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1.
Emerg Microbes Infect ; 9(1): 457-468, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32090689

RESUMO

Porcine deltacoronavirus (PDCoV) is a newly emerging threat to the global porcine industry. PDCoV has been successfully isolated using various medium additives including trypsin, and although we know it is important for viral replication, the mechanism has not been fully elucidated. Here, we systematically investigated the role of trypsin in PDCoV replication including cell entry, cell-to-cell membrane fusion and virus release. Using pseudovirus entry assays, we demonstrated that PDCoV entry is not trypsin dependent. Furthermore, unlike porcine epidemic diarrhea virus (PEDV), in which trypsin is important for the release of virus from infected cells, PDCoV release was not affected by trypsin. We also demonstrated that trypsin promotes PDCoV replication by enhancing cell-to-cell membrane fusion. Most importantly, our study illustrates two distinct spreading patterns from infected cells to uninfected cells during PDCoV transmission, and the role of trypsin in PDCoV replication in cells with different virus spreading types. Overall, these results clarify that trypsin promotes PDCoV replication by mediating cell-to-cell fusion transmission but is not crucial for viral entry. This knowledge can potentially contribute to improvement of virus production efficiency in culture, not only for vaccine preparation but also to develop antiviral treatments.


Assuntos
Fusão Celular , Coronavirus/fisiologia , Fusão de Membrana , Tripsina/metabolismo , Animais , Linhagem Celular , Humanos , Suínos , Internalização do Vírus , Replicação Viral
2.
J Chromatogr A ; 1609: 460507, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31522804

RESUMO

A proteomic workflow for a simple loss-less manual nano-fractionation (300 nL/fraction) for low µg sample amounts which avoids the need to dry down or transfer fractions to autosampler vials is shown to be feasible. It is demonstrated that the conventional procedure of drying samples down followed by reconstitution negatively affects the number of protein and peptide identifications. Furthermore, these losses seem to disproportionately affect hydrophobic peptides from the drying down and reconstitution step. By collecting and concatenating the fractions while the outlet of the column is submerged in a small predefined volume of 0.2% formic acid, the content of acetonitrile in the collecting vials was lowered such that it was compatible with direct injection for the online analysis. This additionally resulted in a time gain of approx. an hour for the total fractionation time. Acetonitrile concentrations up to 7.5% do not seem to compromise the chromatographic performance in the online analysis. Using as little as 2 µg digested HeLa lysate, approx. 7000 protein groups could be easily identified with 2 or more unique peptides. This was the case when fractionation was performed at pH 10 as well as at pH 5.5.


Assuntos
Nanopartículas/química , Proteínas/análise , Proteômica/métodos , Fluxo de Trabalho , Fracionamento Químico , Dessecação , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Peptídeos/análise , Peptídeos/química , Tripsina/metabolismo
3.
Food Chem ; 306: 125581, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31606636

RESUMO

A comprehensive evaluation was conducted to compare the generation of antioxidative peptides produced by alcalase versus trypsin from Atlantic sea cucumber. The in vitro antioxidative peptides were sequenced by de novo sequencing using LC-MS/MS. Key constituent antioxidative amino acids (KCAAA), i.e., Cys, His, Met, Trp and Tyr in the peptides and the molecular interactions between peptides and myeloperoxidase (MPO, a mediator and marker of in vivo oxidative stress), were analyzed by in silico methods. Alcalase-produced protein hydrolysates showed 5-35% higher in vitro antioxidant activity than the trypsin-produced ones. UPLC analysis revealed the total amino acid composition in peptide fractions <2 kDa. Alcalase produced 35.4% of peptides with both KCAAA and potential MPO inhibitory activity, compared with only 30.3% for trypsin. A representative peptide sequence TEFHLL generated by alcalase had intense molecular interactions with MPO active site, predicting a capacity to inhibit in vivo oxidative stress.


