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Alimentary Pharmacology & Therapeutics Oct 2001The anticoagulants, unfractionated heparin and low-molecular-weight heparin, demonstrated anti-inflammatory effects in animal models and in humans. Because of its dual...
BACKGROUND AND AIMS
The anticoagulants, unfractionated heparin and low-molecular-weight heparin, demonstrated anti-inflammatory effects in animal models and in humans. Because of its dual effects, high-dose heparin was proposed as a therapeutic modality for ulcerative colitis. We investigated whether a low dose of low-molecular-weight heparin-enoxaparin (Clexane, Rhône-Poulenc Rorer, France)-ameliorates the inflammatory response in two models of experimental colitis.
METHODS
Colitis was induced in rats by intrarectal administration of dinitrobenzene sulphonic acid. Enoxaparin (40, 80 and 200 microg/kg) or unfractionated heparin (100, 200 and 400 U/kg) were administered subcutaneously immediately after the induction of damage. Enoxaparin, 80 microg/kg, was also administered after induction of colitis by intrarectal administration of iodoacetamide. Rats were sacrificed 1, 3 or 7 days after induction of injury. Colonic damage was assessed macroscopically and histologically. Mucosal prostaglandin E2 generation, myeloperoxidase and nitric oxide synthase activities and tumour necrosis factor-alpha levels in blood were determined.
RESULTS
Enoxaparin and heparin significantly ameliorated the severity of dinitrobenzene sulphonic acid- and iodoacetamide-induced colitis as demonstrated by a decrease in mucosal lesion area, colonic weight and mucosal myeloperoxidase and nitric oxide synthase activities. The dose-response curve had a bell-shaped configuration: enoxaparin, 80 microg/kg, and unfractionated heparin, 200 U/kg, were the optimal doses.
CONCLUSIONS
Low-dose enoxaparin and unfractionated heparin ameliorate the severity of experimental colitis. This effect is related to their anti-inflammatory rather than anticoagulant properties.
Topics: Animals; Anti-Inflammatory Agents; Anticoagulants; Benzenesulfonates; Colitis; Disease Models, Animal; Dose-Response Relationship, Drug; Enoxaparin; Heparin; Iodoacetamide; Male; Nitric Oxide Synthase; Peroxidase; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha
PubMed: 11564011
DOI: 10.1046/j.1365-2036.2001.01079.x -
BMC Biochemistry Dec 2018Generally, proteases in medicinal plants had different therapeutic effects such as anti-inflammatory effect; modulate the immune response and inhibitory effect toward...
BACKGROUND
Generally, proteases in medicinal plants had different therapeutic effects such as anti-inflammatory effect; modulate the immune response and inhibitory effect toward tumor growth. In this study, protease was purified and characterized from miswak roots, as medicinal plant and natural toothbrush.
RESULTS
Physical and chemical characterization of cysteine protease P1 were studied such as pH optimum (6.5), optimum temperature (50 °C), thermal stability (50 °C) and Km (3.3 mg azocasein/ml). The enzyme digested some proteins in the order of caseine > haemoglobin > egg albumin >gelatin > bovine serum albumin. Hg had strong inhibitory effect on enzyme activity compared with other metal ions. Kinetic of inhibition for determination the type of protease was studied. Iodoactamide and p-Hydroximercuribenzaoic acid (p-HMB) caused strong inhibitory effect on enzyme activity indicating the enzyme is cysteine protease.
CONCLUSIONS
The biochemical characterization of this enzyme will be display the suitable conditions for using of this enzyme in toothpaste in the future and the enzyme may be used in other applications.
Topics: Cysteine Proteases; Enzyme Inhibitors; Enzyme Stability; Hydroxymercuribenzoates; Iodoacetamide; Kinetics; Mercury; Plant Proteins; Plant Roots; Recombinant Proteins; Salvadoraceae; Temperature
PubMed: 30509174
DOI: 10.1186/s12858-018-0100-1 -
Drug Design, Development and Therapy 2020Paeoniflorin is a main active component in traditional Chinese medicine. Paeoniae alba radix is widely used as a spasmolytic and pain-relieving agent for abdominal...
