Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 127.279
Filtrar
1.
Clin Sci (Lond) ; 134(17): 2235-2241, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32869854

RESUMO

Human serine protease inhibitors (serpins) are the main inhibitors of serine proteases, but some of them also have the capability to effectively inhibit cysteine proteases. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) main protease (Mpro) is a chymotrypsin-type cysteine protease that is needed to produce functional proteins essential for virus replication and transcription. Serpin traps its target proteases by presenting a reactive center loop (RCL) as protease-specific cleavage site, resulting in protease inactivation. Mpro target sites with its active site serine and other flanking residues can possibly interact with serpins. Alternatively, RCL cleavage site of serpins with known evidence of inhibition of cysteine proteases can be replaced by Mpro target site to make chimeric proteins. Purified chimeric serpin can possibly inhibit Mpro that can be assessed indirectly by observing the decrease in ability of Mpro to cleave its chromogenic substrate. Chimeric serpins with best interaction and active site binding and with ability to form 1:1 serpin-Mpro complex in human plasma can be assessed by using SDS/PAGE and Western blot analysis with serpin antibody. Trapping SARS-CoV-2 Mpro cysteine protease using cross-class serpin cysteine protease inhibition activity is a novel idea with significant therapeutic potential.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Serpinas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/uso terapêutico , Betacoronavirus/enzimologia , Western Blotting , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Eletroforese em Gel de Poliacrilamida , Humanos , Pandemias , Pneumonia Viral/virologia , Serpinas/uso terapêutico , Proteínas não Estruturais Virais/química
2.
Nat Commun ; 11(1): 4070, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792502

RESUMO

Human astroviruses are small non-enveloped viruses with positive-sense single-stranded RNA genomes. Astroviruses cause acute gastroenteritis in children worldwide and have been associated with encephalitis and meningitis in immunocompromised individuals. It is still unknown how astrovirus particles exit infected cells following replication. Through comparative genomic analysis and ribosome profiling we here identify and confirm the expression of a conserved alternative-frame ORF, encoding the protein XP. XP-knockout astroviruses are attenuated and pseudo-revert on passaging. Further investigation into the function of XP revealed plasma and trans Golgi network membrane-associated roles in virus assembly and/or release through a viroporin-like activity. XP-knockout replicons have only a minor replication defect, demonstrating the role of XP at late stages of infection. The discovery of XP advances our knowledge of these important human viruses and opens an additional direction of research into their life cycle and pathogenesis.


Assuntos
Canais Iônicos/metabolismo , Mamastrovirus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Genômica/métodos , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Canais Iônicos/genética , Mamastrovirus/genética , Microscopia de Fluorescência , Plasmídeos/genética , Ribossomos , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Replicação Viral/fisiologia
3.
J Chromatogr A ; 1628: 461443, 2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32822982

RESUMO

Sodium dodecyl sulfate (SDS) in proteomics samples needs to be removed and estimated prior to mass spectrometry (MS)-based analysis and to avoid MS ion-source contamination. Here, we describe an organic solvent free method to remove SDS using a simple apparatus that mainly consists of an agarose gel inside a 1 mL plastic micropipette tip and a voltage power supply with electrodes. A small volume of sample (e.g., 50 µL) is loaded on top of the gel and then voltage (cathode at the sample side) is applied with an acidic solution at the other end of the micropipette tip. Within 25 min, SDS was removed (e.g., ≥99% SDS in 3.5 mM SDS) and the peptides were retained in the sample solution. The strategy was compared to the commercially available and expensive Pierce spin column for the removal of SDS and recovery of peptides from a digested bovine serum albumin sample.


