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1.
Res Vet Sci ; 124: 256-262, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30999161

RESUMO

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of proteins strongly induced downstream of type I interferon signaling. The function of IFITs has been investigated extensively in mammals. IFIT5 is the sole protein in this family found in birds and little information is available about the function of avian IFIT5. In this study, duck IFIT5 was cloned from peripheral blood mononuclear cells. Multiple amino acid sequence alignment and phylogenetic analysis showed that duck IFIT5 is highly homologous to chicken IFIT5. Tissue specificity analysis demonstrated that duck IFIT5 was ubiquitously expressed in all examined tissues of five-day-old ducklings, with the highest expression levels in heart, followed by thymus, cerebrum, liver, and lung; kidney expressed the lowest. Quantitative real-time PCR (qRT-PCR) analysis revealed that duck IFIT5 expression rapidly increased both in vitro and in vivo after stimulation with polyinosinic:polycytidylic acid [poly (I:C)] and infection with virulent duck hepatitis A virus type 3 (DHAV-3), respectively. Altogether, these results indicate that the expression of duck IFIT5 is positively correlated with viral load and may play an important role in the immune response to DHAV-3 infection. This study lays a foundation for further research into the innate antiviral immune responses of ducklings.


Assuntos
Patos/genética , Patos/imunologia , Proteínas de Neoplasias/genética , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos/genética , Animais Recém-Nascidos/imunologia , Proteínas Aviárias/química , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Sequência de Bases , Clonagem Molecular , Vírus da Hepatite do Pato/fisiologia , Hepatite Viral Animal/imunologia , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Fases de Leitura Aberta , Filogenia , Infecções por Picornaviridae/imunologia , Infecções por Picornaviridae/veterinária , Poli I-C/farmacologia , Doenças das Aves Domésticas/imunologia , Alinhamento de Sequência
2.
Gene ; 702: 182-193, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-30910561

RESUMO

Programmed death-1 (PD-1) has a pivotal role in the attenuation of adaptive immune responses and peripheral tolerance. Here we describe the identification of the Pekin duck programmed death-1 orthologue (duPD-1). The duPD-1 cDNA encodes a 283-amino acid polypeptide that has an amino acid identity of 70%, 32% and 31% with chicken, murine and human PD-1, respectively. The duck PD-1 gene shares five conserved exons with chicken, murine and human PD-1 genes. A cluster of putative regulatory elements within the conserved region B (CR-B) of the basal promotor is conserved. Homology modeling was most compatible with the two ß-sheet IgV domain structure of murine PD-1. Contact residues, shown to be critical for binding of the respective human and murine PD-1 ligands are mostly conserved between avian and mammalian species, whereas residues that define the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) are highly conserved across higher vertebrates and frog. Constitutive expression of duPD-1 transcripts was predominantly found in lymphocyte-rich tissues, and mitogen-stimulation of duck peripheral blood mononuclear cells transiently increased duPD-1 mRNA expression. A soluble duPD-1 protein was expressed and shown to engage the identified duck PD-1 ligands. Our observations show considerable evolutionary conservation between mammalian and avian PD-1 orthologues. This work will facilitate further investigation of the role of PD-1 signaling in adaptive immunity in the Pekin duck, a non-mammalian vertebrate and pathogen host with relevance for human and animal health.


Assuntos
Proteínas Aviárias/química , Proteínas Aviárias/genética , Receptor de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/genética , Animais , Proteínas Aviárias/classificação , Mapeamento Cromossômico , Clonagem Molecular , Patos , Expressão Gênica , Ligantes , Modelos Moleculares , Filogenia , Receptor de Morte Celular Programada 1/classificação , Receptor de Morte Celular Programada 1/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Distribuição Tecidual
3.
Proc Natl Acad Sci U S A ; 116(14): 6766-6774, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30877242

RESUMO

Focal adhesion kinase (FAK) is a key signaling molecule regulating cell adhesion, migration, and survival. FAK localizes into focal adhesion complexes formed at the cytoplasmic side of cell attachment to the ECM and is activated after force generation via actomyosin fibers attached to this complex. The mechanism of translating mechanical force into a biochemical signal is not understood, and it is not clear whether FAK is activated directly by force or downstream to the force signal. We use experimental and computational single-molecule force spectroscopy to probe the mechanical properties of FAK and examine whether force can trigger activation by inducing conformational changes in FAK. By comparison with an open and active mutant of FAK, we are able to assign mechanoactivation to an initial rupture event in the low-force range. This activation event occurs before FAK unfolding at forces within the native range in focal adhesions. We are also able to assign all subsequent peaks in the force landscape to partial unfolding of FAK modules. We show that binding of ATP stabilizes the kinase domain, thereby altering the unfolding hierarchy. Using all-atom molecular dynamics simulations, we identify intermediates along the unfolding pathway, which provide buffering to allow extension of FAK in focal adhesions without compromising functionality. Our findings strongly support that forces in focal adhesions applied to FAK via known interactions can induce conformational changes, which in turn, trigger focal adhesion signaling.


