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1.
Int J Biol Macromol ; 181: 793-800, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-33857510

RESUMO

Here, we compare the content and composition of polysaccharides derived from the mycelium (40.4 kDa intracellular polysaccharide, IPS) and culture (27.2 kDa extracellular polysaccharide, EPS) of Penicillium oxalicum. Their chemical structures investigated by IR, NMR, enzymolysis and methylation analysis indicate that both IPS and EPS are galactomannans composed of α-1,2- mannopyranose (Manp) and α-1,6-Manp in a backbone ratio of ~3:1, respectively, both decorated with ß-l,5-galactofuranose (Galf) side chains. A few ß-l,6-Galf residues were also detected in the IPS fraction. EPS and IPS have different molecular weights (Mw) and degrees of branching. IPS obtained by alkaline extraction of P. oxalicum have been reported to be galactofuranans, a composition different from our IPS. Up to now, there have been no reports on the fine structure of EPS. Our results of galectin-mediated hemagglutination demonstrate that IPS exhibits greater inhibitory effects on five galectins compared with EPS. In addition, we find that Galf, a five-membered ring form of galactose, can also inhibit galectins. IPS may provide a new source of galectin inhibitors. These results increase our understanding of structure-activity relationships of polysaccharides as galectin inhibitors.


Assuntos
Espaço Extracelular/química , Polissacarídeos Fúngicos/farmacologia , Galectinas/antagonistas & inibidores , Espaço Intracelular/química , Penicillium/química , Animais , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Ensaios Enzimáticos , Galectinas/metabolismo , Hemaglutinação/efeitos dos fármacos , Hidrólise , Metilação , Peso Molecular , Espectroscopia de Prótons por Ressonância Magnética , Ovinos , Espectroscopia de Infravermelho com Transformada de Fourier
2.
Int J Biol Macromol ; 171: 389-397, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33428960

RESUMO

Zizyphus mauritiana Lam. seeds (ZMS) have been used medicinally as sedative or hypnotic drugs in most of Asian countries. ZMS has significant benefits to the human health. Therefore, we have evaluated immunomodulatory effect of lectin extracted from these ZMSL in both in vitro and in vivo study. Anaphylaxis is a severe life-threatening allergic reaction and Arthus reaction is deposition of immune complex and complement system activation, so we hypothesized that if ZMSL can protect these severe allergic diseases. We have studied the effect of ZMSL on macrophages and Wistar albino rats and confirmed its protective effect against anaphylaxis and Arthus reaction. Results of this study suggest ZMSL have immunostimulatory and antiallergic activity.


Assuntos
Adjuvantes Imunológicos/isolamento & purificação , Antialérgicos/isolamento & purificação , Fatores Imunológicos/isolamento & purificação , Lectinas/isolamento & purificação , Ziziphus/química , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/uso terapêutico , Anafilaxia/prevenção & controle , Animais , Antialérgicos/farmacologia , Antialérgicos/uso terapêutico , Reação de Arthus/prevenção & controle , Antígenos de Grupos Sanguíneos , Inativadores do Complemento/isolamento & purificação , Inativadores do Complemento/farmacologia , Inativadores do Complemento/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Hemaglutinação/efeitos dos fármacos , Humanos , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Lectinas/farmacologia , Lectinas/uso terapêutico , Leucócitos/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Lisossomos/enzimologia , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Plantas Medicinais/química , Coelhos , Ratos Wistar , Sementes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
3.
Protein Pept Lett ; 28(4): 403-413, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32798370

RESUMO

BACKGROUND: The O. tesota lectin PF2 is a tetrameric protein with subunits of 33 kDa that recognizes only complex carbohydrates, resistant to proteolytic enzymes and has insecticidal activity against Phaseolus beans pest. OBJECTIVE: To explore PF2 lectin features at different protein structural levels and to evaluate the effect of temperature and pH on its functionality and conformational stability. METHODS: PF2 lectin was purified by affinity chromatography. Its primary structure was resolved by mass spectrometry and analyzed by bioinformatic tools, including its tertiary structure homology modeling. The effect of temperature and pH on its conformational traits and stability was addressed by dynamic light scattering, circular dichroism, and intrinsic fluorescence. The hemagglutinating activity was evaluated using a suspension of peripheral blood erythrocytes. RESULTS: The proposed PF2 folding comprises a high content of beta sheets. At pH 7 and 25°C, the hydrodynamic diameter (Dh) was found to be 12.3 nm which corresponds to the oligomeric native state of PF2 lectin. Dh increased under the other evaluated pH and temperature conditions, suggesting protein aggregation. At basic pH, PF2 exhibited low conformational stability. The native PF2 (pH 7) retained its full hemagglutinating activity up to 45°C and exhibited one transition state with a melting temperature of 76.8°C. CONCLUSION: PF2 showed distinctive characteristics found in legume lectins. The pH influences the functionality and conformational stability of the protein. PF2 lectin displayed a relatively narrow thermostability to the loss of secondary structure and hemagglutinating activity.


