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Neuroscience Dec 2007There is a gender-related comorbidity of pain-related and inflammatory bowel diseases with psychiatric diseases. Since the impact of experimental gastrointestinal... (Comparative Study)
Comparative Study
There is a gender-related comorbidity of pain-related and inflammatory bowel diseases with psychiatric diseases. Since the impact of experimental gastrointestinal inflammation on the emotional-affective behavior is little known, we examined whether experimental gastritis modifies anxiety, stress coping and circulating corticosterone in male and female Him:OF1 mice. Gastritis was induced by adding iodoacetamide (0.1%) to the drinking water for at least 7 days. Inflammation was assessed by gastric histology and myeloperoxidase activity, circulating corticosterone determined by enzyme immunoassay, anxiety-related behavior evaluated with the elevated plus maze and stress-induced hyperthermia tests, and depression-like behavior estimated with the tail suspension test. Iodoacetamide-induced gastritis was associated with gastric mucosal surface damage and an increase in gastric myeloperoxidase activity, this increase being significantly larger in female mice than in male mice. The rectal temperature of male mice treated with iodoacetamide was enhanced, whereas that of female mice was diminished. The circulating levels of corticosterone were reduced by 65% in female mice treated with iodoacetamide but did not significantly change in male mice. On the behavioral level, iodoacetamide treatment caused a decrease in nocturnal home-cage activity, drinking and feeding. While depression-related behavior remained unaltered following induction of gastritis, behavioral indices of anxiety were significantly enhanced in female but not male mice. There was no correlation between the estrous cycle and anxiety as well as circulating corticosterone. Radiotracer experiments revealed that iodoacetamide did not readily enter the brain, the blood-brain ratio being 20:1. Collectively, these data show that iodoacetamide treatment causes gastritis in a gender-related manner, its severity being significantly greater in female than in male mice. The induction of gastritis in female mice is associated with a reduction of circulating corticosterone and an enforcement of behavioral indices of anxiety. Gastric inflammation thus has a distinct gender-dependent influence on emotional-affective behavior and its neuroendocrine control.
Topics: Alkylating Agents; Animals; Animals, Outbred Strains; Anxiety; Body Weight; Brain; Circadian Rhythm; Corticosterone; Drinking Behavior; Estrous Cycle; Feeding Behavior; Female; Gastric Mucosa; Gastritis; Iodine Radioisotopes; Iodoacetamide; Male; Maze Learning; Mice; Peroxidase; Sex Characteristics; Stress, Psychological; Tomography, Emission-Computed, Single-Photon
PubMed: 17945426
DOI: 10.1016/j.neuroscience.2007.09.024 -
The FEBS Journal Dec 2013Cellular exposure to reactive oxygen species induces rapid oxidation of DNA, proteins, lipids and other biomolecules. At the proteome level, cysteine thiol oxidation is...
Cellular exposure to reactive oxygen species induces rapid oxidation of DNA, proteins, lipids and other biomolecules. At the proteome level, cysteine thiol oxidation is a prominent post-translational process that is implicated in normal physiology and numerous pathologies. Methods for investigating protein oxidation include direct labeling with selective chemical probes and indirect tag-switch techniques. Common to both approaches is chemical blocking of free thiols using reactive electrophiles to prevent post-lysis oxidation or other thiol-mediated cross-reactions. These reagents are used in large excess, and their reactivity with cysteine sulfenic acid, a critical oxoform in numerous proteins, has not been investigated. Here we report the reactivity of three thiol-blocking electrophiles, iodoacetamide, N-ethylmaleimide and methyl methanethiosulfonate, with protein sulfenic acid and dimedone, the structural core of many sulfenic acid probes. We demonstrate that covalent cysteine -SOR (product) species are partially or fully susceptible to reduction by dithiothreitol, tris(2-carboxyethyl)phosphine and ascorbate, regenerating protein thiols, or, in the case of ascorbate, more highly oxidized species. The implications of this reactivity on detection methods for protein sulfenic acids and S-nitrosothiols are discussed.
