			Home About us Contact

Thiol Groups (thiol + groups)

Distribution by Scientific Domains

Chemistry	50%
Life Sciences	25%
Polymers and Materials Science	12%
Medical Sciences	12%

Selected Abstracts

Enhancement of Lysosomal Osmotic Sensitivity Induced by the Photooxidation of Membrane Thiol Groups,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2002
Feng-Yi Wan
ABSTRACT The osmotic lysis of photodamaged lysosomes is a critical event for killing tumor cells. How the photodamage increases lysosomal osmotic sensitivity is still unclear. In this work, the effect of the photooxidation of membrane thiol groups on the lysosomal osmotic sensitivity was studied by measuring the thiol groups with 5,5,-dithio-bis(2-nitrobenzoic acid) and examining the lysosomal ,-hexosaminidase latency loss in a hypotonic sucrose medium. The results show that methylene blue,mediated photooxidation of lysosomes decreased their membrane thiol groups and produced cross-linkage of membrane proteins (molecular weight ranging from 75,000 to 125,000), which was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Simultaneously, the lysosomal osmotic sensitivity increased. These photoinduced alterations of the lysosomes could be recovered by reducing the oxidized thiol groups with dithiothreitol. It indicates that the photooxidation of membrane thiol groups can increase the lysosomal osmotic sensitivity and therefore provides a new explanation for the photoinduced lysosomal lysis. [source]

Probing, Inhibition, and Crystallographic Characterization of Metallo-,-lactamase (IMP-1) with Fluorescent Agents Containing Dansyl and Thiol Groups

CHEMMEDCHEM, Issue 9 2006
Hiromasa Kurosaki Dr.
A series of fluorescent probes, N -(5-(dimethylamino)-1-naphthalenesulfonamido(alkyl)n)-3-thiopropionamide (DansylCnSHs), were rationally designed to detect and inhibit metallo-,-lactamase (IMP-1). These compounds were shown to function as fluorescent probes for and inhibitors of metallo-,-lactamases. The X-ray crystallographic structure shown indicates the potential of these agents for use as a new fluorescent probes for metallo-,-lactamases. [source]

Contribution of exofacial thiol groups in the reducing activity of Lactococcus lactis

FEBS JOURNAL, Issue 10 2010
D. Michelon
Lactococcus lactis can decrease the redox potential at pH 7 (Eh7) from 200 to ,200 mV in oxygen free Man,Rogosa,Sharpe media. Neither the consumption of oxidizing compounds or the release of reducing compounds during lactic acid fermentation were involved in the decrease in Eh7 by the bacteria. Thiol groups located on the bacterial cell surface appear to be the main components that are able to establish a greater exchange current between the Pt electrode and the bacteria. After the final Eh7 (,200 mV) was reached, only thiol-reactive reagents could restore the initial Eh7 value. Inhibition of the proton motive force showed no effect on maintaining the final Eh7 value. These results suggest that maintaining the exofacial thiol (,SH) groups in a reduced state does not depend on an active mechanism. Thiol groups appear to be displayed by membrane proteins or cell wall-bound proteins and may participate in protecting cells against oxidative stress. [source]

Lable-Free Electrochemical DNA Sensor Based on Gold Nanoparticles/Poly(neutral red) Modified Electrode

ELECTROANALYSIS, Issue 6 2010
Keying Zhang
Abstract We present a new strategy for the label-free electrochemical detection of DNA hybridization based on gold nanoparticles (AuNPs)/poly(neutral red) (PNR) modified electrode. Probe oligonucledotides with thiol groups at the 5-end were covalently linked onto the surface of AuNPs/PNR modified electrode via S-Au binding. The hybridization event was monitored by using differential pulse voltammetry (DPV) upon hybridization generates electrochemical changes at the PNR-solution interface. A significant decrease in the peak current was observed upon hybridization of probe with complementary target ssDNA, whereas no obvious change was observed with noncomplementary target ssDNA. And the DNA sensor also showed a high selectivity for detecting one-mismatched and three-mismatched target ssDNA and a high sensitivity for detecting complementary target ssDNA, the detection limit is 4.2×10,12,M for complementary target ssDNA. In addition, the DNA biosensor showed an excellent reproducibility and stability under the DNA-hybridization conditions. [source]

Thiol redox status evaluation in red blood cells by capillary electrophoresis-laser induced fluorescence detection

