our maleimide-PEG Hb adduct with one created using a mono-sulfone PEG. The corresponding mono-sulfone-PEG adduct was significantly more stable when incubated at 37°C for 7days in the presence of 1mM reduced glutathione, 20mg/mL human serum albumin, or human serum. In all cases haemoglobin treated with mono-sulfone-PEG retained >90% of its conjugation whereas maleimide-PEG showed significant deconjugation, especially in the presence of 1mM reduced glutathione where <70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation of Hb seems adequate for an oxygen therapeutic intended for acute use, if longer vascular retention is required reagents such as mono-sulfone-PEG may be more appropriate.

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.

Enzyme‐catalyzed acylation improves gel properties of rapeseed protein isolate

Although rapeseed protein isolate (RPI) possessed some good functional properties, the use of RPI as an ingredient in the food industry is restricted mainly due to its inferior gelation. The purpose of this study was to improve the heat-induced gel properties of RPI using double processes of acylation and additional transglutaminase catalysis. Scanning electron microscopy showed that the gel formed by native RPI exhibited randomly aggregated particulate network structures whereas transglutaminase (TG)-assisted RPI gels significantly improved gelation properties. More importantly, the combined modifications of RPI using TG-assisted acylation can form a gel with unique percolating and small porous structure. Furthermore, TG-catalyzed 5% acylated RPI gel (100 U g-1 , protein basis) exhibited excellent gel properties in terms of gel strength, thermal stability, surface roughness and apparent viscosity compared to non-treated or single modification of RPI gel as determined by texture analyzer, atomic force microscopy and rheometer. Mechanistically, Fourier-transform infrared spectra and gel dissociation test revealed that TG-catalyzed acylation extensively unfolded the hydrophobic and sulfhydryl residues of RPI, in turn, reinforced re-assembly of protein molecules via hydrophobic interactions and disulfide bonds during gel formation. Combined processes of acylation and additional TG catalysis improved the thermal gelation properties by altering inter- and intra-protein structures. Such sequential processes will provide a promising approach to improve the protein gelation that could be potentially applied in the food industry. © 2020 Society of Chemical Industry.

Influence of two different feeding strategies in the dry period on dry matter intake and plasma protein peroxidative and antioxidative profile during dry period and early lactation

BackgroundDairy cows undergo dramatic changes in endocrine and metabolic status around parturition and in early lactation. Meeting the nutritional requirements of transition dairy cows is important for animal health, production and animal wellbeing. Dry cow feeding and managing play an essential role in this. The changes in metabolism of periparturient cows also lead to a rise in the production of oxidising agents, leading to oxidative stress. The relationship between dry cow diet composition and oxidative stress has received little research attention so far. In the present study, the influence of two different dry cow feedings (single diet with medium energy content over the whole dry period versus traditional two-phase diet with a low-energy “far-off” ration and a high energy “close-up” ration) on dry matter intake, energy intake and plasma protein peroxidative and antioxidative profile was investigated.ResultsThe examined parameters revealed a dynamic profile within the experimental period. Dry matter intake (DMI) did not differ between groups. However, there was a time and a group x time interaction effect: Group 1 (“one-phase”) had a very constant DMI with a slow and even decrease until calving. In Group 2 (“two-phase”), an initial increase in DMI two weeks antepartum (a.p.) was followed by a sharp drop at week 1 a.p.. The highest total antioxidant capacity and sulfhydryl residue concentration was noted at partus. In contrast, concentration of formylokinurenine and bityrosine bridges as representatives of protein peroxidation were lowest at parturition. The time course of formylokinurenine and bityrosine bridges showed parallels to the DMI. The contents of sulfhydryl groups, formylokinurenine and total antixoxidant capacity did not differ between groups. In contrast, concentration of bityrosine bridges was always higher in Group 2 compared with Group 1 and these differences were statistically significant at week 3 a.p., week 2 a.p., week 1 a.p. and at parturition.ConclusionThe results of our study suggest time-related changes of pro- and antioxidative plasma parameters. Different dry cow feeding affected antepartal DMI. Furthermore, DMI and diet compositions seemed to have an influence on plasma protein peroxidative profile and activity of antioxidative defence.

