RESUMEN
Structure-selective endonucleases cleave branched DNA substrates. Slx1 is unique among structure-selective nucleases because it can cleave all branched DNA structures at multiple sites near the branch point. The mechanism behind this broad range of activity is unknown. The present study structurally and biochemically investigated fungal Slx1 to define a new protein interface that binds the non-cleaved arm of branched DNAs. The DNA arm bound at this new site was positioned at a sharp angle relative to the arm that was modeled to interact with the active site, implying that Slx1 uses DNA bending to localize the branch point as a flexible discontinuity in DNA. DNA binding at the new interface promoted a disorder-order transition in a region of the protein that was located in the vicinity of the active site, potentially participating in its formation. This appears to be a safety mechanism that ensures that DNA cleavage occurs only when the new interface is occupied by the non-cleaved DNA arm. Models of Slx1 that interacted with various branched DNA substrates were prepared. These models explain the way in which Slx1 cuts DNA toward the 3' end away from the branch point and elucidate the unique ability of Slx1 to cleave various DNA structures.
Asunto(s)
ADN de Hongos/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Candida glabrata/genética , Candida glabrata/metabolismo , Catálisis , Cristalografía por Rayos X , Roturas del ADN de Cadena Simple , Reparación del ADN/genética , ADN de Hongos/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Escherichia coli , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Estructura Cuaternaria de Proteína , Sordariales/genética , Sordariales/metabolismoRESUMEN
Mixed acidic-alkaline refluxate is a major pathogenic factor in chronic esophagitis progressing to Barrett's esophagus (BE). We hypothesized that epidermal growth factor (EGF) can interact with COX-2 and peroxisome proliferator-activated receptor-γ (PPARγ) in rats surgically prepared with esophagogastroduodenal anastomosis (EGDA) with healthy or removed salivary glands to deplete salivary EGF. EGDA rats were treated with 1) vehicle, 2) EGF or PPARγ agonist pioglitazone with or without EGFR kinase inhibitor tyrphostin A46, EGF or PPARγ antagonist GW9662 respectively, 3) ranitidine or pantoprazole, and 4) the selective COX-2 inhibitor celecoxib combined with pioglitazone. At 3 mo, the esophageal damage and the esophageal blood flow (EBF) were determined, the mucosal expression of EGF, EGFR, COX-2, TNFα, and PPARγ mRNA and phospho-EGFR/EGFR protein was analyzed. All EGDA rats developed chronic esophagitis, esophageal ulcerations, and intestinal metaplasia followed by a fall in the EBF, an increase in the plasma of IL-1ß, TNFα, and mucosal PGE2 content, the overexpression of COX-2-, and EGF-EGFR mRNAs, and proteins, and these effects were aggravated by EGF and attenuated by pioglitazone. The rise in EGF and COX-2 mRNA was inhibited by pioglitazone but reversed by pioglitazone cotreated with GW9662. We conclude that 1) EGF can interact with PG/COX-2 and the PPARγ system in the mechanism of chronic esophagitis; 2) the deleterious effect of EGF involves an impairment of EBF and the overexpression of COX-2 and EGFR, and 3) agonists of PPARγ and inhibitors of EGFR may be useful in the treatment of chronic esophagitis progressing to BE.NEW & NOTEWORTHY Rats with EGDA exhibited chronic esophagitis accompanied by a fall in EBF and an increase in mucosal expression of mRNAs for EGF, COX-2, and TNFα, and these effects were exacerbated by exogenous EGF and reduced by removal of a major source of endogenous EGF with salivectomy or concurrent treatment with tyrphostin A46 or pioglitazone combined with EGF. Beneficial effects of salivectomy in an experimental model of BE were counteracted by PPARγ antagonist, whereas selective COX-2 inhibitor celecoxib synergistically with pioglitazone reduced severity of esophageal damage and protected esophageal mucosa from reflux. We propose the cross talk among EGF/EGFR, PG/COX-2, and proinflammatory cytokines with PPARγ pathway in the mechanism of pathogenesis of chronic esophagitis progressing to BE and EAC.