ELECTROPHORESIS, Issue 10 2005
Angelo Zinellu
Abstract Thiols and in particular glutathione (GSH) play a central role in human metabolism, including the detoxification of xenobiotics, cell homeostasis, radioprotection, and antioxidant defence. Here, a new method is provided for the measurement of reduced and total forms of thiols in red blood cells. In order to minimize oxidation of reduced thiols, a water erythrocyte lysis (15 min at 4°C) was performed followed by a protein precipitation step with acetonitrile. The supernatant was rapidly derivatized with 5-iodoacetoamidefluorescein that trapped thiol groups, thus minimizing auto-oxidation. Derivatized samples were separated in a 57 cm × 75 ,m ID capillary by using 5 mmol/L sodium phosphate, 4 mmol/L boric acid as electrolyte solution with 75 mmol/L N -methyl- D -glucamine at pH 11.0. Under these conditions, cysteinylglycine (CysGly), cysteine (Cys), glutathione, and ,-glutamylcysteine (GluCys) were baseline-resolved in , 4 min. Precision tests showed a good repeatability of our method both for migration times (coefficient of variation CV < 0.8%) and areas (CV < 3.3%). Furthermore, a good reproducibility of intrassay and interassay tests was obtained (CV < 5% and CV < 8%, respectively). The method was employed to investigate the effect of acidic precipitation on intracellular thiol concentration. Our data suggest that sample acidification causes a modification of the measured redox thiol status due to the development of a pro-oxidant environment; moreover, the thiol redox status of red blood cells was evaluated in 22 healthy volunteers. [source]

D-2-Hydroxyglutaric acid inhibits creatine kinase activity from cardiac and skeletal muscle of young rats

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 10 2003
C. G. Da Silva
Abstract Background, Tissue accumulation of high amounts of D-2-hydroxyglutaric acid (DGA) is the biochemical hallmark of the inherited neurometabolic disorder D-2-hydroxyglutaric aciduria (DHGA). Patients affected by this disease usually present hypotonia, muscular weakness, hypothrophy and cardiomyopathy, besides severe neurological findings. However, the underlying mechanisms of muscle injury in this disorder are virtually unknown. Materials and methods, In the present study we have evaluated the in vitro role of DGA, at concentrations ranging from 0·25 to 5·0 mm, on total, cytosolic and mitochondrial creatine kinase activities from skeletal and cardiac muscle of 30-day-old Wistar rats. We also tested the effects of various antioxidants on the effects elicited by DGA. Results, We first verified that total creatine kinase (CK) activity from homogenates was significantly inhibited by DGA (22,24% inhibition) in skeletal and cardiac muscle, and that this activity was approximately threefold higher in skeletal muscle than in cardiac muscle. We also observed that CK activities from mitochondrial (Mi-CK) and cytosolic (Cy-CK) preparations from skeletal muscle and cardiac muscle were also inhibited (12,35% inhibition) by DGA at concentrations as low as 0·25 mm, with the effect being more pronounced in cardiac muscle preparations. Finally, we verified that the DGA-inhibitory effect was fully prevented by preincubation of the homogenates with reduced glutathione and cysteine, suggesting that this effect is possibly mediated by modification of essential thiol groups of the enzyme. Furthermore, ,-tocopherol, melatonin and the inhibitor of nitric oxide synthase L-NAME were unable to prevent this effect, indicating that the most common reactive oxygen and nitrogen species were not involved in the inhibition of CK provoked by DGA. Conclusion, Considering the importance of creatine kinase activity for cellular energy homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA might be an important mechanism involved in the myopathy and cardiomyopathy of patients affected by DHGA. [source]

Contribution of exofacial thiol groups in the reducing activity of Lactococcus lactis

Modulation of sarcoplasmic reticulum Ca2+ -ATPase by chronic and acute exposure to peroxynitrite

FEBS JOURNAL, Issue 13 2004
Yolanda Gutiérrez-Martín
The Ca2+ -ATPase of skeletal muscle sarcoplasmic reticulum (SERCA), an integral membrane protein, becomes irreversibly inactivated in vitro by the addition of a single bolus of peroxynitrite with a K0.5 of 200,300 µm, and this results in a large decrease of the ATP-dependent Ca2+ gradient across the sarcoplasmic reticulum (SR) membranes. The inactivation of SERCA is raised by treatment of SR vesicles with repetitive micromolar pulses of peroxynitrite. The inhibition of the SERCA is due to the oxidation of thiol groups and tyrosine nitration. Scavengers that react directly with peroxynitrite, such as cysteine, reduced glutathione, NADH, methionine, ascorbate or Trolox, a water-soluble analog of ,-tocopherol, afforded significant protection. However, dimethyl sulfoxide and mannitol, two hydroxyl radical scavengers, and ,-tocopherol did not protect SERCA from inactivation. Our results showed that the target of peroxynitrite is the cytosolic globular domain of the SERCA and that major skeletal muscle intracellular reductants (ascorbate, NADH and reduced glutathione) protected against inhibition of this ATPase by peroxynitrite. [source]

Assignment of a single disulfide bridge in rat liver methionine adenosyltransferase

FEBS JOURNAL, Issue 1 2000
María L. Martínez-Chantar
Rat liver methionine adenosyltransferase incorporated 8 mol of N -ethylmaleimide per mol of subunit upon denaturation in the presence of 8 m urea, whereas 10 such groups were labelled when dithiothreitol was also included. This observation prompted a re-examination of the state of the thiol groups, which was carried out using peptide mapping, amino acid analysis and N-terminal sequencing. The results obtained revealed a disulfide bridge between Cys35 and Cys61. This disulfide did not appear to be conserved because cysteines homologous to residue 61 do not exist in methionine adenosyltransferases of other origins, therefore suggesting its importance for the differential aspects of the liver-specific enzyme. [source]