Hydrogen Sulfide Mediates Tumor Cell Resistance to Thioredoxin Inhibitor.

Thioredoxin (Trx) is a pro-oncogenic molecule that underlies tumor initiation, progression and chemo-resistance. PX-12, a Trx inhibitor, has been used to treat certain tumors. Currently, factors predicting tumor sensitivity to PX-12 are unclear. Given that hydrogen sulfide (H2S), a gaseous bio-mediator, promotes Trx activity, we speculated that it might affect tumor response to PX-12. Here, we tested this possibility. Exposure of several different types of tumor cells to PX-12 caused cell death, which was reversely correlated with the levels of H2S-synthesizing enzyme CSE and endogenous H2S. Inhibition of CSE sensitized tumor cells to PX-12, whereas addition of exogenous H2S elevated PX-12 resistance. Further experiments showed that H2S abolished PX-12-mediated inhibition on Trx. Mechanistic analyses revealed that H2S stimulated Trx activity. It promoted Trx from the oxidized to the reduced state. In addition, H2S directly cleaved the disulfide bond in PX-12, causing PX-12 deactivation. Additional studies found that, besides Trx, PX-12 also interacted with the thiol residues of other proteins. Intriguingly, H2S-mediated cell resistance to PX-12 could also be achieved through promotion of the thiol activity of these proteins. Addition of H2S-modified protein into culture significantly enhanced cell resistance to PX-12, whereas blockade of extracellular sulfhydryl residues sensitized cells to PX-12. Collectively, our study revealed that H2S mediated tumor cell resistance to PX-12 through multiple mechanisms involving induction of thiol activity in multiple proteins and direct inactivation of PX-12. H2S could be used to predict tumor response to PX-12 and could be targeted to enhance the therapeutic efficacy of PX-12.

Tyrosinase from Penicillium chrysogenum: Characterization and application in phenol removal from aqueous solution.

The tyrosinase of Penicillium chrysogenum strain AUMC 14100 Accession No. MN219732 was purified to homogeneity and chemically modified by N-ethylmaleimide (NEM) and 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride, DC). The inactivation of the purified enzyme obeyed pseudo-first-order reaction kinetics in the presence of NEM and DC (1-5 mM). The rate constants of the enzyme inactivation by NEM and DC were calculated to be 0.083 mol/min and 0.0013 mol/min, respectively. The recovery of enzyme activity by the protective effect of substrate indicates a non-specific modification of the active center. The order of tyrosinase inactivation kinetics and the substrate protection revealed the essentiality of sulfhydryl and lysyl residues in the enzyme active site and its role in the enzyme catalysis. The immobilized tyrosinase on alginate showed a gradual increase in residual activity over the immobilization time until the fourth hour. The desorptivity of tyrosinase was gradually raised with higher sodium dodecyl sulfate (SDS) concentrations. The immobilized enzyme retained about 70% of its original activity after 8 repeated cycles. Thus, immobilized tyrosinase of Penicillium chrysogenum removed 75% of phenol after 8 cycles and thus seems likely to be a good candidate for phenol removal in aqueous solution.

Marine Natural Product Honaucin A Attenuates Inflammation by Activating the Nrf2-ARE Pathway.

The cyanobacterial marine natural product honaucin A inhibits mammalian innate inflammation in vitro and in vivo. To decipher its mechanism of action, RNA sequencing was used to evaluate differences in gene expression of cultured macrophages following honaucin A treatment. This analysis led to the hypothesis that honaucin A exerts its anti-inflammatory activity through activation of the cytoprotective nuclear erythroid 2-related factor 2 (Nrf2)-antioxidant response element/electrophile response element (ARE/EpRE) signaling pathway. Activation of this pathway by honaucin A in cultured human MCF7 cells was confirmed using an Nrf2 luciferase reporter assay. In vitro alkylation experiments with the natural product and N-acetyl-l-cysteine suggest that honaucin A activates this pathway through covalent interaction with the sulfhydryl residues of the cytosolic repressor protein Keap1. Honaucin A presents a potential therapeutic lead for diseases with an inflammatory component modulated by Nrf2-ARE.