Assuntos
Técnicas de Química Analítica/métodos , Técnicas Eletroquímicas , Proteômica/métodos , Dodecilsulfato de Sódio/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Peptídeos/química , Soroalbumina Bovina/química , Dodecilsulfato de Sódio/química
4.
Exp Parasitol ; 217: 107963, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32781092

RESUMO

This study analyzed the large-subunit (60S) ribosomal protein L12 of Eimeria tenella (Et60s-RPL12). A full-length cDNA was cloned, and the recombinant protein was expressed in E. coli BL21 and inoculated in rabbits to produce the polyclonal antibody. Quantitative real-time polymerase chain reaction and western blotting were used to analyze the transcription levels of Et60s-RPL12 and translation levels in different developmental stages of E. tenella. The results showed that the mRNA transcription level of Et60s-RPL12 was highest in second-generation merozoites, whereas the translation level was highest in unsporulated oocysts. Indirect immunofluorescence showed that Et60s-RPL12 was localized to the anterior region and surface of sporozoites, except for the two refractile bodies. As the invasion of DF-1 cells progressed, fluorescence intensity was increased, and Et60s-RPL12 was localized to the parasitophorous vacuole membrane (PVM). The secretion assay results using staurosporine indicated that this protein was secreted, but not from micronemes. The role of Et60s-RPL12 in invasion was evaluated in vitro. The results of the invasion assay showed that polyclonal antibody inhibited host cell invasion by the parasite, which reached about 12%. However, the rate of invasion was not correlated with the concentration of IgG.


Assuntos
Eimeria tenella/genética , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Ceco/parasitologia , Linhagem Celular , Embrião de Galinha , Galinhas , Biologia Computacional , DNA Complementar/genética , DNA Complementar/metabolismo , Eimeria tenella/química , Eletroforese em Gel de Poliacrilamida , Fezes/parasitologia , Fibroblastos , Técnica Indireta de Fluorescência para Anticorpo , Biossíntese de Proteínas , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Ribossômicas/química , Organismos Livres de Patógenos Específicos , Transcrição Genética
5.
Viruses ; 12(7)2020 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-32605306

RESUMO

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), and norovirus (NV) are highly contagious pathogens that threaten human health. Here we focused on the antiviral potential of the medicinal herb, Saxifraga spinulosa (SS). Water-soluble extracts of SS were prepared, and their virus-inactivating activity was evaluated against the human virus pathogens SARS-CoV-2 and IAV; we also examined virucidal activity against feline calicivirus and murine norovirus, which are surrogates for human NV. Among our findings, we found that SS-derived gallocatechin gallate compounds were capable of inactivating all viruses tested. Interestingly, a pyrogallol-enriched fraction (Fr 1C) inactivated all viruses more rapidly and effectively than did any of the component compounds used alone. We found that 25 µg/mL of Fr 1C inactivated >99.6% of SARS-CoV-2 within 10 s (reduction of ≥2.33 log10 TCID50/mL). Fr 1C resulted in the disruption of viral genomes and proteins as determined by gel electrophoresis, electron microscopy, and reverse transcription-PCR. Taken together, our results reveal the potential of Fr 1C for development as a novel antiviral disinfectant.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Vírus da Influenza A/efeitos dos fármacos , Norovirus/efeitos dos fármacos , Extratos Vegetais/farmacologia , Plantas Medicinais , Saxifragaceae , Betacoronavirus/ultraestrutura , Calicivirus Felino/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Genoma Viral/efeitos dos fármacos , Testes de Hemaglutinação , Humanos , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais/efeitos dos fármacos
6.
Wei Sheng Yan Jiu ; 49(3): 453-457, 2020 May.
Artigo em Chinês | MEDLINE | ID: mdl-32693896

RESUMO

OBJECTIVE: To study the digestive stability of 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) protein and phosphinothricina cetyltransferase(PAT) protein in simulated gastric fluid. METHODS: The component of simulated gastric fluid was based on the method of target protein digestive stability in simulative gastric and intestinal in national standard of the People's Republic of China(Published by the Ministry of Agriculture No. 869-2-2007). The test model of stability of different protein to digestion in Simulated Gastric Fluid was established by dodecyl sulfate, sodium salt-polyacrylamide gel electrophoresis(SDS-PAGE)and western blot. The degradation of EPSPS protein and PAT protein in simulated gastric fluid at different digestion time points were analyzed. RESULTS: The experiment showed that EPSPS protein and PAT protein were completely digested within 15 s in simulated gastric fluid, no any remain of protein was detected by SDS-PAGE and Western blot, indicating that EPSPS protein and PAT protein were easily digested in the simulated gastric. CONCLUSION: EPSPS protein and PAT protein do not have immunogenicity after digestion with simulated gastric fluid.


