ABSTRACT
Cecropia pachystachya Tréc., popularly known as embaúba, belongs to the Cecropiaceae family and is used by the native population in the treatment of bronchitis, asthma, high blood pressure, fever, and as a diuretic. The pharmacological actions including anti-inflammatory, antioxidant, cardiotonic and sedative were previously reported. The objective of this study was to (1) isolate and identify bioactive compounds extracted from the ethanolic extract of C. pachystachya roots (ERCP), as well as (2) verify the affinity of these metabolites with the enzymes 5-lipoxygenase (5-LOX) and α-1-antitrypsin through in silico tests. Isolation and/or identification were performed using GC-MS, HPLC, Infrared (IR), and nuclear magnetic resonance (NMR) techniques. After isolation and identification of the active compounds, these substances were subjected to the in silico investigation that proceeded by performing PreADMET simulations and molecular docking calculations. The bioactive compounds identified were 1-(+)-ascorbic acid 2,6-dihexadecanoate, ethyl hexadecanoate, ethyl (9E,12E)-octadec-9,12-dienoate, ethyl (Z)-octadec-9-enoate and ethyl octadecanoate by GC-MS; chlorogenic acid, catechin, epicatechin, syringaldehyde by HPLC; ß-sitosterol, sitostenone, beccaridiol, tormentic acid, lupeol, α- and ß-amyrin by classical chromatography, IR, 1H and 13C NMR techniques. The ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties were determined for each bioactive compound. Tormentic acid demonstrated a greater affinity for 5-LOX enzyme while sitostenone demonstrated a higher affinity for the α-1-antitrypsin enzyme. Our findings demonstrated a diverse range of secondary metabolites isolated from C. pachystachya that showed relevant interactions with the enzymes 5-LOX and α-1-antitrypsin. Thus, "embaúba" may be employed in in vivo experimental studies as a form of alternative treatment for chronic lung diseases.Abbreviations: ADT: Autodock Tools; BBB: Blood-brain barrier; CaCo2: Human colonic adenocarcinoma cells; CC: Classic/open Column; TLC: Thin Layer Chromatography; CD40: Differentiation Cluster 40; CENAUREMN: Centro Nordestino de Aplicação e Uso da Ressonância Magnética Nuclear; GC-MS: Gas Chromatography coupled to mass spectrometry; HPLC: High-Perfomance Liquid Chromatography; CYP2C9, CYP2C19, CYP2D6 and CYP3A4: Cytochrome P450 isoenzymes; COPD: Chronic Obstructive Pulmonary Disease; DRX-500: X-Ray Diffraction - 500; ERCP: Ethanolic extract of the roots of C. pachystachya; FAPEPI: Fundação de Amparo à Pesquisa do Piauí; HIA: Human Intestinal Absorption; IR: Infrared; Ki: Inhibition constant; 5-LOX: 5-Lipoxygenase; mM: miliMolar; nM: nanoMolar; OECD423: acute toxic class method; PDB: Protein Data Bank; P-gP: P-glycoprotein; PM2,5: Small inhalable particles 2,5; PPB: Plasm Protein Binding; PreADMET: Prediction Absorption, Distribution, Metabolization, Excretion and Toxicity; NMR: Nuclear Magnetic Resonance; +S9: with metabolic activation; -S9: no metabolic activation; SisGen: Sistema Nacional de Gestão de Patrimônio Genético e do Conhecimento Tradicional Associado; RT: Retention time; TA100: Ames test with TA100 cells line; TA1535: Ames test with cells of the TA1535 cell line; UESPI: State University of Piauí; V79: lung fibroblast cells; ΔG: Gibbs free energy (Kcal/mol); µM: microMolar.
Subject(s)
Arachidonate 5-Lipoxygenase , Cecropia Plant , alpha 1-Antitrypsin/metabolism , Caco-2 Cells , Cecropia Plant/chemistry , Humans , Molecular Docking Simulation , Plant Extracts/chemistry , Plant Extracts/toxicityABSTRACT
Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN-protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1' residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence-function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4' region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4' region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4' RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs.
Subject(s)
Drug Design , Models, Molecular , Protein C Inhibitor/genetics , Protein Engineering , alpha 1-Antitrypsin/genetics , Animals , Blood Coagulation Tests , Drug Evaluation, Preclinical , HEK293 Cells , Hemophilia A/drug therapy , Humans , Mice , Protein C Inhibitor/metabolism , Protein C Inhibitor/therapeutic use , Substrate Specificity , alpha 1-Antitrypsin/metabolism , alpha 1-Antitrypsin/therapeutic useABSTRACT
α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α1-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.