Assuntos
Trifosfato de Adenosina/química , Proteínas Aviárias/química , Proteína-Tirosina Quinases de Adesão Focal/química , Simulação de Dinâmica Molecular , Desdobramento de Proteína , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Galinhas , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/enzimologia , Adesões Focais/genética , Mecanotransdução Celular/genética , Domínios Proteicos , Relação Estrutura-Atividade
4.
MAbs ; 11(3): 532-545, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30735467

RESUMO

In antibody discovery, in-depth analysis of an antibody library and high-throughput retrieval of clones in the library are crucial to identifying and exploiting rare clones with different properties. However, existing methods have technical limitations, such as low process throughput from the laborious cloning process and waste of the phenotypic screening capacity from unnecessary repetitive tests on the dominant clones. To overcome the limitations, we developed a new high-throughput platform for the identification and retrieval of clones in the library, TrueRepertoire™. This new platform provides highly accurate sequences of the clones with linkage information between heavy and light chains of the antibody fragment. Additionally, the physical DNA of clones can be retrieved in high throughput based on the sequence information. We validated the high accuracy of the sequences and demonstrated that there is no platform-specific bias. Moreover, the applicability of TrueRepertoire™ was demonstrated by a phage-displayed single-chain variable fragment library targeting human hepatocyte growth factor protein.


Assuntos
Proteínas Aviárias , Técnicas de Visualização da Superfície Celular/métodos , Anticorpos de Cadeia Única , Animais , Proteínas Aviárias/biossíntese , Proteínas Aviárias/química , Proteínas Aviárias/genética , Bacteriófagos/genética , Galinhas , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética
5.
Prep Biochem Biotechnol ; 49(2): 192-201, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30734625

RESUMO

In this paper, we report a soluble expression based on Escherichia coli and two-step purification of a novel thioredoxin-tagged chicken interferon-α fusion protein (Trx-rChIFN-α) by using pET32a(+) expression system. The mature ChIFN-α gene was amplified by Reverse transcriptase-polymerase chain reaction (RT-PCR) and subcloned into pET-32a (+) vector prior to transformation into Rosetta (DE3) competent cells. After IPTG induction, the recombinant fusion protein was expressed efficiently in the soluble fraction. The protein purification was performed by nickel affinity chromatography and DEAE anion exchange chromatography. The purified product has a purity of 95% with a yield of 47.3 mg/L of culture. The specific activity of the fusion protein reaches to 2.0 × 107 IU/mg as determined in the CEF/VSV titration system. After excision of the Trx tag by enterokinase, the remaining solo protein was confirmed as rChIFN-α protein by SDS-PAGE, N-terminal sequencing and mass spectrometry. The effects of this Trx-rChIFN-α fusion protein against H9N2 influenza virus infection were also evaluated in ovo. The results showed that the Trx-rChIFN-α protein could significantly reduce the hemagglutination titer of H9N2 virus, and the H9N2 viruses HA gene copy numbers. These findings will enable us to produce large amount and bio-active rChIFN-α protein for future applications.


Assuntos
Antivirais/farmacologia , Proteínas Aviárias/farmacologia , Galinhas/genética , Vírus da Influenza A Subtipo H9N2/efeitos dos fármacos , Influenza Aviária/tratamento farmacológico , Interferon-alfa/farmacologia , Animais , Antivirais/química , Antivirais/isolamento & purificação , Antivirais/metabolismo , Proteínas Aviárias/química , Proteínas Aviárias/genética , Proteínas Aviárias/isolamento & purificação , Escherichia coli/genética , Interferon-alfa/química , Interferon-alfa/genética , Interferon-alfa/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/isolamento & purificação , Tiorredoxinas/farmacologia
6.
Poult Sci ; 98(6): 2360-2370, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30668770