Multifunctional Dendrimer-Templated Antibody Presentation on Biosensor Surfaces for Improved Biomarker Detection

ADVANCED FUNCTIONAL MATERIALS, Issue 3 2010
Hye Jung Han
Abstract Dendrimers, with their well-defined globular shape and high density of functional groups, are ideal nanoscale materials for templating sensor surfaces. This work exploits dendrimers as a versatile platform for capturing biomarkers with improved sensitivity and specificity. The synthesis, characterization, fabrication, and functional validation of the dendrimer-based assay platform are described. Bifunctional hydroxyl/thiol-functionalized G4-polyamidoamine (PAMAM) dendrimer is synthesized and immobilized on the polyethylene-glycol (PEG)-functionalized assay plate by coupling PEG-maleimide and dendrimer thiol groups. Simultaneously, part of the dendrimer thiol groups are converted to hydrazide functionalities. The resulting dendrimer-modified surface is coupled to the capture antibody in the Fc region of the oxidized antibody. This preserves the orientation flexibility of the antigen binding region (Fv) of the antibody. To validate the approach, the fabricated plates are further used as a solid phase for developing a sandwich-type enzyme-linked immunosorbent assay (ELISA) to detect IL-6 and IL-1,, important biomarkers for early stages of chorioamnionitis. The dendrimer-modified plate provides assays with significantly enhanced sensitivity, lower nonspecific adsorption, and a detection limit of 0.13,pg,mL,1 for IL-6 luminol detection and 1.15,pg,mL,1 for IL-1, TMB detection, which are significantly better than those for the traditional ELISA. The assays were validated in human serum samples from a normal (nonpregnant) woman and pregnant women with pyelonephritis. The specificity and the improved sensitivity of the dendrimer-based capture strategy could have significant implications for the detection of a wide range of cytokines and biomarkers since the capture strategy could be applied to multiplex microbead assays, conductometric immunosensors, and field-effect biosensors. [source]

Electroactive Films of Multicomponent Building Blocks,

ADVANCED FUNCTIONAL MATERIALS, Issue 5 2007
I. Yildiz
Abstract A ligand consisting of a 2,2,-bipyridine core and two 4,4,-bipyridinium arms terminated by a thiol group is prepared following a multistep synthetic procedure. Two of these ligands assemble around a single CuI center as a result of the tetrahedral coordination of their 2,2,-bipyridine cores by the metal. Both the ligand and the complex adsorb spontaneously on the surface of polycrystalline-gold electrodes. The surface coverage of the films prepared by immersing a gold substrate into a solution of the ligand increases from monolayer to multilayer values with immersion time. Instead, the complex can only form monolayers. The cyclic voltammograms of the resulting films show the characteristic response for the reversible reduction of the 4,4,-bipyridinium dications to their radical cations. In the case of the complex, a wave for the monoelectronic oxidation of the metal center can also be observed. The back reduction wave, however, is markedly broader and appears at significantly lower potentials. Model studies in solution indicate that this response is a result of the presence of free thiol groups and is consistent with a change in the coordination geometry of the metal. Specifically, the oxidation of the CuI center to a CuII ion is, presumably, accompanied by the folding of one of the thiol groups back to interact with the metal. Thus, oxidation/reduction cycles of the metal center can, in principle, be exploited to control reversibly large amplitude molecular motions at the electrode/solution interface in the shape of the folding/unfolding of oligomethylene chains. [source]

Synthesis and metal transport ability of a new series of thiamacrocycles containing thiol and disulfide groups inside the ring

HETEROATOM CHEMISTRY, Issue 4 2001
Tatsuya Nabeshima
Synthetic methods for thiamacrocycles containing two thiol groups or a disulfide linkage and their abilities to effect single heavy-metal-ion transport across a liquid membrane were examined. High Ag+ selectivity was accomplished by the thiol or disulfide hosts, although all the corresponding crown ether analogs bearing a disulfide group showed no Ag+ selectivity. The difference of the transport preferences among the thiacrown ethers prepared here is considered to be reflected by the position and the number of the sulfur atoms ligating to Ag+ and the cavity size of the hosts. © 2001 John Wiley & Sons, Inc. Heteroatom Chem 12:276,281, 2001 [source]

Inhibition of creatine kinase activity by 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one in the cerebral cortex and cerebellum of young rats

JOURNAL OF APPLIED TOXICOLOGY, Issue 6 2010
Rodrigo Binkowski de Andrade
Abstract In the present study, we investigated the potential in vitro toxicity of the tellurium compound 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one on creatine kinase activity in cerebral cortex and cerebellum of 30-day-old Wistar rats. First, enriched mitochondrial and cytosolic fractions from the two tissues were pre-incubated for 30,min in the presence or absence of 1, 5 or 20,µm of organotellurium and the creatine kinase activity was measured. The organochalcogen reduced creatine kinase activity in a concentration-dependent pattern in the two tissues studied. Furthermore, the enzyme activity was performed after pre-incubation for 30, 60 or 90,min in the presence of 5,µm of the organotellurium. The compound inhibited creatine kinase activity in a time-dependent way in the enriched mitochondrial fraction of both tissues, but not in the cytosolic fraction, indicating different mechanisms for the organochalcogen in the mitochondrial and in the cytosolic creatine kinase. Pre-incubation of tellurium compound with reduced glutathione suggests that creatine kinase activity inhibition might be caused by direct interaction with thiol groups or by oxidative stress. Our findings suggest that creatine kinase inhibition may be one of the mechanisms by which this organotellurium could cause toxicity to the rat brain. Copyright © 2010 John Wiley & Sons, Ltd. [source]

