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To investigate the effects of Luhong Yixin Granules on myocardial fibrosis in rats with heart failure and its possible mechanism, a total of 60 male Wistar rats were randomly divided into the control group, model group, and low-, medium-and high-dose Luhong Yixin Granules groups, with 12 rats in each group. Except for those in the control group, rats in the other groups were induced by intraperitoneal injection of doxorubicin(DOX) into a rat model. After the Luhong Yixin Granules were dissolved in the same amount of normal saline, they were given by gavage at low, medium and high doses(2.8, 5.6, 11.2 g·kg~(-1)·d~(-1)), and the control group and the model group were given the same amount of normal saline by gavage for 40 days. After the end of dosing, echocardiography was used to measure left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS). Rat body weight(BW) and heart weight(HW) were calculated as HW/BW. Enzyme-linked immunosorbent assay was used to measure the levels of interleukin-6(IL-6), interleukin-17(IL-17), tumor necrosis factor-α(TNF-α), transforming growth factor-ß1(TGF-ß1), growth stimulation expressed gene 2 protein(ST2), N-terminal pro-B-type natriuretic peptide(NT-proBNP), galectin-3(Gal-3) and creatine kinase isoenzyme(CK-MB) in serum. Hematoxylin-eosin(HE) staining and Masson staining were used to observe the pathological morphology of myocardial tissue. Western blot and quantitative real-time polymerase chain reaction were used to detect the protein and mRNA expression levels of IL-6, IL-17, TNF-α, TGF-ß1, Smad3, Smad7, α-smooth muscle actin(α-SMA), and collagen â (COL-â ), respectively. RESULTS:: showed that compared with those in the control group, LVEF, LVFS, and HW/BW in the model group were decreased(P<0.05), and the levels of IL-6, IL-17, TNF-α, TGF-ß1, ST2, NT-proBNP, Gal-3, and CK-MB were increased(P<0.05). HE staining showed inflammatory changes in myocardial tissue; Masson staining showed decreases in the cross-sectional area and ventricular cavity area of the heart, and myocardial fibrosis of varying degrees(P<0.05). The protein and mRNA expression of IL-6, IL-17, TNF-α, TGF-ß1, Smad3, α-SMA, and COL-â were increased(P<0.05), and the protein and mRNA expression of Smad7 protein was decreased(P<0.01). Compared with those in the model group, LVEF, LVFS and HW/BW of the low-, medium-and high-dose Luhong Yixin Granules groups were increased(P<0.05), and the levels of IL-6, IL-17, TNF-α, TGF-ß1, ST2, NT-proBNP, Gal-3 and CK-MB were decreased(P<0.05). HE staining showed gradually reduced inflammatory changes of myocardial tissue, and Masson staining showed increased cross-sectional area and ventricular cavity area of the heart and decreased area of myocardial fibrosis(P<0.05). The protein and mRNA expression levels of IL-6, IL-17, TNF-α, TGF-ß1, Smad3, α-SMA, and COL-â were decreased(P<0.05), while the protein and mRNA expression levels of Smad7 were increased(P<0.05). Luhong Yixin Granules may be of great value in the treatment of heart failure by regulating the TGF-ß1/Smads signaling pathway, inhibiting the expression of inflammation-related proteins, reducing the deposition of extracellular matrix, and alleviating myocardial fibrosis.
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Medicamentos de Ervas Chinesas , Fibrose , Insuficiência Cardíaca , Miocárdio , Ratos Wistar , Transdução de Sinais , Proteínas Smad , Fator de Crescimento Transformador beta1 , Animais , Masculino , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/genética , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/farmacologia , Ratos , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Transdução de Sinais/efeitos dos fármacos , Miocárdio/patologia , Miocárdio/metabolismo , Proteínas Smad/metabolismo , Proteínas Smad/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , HumanosRESUMO
Polysaccharides and flavonoids have excellent antioxidant properties and tyrosinase inhibitory effects. In this paper, the antioxidant capacity of Inonotus sanghuang extract and its inhibition kinetics on mushroom tyrosinase were investigated to determine the preparation process of Inonotus sanghuang primary whitening gel. By conducting experimental studies on the effects of water extract and alcohol extract of Inonotus sanghuang on antioxidant capacity and tyrosinase activity, their inhibitory ability and types of inhibitory effects were determined. The single factor experiment was used to determine the preparation process of Sanghuang primary whitening gel. This study has proven that the extract of Inonotus sanghuang possesses antioxidant and tyrosinase inhibitory capabilities. It also identified the preparation process for the primary whitening gel of Inonotus sanghuang, thereby providing a theoretical and experimental basis for the development of whitening products utilising Inonotus sanghuang.