Assuntos
Fabaceae/química , Lectinas de Plantas/química , Eritrócitos/química , Hemaglutinação , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Domínios Proteicos , Estabilidade Proteica , Relação Estrutura-Atividade
4.
J Infect Chemother ; 27(3): 434-438, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33077365

RESUMO

INTRODUCTION: This study aimed to examine the immunity level against rubella in pregnant women of different birth cohorts. METHODS: In total, 512 pregnant women who visited a primary clinic between May 2019 and March 2020 were enrolled. Information in terms of the patients' hemagglutination inhibition (HI) titers, birthdate, obstetrical history, and vaccination history were collected. Participants were divided into three generational groups according to the vaccination policy in Japan. Publicly funded vaccination was administered twice as part of a routine program in group A (n = 11), once as part of a routine program and once in a catch-up program in group B (n = 181), and once in group C (n = 320). RESULTS: All groups had some women with negative rubella HI antibody titers (7.6% of all the women, 18.2% of group A, 9.4% of group B, and 6.3% of group C) and those with rubella HI antibody titers of ≤1:16 (45.1% of all women, 90.9% of group A, 56.4% of group B, and 37.2% of group C). Rubella HI antibody titers differed between the groups; group C had higher titers than that in group B. In groups B and C, the proportions of women with rubella HI antibody titers of ≤1:16 were not statistically different between primipara and multipara. CONCLUSIONS: Our study showed that an increase in immunity to rubella, a vaccine-preventable disease, is nevertheless required among childbearing women to prevent rubella and congenital rubella syndrome.


Assuntos
Hemaglutinação , Rubéola (Sarampo Alemão) , Anticorpos Antivirais , Feminino , Testes de Inibição da Hemaglutinação , Humanos , Japão/epidemiologia , Gravidez , Rubéola (Sarampo Alemão)/epidemiologia , Rubéola (Sarampo Alemão)/prevenção & controle , Vacinação
5.
Methods Mol Biol ; 2210: 97-112, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32815131

RESUMO

Porphyromonas gingivalis is a gram-negative, rod-shaped, nonmotile bacterium belonging to the phylum Bacteroidetes. It produces abundant amounts of proteases in both cell-associated and secretory forms, including a group of cysteine proteases referred to as gingipains, which have attracted much attention due to their high proteolytic activity associated with pathogenicity. Gingipains are grouped into arginine (R)-specific (RgpA and RgpB) and lysine (K)-specific (Kgp) types. Both Rgp (collective term for RgpA and RgpB) and Kgp gingipains play crucial roles in the virulence of P. gingivalis, including the degradation of host periodontal tissues, disruption of host defense mechanisms, and loss of viability in host cells, such as fibroblasts and endothelial cells. In addition to their function in virulence, gingipains are also essential for the growth and survival of P. gingivalis in periodontal pockets through the acquisition of amino acids and heme groups. Furthermore, Rgp and Kgp gingipains are critical in processing fimbriae and several bacterial proteins that contribute to hemagglutination, coaggregation, and hemoglobin binding. This chapter describes the methods used to analyze gingipains.


Assuntos
Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases Gingipaínas/metabolismo , Porphyromonas gingivalis/metabolismo , Animais , Arginina/metabolismo , Cisteína Endopeptidases/metabolismo , Células Endoteliais/metabolismo , Feminino , Fibroblastos/metabolismo , Fímbrias Bacterianas/metabolismo , Cobaias , Hemaglutinação/fisiologia , Hemaglutininas/metabolismo , Lisina/metabolismo , Virulência/fisiologia
6.
Methods Mol Biol ; 2210: 123-133, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32815133

RESUMO

The type IX secretion system (T9SS) is a protein secretion system for gingipain proteases and is found on the cell surface of Porphyromonas gingivalis. Proteins secreted by T9SS contain a signal peptide, functional domains, an immunoglobulin (Ig)-like domain, and a C-terminal domain (CTD). Thirty genes on the P. gingivalis chromosome encode proteins that possess the CTD, which is important for T9SS-mediated translocation to the cell surface across the outer membrane. In T9SS mutant strains, proteins accumulate as precursors in the cell and therefore exhibit a phenotype similar to that of secreted protein-deficient mutants. Black pigment productivity and hemagglutination are phenotypic features of P. gingivalis associated with the activity of gingipains. In P. gingivalis T9SS mutants, unprocessed gingipains with high molecular weights accumulate in the cell, and colony pigmentation and hemagglutination are not observed in the same phenotype as a gingipain null mutant.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Porphyromonas gingivalis/metabolismo , Adesinas Bacterianas/metabolismo , Animais , Cisteína Endopeptidases Gingipaínas/metabolismo , Hemaglutinação/fisiologia , Coelhos , Fatores de Virulência/metabolismo
7.
Sci Rep ; 10(1): 21109, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33273542