Sulfhydryl-Based Inhibition of δ-ALA-D and Na+ , K+ -ATPase Activities Depends on the Organoselenium Group Bonded to the Isoquinoline.

Organoselenium compounds and isoquinoline derivatives have their toxicity linked to induction of pro-oxidant situations. δ-Aminolevulinate dehydratase (δ-ALA-D) and Na+ , K+ -ATPase have sulfhydryl groups susceptible to oxidation. Thus, we investigated toxicological effects of 4-organoseleno-isoquinoline derivatives, cerebral monoamine oxidase B inhibitors, on rat cerebral δ-ALA-D and Na+ , K+ -ATPase activities and the involvement of sulfhydryl groups in vitro. Compounds substituted with fluoro (4-(4-fluorophenylseleno)-3-phenylisoquinoline), chloro (4-(4-chlorophenylseleno)-3-phenylisoquinoline) and trifluoro (4-(3-trifluoromethylphenylseleno)-3-phenylisoquinoline) at the selenium-bonded aromatic ring inhibited δ-ALA-D (IC50 values: 78.42, 92.27, 44.98 µM) and Na+ , K+ -ATPase (IC50 values: 41.36, 89.43, 50.66 µM) activities, possibly due to electronic effects induced by these groups. 3-Phenyl-4-(phenylseleno) isoquinoline (without substitution at the selenium-bonded aromatic ring) and 4-(4-methylphenylseleno)-3-phenylisoquinoline (with a methyl group substituted at the selenium-bonded aromatic ring) did not alter the activity of these enzymes. Dithiothreitol, a reducing agent, restored the enzymatic activities inhibited by 4-(4-fluorophenylseleno)-3-phenylisoquinoline, 4-(4-chlorophenylseleno)-3-phenylisoquinoline and 4-(3-trifluoromethylphenylseleno)-3-phenylisoquinoline, suggesting the involvement of sulfhydryl residues in this effect. However, the release of essential zinc seems not to be related to the δ-ALA-D inhibition by these compounds. According to these data, the effect of oral administration (300 mg/kg, intragastric) of 3-phenyl-4-(phenylseleno) isoquinoline on markers of systemic toxicity in Wistar rats was evaluated. None signs of toxicity was observed during or after treatment. This study suggests that the insertion of electron-withdrawing groups in the aromatic ring bonded to the selenium atom of isoquinolines tested increased its inhibitory effect on sulfhydryl enzymes in vitro. 3-Phenyl-4-(phenylseleno) isoquinoline, which has documented pharmacological properties, had no toxicological effects on the parameters evaluated in this study. J. Cell. Biochem. 118: 1144-1150, 2017. © 2016 Wiley Periodicals, Inc.

Effects of power ultrasound on oxidation and structure of beef proteins during curing processing

The aim of this study was to evaluate the effects of power ultrasound intensity (PUS, 2.39, 6.23, 11.32 and 20.96Wcm−2) and treatment time (30, 60, 90 and 120min) on the oxidation and structure of beef proteins during the brining procedure with 6% NaCl concentration. The investigation was conducted with an ultrasonic generator with the frequency of 20kHz and fresh beef at 48h after slaughter. Analysis of TBARS (Thiobarbituric acid reactive substances) contents showed that PUS treatment significantly increased the extent of lipid oxidation compared to static brining (P<0.05). As indicators of protein oxidation, the carbonyl contents were significantly affected by PUS (P<0.05). SDS–PAGE analysis showed that PUS treatment increased protein aggregation through disulfide cross-linking, indicated by the decreasing content of total sulfhydryl groups which would contribute to protein oxidation. In addition, changes in protein structure after PUS treatment are suggested by the increases in free sulfhydryl residues and protein surface hydrophobicity. Fourier transformed infrared spectroscopy (FTIR) provided further information about the changes in protein secondary structures with increases in β-sheet and decreases in α-helix contents after PUS processing. These results indicate that PUS leads to changes in structures and oxidation of beef proteins caused by mechanical effects of cavitation and the resultant generation of free radicals.