RESUMO

Antioxidant peptides are increasingly attracting researchers in medicine and foods. In the present study, egg white hydrolysates of embryonated eggs hatched on the sixth day (EWHD) were segmented consecutively by ultrafiltration membranes with small tangential flow ultrafiltration system. Four segments with more than 30, 30 to 10 kDa, 10 to 5 kDa, and less than 5 kDa of molecular weight cut-off (MWCO) values were separated and were labeled as MWCOI, MWCOII, MWCOIII, and MWCOIV, respectively. The antioxidant activities of segments were investigated by performing DPPH•, •OH radical scavenging, ultra oxygen anion (O2-•), total antioxidant capacity, and reducing power experiments. The results indicated that MWCOI has the strongest scavenging activities on DPPH• radical. However, MWCOIV has the strongest scavenging activities on •OH, O2-•, total antioxidant capacity, and reducing power, which revealed that MWCOIV has strong antioxidant activity. MWCOIV was further separated into 14 fractions via semipreparative reverse-phased high-performance liquid chromatography (RP-HPLC), and their antioxidant activity was evaluated by different antioxidant assays in vitro. The fractions 10 and 7 had strong antioxidant activities. The purities of these 2 fractions were determined by analytical RP-HPLC. Moreover, both fractions 10 and 7 displayed high purity levels, and they were identified by quadrupole time-of-flight tandem mass spectrometry. Only fraction 10, with a molecular weight of 204 Da, can be identified to be Ser-Val. EWHD can be considered as a promising source of natural food antioxidants for the development of functional food.


Assuntos
Antioxidantes/isolamento & purificação , Proteínas Aviárias/isolamento & purificação , Embrião de Galinha/química , Peptídeos/isolamento & purificação , Animais , Antioxidantes/química , Proteínas Aviárias/química , Galinhas , Cromatografia Líquida de Alta Pressão/veterinária , Clara de Ovo/química , Óvulo/química , Peptídeos/química , Hidrolisados de Proteína/química , Hidrolisados de Proteína/isolamento & purificação , Espectrometria de Massas em Tandem/veterinária
7.
Br Poult Sci ; 60(2): 94-104, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30595037

RESUMO

1. Melanoma differentiation-associated gene 5 (MDA5) is a critical member of cytosolic pattern recognition receptors (PRRs) that recognise viral RNA and mediate type I interferon secretion in host cells. 2. The objective of the present study was to identify and characterise the structure and expression of pigeon MDA5. 3. The full-length MDA5 cDNA was cloned from pigeon spleen using RT-PCR and RACE. The distribution and expression level of pigeon MDA5 in different tissues were determined by QRT-PCR. 4. The results showed that the full-length pigeon MDA5 cDNA had 3858 nucleotides (containing a 210-bp 5'-UTR, a 3030-bp open reading frame and a 618-bp 3'-UTR) encoding a polypeptide of 1009 amino acids. The deduced amino acid sequence contained six conserved structural domains typical of RIG-I-like receptor (RLR), including two tandem arranged N-terminal caspase activation and recruitment domains (CARDs), a DEAH/DEAD box helicase domain (DExDc), a helicase superfamily c-terminal domain (HELICc), a type III restriction enzyme (ResIII) and a C-terminal regulatory domain (RD). 5. The pigeon MDA5 showed 84.8%, 87.3%, 87.9% and 87.2% amino acid sequence identities with previously described homologues from chicken, duck, goose and Muscovy ducks, respectively, and phylogenetic analysis revealed a close relationship among these MDA5. 6. Pigeon MDA5 transcript was ubiquitously expressed in all seven tissues tested in healthy pigeons and showed a high level in the thymus gland and kidney. 7. These findings lay the foundation for further research on the function and mechanism of MDA5 in innate immune responses related to vaccinations and infectious diseases in the pigeon.


Assuntos
Proteínas Aviárias/genética , Columbidae/genética , Helicase IFIH1 Induzida por Interferon/genética , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , Sequência de Bases , Columbidae/metabolismo , Perfilação da Expressão Gênica/veterinária , Helicase IFIH1 Induzida por Interferon/química , Helicase IFIH1 Induzida por Interferon/metabolismo , Filogenia , Alinhamento de Sequência/veterinária
8.
Poult Sci ; 97(9): 3343-3357, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-29796647