Topics: Ascorbic Acid; Cyclohexanones; Cysteine; Dithiothreitol; Ethylmaleimide; Iodoacetamide; Methyl Methanesulfonate; Oxidation-Reduction; Proteins; Reactive Oxygen Species; Spectrometry, Mass, Electrospray Ionization; Sulfenic Acids; Sulfhydryl Compounds
PubMed: 24103186
DOI: 10.1111/febs.12535 -
Journal of Chromatography. A Oct 2023Enantioselective amino acid analysis is gaining increasing importance in pharmaceutical, biomedical and food sciences. While there are many methods available for...
Automated sample preparation with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate and iodoacetamide derivatization reagents for enantioselective liquid chromatography tandem mass spectrometry amino acid analysis.
Enantioselective amino acid analysis is gaining increasing importance in pharmaceutical, biomedical and food sciences. While there are many methods available for enantiomer separation of amino acids, the simultaneous analysis of all chiral proteinogenic amino acids by a single method with one column and a single condition is still challenging. Herein, we report an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS) assay using Chiralpak QN-AX as chiral column. With 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate (AQC) as derivatization reagent, efficient enantioselective separation of D- and L-amino acids using HPLC has become possible. Thiol-containing amino acids like Cys are alkylated prior to AQC-labelling. A protocol for automated sample preparation including both derivatization step and calibrator preparation is presented. For compensating matrix effects, u-CN-labelled internal standards (IS) were employed. The method was validated and applied to the enantioselective analysis of amino acids in a bacterial fermentation broth.
Topics: Amino Acids; Iodoacetamide; Stereoisomerism; Tandem Mass Spectrometry; Chromatography, Liquid; Carbamates
PubMed: 37696129
DOI: 10.1016/j.chroma.2023.464349 -
Nature Chemical Biology Apr 2018In this article we describe the production and screening of a genetically encoded library of 10 lanthipeptides in Escherichia coli using the substrate-tolerant...
In this article we describe the production and screening of a genetically encoded library of 10 lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.
Topics: Crystallography, X-Ray; DNA-Binding Proteins; Endosomal Sorting Complexes Required for Transport; ErbB Receptors; Escherichia coli; Escherichia coli Proteins; Ethylmaleimide; Gene Library; HEK293 Cells; HIV; HeLa Cells; Humans; Inhibitory Concentration 50; Iodoacetamide; Peptide Hydrolases; Peptide Synthases; Peptides; Plasmids; Protein Domains; Protein Interaction Mapping; Substrate Specificity; Transcription Factors; gag Gene Products, Human Immunodeficiency Virus
PubMed: 29507389
DOI: 10.1038/s41589-018-0008-5 -
The Biochemical Journal Apr 1973At a glucose concentration of 3mm or less, iodoacetamide had no effect on the release of insulin from microdissected pancreatic islets of ob/ob-mice. At higher glucose...
At a glucose concentration of 3mm or less, iodoacetamide had no effect on the release of insulin from microdissected pancreatic islets of ob/ob-mice. At higher glucose concentrations, iodoacetamide exerted both an initial stimulatory and a subsequent inhibitory action. When islets were perifused with 1mm-iodoacetamide and 17mm-glucose the inhibitory action predominated after about 15min of transient stimulation. With decreasing concentrations of iodoacetamide the stimulatory phase was gradually prolonged, and with 0.003-0.1mm-iodoacetamide stimulation only was observed for 75min. Prolonged stimulation was also noted after a short pulse of iodoacetamide. Similar responses to 0.1mm-iodoacetamide were observed with islets from normal mice. With islets from ob/ob-mice the effect of 0.1mm-iodoacetamide was reproduced with 0.1mm-iodoacetate, whereas 0.1mm-acetamide had no apparent effect. Iodoacetamide increased the V(max.) of glucose-stimulated insulin release without altering the apparent K(m) for glucose. Leucine, glibenclamide or theophylline could not replace glucose in this synergistic action with iodoacetamide. Iodoacetamide rather inhibited the insulin-releasing action of theophylline. Iodoacetamide-induced potentiation of the glucose-stimulated insulin release was rapidly and reversibly inhibited by mannoheptulose, adrenaline, or calcium deficiency. The potentiating effect on insulin release was not paralleled by effects on glucose oxidation or on islet fructose 1,6-diphosphate. However, the inhibitory action of iodoacetamide might be explained by inhibition of glycolysis as evidenced by an inhibition of glucose oxidation and a rise of fructose 1,6-diphosphate. The results support our previous hypothesis that thiol reagents can stimulate insulin release by acting on relatively superficial thiol groups in the beta-cell plasma membrane. Glycolysis seems to be necessary in order for iodoacetamide to stimulate in this way.