RESUMO

Porphyromonas gingivalis, a periodontal pathogen, translocates many virulence factors including the cysteine proteases referred to as gingipains to the cell surface via the type IX secretion system (T9SS). Expression of the T9SS component proteins is regulated by the tandem signaling of the PorXY two-component system and the ECF sigma factor SigP. However, the details of this regulatory pathway are still unknown. We found that one of the T9SS conserved C-terminal domain-containing proteins, PGN_0123, which we have designated PorA, is involved in regulating expression of genes encoding T9SS structural proteins and that PorA can be translocated onto the cell surface without the T9SS translocation machinery. X-ray crystallography revealed that PorA has a domain similar to the mannose-binding domain of Escherichia coli FimH, the tip protein of Type 1 pilus. Mutations in the cytoplasmic domain of the sensor kinase PorY conferred phenotypic recovery on the ΔporA mutant. The SigP sigma factor, which is activated by the PorXY two-component system, markedly decreased in the ΔporA mutant. These results strongly support a potential role for PorA in relaying a signal from the cell surface to the PorXY-SigP signaling pathway.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Porphyromonas gingivalis/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Mutação com Ganho de Função , Cisteína Endopeptidases Gingipaínas/farmacologia , Hemaglutinação/efeitos dos fármacos , Lipopolissacarídeos/metabolismo , Modelos Biológicos , Mapeamento de Peptídeos , Pigmentação/efeitos dos fármacos , Porphyromonas gingivalis/efeitos dos fármacos , Domínios Proteicos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
8.
J Bacteriol ; 203(2)2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33106345

RESUMO

Streptococcus gordonii is a commensal oral organism. Harmless in the oral cavity, S. gordonii is an opportunistic pathogen. S. gordonii adheres to body surfaces using surface adhesive proteins (adhesins), which are critical to subsequent formation of biofilm communities. As in most Gram-positive bacteria, S. gordonii surface proteins containing the C-terminal LPXTG motif cleavage sequence are processed by sortase A (SrtA) to become covalently attached to the cell wall. To characterize the functional diversity and redundancy in the family of SrtA-processed proteins, an S. gordonii DL1 markerless deletion mutant library was constructed of each of the 26 putative SrtA-processed proteins. Each library member was evaluated for growth in rich medium, biofilm formation on plastic, saliva and salivary fractions, cell surface hydrophobicity (CSH), hemagglutination, and integration into an ex vivo plaque biofilm community. Library members were compared to the non-SrtA-processed adhesins AbpA and AbpB. While no major growth differences in rich medium were observed, many S. gordonii LPXTG/A proteins impacted biofilm formation on one or more of the substrates. Several mutants showed significant differences in hemagglutination, hydrophobicity, or fitness in the ex vivo plaque model. From the identification of redundant and unique functions in these in vitro and ex vivo systems, functional stratification among the LPXTG/A proteins is apparent.IMPORTANCE S. gordonii interactions with its environment depend on the complement of cell wall proteins. A subset of these cell wall proteins requires processing by the enzyme sortase A (SrtA). The identification of SrtA-processed proteins and their functional characterization will help the community to better understand how S. gordonii engages with its surroundings, including other microbes, integrates into the plaque community, adheres to the tooth surface, and hematogenously disseminates to cause blood-borne infections. This study identified 26 putative SrtA-processed proteins through creation of a markerless deletion mutant library. The library was subject to functional screens that were chosen to better understand key aspects of S. gordonii physiology and pathogenesis.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Cisteína Endopeptidases/metabolismo , Streptococcus gordonii/fisiologia , Aminoaciltransferases/química , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/química , Placa Dentária/microbiologia , Deleção de Genes , Hemaglutinação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Boca/microbiologia , Saliva/microbiologia , Ovinos/sangue , Streptococcus gordonii/genética , Streptococcus gordonii/crescimento & desenvolvimento
9.
J Virol ; 94(23)2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-32907980