RESUMO

Beta-keratin in poultry feathers is a structural protein that is resistant to degradation due to disulfide and hydrogen bonds. Feather meal can be a valuable feed compound if the digestibility can be increased. The objective of the present study was to analyze the effects of chemical, enzymatic, and pressure-thermic treatments for chicken feathers on solubility, in vitro protein digestibility (IVPD), and amino acid composition of solubilized and residual fractions. Two experiments were conducted. In experiment 1, models for solubility and IVPD were developed including the above factors applying a central composite face-centered design. Addition of sodium hydroxide (NaOH) and sodium sulfite (Na2SO3), and autoclaving time affected solubility and IVPD of the feather hydrolysates, but not addition of keratinolytic enzyme. In experiment 2, 7 combinations of the hydrolysis factors NaOH, Na2SO3, and autoclaving time with a predicted IVPD of 900 g/kg of DM, calculated for the sum of solubilized and residual feather fractions, were included to measure effects on IVPD and amino acid composition in each fraction. The IVPD values were higher for solubilized than residual fractions when treated with NaOH and autoclaving, but no differences were found when treated with Na2SO3 and autoclaving. Losses of cystine were substantial for all treatments, but lower for Na2SO3 than for NaOH. Furthermore, use of lower Na2SO3 concentration and longer autoclaving time reduced losses of cystine. Compared with NaOH treatments, Na2SO3 gave lower losses of threonine, arginine, serine, and tyrosine. With reference to the ideal protein profile for Atlantic salmon (Salmo salar L.), the treatments with 60 or 90 min autoclaving and 0.36 or 0.21% Na2SO3 had the highest chemical scores. The scores were generally higher for amino acids in residual than solubilized fractions, but with 90 min autoclaving and 0.21% Na2SO3 differences were small. In conclusion, hydrolysis of chicken feathers with low concentrations of Na2SO3 combined with autoclaving results in feather meal with high nutritional value for Atlantic salmon; separation of solubilized and residual fractions is not necessary.


Assuntos
Aminoácidos/química , Ração Animal/análise , Aquicultura/métodos , Proteínas Aviárias/química , Galinhas , Plumas/química , Pepsina A/química , Animais , Proteólise , Salmo salar , Solubilidade
9.
Int J Biol Macromol ; 116: 893-900, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29775716

RESUMO

Egg ovalbumin (OVA) as a macromolecular carrier has the potential to improve the solubility and stability of insolubility bioactive molecules, however, their binding behavior and the mechanism is still ambiguous. In this work, the curcumin was selected as the target to study the interaction and binding mechanism between curcumin and OVA by thermodynamic titration technique in combination with molecular dynamic simulation. The results suggested that the binding included two steps: first, curcumin molecule entered into the hydrophobic pocket of OVA by hydrophobic interaction; and second the interaction was enhanced via hydrogen bonds, resulting in static fluorescence quenching and secondary structural change of OVA. This study provided further evidence in support of the proposed mechanism of the polyphenol-protein binding by the "Hands-gloves" model. Furthermore, when the OVA was as a carrier, the solubility of curcumin has been increased ~370 times to 32.73 µg/mL compared to that of free curcumin at pH 7.0. The photostability was enhanced significantly indicating that it is an efficient way to improve the stability of curcumin in contributing to its application in nutritional supplements or functional foods.


Assuntos
Proteínas Aviárias/química , Curcumina/química , Portadores de Fármacos/química , Ovalbumina/química , Animais , Galinhas , Estabilidade de Medicamentos
10.
Int J Biol Macromol ; 116: 955-965, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29778879

RESUMO

The aim of present investigation is to explore the effect of zinc oxide nanoparticles (ZnONP, 30 nm) interface on conformational dynamics and stability of lysozyme, at pH 7.4 and pH 9.0. Lysozyme adopts partially disordered conformation at pH 9.0, which adopts fibril morphology in presence of sodium dodecyl sulfate (SDS), compared to the conformation adopted at pH 7.4. However, the presence of ZnONP interface renders partially disordered lysozyme relatively regular and non-amyloidogenic conformation, and enhances the functional efficacy of lysozyme at pH 9.0. Additionally, the thermograms reveal a non-cooperative unfolding of the pH 9.0 lysozyme conformation, which accompanied with intermediate conformations that increased with increase in the interface concentration. The binding thermodynamics indicate that at pH 9.0, lysozyme conformation preferentially binds to ZnONP interface than SDS interface. The preferential binding is attributed for the resulting anti-fibrillation propensity of ZnONP interface. The data, altogether, suggest that the presence of ZnONP interface resulted in conformational rearrangements in the partially disordered lysozyme at pH 9.0 causing accumulation of non-amyloidogenic and functionally active intermediates, thus shielding the lysozyme from SDS induced fibrillation and cytotoxicity.


Assuntos
Proteínas Aviárias , Muramidase , Nanopartículas/química , Óxido de Zinco , Animais , Proteínas Aviárias/química , Proteínas Aviárias/farmacologia , Linhagem Celular Tumoral , Galinhas , Citotoxinas/química , Citotoxinas/farmacologia , Humanos , Muramidase/química , Muramidase/farmacologia , Óxido de Zinco/química , Óxido de Zinco/farmacologia
11.
Poult Sci ; 97(7): 2258-2266, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29688456