Assuntos
Antioxidantes/metabolismo , Peptídeos/metabolismo , Pepinos-do-Mar/metabolismo , Subtilisinas/metabolismo , Tripsina/metabolismo , Animais , Cromatografia Líquida , Estresse Oxidativo , Espectrometria de Massas em Tandem
4.
Arch Insect Biochem Physiol ; 103(1): e21637, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31625209

RESUMO

Anticarsia gemmatalis represents a relevant factor for lowering soybean and other legume crop productivities. Protease inhibitors affect protein degradation and reduce the availability of amino acids, impairing the development and survival of insect pests. To evaluate the possible use of proteinaceous protease inhibitors in the management of this pest, the activities of midgut proteases and the growth and development of A. gemmatalis larvae exposed to soybean Bowman-Birk trypsin-chymotrypsin inhibitor (SBBI) and soybean Kunitz trypsin inhibitor (SKTI) were determined. The survival curves obtained using Kaplan-Meier estimators indicated that SKTI and SBBI stimulated larval survival. However, the development of A. gemmatalis was delayed, and prepupal weight decreased in the presence of both inhibitors. The results showed that SKTI and SBBI inhibited the trypsin-like and total proteolytic activities of larvae on the 12th day after eclosion. On the 15th day after eclosion, larvae exposed to SKTI increased the activities of trypsin and total proteases. Although SKTI and SBBI did not affect the survival of the insect, they had effects on midgut proteases in a stage wherein A. gemmatalis fed voraciously, increased the larval cycle, and decreased prepupal weight. These findings provide baseline information about the potential of proteinaceous protease inhibitors to manage the velvetbean caterpillar, avoiding chemical pesticides.


Assuntos
Mariposas/efeitos dos fármacos , Inibidor da Tripsina de Soja de Bowman-Birk/farmacologia , Inibidor da Tripsina de Soja de Kunitz/farmacologia , Animais , Trato Gastrointestinal/enzimologia , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/crescimento & desenvolvimento , Mariposas/enzimologia , Mariposas/crescimento & desenvolvimento , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Soja/enzimologia , Tripsina/metabolismo
5.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1134-1135: 121856, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31786420

RESUMO

Renin is the rate-limiting step within the renin-angiotensin-aldosterone system, but the reliable quantification of human endogenous renin levels by liquid chromatography coupled with mass spectrometry remains challenging. The complex sample matrix triggering ion suppression and the detection of the low-abundance as well as the proteolytical-resistant renin make a hybrid approach using immunocapture coupled with LC-HRMS a promising method for investigation. Therefore, in-silico digestion and BLAST® experiments were conducted in order to identify the unique amino acid sequence for mass spectrometric detection. To enhance mass spectrometric response, impacting parameters within the denaturation, alkylation, and digestion experiments were identified and optimized by a multistep Design of Experiments process. The optimal denaturation buffer consisted of RapiGest® and urea, leading to a signature peptide intensity increase of 56% at 20 °C, whereas the optimal reducing agent improved intensity by 27%. The most effective generation of signature peptide I was achieved using a high trypsin concentration and a low incubation temperature enhancing digestion by 75%. The applicability of this hybrid approach was confirmed in human matrix and allowed for a fivefold reduction in total assay procedure time without limiting the reliable quantification if compared to a conventional digestion approach.


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Renina , Simulação por Computador , Humanos , Renina/sangue , Renina/química , Renina/metabolismo , Projetos de Pesquisa , Extração em Fase Sólida , Temperatura Ambiente , Tripsina/metabolismo
6.
Analyst ; 144(21): 6270-6275, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31566639

RESUMO

Enhanced-fluidity, reversed-phase liquid chromatography was developed using custom instrumentation for separation and characterization of intact KRas proteins and tryptic peptides. The KRas, HRas and NRas function as GDP-GTP regulated binary switches in many signalling pathways, and mutations in Ras proteins are frequently found in human cancers and represent poor prognosis markers for patients. Mutations of the KRas isoform constitute some of the most common aberrations among all human cancers and intensive drug discovery efforts have been directed toward targeting the KRas protein. Separation and characterization of the KRas protein and tryptic peptides are helpful for exploring targeting, which has not been fully investigated using liquid chromatography-tandem mass spectrometry. EFLC-MS provided improved chromatographic performance compared to traditional HPLC-MS in terms of shorter analysis time, increased ion intensity and a shift to higher charge states for intact KRas proteins.