Subject(s)
Drug Design , Protein Folding , alpha 1-Antitrypsin/metabolism , Crystallization , Drug Development/methods , Drug Evaluation, Preclinical , Endoplasmic Reticulum/metabolism , Gene Library , Hepatocytes/metabolism , Humans , Models, Molecular , Protein Conformation , alpha 1-Antitrypsin/geneticsABSTRACT
Alpha1-antitrypsin (AAT) deficiency predisposes individuals to emphysema and liver diseases such as cirrhosis and hepatocellular carcinoma. The deficiency results from mutations in the SERPIN1A gene encoding AAT molecules that cause hepatotoxic retention within the endoplasmic reticulum. Since the E342K mutation is the basis for destabilization leading to lung and liver pathologies, we used the crystal structure of the mutated AAT as the basis for molecular docking selection of candidate compounds that may bind and stabilize the 342K structural pocket. We identified compounds that inhibited intracellular accumulation of AAT in hepatocytes in vitro. These data suggest that drug binding to a structural site encoded by a mutation associated with AAT deficiency has the potential for clinical utility by modulating conformational transitions.
Subject(s)
Liver Diseases/complications , Liver Diseases/drug therapy , Molecular Targeted Therapy , Mutation , alpha 1-Antitrypsin Deficiency/complications , alpha 1-Antitrypsin/genetics , Cell Line , Drug Evaluation, Preclinical , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Liver Diseases/genetics , Molecular Docking Simulation , Protein Conformation , alpha 1-Antitrypsin/chemistry , alpha 1-Antitrypsin/metabolismABSTRACT
Selenium (Se), as a nutritionally essential trace element, has been shown to decrease with age and is closely related to Alzheimer's disease (AD). To probe the effects of Se on AD pathology, two-dimensional fluorescence difference gel electrophoresis was applied to the serum samples collected from the wild-type (WT) mice and the triple transgenic (PS1M146V/AßPPSwe/TauP301L) AD mice (3xTg-AD), treated with or without sodium selenate in drinking water for 4 months beginning at 2 months of age. Proteomics results revealed 17 differentially expressed proteins between WT and 3xTg-AD mice. It was found that the administration of selenate reversed the alterations of the differentially expressed serum proteins by up-regulating 13 proteins and down-regulating 2 proteins which were reported to be involved in the key pathogenesis of AD, including regulation of Aß production, lipid metabolism regulation, and anti-inflammation. These results suggested that a dietary supplement with selenate is effective for prevention and treatment of AD, and the mechanism was maybe related to its role in Aß regulation, lipid metabolism, and anti-inflammation. Moreover, we also presented that α-2 macroglobulin, transthyretin, haptoglobin, alpha-2-HS-glycoprotein, and alpha-1-antitrypsin in the serum can be used to evaluate the effect of selenate on AD pathology.
Subject(s)
Alzheimer Disease/drug therapy , Disease Models, Animal , Proteomics , Selenic Acid/pharmacology , Alzheimer Disease/blood , Alzheimer Disease/pathology , Animals , Glycoproteins/antagonists & inhibitors , Glycoproteins/blood , Haptoglobins/analysis , Haptoglobins/antagonists & inhibitors , Mice , Mice, Inbred Strains , Mice, Transgenic , Prealbumin/analysis , Prealbumin/antagonists & inhibitors , Pregnancy-Associated alpha 2-Macroglobulins/analysis , Pregnancy-Associated alpha 2-Macroglobulins/antagonists & inhibitors , alpha 1-Antitrypsin/blood , alpha 1-Antitrypsin/metabolismABSTRACT
Elastin is a unique protein providing deformability and resilience to dynamic tissues, such as arteries and lungs. It is an absolute basic requirement for circulation and respiration. Elastin can be degraded by elastases and has a high calcium affinity. Elastin calcification and elastin degradation are two pathological processes that impair elastin's functioning. Furthermore, elastin degradation can be associated to elastin calcification. Matrix Gla Protein (MGP) is probably the most potent natural inhibitor of elastin calcification and requires vitamin K for its activation. Measuring circulating levels of inactive MGP (dp-ucMGP) is a frequently used method to assess vitamin K status. Dp-ucMGP reflects the burden of vitamin K-dependent proteins that have not been activated by vitamin K and could therefore best be regarded as a biomarker of a vitamin K deficit. Dp-ucMGP levels decrease after vitamin K supplementation. Since the amino acids desmosine and isodesmosine (DES) are unique to crosslinked elastin fibers, systemic elastin degradation can be assessed with the plasma DES assay. Recently, we discovered a strong correlation between plasma dp-ucMGP and plasma DES levels in both patients with chronic obstructive pulmonary disease (COPD) and controls. The 'Vitamin K deficit and elastolysis theory' posits that elastin degradation causes a rise in the vitamin K deficit and implies that vitamin K supplementation could be preventing elastin degradation. If this hypothesis holds true and is universally found in every state and condition, it will have an unprecedented impact on the management of every single pulmonary disease characterized by accelerated elastin degradation, such as alpha-1 antitrypsin deficiency, bronchiectasis, COPD and cystic fibrosis. Theoretically, a plasma dp-ucMGP concentration of zero would be associated with a near-complete standstill of elastin degradation and disease progression in patients with any of these debilitating conditions.