Improvement in radiolabelling proteins with the 99mTc-tricarbonyl-core [99mTc(CO)3]+, by thiol-derivatization with iminothiolane: application to ,-globulins and annexin V

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 12 2005
Marie-Laure Biechlin
Abstract The aim of this study was to improve the radiolabelling of proteins with the 99mTc-tricarbonyl-[Tc(I)(CO)3]+ core by introducing thiol groups to their structure. To achieve this goal, ,-globulins and annexin V were derivatized with mercaptobutyrimidyl groups (MBG) after reaction with 2-iminothiolane. The optimal conditions permitted attachment of an average of 3.3 thiol groups on ,-globulins and 1.0 to annexin V. The radiolabelling assays were carried out by incubating 3.2 nmol of either ,-globulin-SH or unmodified ,-globulin with 60 MBq 99mTc-tricarbonyl produced from an Isolink® kit (Mallinckrodt) under different reaction conditions. Results clearly showed that the introduction of three MBG could double the radiolabelling yields to more than 90% in a short time (30 mn, 37°C). Such results would never have been reached with unmodified ,-globulins alone. Under the same conditions when using 1,2 nmol derivatized annexin V, the average radiolabelling yield was 55% against 19% with the unmodified protein. The 99mTc-tricarbonyl-conjugates were challenged with cysteine or histidine and showed good stability. Copyright © 2005 John Wiley & Sons, Ltd. [source]

Evidence that 3-hydroxy-3-methylglutaric acid promotes lipid and protein oxidative damage and reduces the nonenzymatic antioxidant defenses in rat cerebral cortex

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2008
Guilhian Leipnitz
Abstract In the present work we investigated the in vitro effect of 3-hydroxy-3-methylglutarate (HMG) that accumulates in 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (HMGLD) on important parameters of oxidative stress in rat cerebral cortex. It was observed that HMG induced lipid peroxidation by significantly increasing chemiluminescence and levels of thiobarbituric acid-reactive substances (TBA-RS). This effect was prevented by the antioxidants ,-tocopherol, melatonin, N-acetylcysteine, and superoxide dismutase plus catalase, suggesting that free radicals were involved in the lipid oxidative damage. On the other hand, HMG did not change TBA-RS levels in intact or disrupted mitochondrial preparations, indicating that generation of oxidants by this organic acid was dependent on cytosolic mechanisms. HMG also induced protein oxidative damage in cortical supernatants, which was reflected by increased carbonyl content and sulfhydryl oxidation. Furthermore, HMG significantly reduced the nonenzymatic antioxidant defenses total-radical trapping antioxidant potential, total antioxidant reactivity, and reduced glutathione (GSH) levels in rat cerebral cortex. HMG-induced GSH reduction was totally blocked by melatonin pretreatment. We also verified that the decrease of GSH levels provoked by HMG in cortical supernatants was not due to a direct oxidative effect of this organic acid, because exposition of commercial GSH and purified membrane protein-bound thiol groups to HMG in the absence of cortical supernatants did not decrease the reduced sulfhydryl groups. Finally, the activities of the main antioxidant enzymes were not altered by HMG exposure. Our data indicate that oxidative stress elicited in vitro by HMG may possibly contribute at least in part to the pathophysiology of the brain injury in HMGLD. © 2007 Wiley-Liss, Inc. [source]

In vivo evaluation of a nasal insulin delivery system based on thiolated chitosan

JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 11 2006
Alexander H. Krauland
Abstract The aim of this study was the preparation and in vivo evaluation of a nasal insulin delivery system based on thiolated chitosan. 2-Iminothiolane was covalently attached to chitosan. The resulting conjugate (chitosan-TBA) exhibited 304.9,±,63.5 µmol thiol groups per gram polymer. Microparticles were prepared via a new precipitation-micronization technique. The microparticulate delivery system comprised insulin, reduced glutathione and chitosan-TBA (Chito-TBA/Ins) or unmodified chitosan (Chito/Ins) and control microparticles were composed of insulin and mannitol (Mannitol/Ins). Due to a hydration process the size of Chito-TBA/Ins and Chito/Ins microparticles increased in phosphate buffer pH 6.8 2.6- and 2.2-fold, respectively. Fluorescent-labeled insulin-loaded chitosan-TBA microparticles showed a controlled release over 4 h. Chito-TBA/Ins administered nasally to rats led to an absolute bioavailability of 6.9,±,1.5%. The blood glucose level decreased for more than 2 h and the calculated absolute pharmacological efficacy was 4.9,±,1.4%. Chito/Ins, in comparison, displayed a bioavailability of 4.2,±,1.8% and a pharmacological efficacy of 0.7,±,0.6%. Mannitol/Ins showed a bioavailability of 1.6,±,0.4% and no reduction of the blood glucose level at all. According to these findings microparticles comprising chitosan-TBA seem to have substantial higher potential for nasal insulin administration than unmodified chitosan. © 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:2463,2472, 2006 [source]