RESUMO

In mammals, fibroblast growth factor 23 (FGF23) regulates phosphate homeostasis in kidney by binding α-Klotho, a coreceptor of FGF23. FGF23 mRNA is highly expressed in bone and slightly expressed in liver, and is regulated by dietary phosphorus. Little is known about distribution and regulation of FGF23 mRNA in avian lineage. The expression of FGF23 and its coreceptor α-Klotho in chicken and embryo were investigated by real-time quantitative PCR. The effect of dietary phosphorus on FGF23 expression was measured. 36 laying hens at 25 wk were randomly assigned to three dietary available phosphorus (AP) treatments for 11 days: 0.15% AP (LP), 0.40% AP (MP), and 0.80% AP (HP). We first cloned the full coding sequence of FGF23 by the reverse transcription PCR from chicken liver and calvaria. Bioinformatics analysis indicated that the deduced amino acid sequence was 57-87% identical to FGF23 of other species. In adult chicken FGF23 mRNA was expressed at unexpected higher level in liver than other tissues evaluated, including calvaria, femur, tibia, medullary bone, brain, spleen, duodenum, jejunum, ileum, heart and kidney (P < 0.0001), and α-Klotho was expressed at highest level in kidney. However, in 18-d chicken embryos, FGF23 mRNA level was much higher in tibia than in liver, heart and jejunum (P < 0.0001). Chickens at 2, 25, 50 and 80 wk had higher FGF23 expression in liver than 18-d chicken embryos, whereas chickens at 25 wk had lower FGF23 expression in tibia than 18-d chicken embryos and 2-wk-old chickens. HP diets significantly increased serum inorganic phosphorus level (P < 0.001) and FGF23 expression (P < 0.05) in bone tissue compared with LP diets, however, FGF23 mRNA abundance in liver was not changed significantly (P > 0.05) by dietary phosphorus treatments. In conclusion, FGF23 mRNA expression pattern in chicken was clearly different from that in mammals and dietary phosphorus regulated the expression of FGF23 in a tissue-specific way.


Assuntos
Proteínas Aviárias/genética , Galinhas/genética , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica , Fósforo na Dieta/metabolismo , Transcriptoma , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , Sequência de Bases , Osso e Ossos/metabolismo , Galinhas/metabolismo , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Perfilação da Expressão Gênica/veterinária , Fígado/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória , Alinhamento de Sequência/veterinária
12.
Ecotoxicol Environ Saf ; 157: 128-133, 2018 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-29617632

RESUMO

Muscle protein was one of critical accumulation protein for anthropogenic chemicals. However, few predictive models were constructed for muscle protein up to now. In addition, some ionizable chemicals classes e.g. sulfonates were not successfully modeled in previously models, indicating considerable work would be needed. The major objective of this study was to develop quantitative structure-activity relationship (QSAR) models for predicting the muscle protein-water partition coefficient (logKMP/w) of chicken and fish. In the modeling, the n-octanol/water distribution coefficient (logD), functional groups, atom-centred fragments and chemical form adjusted descriptors were used to construct the models. The application domain of the derived models was defined by the Euclidean distance-based method and Williams plot. The modeling results indicated that the determination coefficient (R2), leave-one out cross validation Q2 (Q2LOO) and bootstrapping coefficient (Q2BOOT) of the QSAR models for chicken and fish were 0.882 and 0.929, 0.844 and 0.906, 0.779 and 0.792, respectively, implying the models had good goodness-of-fit and robustness. The coefficient determination (R2EXT) and external validation coefficient (Q2EXT) of the validation set for the two models were 0.874 and 0.937, 0.869 and 0.915, respectively, indicating the models had good predictive ability. The predictor variables selected to construct the logKMP/w models of chicken and fish included logD, the function groups, and the fraction of the ionized species (δI). Considering the molecular descriptors used here can be calculated from their molecular structures directly, the developed models could be easily used to fill the logKMP/w data gap for other chemicals within the applicability domain.


Assuntos
Modelos Biológicos , Proteínas Musculares/química , Animais , Proteínas Aviárias/química , Galinhas , Proteínas de Peixes/química , Íons , Compostos Orgânicos/química , Relação Quantitativa Estrutura-Atividade , Água/química
13.
PLoS One ; 13(3): e0193265, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29494674