Subject(s)
Calcium-Binding Proteins/metabolism , Elastin/metabolism , Extracellular Matrix Proteins/metabolism , Lung Diseases/metabolism , Vitamin K Deficiency/metabolism , Biomarkers/metabolism , Calcium/metabolism , Desmosine/blood , Elasticity , Humans , Isodesmosine/blood , Models, Biological , Vitamin K/therapeutic use , alpha 1-Antitrypsin/metabolism , Matrix Gla ProteinABSTRACT
OBJECTIVE: To explore the mechanism of pulmonary involvement in ulcerative colitis (UC) patients by observing the correlation between pulmonary functions and levels of alpha1-antitrypsin (A1AT) in serum and colon tissue in UC patients. METHODS: Totally 90 patients with confirmed UC were assigned to different groups according to the extent of disease, the disease activity, the staging of severity, and course of disease. The serum level of A1AT in UC patients with different extent of disease, the disease activity, the staging of severity, and course of disease were compared. And 30 healthy volunteers were recruited as the control group. The serum renal and hepatic functions, pulmonary functions, and serum levels of A1AT were detected in the UC group and the control group. The correlation between A1AT and each pulmonary function index in UC patients was analyzed. The A1AT content in the colon tissue was detected with immunohistochemical assay in 20 UC patients as well as in 10 healthy volunteers. RESULTS: Of the 90 UC patients, 54 patients were accompanied with pulmonary function abnormality (60.0%), and 24 with extraintestinal manifestations (26.7%). Compared with the control group, the serum level of A1AT was significantly lower in the UC group (P < 0.05). The serum level of A1AT was significantly higher in those with proctitis than in those with distal colonitis and pancolitis (P < 0.05). The serum level of A1AT was lower in patients with the course of disease 5 years and more than 5 years than in those with the course of disease less than 5 years (P < 0.05). Vital capacity (VC), forced vital capacity (FVC), forced expiratory volume in one second (FEV1.0), total lung capacity (TLC), function residual volume (FRV), and the ratio of diffusion capacity for carbon monoxide of lung (DLCO) were much lower in those with proctitis than in those with distal colonitis and pancolitis (P < 0.05). The ratio of FVC was negatively linear correlated with the course of disease (r = -0.23, P = 0.018). There was a positive correlation between the serum level of A1AT and peak expiratory flow (PEF) (r = 0.22, P = 0.03). The level of A1AT in the colon tissue was obviously lower in the UC patients than in those of the control group (P < 0.05). Mild and moderate UC patients had increased levels of A1AT in the colon tissue, when compared with severe UC patients (P < 0.05). The level of A1AT in the colon tissue was higher in those with proctitis than in those with distal colonitis and pancolitis (P < 0.05). CONCLUSIONS: The prevalence of pulmonary function impairment was higher than other extraintestinal manifestations in UC patients. The pulmonary function test was helpful to screen the pulmonary impairment of UC patients. The A1AT level in the serum and the colon tissue obviously decreased in UC patients, indicating the pulmonary function impairment of UC patients might manifest as decreased A1AT levels correlated chronic airway inflammation, remodeling of airway, and obstructive changes.
Subject(s)
Colitis, Ulcerative/metabolism , Lung/physiopathology , alpha 1-Antitrypsin/metabolism , Adult , Aged , Case-Control Studies , Colitis, Ulcerative/pathology , Colitis, Ulcerative/physiopathology , Colon/metabolism , Female , Humans , Male , Middle Aged , Young Adult , alpha 1-Antitrypsin/bloodABSTRACT
Exposure to arsenic in drinking water is associated with increased respiratory disease. Alpha-1 antitrypsin (AAT) protects the lung against tissue destruction. The objective of this study was to determine whether arsenic exposure is associated with changes in airway AAT concentration and whether this relationship is modified by selenium. A total of 55 subjects were evaluated in Ajo and Tucson, Arizona. Tap water and first morning void urine were analyzed for arsenic species, induced sputum for AAT and toenails for selenium and arsenic. Household tap-water arsenic, toenail arsenic and urinary inorganic arsenic and metabolites were significantly higher in Ajo (20.6±3.5 µg/l, 0.54±0.77 µg/g and 27.7±21.2 µg/l, respectively) than in Tucson (3.9±2.5 µg/l, 0.16±0.20 µg/g and 13.0±13.8 µg/l, respectively). In multivariable models, urinary monomethylarsonic acid (MMA) was negatively, and toenail selenium positively associated with sputum AAT (P=0.004 and P=0.002, respectively). In analyses stratified by town, these relationships remained significant only in Ajo, with the higher arsenic exposure. Reduction in AAT may be a means by which arsenic induces respiratory disease, and selenium may protect against this adverse effect.