Evaluation of the inhibition effect of thiolated poly(acrylates) on vaginal membrane bound aminopeptidase N and release of the model drug LH-RH

JOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 5 2002
Claudia Valenta
The purpose of this study was to evaluate the inhibitory effect of thiolated carbopol 974P (carbcys) on the enzymatic activity of vaginal aminopeptidase N in-vitro. Mediated by a carbodiimide, L-cysteine was covalently linked to carbopol 974P. Depending on the weight ratio of polymer to cysteine during the coupling reaction, resulting conjugates displayed 31.3,54.4 ,mol thiol groups per g polymer. The inhibitory effect of carb-cys conjugates was evaluated towards isolated aminopeptidase N and aminopeptidase-N-like activity of excised vaginal mucosa covered with native mucus, respectively. Enzymatic activity was assayed spectrophotometrically using L-leucine- p -nitroanilide (L-leu-pNA) as a synthetic substrate. Carb-cys thereby showed a significantly higher inhibitory effect than unmodified polymer towards both isolated enzyme and vaginal mucosa. Moreover, enzyme inhibition was strongly dependent on the amount of thiol groups being immobilised. The more thiol groups available the higher was the inhibitory effect. Due to its additional high cohesive properties and the possibility of a sustained drug release, which could be shown for the model drug LH-RH, carb-cys appears interesting for the development of vaginal peptide drug-delivery systems. [source]

Stability and utility of pyridyl disulfide functionality in RAFT and conventional radical polymerizations

JOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 21 2008
Cyrille Boyer
Abstract Two RAFT agents, suitable for inducing living radical polymerization in water, have been synthesized. Both RAFT agents were shown to be effective over the temperature range 25,70 °C. One RAFT agent was functionalized with a pyridyl disulfide group. RAFT efficacy was demonstrated for the polymerizations of N -isopropyl acrylamide (NIPAAM) and poly(ethylene oxide)-acrylate (PEG-A) in both water and acetonitrile. The kinetic data indicates that the pyridyl disulfide functionality is largely benign in free radical polymerizations, remaining intact for subsequent reaction with thiol groups. This result was confirmed by studying conventional radical polymerizations in the presence of hydroxyethyl pyridyl disulfide. The utility of the pyridyl disulfide functionality at the terminus of the polymers was demonstrated by synthesizing polymer-BSA conjugates. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 7207,7224, 2008 [source]

Effect of plant phenolics, tocopherol and ascorbic acid on oxidative stability of pork patties

JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 8 2009
Lindsey Haak
Abstract BACKGROUND: There is great interest in the use of naturally occurring antioxidants to delay oxidation in meat products. The effect of rosemary extract (RE), green tea extract (TE), tocopherol, trolox, ascorbic acid (AA) and ascorbyl palmitate (AP), at levels of 50,200 ppm of antioxidant components, on colour (CIE L*a*b*), lipid (TBARS) and protein oxidation (thiol groups) in fresh, frozen and cooked pork patties during illuminated chill storage was investigated. Individual components of RE and TE were also tested. RESULTS: RE, TE, AP, tocopherol and trolox equally inhibited lipid oxidation in fresh and frozen patties, whereas for cooked patties RE was most effective. AA stimulated lipid oxidation. No dose effect in the range of 50,200 ppm was found for fresh and frozen patties, whereas for cooked patties higher doses of RE and TE more efficiently prevented lipid oxidation. Protein oxidation was hardly influenced by antioxidant treatment. Colour stability decreased as follows: tocopherol, AA and AP > RE and TE > trolox. Antioxidant properties of the extracts and their major antioxidant components were comparable. CONCLUSION: The relative effect of the antioxidants depends on the oxidation parameter assessed, the applied dose and the hydrophilic/lipophilic character. Copyright © 2009 Society of Chemical Industry [source]

The treatment of inflammatory bowel disease with 6-mercaptopurine or azathioprine

ALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 11 2001
O. H. Nielsen
The thioguanine derivative, azathioprine, is a prodrug of 6-mercaptopurine that is further metabolized by various enzymes present in the liver and gut. Azathioprine and 6-mercaptopurine have been used in the treatment of inflammatory bowel disease, i.e. ulcerative colitis and Crohn's disease, for more than 30 years. However, widespread use of azathioprine or 6-mercaptopurine in inflammatory bowel disease is of more recent origin, the primary reason being a long-standing debate on the efficacy of these agents in inflammatory bowel disease. Both drugs are slow acting, which is why clinical efficacy cannot be expected until several weeks or even months of treatment have elapsed. Consequently, azathioprine and 6-mercaptopurine have no place as monotherapy in the treatment of acute relapsing inflammatory bowel disease. Today, azathioprine and 6-mercaptopurine are the most commonly used immunomodulatory drugs in the treatment of inflammatory bowel disease. Their clinical effects are probably identical, although their exact mode of action is still unknown. The mode of action of azathioprine is thought to be multifactorial, including conversion to 6-mercaptopurine (which acts as a purine antimetabolite), possible blockade of thiol groups by alkylation, inhibition of several pathways in nucleic acid biosynthesis (preventing proliferation of cells involved in the determination and amplification of the immune response) and damage to DNA through the incorporation of thiopurine analogues. However, 6-thioguanine nucleotides may accumulate in toxic doses in myeloid precursor cells, resulting in life-threatening myelosuppression. Azathioprine and 6-mercaptopurine are further known to alter lymphocyte function, reduce the number of lamina propria plasma cells and affect natural killer cell function. The purpose of this comprehensive review is to suggest guidelines for the application of azathioprine and 6-mercaptopurine in the treatment of inflammatory bowel disease. [source]

Staphylococcal NreB: an O2 -sensing histidine protein kinase with an O2 -labile iron,sulphur cluster of the FNR type

MOLECULAR MICROBIOLOGY, Issue 3 2004
Annegret Kamps
Summary The nreABC (nitrogen regulation) operon encodes a new staphylococcal two-component regulatory system that controls dissimilatory nitrate/nitrite reduction in response to oxygen. Unlike other two-component sensors NreB is a cytosolic protein with four N-terminal cysteine residues. It was shown that both the NreB,cysteine cluster and Fe ions are required for function. Isolated NreB was converted to the active form by incubation with cysteine desulphurase, ferrous ions and cysteine. This activation is typical for FeS-containing proteins and was reversed by oxygen. During reconstitution an absorption band at 420 nm and a yellow-brownish colour (typical for an FNR-type iron,sulphur cluster formation) developed. After alkylation of thiol groups in NreB and in the cysteine mutant NreB(C62S) almost no iron,sulphur cluster was incorporated; both findings corroborated the importance of the cysteine residues. Comparison of the kinase activity of (i) the reconstituted (ii) the unreconstituted, and (iii) the unreconstituted and deferrated NreB,His indicated that NreB kinase activity depended on iron availability and was greatly enhanced by reconstitution. NreB is the first direct oxygen-sensing protein described in staphylococci so far. Reconstituted NreB contains 4,8 acid-labile Fe and sulphide ions per NreB which is in agreement with the presence of 1,2 iron,sulphur [4Fe-4S]2+ clusters of the FNR-type. Unlike FNR, NreB does not act directly as transcriptional activator, but transfers the phosphoryl group to the response regulator NreC. [source]

Enhancement of Lysosomal Osmotic Sensitivity Induced by the Photooxidation of Membrane Thiol Groups,

Redox enzymes in the plant plasma membrane and their possible roles

PLANT CELL & ENVIRONMENT, Issue 12 2000
A. Bérczi
ABSTRACT Purified plasma membrane (PM) vesicles from higher plants contain redox proteins with low-molecular-mass prosthetic groups such as flavins (both FMN and FAD), hemes, metals (Cu, Fe and Mn), thiol groups and possibly naphthoquinone (vitamin K1), all of which are likely to participate in redox processes. A few enzymes have already been identified: Monodehydroascorbate reductase (EC 1.6.5.4) is firmly bound to the cytosolic surface of the PM where it might be involved in keeping both cytosolic and, together with a b -type cytochrome, apoplastic ascorbate reduced. A malate dehydrogenase (EC 1.1.1.37) is localized on the inner side of the PM. Several NAD(P)H-quinone oxidoreductases have been purified from the cytocolic surface of the PM, but their function is still unknown. Different forms of nitrate reductase (EC 1.6.6.1,3) are found attached to, as well as anchored in, the PM where they may act as a nitrate sensor and/or contribute to blue-light perception, although both functions are speculative. Ferric-chelate-reducing enzymes (EC 1.6.99.13) are localized and partially characterized on the inner surface of the PM but they may participate only in the reduction of ferric-chelates in the cytosol. Very recently a ferric-chelate-reducing enzyme containing binding sites for FAD, NADPH and hemes has been identified and suggested to be a trans -PM protein. This enzyme is involved in the reduction of apoplastic iron prior to uptake of Fe2+ and is induced by iron deficiency. The presence of an NADPH oxidase, similar to the so-called respiratory burst oxidase in mammals, is still an open question. An auxin-stimulated and cyanide-insensitive NADH oxidase (possibly a protein disulphide reductase) has been characterized but its identity is still awaiting independent confirmation. Finally, the only trans -PM redox protein which has been partially purified from plant PM so far is a high-potential and ascorbate-reducible b -type cytochrome. In co-operation with vitamin K1 and an NAD(P)H-quinone oxidoreductase, it may participate in trans -PM electron transport. [source]

CxxS: Fold-independent redox motif revealed by genome-wide searches for thiol/disulfide oxidoreductase function