RESUMO

Humoral immune responses to influenza virus vaccines in elderly individuals are poorly adapted toward new antigenically drifted influenza virus strains. Instead, older individuals respond in an original antigenic sin fashion and produce much more cross-reactive but less potent antibodies. Here, we investigated four influenza B virus hemagglutinin (HA) head specific, hemagglutination inhibition-inactive monoclonal antibodies (MAbs) from elderly individuals. We found that they were broadly reactive within the B/Victoria/2/1987-like lineage, and two were highly cross-reactive with B/Yamagata/16/1988-like lineage viruses. The MAbs were found to be neutralizing, to utilize Fc effector functions, and to be protective against lethal viral challenge in a mouse model. In order to identify residues on the influenza B virus hemagglutinin interacting with the MAbs, we generated escape mutant viruses. Interestingly, escape from these MAbs led to numerous HA mutations within the head domain, including in the defined antigenic sites. We observed that each individual escape mutant virus was able to avoid neutralization by its respective MAb along with other MAbs in the panel, although in many cases binding activity was maintained. Point mutant viruses indicated that K90 is critical for the neutralization of two MAbs, while escape from the other two MAbs required a combination of mutations in the hemagglutinin. Three of four escape mutant viruses had increased lethality in the DBA2/J mouse model. Our work indicates that these cross-reactive antibodies have the potential to cause antigenic drift in the viral population by driving mutations that increase virus fitness. However, binding activity and cross-neutralization were maintained by a majority of antibodies in the panel, suggesting that this drift may not lead to escape from antibody-mediated protection.IMPORTANCE Understanding the immune response that older individuals mount to influenza virus vaccination and infection is critical in order to design better vaccines for this age group. Here, we show that older individuals make broadly neutralizing antibodies that have no hemagglutination-inhibiting activity and are less potent than strain-specific antibodies. These antibodies could drive viral escape from neutralization but did not result in escape from binding. Given their different mechanisms of action, they might retain protective activity even against escape variants.


Assuntos
Anticorpos Antivirais/imunologia , Testes de Inibição da Hemaglutinação/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Hemaglutininas/imunologia , Vírus da Influenza B/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos Virais/imunologia , Reações Cruzadas , Modelos Animais de Doenças , Feminino , Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza B/genética , Camundongos , Camundongos Endogâmicos DBA , Mutação , Testes de Neutralização
10.
Front Immunol ; 11: 1565, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32849530

RESUMO

Peripheral tolerance is essential for silencing weakly autoreactive B cells that have escaped central tolerance, but it is unclear why these potentially pathogenic B cells are retained rather than being eliminated entirely. Release from peripheral tolerance restraint can occur under certain circumstances (i.e., strong TLR stimulus), that are present during infection. In this regard, we hypothesized that autoreactive B cells could function as a reserve population that can be activated to contribute to the humoral immune response, particularly with pathogens, such as HIV-1, that exploit immune tolerance to avoid host defense. In this study, we identify a population of autoreactive B cells with the potential to neutralize HIV-1 and experimentally release them from the functional restrictions of peripheral tolerance. We have previously identified murine monoclonal antibodies that displayed autoreactivity against histone H2A and neutralized HIV-1 in vitro. Here, we identify additional H2A-reactive IgM monoclonal antibodies and demonstrate that they are both autoreactive and polyreactive with self and foreign antigens and are able to neutralize multiple clades of tier 2 HIV-1. Flow cytometric analysis of H2A-reactive B cells in naïve wildtype mice revealed that these B cells are present in peripheral B cell populations and we further document that murine H2A-reactive B cells are restrained by peripheral tolerance mechanisms. Specifically, we show endogenous H2A-reactive B cells display increased expression of the inhibitory mediators CD5 and phosphatase and tensin homolog (PTEN) phosphatase and fail to mobilize calcium upon immunoreceptor stimulation; all characterized markers of anergy. Moreover, we show that toll-like receptor stimulation or provision of CD4 T cell help induces the in vitro production of H2A-reactive antibodies, breaking tolerance. Thus, we have identified a novel poly/autoreactive B cell population that has the potential to neutralize HIV-1 but is silenced by immune tolerance.


Assuntos
Alérgenos/imunologia , Autoimunidade , Linfócitos B/imunologia , Linfócitos B/metabolismo , Infecções por HIV/imunologia , HIV-1/imunologia , Histonas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Autoantígenos/imunologia , Células COS , Linhagem Celular , Chlorocebus aethiops , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Hemaglutinação , Interações Hospedeiro-Patógeno , Humanos , Imunização , Imunoglobulina M/imunologia , Camundongos , Tolerância Periférica , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Receptores Toll-Like/metabolismo
11.
Acta Biochim Biophys Sin (Shanghai) ; 52(10): 1081-1092, 2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-32852549