Assuntos
Cromatografia Líquida/métodos , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Tripsina/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Solventes/química
7.
Biochimie ; 166: 203-213, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31518617

RESUMO

Influenza A virus (IAV) is one of the most common infectious pathogen and associated with significant morbidity and mortality. Although processing the IAV hemagglutinin (HA) envelope glycoprotein precursor is a pre-requisite for viral membrane fusion activity, viral entry and transmission, HA-processing protease is not encoded in the IAV genome and thus the cellular trypsin-type serine HA-processing proteases determine viral infectious tropism and viral pathogenicity. The initial process of IAV infection of the airway is followed by marked upregulation of ectopic trypsin in various organs and endothelial cells through the induction of various proinflammatory cytokines, and this process has been termed the "influenza virus-cytokine-trypsin" cycle. In the advanced stage of IAV infection, the cytokine storm induces disorders of glucose and lipid metabolism and the "metabolic disorders-cytokine" cycle is then linked with the "influenza virus-cytokine-trypsin" cycle, to advance the pathogenic process into energy crisis and multiple organ failure. Application of protease inhibitors and treatment of metabolic disorders that break these cycles and their interconnection is therefore a promising therapeutic approach against influenza. This review discusses IAV pathogenicity on trypsin type serine HA-processing proteases, cytokines, metabolites and therapeutic options.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A , Influenza Humana , Serina Proteases/fisiologia , Internalização do Vírus/efeitos dos fármacos , Animais , Galinhas/virologia , Citocinas/metabolismo , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/patogenicidade , Influenza Aviária/tratamento farmacológico , Influenza Aviária/virologia , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/patogenicidade , Tripsina/metabolismo
8.
Chemosphere ; 237: 124561, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31549663

RESUMO

With the development of nanotechnology and increased nanomaterial application, TiO2 nanoparticles have been released into the aquatic environment, causing potential ecotoxicological effects on aquatic organisms. Ocean acidification caused by anthropogenic CO2 is one of the most common environmental stressors, occurring simultaneously with marine contaminants, e.g., nanoparticles. Marine bivalves can accumulate nanoparticles and their digestive functions may be affected. In this study, we investigated the potential influences of TiO2 nanoparticles on the digestive enzyme activities of marine mussels Mytilus coruscus under ocean acidification. Mussels were exposed to combined treatments with three concentrations of nano-TiO2 (0, 2.5, 10 mg/L) and two pH values (8.1, 7.3) for 14 days, and then recovered under ambient condition (pH 8.1 and no nanoparticle) for 7 days. Samples were taken on the 1st, 3rd, 7th, 14th, and 21st day, the digestive enzymes, including amylase, pepsin, trypsin, lipase, and lysozyme, were investigated. Our results showed that nano-TiO2 and low pH had negative effects on amylase, pepsin, trypsin, and lipase, while both of them led an increase in lysozyme activity. Nano-TiO2 showed greater effects on the digestive capacity of M. coruscus rather than low pH. Moreover, a recovery period of 7 days was not sufficient for these enzymes to fully recover.