INTRODUCTION
Paeoniflorin is a main active component in traditional Chinese medicine. Paeoniae alba radix is widely used as a spasmolytic and pain-relieving agent for abdominal spasmodic pain. Functional dyspepsia (FD) is characterized by pain or burning in the epigastrium, fullness, bloating and nausea. However, limited information is available about the effect of paeoniflorin on FD.
MATERIALS AND METHODS
In this study, iodoacetamide or clonidine-induced FD rat models were established to investigate the impacts of paeoniflorin on FD induced by different pathophysiologic disturbances.
RESULTS
We found the therapeutic effect of paeoniflorin through assessing the gastric emptying, gastric accommodation and visceral hypersensitivity. This function of paeoniflorin was related to the release of acetylcholine (ACh), which was accompanied by reduced acetylcholinesterase (AchE) activity in stomach and hypothalamus. Paeoniflorin administration inhibited the cyclo-oxygenase-2 (COX-2) expression and increased the level of ghrelin in the stomach. Besides, the levels of occludin and ZO-1 were elevated in the duodenum from paeoniflorin-treated rats, suggesting the impaired duodenal barrier was ameliorated.
DISCUSSION
These results indicate that paeoniflorin possesses the ability to alleviate functional dyspepsia.
Topics: Acetylcholine; Animals; Clonidine; Disease Models, Animal; Dyspepsia; Glucosides; Iodoacetamide; Male; Monoterpenes; Rats; Rats, Sprague-Dawley
PubMed: 33376306
DOI: 10.2147/DDDT.S260703 -
The Journal of Biological Chemistry Apr 2005Hsp31, the Escherichia coli hcha gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperature. Its crystal structure reveals a putative...
Hsp31, the Escherichia coli hcha gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperature. Its crystal structure reveals a putative Cys(184), His(185), and Asp(213) catalytic triad similar to that of the Pyrococcus horikoshii protease PH1704, suggesting that it should display a proteolytic activity. A preliminary report has shown that Hsp31 has an exceedingly weak proteolytic activity toward bovine serum albumin and a peptidase activity toward two peptide substrates with small amino acids at their N terminus (alanine or glycine), but the physiological significance of this observation remains unclear. In this study, we report that Hsp31 does not diplay any significant proteolytic activity but has peptidolytic activity. The aminopeptidase cleavage preference of Hsp31 is Ala > Lys > Arg > His, suggesting that Hsp31 is an aminopeptidase of broad specificity. Its aminopeptidase activity is inhibited by the thiol reagent iodoacetamide and is completely abolished in a C185A mutant, which is consistent with Hsp31 being a cysteine peptidase. The aminopeptidase activity of Hsp31 is also inhibited by EDTA and 1,10-phenanthroline, in concordance with the importance of the putative His(85), His(122), and Glu(90) metal-binding site revealed by crystallographic studies. An Hsp31-deficient mutant accumulates more 8-12-mer peptides than its parental strain, and purified Hsp31 can transform these peptides into smaller peptides, suggesting that Hsp31 has an important peptidase function both in vivo and in vitro. Proteins interacting with Hsp31 have been identified by reverse purification of a crude E. coli extract on an Hsp31-affinity column, followed by SDS-polyacrylamide electrophoresis and mass spectrometry. The ClpA component of the ClpAP protease, the chaperone GroEL, elongation factor EF-Tu, and tryptophanase were all found to interact with Hsp31, thus substantiating the role of Hsp31 as both chaperone and peptidase.
Topics: Adenosine Triphosphate; Alanine; Arginine; Catalysis; Cations; Chromatography; Chromatography, High Pressure Liquid; Crystallography, X-Ray; Cysteine Endopeptidases; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli Proteins; Histidine; Hydrogen-Ion Concentration; Hydrolysis; Iodoacetamide; Kinetics; Lysine; Mass Spectrometry; Molecular Chaperones; Mutation; Peptide Elongation Factor Tu; Peptides; Phenanthrolines; Protein Binding; Substrate Specificity; Temperature; Tryptophanase
PubMed: 15550391
DOI: 10.1074/jbc.M408296200 -
Nature Communications Apr 2018Reactive oxygen species (ROS) are increasingly recognised as important signalling molecules through oxidation of protein cysteine residues. Comprehensive identification...
