RESUMEN
We advance the quality of first-principles calculations of protein electronic circular dichroism (CD) through an amelioration of a key deficiency of a previous procedure that involved diabatization of electronic states on the amide chromophore (to obtain interamide couplings) in a ß-strand conformation of a diamide. This yields substantially improved calculated far-ultraviolet (far-UV) electronic circular dichroism (CD) spectra for ß-sheet conformations. The interamide couplings from the diabatization procedure for 13 secondary structural elements (13 diamide structures) are applied to compute the CD spectra for seven example proteins: myoglobin (α helix), jacalin (ß strand), concanavalin A (ß type I), elastase (ß type II), papain (α + ß), 310-helix bundle (310-helix) and snow flea antifreeze protein (polyproline). In all cases, except concanavalin A and papain, the CD spectra computed using the interamide couplings from the diabatization procedure yield improved agreement with experiment with respect to previous first-principles calculations.
Asunto(s)
Dicroismo Circular , Concanavalina A , Concanavalina A/química , Mioglobina/química , Proteínas/química , Papaína/química , Papaína/metabolismo , Péptidos/química , Electrones , Elastasa Pancreática/química , Elastasa Pancreática/metabolismo , Estructura Secundaria de ProteínaRESUMEN
As a clinical anti-glioma agent, the therapeutic effect of carmustine (BCNU) was largely decreased because of the drug resistance mediated by O6-alkylguanine-DNA alkyltransferase (AGT) and the blood-brain barrier (BBB). To overcome these obstacles, we synthesized a BCNU-loaded hypoxia/esterase dual stimulus-activated nanomicelle, abbreviated as T80-HACB/BCNU NPs. In this nano-system, Tween 80 acts as the functional coating on the surface of the micelle to facilitate transport across the BBB. Hyaluronic acid (HA) with active tumor-targeting capability was linked with the hypoxia-sensitive AGT inhibitors (O6-azobenzyloxycarbonyl group) via an esterase-activated ester bond. The obtained T80-HACB/BCNU NPs had an average particle size of 232.10 ± 10.66 nm, the zeta potential of -18.13 ± 0.91 mV, and it showed high drug loading capacity, eximious biocompatibility and dual activation of hypoxia/esterase drug release behavior. The obtained T80-HACB/BCNU NPs showed enhanced cytotoxicity against hypoxic T98G and SF763 cells with IC50 at 132.2 µM and 133.1 µM, respectively. T80 modification improved the transportation of the micelle across an in vitro BBB model. The transport rate of the T80-HACB/Cou6 NPs group was 12.37 %, which was 7.6-fold (p<0.001) higher than the micelle without T80 modification. T80-HACB/BCNU NPs will contribute to the development of novel CENUs chemotherapies with high efficacy.
Asunto(s)
Antineoplásicos Alquilantes , Carmustina , Hipoxia de la Célula , Nanopartículas , Elastasa Pancreática , Polisorbatos , Polisorbatos/química , Micelas , Elastasa Pancreática/química , Elastasa Pancreática/metabolismo , Carmustina/síntesis química , Carmustina/química , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacología , Nanopartículas/química , Nanopartículas/toxicidad , Ácido Hialurónico/química , Humanos , Línea Celular Tumoral , Dextranos/química , Sistemas de Liberación de Medicamentos , Apoptosis/efectos de los fármacosRESUMEN
INTRODUCTION: Quantitative fecal fat estimation is the gold standard test to diagnose steatorrhea (fecal fat >7 g/day) in chronic pancreatitis (CP), but cumbersome and inconvenient. So, fecal elastase-1 (FE) is proposed as a good alternative but the data on the diagnostic utility of FE to diagnose steatorrhea is variable. METHODS: This retrospective study included adult CP patients evaluated with both 24-h fecal-fat and FE tests within a 3-month period. The objective was to evaluate the diagnostic performance of FE to diagnose steatorrhea and to evaluate the FE progression over 9-month period. RESULTS: Among the 147 included patients, the frequency of steatorrhea (fecal fat >7 g/day) was 34%. The sensitivity, specificity, and negative likelihood ratio (LR) of FE was 90%, 28.9% and 0.35 at cut-off of <100 µg/g stool to diagnose steatorrhea; and 96%, 11.3% and 0.35 at cut-off of <200 µg/g stool, respectively. The optimal cut-off of FE was <20 on receiver operating characteristic curve (sensitivity 66%; specificity 69%; positive LR 2.14). There was no statistically significant variation in FE levels over 9 months interval among a hundred patients. CONCLUSION: Compared to FE ≥ 200 µg/g stool, FE ≥ 100 can used to exclude steatorrhea (better specificity and negative LR). FE < 20 alone cannot replace fecal fat estimation to confirm steatorrhea but to be interpreted with clinical features. Repeat FE testing for exocrine insufficiency progression can be done at least a year later.