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The electrochemical nitrate reduction reaction (ENO3RR) is a green ammonia synthesis method under ambient conditions relative to the traditional Haber-Bosch technology, which does not require high-temperature or high-pressure conditions and can convert nitrate pollutants in the environment into value-added NH3, thus achieving a dual purpose. However, more electrocatalysts with a remarkable performance towards high-efficiency ENO3RR need to be developed. In this work, a Cu/NiO-NF composite electrocatalyst with a nanorod structure on nickel foam was successfully fabricated, which contains heterogeneous interfaces between Cu and NiO toward selective electrocatalytic nitrate reduction for ammonia synthesis. The steric nanorod morphology of the catalyst can significantly increase the surface area, expose more active sites, and improve the reaction activity. Moreover, the construction of the composite and the interface effectively boosts the synergistic effect of the active species Cu and NiO, which can regulate the electronic structure of the catalyst, expose more active sites, enhance the conductivity of the material, and accelerate the interfacial electron transfer, thereby further promoting the ENO3RR performance. This Cu/NiO-NF composite exhibits a high NH3 yield of 0.6 mmol h-1 cm-2 and up to 97.81% faradaic efficiency at the optimal applied potential of -1.0 V (vs. RHE) in a concentration of 0.1 M NO3--containing 0.1 M PBS. Furthermore, it demonstrates excellent electrochemical cycle stability. This work provides insights into the rational design and fabrication of ENO3RR electrocatalysts for potential electrocatalytic applications.
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The beneficial microorganisms in food are diverse and complex in structure. These beneficial microorganisms can produce different and unique flavors in the process of food fermentation. The unique flavor of these fermented foods is mainly produced by different raw and auxiliary materials, fermentation technology, and the accumulation of flavor substances by dominant microorganisms during fermentation. The succession and metabolic accumulation of microbial flora significantly impacts the distinctive flavor of fermented foods. The investigation of the role of microbial flora changes in the production of flavor substances during fermentation can reveal the potential connection between microbial flora succession and the formation of key flavor compounds. This paper reviewed the evolution of microbial flora structure as food fermented and the key volatile compounds that contribute to flavor in the food system and their potential relationship. Further, it was a certain guiding significance for food industrial production.
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The release of flavor compounds is a critical factor that influences the quality of fermented foods. A recent study investigated the interactions between four fermentation-stinky compounds (indole, isovaleric acid, dimethyl disulfide, and dibutyl phthalate) and myofibrillar proteins (MPs). The results indicated that all four fermentation-stinky compounds had different degrees of binding to MPs, with dibutyl phthalate and dimethyl disulfide exhibiting stronger interactions. Reduced hydrophobicity enhanced these interactions. Multi-spectroscopy showed that static fluorescence quenching was dominant in the MPs-fermentation-stinky compound complexes. The interaction altered the secondary structure of MPs, predominantly transitioning from ß-sheets to α-helix or random coil structures via hydrogen bond interactions. Molecular docking confirmed that these complexes maintained steady states due to stronger hydrogen bonds, van der Waals forces, ionic bonds, conjugate systems, and lower hydrophobicity interactions. Hence, it is a novel sight that the addition of hydrophobic bond-disrupting agents could improve the flavor of fermented foods.
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CONTEXT: Leukaemia has become a serious threat to human health. Although tyrosine kinase inhibitors (TKIs) have been developed as targets for the remedy of leukaemia, drug resistance occurs. Research demonstrated that the simultaneous targeting of sphingosine kinase 1 (Sphk1) and Sirtuin 1 (Sirt1) can downregulate myeloid cell leukaemia-1 (MCL-1), overcome the resistance of tyrosine kinase inhibitors, and play a synergistic inhibitory impact on leukaemia treatment. METHODS: In this study, virtual screening of 7.06 million small molecules was done by sphingosine kinase 1 and Sirtuin 1 pharmacophore models using Schrödinger version 2019; after that, ADME and Toxicity molecule properties were predicted using Discovery Studio. Molecular docking using Schrödinger selected five molecules, which have the best binding affinity with sphingosine kinase 1 and Sirtuin 1. The five molecules and reference inhibitors were constructed with a total of 12 systems with GROMACS that carried out 100 ns molecular dynamics simulation and molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) calculation. Due to compound 3 has the lowest binding energy, its structure was modified. A series of compounds docked with sphingosine kinase 1 and Sirtuin 1, respectively. Among them, QST-LC03, QST-LD05, QST-LE03, and QST-LE04 have the better binding affinity than reference inhibitors. Moreover, the SwissADME and PASS platforms predict that 1, 3, QST-LC03, and QST-LE04 have further study value.