NO Better Way to Protect the Heart during Ischemia-Reperfusion: To be in the Right Place at the Right Time.

Acute myocardial infarction (MI) is one of the leading causes of mortality worldwide. MI is the heart muscle irreversible death secondary to prolonged ischemia. Over the last few decades, medical progress in how and when to restore blood flow to the ischemic area have markedly improved patient survival. Although early heart reperfusion is acknowledged to be the most effective way to limit infarct size, post-ischemic reperfusion is associated with detrimental effects, such as myocardial stunning, ventricular arrhythmias, microvascular dysfunction, and cell death. The molecular mechanisms of these reperfusion injuries remain to be elucidated and their management is very challenging. Among the various therapeutic molecular approaches proposed by experimental studies, nitric oxide (NO) role in protecting heart against MI and reperfusion injuries has been widely assessed and discussed (1–4). NO is a gasotransmitter that is abundantly produced in the cardiovascular system mainly by the NO synthase (NOS) enzymes system. Two isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS), are constitutively expressed in both myocardium and vessels, whereas inducible NOS (iNOS) is detected only in pathological conditions, such as inflammatory and/or oxidative stress. Both eNOS and nNOS are low-NO output Ca2+-dependent enzymes, while iNOS is a high-NO output Ca2+-independent enzyme. In physiological conditions, NOS form homodimers (“coupled” NOS) that catalyze NO production from l-arginine and O2 through electron transfer from NADPH on the reductase domain of one monomer to the oxidase domain of the second monomer. In pathological conditions, such as in the absence of the essential cofactor tetrahydrobiopterin (BH4), eNOS can be “uncoupled” to produce O2− instead of NO. In stress conditions, NO protects tissues through two distinct pathways. In the first one, NO activates the soluble guanylate cyclase (sGC) that initiates cyclic guanosine monophosphate (cGMP) production, leading to the activation of protein kinase G (PKG). As sGC is the major cell receptor for NO and the NO/sGC/cGMP/PKG pathway plays a critical role in both myocardium excitation–contraction coupling and cardiovascular function regulation (5–8), NO cardioprotective role was first attributed to PKG activation (9–11). However, a second pathway in which proteins are directly modified by NO addition to sulfhydryl residues, a process known as S-nitrosylation (SNO), has recently emerged in the scientific literature. Although PKG activation pathway has been largely involved in NO-mediated cardioprotection (11–13), SNO is now taking the front stage and is considered to be a key player in cardioprotection through (i) the transient modification of protein activity and/or (ii) their protection from irreversible oxidation (14–17). Indeed, Sun et al. (18) showed that reduced heart vulnerability to ischemia–reperfusion (IR) following acute ischemic preconditioning is mainly related to SNO signaling and not to PKG activation through the NO–SGC–cGMP pathway. Accordingly, we found that in exercise training-induced cardioprotection against IR injuries, protein SNO level, but not cGMP level, increased during early reperfusion (19). The same year, Methner et al. (20), using a Cre/loxP approach to selectively ablate type I PKG in cardiomyocytes, demonstrated that ischemic post-conditioning reduced infarct size in these mice like in wild type controls. Moreover, they showed that the cardioprotective effect against IR injury of mitochondria-targeted S-nitrosothiol (MitoSNO), which allows NO and S-nitrosothiol accumulation in mitochondria, was comparable in mice that specifically lack PKG in cardiomyocytes and in controls. This indicates that MitoSNO cardioprotective effect is independent of PKG. The current literature strongly supports NO implication in cardioprotection. However, the mechanism is still debated and whether increased NO availability during IR is cytoprotective remains to be demonstrated. Here, we discuss how NO might contribute to protect heart and particularly the importance of NO (i) localization, (ii) concentration, and (iii) time of availability during IR.

Close relationship between redox state of human serum albumin and serum cysteine levels in non-diabetic CKD patients with various degrees of renal function.