Asunto(s)
Insuficiencia Pancreática Exocrina , Elastasa Pancreática , Pancreatitis Crónica , Adulto , Humanos , Insuficiencia Pancreática Exocrina/diagnóstico , Heces , Elastasa Pancreática/química , Pancreatitis Crónica/complicaciones , Estudios Retrospectivos , Esteatorrea/diagnósticoRESUMEN
In order for the intestinal mucosa to absorb dietary proteins they have to be digested into single amino acids or very short peptides of a length of not more than four amino acids. In order to study the efficiency of the digestive endopeptidases to digest folded proteins we have analyzed several target proteins under different conditions, native proteins, heat denatured and acid treated. The three pancreatic serine proteases, trypsin, chymotrypsin, and pancreatic elastase, were found to be remarkable inefficient in cleaving native folded proteins whereas pepsin, which acts at a very low pH (pH 1.2) was much more efficient, possibly due to the denaturing conditions and thereby better accessibility to internal cleavage sites at the low pH. Heat treatment improved the cleavage considerably by all three pancreatic enzymes, but acid treatment followed by return to neutral pH did not have any major effect. Cleavage at the low pH when the protein is in a denatured state, is apparently very efficient. This indicates that pepsin is the prime enzyme cleaving the properly folded native proteins and that the pancreatic enzymes primarily are involved in generating single amino acids or very short peptides for efficient uptake by the intestinal mucosa.
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Quimotripsina/química , Elastasa Pancreática/química , Pepsina A/química , Tripsina/química , Animales , Bovinos , Quimotripsina/metabolismo , Mucosa Gástrica/enzimología , Páncreas/enzimología , Elastasa Pancreática/metabolismo , Pepsina A/metabolismo , Pliegue de Proteína , Porcinos , Tripsina/metabolismoRESUMEN
In this study, a fluorescence detection strategy is reported for the peptidase activity assay, which is based on fluorescence resonance energy transfer (FRET) from a fluorophore-labelled peptide to graphene oxide (GO). By the hydrolysis of the peptide, the fluorophore-labelled peptide releases the fluorophore 5-carboxyfluorescein, which can avoid quenching from GO. Thus, the increased intensity of the obtained fluorescence signal in the assay is directly dependent on the peptidase activity. As a model case of the developed strategy, the activity determination of pancreatic elastase (PE) is performed. Under the optimal experimental conditions at an excitation wavelength of 494 nm, the activity of PE can be determined in the range from 0.003 to 0.10 U/mL, with a detection limit of 0.001 U/mL at the emission wavelength of 518 nm. This is ultra-sensitive for the determination of PE. The specificity of the method is demonstrated by the analysis of PE under complex conditions using fetal bovine serum as the substrate. Hence, the developed method might provide an intrinsically convenient, sensitive platform for the PE activity assay and related biochemical studies due to its homogeneous, and fluorescence-based detection strategy.
Asunto(s)
Fluoresceínas/química , Transferencia Resonante de Energía de Fluorescencia , Grafito/química , Elastasa Pancreática/química , Péptidos/química , Animales , PorcinosRESUMEN
The biological properties of elastase and Aspergillus flavus elastase inhibitor (AFLEI) from A. flavus were examined. Pathogenicity of elastase was investigated in mice immunocompromised with cyclophosphamide, cyclosporine, prednisolone and carrageenan. Compared to cyclophosphamide immunocompromised mice treated with the spores of elastase nonproducing strain, cyclophosphamide immunocompromised mice treated with the spores of elastase producing strain had a significantly shorter survival rate. Molecular mass of AFLEI was determined to be 7525.8 Da. The elastolytic activity of elastases from A. flavus, and human leukocytes were inhibited by AFLEI. The primary structure of AFLEI was determined by the Edman sequencing procedure. The search for amino acid homology with other proteins demonstrated that amino acid residues 1 to 68 of AFLEI are 100% identical to residues 20 to 87 of the hypothetical protein AFUA_3G14940 of A. fumigatus. When immunocompromised mice administered of cyclophosphamide were infected by inhalation of A. flavus then administered amphotericin B (AMPH) alone or in combination with AFLEI, survival rate tended to be higher with combination treatment than with AMPH alone. Moreover, although extensive bleeding was seen in pathology sections taken from rat lung resected 24 h after elastase was administered to the lung via the bronchus, this bleeding was inhibited by AFLEI. The X-ray analysis has revealed that the structure of this inhibitor was wedge shaped and composed of a binding loop and a scaffold protein core. As synthetic-inhibitor strongly inhibited cytotoxicity induced by elastase in human-derived cells, it could prove beneficial for the treatment of pulmonary aspergillosis.