RESUMO

Plant lectins are carbohydrate-binding proteins with nonimmune origin, which can reversibly bind with carbohydrates, agglutinate cells, and precipitate polysaccharides and glycoconjugates. Plant lectins have attracted much attention for their anti-virus, anti-proliferation, and pro-apoptosis properties. Thus the exploration of new lectins has received special attention. Here we purified a mannose-binding lectin from the rhizomes of Liparis nervosa by ion exchange chromatography on DEAE-Sepharose, affinity chromatography on Mannose-Sepharose 4B, and gel filtration chromatography on Sephacryl S-100. The purified L. nervosa lectin (LNL) was identified to be a monomeric protein with a molecular mass of 13 kDa. LNL exhibited hemagglutinating activity towards rabbit erythrocytes, and its activity could be strongly inhibited by D-mannose, N-acetyl glucosamine and thyroglobulin. In vitro experiments showed that LNL exhibited a comparable anti-fungal activity against Piricularia oryzae (Cavara), Bipolaris maydis, Fusarium graminearum, and Sclerotium rolfsii, and anti-proliferation activity against tumor cells by inducing apoptosis. The full-length cDNA sequence of LNL is 715 bp in length and contains a 525 bp open reading frame (ORF) encoding a 110-residue mature protein. It was predicted to have three mannose-binding conserved motifs 'QXDXNXVXY'. The binding pattern of LNL was further revealed by homology modeling and molecular docking. We demonstrated that LNL is not only a potential therapeutic candidate against tumor but also a new anti-fungal agent.


Assuntos
Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Lectinas de Ligação a Manose/farmacologia , Orchidaceae/química , Lectinas de Plantas/farmacologia , Sequência de Aminoácidos , Animais , Antifúngicos/química , Antifúngicos/isolamento & purificação , Antifúngicos/metabolismo , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Basidiomycota/efeitos dos fármacos , Bipolaris/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Fusarium/efeitos dos fármacos , Hemaglutinação/efeitos dos fármacos , Humanos , Manose/metabolismo , Lectinas de Ligação a Manose/química , Lectinas de Ligação a Manose/isolamento & purificação , Lectinas de Ligação a Manose/metabolismo , Simulação de Acoplamento Molecular , Peso Molecular , Orchidaceae/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Lectinas de Plantas/metabolismo , Coelhos , Homologia de Sequência de Aminoácidos
12.
PLoS One ; 15(7): e0236172, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726321

RESUMO

There are several broadly neutralizing monoclonal antibodies that neutralize influenza viruses with different mechanisms from traditional polyclonal antibodies induced by vaccination. CT149, which is one of the broadly neutralizing antibodies, was also previously reported to neutralize group 2 and some of group 1 influenza viruses (13 out of 13 tested group 2 viruses and 5 out of 11 group 1 viruses). In this study, we developed another antibody with the aim of compensating partial coverage of CT149 against group 1 influenza viruses. CT120 was screened among different antibody candidates and mixed with CT149. Importantly, although the binding sites of CT120 and CT149 are close to each other, the two antibodies do not interfere. The mixture of CT120 and CT149, which we named as CT-P27, showed broad efficacy by neutralizing 37 viruses from 11 different subtypes, of both group 1 and 2 influenza A viruses. Moreover, CT-P27 showed in vivo therapeutic efficacy, long prophylactic potency, and synergistic effect with oseltamivir in influenza virus-challenged mouse models. Our findings provide a novel therapeutic opportunity for more efficient treatment of influenza.


Assuntos
Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/farmacologia , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos/imunologia , Antígenos Virais/imunologia , Hemaglutinação/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Camundongos , Testes de Neutralização , Vacinação
13.
Fish Shellfish Immunol ; 106: 1131-1138, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32702482

RESUMO

An O-acetyl sialic acid specific lectin was purified from the hemolymph of the marine crab Atergatis integerrimus by affinity chromatography using BSM (Bovine Submaxillary Mucin) coupled to cyanogen bromide activated Sepharose 4B and biospecific adsorption using formalinized buffalo erythrocytes. The purified AiL (Atergatis integerrimus lectin) showed an 1218 fold increase in specific activity when compared to the crude hemolymph agglutinin. The lectin, on non - denaturing PAGE showed a single band of 216 kDa and when subjected to SDS - PAGE, the lectin resolved into three subunits of molecular weight 70, 72 and 74 kDa. Physico chemical characterization revealed the lectin as pH and temperature sensitive, calcium dependent and sensitive to calcium chelators. Based on the calcium dependency of the lectin, AiL could be classified as a C-type lectin. The purified lectin agglutinated buffalo erythrocytes with greater avidity and was inhibited by the glycoproteins BSM, thyroglobulin, fetuin, PSM, and sugars raffinose, trehalose, l - fucose, α - Lactose, melibiose and GluNAc suggesting the affinity of the lectin to sialic acid. Reduction in HA with asialo buffalo erythrocytes and HAI titer with desialylated BSM, confirms the sialic acid specificity of the lectin. The reduction in HAI following de - O - acetylation confirms the specificity of the lectin for O - acetyl sialic acid. FTIR analysis confirms the purified lectin as a glycoprotein with spectral bands corresponding to amide bands and saccharides. Thus this study paves way to assess the therapeutic application of this lectin that could be targeted to modified sialic acid moieties that are expressed on the malignant cells and pathogenic microbes and also deduce the crystal structure of the lectin.