Assuntos
Digestão , Proteínas , Western Blotting , China , Eletroforese em Gel de Poliacrilamida
7.
PLoS One ; 15(7): e0228607, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645009

RESUMO

Among the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectrometry. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LO in vitro.


Assuntos
Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Cálcio/metabolismo , Proteínas Ativadoras de 5-Lipoxigenase/química , Ácido Araquidônico/química , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Nanoestruturas/ultraestrutura , Ligação Proteica , Conformação Proteica , Sacarose
8.
J Sports Sci ; 38(20): 2390-2395, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32602402

RESUMO

The purpose of the present study was to compare the myosin heavy chain (MHC) isoform composition of the deltoid and vastus lateralis muscles of the dominant and non-dominant limbs in handball players. Eleven male Greek elite handball players (age 22.6 ± 1.9 yrs, training experience 10.6 ± 2.1 yrs, height 184.1 ± 4.1 cm, and weight 81.0 ± 12.5 kg) participated in the study. Four muscle biopsies were obtained from the dominant and non-dominant deltoid and vastus lateralis muscles during the in-season period. The MHC composition was determined using SDS-PAGE. No significant difference was found between the dominant and non-dominant muscles; Deltoid muscle: MHC I [(95%CI = -1.22, 0.33), P = 0.228], MHC ΙΙa [(95%CI = -0.32, 1.59), P = 0.168] and MHC IIx [(95%CI = -1.49, 1.10), P = 0.749]; Vastus lateralis muscle: MHC I [(95%CI = -0.38, 0.63), P = 0.586], MHC ΙΙa [(95%CI = -0.50, 0.65), P = 0.783] and MHC IIx [(95%CI = -1.08, 0.42), P = 0.355]. The findings of the present study indicate that the greater use of the dominant limbs for throwing actions and body movements in handball do not lead to altered MHC isoform composition compared to the non-dominant limbs.


Assuntos
Músculo Deltoide/química , Cadeias Pesadas de Miosina/análise , Músculo Quadríceps/química , Esportes/fisiologia , Eletroforese em Gel de Poliacrilamida , Humanos , Masculino , Cadeias Pesadas de Miosina/química , Isoformas de Proteínas/análise , Adulto Jovem
9.
Toxicon ; 184: 19-27, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32479836

RESUMO

Bothrops brazili is a pitviper from Amazonian region, responsible for many accidents in Peru. Despite its relevance, its venom has not been extensively characterized. In the present work, Bothrops brazili venom (BbV) components were analyzed by RP-HPLC, SDS-PAGE and MALDI-TOF/TOF. Approximately 37 proteins were identified, belonging to 7 families. Snake venom metalloproteinases (SVMPs) were the most abundant proteins of the venom (33.05%), followed by snake venom serine proteinases (SVSPs, 26.11%), phospholipases A2 (PLA2, 25.57%), snake C-type lectins (CTLs, 9.61%), L-aminoacid oxidase (LAAO, 3.80%), cystein-rich secretory proteins (CRISP, 1.67%) and Bradykinin-potentiating peptide (BPP, 0.20%). In vitro enzymatic activities of BbV showed high levels of SVMP activity and reduced Hyal activity in comparison with other bothropic venoms. Furthermore, BbV reduced VERO cells viability. ELISA and Western Blotting showed that both Peruvian and Brazilian bothropic antivenoms were able to recognize BbV components. This work provides an overview of BbV venom content and indicates a potential efficiency of Peruvian and Brazilian antivenoms to treat accidents with this species.