Subject(s)
Arsenic/toxicity , Environmental Exposure , Selenium/pharmacology , Sputum/metabolism , Water Pollutants, Chemical/toxicity , alpha 1-Antitrypsin/metabolism , Aged , Aged, 80 and over , Female , Humans , Male , Middle AgedABSTRACT
Aquatic organisms may suffer from exposure to high Cu concentrations, since this metal is widely used in feed supplementation, in pesticide formulation and as antifouling. Chronic exposure to Cu, even at sub-lethal doses, may strongly affect fish physiology. To date, several biomarkers have been used to detect Cu exposure in fish producing contrasting results. Therefore, we used a proteomic approach to clarify how Cu exposure may affect the serum proteome of gilthead sea bream (Sparus aurata), since serum could be considered a good source of early-biomarkers of Cu toxicosis. For this purpose we exposed juvenile gilthead sea bream to waterborne Cu (0.5 mg/L). Our results indicate that fish tightly regulate circulating Cu levels, which are not affected by metal exposure. This homeostatic control is mainly achieved by the liver, able to excrete high amounts of the metal via bile. Cu exposure caused differential expression of several serum proteins, 10 of which were identified by Mascot and BLAST search. All these proteins, with the exception of growth hormone receptor and γ-glutamyl-carboxylase, can be related to: 1) Cu-induced hepatotoxicity (cytochrome oxidase subunit I, alanine aminotransferase, glutathione S-transferase); 2) potential immunosuppression due to interference of Cu with the inflammation/immunity network (α-1 antitrypsin, angiotensinogen, complement component C3, recombination-activating protein-1 and warm temperature acclimation-related 65 kDa protein).
Subject(s)
Blood Proteins/metabolism , Copper/toxicity , Gene Expression Regulation/immunology , Liver/metabolism , Proteomics/methods , Sea Bream/immunology , Water Pollutants, Chemical/toxicity , Alanine Transaminase/metabolism , Angiotensinogen/metabolism , Animals , Bile/chemistry , Complement C3/metabolism , Computational Biology , Electron Transport Complex IV/metabolism , Gene Expression Regulation/drug effects , Glutathione Transferase/metabolism , Liver/drug effects , Sea Bream/blood , alpha 1-Antitrypsin/metabolismABSTRACT
No medication exists that clearly improves the mortality of chronic obstructive pulmonary disease (COPD). Oxidative molecules, in particular superoxide anions, play important roles in the COPD-associated abnormal inflammatory response and pulmonary emphysema, which arises because of an imbalance in proteases and antiproteases and increased apoptosis. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide anions. Lecithinized human Cu/Zn- SOD (PC-SOD) has overcome a number of the clinical limitations of SOD, including low tissue affinity and low stability in plasma. In this study, we examine the effect of PC-SOD on elastase-induced pulmonary emphysema, an animal model of COPD. The severity of the pulmonary inflammatory response and emphysema in mice was assessed by various criteria, such as the number of leukocytes in the bronchoalveolar lavage fluid and the enlargement of airspace. Not only intravenous administration but also inhalation of PC-SOD suppressed elastase-induced pulmonary inflammation, emphysema, and dysfunction. Inhalation of PC-SOD suppressed the elastase-induced increase in the pulmonary level of superoxide anions and apoptosis. Inhalation of PC-SOD also suppressed elastase-induced activation of proteases and decreased in the level of antiproteases and expression of proinflammatory cytokines and chemokines. We also found that inhalation of PC-SOD suppressed cigarette smoke-induced pulmonary inflammation. The results suggest that PC-SOD protects against pulmonary emphysema by decreasing the pulmonary level of superoxide anions, resulting in the inhibition of inflammation and apoptosis and amelioration of the protease/antiprotease imbalance. We propose that inhalation of PC-SOD would be therapeutically beneficial for COPD.