Asunto(s)
Esófago de Barrett/metabolismo , Ciclooxigenasa 2/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Mucosa Esofágica/metabolismo , Esofagitis/metabolismo , PPAR gamma/metabolismo , Animales , Esófago de Barrett/tratamiento farmacológico , Esófago de Barrett/genética , Esófago de Barrett/patología , Ciclooxigenasa 2/genética , Inhibidores de la Ciclooxigenasa 2/farmacología , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Mucosa Esofágica/efectos de los fármacos , Mucosa Esofágica/patología , Esofagitis/tratamiento farmacológico , Esofagitis/genética , Esofagitis/patología , Interleucina-1beta/metabolismo , Masculino , PPAR gamma/agonistas , PPAR gamma/genética , Pioglitazona/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de la Bomba de Protones/farmacología , Ratas Wistar , Transducción de Señal , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Melatonin is a tryptophan-derived molecule with pleiotropic activities which is produced in all living organisms. This "sleep" hormone is a free radical scavenger, which activates several anti-oxidative enzymes and mechanisms. Melatonin, a highly lipophilic hormone, can reach body target cells rapidly, acting as the circadian signal to alter numerous physiological functions in the body. This indoleamine can protect the organs against a variety of damaging agents via multiple signaling. This review focused on the role played by melatonin in the mechanism of esophagoprotection, starting with its short-term protection against acute reflux esophagitis and then investigating the long-term prevention of chronic inflammation that leads to gastroesophageal reflux disease (GERD) and Barrett's esophagus. Since both of these condition are also identified as major risk factors for esophageal carcinoma, we provide some experimental and clinical evidence that supplementation therapy with melatonin could be useful in esophageal injury by protecting various animal models and patients with GERD from erosions, Barrett's esophagus and neoplasia. The physiological aspects of the synthesis and release of this indoleamine in the gut, including its release into portal circulation and liver uptake is examined. The beneficial influence of melatonin in preventing esophageal injury from acid-pepsin and acid-pepsin-bile exposure in animals as well as the usefulness of melatonin and its precursor, L-tryptophan in prophylactic and supplementary therapy against esophageal disorders in humans, are also discussed.
Asunto(s)
Adenocarcinoma/prevención & control , Esófago de Barrett/prevención & control , Neoplasias Esofágicas/prevención & control , Esofagitis Péptica/prevención & control , Melatonina/uso terapéutico , Animales , Esófago/efectos de los fármacos , Esófago/metabolismo , Esófago/patología , Humanos , Melatonina/metabolismo , Melatonina/farmacología , Modelos Biológicos , Sustancias Protectoras/farmacología , Sustancias Protectoras/uso terapéuticoRESUMEN
Despite the fact that there is a growing awareness to the callosal connections between hemispheres the two hemispheres of the brain are commonly treated as independent structures when peripheral or cortical manipulations are applied to one of them. The contralateral hemisphere is often used as a within-animal control of plastic changes induced onto the other side of the brain. This ensures uniform conditions for producing experimental and control data, but it may overlook possible interhemispheric interactions. In this paper we provide, for the first time, direct proof that cortical, experience-dependent plasticity is not a unilateral, independent process. We mapped metabolic brain activity in rats with 2-[14C] deoxyglucose (2DG) following experience-dependent plasticity induction after a month of unilateral (left), partial whiskers deprivation (only row B was left). This resulted in â¼45% widening of the cortical sensory representation of the spared whiskers in the right, contralateral barrel field (BF). We show that the width of 2DG visualized representation is less than 20% when only contralateral stimulation of the spared row of whiskers is applied in immobilized animals. This means that cortical map remodeling, which is induced by experience-dependent plasticity mechanisms, depends partially on the contralateral hemisphere. The response, which is observed by 2DG brain mapping in the partially deprived BF after standard synchronous bilateral whiskers stimulation, is therefore the outcome of at least two separately activated plasticity mechanisms. A focus on the integrated nature of cortical plasticity, which is the outcome of the emergent interactions between deprived and non-deprived areas in both hemispheres may have important implications for learning and rehabilitation. There is also a clear implication that there is nothing like "control hemisphere" since any plastic changes in one hemisphere have to have influence on functioning of the opposite one.
RESUMEN
Studies of cortical function-recovery require a comparison between normal and post-stroke conditions that lead to changes in cortical metaplasticity. Focal cortical stroke impairs experience-dependent plasticity in the neighboring somatosensory cortex and usually evokes periinfarct depolarizations (PiDs) - spreading depression-like waves. Experimentally induced spreading depressions (SDs) affect gene expression and some of these changes persist for at least 30â¯days. In this study we compare the effects of non-stroke depolarizations that impair cortical experience-dependent plasticity to the effects of stroke, by inducing experience-dependent plasticity in rats with SDs or PiDs by a month of contralateral partial whiskers deprivation. We found that whiskers' deprivation after SDs resulted in normal cortical representation enlargement suggesting that SDs and PiDs depolarization have no influence on experience-dependent plasticity cortical map reorganization. PiDs and the MMP-9, -3, -2 or COX-2 proteins, which are assumed to influence metaplasticity in rats after stroke were compared between SDs induced by high osmolarity KCl solution and the PiDs that followed cortical photothrombotic stroke (PtS). We found that none of these factors directly caused cortical post-stroke metaplasticity changes. The only significant difference between stoke and induced SD was a greater imbalance in interhemispheric activity equilibrium after stroke. The interhemispheric interactions that were modified by stroke may therefore be promising targets for future studies of post-stroke experience-dependent plasticity and of recuperation studies.