Topics: Amides; Animals; Calcium; Carbon Isotopes; Dithiothreitol; Epinephrine; Female; Fructosephosphates; Glucose; Glyburide; In Vitro Techniques; Insulin; Insulin Secretion; Iodine Isotopes; Iodoacetates; Islets of Langerhans; Leucine; Male; Mice; Obesity; Radioimmunoassay; Theophylline; Tritium
PubMed: 4198516
DOI: 10.1042/bj1320775 -
The Journal of Biological Chemistry Aug 2001A thiol/disulfide oxidoreductase component of the GSH system, glutaredoxin (Grx), is involved in the reduction of GSH-based mixed disulfides and participates in a...
A thiol/disulfide oxidoreductase component of the GSH system, glutaredoxin (Grx), is involved in the reduction of GSH-based mixed disulfides and participates in a variety of cellular redox pathways. A single cytosolic Grx (Grx1) was previously described in mammals. We now report identification and characterization of a second mammalian Grx, designated Grx2. Grx2 exhibited 36% identity with Grx1 and had a disulfide active center containing the Cys-Ser-Tyr-Cys motif. Grx2 was encoded in the genomes of mammals and birds and expressed in a variety of cell types. The gene for human Grx2 consisted of four exons and three introns, spanned 10 kilobase pairs, and localized to chromosome 1q31.2-31.3. The coding sequence was present in all exons, with the first exon encoding a mitochondrial signal peptide. The mitochondrial leader sequence was also present in mouse and rat Grx2 sequences and was shown to direct either Grx2 or green fluorescent protein to mitochondria. Alternative splicing forms of mammalian Grx2 mRNAs were identified that differed in sequences upstream of exon 2. To functionally characterize the new protein, human and mouse Grx2 proteins were expressed in Escherichia coli, and the purified proteins were shown to reduce mixed disulfides formed between GSH and S-sulfocysteine, hydroxyethyldisulfide, or cystine. Grx1 and Grx2 were sensitive to inactivation by iodoacetamide and H(2)O(2) and exhibited similar pH dependence of catalytic activity. However, H(2)O(2)-inactivated Grx2 could only be reactivated with 5 mm GSH, whereas Grx1 could also be reactivated with dithiothreitol or thioredoxin/thioredoxin reductase. The Grx2 structural model suggested a common reaction mechanism for this class of proteins. The data provide the first example of a mitochondrial Grx and also indicate the occurrence of a second functional Grx in mammals.
Topics: 3T3 Cells; Alternative Splicing; Amino Acid Sequence; Animals; Catalysis; Chromosome Mapping; Chromosomes, Human, Pair 1; Disulfides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Escherichia coli; Exons; Expressed Sequence Tags; Glutaredoxins; Glutathione Transferase; Green Fluorescent Proteins; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Introns; Iodoacetamide; Kinetics; Luminescent Proteins; Mice; Microscopy, Confocal; Mitochondria; Models, Molecular; Molecular Sequence Data; Oxidation-Reduction; Oxidoreductases; Protein Binding; Protein Sorting Signals; Protein Structure, Tertiary; Proteins; Rats; Recombinant Fusion Proteins; Sequence Homology, Amino Acid; Software; Substrate Specificity; Thioredoxin-Disulfide Reductase
PubMed: 11397793
DOI: 10.1074/jbc.M100020200 -
The Journal of Biological Chemistry Sep 2014The rise of multidrug-resistant and totally drug-resistant tuberculosis and the association with an increasing number of HIV-positive patients developing tuberculosis...