Assuntos
Bothrops , Venenos de Crotalídeos/toxicidade , Animais , Antivenenos , Western Blotting , Brasil , Chlorocebus aethiops , Cromatografia Líquida de Alta Pressão , Venenos de Crotalídeos/metabolismo , Eletroforese em Gel de Poliacrilamida , L-Aminoácido Oxidase/metabolismo , Peru , Fosfolipases A2/química , Proteômica , Serina Proteases/metabolismo , Células Vero
10.
Toxicon ; 184: 94-98, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32533959

RESUMO

The emergence of novel venom extraction techniques over the last half-century has greatly facilitated advances in the field of cnidarian research. A new recovery protocol utilizing ethanol as the primary stimulant in nematocyst discharge was recently published, however in vitro examination of the venom on organic models was not performed. This present study reports an original comparison of the chemically-induced discharge technique in vitro with a commonly used saltwater extraction method. Size-exclusion chromatography revealed distinct differences in venom profiles between the two methods: the saltwater recovery method FPLC profile and SDS-PAGE gel were similar to previously published results, whereas the ethanol-induced method was not. SDS-PAGE gel revealed distinct 40-55 kDa bands of previously identified cardiotoxic proteins recovered from the saltwater method, whereas the ethanol-induced method yielded degraded venom protein bands. A concentration-response curve generated through xCELLigence Real-Time Cell Analysis (RTCA) revealed a dramatic decrease in human cardiomyocyte activity when venom recovered via saltwater discharge was applied to these cells. With the exception of one sample, all ethanol-induced recovered venom failed to prompt a concentration-dependent decrease in cell survival when applied to human cardiomyocytes, resulting in a significant difference in IC50 concentrations between the compared venom samples. The data presented here facilitates an improved understanding of the parameters and analyses that are essential when developing and utilizing novel techniques for future cnidarian venom extraction research and supports the conclusion that recovery of venom from the tentacles of the box jellyfish Chironex fleckeri by ethanol is not an effective, efficient, or comprehensive extraction method compared to the published method of saltwater degradation of tentacles and bead mill extraction.


Assuntos
Venenos de Cnidários/análise , Cubomedusas , Animais , Sobrevivência Celular , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Miócitos Cardíacos , Nematocisto
11.
Food Chem ; 329: 127193, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32516711

RESUMO

This study was aimed to produce bioactive peptides from optimally fermented tempe, and map their overall bioactivities. There were three preparative methods utilized for producing tempe-based peptides, such as water-facilitated extraction, alcalase, and papain hydrolysis, and in combination with membrane filtration. Fermenting soybean at 144 h was selected as the optimum time based on protein content and degree of hydrolysis. Through SDS-PAGE analysis, an increased degree of hydrolysis with longer fermentation time was confirmed. The best preparative method for producing bioactive peptides was through papain hydrolysis and followed by 5 kDa membrane filtration. By this, the enhancement was distinct for antioxidant activity, ACE-, α-glucosidase-, and Kunitz trypsin-inhibitory activity. The annotated peptide sequences resulting from Nano LC Ultimate 3000 Series System tandem Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer were matched with the BIOPEP database. The major bioactivities of tempe peptides obtained were as an ACE inhibitor, antioxidant, and antithrombotic.


Assuntos
Peptídeos/química , Alimentos de Soja/análise , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Antioxidantes/química , Cromatografia Líquida de Alta Pressão , Bases de Dados de Proteínas , Eletroforese em Gel de Poliacrilamida , Fermentação , Filtração , Farinha/análise , Hidrólise , Espectrometria de Massas , Papaína/metabolismo , Peptídeos/metabolismo , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Solubilidade , Soja/metabolismo , Subtilisinas/metabolismo
12.
Food Chem ; 330: 127319, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32569936

RESUMO

The influence of fresh egg white (EW) addition on the quality characteristics and protein aggregation in oat noodles containing wheat flour and gluten was studied. EW addition decreased cooking loss and increased cooking time of 70% oat noodles. The hardness, chewiness, tensile force and tensile distance improved significantly. A smooth surface and continuous protein network were observed by scanning electron microscopy (SEM) after adding EW. After cooking, the peak area in SE-HPLC profile of 70% oat noodles with EW decreased obviously. The extractabilities of protein in sodium dodecyl sulfate containing medium (SDSEP) of cooked wheat and oat noodles under non-reducing condition were lower than those of samples under reducing condition. The protein bands changes in SDS-PAGE profiles showed that EW could induce disulfide cross-linking of proteins in noodles. EW addition promoted proteins interaction and improved the cooking and texture properties of oat noodles.