Asunto(s)
Aspergillus flavus/química , Aspergillus flavus/patogenicidad , Inhibidores Enzimáticos/farmacología , Elastasa Pancreática/efectos adversos , Anfotericina B/administración & dosificación , Animales , Aspergillus flavus/enzimología , Aspergillus flavus/genética , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Hemorragia/tratamiento farmacológico , Hemorragia/etiología , Humanos , Huésped Inmunocomprometido , Enfermedades Pulmonares/tratamiento farmacológico , Enfermedades Pulmonares/etiología , Ratones , Elastasa Pancreática/química , Elastasa Pancreática/aislamiento & purificación , Aspergilosis Pulmonar/tratamiento farmacológico , RatasRESUMEN
Chia (Salvia hispanica) seed peptides have drawn attention because of their antioxidant, antihypertensive and anti-inflammatory activities, making them ideal candidates for development of cosmeceutical skin products. However, there are no preceding reports that address their aging-related enzyme inhibitory activities. The aim of this study was to investigate the in vitro and in silico inhibitory activity of chia seed peptides towards the main aging-related enzymes. Enzyme-inhibition activity of < 3 kDa chia seed peptides towards collagenase, hyaluronidase, tyrosinase, and elastase was evaluated. Further fractions were obtained by size exclusion chromatography (SEC) and re-tested for enzyme inhibitory activity. Peptide sequences were identified from the most effective fraction and used for in silico analysis. The < 3 kDa peptides exhibited inhibitory activities towards elastase (65.32%, IC50 = 0.43 mg/mL), tyrosinase (58.74%, IC50 = 0.66 mg/mL), hyaluronidase (26.96%, IC50 = 1.28 mg/mL), and collagenase (28.90%, IC50 = 1.41 mg/mL). They showed mixed-type inhibition patterns towards elastase and hyaluronidase, while a non-competitive inhibition pattern was observed towards collagenase and tyrosinase. Fraction II obtained by SEC, showed higher enzyme inhibitory activity. Seven peptides were identified in this fraction (APHWYTN, DQNPRSF, GDAHWAY, GDAHWTY, GDAHWVY, GFEWITF, and KKLKRVYV), which according to in silico analysis, possess 19-29 enzyme-peptide pair interactions towards elastase and three peptide sequences shared homology sequence (GDAHW). These results demonstrate that peptides from chia seeds may contribute in the improvement of skin health by offering protection against aging-related enzymes by preventing degradation of the protein matrix on the skin; however, further in vivo studies are needed to evaluate its actual capability.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Elastasa Pancreática/antagonistas & inhibidores , Péptidos/farmacología , Salvia/química , Semillas/química , Envejecimiento de la Piel/efectos de los fármacos , Inhibidores Enzimáticos/química , Hialuronoglucosaminidasa/antagonistas & inhibidores , Hialuronoglucosaminidasa/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz/química , Inhibidores de la Metaloproteinasa de la Matriz/aislamiento & purificación , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Modelos Moleculares , Monofenol Monooxigenasa/antagonistas & inhibidores , Elastasa Pancreática/química , Péptidos/química , Péptidos/aislamiento & purificaciónRESUMEN
BACKGROUND: Although exocrine pancreatic insufficiency (EPI) has been described in patients with neuroendocrine neoplasia (NEN) treated with somatostatin analogs (SSAs), its role in the therapeutic management of these patients is not well established. AIM: To determine the frequency of EPI in patients with NEN long-term treated with SSAs. METHODS: This is a prospective single-center study evaluating 35 patients treated with SSAs for >12 months due to unresectable/advanced nonpancreatic well-differentiated NEN. Clinical evaluation, biochemical parameters, and fecal elastases 1 (FE-1) were assessed to diagnose EPI. RESULTS: A total of 7 patients (20%) had EPI, given the presence of abdominal symptoms and a median FE-1 value of 180 mcg/g stool (150-198). No patient had severe EPI, defined as FE-1 < 100 mcg/g stool. Elevated glycated Hb levels were a significant predictor for developing EPI (OR 4.81, p = 0.01). No significant difference in terms of duration of SSA treatment was observed between patients with or without EPI diagnosed (84 months and 72 months, respectively; p = 0.950). CONCLUSIONS: Mild-moderate EPI is a relatively common condition in patients receiving long-term treatment with SSAs. Specific clinical and biochemical evaluations, including FE-1, should be planned in these patients to diagnose this relevant condition early, which may deteriorate quality of life and cause malnutrition.