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Leucemia , Sirtuína 1 , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica MolecularRESUMO
While the rupture rate of cerebral aneurysms is only 1% per year, ruptured aneurysms are associated with significant morbidity and mortality, while aneurysm treatments have their own associated risk of morbidity and mortality. Conventional markers for aneurysm rupture include patient-specific and aneurysm-specific characteristics, with the development of scoring systems to better assess rupture risk. These scores, however, rely heavily on aneurysm size, and their accuracy in assessing risk in smaller aneurysms is limited. While the individual risk of rupture of small aneurysms is low, due to their sheer number, the largest proportion of ruptured aneurysms are small aneurysms. Conventional imaging techniques are valuable in characterizing aneurysm morphology; however, advanced imaging techniques assessing the presence of inflammatory changes within the aneurysm wall, hemodynamic characteristics of blood flow within aneurysm sacs, and imaging visualization of irregular aneurysm wall motion have been used to further determine aneurysm instability that otherwise cannot be characterized by conventional imaging techniques. The current manuscript reviews conventional imaging techniques and their value and limitations in cerebral aneurysm characterization, and evaluates the applications, value and limitations of advanced aneurysm imaging and post-processing techniques including intracranial vessel wall MRA, 4D-flow, 4D-CTA, and computational fluid dynamic simulations.
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Aneurisma Roto , Aneurisma Intracraniano , Humanos , Aneurisma Intracraniano/diagnóstico por imagem , Aneurisma Intracraniano/complicações , Angiografia Cerebral/métodos , Aneurisma Roto/diagnóstico por imagem , Aneurisma Roto/complicações , Hemodinâmica/fisiologiaRESUMO
Triple-negative breast cancer (TNBC) and HER2-positive breast cancer are particularly aggressive and the effectiveness of current therapies for them is limited. TNBC lacks effective therapies and HER2-positive cancer is often resistant to HER2-targeted drugs after an initial response. The recent studies have demonstrated that the combination of JAK2 inhibitors and SMO inhibitors can effectively inhibit the growth and metastasis of TNBC and HER2-positive drug resistant breast cancer cells. In this study, deep reinforcement learning was used to learn the characteristics of existing small molecule inhibitors of JAK2 and SMO, and to generate a novel library of small molecule compounds that may be able to inhibit both JAK2 and SMO. Subsequently, the molecule library was screened by molecular docking and a total of 7 compounds were selected out as dual inhibitors of JAK2 and SMO. Molecular dynamics simulations and binding free energies showed that the top three compounds stably bound to both JAK2 and SMO proteins. The binding free energies and hydrogen bond occupancy of key amino acids indicate that A8976 and A10625 has good properties and could be a potential dual-target inhibitor of JAK2 and SMO.
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Inibidores de Janus Quinases , Neoplasias de Mama Triplo Negativas , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neoplasias de Mama Triplo Negativas/patologia , Receptor Smoothened , Janus Quinase 2/metabolismoRESUMO
Background: Since December 2019, SARS-CoV-2 has continued to spread rapidly around the world. The effective drugs may provide a long-term strategy to combat this virus. The main protease (Mpro) and papain-like protease (PLpro) are two important targets for the inhibition of SARS-CoV-2 virus replication and proliferation. Materials & methods: In this study, deep reinforcement learning, covalent docking and molecular dynamics simulations were used to identify novel compounds that have the potential to inhibit both Mpro and PLpro. Results & conclusion: Three compounds were identified that can effectively occupy the Mpro protein cavity with the PLpro protein cavity and form high-frequency contacts with key amino acid residues (Mpro: His41, Cys145, Glu166; PLpro: Cys111). These three compounds can be further investigated as potential lead compounds for SARS-CoV-2 inhibitors.