The rise of multidrug-resistant and totally drug-resistant tuberculosis and the association with an increasing number of HIV-positive patients developing tuberculosis emphasize the necessity to find new antitubercular targets and drugs. The antigen 85 (Ag85) complex from Mycobacterium tuberculosis plays important roles in the biosynthesis of major components of the mycobacterial cell envelope. For this reason, Ag85 has emerged as an attractive drug target. Recently, ebselen was identified as an effective inhibitor of the Ag85 complex through covalent modification of a cysteine residue proximal to the Ag85 active site and is therefore a covalent, allosteric inhibitor. To expand the understanding of this process, we have solved the x-ray crystal structures of Ag85C covalently modified with ebselen and other thiol-reactive compounds, p-chloromercuribenzoic acid and iodoacetamide, as well as the structure of a cysteine to glycine mutant. All four structures confirm that chemical modification or mutation at this particular cysteine residue leads to the disruption of the active site hydrogen-bonded network essential for Ag85 catalysis. We also describe x-ray crystal structures of Ag85C single mutants within the catalytic triad and show that a mutation of any one of these three residues promotes the same conformational change observed in the cysteine-modified forms. These results provide evidence for active site dynamics that may afford new strategies for the development of selective and potent Ag85 inhibitors.
Topics: Acyltransferases; Allosteric Regulation; Anti-Inflammatory Agents, Non-Steroidal; Antigens, Bacterial; Azoles; Biocatalysis; Catalytic Domain; Chloromercuribenzoates; Crystallography, X-Ray; Cysteine; Enzyme Activation; Enzyme Inhibitors; Hydrogen Bonding; Iodoacetamide; Isoindoles; Models, Molecular; Molecular Structure; Mutation; Organoselenium Compounds; Protein Conformation; Protein Structure, Secondary
PubMed: 25028518
DOI: 10.1074/jbc.M114.582445 -
The Journal of Cell Biology Jun 1984ATP-depleted human erythrocytes lose their smooth discoid shape and adopt a spiny, crenated form. This shape change coincides with the conversion of...
ATP-depleted human erythrocytes lose their smooth discoid shape and adopt a spiny, crenated form. This shape change coincides with the conversion of phosphatidylinositol-4,5-bisphosphate to phosphatidylinositol and phosphatidic acid to diacylglycerol. Both crenation and lipid dephosphorylation are accelerated by iodoacetamide, and both are reversed by nutrient supplementation. The observed changes in lipid populations should shrink the membrane inner monolayer by 0.6%, consistent with estimates of bilayer imbalance in crenated cells. These observations suggest that metabolic crenation arises from a loss of inner monolayer area secondary to the degradation of phosphatidylinositol-4,5-bisphosphate and phosphatidic acid. A related process, crenation after Ca2+ loading, appears to arise from a loss inositides by a different pathway.
Topics: Adenosine Triphosphate; Erythrocyte Membrane; Erythrocytes; Humans; Iodoacetamide; Kinetics; Microscopy, Electron, Scanning; Phosphatidylinositols; Phospholipids; Phosphorus Radioisotopes
PubMed: 6327723
DOI: 10.1083/jcb.98.6.1992 -
The Journal of Biological Chemistry Nov 1996Human placental S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1. 1) was inactivated by 5',5-dithiobis(2-nitrobenzoic acid) following pseudo-first-order kinetics....