Assuntos
Enzimas/metabolismo , Nanopartículas Metálicas/toxicidade , Mytilus/efeitos dos fármacos , Água do Mar/química , Titânio/toxicidade , Poluentes Químicos da Água/toxicidade , Amilases/metabolismo , Animais , Ecotoxicologia , Concentração de Íons de Hidrogênio , Lipase/metabolismo , Muramidase/metabolismo , Mytilus/fisiologia , Pepsina A/metabolismo , Tripsina/metabolismo , Poluentes Químicos da Água/química
9.
An Acad Bras Cienc ; 91(3): e20180735, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31553366

RESUMO

Candida haemulonii complex (C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonii) consists of emergent multidrug-resistant pathogens that cause bloodstream and deep-seated infections. However, little is known about their virulence factors. Herein, we evaluated the presence of extracellular serine peptidases in this fungal complex. Serine peptidase activity was measured by spectrophotometry using chromogenic peptide substrates to the S1 family. Chymotrypsin-, trypsin- and elastase-like activities were detected in all fungal isolates. Since higher chymotrypsin- and trypsin-like activities were observed from the cleavage of N-succinyl-Ala-Ala-Pro-Phe-pNa and N-benzoyl-Phe-Val-Arg-pNa, respectively, these substrates were selected for further experiments. Overall, pHs 7.0 and 9.0 were those in which higher chymotrypsin- and trypsin-like activities were observed, respectively, displaying higher hydrolytic activities at 37-45°C. Additionally, the serine peptidases produced by C. haemulonii complex were inhibited by PMSF and AEBSF in a typically concentration-dependent manner. Although the Michaelis constant (Km) values obtained for chymotrypsin-like peptidases were similar, greater differences were observed for trypsin-like enzymes secreted by the different fungal isolates. This is the first time that peptidases belonging to the S1 family are described in the C. haemulonii species complex. Thus, these data open the doors for more detailed studies into potential roles of these peptidases in fungal virulence.


Assuntos
Candida/enzimologia , Quimotripsina/metabolismo , Farmacorresistência Fúngica Múltipla , Tripsina/metabolismo , Candida/classificação , Meios de Cultura , Espectrofotometria , Temperatura Ambiente
10.
Anal Bioanal Chem ; 411(27): 7087-7094, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31471684

RESUMO

Accurate measurement and understanding of therapeutic uptake and metabolism is key in the drug development process. This work examines the amount of doxorubicin that can penetrate into spheroids after being encapsulated in a liposomal configuration in comparison with free drug. Through a process known as serial trypsinization, three distinct cellular populations of a spheroid were successfully separated and a small molecule extraction was used to isolate the chemotherapeutic. Doxorubicin showed a time-dependent permeability into spheroids with the most drug accumulating in the core at 24 h of treatment. Entrapment of the chemotherapeutic delayed the permeability of the drug and resulted in reduced amounts quantified at the earlier time points. These findings validate the claim that liposomal therapeutics have the ability to alter the pharmacokinetics and pharmacodynamics profiles of a drug while also demonstrating the combined power of mass spectrometry and three-dimensional cell cultures to evaluate drug penetration and metabolism. Graphical abstract.


Assuntos
Antibióticos Antineoplásicos/metabolismo , Doxorrubicina/análogos & derivados , Esferoides Celulares/metabolismo , Antibióticos Antineoplásicos/farmacocinética , Doxorrubicina/metabolismo , Doxorrubicina/farmacocinética , Células HCT116 , Humanos , Espectrometria de Massas , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacocinética , Tripsina/metabolismo
11.
Mol Immunol ; 114: 278-288, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31419704

RESUMO

Protease-activated receptors (PARs) have been described in a wide diversity of vertebrate cells, including human immune cells. Macrophages are pivotal cells in the host-pathogen interaction and their polarization in M1 or M2 cells has been described as a new central paradigm in the immune response to pathogens. In this context, we explored the involvement of PAR activation by serine proteases on M1/M2 macrophage differentiation and their impact on the Th1/Th2 cytokine profile in response to Mycobacterium tuberculosis antigen. Our results demonstrate that the serine proteases, thrombin and trypsin, induce interleukin (IL)-4 release from human monocytes, together with upregulation of the macrophage mannose receptor (CD206) in the same way that alternative M2a differentiated cells with M-CSF/IL-4. Protease stimulation of monocytes in the presence of PAR-1 (SCH-79797) or PAR-2 (FSLLRY-NH2) antagonists abolished IL-4 release from monocytes, whereas the use of the peptide agonist for PAR-1 (SFLLRNPNDKYEPF-NH2) or PAR-2 (SLIGKV-NH2) induced the secretion of IL-4 at a level comparable to thrombin or trypsin. When these protease-induced M2 macrophages from healthy human PPD + donors were co-cultured with autologous lymphocyte population in the presence of Mycobacterium tuberculosis antigen, we found a consistent inhibition of IFN-γ/IL-12 release together with persistent IL-4 expression, in contrast to the expected Th1 profile obtained with M2a macrophages. To our knowledge, this is the first observation that proteolytic activation of PAR1/2 receptors in monocytes induces M2-like macrophages with impaired plasticity and their implication in the driving of the Th1/Th2 cytokine profile.