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Antivirais/farmacologia , Aprendizado Profundo , Avaliação Pré-Clínica de Medicamentos , SARS-CoV-2/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/farmacologiaRESUMO
Predator-prey interactions play important roles in the cycling of marine organic matter. Here we show that a Gram-negative bacterium isolated from marine sediments (Pseudoalteromonas sp. strain CF6-2) can kill Gram-positive bacteria of diverse peptidoglycan (PG) chemotypes by secreting the metalloprotease pseudoalterin. Secretion of the enzyme requires a Type II secretion system. Pseudoalterin binds to the glycan strands of Gram positive bacterial PG and degrades the PG peptide chains, leading to cell death. The released nutrients, including PG-derived D-amino acids, can then be utilized by strain CF6-2 for growth. Pseudoalterin synthesis is induced by PG degradation products such as glycine and glycine-rich oligopeptides. Genes encoding putative pseudoalterin-like proteins are found in many other marine bacteria. This study reveals a new microbial interaction in the ocean.
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Proteínas de Bactérias/metabolismo , Sedimentos Geológicos/microbiologia , Bactérias Gram-Positivas/fisiologia , Interações Microbianas/fisiologia , Pseudoalteromonas/fisiologia , Alanina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Ácido Glutâmico/metabolismo , Metaloproteases/química , Metaloproteases/genética , Metaloproteases/metabolismo , Simulação de Acoplamento Molecular , Mutação , Peptidoglicano/metabolismo , Água do Mar/microbiologia , Staphylococcus/fisiologia , Sistemas de Secreção Tipo II/metabolismoRESUMO
Chiral solid membranes of cellulose, sodium alginate, and hydroxypropyl-ß-cyclodextrin were prepared for chiral dialysis separations. After optimizing the membrane material concentrations, the membrane preparation conditions and the feed concentrations, enantiomeric excesses of 89.1%, 42.6%, and 59.1% were obtained for mandelic acid on the cellulose membrane, p-hydroxy phenylglycine on the sodium alginate membrane, and p-hydroxy phenylglycine on the hydroxypropyl-ß-cyclodextrin membrane, respectively. To study the optical resolution mechanism, chiral discrimination by membrane adsorption, solid phase extraction, membrane chromatography, high-pressure liquid chromatography ultrafiltration were performed. All of the experimental results showed that the first adsorbed enantiomer was not the enantiomer that first permeated the membrane. The crystal structures of mandelic acid and p-hydroxy phenylglycine are the racematic compounds. We suggest that the chiral separation mechanism of the solid membrane is "adsorption - association - diffusion," which is able to explain the optical resolution of the enantioselective membrane. This is also the first report in which solid membranes of sodium alginate and hydroxypropyl-ß-cyclodextrin were used in the chiral separation of p-hydroxy phenylglycine.
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2-Hidroxipropil-beta-Ciclodextrina/química , Alginatos/química , Celulose/química , Membranas Artificiais , Fenômenos Ópticos , Cromatografia Líquida de Alta Pressão , Ácido Glucurônico/química , Glicina/análogos & derivados , Glicina/química , Glicina/isolamento & purificação , Ácidos Hexurônicos/química , EstereoisomerismoRESUMO
Pseudolysin is the most abundant protease secreted by Pseudomonas aeruginosa and is the major extracellular virulence factor of this opportunistic human pathogen. Pseudolysin destroys human tissues by solubilizing elastin. However, the mechanisms by which pseudolysin binds to and degrades elastin remain elusive. In this study, we investigated the mechanism of action of pseudolysin on elastin binding and degradation by biochemical assay, microscopy and site-directed mutagenesis. Pseudolysin bound to bovine elastin fibers and preferred to attack peptide bonds with hydrophobic residues at the P1 and P1' positions in the hydrophobic domains of elastin. The time-course degradation processes of both bovine elastin fibers and cross-linked human tropoelastin by pseudolysin were further investigated by microscopy. Altogether, the results indicate that elastin degradation by pseudolysin began with the hydrophobic domains on the fiber surface, followed by the progressive disassembly of macroscopic elastin fibers into primary structural elements. Moreover, our site-directed mutational results indicate that five hydrophobic residues in the S1-S1' sub-sites played key roles in the binding of pseudolysin to elastin. This study sheds lights on the pathogenesis of P. aeruginosa infection.