Subject(s)
Lecithins/chemistry , Lecithins/pharmacology , Pulmonary Emphysema/drug therapy , Superoxide Dismutase/chemistry , Superoxide Dismutase/pharmacology , Animals , Bronchoalveolar Lavage Fluid/cytology , Cell Count , Cell Death/drug effects , Chemokines/metabolism , Cytokines/metabolism , Enzyme Activation/drug effects , Immunohistochemistry , In Situ Nick-End Labeling , Male , Mice , Mice, Inbred ICR , Pancreatic Elastase/antagonists & inhibitors , Peptide Hydrolases/metabolism , Phosphatidylcholines/chemistry , Pneumonia/drug therapy , Pneumonia/pathology , Pulmonary Disease, Chronic Obstructive/drug therapy , Respiratory Function Tests , Reverse Transcriptase Polymerase Chain Reaction , Superoxides/metabolism , alpha 1-Antitrypsin/metabolismABSTRACT
Aging is accompanied by changes in the redox balance that is additionally modified by alcohol. Ethanol metabolism is connected with generation of free radicals which can damage cell components especially when antioxidant mechanisms are not able to neutralize them. In connection with the necessity of prevention against oxidative consequences, natural antioxidants are looked for. A natural and commonly used component of the diets with antioxidant properties are teas, especially the black tea. This study provides evidence of the role of black tea in the protection of rat plasma proteins and lipids against oxidative stress caused by aging and ethanol intoxication. For 5 weeks, the rats (2-, 12-, and 24-months old) used for the experiment received a black tea beverage (3 g/l) without or with alcohol (given for 4 weeks). The decrease in antioxidant abilities determined as total antioxidant status during aging and ethanol intoxication resulted in enhanced lipid and protein oxidation (determined as malondialdehyde, carbonyl groups, dityrosine, tryptophan and sulfhydryl groups level). In consequence the decrease in anti-proteases (alpha-1-antitrypsin, alpha-2-macroglobulin) activity and the increase in proteases (elastase and cathepsin G) activity were observed. Black tea protected the plasma antioxidants and prevented oxidative modifications of lipid and protein observed during aging as well as ethanol intoxication. The results indicate that a shift into plasma proteolytic activity results from a decrease in antioxidant abilities, so the use of black tea appears to be beneficial in reducing oxidative stress caused by ethanol and/or aging.
Subject(s)
Antioxidants/pharmacology , Ethanol/toxicity , Tea , Age Factors , Animals , Antioxidants/metabolism , Cathepsin G/metabolism , Ethanol/administration & dosage , Ethanol/metabolism , Homeostasis/drug effects , Lipid Peroxidation/drug effects , Male , Malondialdehyde/metabolism , Oxidation-Reduction/drug effects , Pancreatic Elastase/metabolism , Rats , Rats, Wistar , Tryptophan/metabolism , Tyrosine/analogs & derivatives , alpha 1-Antitrypsin/metabolism , alpha-Macroglobulins/metabolismABSTRACT
Protein-losing gastroenteropathy (PLE) consists on an active digestive tract protein loss syndrome and it is related to some diseases. After a wide research into bibliography (MEDLINE - Pubmed),we have found few references to this gastroenteropathy as a cause of hypoalbuminemia related to malnutrition. This has motivated us to review this entity, detailing some recent clinical cases of our experience.
Subject(s)
Gastrointestinal Diseases/complications , Gastrointestinal Diseases/therapy , Hypoalbuminemia/etiology , Hypoalbuminemia/therapy , Proteins/metabolism , Adult , Aged, 80 and over , Azathioprine/therapeutic use , Diarrhea/etiology , Dietary Supplements , Diuretics/therapeutic use , Enteral Nutrition , Female , Gastrointestinal Diseases/diagnosis , Humans , Hypoalbuminemia/diagnosis , Hypoproteinemia/etiology , Male , Serum Albumin/analysis , Serum Albumin/metabolism , Tuberculosis, Pulmonary/complications , alpha 1-Antitrypsin/metabolismABSTRACT
The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.
Subject(s)
Caenorhabditis elegans/metabolism , Drug Evaluation, Preclinical/methods , Microscopy, Fluorescence/methods , alpha 1-Antitrypsin/metabolism , Animals , Autophagy/drug effects , Caenorhabditis elegans/drug effects , Caenorhabditis elegans/genetics , Cantharidin/pharmacology , Cell Survival/drug effects , Dopamine Antagonists/pharmacology , Enzyme Inhibitors/pharmacology , Fluphenazine/pharmacology , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Models, Animal , Pimozide/pharmacology , Sodium Azide/pharmacology , alpha 1-Antitrypsin/geneticsABSTRACT
Z alpha(1)-antitrypsin (ZAAT) deficiency is a disease associated with emphysematous lung disease and also with liver disease. The liver disease of AAT deficiency is associated with endoplasmic reticulum (ER) stress. SEPS1 is a selenoprotein that, through a chaperone activity, decreases ER stress. To determine the effect of SEPS1 on ER stress in ZAAT deficiency, we measured activity of the grp78 promoter and levels of active ATF6 as markers of the unfolded protein response in HepG2 cells transfected with the mutant form of AAT, a ZAAT transgene. We evaluated levels of NFkappaB activity as a marker of the ER overload response. To determine the effect of selenium supplementation on the function of SEPS1, we investigated glutathione peroxidase activity, grp78 promoter activity, and NFkappaB activity in the presence or absence of selenium. SEPS1 reduced levels of active ATF6. Overexpression of SEPS1 also inhibited grp78 promoter and NFkappaB activity, and this effect was enhanced in the presence of selenium supplementation. This finding demonstrates a role for SEPS1 in ZAAT deficiency and suggests a possible therapeutic potential for selenium supplementation.