RESUMO

Since the late 1990s, high mortality and declining populations have been reported among sea birds including Herring gulls (Larus argentatus) from the Baltic Sea area in Northern Europe. Repeated BoNT type C/D botulism outbreaks have occurred, but it remains unclear whether this is the sole and primary cause of mortality. Thiamine deficiency has also been suggested as a causal or contributing factor. With this study, we aimed to investigate gross and microscopic pathology in Herring gulls from affected breeding sites in Sweden in search of contributing diseases. Herring gulls from Iceland served as controls. Necropsies and histopathology were performed on 75 birds, of which 12 showed signs of disease at the time of necropsy. Parasites of various classes and tissues were commonly observed independent of host age, e.g. oesophageal capillariosis and nematode infection in the proventriculus and gizzard with severe inflammation, air sac larid pentastomes and bursal trematodiasis in pre-fledglings. Gross and microscopic findings are described. Notably, amyloidosis was diagnosed in 93 and 33% of the adult birds from Sweden and Iceland, respectively (p<0.001), with more pronounced deposits in Swedish birds (p<0.001). Gastrointestinal deposits were observed in the walls of arteries or arterioles, and occasionally in villi near the mucosal surface. Amyloid was identified within the intestinal lumen in one severely affected gull suggesting the possibility of oral seeding and the existence of a primed state as previously described in some mammals and chickens. This could speculatively explain the high occurrence and previously reported rapid onset of amyloidosis upon inflammation or captivity in Herring gulls. Amyloid-induced malabsorbtion is also a possibility. The Herring gull SAA/AA protein sequence was shown to be highly conserved but differed at the N-terminus from other avian species.


Assuntos
Amiloidose/diagnóstico , Doenças das Aves/diagnóstico , Sequência de Aminoácidos , Amiloidose/epidemiologia , Amiloidose/parasitologia , Animais , Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , Doenças das Aves/epidemiologia , Doenças das Aves/parasitologia , Bolsa de Fabricius/parasitologia , Bolsa de Fabricius/patologia , Charadriiformes , Surtos de Doenças , Feminino , Trato Gastrointestinal/parasitologia , Trato Gastrointestinal/patologia , Masculino , Alinhamento de Sequência , Suécia/epidemiologia
14.
Poult Sci ; 97(6): 2218-2229, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29514309

RESUMO

The objective of this study was to discover the relationship between the ultrasound probe treatment (UPT) on egg white proteins (EWPs) before EWPs hydrolysis by different proteases, and the functional properties of the obtained hydrolysates. To fulfill this goal, the protein solubility, foaming, and emulsifying properties were studied as a function of the UPT time and then related to the surface characteristics and structural properties. The changes in the hydrolysates microstructures and macromolecular conformation, induced by the UPT, were followed using scanning electron microscope analyzis (SEM) and Fourier transforms infrared spectroscopy (FTIR). The results showed that UPT influenced (P < 0.05) the proteolysis of egg white proteins for all examined treatment times. Alcalase hydrolysates (AHs) and papain hydrolysates (PHs) were found to have a higher solubility, as a consequence of their relatively higher foaming, and emulsifying properties compared to the untreated hydrolysates. The changes in surface hydrophobicity, sulfhydryl content and surface charge of AHs and PHs indicated unfolding of EWPs affected by ultrasound. SEM analyzis showed that UPT destroyed the microstructures of AHs and PHs, while FTIR spectra indicated remarkable changes in the macromolecular conformation of AHs and PHs after UPT. This study revealed that by combining ultrasound pre-hydrolysis treatment under controlled conditions with thoughtful proteases selection, hydrolysates with improved functional properties could be produced, enhancing utilization of EWPs in food products.


Assuntos
Proteínas Aviárias/química , Proteínas do Ovo/química , Papaína/química , Hidrolisados de Proteína/química , Subtilisinas/química , Ultrassonografia/instrumentação , Ultrassonografia/métodos , Animais , Galinhas , Microscopia Eletrônica de Varredura/veterinária , Espectroscopia de Infravermelho com Transformada de Fourier/veterinária
15.
Poult Sci ; 97(5): 1852-1860, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29462461

RESUMO

Soups and broths are popular in the world due to their nutrition and flavor, and flavor compounds tend to be bound by the proteins in the soups and broth, influencing the flavor perception. Thus, identification of the major proteins in meat-based broth may present a basis for understanding protein adsorption of flavor compounds. The present study aimed to identify the major proteins in traditional Chinese chicken broth and to describe the structural changes of proteins during stewing (1, 2, or 3 h). As stewing time increased, protein content in the broth significantly increased. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the macro-molecule proteins (>10 kDa) in the broth were mainly gelatin and actin and that the micro-molecule proteins fractions (<10 kDa) increased substantially. The gelatin had an ordered structure even after 3 h of stewing, as seen by circular dichroism (CD) spectroscopy. The presence of reactive sulfhydryl groups increased remarkably with stewing time. The surface hydrophobicity of the proteins significantly increased within 2 h then deceased slightly after 3 h. The intermolecular crosslinks, as indicated by dispersion index, increased remarkably, consistent with the result of atomic force microscopy (AFM), which together suggested that protein aggregation increased during stewing. These findings suggested that gelatin was the structural protein in the broth system and that intermolecular crosslinks functioned to maintain the broth system.