PROTEIN SCIENCE, Issue 10 2002
Dmitri E. Fomenko
Abstract Redox reactions involving thiol groups in proteins are major participants in cellular redox regulation and antioxidant defense. Although mechanistically similar, thiol-dependent redox processes are catalyzed by structurally distinct families of enzymes, which are difficult to identify by available protein function prediction programs. Herein, we identified a functional motif, CxxS (cysteine separated from serine by two other residues), that was often conserved in redox enzymes, but rarely in other proteins. Analyses of complete Escherichia coli, Campylobacter jejuni, Methanococcus jannaschii, and Saccharomyces cerevisiae genomes revealed a high proportion of proteins known to use the CxxS motif for redox function. This allowed us to make predictions in regard to redox function and identity of redox groups for several proteins whose function previously was not known. Many proteins containing the CxxS motif had a thioredoxin fold, but other structural folds were also present, and CxxS was often located in these proteins upstream of an ,-helix. Thus, a conserved CxxS sequence followed by an ,-helix is typically indicative of a redox function and corresponds to thiol-dependent redox sites in proteins. The data also indicate a general approach of genome-wide identification of redox proteins by searching for simple conserved motifs within secondary structure patterns. [source]

Reversed-phase liquid chromatography method for the determination of total plasma thiols after derivatization with 1-benzyl-2-chloropyridinium bromide

BIOMEDICAL CHROMATOGRAPHY, Issue 7 2009
Krzysztof Ku, mierek
Abstract A high-performance liquid chromatography method was developed for simultaneous detection and quantitation of total cysteine, glutathione, homocysteine and cysteinylglycine in human plasma. The two key steps in the analysis are reduction of disulfides and treatment with 1-benzyl-2-chloropyridinium bromide, which rapidly and quantitatively reacts with thiol groups to form stable S- pyridinium derivatives with intense UV absorption. The derivatives are well separated on a Zorbax SB C18 column using reversed-phase high-performance liquid chromatography and monitored at 315 nm. The calibration graphs were linear over concentration ranges covering most experimental and clinical cases with a regression coefficients better than 0.999. The detection and quantitation limits for all analytes were 0.2 and 0.5 µmol/L, respectively. The recoveries were 99.25,101.68%. The intra- and interassay imprecisions were 0.88,4.24 and 1.68,5.14%, respectively. The method was applied for plasma samples donated by apparently healthy volunteers. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Thiopropyl-agarose as a solid phase reducing agent for chemical modification of IgG and F(ab,)2

BIOTECHNOLOGY PROGRESS, Issue 5 2008
Natalia Ferraz
Abstract Selective reduction of native disulfide bonds in immunoglobulins is one of the best methods for introducing reactive groups on to the protein surface. Additionally, the thiol groups so generated may allow oriented conjugation at a specific site of the immunoglobulin. Solid-phase reducing agents have many advantages over soluble ones (including ease of separation of excess reagent from reduced protein by filtration, and the potential for regeneration and multiple reuse). In this work we report a comparative study of the reduction of rabbit IgG and its F(ab,)2 fragments, with mercaptohydroxypropylether-agarose (thiopropyl-agarose), a solid phase reducing agent, and dithiothreitol. The effect of different parameters on the process, such as the amount of reducing agent, incubation period, and temperature, was assessed by titration of thiol groups and SDS-PAGE analysis. Optimized reduction with thiopropyl-agarose introduced six thiol groups in the F(ab,)2 fragment (mol/mol). Native IgG was less reactive, probably due to steric effects, as only an average of three thiol groups were introduced. However, by increasing reaction temperature from 22 to 37°C, six thiol groups could be introduced in native IgG (mol/mol). Reduction with dithiothreitol also introduced six thiol groups in F(ab,)2 fragments (mol/mol) but led to higher thiol content for the whole IgG. These results demonstrated that thiopropyl-agarose can be a very useful tool for exercising more precise control over the reduction treatment, and for selecting which disulfide bridges are to be broken. After 6 h incubation with reducing agent containing 8 and 16 ,moles SH per mg of protein, the resulting reduced IgG retained the same biological activity as the native immunoglobulin. The controlled modification of native disulfides achieved with thiopropyl-agarose will be useful for the development of soluble and insoluble immunoglobulin conjugates. [source]

A Resonance Energy Transfer Immunoassay Based on a Thiol-Reactive Ruthenium Donor Dye and a Longwave-Emitting Acceptor

CHEMBIOCHEM, Issue 1 2007
Jochen Weh
Abstract A novel immunoassay is described that applies a thiol-reactive ruthenium metal,ligand complex as the donor dye in a luminescence energy transfer (LET) detection scheme. Unlike amine-reactive labels, the LET with a thiol label allows improved specificity and better reproducibility of labelling positions on proteins, because the number of reactive thiol groups of proteins is distinctly smaller. This helps to reduce the risk of over-labelling and self-quenching of the fluorophore. The synthesis of the thiol label was significantly improved, resulting in almost quantitative yields of pure product. The absorption and emission maxima of the ruthenium donor dye are at 460 nm and 600 nm, respectively, and a Stokes' shift of 140 nm warrants distinct separation of excitation and emission wavelengths even in turbid samples. A cyanine dye with an absorption maximum at 642 nm was chosen as the acceptor label because it has good overlap with the emission spectrum of the donor label. The emission of the acceptor peaks at 660 nm, thus further increasing the Stokes' shift (to an overall 200 nm). The quantification of anti-HSA with the LET immunoassay is possible with this new approach at concentrations as low as 220 pmol,L,1. [source]