Subject(s)
Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Selenoproteins/metabolism , alpha 1-Antitrypsin Deficiency/metabolism , alpha 1-Antitrypsin/metabolism , Base Sequence , Cell Line , DNA Primers/genetics , Endoplasmic Reticulum Chaperone BiP , Gene Expression , Genetic Variation , Glutathione Peroxidase/metabolism , Heat-Shock Proteins/genetics , Humans , NF-kappa B/metabolism , Promoter Regions, Genetic , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Selenium/administration & dosage , Selenium/blood , Stress, Physiological , Transfection , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin Deficiency/drug therapy , alpha 1-Antitrypsin Deficiency/geneticsABSTRACT
Postoperative recurrence after ileo-colonic resection is a feature of Crohn's Disease (CD), almost 73% of patients show endoscopic recurrence at 1 year and 90% at 3 years. After surgical resection for CD, symptoms may be related to the surgical resection itself. Moreover, the development of an early severe endoscopic recurrence within 1 year represents a risk factor for early clinical recurrence. On the basis of these observations, the early detection and assessment of asymptomatic endoscopic recurrence may allow a timely and appropriate treatment of CD patients after ileo-colonic resection. At this purpose, conventional colonoscopy with ileoscopy currently represents the gold standard for assessing CD recurrence, graded according to the Rutgeerts' score. Lesions compatible with CD recurrence can be also detected by conventional radiology, including small bowel follow through and enema, both associated with a high radiation exposure. Due to the ineluctable course of CD after resection, and to the need of a proper follow up for assessing CD recurrence, several alternative, non invasive techniques have been searched in order to assess the post-operative recurrence, including: faecal alpha 1-antitrypsin clearance, faecal calprotectin, 99Tc-HMPAO scintigraphy, virtual colonoscopy, ultrasonography and, more recently, wireless capsule endoscopy (WCE) and Small Intestine Contrast Ultrasonography (SICUS). Among these, current evidences suggest that in experienced hands, ultrasound examination by SICUS represents a non-invasive technique useful for assessing recurrence in CD patients under regular follow up after surgery. The same findings are suggested for WCE, although the impact risk related to the recurrence or to the surgical anastomosis itself limits the use of this non-invasive technique for assessing CD recurrence after surgery.
Subject(s)
Crohn Disease/diagnostic imaging , Crohn Disease/diagnosis , Biomarkers/metabolism , Capsule Endoscopy , Contrast Media , Crohn Disease/pathology , Feces/chemistry , Humans , Leukocyte L1 Antigen Complex/metabolism , Recurrence , Ultrasonography , alpha 1-Antitrypsin/metabolismABSTRACT
INTRODUCTION: Activated protein C (APC) is well-established as a physiologically important anticoagulant. During development, plasma concentrations of protein C and alpha(2)macroglobulin, factors involved in APC generation, differ from adult levels. Chemotherapy drugs can perturb endothelial expression of PC-activating receptors. This study examines the effect of chemotherapy treatment of endothelium on APC generation in newborn and adult plasma. MATERIALS AND METHODS: APC generations were initiated on endothelial cells treated with vincristine or media by recalcifying defibrinated plasma with buffer containing thromboplastin. APC generation was terminated by mixing timed subsamples into FFRCMK-EDTA or heparin, followed by EDTA. APC-PCI and APC-alpha(1)AT were assayed by ELISA. APC-alpha(2)M was measured chromogenically. Since heparin converts free APC to APC-PCI, the difference between APC-PCI detected in heparin subsamples and APC-PCI detected in FFRCMK-EDTA subsamples gave the free APC. Cellular expression of EPCR and TM were measured by flow cytometry and Western blot. RESULTS: Vincristine-treated endothelium decreased free APC generation in newborn plasma to a greater degree than in adult plasma. APC-PCI levels in both adult and newborn plasma were unaffected by chemotherapy. Vincristine treatment reduced levels of APC-alpha(1) AT and APC-alpha(2) M to a greater degree in newborn plasma versus adult plasma. Expression of EPCR was reduced in cells treated with vincristine. Conversely, TM was reduced on the cell surface, but increased in whole cell lysates. CONCLUSIONS: The differential response of newborn and adult plasma PC components to chemotherapy-mediated changes in cell surface components may be a factor in the increased risk of thrombosis in children receiving chemotherapy.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Blood Proteins/pharmacology , Endothelial Cells/drug effects , Protein C/metabolism , Thrombosis/prevention & control , Vincristine/pharmacology , Adult , Age Factors , Cells, Cultured , Endothelial Cells/cytology , Endothelial Cells/metabolism , Humans , Infant, Newborn , Membrane Proteins/metabolism , Plasma , Protein Binding/drug effects , Protein C Inhibitor/metabolism , Thrombomodulin/metabolism , Thrombosis/chemically induced , Thrombosis/metabolism , Umbilical Veins/cytology , alpha 1-Antitrypsin/metabolism , alpha-Macroglobulins/metabolismABSTRACT