Asunto(s)
Depresión de Propagación Cortical , Accidente Cerebrovascular , Animales , Depresión , Ratas , Corteza Somatosensorial , VibrisasRESUMEN
Holliday junctions (HJs) are four-way DNA structures that occur in DNA repair by homologous recombination. Specialized nucleases, termed resolvases, remove (i.e., resolve) HJs. The bacterial protein RuvC is a canonical resolvase that introduces two symmetric cuts into the HJ. For complete resolution of the HJ, the two cuts need to be tightly coordinated. They are also specific for cognate DNA sequences. Using a combination of structural biology, biochemistry, and a computational approach, here we show that correct positioning of the substrate for cleavage requires conformational changes within the bound DNA. These changes involve rare high-energy states with protein-assisted base flipping that are readily accessible for the cognate DNA sequence but not for non-cognate sequences. These conformational changes and the relief of protein-induced structural tension of the DNA facilitate coordination between the two cuts. The unique DNA cleavage mechanism of RuvC demonstrates the importance of high-energy conformational states in nucleic acid readouts.
Asunto(s)
ADN Bacteriano/química , ADN Cruciforme/química , Thermus thermophilus/metabolismo , Arginina/química , Proteínas Bacterianas/química , Emparejamiento Base , Secuencia de Bases , Biocatálisis , ADN Bacteriano/metabolismo , Simulación de Dinámica MolecularRESUMEN
This review was designed to provide an update on the role of asymmetric arginine (ADMA), the endogenous inhibitor of nitric oxide (NO) synthase in the pathophysiology of the upper gastrointestinal (GI) tract. Numerous studies in the past confirmed that NO is a multifunctional endogenous gas molecule involved in most of the body organs' functional and metabolic processes including the regulation of gastrointestinal (GI) secretory functions, motility, maintenance of GI integrity, gastroprotection and ulcer healing. NO is metabolized from L-arginine by enzymatic reaction in the presence of constitutive NO synthase. In upper GI tract, NO acts as a potent vasodilator known to increase gastric mucosa blood flow, regulates the secretion of mucus and bicarbonate, inhibits the gastric secretion and protects the gastric mucosa against the damage induced by a variety of damaging agents and corrosive substances. In contrast, ADMA first time described by Vallance and coworkers in 1992, is synthesized by the hydrolysis of proteins containing methylated arginine amino acids located predominantly within the nucleus of cells. This molecule has been shown to competitively inhibit NO synthase suggesting its regulatory role in the functions of vascular endothelial cells and systemic circulation in humans and experimental animals. Nowadays, ADMA is a potentially important risk factor for coronary artery diseases and a marker of cardiovascular risk. Increased plasma levels of ADMA have been documented in several conditions that are characterized by endothelial dysfunction, including hypertension, hypercholesterolemia, hyperglycemia, renal failure and tobacco exposure. The role of ADMA in other systems including GI-tract has been so far less documented. Nevertheless, ADMA was shown to directly induce oxidative stress and cell apoptosis in gastric mucosal cells in vitro and to contribute to the inflammatory reaction associated with major human pathogen to gastric mucosa, Helicobacter pylori (H.pylori). Infection of gastric mucosa with this germ or H. pylori water extract led to marked increase in the plasma concentration of ADMA and significantly inhibited bicarbonate secretion, considered as one of the important components of upper GI-tract defense system. When administered to rodents, ADMA aggravated gastric mucosal lesions injury induced by cold stress, ethanol and indomethacin and this worsening effect on gastric lesions was accompanied by the significant increase in the plasma level of ADMA. This exaggeration of gastric lesions by ADMA was coincided with the inhibition of NO, the suppression of gastric blood flow and excessive release of proinflammatory cytokine TNF-α. This metabolic analog of L-arginine applied to rats was exposed to water immersion and restraint stress and ischemia-reperfusion, causing an elevation of plasma levels of ADMA and gastric MDA content, which is the marker of lipid peroxidation. These effects, including the rise in the plasma levels of ADMA in rats with stress and ischemia-reperfusion-induced gastric lesions, were attenuated by concomitant treatment with L-arginine, the substrate for NO-synthase, and superoxide dismutase (SOD), a reactive oxygen metabolite scavenger added to ADMA. We conclude that ADMA could be considered as an important factor contributing to the pathogenesis of gastric mucosal damage and inflammatory reaction in H. pylori-infected stomach due to inhibition of NO, suppression of GI microcirculation, and the proinflammatory and proapoptotic actions of this arginine analog.