Assuntos
Avena , Clara de Ovo/química , Farinha , Qualidade dos Alimentos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Culinária , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Farinha/análise , Indústria de Processamento de Alimentos , Glutens , Dureza , Microscopia Eletrônica de Varredura , Agregados Proteicos , Triticum
13.
Food Chem ; 330: 127324, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32569938

RESUMO

Enzymes currently used in cheesemaking have various drawbacks, and there is a continual need to find new coagulants. This study describes the extraction and biochemical characterization of two proteases from the red alga Gracilaria edulis. The proteases were extracted with phosphate buffer and partially purified by ammonium sulphate precipitation and dialysis. The enzymes exhibited optimum caseinolytic activity at 60 °C and a pH range of 6-8. They showed a high ratio of milk-clotting over caseinolytic activity, indicating they had an excellent milk-clotting ability. The proteases were confirmed to be serine protease and metalloprotease with molecular weight (MW) of 44 and 108 kDa. They exhibited high hydrolytic activity on κ-caseins, cleaving κ-casein at four main sites, one of which being the same as that of calf rennet, which is the first reported for an algal protease. The findings demonstrated that the proteases could potentially be used as a milk coagulant in cheesemaking.


Assuntos
Caseínas/metabolismo , Gracilaria/enzimologia , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/metabolismo , Alga Marinha/enzimologia , Sulfato de Amônio , Animais , Caseínas/química , Fracionamento Químico , Quimosina/metabolismo , Eletroforese em Gel de Poliacrilamida , Gracilaria/química , Concentração de Íons de Hidrogênio , Hidrólise , Leite/química , Leite/metabolismo , Peso Molecular , Peptídeo Hidrolases/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/metabolismo , Alga Marinha/química , Serina Proteases/química , Serina Proteases/isolamento & purificação , Serina Proteases/metabolismo , Espectrometria de Massas em Tandem , Temperatura
14.
Am J Respir Crit Care Med ; 202(6): 812-821, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32584597

RESUMO

Rationale: Coronavirus disease (COVID-19) is a global threat to health. Its inflammatory characteristics are incompletely understood.Objectives: To define the cytokine profile of COVID-19 and to identify evidence of immunometabolic alterations in those with severe illness.Methods: Levels of IL-1ß, IL-6, IL-8, IL-10, and sTNFR1 (soluble tumor necrosis factor receptor 1) were assessed in plasma from healthy volunteers, hospitalized but stable patients with COVID-19 (COVIDstable patients), patients with COVID-19 requiring ICU admission (COVIDICU patients), and patients with severe community-acquired pneumonia requiring ICU support (CAPICU patients). Immunometabolic markers were measured in circulating neutrophils from patients with severe COVID-19. The acute phase response of AAT (alpha-1 antitrypsin) to COVID-19 was also evaluated.Measurements and Main Results: IL-1ß, IL-6, IL-8, and sTNFR1 were all increased in patients with COVID-19. COVIDICU patients could be clearly differentiated from COVIDstable patients, and demonstrated higher levels of IL-1ß, IL-6, and sTNFR1 but lower IL-10 than CAPICU patients. COVID-19 neutrophils displayed altered immunometabolism, with increased cytosolic PKM2 (pyruvate kinase M2), phosphorylated PKM2, HIF-1α (hypoxia-inducible factor-1α), and lactate. The production and sialylation of AAT increased in COVID-19, but this antiinflammatory response was overwhelmed in severe illness, with the IL-6:AAT ratio markedly higher in patients requiring ICU admission (P < 0.0001). In critically unwell patients with COVID-19, increases in IL-6:AAT predicted prolonged ICU stay and mortality, whereas improvement in IL-6:AAT was associated with clinical resolution (P < 0.0001).Conclusions: The COVID-19 cytokinemia is distinct from that of other types of pneumonia, leading to organ failure and ICU need. Neutrophils undergo immunometabolic reprogramming in severe COVID-19 illness. Cytokine ratios may predict outcomes in this population.