Human mercaptalbumin (HMA) acts as a covalent-carrier protein for sulfur-containing amino acids, cysteine, and glutathione. In addition, its sulfhydryl residue reacts with peroxyl radicals. In this study, we evaluated the redox state of human serum albumin and its relationship with serum amino thiol levels in non-diabetic chronic kidney disease (CKD) patients with various degrees of renal function. High performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring were used to analyze serum samples collected from 37 non-dialysis patients with primary glomerulonephritis without diabetes mellitus (DM) or other systemic diseases (non-diabetic CKD patients). Total cysteine and homocysteine plasma levels increased with decreasing renal function and showed a significant negative correlation with glomerular filtration rate. The protein-bound ratio of serum cysteine also changed with the degree of renal dysfunction. The HPLC fraction of human mercaptalbumin (HMA) (%) was significantly lower in nondiabetic CKD patients than in healthy subjects. The redox state of human serum albumin (i.e., HMA %) correlated significantly with the total serum cysteine level. The HPLC fraction of HMA (%) closely correlated with the serum cysteine level in non-diabetic CKD patients. An increase in oxidized cysteine with impaired renal function and a reduced plasma redox capacity associated with a decrease in the reduced form of serum albumin (HMA %) may be important risk factors for promoting long-term complications in patients with renal dysfunction.

Measurement and characterization of superoxide generation from xanthine dehydrogenase: a redox-regulated pathway of radical generation in ischemic tissues.

The enzyme xanthine oxidoreductase (XOR) is an important source of oxygen free radicals and related postischemic injury. Xanthine dehydrogenase (XDH), the major form of XOR in tissues, can be converted to xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. The conversion of XDH to XO has been assumed to be required for radical generation and tissue injury. It is also possible that XDH could generate significant quantities of superoxide, •O2–, for cellular signaling or injury; however, this possibility and its potential ramifications have not been previously considered. To unambiguously determine if XDH can be a significant source of •O2–, experiments were performed to measure and characterize •O2– generation using XDH from chicken liver that is locked in the dehydrogenase conformation. Electron paramagnetic resonance spin trapping experiments with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide demonstrated that XDH in the presence of xanthine produces significant amounts of •O2–. NAD+ and NADH inhibited the generation of •O2– from XDH in a dose-dependent manner, with NAD+ exhibiting stronger inhibition than NADH at low physiological concentrations. Decreased amounts of NAD+ and NADH, which occur during and following tissue ischemia, enhanced the generation of •O2– from XDH in the presence of xanthine. It was observed that XDH-mediated oxygen radical generation markedly depressed Ca2+-ATPase activity of isolated sarcoplasmic reticulum vesicles from cardiac muscle, and this was modulated by NAD+ and NADH. Thus, XDH can be an important redox-regulated source of •O2– generation in ischemic tissue, and conversion to XO is not required to activate radical formation and subsequent tissue injury.

Dietary macronutrients modulate the fatty acyl composition of rat liver mitochondrial cardiolipins

The interaction of dietary fats and carbohydrates on liver mitochondria were examined in male FBNF1 rats fed 20 different low-fat isocaloric diets. Animal growth rates and mitochondrial respiratory parameters were essentially unaffected, but mass spectrometry-based mitochondrial lipidomics profiling revealed increased levels of cardiolipins (CLs), a family of phospholipids essential for mitochondrial structure and function, in rats fed saturated or trans fat-based diets with a high glycemic index. These mitochondria showed elevated monolysocardiolipins (a CL precursor/product of CL degradation), elevated ratio of trans-phosphocholine (PC) (18:1/18:1) to cis-PC (18:1/18:1) (a marker of thiyl radical stress), and decreased ubiquinone Q9; the latter two of which imply a low-grade mitochondrial redox abnormality. Extended analysis demonstrated: i) dietary fats and, to a lesser extent, carbohydrates induce changes in the relative abundance of specific CL species; ii) fatty acid (FA) incorporation into mature CLs undergoes both positive (>400-fold) and negative (2.5-fold) regulation; and iii) dietary lipid abundance and incorporation of FAs into both the CL pool and specific mature tetra-acyl CLs are inversely related, suggesting previously unobserved compensatory regulation. This study reveals previously unobserved complexity/regulation of the central lipid in mitochondrial metabolism.