Asunto(s)
Antineoplásicos Hormonales/uso terapéutico , Insuficiencia Pancreática Exocrina/epidemiología , Insuficiencia Pancreática Exocrina/etiología , Tumores Neuroendocrinos/complicaciones , Tumores Neuroendocrinos/tratamiento farmacológico , Somatostatina/análogos & derivados , Somatostatina/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Heces/química , Femenino , Hemoglobina Glucada/análisis , Humanos , Incidencia , Masculino , Desnutrición/etiología , Persona de Mediana Edad , Elastasa Pancreática/química , Valor Predictivo de las Pruebas , Estudios ProspectivosRESUMEN
Elastase-1 is one member of serine protease family, distributes in organisms widely and plays a crucial role in the invasion and development of Trichinella spiralis. In order to identify the binding of T. spiralis elastase-1 (TsEla) with host's intestinal epithelial cells (IECs) and its role in Trichinella larval intrusion, TsEla gene was cloned and expressed in our previous study. The recombinant TsEla (rTsEla) has the enzymatic activity to degrade specific peptide substrate. A specific binding between rTsEla and IECs was detected by Far Western blot and ELISA. In an in vitro invasion assay, rTsEla promoted the larval intrusion, whereas anti-rTsEla serum inhibited the larval penetration. The larval intrusion was also suppressed after the silencing of TsEla by siRNA. Silencing of TsEla gene by siRNA-291 meditated RNA interference suppressed TsEla protein expression, reduced the worm infectivity, development and reproductive capacity. These results indicated that TsEla plays an important role in the T. spiralis intrusion of host's intestinal epithelia, and it could be a prospective vaccine molecular target against T. spiralis infection.
Asunto(s)
Enterocitos/fisiología , Proteínas del Helminto/metabolismo , Mucosa Intestinal/fisiología , Elastasa Pancreática/metabolismo , Trichinella spiralis/enzimología , Triquinelosis/parasitología , Animales , Enterocitos/inmunología , Células Epiteliales , Regulación Enzimológica de la Expresión Génica , Proteínas del Helminto/inmunología , Intestinos , Larva/fisiología , Ratones , Ratones Endogámicos BALB C , Elastasa Pancreática/química , Elastasa Pancreática/genética , Estudios Prospectivos , Trichinella spiralis/genética , Triquinelosis/inmunologíaRESUMEN
The temporomandibular joint disc is a fibrocartilaginous structure, composed of collagen fibers, elastin fibers, and proteoglycans. Despite the crucial role of elastin fibers in load-bearing properties of connective tissues, its contribution in temporomandibular joint disc biomechanics has been disregarded. This study attempts to characterize the structural-functional contribution of elastin in the temporomandibular joint disc. Using elastase, we selectively perturbed the elastin fiber network in porcine temporomandibular joint discs and investigated the structural, compositional, and mechanical regional changes through: (a) analysis of collagen and elastin fibers by immunolabeling and transmission electron microscopy; (b) quantitative analysis of collagen tortuosity, cell shape, and disc volume; (c) biochemical quantification of collagen, glycosaminoglycan and elastin content; and (d) cyclic compression test. Following elastase treatment, microscopic examination revealed fragmentation of elastin fibers across the temporomandibular joint disc, with a more pronounced effect in the intermediate regions. Also, biochemical analyses of the intermediate regions showed significant depletion of elastin (50%), and substantial decrease in collagen (20%) and glycosaminoglycan (49%) content, likely due to non-specific activity of elastase. Degradation of elastin fibers affected the homeostatic configuration of the disc, reflected in its significant volume enlargement accompanied by remarkable reduction of collagen tortuosity and cell elongation. Mechanically, elastase treatment nearly doubled the maximal energy dissipation across the intermediate regions while the instantaneous modulus was not significantly affected. We conclude that elastin fibers contribute to the restoration and maintenance of the disc resting shape and actively interact with collagen fibers to provide mechanical resilience to the temporomandibular joint disc.