Reactive oxygen species (ROS) are increasingly recognised as important signalling molecules through oxidation of protein cysteine residues. Comprehensive identification of redox-regulated proteins and pathways is crucial to understand ROS-mediated events. Here, we present stable isotope cysteine labelling with iodoacetamide (SICyLIA), a mass spectrometry-based workflow to assess proteome-scale cysteine oxidation. SICyLIA does not require enrichment steps and achieves unbiased proteome-wide sensitivity. Applying SICyLIA to diverse cellular models and primary tissues provides detailed insights into thiol oxidation proteomes. Our results demonstrate that acute and chronic oxidative stress causes oxidation of distinct metabolic proteins, indicating that cysteine oxidation plays a key role in the metabolic adaptation to redox stress. Analysis of mouse kidneys identifies oxidation of proteins circulating in biofluids, through which cellular redox stress can affect whole-body physiology. Obtaining accurate peptide oxidation profiles from complex organs using SICyLIA holds promise for future analysis of patient-derived samples to study human pathologies.
Topics: Adaptation, Physiological; Animals; Cells, Cultured; Cysteine; Fumarate Hydratase; Hydrogen Peroxide; Iodoacetamide; Isotope Labeling; Kidney; Male; Mice; Mice, Knockout; Oxidation-Reduction; Oxidative Stress; Proteome; Proteomics
PubMed: 29679077
DOI: 10.1038/s41467-018-04003-3 -
The Biochemical Journal May 1997Extracellular proteases of Porphyromonas gingivalis specific for arginyl peptide bonds are considered to be important virulence factors in periodontal disease. In order... (Comparative Study)
Comparative Study
Extracellular proteases of Porphyromonas gingivalis specific for arginyl peptide bonds are considered to be important virulence factors in periodontal disease. In order to determine the number, inter-relationship and kinetic properties of these proteases, extracellular enzymes with this peptide-bond specificity were purified and characterized from P. gingivalis W50. Three forms, which we denote RI, RI-A and RI-B, accounted for all of the activity in the supernatant. All three enzymes contain an alpha chain of approximately 54 kDa with the same N-terminal amino acid sequence. RI is a heterodimer of non-covalently linked alpha and beta chains which migrate to the same position on SDS/PAGE but which can be resolved by 8 M urea/PAGE. RI-A and RI-B are both monomeric, but the molecular mass of RI-B (70-80 kDa) is significantly increased due to post-translational modification with lipopolysaccharide. All forms show absolute specificity for peptide bonds with Arg in the P1 position and are also capable of hydrolysing N-terminal Arg and C-terminal Arg-Arg peptide bonds. Thus they show limited amino- and carboxy-peptidase activity. For the hydrolysis of Nalpha-benzoyl-L-Arg-p-nitroanilide, the pH optimum is 8.0 at 30 degrees C. The Vmax for all three enzymes is controlled by ionization of two residues with apparent pKas at 30 degrees C of 6. 5+/-0.05 and 9.7+/-0.05, and DeltaH values of approximately 29 kJ/mol and approximately 24 kJ/mol in the enzyme-substrate complex. By analogy with papain, the pKa of 6.5 could be ascribed to a Cys and the pKa of 9.7 to a His residue. E-64 [L-trans-epoxysuccinyl-leucylamide-4-(4-guanidino)butane] is a competitive inhibitor of RI, RI-A and RI-B. Based on physical properties and kinetic behaviour, RI-A appears to be analogous to gingipain from P. gingivalis HG66. However the alpha/beta structure of RI differs significantly from that of the high-molecular-mass multimeric complex of gingipain containing four haemagglutinins described by others. Since the genes for RI and high-molecular-mass gingipain are identical, the data indicate that an alternative processing pathway is involved in the formation of RI from the initial precursor. Furthermore, the identical N-termini and enzymic properties of the catalytic component of RI, RI-A and RI-B suggest that the maturation pathway of the RI precursor may also give rise to RI-A and RI-B. The physiological functions of these isoforms and their role in the disease process may become more apparent through examination of their interactions with host proteins.