Assuntos
Proteínas Aviárias/análise , Galinhas , Culinária , Produtos da Carne/análise , Animais , Proteínas Aviárias/química , Galinhas/fisiologia , Cor , Eletroforese em Gel de Poliacrilamida , Plumas/fisiologia
16.
Poult Sci ; 97(5): 1730-1737, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29462487

RESUMO

Chicken egg white protein ovalbumin (OVA) undergoes a conversion to a more thermostable form by alkali treatment, which is assumed to be involved in the physiological functions of OVA. Ovalbumin-related protein X (OVAX), a chicken egg white protein, has 77% sequence similarity to OVA and binds to heparin. In this study, structure characteristics and heparin-binding affinity of alkali-treated OVAX were investigated. Cation-exchange chromatography using SP Sepharose resin showed that alkali treatment (pH 10, 55°C) of OVAX induces the occurrence of a distinct OVAX form with a less positive-charge (acidic OVAX). Circular dichroism and tryptophan-fluorescence analyses showed that the newly-formed acidic OVAX form has an 8% lower α-helical content than its native counterpart, while there is no significant difference in steric environments around tryptophan residues between the 2 forms. The OVAX structure built by homology-modeling showed that OVAX possess a basic cluster domain with α-helix equivalent to 7% of total secondary structures, which does not contain any tryptophan residues. These results suggest that, during alkali treatment, OVAX undergoes mainly a conformational change of the α-helical basic cluster domain and thereby forms acidic OVAX. Acidic OVAX induced by alkali treatment exhibited weaker interactions with Heparin Sepharose resin than native OVAX did. Our results suggest that OVAX basic cluster domain is likely a specific binding site of heparin. Consequently, it is suggested that alkali treatment causes the collapse of the OVAX heparin binding site, which might participate in regulating the functions of heparin.


Assuntos
Álcalis/química , Proteínas Aviárias/química , Galinhas , Ovalbumina/química , Animais , Dicroísmo Circular/veterinária , Heparina/química , Ligação Proteica , Estrutura Secundária de Proteína , Triptofano/química
17.
Bioprocess Biosyst Eng ; 41(5): 707-714, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29470707

RESUMO

Miniaturized systems based on the principles of microfluidics are widely used in various fields, such as biochemical and biomedical applications. Systematic design processes are demanded the proper use of these microfluidic devices based on mathematical simulations. Aggregated proteins (e.g., inclusion bodies) in solution with chaotropic agents (such as urea) at high concentration in combination with reducing agents are denatured. Refolding methods to achieve the native proteins from inclusion bodies of recombinant protein relying on denaturant dilution or dialysis approaches for suppressing protein aggregation is very important in the industrial field. In this paper, a modeling approach is introduced and employed that enables a compact and cost-effective method for on-chip refolding process. The innovative aspect of the presented refolding method is incorporation dialysis and dilution. Dilution-dialysis microfluidic chip (DDMC) increases productivity folding of proteins with the gradual reduction of the amount of urea. It has shown the potential of DDMC for performing refolding of protein trials. The principles of the microfluidic device detailed in this paper are to produce protein on the dilution with slow mixing through diffusion of a denatured protein solution and stepwise dialysis of a refolding buffer flowing together and the flow regime is creeping flow. The operation of DDMC was modeled in two dimensions. This system simulated by COMSOL Multiphysics Modeling Software. The simulation results for a microfluidic refolding chip showed that DDMC was deemed to be perfectly suitable for control decreasing urea in the fluid model. The DDMC was validated through an experimental study. According to the results, refolding efficiency of denaturant Hen egg white lysozyme (HEWL) (EC 3.2.1.17) used as a model protein was improved. Regard to the remaining activity test, it was increased from 42.6 in simple dilution to 93.7 using DDMC.


Assuntos
Proteínas Aviárias/química , Técnicas Analíticas Microfluídicas , Modelos Químicos , Muramidase/química , Redobramento de Proteína , Animais , Galinhas , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos
18.
J Inorg Biochem ; 181: 11-17, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29353085