Human placental S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1. 1) was inactivated by 5',5-dithiobis(2-nitrobenzoic acid) following pseudo-first-order kinetics. Modification of three of the 10 cysteine residues per enzyme subunit resulted in complete inactivation of the enzyme. The three modified cysteine residues were identified as Cys113, Cys195, and Cys421, respectively, by protein sequencing after modification with [1-14C]iodoacetamide. Of the three modifiable cysteines, Cys113 and Cys195 could be protected from modification in the presence of the substrate adenosine (Ado), which also protected the enzyme from inactivation. On the other hand, Cys421 was not protected by Ado, and modification of Cys421 alone did not affect the enzyme activity. To verify whether some of these cysteine residues are important for the enzyme catalysis, these three cysteine residues were replaced by either serine or aspartic acid using site-directed mutagenesis. Mutants of both Cys113 (C113S and C113D) and Cys421 (C421S and C421D) had enzyme activities similar to that of the wild-type enzyme, and only slight changes were observed in the steady-state kinetics measured in both the synthetic and hydrolytic directions. However, mutants of Cys195 (C195D and C195S) displayed drastically reduced enzyme activities, and kcat values were only 7 and 12% of that of the wild-type enzyme, respectively, resulting in a calculated loss in binding energy (DeltaDeltaG) of approximate 1 Kcal/mol. The Cys195 mutants were capable of catalyzing both the 3'-oxidative and 5'-hydrolytic reactions, as evidenced by the reduction of E.NAD+ to NADH and formation of the 5'-hydrolytic product when incubated with (E)-5', 6'-didehydro-6'-deoxy-6'-chlorohomoadenosine at rates comparable with those catalyzed by the wild-type enzyme. However, mutations of the Cys195 severely altered the 3'-reduction potential as evidenced by the drastic reduction in the rate of [2,8-3H]Ado release from the E-NADH.[2,8-3H]3'-keto-Ado complex. Circular dichroism studies of the Cys195 mutants confirmed that the observed effects are not due to changes in secondary structure. These results suggested that the Cys195 is involved in the catalytic center and may play an important role in maintaining the 3'-reduction potential for effective release of the reaction products and regeneration of the active form (NAD+ form) of the enzyme; the Cys113 is located in or near the substrate binding site, but plays no role beneficial to the catalysis; and the Cys421 is a nonessential residue, which also explains why Cys421 does not occur in any other known AdoHcy hydrolases.
Topics: Adenosylhomocysteinase; Amino Acid Sequence; Chromatography, High Pressure Liquid; Dithionitrobenzoic Acid; Humans; Hydrolases; Iodoacetamide; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Placenta; Trypsin
PubMed: 8910410
DOI: 10.1074/jbc.271.45.28009 -
Isolation and characterization of acetylated histones H3 and H4 and their assembly into nucleosomes.The Journal of Biological Chemistry Nov 1990Nucleosome and chromatin structure/function relationships of histone acetylations are not understood. To address these questions we have developed chromatographic...
Nucleosome and chromatin structure/function relationships of histone acetylations are not understood. To address these questions we have developed chromatographic procedures that separate subtypes of H3 and the acetylated states of histone H3 and H4 in exceptionally pure forms. The sites of acetylation of the intermediately acetylated states of H3 have been determined and show a specific pattern of acetylation. An unexpected finding was the identification of a fifth site of acetylation in H3 at lysine 27. Nucleosome particles with fully acetylated H3 and H4 have been assembled on the Lytechinus variegatus 5 S rRNA DNA phasing sequence and characterized. These defined acetylated H3 and H4 particles migrate more slowly in polyacrylamide nucleoprotein particle gels than the control particles indicating a subtle effect of acetylation in nucleosome structure. However, DNA footprinting of these particles using DNase I show only small changes when compared to control particles over the core particle DNA length. It is shown further that H3 cysteines in the particle containing fully acetylated H3 and H4 were not accessible to iodoacetamide indicating that protein factors additional to H3 and H4 acetylation are required to make H3 cysteines accessible to the label. These findings are consistent with the proposal that histones H3, H4 acetylations exert their major effects outside of the core particle 146-base pair DNA, either on the DNA segment entering and leaving the nucleosome or possibly on the internucleosome interactions that involve the amino-terminal domains of the core histones in organization and stability of higher order chromatin structures.
Topics: Acetylation; Binding Sites; Cell Nucleus; Chromatography, High Pressure Liquid; Cysteine; DNA; Deoxyribonuclease I; Electrophoresis, Polyacrylamide Gel; HeLa Cells; Histones; Humans; Iodoacetamide; Lysine; Nucleosomes
PubMed: 2123192
DOI: No ID Found