Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease
Abstract
BACKGROUND & AIMS: Inducible nitric oxide synthase (iNOS) is generated in several cell types by treatment with lipopolysaccharides or cytokines. Earlier studies suggested that ulcerative colitis is associated with increased NO produced by iNOS; however, the cellular source of the NO synthesis was not identified. A possible mechanism of NO-induced cellular damage is through its interaction with superoxide to produce peroxynitrite, which reacts with tyrosine to form nitrotyrosine in cellular proteins. METHODS: Using immunoperoxidase microscopy with a new monospecific human iNOS antibody (NO-53), the cellular distribution of iNOS and nitrotyrosine was examined using human colonic mucosa from normal bowel, ulcerative colitis, Crohn's disease, and diverticulitis. RESULTS: Intense focal iNOS labeling was localized to the inflamed colonic epithelium in ulcerative colitis, Crohn's disease, and diverticulitis but was not detectable in the uninflamed epithelium. Nitrotyrosine labeling was also observed in the inflamed colonic epithelium and was associated with nearby iNOS staining; nitrotyrosine was undetectable in normal mucosal epithelium. iNOS and nitrotyrosine were also detected in lamina propria mononuclear cells and neutrophils. CONCLUSIONS: These findings suggest that iNOS is induced in the inflamed human colonic epithelium and is associated with the formation of peroxynitrite and the nitration of cellular proteins. (Gastroenterology 1996 Oct;111(4):871-85)
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Recombinant protein expression: Challenges in production and folding related matters
2023, International Journal of Biological MacromoleculesProtein folding is a biophysical process by which proteins reach a specific three-dimensional structure. The amino acid sequence of a polypeptide chain contains all the information needed to determine the final three-dimensional structure of a protein. When producing a recombinant protein, several problems can occur, including proteolysis, incorrect folding, formation of inclusion bodies, or protein aggregation, whereby the protein loses its natural structure. To overcome such limitations, several strategies have been developed to address each specific issue. Identification of proper protein refolding conditions can be challenging, and to tackle this high throughput screening for different recombinant protein folding conditions can prove a sound solution. Different approaches have emerged to tackle refolding issues. One particular approach to address folding issues involves molecular chaperones, highly conserved proteins that contribute to proper folding by shielding folding proteins from other proteins that could hinder the process. Proper protein folding is one of the main prerequisites for post-translational modifications. Incorrect folding, if not dealt with, can lead to a buildup of protein misfoldings that damage cells and cause widespread abnormalities. Said post-translational modifications, widespread in eukaryotes, are critical for protein structure, function and biological activity. Incorrect post-translational protein modifications may lead to individual consequences or aggregation of therapeutic proteins. In this review article, we have tried to examine some key aspects of recombinant protein expression. Accordingly, the relevance of these proteins is highlighted, major problems related to the production of recombinant protein and to refolding issues are pinpointed and suggested solutions are presented. An overview of post-translational modification, their biological significance and methods of identification are also provided. Overall, the work is expected to illustrate challenges in recombinant protein expression.
A20 Restricts NOS2 Expression and Intestinal Tumorigenesis in a Mouse Model of Colitis-Associated Cancer
2023, Gastro Hep AdvancesColon cancer can occur sporadically or in the setting of chronic inflammation, such as in patients with inflammatory bowel disease. We previously showed that A20, a critical negative regulator of tumor necrosis factor signal transduction, could regulate sporadic colon cancer development. In this report, we investigate whether A20 also acts as a tumor suppressor in a model of colitis-associated cancer.
Colitis and colitis-associated tumors were induced in wild-type and A20 intestinal epithelial cell-specific knockout (A20dIEC) mice using dextran sodium sulfate and azoxymethane. Clinicopathologic markers of inflammation were assessed in conjunction with colonic tumor burden. Gene expression analyses and immunohistochemistry were performed on colonic tissue and intestinal enteroids. Nitric oxide (NO) production and activity were assessed in whole colonic lysates and mouse embryonic fibroblasts.
A20dIEC mice develop larger tumors after treatment with dextran sodium sulfate and azoxymethane than wild-type mice. In addition to elevated markers of inflammation, A20dIEC mice have significantly enhanced expression of inducible nitric oxide synthase (iNOS), a well-known driver of neoplasia. Enhanced iNOS expression is associated with the formation of reactive nitrogen species and DNA damage. Loss of A20 also enhances NO-dependent cell death directly.
Mechanistically, we propose that A20 normally restricts tumor necrosis factor–induced nuclear factor kappa B–dependent production of iNOS in intestinal epithelial cells, thereby protecting against colitis-associated tumorigenesis. We also propose that A20 plays a direct role in regulating NO-dependent cell death.