RESUMO

The tetranuclear Pt complex (PtL)4 (where L2- is the anion derived from para-isopropyl thiosemicarbazone) was first described in A.G. Quiroga et al., J. Med. Chem. 41, 1998, 1399-1408. (PtL)4 manifests antiproliferative properties toward various cancer cell lines being a promising anticancer drug candidate. Yet, details of its reactivity with biomolecules have not been elucidated. To this end, we investigated the reactions of (PtL)4 with a few model proteins, i.e. bovine pancreatic ribonuclease (RNase A), cytochrome c (Cyt c) and hen egg white lysozyme (Lysozyme), through electrospray ionization mass spectrometry and other biophysical methods. A rich reactivity of (PtL)4 with the above-mentioned model proteins is observed, leading to the formation of numerous metallodrug-protein adducts. The tetranuclear complex breaks down and various fragments bind proteins up to high metal/protein ratios; this typically results into very complicated mass spectral patterns. However, some of the main mass peaks could be assigned in the case of the Lysozyme adduct. In addition, crystallographic data were obtained for the (PtL)4/Lysozyme and (PtL)4/RNase A adducts pointing at His side chains as the primary binding sites for monometallic Pt fragments. Notably, a few selected features of the interactions observed in the (PtL)4/protein adducts were reproduced by reacting (PtL)4 with a small molecule, i.e. N-methylimidazole. In conclusion, the present study confirms the prodrug nature of the tetraplatinum complex, clarifies one possible pathway for its activation through cluster disassembly and allows initial identification of adducts formed with a representative protein.


Assuntos
Citocromos c/metabolismo , Modelos Moleculares , Muramidase/metabolismo , Platina/metabolismo , Ribonuclease Pancreático/metabolismo , Tiossemicarbazonas/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , Sítios de Ligação , Bovinos , Quelantes/química , Quelantes/metabolismo , Galinhas , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Cristalografia por Raios X , Citocromos c/química , Cavalos , Ligantes , Conformação Molecular , Estrutura Molecular , Peso Molecular , Muramidase/química , Platina/química , Ribonuclease Pancreático/química , Tiossemicarbazonas/química
19.
J Biochem Mol Toxicol ; 32(3): e22034, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29350485

RESUMO

The use of quail meat and eggs has made this animal important in recent years, with its low cost and high yields. Glutathione S-transferases (GST, E.C.2.5.1.18) are an important enzyme family, which play a critical role in detoxification system. In our study, GST was purified from quail liver tissue with 47.88-fold purification and 12.33% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by SDS-PAGE method and showed a single band. In addition, inhibition effects of (3aR,4S,7R,7aS)-2-(4-((E)-3-(aryl)acryloyl)phenyl)-3a,4,7,7a-tetrahydro-1H-4,7methanoisoindole-1,3(2H)-dion derivatives (1a-g) were investigated on the enzyme activity. The inhibition parameters (IC50 and Ki values) were calculated for these compounds. IC50 values of these derivatives (1a-e) were found as 23.00, 15.75, 115.50, 10.00, and 28.75 µM, respectively. Ki values of these derivatives (1a-e) were calculated in the range of 3.04 ± 0.50 to 131.50 ± 32.50 µM. However, for f and g compounds, the inhibition effects on the enzyme were not found.


Assuntos
Proteínas Aviárias , Inibidores Enzimáticos/química , Glutationa Transferase , Fígado/enzimologia , Codorniz , Animais , Proteínas Aviárias/antagonistas & inibidores , Proteínas Aviárias/química , Proteínas Aviárias/isolamento & purificação , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/química , Glutationa Transferase/isolamento & purificação
20.
Reprod Biol ; 18(1): 60-65, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29336947

RESUMO

Spermine synthase (SPMS), which converts spermidine into spermine, is essential for normal cell growth and development processes in humans and other mammals, but the molecular characterization and expression profiling of the SPMS gene remain undetermined in goose tissues and ovarian follicles. In this study, the SPMS cDNA sequence of the Sichuan white goose was cloned and analysed, and SPMS mRNA expression was profiled in various tissues and ovarian follicles. The results showed that the open reading frame of the SPMS cDNA sequence was 1092 bp in length, encoding 363 amino acids with a molecular weight of 41 kDa. Among all the examined tissues, SPMS expression was highest in the spleen and cerebrum and lowest in the breast and thigh muscles. SPMS expression in the F1 follicle was significantly higher than that in the POF (except for POF2) (P < 0.05). Our results indicate that SPMS might play an important role in follicular development and ovulation.


Assuntos
Proteínas Aviárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ovário/enzimologia , RNA Mensageiro/metabolismo , Espermina Sintase/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Sequência de Bases , Cérebro/enzimologia , Cérebro/metabolismo , China , Biologia Computacional , DNA Complementar/química , DNA Complementar/metabolismo , Feminino , Gansos , Perfilação da Expressão Gênica/veterinária , Peso Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/enzimologia , Neurônios/metabolismo , Fases de Leitura Aberta , Especificidade de Órgãos , Folículo Ovariano/enzimologia , Folículo Ovariano/metabolismo , Ovário/metabolismo , Filogenia , RNA Mensageiro/química , Alinhamento de Sequência/veterinária , Homologia de Sequência , Espermina Sintase/química , Espermina Sintase/genética , Baço/enzimologia , Baço/metabolismo
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