3224: Chitosan in the treatment of dry eye

ACTA OPHTHALMOLOGICA, Issue 2010
L SCHMETTERER
Purpose To demonstrate the safety and efficacy of a new eye drop formulation containing a novel thiolated biopolymer, namely chitosan-N-acetylcysteine, in a number of pre-clinical tests. It is postulated that interaction between thiol groups of the topically applied chitosan-N-acetylcysteine and cysteine-rich mucin (MUC5AC) increases polymer residence time on the ocular surface and tear film stability. Methods The efficacy of isotonic and buffered chitosan-N-acetylcysteine eye drops was evaluated in two different dry eye mouse models. The dosage dependent ocular residence time and biodistribution were investigated in a rabbit model using microPET technology. Long-term irritation and delayed-type hypersensitivity tests with chitosan-N-acteylcysteine eye drop formulation were conducted in rabbits. Results In both dry eye studies mice treated with chitosan-N-acetylcysteine showed decreased expression of ocular surface mRNA of IL-,, IL-10, IL-12,, and TNF, indicating that the formulation may have protective ocular surface properties. The residence time of chitosan-N-acetylcysteine eye drops on the ocular surface of rabbits was increased (detection up to 22h). Results of a long-term ocular irritation study in rabbits demonstrate that the novel formulation is well tolerated and non-irritant to the eye. Conclusion Based on the promising pre-clinical study results both in terms of efficacy and safety a clinical phase 1 trial is scheduled in the near future. Commercial interest [source]

Gold Manno -Glyconanoparticles: Multivalent Systems to Block HIV-1 gp120 Binding to the Lectin DC-SIGN,

CHEMISTRY - A EUROPEAN JOURNAL, Issue 38 2009
Olga Martínez-Ávila Dr.
Abstract The HIV envelope glycoprotein gp120 takes advantage of the high-mannose clusters on its surface to target the C-type lectin dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) on dendritic cells. Mimicking the cluster presentation of oligomannosides on the virus surface is a strategy for designing carbohydrate-based antiviral agents. Bio-inspired by the cluster presentation of gp120, we have designed and prepared a small library of multivalent water-soluble gold glyconanoparticles (manno- GNPs) presenting truncated (oligo)mannosides of the high-mannose undecasaccharide Man9GlcNAc2 and have tested them as inhibitors of DC-SIGN binding to gp120. These glyconanoparticles are ligands for DC-SIGN, which also interacts in the early steps of infection with a large number of pathogens through specific recognition of associated glycans. (Oligo)mannosides endowed with different spacers ending in thiol groups, which enable attachment of the glycoconjugates to the gold surface, have been prepared. manno- GNPs with different spacers and variable density of mannose (oligo)saccharides have been obtained and characterized. Surface plasmon resonance (SPR) experiments with selected manno -GNPs have been performed to study their inhibition potency towards DC-SIGN binding to gp120. The tested manno -GNPs completely inhibit the binding from the micro- to the nanomolar range, while the corresponding monovalent mannosides require millimolar concentrations. manno- GNPs containing the disaccharide Man,1-2Man, are the best inhibitors, showing more than 20,000-fold increased activity (100,% inhibition at 115,nM) compared to the corresponding monomeric disaccharide (100,% inhibition at 2.2,mM). Furthermore, increasing the density of dimannoside on the gold platform from 50 to 100,% does not improve the level of inhibition. [source]

Mechanistic Study of the Reaction of Thiol-Containing Enzymes with ,,,-Unsaturated Carbonyl Substrates by Computation and Chemoassays

CHEMMEDCHEM, Issue 6 2010
Alexander Paasche
Abstract We investigated the reactions between substituted ,,,-unsaturated carbonyl compounds (Michael systems) and thiols by computations as well as chemoassays. The results give insight into variations in the underlying mechanisms as a function of the substitution pattern. This is of interest for the mechanisms of inhibition of the SARS coronavirus main protease (SARS-CoV Mpro) by etacrynic acid derivatives as well as for the excess toxicity of substituted ,,,-unsaturated carbonyl compounds. This study compares possible reaction courses including 1,4-addition followed by a ketonization step, and underscores the importance of a base-catalyzed step for the reactivity of thiol groups in enzymes. Phenyl and methyl substituents at the Michael system decrease the reactivity of the electrophilic compound, but chlorophenyl substituents partly recover the reactivity. Computations also indicate that electron-pushing substituents lead to a change in the reaction mechanism. The conformation of the Michael system is also found to significantly influence reactivity: the s - cis conformation leads to higher reactivity than the s - trans conformation. The computed data explain the trends in measured inhibition potencies of substituted ,,,-unsaturated carbonyl compounds and of reaction rates in chemical assays. They also indicate that the reversibility of inhibition does not stand in contrast to the formation of a new covalent bond between inhibitor and protease. [source]