Assuntos
Braquiúros , Hemolinfa/química , Lectinas/isolamento & purificação , Animais , Búfalos , Cães , Eritrócitos , Glicoproteínas/química , Cobaias , Hemaglutinação , Humanos , Concentração de Íons de Hidrogênio , Lectinas/química , Camundongos , Ácido N-Acetilneuramínico , Neuraminidase/química , Coelhos , Ratos , Temperatura
14.
Int J Biol Macromol ; 163: 431-441, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32645492

RESUMO

Lectins are a specialized group of proteins with immense biological properties and applications. This study describes the purification and characterization of a lectin from Leucaena leucocephala seeds, a plant belonging to the Fabaceae family. Leucaena leucocephala lectin (LLL) was purified by a two-step purification method involving DEAE-cellulose anion exchange chromatography and Sephadex G-75 size exclusion chromatography. The isolated lectin displayed a high haemagglutination titre upon treatment with rabbit erythrocytes. SDS-PAGE and Reverse-Phase High performance liquid chromatography (RP-HPLC) analysis experimentally revealed the presence of three bands corresponding to 37, 27 and 20 kDa indicating the presence of isolectins. Periodic Acid Schiff's (PAS) staining of LLL confirmed the presence of glycoprotein. Various biochemical parameters were analysed to study its effect on the haemagglutination activity. Sugar inhibition studies experimentally revealed that Glucose was the most potent inhibitor. Fluorescence spectrometric analysis of LLL and Glucose indicated a strong interaction with an association constant of 0.159 × 103 M-1. Circular Dichroism spectroscopy indicated a higher alpha helical content (25.27%). LLL was observed to possess mitogenic activity against Peripheral blood mononuclear cells (PBMC). The present investigation reports the isolation of a novel lectin from this plant which could contribute towards the diagnostic studies of certain diseases and for its therapeutic potential.


Assuntos
Fabaceae/química , Glucose/química , Lectinas/química , Lectinas/farmacologia , Linfócitos/efeitos dos fármacos , Mitógenos/química , Mitógenos/farmacologia , Sementes/química , Animais , Carboidratos/química , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Eritrócitos/metabolismo , Glucose/metabolismo , Hemaglutinação/efeitos dos fármacos , Testes de Hemaglutinação , Humanos , Concentração de Íons de Hidrogênio , Íons/química , Lectinas/isolamento & purificação , Lectinas/metabolismo , Linfócitos/metabolismo , Metais/química , Mitógenos/isolamento & purificação , Peso Molecular , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Ligação Proteica , Coelhos , Temperatura
15.
J Clin Microbiol ; 58(9)2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32493784

RESUMO

We compared titers of antibodies against A/H1N1, A/H3N2, and B influenza virus strains collected pre- and postvaccination using hemagglutination inhibition (HI) and microneutralization (MN) assays and data from two vaccine trials: study 1, performed with a cell-grown trivalent influenza vaccine (TIVc) using cell-grown target virus in both assays, and study 2, performed with an egg-grown adjuvanted quadrivalent influenza vaccine (aQIVe) using egg-grown target virus. The relationships between HI- and MN-derived log-transformed titers were examined using different statistical techniques. Deming regression analyses showed point estimates for slopes generally close to 1 across studies and strains. The slope of regression was closest to 1 for A/H3N2 strain when either cell- or egg-grown viral target virus was used. Bland-Altman plots indicated a very small percentage of results outside 2 and 3 standard deviations. The magnitudes and directions of differences between titers in the two assays varied by study and strain. Mean differences favored the MN assay for A/H1N1 and B strains in study 1, whereas the titers determined by HI were higher than those determined by MN against the A/H3N2 strain. In study 2, mean differences favored the MN assay for A/H3N2 and B strains. Overall, the directions and magnitudes of the mean differences were similar between the two vaccines. The concordance correlation coefficient values ranged from 0.74 (A/H1N1 strain, study 1) to 0.97 (A/H3N2 strain, study 1). The comparative analysis demonstrates an overall strong positive correlation between the HI and MN assays. These data support the use of the MN assay to quantify the immune response of influenza vaccines in clinical studies, particularly for the A/H3N2 strain.