Assuntos
Polaridade Celular/fisiologia , Macrófagos/metabolismo , Macrófagos/fisiologia , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Diferenciação Celular/fisiologia , Plasticidade Celular , Células Cultivadas , Citocinas/metabolismo , Humanos , Interleucina-4/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/fisiologia , Ativação de Macrófagos/fisiologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Monócitos/metabolismo , Monócitos/fisiologia , Mycobacterium tuberculosis/patogenicidade , Tripsina/metabolismo , Tuberculose/metabolismo , Regulação para Cima/fisiologia
12.
Food Chem ; 299: 125038, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31284248

RESUMO

Wheat is one of the world's most widely consumed staple food. However, the number of people suffering from wheat-related disorders has increased drastically. Amylase-trypsin inhibitors (ATIs) have recently been identified as one of the main triggers of non-celiac wheat sensitivity (NCWS). In this study, an enzymatic assay for the determination of trypsin inhibition activity in hexaploid wheat was developed. This method was optimized with respect to several parameters, such as extraction and incubation procedures, and was validated according to international standards, concerning accuracy, precision and robustness of the method. Results revealed that linear inhibition and thus accuracy occurred only in a narrow concentration range. However, after optimization of settings the novel method was found to be satisfactory for accurate determination of trypsin inhibition in wheat. Purification of the wheat extract with immobilized trypsin beads led to the identification of CM inhibitors (chloroform/methanol soluble proteins) as main contributors of trypsin inhibition.


Assuntos
Amilases/farmacologia , Ensaios Enzimáticos/métodos , Triticum/enzimologia , Inibidores da Tripsina/farmacologia , Tripsina/metabolismo , Alérgenos/farmacologia , Humanos
13.
Biomed Res Int ; 2019: 6302950, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31317034

RESUMO

This study aimed to investigate the effects of isoleucine (Ile) on the synthesis and secretion of digestive enzymes and cellular signalling in the pancreatic tissue of dairy goats. The pancreatic tissues were incubated in buffer containing 0, 0.40, 0.80, and 1.60 mM Ile. High levels of Ile significantly increased the buffer release and total concentration of ɑ-amylase in the tissues (P < 0.001). The total trypsin and chymotrypsin concentrations in each of the Ile groups were significantly higher than those in the control group (P < 0.05); however, lipase was not affected. High levels of Ile significantly increased ɑ-amylase mRNA expression (P < 0.001) but had no effect on the mRNA expression of trypsin, chymotrypsin, or lipase. Ile did not affect S6K1 phosphorylation levels. High levels of Ile significantly increased the expression of the γ isoform of 4EBP1 (P < 0.001), which indicated that the phosphorylation of 4EBP1 was significantly increased. The phosphorylation level of eEF2 gradually decreased with the addition of Ile (P < 0.001). These results suggested that high doses of Ile can regulate the excretion of enzymes, especially ɑ-amylase, in the pancreatic tissues of dairy goats by modulating mTOR signalling, and this regulation is independent of the mTOR-S6K1 pathway.


Assuntos
Cabras/metabolismo , Isoleucina/metabolismo , Pâncreas/enzimologia , alfa-Amilases/biossíntese , Animais , Quimotripsina/biossíntese , Quimotripsina/metabolismo , Quinase do Fator 2 de Elongação/genética , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica/genética , Lipase/biossíntese , Lipase/metabolismo , Pâncreas/metabolismo , Fosforilação , RNA Mensageiro/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Tripsina/biossíntese , Tripsina/metabolismo , alfa-Amilases/metabolismo
14.
Plant Foods Hum Nutr ; 74(3): 414-420, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31278561

RESUMO

The amount of cold press oil manufacture is globally rising, which in turn leads to the accumulation of deoiled plant seeds at significant quantities and consequent manufacture of plant protein products. In this study, we made an attempt to analyze the protein profile of black cumin seed protein concentrates prepared by the alkali extraction-acid precipitation technique (AE-IP). The analytical strategy relied on gel-based proteome mapping which included two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF/TOF). 14 different protein bands were identified, and in gel-trypsinolysis was carried out for the corresponding gel spots. Using the MASCOT database, current findings on 10 proteins were compared with the existing data. The highest similarity was 46 which was obtained between the highest pI black cumin protein observed here and the cyclin dependent kinase inhibitor of Arabidopsis thaliana. The molecular mass of the intact protein was determined by linear MALDI-TOF/TOF-MS as 23,711.2186 Da. The peptide constructs of this protein have been further studied in order to identify potential biological activity. Matching sequences generated bioactive peptides in silico such as IR, AL, and SL dipeptides during sequential enzymatic digestion with pepsin and trypsin. Since the majority of bioactivity investigations on black cumin seeds have been related to black cumin oil and its oil soluble components, the structure and bioactivities of black cumin proteins deserve further research.


Assuntos
Nigella sativa/metabolismo , Peptídeos/análise , Proteínas de Plantas/análise , Proteoma , Eletroforese em Gel Bidimensional , Peso Molecular , Proteômica , Sementes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismo
15.
J Sep Sci ; 42(17): 2788-2795, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31218807

RESUMO

With the explosive growth of the bioscience and biopharmaceuticals, the demand for high efficient analysis and separation of proteins is urgent. High-performance liquid chromatography is an appropriate technology for this purpose, and the stationary phase is the kernel to the separation efficiency. In this study, flow-through poly(styrene-co-divinylbenzene) microspheres characteristic of the binary pores, i.e. flow-through pores and mesopores, were synthesized; this special porous structure would benefit the convective mass transfer while guarantee the high specific surface area. Owing to the hydrophobic nature, poly(styrene-co-divinylbenzene) microspheres were suitable as the reversed-phase stationary phase for separation of proteins. For the high permeability of the poly(styrene-co-divinylbenzene) microspheres packed column, fast separation of the studied six proteins in ∼2 min was achieved. The recoveries of studied proteins were acceptable in the range of 79.0-99.4%. The proposed column had good pH stability of 1-13 and repeatability. Moreover, the column was applied for egg white fast separation, further demonstrating its applicability for complex bio-sample separation. The flow-through poly(styrene-co-divinylbenzene) microspheres were promising for fast separation of large molecules.


Assuntos
Cromatografia de Fase Reversa , Microesferas , Poliestirenos/química , Animais , Bovinos , Citocromos c/química , Citocromos c/isolamento & purificação , Lactoglobulinas/química , Lactoglobulinas/isolamento & purificação , Muramidase/química , Muramidase/isolamento & purificação , Muramidase/metabolismo , Ribonuclease Pancreático/química , Ribonuclease Pancreático/isolamento & purificação , Ribonuclease Pancreático/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/isolamento & purificação , Suínos , Transferrina/química , Transferrina/isolamento & purificação , Tripsina/química , Tripsina/isolamento & purificação , Tripsina/metabolismo
16.
PLoS One ; 14(6): e0218374, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31246970