Assuntos
Reação de Fase Aguda/imunologia , Proteínas de Transporte/metabolismo , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/metabolismo , Citocinas/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ácido Láctico/metabolismo , Proteínas de Membrana/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/metabolismo , Hormônios Tireóideos/metabolismo , alfa 1-Antitripsina/imunologia , Reação de Fase Aguda/metabolismo , Adulto , Idoso , Betacoronavirus , Western Blotting , Estudos de Casos e Controles , Infecções Comunitárias Adquiridas/imunologia , Infecções Comunitárias Adquiridas/metabolismo , Infecções por Coronavirus/mortalidade , Infecções por Coronavirus/fisiopatologia , Estado Terminal , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Hospitalização , Humanos , Unidades de Terapia Intensiva , Interleucina-10/imunologia , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Interleucina-8/imunologia , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Neutrófilos/metabolismo , Pandemias , Fosforilação , Pneumonia/imunologia , Pneumonia/metabolismo , Pneumonia Viral/mortalidade , Pneumonia Viral/fisiopatologia , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Índice de Gravidade de Doença , alfa 1-Antitripsina/metabolismo
15.
Food Chem ; 324: 126894, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32361094

RESUMO

This study aims to extract acorn protein isolate (API) from locally abundant waste acorn fruit and investigate its emulsification behavior by mixing different protein (0.1-2% w/v) and oil volume concentrations (5-45% v/v). Significant decrease in emulsifying activity index (EAI) and an increase in emulsifying stability index (ESI) were observed with an increase in API concentrations (P < 0.05). Droplet sizes of emulsions and viscosity were observed to decrease significantly (P < 0.05) with increase in API concentration while the increase was observed in interfacial protein concentration (Г). In contrast, increase in oil volume concentration results in increase of droplet sizes, packing fractions and viscosity, while decrease in Г values was observed. The results reveal that main fractions of API (66.2-14.4 kDa) were migrated to oil-water interface for emulsion stabilization. These results demonstrate the potential application of API in food formulation and development.


Assuntos
Emulsificantes/química , Óleos/química , Proteínas de Plantas/química , Quercus/metabolismo , Eletroforese em Gel de Poliacrilamida , Emulsões/química , Frutas/metabolismo , Viscosidade
16.
Invest Ophthalmol Vis Sci ; 61(5): 28, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32421148

RESUMO

Purpose: Bestrophinopathies are a group of untreatable inherited retinal dystrophies caused by mutations in the retinal pigment epithelium (RPE) Cl- channel bestrophin 1. We tested whether sodium phenylbutyrate (4PBA) could rescue the function of mutant bestrophin 1 associated with autosomal dominant and recessive disease. We then sought analogues of 4PBA with increased potency and determined the mode of action for 4PBA and a lead compound 2-naphthoxyacetic acid (2-NOAA). Lastly, we tested if 4PBA and 2-NOAA could functionally rescue bestrophin 1 function in RPE generated from induced pluripotent stem cells (iPSC-RPEs) derived from patients with a dominant or recessive bestrophinopathy. Methods: Global and plasma membrane expression was determined by Western blot and immunofluorescent microscopy, respectively. The effect of 4PBA and 2-NOAA on transcription was measured by quantitative RT-PCR and the rate of protein turnover by cycloheximide chase and Western blot. Channel function was measured by whole-cell patch clamp. Results: 4PBA and 2-NOAA can rescue the global and membrane expression of mutant bestrophin 1 associated with autosomal dominant disease (Best vitelliform macular dystrophy [BVMD]) and autosome recessive bestrophinopathy (ARB), and these small molecules have different modes of action. Both 4PBA and 2-NOAA significantly increased the channel function of mutant BVMD and ARB bestrophin 1 in HEK293T and iPSC-RPE cells derived from patients with BVMD and ARB. For 4PBA, the increased mutant channel function in BVMD and ARB iPSC-RPE was equal to that of wild-type iPSC-RPE bestrophin 1. Conclusions: The restoration of bestrophin 1 function in patient-derived RPE confirms the US Food and Drug Administration-approved drug 4PBA as a promising therapeutic treatment for bestrophinopathies.