Conformational diseases include heterogeneous disorders sharing a similar pathological mechanism, leading to intracellular aggregation of proteins with toxic effects. Serpins are commonly involved in these diseases. These are structurally sensitive molecules that modify their folding under even minor genetic or environmental variations. Indeed, under normal conditions, the rate of misfolding of serpins is high and unfolded serpins must be degraded by the proteasome system. Our aim was to study the effects of bortezomib, a proteasome inhibitor, on conformationally sensitive serpins. The effects of bortezomib were analysed in patients with multiple myeloma, HepG2 cells, and Swiss mice, as well as in vitro. Levels, anti-FXa activity, heparin affinity, and conformational features of antithrombin, a relevant anticoagulant serpin, were analysed. Histological, ultrastructural features and immunohistological distribution of antithrombin and alpha1-antitrypsin (another hepatic serpin) were evaluated. We also studied the intracellular accumulation of conformationally sensitive (fibrinogen) or non-sensitive (prothrombin) hepatic proteins. The inhibition of the proteasome caused intracellular accumulation and aggregation of serpins within the endoplasmic reticulum that was associated with confronting cisternae and Mallory body formation. These effects were accompanied by a heat stress response. Bortezomib also increased the levels of intracellular fibrinogen, but has no significant effect on prothrombin. Finally, bortezomib had only minor effects on the mature circulating antithrombin, with increased amounts of latent antithrombin in plasma. These results suggest that the impairment of proteasomal activities leads to an intracellular accumulation of conformationally sensitive proteins and might facilitate the release of misfolded serpins into circulation where they adopt more stable conformations.
Subject(s)
Antineoplastic Agents/pharmacology , Antithrombins/metabolism , Boronic Acids/pharmacology , Liver/metabolism , Proteasome Inhibitors , Pyrazines/pharmacology , Serpins/metabolism , Alleles , Animals , Antithrombins/genetics , Antithrombins/ultrastructure , Boronic Acids/administration & dosage , Bortezomib , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/pathology , Case-Control Studies , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Evaluation, Preclinical , Endoplasmic Reticulum/metabolism , Factor Xa Inhibitors , Fibrinogen/biosynthesis , Humans , Immunohistochemistry , Injections, Intravenous , Leukocyte Elastase/adverse effects , Leukocyte Elastase/blood , Leukocyte Elastase/genetics , Leukocyte Elastase/immunology , Leukocyte Elastase/metabolism , Leukocyte Elastase/ultrastructure , Liver/pathology , Liver/ultrastructure , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Male , Mice , Molecular Chaperones/metabolism , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Proteins/metabolism , Pyrazines/administration & dosage , Serpins/biosynthesis , Serpins/genetics , Ubiquitin/metabolism , alpha 1-Antitrypsin/adverse effects , alpha 1-Antitrypsin/blood , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin/immunology , alpha 1-Antitrypsin/metabolism , alpha 1-Antitrypsin/ultrastructureABSTRACT
OBJECTIVE: To observe the effect of Dansen injection on the experimental emphysema in rabbits. METHOD: Thirty-six rabbits were randomized into emphysema group (n = 12), Dansen injection treated group (n = 12) and alpha1-antitrypsin(alpha1-AT) treated group (n = 12). The animal model of emphysema was induced by intratracheal instillation of porcine pancreatic elastase. Dansen injection and alpha1-ATwere instilled intratracheal in two treated group after 14 days with porcine pancreatic elastase, respectively, once a week, to continue for four weeks. The level of alpha1-AT in serum and in bronchoalveolar lavage fluid (BALF) were analyzed in different times. The mean linear intercept value (MLIV) and the numbers of alveolar per square (NAPS) of all groups were compared after eight weeks with porcine pancreatic elastase. RESULT: The levels of alpha1-AT in BALF were significantly different between treated groups and emphysema group after two weeks treatment, alpha1-AT levels of treated groups were more increased than those of emphysema group (P < 0.01). The levels of alpha1-AT in serum were similar at same times in different groups (P > 0.05), but were great different in different times. The MLIV and the NAPS were significantly different from emphysema group to treated groups in sixth and eighth weeks (P < 0.01), there is no difference between dancen group and alpha1-AT group. CONCLUSION: The contents of alpha1-AT in local pulmonary tissue could be improved by Dansen injection through intratracheal instillation during the information of emphysema in rabbits. The effect of Dansen injection and alpha1-AT on preventing formation of emphysema is similar.