Topics: Adhesins, Bacterial; Amino Acid Sequence; Arginine; Bacterial Proteins; Binding Sites; Cysteine Endopeptidases; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Evolution, Molecular; Gingipain Cysteine Endopeptidases; Hemagglutinins; Hydrogen-Ion Concentration; Iodoacetamide; Iodoacetates; Iodoacetic Acid; Kinetics; Leucine; Molecular Sequence Data; Molecular Weight; Porphyromonas gingivalis; Protease Inhibitors; Substrate Specificity; Virulence
PubMed: 9169603
DOI: 10.1042/bj3230701 -
Journal of Agricultural and Food... Aug 2023Quantifying sulforaphane (SFN) and its thiol metabolites in biological samples using liquid chromatography-tandem mass spectrometry is complicated by SFN's electrophilic...
Quantifying sulforaphane (SFN) and its thiol metabolites in biological samples using liquid chromatography-tandem mass spectrometry is complicated by SFN's electrophilic nature and the facile dissociation of SFN-thiol conjugates. SFN can be lost during sample preparation due to conjugation with protein thiols, which are precipitated and discarded. We observe that only 32 ± 3% of SFN is recovered 2 h after spiking into fetal bovine serum. The SFN-glutathione conjugate prepared at 10 mM in 0.1% formic acid in water (pH 3) dissociated by approximately 95% to free SFN, highlighting the difficulty in preparing thiol metabolite standards. We used the alkylating agent iodoacetamide (IAA) to both release SFN from protein thiols and force the dissociation of SFN metabolites. This thiol-blocking method increased SFN percent recovery from serum from 32 to 94 ± 5%, with a 4.7 nM method limit of quantitation. Applying the method to clinical samples, SFN concentrations were on average 6 times greater than when IAA was omitted. The IAA thiol-blocking method streamlines the analysis of bioavailable SFN in plasma samples.
Topics: Chromatography, Liquid; Tandem Mass Spectrometry; Sulfhydryl Compounds; Isothiocyanates; Sulfoxides; Iodoacetamide
PubMed: 37584212
DOI: 10.1021/acs.jafc.3c01367 -
The Journal of Clinical Investigation Jun 1981A proteolytic procoagulant has been identified in extracts of human and animal tumors and in cultured malignant cells. It directly activated Factor X but its similarity...
A proteolytic procoagulant has been identified in extracts of human and animal tumors and in cultured malignant cells. It directly activated Factor X but its similarity to other Factor S-activating serine proteases was not clear. This study describes work done to determine whether this enzyme, cancer procoagulant, is a serine or cysteine protease. Purified cancer procoagulant from rabbit V2 carcinoma was bound to a p-chloromercurialbenzoate-agarose affinity column and was eluted with dithiothreitol. The initiation of recalcified, citrated plasma coagulation activity by cancer procoagulant was inhibited by 5 mM diisopropylfluorophosphate, 1 mM phenylmethylsulfonylfluoride, 0.1 mM HgCl2, and 1 mM iodoacetamide. Activity was restored in the diisopropylfluorophosphate-, phenylmethylsulfonylfluoride-, and HgCl2-inhibited samples by 5 mM dithiothreitol; iodoacetamide inhibition was irreversible. Russell's viper venom, a control Factor X-activating serine protease, was not inhibited by either 0.1 mM HgCl2 or 1 mM iodoacetamide. The direct activation of Factor X by cancer procoagulant in a two-stage assay was inhibited by diisopropylfluorophosphate and iodoacetamide. Diisopropylfluorophosphate inhibits serine proteases, and an undefined impurity in most commercial preparations inhibits cysteine proteases. Hydrolysis of diisopropylfluorophosphate with CuSO4 and imidazole virtually eliminated inhibition of thrombin, but cancer procoagulant inhibition remained complete, suggesting that cancer procoagulant was inhibited by the undefined impurity. These results suggest that cancer procoagulant is a cysteine endopeptidase, which distinguishes it from other coagulation factors including tissue factor. This and other data suggest that neoplastic cells produce this unique cysteine protease which may initiate blood coagulation.
Topics: Animals; Blood Coagulation; Chromatography, Affinity; Cysteine Endopeptidases; Endopeptidases; Enzyme Activation; Factor X; Iodoacetamide; Isoflurophate; Neoplasm Proteins; Neoplasms, Experimental; Phenylmethylsulfonyl Fluoride; Protease Inhibitors; Rabbits
PubMed: 7016920
DOI: 10.1172/jci110203 -
Chembiochem : a European Journal of... Aug 2017We have investigated 4-halopyridines as selective, tunable, and switchable covalent protein modifiers for use in the development of chemical probes. Nonenzymatic...
We have investigated 4-halopyridines as selective, tunable, and switchable covalent protein modifiers for use in the development of chemical probes. Nonenzymatic reactivity of 4-chloropyridine with amino acids and thiols was ranked with respect to common covalent protein-modifying reagents and found to have reactivity similar to that of acrylamide, but could be switched to a reactivity similar to that of iodoacetamide upon stabilization of the positively charged pyridinium. Diverse, fragment-sized 4-halopyridines inactivated human dimethylarginine dimethylaminohydrolase-1 (DDAH1) through covalent modification of the active site cysteine, acting as quiescent affinity labels that required off-pathway catalysis through stabilization of the protonated pyridinium by a neighboring aspartate residue. A series of 2-fluoromethyl-substituted 4-chloropyridines demonstrated that the pK and k /K values could be predictably varied over several orders of magnitude. Covalent labeling of proteins in an Escherichia coli lysate was shown to require folded proteins, indicating that alternative proteins can be targeted, and modification is likely to be catalysisdependent. 4-Halopyridines, and quiescent affinity labels in general, represent an attractive strategy to develop reagents with switchable electrophilicity as selective covalent protein modifiers.
Topics: Acrylamide; Affinity Labels; Amidohydrolases; Cysteine; Escherichia coli; Glutathione; Humans; Iodoacetamide; Phenols; Proteome; Pyridines; Pyridinium Compounds; Sulfhydryl Compounds
PubMed: 28470883
DOI: 10.1002/cbic.201700104 -
Life Sciences Jan 2011Vascular endothelial growth factor (VEGF) and pathologic angiogenesis have been demonstrated to play a pathogenic role in the development and progression of inflammatory...
AIMS
Vascular endothelial growth factor (VEGF) and pathologic angiogenesis have been demonstrated to play a pathogenic role in the development and progression of inflammatory bowel disease. Thus, we hypothesized that the potent anti-angiogenic factor endostatin might play a beneficial role in experimental ulcerative colitis (UC).
MAIN METHODS
We used three animal models of UC: (1) induced by 6% iodoacetamide (IA) in rats, or (2) by 3% dextran sulfate sodium (DSS) in matrix metalloproteinase-9 (MMP-9) knockout (KO) and wild-type mice, and (3) interleukin-10 (IL-10) KO mice. Groups of MMP-9 KO mice with DSS-induced UC were treated with endostatin or water for 5days.
KEY FINDINGS
We found concomitant upregulation of VEGF, PDGF, MMP-9 and endostatin in both rat and mouse models of UC. A positive correlation between the levels of endostatin or VEGF and the sizes of colonic lesions was seen in IA-induced UC. The levels and activities of MMP-9 were also significantly increased during UC induced by IA and IL-10 KO. Deletion of MMP-9 decreased the levels of endostatin in both water- and DSS-treated MMP-9 KO mice. Treatment with endostatin significantly improved DSS-induced UC in MMP-9 KO mice.
SIGNIFICANCE
1) Concomitantly increased endostatin is a defensive response to the increased VEGF in UC, 2) MMP-9 is a key enzyme to generate endostatin which may modulate the balance between VEGF and endostatin during experimental UC, and 3) endostatin treatment plays a beneficial role in UC. Thus, anti-angiogenesis seems to be a new therapeutic option for UC.
Topics: Animals; Blotting, Western; Colitis, Ulcerative; Colon; Dextran Sulfate; Disease Models, Animal; Endostatins; Enzyme-Linked Immunosorbent Assay; Female; Iodoacetamide; Matrix Metalloproteinase 9; Mice; Mice, Knockout; Platelet-Derived Growth Factor; Rats; Up-Regulation; Vascular Endothelial Growth Factor A
PubMed: 21047522
DOI: 10.1016/j.lfs.2010.10.026