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Proteínas de Bactérias/metabolismo , Elastina/metabolismo , Metaloendopeptidases/metabolismo , Pseudomonas aeruginosa/metabolismo , Fatores de Virulência/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Biocatálise , Bovinos , Elastina/química , Elastina/genética , Humanos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Metaloendopeptidases/química , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Tropoelastina/genética , Tropoelastina/metabolismoRESUMO
Elastases have been widely studied because of their important uses as medicine and meat tenderizers. However, there are relatively few studies on marine elastases. Myroilysin, secreted by Myroides profundi D25 from deep-sea sediment, is a novel elastase. In this study, we examined the elastin degradation mechanism of myroilysin. When mixed with insoluble bovine elastin, myroilysin bound hydrophobically, suggesting that this elastase may interact with the hydrophobic domains of elastin. Consistent with this, analysis of the cleavage pattern of myroilysin on bovine elastin and recombinant tropoelastin revealed that myroilysin preferentially cleaves peptide bonds with hydrophobic residues at the P1 and/or P1' positions. Scanning electron microscopy (SEM) of cross-linked recombinant tropoelastin degraded by myroilysin showed preferential damages of spherules over cross-links, as expected for a hydrophobic preference. The degradation process of myroilysin on bovine elastin fibres was followed by light microscopy and SEM, revealing that degradation begins with the formation of crevices and cavities at the fibre surface, with these openings increasing in number and size until the fibre breaks into small pieces, which are subsequently fragmented. Our results are helpful for developing biotechnological applications for myroilysin.
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Elastina/metabolismo , Flavobacteriaceae/enzimologia , Sedimentos Geológicos/microbiologia , Metaloproteases/metabolismo , Animais , Bovinos , Interações Hidrofóbicas e Hidrofílicas , Metaloproteases/isolamento & purificação , Microscopia Eletrônica de Varredura , Tropoelastina/metabolismoRESUMO
BACKGROUND: The H7N9 strain of bird flu is a new type of avian flu that was identified at the end of March 2013. The disease is concerning because most patients have become severely ill. PURPOSE: To study the X-ray and computed tomography (CT) findings of early H7N9 avian influenza cases. MATERIAL AND METHODS: Chest radiography and CT were performed in six patients with H7N9 avian influenza within 1-20 days after onset. The CT examinations included conventional spiral CT and high-resolution CT. The findings on the radiography and CT images were analyzed. RESULTS: Abnormal X-ray and CT findings were present in all of the patients. All of the cases had acute onset. In the early stage, the right lung was more commonly affected (particularly in the right upper and middle lobes). The lesions rapidly expanded to the entire lungs and were characterized primarily by ground-glass opacities (GGOs) combined with consolidation. Diffuse GGO was observed in all six cases (1 was symmetric, and 5 were non-symmetric). Local consolidation was found in four cases, and lobar consolidation was found in two cases. Normal lung tissue was observed between the lesions. Pleural thickening was common and was combined with pleural/pericardial effusion or mediastinal lymph node enlargement. Reticular changes, centrilobular nodules, and the tree-in-bud sign were observed in some cases, but reticular changes, bronchial wall thickening, and hyperinflation were not found. CONCLUSION: Radiological changes associated with both acute pneumonia and acute interstitial inflammation were observed in early H7N9 avian influenza cases. Serial chest X-rays were useful for the diagnosis and severity assessment of the disease. CT may provide a more accurate assessment of the lung pathology.
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Subtipo H7N9 do Vírus da Influenza A , Influenza Humana/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Radiografia Torácica/métodos , Tomografia Computadorizada por Raios X/métodos , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tomografia Computadorizada Espiral/métodosRESUMO
Proteases in the M23 family have speciï¬c activities toward elastin and bacterial peptidoglycan. The peptidoglycan-degrading property makes these proteases have potential as novel antimicrobials. Because M23 proteases cannot be maturely expressed in Escherichia coli, it is significant to improve the production of these enzymes in their wild strains. Pseudoalterin is a new M23 protease secreted by the deep-sea bacterium Pseudoalteromonas sp. CF6-2. In this study, the fermentation conditions of strain CF6-2 for pseudoalterin production were optimized using single factor experiments and response surface methodology to improve the enzyme yield. To reduce the fermentation cost, bovine artery powder instead of elastin was determined as a cheap and efficient inducer. Based on single factor experiments, artery powder content, culture temperature and culture time were determined as the main factors influencing pseudoalterin production and were further optimized by the central composite design. The optimal values of these factors were determined as: artery powder of 1.2%, culture temperature of 20.17 °C and culture time of 28.04 h. Under the optimized conditions, pseudoalterin production reached 100.02±9.0 U/mL, more than twice of that before optimization. These results lay a good foundation for developing the biotechnological potential of pseudoalterin.
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Artérias/química , Proteínas de Bactérias/biossíntese , Endopeptidases/biossíntese , Pseudoalteromonas/metabolismo , Animais , Organismos Aquáticos , Bovinos , Meios de Cultura/química , Elastina/metabolismo , Elastina/farmacologia , Análise Fatorial , Fermentação/efeitos dos fármacos , Pós/farmacologia , Pseudoalteromonas/efeitos dos fármacosRESUMO
A number of proteases in the subtilisin family derived from environmental or pathogenic microorganisms have been reported to be collagenolytic serine proteases. However, their collagen degradation mechanisms remain unclear. Here, the degradation mechanism of type I collagen fibres by the S8 collagenolytic protease MCP-01, from Pseudoalteromonas sp. SM9913, was studied. Atomic force microscopy observation and biochemical analysis confirmed that MCP-01 progressively released single fibrils from collagen fibres and released collagen monomers from fibrils mainly by hydrolysing proteoglycans and telopeptides in the collagen fibres. Structural and mutational analyses indicated that an enlarged substrate-binding pocket, mainly composed of loops 7, 9 and 11, is necessary for collagen recognition and that the acidic and aromatic residues on these loops form a negatively charged, hydrophobic environment for collagen binding. MCP-01 displayed a non-strict preference for peptide bonds with Pro or basic residues at the P1 site and/or Gly at the P1' site in collagen. His211 is a key residue for the P1-basic-residue preference of MCP-01. Our study gives structural and mechanistic insights into collagen degradation of the S8 collagenolytic protease, which is helpful in developing therapeutics for diseases with S8 collagenolytic proteases as pathogenic factors and in studying environmental organic nitrogen degradation mechanisms.
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Aminoácidos/metabolismo , Colágeno Tipo I/metabolismo , Endopeptidases/química , Endopeptidases/metabolismo , Proteoglicanas/metabolismo , Pseudoalteromonas/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Microscopia de Força Atômica , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pseudoalteromonas/química , Pseudoalteromonas/classificação , Especificidade por Substrato , Subtilisina/química , Subtilisina/metabolismoRESUMO
Marine xylanases are rather less studied compared to terrestrial xylanases. In this study, a new xylanase gene, xynB, was cloned from the marine bacterium, Glaciecola mesophila KMM241, and expressed in Escherichia coli. xynB encodes a multi-domain xylanase XynB of glycoside hydrolase (GH) family 8. The recombinant XynB comprises an N-terminal domain (NTD) with unknown function and a catalytic domain, which is structurally novel among the characterized xylanases of GH family 8. XynB has the highest identity (38%) to rXyn8 among the characterized xylanases. The recombinant XynB showed maximal activity at pH 6-7 and 35 °C. It is thermolabile and salt-tolerant. XynB is an endo-xylanase that demands at least five sugar moieties for effective cleavage and to hydrolyze xylohexaose and xylopentaose into xylotetraose, xylotriose and xylobiose. NTD was expressed in Escherichia coli to analyze its function. The recombinant NTD exhibited a high binding ability to insoluble xylan and avicel and little binding ability to chitosan and chitin. Since the NTD shows no obvious homology to any known carbohydrate-binding module (CBM) sequence in public databases, XynB may contain a new type of CBM.