Assuntos
Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Anticorpos Antivirais , Hemaglutinação , Testes de Inibição da Hemaglutinação , Humanos , Vírus da Influenza A Subtipo H3N2 , Influenza Humana/prevenção & controle , Estações do Ano
16.
Int J Biol Macromol ; 161: 1079-1085, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32561277

RESUMO

Lectins are proteins of non-immunological origin that may play several biological applications, of which we can highlight the anti-inflammatory and antinociceptive activities. In this work, we evaluated the possible effect of orofacial antinociceptive activity of three plant lectins, Dioclea violacea (DVL - Man/Glc-binding), Vatairea macrocarpa (VML - Gal-binding) and PPL (Parkia platycephala - Man/Glc-binding) in adult zebrafish. Acute nociception was induced by menthol (1.2 µM), or capsaicin (4.93 µM) applied into in the upper lip (5.0 µL) of adult wild zebrafish. Zebrafish were pretreated by intraperitoneal injection (20 µL) with vehicle (Control) or lectins (0.025; 0.05 or 0.1 mg/mL) 30 min before induction. The effect of lectins on zebrafish locomotor behavior was evaluated with the open field test. Naive groups (n = 8) were included in all tests. Our results indicate that only PPL presented antinociceptive induced by capsaicin, suggesting the potential clinical application of PPL as inhibitor of orofacial nociception and that this effect may be due to the modulation of TRPV1 channel. In conclusion, lectins that exhibit affinity to the same or different carbohydrates do not necessarily have an antinociceptive effect on the orofacial nociception model, indicating that the glycan carbohydrate binding pattern may be related to the effect on nociception inhibition.


Assuntos
Lectinas/química , Lectinas/farmacologia , Monossacarídeos/química , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Fracionamento Químico , Cromatografia em Gel , Hemaglutinação , Testes de Hemaglutinação , Lectinas/isolamento & purificação , Vasoconstritores/química , Vasoconstritores/farmacologia , Peixe-Zebra
17.
Int J Biol Macromol ; 161: 417-430, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32526302

RESUMO

In the present study, a novel lectin was purified from the newly isolated cyanobacterium, Lyngabya confervoides MK012409 and tested for its antiviral and anticancer activity. Out of 30 isolates, Mabroka-s isolate which identified as Lyngabya confervoides MK012409 showed the highest agglutination titer. Lyngabyal lectin showed the greatest haemagglutination activity with pigeon/rabbit erythrocytes with a minimum concentration of 2.4 µg/ml. Physical characterization of Lyngabyal lectin showed ability to keep the activity at a higher temperature up to 80 °C with stability over a wide pH range (4-8) as well as its stability toward chemical denaturants. Carbohydrate specificity test revealed that the sugar alcohols completely inhibited the lectin haemagglutination activity. The electrophoretic analysis revealed that the lyngabyal lectin is a 140 kDa composed of two 70 kDa subunits. Lyngabyal lectin was able to inhibit the proliferation of MCF-7 and Caco-2 cancer cell lines with IC50 values of 246 ± 0.17 and 376.4 ± 0.34 µg/ml, respectively. Lyngabyal lectin also showed virucidal activity against HSV-1 with EC50 of 167 ± 0.52 ng/ml and inhibited plaque formation in the HSV-1 infected Vero cells with EC50 of 84.94 ± 0.34 ng/ml. These findings emphasize the ability of the lyngabyal lectin to fight breast and colon cancer besides it represents a promising antiviral agent.


Assuntos
Antineoplásicos/farmacologia , Antivirais/farmacologia , Cianobactérias/química , Hemaglutinação/efeitos dos fármacos , Lectinas/farmacologia , Animais , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Galinhas , Chlorocebus aethiops , Columbidae , Testes de Hemaglutinação/métodos , Herpesvirus Humano 1/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Células MCF-7 , Coelhos , Células Vero
18.
Carbohydr Polym ; 240: 116288, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32475569

RESUMO

Global increase of antibiotic-resistant pathogens as well as elevated content of drug residues in the foodstuffs and the environment urgently calls for new biocompatible antimicrobial biomaterials. Yeast mannans represent readily available source of biodegradable materials for tailor-made derivatives that could be effective in biomedical applications. Here, antimicrobial properties of quaternized mannans (DSQ 0.12, 0.24, 0.30, 0.62) from Candida albicans against clinical multi-resistant strains of Staphylococcus aureus are confronted with possible cytotoxicity against human cells. As expected, both effects increase with increasing degree of quaternization. However, it is possible to define the "window", at quaternized mannan with DSQ 0.30 with good anti-microbial effectiveness and low cytotoxicity. This derivative exhibit minimum inhibitory (MIC) and minimum bactericidal (MBC) concentration from 62.5 to 250 µg/mL and demonstrate good biofilm inhibition effect. Also acceptable values were obtained in hemagglutination and hemolytic activity assays and also in cytotoxicity tests on human fibroblasts.