Electrochemical study of pyrite–ovalbumin interaction in relation to flotation

Ovalbumin (OVA), chicken egg albumin, is an environmentally friendly, non-toxic, cheap surface active agent. It has electrochemically active sulfhydryl residues in molecular structure. OVA adsorption on pyrite as an alternative depressant was investigated by cyclic voltammetry, FTIR spectroscopy and flotation. Tests were conducted in a wide potential range in alkaline pH to clarify the role of electrochemical condition on the adsorption mechanisms and the rate of depression. In the absence of OVA, the floatability reached its maxima around slightly oxidizing condition. Beyond the range, it decreased presumably due to the formation of Fe-oxyhydroxides together with the hydrophilic S-containing species. OVA–pyrite interaction occurred in the whole examined potential range. Depressing effect of OVA increased gradually from reducing to slightly oxidizing potential, open circuit potential (OCP) of pyrite, mainly due to weak conformational changes in OVA molecules together with the hydrophobic interaction around OCP. The rate of depression reached its maxima at moderately oxidizing potentials, which was referred to electrostatic attraction. This level was almost kept at higher potentials owing to OVA adsorption as metal–chelates.

Abstract LB-175: Phase II study of disulfiram and chelated Zn for the treatment of disseminated metastatic melanoma.

Abstract Background: Disulfiram, when complexed with metal ions, is capable of inactivating key oncoproteins. It is thought to accomplish this via glutathionylation of sulfhydryl residues. In human experience thus far is limited. Despite recent advances in treatment including immunologic checkpoint blockade, and b-raf inhibition Stage IV melanoma remains a near-uniformly fatal disease. This study was designed to determine the response rate following treatment with disulfiram and chelated Zn in patients with stage IV melanoma with secondary endpoints of progression free survival, overall survival, pharmacodynamics and toxicity of the combination. Methods: Eligible patients had biopsy proven Stage IV melanoma. Patients with brain metastases were allowed provided the brain metastases were treated, and the patient was on stable doses of steroids. Disulfiram was dosed at 250 mg orally at bedtime, while chelated Zn was given thrice daily at 50 mg elemental Zn orally with each meal. A treatment cycle consisted of 28 days. Patients were scheduled for response evaluation after two cycles of therapy. Serum was sampled at baseline, and at 1 week from the start of therapy for pharmacodynamic analysis. Results: Enrollment for this study began 8/3/2010, with the final patient enrolling on 1/16/2013. In total, 12 patients were enrolled, with ten being evaluable for response for this interim futility analysis. 10 of the 12 patients were tested for b-raf mutation status, with three patients harboring a b-raf mutation. One patient experienced a partial response not meeting RECIST criteria with one target lesion decreasing 27% from baseline measurements. Average time on treatment for evaluable patients was 55 days (range 20-168). Progression free survival and overall survival of evaluable patients was 53 days and 203 days respectively. Overall safety data has shown this regimen to be well tolerated with only one Gr 3 or greater drug related adverse event recorded consisting of a confusional episode which resolved following discontinuation of study medication. Other Gr 1 and 2 toxicities have included fatigue, dysgeusia, nausea, vomiting, diarrhea, ataxia, renal insufficiency, and LFT elevations. Pharmacodynamic analysis is pending. Conclusions: Disulfiram dosed at 250 mg daily together with chelated Zn at doses of 50 mg three times daily is well tolerated in patients with advanced stage IV melanoma; the combination is capable of producing tumor shrinkage. The interim futility analysis endpoint of one responding patient was not met and the study will not expand to the full Phase II cohort of 29 patients. Future work with this combination may be warranted in other cancers given the favorable safety profile, and may also be considered in combination with other therapies actively in use for stage IV melanoma. Citation Format: Kenneth F. Grossmann, Moesis Terrazas, Hung T. Khong, Wallace Akerley, Ken Kosak, Ken Boucher, Thomas P. Kennedy, Paul J. Shami. Phase II study of disulfiram and chelated Zn for the treatment of disseminated metastatic melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-175. doi:10.1158/1538-7445.AM2013-LB-175