Assuntos
Antineoplásicos/farmacologia , Bestrofinas/genética , Oftalmopatias Hereditárias/tratamento farmacológico , Regulação da Expressão Gênica/fisiologia , Glicolatos/farmacologia , Fenilbutiratos/farmacologia , Doenças Retinianas/tratamento farmacológico , Epitélio Pigmentado da Retina/efeitos dos fármacos , Western Blotting , Membrana Celular/metabolismo , Canais de Cloreto/metabolismo , Cicloeximida/farmacologia , Eletroforese em Gel de Poliacrilamida , Oftalmopatias Hereditárias/genética , Oftalmopatias Hereditárias/metabolismo , Genes Recessivos , Células HEK293/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia de Fluorescência , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase em Tempo Real , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transfecção
17.
Nat Med ; 26(7): 1033-1036, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32398876

RESUMO

Here, we describe a serological enzyme-linked immunosorbent assay for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma and is amenable to scaling. Serological assays are of critical importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy and investigate correlates of protection.


Assuntos
Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/imunologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/imunologia , Soroconversão , Adulto , Betacoronavirus/patogenicidade , Estudos de Casos e Controles , Infecções por Coronavirus/sangue , Infecções por Coronavirus/terapia , Infecções por Coronavirus/virologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização Passiva , Estudos Longitudinais , Pessoa de Meia-Idade , Testes de Neutralização , Pandemias , Pneumonia Viral/terapia , Pneumonia Viral/virologia , Adulto Jovem
18.
J Food Sci ; 85(6): 1707-1716, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32449946

RESUMO

In this paper, we studied the effect of glycosylation reaction on the molecular structure and functional properties of whey protein isolate (WPI), and studied the effect of reaction temperature (50 to 90 °C) on the molecular structure and functional properties of WPI-dextran conjugates (WPI-D). The results of the extent of glycation (EG) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the formation of WPI-D. Circular dichroism (CD), Fourier transform infrared spectrum, and fluorescence spectroscopy indicated that the molecular structure of WPI was changed after glycosylation-the ß-sheet content was decreased and the tryptophan content was increased. The emulsifying properties and the ability to encapsulate ß-carotene of WPI-D were improved compared with WPI (P < 0.05). When the reaction temperature was 70 and 80 °C, the EG and the ability to encapsulate ß-carotene of WPI-D were better (P < 0.05), which was related to protein unfolding. However, due to the polymerization between the WPI molecules, the emulsion activity index of WPI-D and the ability to encapsulate ß-carotene were lowered at 90 °C (P < 0.05). Therefore, the glycosylation reaction can change the molecular structure and functional properties of WPI; the emulsifying properties and the ability to encapsulate ß-carotene of WPI-D can be changed by controlling the reaction temperature of glycosylation. PRACTICAL APPLICATION: The glycosylation reaction can change the molecular structure and functional properties of Whey protein isolate; the emulsifying properties and the ability to encapsulate ß-carotene of WPI-dextran conjugates can be changed by controlling the reaction temperature of glycosylation.


Assuntos
Dextranos/química , Proteínas do Soro do Leite/química , beta Caroteno/química , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Emulsões/química , Glicosilação , Conformação Proteica , Temperatura
19.
J Parasitol ; 106(2): 276-282, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32294759