RESUMO

Trypsin is by far the most commonly used protease in proteomics. Even though the amount of protease used in each experiment is very small, digestion of large amounts of protein prior to enrichment can be rather costly. The price of commercial trypsin is highly dependent on the quality of the enzyme, which is determined by its purity, activity, and chemical modifications. In this study we evaluated several strategies for improving the quality of crude trypsin by reductive methylation and affinity purification. We present a protocol applicable to most proteomics laboratories for obtaining a highly stable and pure trypsin preparation using reductive methylation and purification by benzamidine-sepharose. The entire workflow can be performed within a day and yields ~4 mg per batch but is completely scalable. The methylated product was benchmarked against sequencing grade trypsin from Promega and they were found to be comparable for one hour digestions at elevated temperatures, where residual chymotryptic activity was found to be negligible.


Assuntos
Proteômica , Tripsina/química , Cromatografia Líquida , Estabilidade Enzimática , Células HeLa , Temperatura Alta , Humanos , Peptídeos/química , Proteólise , Proteômica/economia , Proteômica/métodos , Espectrometria de Massas em Tandem , Tripsina/isolamento & purificação , Tripsina/metabolismo , Fluxo de Trabalho
17.
Parasitol Res ; 118(7): 2223-2233, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31187225

RESUMO

Blood coagulation in vertebrates is a complex mechanism that involves the precisely coordinated and regulated action of a cascade of factors in order to prevent excessive blood loss upon wounding. Any blood sucking ectoparasite, however, has to circumvent this mechanism to ensure the uptake of an adequate blood meal. Inhibitors of blood coagulation in the saliva are hence widespread among these animals. Thrombin as a key factor of blood coagulation is a prominent target of such inhibitors, and hirudin is probably the best known among the thrombin inhibitors. Hirudin was originally described in the genus Hirudo, but occurs in other leech genera like Hirudinaria and Macrobdella as well. Besides several isoforms of hirudin, a new class of putative leech saliva components, the hirudin-like factors (HLFs), was identified in both genera Hirudo and Hirudinaria. Here, we describe the expression, purification, and functional characterization of three HLFs (HLF5, 6, and 8, respectively) and two additional hirudins (HM3 and HM4) of Hirudinaria manillensis. While HLF6 lacked any inhibitory activity on thrombin, HLF5 as well as HLF8 clearly exhibited anticoagulatory properties. The inhibitory activity of HLF5 and HLF8, however, was much lower compared with both HM3 and HM4 of Hirudinaria manillensis as well as the hirudin variants 1 (HV1) and 2 (HV2) of Hirudo medicinalis. Neither an inhibition of trypsin nor a platelet aggregation was caused by HLF8. Our data indicates the presence of two classes (rather than isoforms) of hirudins in Hirudinaria manillensis with markedly different inhibitory activity on human thrombin.


Assuntos
Antitrombinas/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Hirudinas/metabolismo , Hirudo medicinalis/metabolismo , Trombina/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Humanos , Proteínas Recombinantes/metabolismo , Tripsina/metabolismo
18.
J Chem Phys ; 150(22): 220901, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31202243

RESUMO

As molecular scientists have made progress in their ability to engineer nanoscale molecular structure, we face new challenges in our ability to engineer molecular dynamics (MD) and flexibility. Dynamics at the molecular scale differs from the familiar mechanics of everyday objects because it involves a complicated, highly correlated, and three-dimensional many-body dynamical choreography which is often nonintuitive even for highly trained researchers. We recently described how interactive molecular dynamics in virtual reality (iMD-VR) can help to meet this challenge, enabling researchers to manipulate real-time MD simulations of flexible structures in 3D. In this article, we outline various efforts to extend immersive technologies to the molecular sciences, and we introduce "Narupa," a flexible, open-source, multiperson iMD-VR software framework which enables groups of researchers to simultaneously cohabit real-time simulation environments to interactively visualize and manipulate the dynamics of molecular structures with atomic-level precision. We outline several application domains where iMD-VR is facilitating research, communication, and creative approaches within the molecular sciences, including training machines to learn potential energy functions, biomolecular conformational sampling, protein-ligand binding, reaction discovery using "on-the-fly" quantum chemistry, and transport dynamics in materials. We touch on iMD-VR's various cognitive and perceptual affordances and outline how these provide research insight for molecular systems. By synergistically combining human spatial reasoning and design insight with computational automation, technologies such as iMD-VR have the potential to improve our ability to understand, engineer, and communicate microscopic dynamical behavior, offering the potential to usher in a new paradigm for engineering molecules and nano-architectures.