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Alteromonadaceae/enzimologia , Clonagem Molecular , Endo-1,4-beta-Xilanases/metabolismo , Escherichia coli/enzimologia , beta-Glucosidase/metabolismo , Alteromonadaceae/genética , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/isolamento & purificação , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Concentração de Íons de Hidrogênio , Tolerância ao Sal , Temperatura , beta-Glucosidase/genética , beta-Glucosidase/isolamento & purificaçãoRESUMO
Elastin is a common insoluble protein that is abundant in marine vertebrates, and for this reason its degradation is important for the recycling of marine nitrogen. It is still unclear how marine elastin is degraded because of the limited study of marine elastases. Here, a novel protease belonging to the M23A subfamily, secreted by Pseudoalteromonas sp. CF6-2 from deep-sea sediment, was purified and characterized, and its elastolytic mechanism was studied. This protease, named pseudoalterin, has low identities (<40%) to the known M23 proteases. Pseudoalterin has a narrow specificity but high activity toward elastin. Analysis of the cleavage sites of pseudoalterin on elastin showed that pseudoalterin cleaves the glycyl bonds in hydrophobic regions and the peptide bonds Ala-Ala, Ala-Lys, and Lys-Ala involved in cross-linking. Two peptic derivatives of desmosine, desmosine-Ala-Ala and desmosine-Ala-Ala-Ala, were detected in the elastin hydrolysate, indicating that pseudoalterin can dissociate cross-linked elastin. These results reveal a new elastolytic mechanism of the M23 protease pseudoalterin, which is different from the reported mechanism where the M23 proteases only cleave glycyl bonds in elastin. Genome analysis suggests that M23 proteases may be popular in deep-sea sediments, implying their important role in elastin degradation. An elastin degradation model of pseudoalterin was proposed, based on these results and scanning electron microscopic analysis of the degradation by pseudoalterin of bovine elastin and cross-linked recombinant tropoelastin. Our results shed light on the mechanism of elastin degradation in deep-sea sediment.
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Metaloproteases/química , Metaloproteases/genética , Pseudoalteromonas/enzimologia , Pseudoalteromonas/genética , Água do Mar/microbiologia , Microbiologia da Água , Sequência de Aminoácidos , Animais , Bovinos , Elastina/química , Metaloproteases/metabolismo , Dados de Sequência Molecular , Oceanos e MaresRESUMO
The enzymes currently used to increase meat tenderness are all mesophilic or thermophilic proteases. This study provides insight into the tenderization effect and the mechanism of a cold-adapted collagenolytic enzyme MCP-01 on beef meat at low temperatures. MCP-01 (10 U of caseinolytic activity) reduced the meat shear force by 23% and increased the relative myofibrillar fragmentation index of the meat by 91.7% at 4 °C, and it also kept the fresh colour and moisture of the meat. Compared to the commercially used tenderizers papain and bromelain, MCP-01 showed a unique tenderization mechanism. MCP-01 had a strong selectivity for degrading collagen at 4 °C, showed a distinct digestion pattern on the myofibrillar proteins, and had a different disruption pattern on the muscle fibres under scanning electron micrograph. These results suggest that the cold-adapted collagenolytic protease MCP-01 may be promising for use as a meat tenderizer at low and moderate temperatures.
Assuntos
Colágeno/química , Endopeptidases/química , Manipulação de Alimentos/métodos , Carne/análise , Músculo Esquelético/química , Papaína/química , Animais , Bovinos , Temperatura Baixa , Combinação de Medicamentos , Manipulação de Alimentos/instrumentação , Sódio na DietaRESUMO
A protease-producing marine bacterium, designated CF12-14(T), was isolated from sediment of the South China Sea. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain CF12-14(T) formed a separate lineage within the genus Idiomarina (Gammaproteobacteria). The isolate showed the highest 16S rRNA gene sequence similarity with Idiomarina salinarum ISL-52(T) (94.7â%), Idiomarina seosinensis CL-SP19(T) (94.6â%) and other members of the genus Idiomarina (91.9-94.6â%). Cells were gram-negative, aerobic, flagellated, straight or slightly curved, and often formed buds and prosthecae. Strain CF12-14(T) grew at 4-42 °C (optimum 30-35 °C) and with 0.1-15â% (w/v) NaCl (optimum 2-3â%). The isolate reduced nitrate to nitrite and hydrolysed DNA, but did not produce acids from sugars. The predominant cellular fatty acids were iso-C(15â:â0) (27.4â%), iso-C(17â:â0) (16.0â%) and iso-C(17â:â1)ω9c (15.8â%). The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The major respiratory quinone was ubiquinone 8. The DNA G+C content was 50.4 mol%. The phylogenetic, phenotypic and chemotaxonomic data supported the conclusion that CF12-14(T) represents a novel species of the genus Idiomarina, for which the name Idiomarina maris sp. nov. is proposed. The type strain is CF12-14(T) (â=âCCTCC AB 208166(T)â=âKACC 13974(T)).