Asunto(s)
Elastasa Pancreática/química , Disco de la Articulación Temporomandibular/anatomía & histología , Animales , Forma de la Célula , Colágeno , Elastina , Glicosaminoglicanos , Fenómenos Mecánicos , Estrés Mecánico , Porcinos , Disco de la Articulación Temporomandibular/química , Disco de la Articulación Temporomandibular/citología , Resistencia a la Tracción , Soporte de PesoRESUMEN
A new series of 4H-chromene-3-carboxylate derivatives were synthesized using multicomponent reaction of salicylaldehyde, ethyl acetoacetate and dimedone in ethanol with K3PO4 as a catalyst at 80 °C. The structures of all newly synthesized compounds were confirmed by spectral techniques viz. IR, 1H NMR, 13C NMR, and LCMS analysis. The newly synthesized compounds 4a to 4j were screened against elastase enzyme. Interestingly, all these compounds found to be potent elastase inhibitors with much lower IC50 value. The compound 4b was found to be most potent elastase inhibitor (IC50 = 0.41 ± 0.01 µM) amongst the synthesized series against standard Oleanolic Acid (IC50 value = 13.45 ± 0.0 µM). The Kinetics mechanism for compound 4b was analyzed by Lineweaver-Burk plots which revealed that compound inhibited elastase competitively by forming an enzyme-inhibitor complex. Along with this, all the synthesized compounds (4a - 4j) exhibits excellent DPPH free radical scavenging ability. The inhibition constant Ki for compound 4b was found to be 0.6 µM. The computational study was comprehensible with the experimental results with good docking energy values (Kcal/mol). Therefore, these molecules can be considered as promising medicinal scaffolds for the treatment of skin-related maladies.
Asunto(s)
Benzopiranos/química , Benzopiranos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Elastasa Pancreática/antagonistas & inhibidores , Animales , Benzopiranos/síntesis química , Ácidos Carboxílicos/síntesis química , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacología , Quimioinformática , Inhibidores Enzimáticos/síntesis química , Simulación del Acoplamiento Molecular , Páncreas/enzimología , Elastasa Pancreática/química , Elastasa Pancreática/metabolismo , PorcinosRESUMEN
Hydrogen bonds are essential for protein structure and function, making experimental access to long-range interactions between amide protons and heteroatoms invaluable. Here we show that measuring distance restraints involving backbone hydrogen atoms and carbonyl- or α-carbons enables the identification of secondary structure elements based on hydrogen bonds, provides long-range contacts and validates spectral assignments. To this end, we apply specifically tailored, proton-detected 3D (H)NCOH and (H)NCAH experiments under fast magic angle spinning (MAS) conditions to microcrystalline samples of SH3 and GB1. We observe through-space, semi-quantitative correlations between protein backbone carbon atoms and multiple amide protons, enabling us to determine hydrogen bonding patterns and thus to identify ß-sheet topologies and α-helices in proteins. Our approach shows the value of fast MAS and suggests new routes in probing both secondary structure and the role of functionally-relevant protons in all targets of solid-state MAS NMR.
Asunto(s)
Enlace de Hidrógeno , Resonancia Magnética Nuclear Biomolecular/métodos , Estructura Secundaria de Proteína , Amiloide/química , Elastasa Pancreática/química , Pliegue de Proteína , Protones , Dominios Homologos srcRESUMEN
Endovascular techniques for treating cerebral aneurysms are rapidly advancing and require testing to optimize device configurations. The purpose of this work was to customize tissue-engineered aneurysm "blood vessel mimics" (aBVMs) for early stage in vitro assessment of vascular cell responses to flow diverters and other devices. Aneurysm scaffolds with varying neck size and height were created through solid modeling, mold fabrication, mandrel creation, and electrospinning. Scaffold dimensions and fiber morphology were characterized. aBVMs were created by depositing human smooth muscle and endothelial cells within scaffolds, and cultivating within perfusion bioreactors. These vessels were left untreated or used for flow diverter implantation. Cellular responses to flow diverters were evaluated at 3 days. Custom scaffolds were created with aneurysm neck diameters of 2.3, 3.5, and 5.5 mm and with aneurysm heights of 2, 5, and 8 mm. A set of scaffolds with varying neck size was used for aBVM creation, and dual-sodding of endothelial and smooth muscle cells resulted in consistent and confluent cellular linings. Flow diverters were successfully implanted in a subset of aBVMs, and initial cell coverage over devices was seen in the parent vessel at 3 days. Direct visualization of the device over the neck region was feasible, supporting the future use of these models for evaluating and comparing flow diverter healing. Tissue-engineered aneurysm models can be created with custom neck sizes and heights, and used to evaluate cellular responses to flow diverters and other endovascular devices.
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Prótesis Vascular , Aneurisma Intracraneal/fisiopatología , Ingeniería de Tejidos/métodos , Andamios del Tejido , Cicatrización de Heridas/fisiología , Animales , Reactores Biológicos , Endotelio Vascular/patología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Modelos Cardiovasculares , Miocitos del Músculo Liso/metabolismo , Elastasa Pancreática/química , Diseño de Prótesis , ConejosRESUMEN
3-Oxo-ß-sultams are four-membered ring ambident electrophiles that can react with nucleophiles either at the carbonyl carbon or at the sulfonyl sulfur atoms, and that have been reported to inhibit serine hydrolases via acylation of the active-site serine residue. We have developed a panel of 3-oxo-ß-sultam inhibitors and show, through crystallographic data, that they are regioselective sulfonylating electrophiles, covalently binding to the catalytic serine of human and porcine elastases through the sulfur atom. Application of 3-oxo-ß-sultam-derived activity-based probes in a human proteome revealed their potential to label disease-related serine hydrolases and proteasome subunits. Activity-based protein profiling applications of 3-oxo-ß-sultams should open up new opportunities to investigate these classes of enzymes in complex proteomes and expand the toolbox of available sulfur-based covalent protein modifiers in chemical biology.
Asunto(s)
Inhibidores Enzimáticos/química , Compuestos Heterocíclicos con 1 Anillo/química , Elastasa Pancreática/antagonistas & inhibidores , Proteoma/química , Sulfonamidas/química , Animales , Línea Celular Tumoral , Teoría Funcional de la Densidad , Células HEK293 , Humanos , Modelos Químicos , Elastasa Pancreática/química , Proteómica/métodos , Serina/química , PorcinosRESUMEN
BACKGROUND: Trichinella spiralis muscle larval (ML) excretion/secretion (ES) antigen is the most widely used diagnostic antigen of trichinellosis, but preparation of ES antigen requires collecting worms from infected animals, and detection of specific IgG against ML ES antigen may result in a false negative at the early stage of infection. The aim of the study was to characterize T. spiralis elastase-1 (TsEla) and to evaluate its potential as diagnostic antigen for trichinellosis. METHODS: The complete cDNA sequences of the TsEla gene were cloned and expressed, and recombinant (rTsEla) was purified. TsEla transcription and expression in different T. spiralis life-cycle stages was investigated by qPCR and western blotting, and its location in the nematodes was evaluated using an immunofluorescence assay (IFA). The antigenicity of rTsEla was investigated by western blotting analysis and ELISA. Anti-Trichinella IgG, IgM and IgE of experimentally infected mice and specific IgG antibodies of trichinellosis patients were assayed by rTsEla-ELISA and ES-ELISA. RESULTS: The results of the qPCR and western blotting showed that TsEla was expressed in various T. spiralis life stages. Natural TsEla was detected in the soluble proteins and ES proteins of different life stages. IFA revealed that TsEla was identified in the whole nematodes of various stages, especially in the cuticle, stichosome and genital primordium of the parasite. Serum anti-Trichinella IgM, IgG and IgE in infected mice was first detected by rTsEla-ELISA at 6, 10 and 12 days post-infection (dpi), and reached 100% at 8, 14 and 14 dpi, respectively. When rTsEla-ELISA and ES-ELISA were used to detect anti-Trichinella IgG in sera of trichinellosis patients, the sensitivity was 97.37% (37/38) and 89.74% (34/38) (P > 0.05), and the specificity was 99.10% (220/222) and 98.20% (218/222), respectively (P > 0.05). The rTsEla cross-reacted with only one serum sample out of 20 samples from paragonimiasis patients and 7 samples from clonorchiasis patients. CONCLUSIONS: rTsEla is valuable to early diagnosis of trichinellosis and could be an alternative diagnostic antigen to the ML ES antigens.
Asunto(s)
Antígenos Helmínticos/genética , Antígenos Helmínticos/inmunología , Elastasa Pancreática/química , Elastasa Pancreática/inmunología , Trichinella spiralis/enzimología , Triquinelosis/diagnóstico , Secuencia de Aminoácidos , Animales , Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/química , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Femenino , Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Humanos , Estadios del Ciclo de Vida , Ratones Endogámicos BALB C , Elastasa Pancreática/genética , Alineación de Secuencia , Trichinella spiralis/genética , Trichinella spiralis/crecimiento & desarrollo , Trichinella spiralis/inmunología , Triquinelosis/sangre , Triquinelosis/inmunología , Triquinelosis/parasitologíaRESUMEN
BACKGROUND: Oleic acid (OA) is reported to show anti-inflammatory activity toward activated neutrophils. It is also an important material in nanoparticles for increased stability and cellular internalization. We aimed to evaluate the anti-inflammatory activity of injectable OA-based nanoparticles for treating lung injury. Different sizes of nanocarriers were prepared to explore the effect of nanoparticulate size on inflammation inhibition. RESULTS: The nanoparticles were fabricated with the mean diameters of 105, 153, and 225 nm. The nanocarriers were ingested by isolated human neutrophils during a 5-min period, with the smaller sizes exhibiting greater uptake. The size reduction led to the decrease of cell viability and the intracellular calcium level. The OA-loaded nanosystems dose-dependently suppressed the superoxide anion and elastase produced by the stimulated neutrophils. The inhibition level was comparable for the nanoparticles of different sizes. In the ex vivo biodistribution study, the pulmonary accumulation of nanoparticles increased following the increase of particle size. The nanocarriers were mainly excreted by the liver and bile clearance. Mice were exposed to intratracheal lipopolysaccharide (LPS) to induce acute respiratory distress syndrome (ARDS), like lung damage. The lipid-based nanocarriers mitigated myeloperoxidase (MPO) and cytokines more effectively as compared to OA solution. The larger nanoparticles displayed greater reduction on MPO, TNF-α, and IL-6 than the smaller ones. The histology confirmed the decreased pulmonary neutrophil recruitment and lung-architecture damage after intravenous administration of larger nanoparticles. CONCLUSIONS: Nanoparticulate size, an essential property governing the anti-inflammatory effect and lung-injury therapy, had different effects on activated neutrophil inhibition and in vivo therapeutic efficacy.
Asunto(s)
Antiinflamatorios/química , Lípidos/química , Nanocápsulas/química , Neutrófilos/efectos de los fármacos , Ácido Oléico/química , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Animales , Antiinflamatorios/farmacología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Liberación de Fármacos , Humanos , Lipopolisacáridos/química , Pulmón , Ratones , Ratones Endogámicos C57BL , Activación Neutrófila/efectos de los fármacos , Elastasa Pancreática/química , Tamaño de la Partícula , Peroxidasa/metabolismo , Superóxidos/química , Propiedades de Superficie , Distribución Tisular , Resultado del TratamientoRESUMEN
We found an elastolytic activity in the culture supernatant of Streptomyces sp. P-3, and the corresponding enzyme (streptomycetes elastase, SEL) was purified to apparent homogeneity from the culture supernatant. The molecular mass of purified SEL was approximately 18 kDa as judged by SDS-PAGE analysis and gel-filtration chromatography. Utilizing information from N-terminal amino acid sequencing of SEL and mass spectrometry of SEL tryptic fragments, we succeeded in cloning the gene-encoding SEL. The cloned SEL gene contains a 726 bp ORF, which encodes a 241 amino acid polypeptide containing a putative signal peptide for secretion (28 amino acid) and pro-sequence (14 amino acid). Although the deduced primary structure of SEL has sequence similarity to proteins in the S1 protease family, the amino acid sequence shares low identity (< 31.5 %) with any known elastase. SEL efficiently hydrolyses synthetic peptides having Ala or Val in the P1 position such as N-succinyl-Ala-Ala-(Pro or Val)-Ala-p-nitroanilide (pNA), whereas reported proteases by streptomycetes having elastolytic activity prefer large residues, such as Phe and Leu. Compared of kcat/Km ratios for Suc-Ala-Ala-Val-Ala-pNA and Suc-Ala-Ala-Pro-Ala-pNA with subtilisin YaB, which has high elastolytic activity, Streptomyces sp. P-3 SEL exhibits 12- and 121-fold higher, respectively. Phylogenetic analyses indicate that the predicted SEL protein, together with predicted proteins in streptomycetes, constitutes a novel group within the S1 serine protease family. These characteristics suggest that SEL-like proteins are new members of the S1 serine protease family, which display elastolytic activity.
Asunto(s)
Elastasa Pancreática , Serina Proteasas , Streptomyces/enzimología , Genes Bacterianos , Elastasa Pancreática/biosíntesis , Elastasa Pancreática/química , Elastasa Pancreática/genética , Elastasa Pancreática/aislamiento & purificación , Filogenia , Serina Proteasas/biosíntesis , Serina Proteasas/química , Serina Proteasas/genética , Serina Proteasas/aislamiento & purificaciónRESUMEN
The response of porcine pancreatic elastase (PPE) to propanol was examined by various techniques including UV-vis spectrophotometry, spectrofluorometry and circular dichroism, as well as molecular docking and molecular simulation. These techniques were used to investigate the structural changes and elastase activity in the presence of propanol. This work was performed at three temperatures of 303, 313 and 323 K, with the pH value of 8.5 (Tris buffer). The results of the UV-vis spectrophotometry indicated the transfer of tryptophan to an environment with low hydrophobicity. Fluorescence measurements also revealed the quenching of fluorescence intensity was induced by propanol, and dynamic quenching was the proposed quenching mechanism. Kinetic studies also suggested the inhibitory effect (noncompetitive) of propanol on elastase. Further, Circular Dichroism (CD) spectra showed that propanol caused slight alterations in the secondary structures of PPE (0.3% increase for the α-helix and 0.5% decrease for the ß-sheet). Addition of propanol decreased the Tm (Melting Temperature) parameter from 332.8 K to 330.1 K.
Asunto(s)
1-Propanol/química , 1-Propanol/farmacología , Simulación del Acoplamiento Molecular/métodos , Elastasa Pancreática/química , Elastasa Pancreática/efectos de los fármacos , Elastasa Pancreática/metabolismo , Animales , Fenómenos Químicos , Dicroismo Circular , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Desnaturalización Proteica/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Espectrometría de Fluorescencia , Porcinos , Temperatura , Triptófano/metabolismoRESUMEN
The aim of this study was to investigate the spermine effect on the thermal denaturation, conformation and activity of elastase at three temperatures of 303, 313 and 323 K in the Tris buffer, at pH 8.5, using UV-vis spectrophotometry, spectrofluorometry and circular dichroism as well as molecular docking and molecular simulation. The increased absorption of elastase in the presence of spermine suggested a change in the environment of tryptophan. It was found that under the influence of spermine, the emission intensity of elastase extremely was reduced, and the use of the Stern-Volmer equation showed that some static quenching had occurred. The thermodynamic parameters values (enthalpy and entropy) and the molecular docking technique also revealed that van der Waals forces or hydrogen bonding interactions played an important role in the binding process. The spermine-elastase complex formation led to increasing the value of the catalytic constant (kcat). So it could be considered as an activator. Slight changes were observed in the second structure of elastase (1.06% increase for the α-helix and 0.048% decrease the ß-sheet) and the thermal stability effect. Molecular docking results also demonstrated that spermine could bind to porcine pancreatic elastase, and van der Waals forces or hydrogen bonding interactions played the major role in the binding process. Overall, our results showed that spermine could induce structural alterations in elastase, acting as a partial stabilizer and an activator for the enzyme.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Elastasa Pancreática/química , Espermina/química , Algoritmos , Animales , Catálisis , Conformación Molecular , Estructura Molecular , Unión Proteica , Análisis Espectral , Espermina/farmacología , Porcinos , TermodinámicaRESUMEN
Cold-adapted enzymes from psychrophilic species achieve their high catalytic efficiency at low temperature by a different partitioning of the activation free energy into its enthalpic and entropic components, compared to orthologous mesophilic enzymes. Their lower activation enthalpy, partly compensated by an increased entropic penalty, has been suggested to originate from changes in flexibility of the protein surface. Multiple sequence alignments of psychrophilic and mesophilic enzymes also show characteristic motifs located in surface loops of the protein. Here, we use computer simulations to examine the effects of a number of designed surface mutations of psychrophilic and mesophilic elastases on the temperature dependence of the catalyzed peptide cleavage reaction. For each of 14 mutant enzyme variants we report calculations of their thermodynamic activation parameters. The results show that substitution of psychrophilic loop residues into the mesophilic enzyme consistently changes both the activation parameters and loop flexibilities towards the former, and vice versa for opposite substitutions.