Assuntos
Simulação de Dinâmica Molecular , Software , Realidade Virtual , Benzamidinas/metabolismo , Ciclofilina A/química , Humanos , Subtipo H7N9 do Vírus da Influenza A/enzimologia , Relações Interpessoais , Ligantes , Neuraminidase/metabolismo , Compostos Orgânicos/química , Oseltamivir/metabolismo , Ligação Proteica , Conformação Proteica , Teoria Quântica , Tripsina/metabolismo
19.
Int J Biol Macromol ; 136: 404-409, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31202843

RESUMO

Chemical, thermal and mechanical collagen characteristics of intramuscular perimysial connective tissue (IMCT) from bovine Semitendinosus (ST) and Pectoralis profundus (PP) muscles were studied. Furthermore, these collagen characteristics in presence/absence of other extracellular matrix components were analyzed for both muscles. Differences between muscles were observed for collagen content; all IMCT-PP perimysial samples were higher than ST samples. In addition, for both muscles, IMCT-alkali resistant samples allowed the highest trypsin soluble collagen. The main differences between muscles were recorder for thermal and mechanical properties. The denaturation of collagen in the perimysium evidenced differences in total denaturation energy (ΔH) and peak temperatures (Tp). The ΔH resulted higher for IMCT-PP than for IMCT-ST tissues in all samples. By the tensile test it was observed that the maximum loads were constant and higher in all PP samples. In the FTIR assay, the peaks for the main amides were registered in both tissues. However, slight differences between ST and PP-IMCT were detected on hydrogen bond interactions and in secondary structure of the protein. The results reinforce the hypothesis of the presence of different IMCT-perimysial-collagen pools. In this study, chemical, thermal and mechanical characteristics were considered and quantified. However, the mechanical function and development of muscle in-vivo could be the main influence on the extracellular collagen characteristics as well as its interactions with non-collagen compounds. Its formation is essential for muscle function.


Assuntos
Fenômenos Químicos , Colágeno/química , Colágeno/metabolismo , Tecido Conjuntivo/metabolismo , Fenômenos Mecânicos , Músculos/metabolismo , Temperatura Ambiente , Animais , Fenômenos Biomecânicos , Bovinos , Hidrólise , Resistência à Tração , Tripsina/metabolismo
20.
Iran Biomed J ; 23(6): 395-403, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31104399

RESUMO

Background: It is believed that the loading value of anticancer drug conjugated to the monoclonal antibody, called drug-to-antibody ratio (DAR), is the main quality feature of antibody-drug conjugates. Methods: In this study, matrix assisted laser desorption/ionization mass spectrometry was used to determine the average molecular weight of trastuzumab and its three conjugated forms. The differences in the measured masses for each conjugate and unconjugated trastuzumab were compared to the expected mass change through the conjugation of one mole of related drug-linker, in order to measure DAR. Results: There was a consistency between the loading results of mass spectrometry and the measurements of UV spectrometry in most cases. Conclusion: According to our findings, the MALDI-MS method for determining the loading values can be used rapidly and reliably to estimate the covalently bound drugs conjugated to antibodies when ESI-TOF-MS is unavailable.


Assuntos
Anticorpos Monoclonais/análise , Antineoplásicos/análise , Imunoconjugados/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Peso Molecular , Peptídeos/análise , Reprodutibilidade dos Testes , Espectrofotometria Ultravioleta , Trastuzumab/análise , Trastuzumab/química , Tripsina/metabolismo
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