Subject(s)
Drugs, Chinese Herbal/pharmacology , Pulmonary Emphysema/pathology , Salvia miltiorrhiza , alpha 1-Antitrypsin/metabolism , Animals , Bronchoalveolar Lavage Fluid/chemistry , Drugs, Chinese Herbal/administration & dosage , Drugs, Chinese Herbal/isolation & purification , Injections , Lung/pathology , Male , Pancreatic Elastase , Plants, Medicinal/chemistry , Pulmonary Emphysema/chemically induced , Pulmonary Emphysema/metabolism , Rabbits , Random Allocation , Salvia miltiorrhiza/chemistry , alpha 1-Antitrypsin/blood , alpha 1-Antitrypsin/pharmacologyABSTRACT
In an infection, polymorphonuclear neutrophils (PMN) become activated and they produce oxidizing compounds and elastase in the extracellular medium. Alpha-1-proteinase inhibitor (alpha1PI), a protease inhibitor which is inactivated by oxidants, is the main endogenous inhibitor of elastase helping to limit excessive elastase activity. This study evaluates the ability of a plant extract, Cola nitida nuts, to protect alpha1PI from inactivation by oxidizing compounds as reactive oxygen species. On the one hand, we have evaluated the direct effect of cola nut extract on neutrophil elastase, and on the H(2)O(2) and myeloperoxidase (MPO)-H(2)O(2) system via cell-free systems. Results showed that cola nut extract scavenges H(2)O(2) and therefore protects alpha1PI from HOCl which is produced from the MPO-H(2)O(2) system. Experiments also showed that cola extract has the capacity to limit elastase activity. On the other hand, we have worked on cellular systems including isolated PMN with the aim to study the effect of cola extract on PMN metabolism. PMN were stimulated with PMA, calcium ionophore or fMLP. Each stimulant possesses its own stimulation pathway. According to the inhibitory concentration obtained at 50%, the results on cellular systems led to the conclusion that cola extract can reduce elastase liberation from PMN. It can then be concluded that cola nut extract can protect alpha1PI from inactivation, and has an effect both on elastase liberation and elastase activity. The cola nut extract effect is rather biased towards a reduction in elastase release, thus limiting the injurious effects caused by this enzyme.
Subject(s)
Cola , Leukocyte Elastase/antagonists & inhibitors , N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , alpha 1-Antitrypsin/metabolism , Caffeine , Calcimycin/pharmacology , Cell Survival/drug effects , Enzyme Activators/pharmacology , Free Radical Scavengers/pharmacology , Humans , Hydrogen Peroxide/metabolism , In Vitro Techniques , Ionophores/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/cytology , Neutrophils/metabolism , Nuts , Plant Extracts/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Type C Phospholipases/metabolismABSTRACT
Protein ectodomain shedding is a specialized type of regulated proteolysis that releases the extracellular domain of transmembrane proteins. The metalloprotease disintegrin tumor necrosis factor-alpha-converting enzyme (TACE) has been convincingly shown to play a central role in ectodomain shedding, but despite its broad interest, very little is known about the mechanisms that regulate its activity. An analysis of the biosynthesis of TACE in mutant cell lines that have a gross defect in ectodomain shedding (M1 and M2) shows a defective removal of the prodomain that keeps TACE in an inactive form. Using LoVo, a cell line that lacks of active furin, and alpha1-Antitrypsin Portland, a protein inhibitor of proprotein convertases, we show that TACE is normally processed by furin and other proprotein convertases. The defect in M1 and M2 cells is due to a blockade of the exit of TACE from the endoplasmic reticulum. The processing of other zinc-dependent metalloproteases, previously suggested to participate in activated ectodomain shedding is normal in the mutant cells, indicating that the component mutated is highly specific for TACE. In summary, the characterization of shedding-defective somatic cell mutants unveils the existence of a specific mechanism that directs the proteolytic activation of TACE through the control of its exit from the ER.