Assuntos
Antibacterianos/farmacologia , Candida albicans , Mananas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Hemaglutinação/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/fisiologia
19.
Arq. bras. med. vet. zootec. (Online) ; 72(3): 915-920, May-June, 2020. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1129595

RESUMO

Nabumetone is used to reduce the pain and inflammation in rheumatoid arthritis. In the current study, immunomodulatory effect of Nabumetone is investigated in mice. The control group was administered normal saline orally as placebo. Nabumetone was administered orally via gavage in two treatment groups at 14mg/kg.b.w. doses and 28mg/kgb.w., respectively. Haemagglutination (HA) assay, Jerne hemolytic plaque and mice lethality assays were applied. In HA assay, the titer was significantly decreased in Nabumetone treatment groups (P< 0.001). In Jerne hemolytic plaque formation assay, there was a significant reduction (P< 0.001) in number of plaques in Nabumetone treated groups when compared with control. In mice lethality assay, there was a significant difference in mortality ratio of mice in control and Nabumetone treated groups (P< 0.001). Therefore, it is concluded that Nabumetone suppresses the humoral immune response in mice.(AU)


A nabumetona é usada na redução da dor e inflamação da artrite reumática. No presente estudo, o efeito imunomodulador é investigado em camundongos. O grupo de controle recebeu solução salina via oral como placebo. Nabumetona foi administrada oralmente via gavagem em dois grupos de tratamentos com doses de 14mg/kg.b.w. e 28mg/kgb.w., respectivamente. Foram realizados ensaios de hemaglutinação (HA), placa hemolítica de Jerne e letalidade dos camundongos. No ensaio HA, o grau foi significativamente menor nos grupos de tratamento com nabumetoma (P< 0.001). No ensaio de formação de placa hemolítica de Jerne houve redução significativa (P< 0.001) no número de placas em grupos tratados com nabumetoma comparado ao controle. No ensaio de letalidade dos camundongos houve diferença significativa no grau de mortalidade de camundongos no grupo de controle e grupos tratados com nabumetoma (P< 0.001). Portanto, conclui-se que a Nabumetoma suprime a resposta imune humoral em camundongos.(AU)


Assuntos
Animais , Camundongos , Imunidade Humoral/efeitos dos fármacos , Nabumetona/administração & dosagem , Fatores Imunológicos/análise , Artrite Reumatoide/veterinária , Solução Salina , Hemaglutinação
20.
Int J Biol Macromol ; 159: 455-460, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32437801

RESUMO

Pollen has been defined as dietary supplement used to supplement the diet in many countries, but the primary structure and activity of Camellia japonica pollen polysaccharide remain unclear. In this study, the water-soluble polysaccharide extracted from Camellia japonica pollen (WCPP) was fractionated into one neutral fraction (WCPP-N) and two acidic fractions (WCPP-A1 and WCPP-A2) by DEAE-cellulose column, and WCPP-A2 was further fractionated into two homogeneous sub-fractions (WCPP-A2a and WCPP-A2b) by Sepharose CL-6B column. Monosaccharide composition results showed that WCPP-N might mainly contain starch-like glucan as well as some arabinogalactan, while WCPP-A1, WCPP-A2 and its sub-fractions might mainly composed of rhamnogalacturonan I (RG-I) pectic polysaccharide domain backbone with some different types of side chains, including arabinan, galactan, and/or arabinogalactan. The primary structure analysis of WCPP-A2a by NMR spectra analysis suggested that WCPP-A2a was an RG-I-like pectic polysaccharide, branched at the O-4 of Rha residues in the backbone, with α-(1 â†’ 3,5)-L-arabinan as well as type-II arabinogalactan side chain to which were attached. The results of galectin-3-mediated hemagglutination assay indicated that WCPP-A2a exhibited the strongest inhibitory effect on galectin-3 with MIC value around 0.27 µg/mL. These results suggested the potential use of Camellia japonica pollen polysaccharide as a galectin3 inhibitor in functional foods.


Assuntos
Camellia/química , Galectina 3/antagonistas & inibidores , Pólen/química , Polissacarídeos/química , Polissacarídeos/farmacologia , Fracionamento Químico , Galectina 3/química , Hemaglutinação , Testes de Hemaglutinação , Espectroscopia de Ressonância Magnética , Peso Molecular , Monossacarídeos , Polissacarídeos/isolamento & purificação , Solubilidade , Água
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