Molsidomine alleviates acetic acid-induced colitis in rats by reducing oxidative stress, inflammation and apoptosis
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Effects of Salmonella enterica serovar typhimurium sseK1 on macrophage inflammation-related cytokines and glycolysis
2021, CytokineSalmonella enterica serovar Typhimurium (S. Typhimurium), an important virulent intracellular pathogen, causes inflammatory gastroenteritis or typhoid. Macrophages play a key role in innate immunity against Salmonella. Salmonella secreted effector K1 (SseK1) encoded by SPI2 has been identified a novel translocated protein. To investigate the role of Salmonella enterica serovar Typhimurium sseK1 about the inflammation and glycolysis in macrophages, the levels of IL-1β, IL-2, IL-4, IL-6, IFN-γ and Nitric Oxide in macrophages infected by S. Typhimurium SL1344 wild-type (WT) group, ΔsseK1 mutant group and sseK1-complemented group were measured. And the glycolysis level was determined in RAW 264.7 cells infected with these different Salmonella strains. The results showed that groups infected by wild-type strain, sseK1 mutant and sseK1-complemented strain upregulated the production of IL-1β, IL-2, IL-4, IL-6, IFN-γ and NO at 3 h, 6 h and 12 h, respectively. The production of IL-1β, IL-2, IL-4, IL-6, IFN-γ and NO in wild-type strain group were significantly decreased compared with the ΔsseK1 mutant group, which suggested that sseK1 down-regulated the production of related inflammatory factors. Moreover, hexokinase, lactic acid and pyruvic acid levels significantly decreased by infection with sseK1 mutant compared to the wild-type strain. The ATP level of ΔsseK1 mutant group was remarkably increased than WT group and sseK1-complemented group. These indicated that the sseK1 enhanced the level of glycolysis of macrophages infected by S. Typhimurium. In summary, the results demonstrated that sseK1 can down-regulate the inflammation-related cytokines and enhance the glycolysis level in macrophages infected by S. Typhimurium, which may be beneficial for S. typhimurium survival in macrophages.
Nuclear DNA damages generated by reactive oxygen molecules (ROS) under oxidative stress and their relevance to human cancers, including ionizing radiation-induced neoplasia part II: Relation between ROS-induced DNA damages and human cancer
2020, Radiation Medicine and ProtectionOxidative stress (OS) occurs when the production of reactive oxygen species (ROS) overrides the body’s natural defence. When the cell nucleus represents the target, macromolecular damage may result in mutations. Cancer is a disease of mutations, and DNA damages that are not repaired or mis-repaired during cell proliferation are necessary but not sufficient for cancer development. A role of ROS for cancer initiation depends on the likelihood of interaction between reactive electrophilic molecules and nuclear DNA. As described in part one of this presentation, the physico-chemical properties of the ROS involved in OS and of the ensuing DNA lesions are of major importance. Current knowledge dictates that emphasis should be shifted from oxidative DNA damages of low genotoxicity towards pro-mutagenic lesions induced by reaction products of nitrogen monoxide and complex highly reactive carbonyls, e.g. from the peroxidation of lipids.
Based on the determination of pro-mutagenic DNA adducts in human tissues there is compelling evidence for a causal relation between OS and cancers of the liver, colon/rectum, cervix, pancreas and stomach. However, modulation by the simultaneous presence of an ubiquitous high background of potent pro-carcinogenic DNA adducts, which are not generated by ROS should be taken into account.
Ionizing radiation is established human carcinogenic agent, and generate some of the same oxidative ROS as those involved in OS. However, the cancer spectrum from whole body radiation exposure differs in some important respects from that associated with OS. The scientific support for a causal link between exposure to non-ionizing electromagnetic radiation and human cancer is judged to be insufficient.
As exemplified by diabetes, a common shortcoming when assessing the role of OS in disease is the failure to distinguish between cause and effect - i.e. could the indicators of harmful oxidative stress be the result of the pathological condition in question, rather than its cause.
A designed whole-cell biosensor for live diagnosis of gut inflammation through nitrate sensing
2020, Biosensors and BioelectronicsCitation Excerpt :RNS also provide Enterobacteria with TEA for anaerobic respiration (Simmonds and Rampton, 1993). Gut inflammation induces the expression of nitric oxide synthase (iNOS) at high levels and increases the production of nitric oxide (NO) (Lundberg et al., 1994; Singer et al., 1996; Stanek et al., 2008). Genetic circuits capable of detecting and responding to NO have been generated previously using the NO reduction and detoxification regulator NorR that activates the transcription of NO-detoxifying flavorubredoxin (Archer et al., 2012), as well as the nitrate-sensitive repressor NsrR that regulates the expression of genes responsible for cell protection against NO (McKay et al., 2018).
Microbes reprogrammed using advanced genetic circuits are envisaged as emerging living diagnostics for a wide range of diseases and play key roles in regulating gut microbiota to treat disease-associated symptoms in a non-invasive manner. Here, we developed a designer probiotic Escherichia coli that senses and responds to nitrate, a biomarker of gut inflammation. To this end, we first employed the NarX-NarL two-component regulatory system in E. coli to construct a nitrate-responsive genetic circuit. Next, we optimized the nitrate biosensor for the best performance using measures of sensitivity and specificity. We then introduced this genetic circuit into a probiotic E. coli Nissle 1917. We demonstrated that the designed biosensor can sense elevated nitrate levels during gut inflammation in mice with native gut microbiota. Moreover, using Boolean AND gate, we generated a genetically encoded biosensor for simultaneous sensing of the thiosulfate and nitrate biomarkers, thus increasing the tool's specificity for diagnosing gut inflammation. The nitrate-responsive genetic circuit will enable new approaches for non-invasive diagnostics of inflammation-associated diseases.