Asunto(s)
Arginina/análogos & derivados , Mucosa Gástrica/patología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Animales , Apoptosis , Arginina/metabolismo , Mucosa Gástrica/microbiología , Tracto Gastrointestinal/microbiología , Tracto Gastrointestinal/fisiopatología , Infecciones por Helicobacter/patología , Helicobacter pylori/aislamiento & purificación , Humanos , Inflamación/microbiología , Inflamación/fisiopatología , Óxido Nítrico/metabolismo , Ratas , Factores de RiesgoRESUMEN
Previous studies revealed that prostaglandins contribute to the mechanism of maintenance of gastrointestinal integrity and mediate various physiological aspects of mucosal defense. The suppression of prostaglandin synthesis in the stomach is a critical event in terms of the development of mucosal injury after administration of various NSAID including aspirin (ASA). A worldwide use of ASA is now accepted due to its remarkable analgesic, antipyretic and anti-thrombotic prophylactics against myocardial infarct and coronary disorders despite the fact that the use of NSAIDs is associated with the risk of gastrointestinal bleedings, haemorrhagic lesions and ulcerations. It has become clear that other mediators besides prostaglandins can similarly act to protect the gastrointestinal mucosa of experimental animals and humans from injury induced by ASA. For instance, nitric oxide (NO) released from vascular epithelium, epithelial cells of gastrointestinal tract and sensory nerves can influence many of the same components of mucosal defense as do prostaglandins. This review was designed to provide an updated overview based on the experimental and clinical evidence on the involvement COX-2 derived products, lipoxins in the mechanism of gastric defense, gastroprotection and gastric adaptation to ASA. Lipoxins were recently considered as another group of lipid mediators that can protect the stomach similarly as NO-donors known to exert protective influence on the stomach from the injury under condition where the mucosal prostaglandin levels are suppressed. The new class of NO-releasing NSAIDs, including NO-aspirin or NO-naproxen, represent a very promising approach to reducing the toxicity of their parent NSAIDs. Aspirin-triggered lipoxin (ATL) synthesis, via COX-2, acts to reduce the severity of damage induced by this NSAID. Lipoxin analogues may prove to be useful for preventing mucosal injury and for modulating mucosal inflammation. Evidence presented in this review documents that ATL also play in important role in gastric adaptation during chronic ASA administration. Suppression of COX-2 activity by selective COX-2 inhibitors such as rofecoxib or celecoxib was shown to abolish the production of ATL and to diminish the gastric tolerability of ASA and gastric adaptation developed in response to repetitive administration of this NSAID. Synthetic analogues of lipoxins as well as newer class of NSAIDs releasing NO may be used in the future as the therapeutic approach to counteract adverse effects in the stomach associated with NSAIDs ingestion.
Asunto(s)
Adaptación Fisiológica , Aspirina/efectos adversos , Lipoxinas/fisiología , Úlcera Gástrica/prevención & control , Estómago/efectos de los fármacos , Humanos , Estómago/fisiopatología , Úlcera Gástrica/fisiopatologíaRESUMEN
Orexigenic peptides are group of endocrine hormones exerting a pleiotropic influence on many physiological functions including regulation of the feeding behaviour and energy expenditure, release of growth hormone (GH) and inotropic effects on the heart. Some of these peptides such as ghrelin, originally identified in the gastric mucosa, has been involved not only in control of food intake and growth hormone release but also exerts the immunomodulatory and anti-inflammatory properties. This review summarizes the recent attempts to prove the concept that orexigenic peptides such as ghrelin, orexin-A and obestatin besides playing an important role in the mechanism of food intake, exhibit a potent gastroprotective action against the formation of acute gastric mucosal injury induced by various ulcerogens. This protective effect depends upon vagal activity and hyperemia mediated by NOS/NO and COX/PG systems and CGRP released from sensory afferent nerves. In addition, the appetite peptides such as ghrelin and orexin-A are implicated in the mechanism of the healing of preexisting gastric ulcers due to an activation of specific GHS-R1a and OX-R1 receptors and PG/COX system.