RESUMO

Heterobothrium okamotoi, a monogenean gill parasite, exhibits high host specificity for the tiger puffer, Takifugu rubripes, and it has been experimentally verified that the parasite cannot colonize either closely related species such as the grass puffer Takifugu niphobles or distantly related fish such as the red seabream Pagrus major. Previously, we demonstrated in T. rubripes that immunoglobulin M (IgM) with d-mannose affinity induced deciliation of the oncomiracidia, the first step of parasitism, indicating that the parasite utilizes the molecule as a receptor for infection. In the present study, we purified mannose-specific IgM from 2 nonhost species, T. niphobles and P. major, by affinity and gel-filtration chromatography techniques and compared their deciliation-inducing activity against H. okamotoi oncomiracidia. The IgM of the former showed activity, whereas the latter had no effect, suggesting that in addition to d-mannose-binding ability, the crystallizable fragment domain of IgM, which is not part of the antigen-binding domain, plays an important role in host recognition by the oncomiracidia, such as direct binding to the parasites. It also suggests that the host specificity of H. okamotoi is relatively low upon initial recognition, and the specificity is established by exclusion in nonhosts during a later stage.


Assuntos
Ectoparasitoses/veterinária , Doenças dos Peixes/parasitologia , Imunoglobulina M/fisiologia , Manose/imunologia , Platelmintos/imunologia , Takifugu/parasitologia , Sequência de Aminoácidos , Animais , Western Blotting , Cílios/imunologia , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/imunologia , Ectoparasitoses/imunologia , Ectoparasitoses/parasitologia , Eletroforese em Gel de Poliacrilamida , Doenças dos Peixes/imunologia , Expressão Gênica , Brânquias/parasitologia , Especificidade de Hospedeiro , Concentração de Íons de Hidrogênio , Imunoglobulina M/sangue , Imunoglobulina M/genética , Imunoglobulina M/isolamento & purificação , Membrana Mucosa/química , Membrana Mucosa/imunologia , Membrana Mucosa/parasitologia , Platelmintos/patogenicidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Takifugu/imunologia , Infecções por Trematódeos/imunologia , Infecções por Trematódeos/parasitologia , Infecções por Trematódeos/veterinária
20.
J Parasitol ; 106(2): 283-290, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32296849

RESUMO

The erythrocytic-stage surface protein equi merozoite antigen 1 (EMA-1) of Theileria equi is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In this study, BALB/c mice were immunized with purified recombinant EMA-1 to prepare monoclonal antibody (mAb) against T. equi EMA-1, and 1 mAb 5H2 was obtained that showed good reaction with infected red blood cells (RBC) in the indirect immunofluorescence assay (IFA). To develop a rapid serological detection method for T. equi infection in Xinjiang Uygur Autonomous Region, China, recombinant EMA-1 originating from the local T. equi strain and the mAb to EMA-1 were employed to develop an immunochromatographic test (ICT) to detect antibodies to T. equi in horse sera. The ICT showed high sensitivity and specificity and no cross-reaction with Babesia caballi. Ninety-two horse serum samples collected from Ili, Xinjiang, were tested by ICT and compared with the detection results of a commercial ELISA kit. The results showed that 56 of 92 (61%) serum samples were seropositive according to the ICT assay, and 50 (54%) samples were seropositive according to the ELISA kit. The ICT had a high coincidence (91.3%) but was more sensitive than the reference ELISA kit. To confirm whether the horses were infected by T. equi, 30 blood DNA samples from 92 horses were examined by PCR. The results showed that 14 of 30 (47%) horses were confirmed to be infected with T. equi by PCR, while 16 of 30 (53%) horses were seropositive by ICT. All PCR-positive horses were ICT-positive. The findings indicate that T. equi is endemic in Ili, Xinjiang, and that the ICT is reliable as a serological diagnosis method. The ICT developed in this study could be an efficient diagnostic tool to detect T. equi infection in horses in the Xinjiang area.


Assuntos
Antígenos de Protozoários/imunologia , Doenças dos Cavalos/parasitologia , Proteínas de Protozoários/imunologia , Theileria/isolamento & purificação , Theileriose/parasitologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/isolamento & purificação , Western Blotting , China , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Eritrócitos/parasitologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Doenças dos Cavalos/sangue , Doenças dos Cavalos/diagnóstico , Cavalos , Hibridomas/citologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Sensibilidade e Especificidade , Baço/citologia , Baço/imunologia , Theileria/imunologia , Theileriose/sangue , Theileriose/diagnóstico , Células Tumorais Cultivadas
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA