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1.
Theriogenology ; 205: 79-86, 2023 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-37094460

RESUMO

Extracellular vesicles (EVs) are membrane-bound nanoparticles that are released by different cell types and play a crucial role in the intercellular communication. They carry various biomolecular compounds such as DNA, RNA, proteins, and lipids. Given that EVs are a new element of the communication within the ovarian follicle, extensive research is needed to optimize method of their isolation. The aim of the study was to assess size-exclusion chromatography (SEC) as a tool for effective EVs isolation from porcine ovarian follicular fluid. The characterization of EVs was performed by nanoparticle tracking analysis, transmission electron microscopy, atomic force microscopy, mass spectrometry and Western blot. We determined EVs concentration, size distribution, zeta potential, morphology, purity, and marker proteins. Our results show that SEC is an effective method for isolation of EVs from porcine follicular fluid. They displayed predominantly exosome properties with sufficient purity and possibility for further functional analyses, including proteomics.


Assuntos
Exossomos , Vesículas Extracelulares , Feminino , Animais , Suínos , Líquido Folicular , Vesículas Extracelulares/química , Exossomos/metabolismo , Cromatografia em Gel/veterinária , Proteínas/metabolismo
2.
Vet Comp Oncol ; 21(1): 36-44, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36111535

RESUMO

Extracellular vesicles (EVs) are cell-derived membrane-bound vesicles involved in many biological processes such as tumour progression. For years, ultracentrifugation (UC) has been considered the gold standard for EV isolation but limited purity and integrity allowed the diffusion of alternative techniques. In this study, EVs were isolated from a canine mammary tumour cell line using UC and size exclusion chromatography (SEC) and analysed for size and concentration by nanoparticle tracking analysis (NTA) and for protein expression by western blot (WB). EV autocrine effect on cell proliferation, migration and invasiveness was then evaluated in vitro. In all samples, particles were in the EV size range (50-1000 nm), with a higher concentration in UC than in SEC samples (1011 and 1010 particles/ml respectively), and expressed EV markers (Alix, CD9). Functional assays did not show statistically significant difference among conditions, but EV treatment slightly increased cell proliferation and invasiveness and treatment with SEC-isolated EVs slightly enhanced cell migration compared to UC-isolated EVs. In conclusion, the main differences between the two isolation techniques are the quantity of the final EV-product and slight differences on EV functionality, which should be further explored to better highlight the real autocrine effect of tumoral EVs.


Assuntos
Doenças do Cão , Vesículas Extracelulares , Neoplasias Mamárias Animais , Animais , Cães , Doenças do Cão/metabolismo , Cromatografia em Gel/veterinária , Ultracentrifugação/métodos , Ultracentrifugação/veterinária , Neoplasias Mamárias Animais/metabolismo , Linhagem Celular Tumoral
3.
J Parasitol ; 108(1): 10-21, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34995354

RESUMO

Globally, parasites are sensitive toward environmental changes, and, in some cases, they are even more sensitive than their hosts. However, there is limited knowledge on the physiological responses of parasites and their effects on their hosts in relation to environmental degradation. In this study, metallothioneins (MTs) were isolated and compared between the ectoparasite Lamproglena clariae and its host fish Clarias gariepinus. Differences in the levels of MTs in the parasite and host were compared to physicochemical water quality variables and metals to determine if MT expression was linked with changes in water quality. Clarias gariepinus individuals were sampled from 2 sites of differing water quality along the Vaal River using gill nets and assessed for L. clariae. Gill, muscle, and liver tissue of the host and L. clariae were collected and stored in liquid nitrogen for analysis of MT. Water and sediment samples were collected for metal analysis by inductively coupled plasma-optical emission spectrometry and inductively coupled plasma-mass spectrometry. Nutrient levels and water hardness in water samples were assessed using spectrophotometry. MTs were quantified using spectrophotometry and size exclusion chromatography in the host and parasite, respectively. Infections by L. clariae differed between sites, with higher parasite intensity at the unpolluted Vaal Dam site. Concentrations of MT in host tissues and L. clariae were significantly higher at the polluted site, below the Vaal River Barrage, compared to the Vaal Dam site. Parasite MT concentrations were significantly lower compared to concentrations in the liver and gill tissue of C. gariepinus individuals. In conclusion, differences in the concentrations of MT and infection biology of L. clariae reflected the state of the environment and support the usefulness of this parasite and other Lamproglena spp. as bioindicators.


Assuntos
Peixes-Gato/parasitologia , Copépodes/metabolismo , Ectoparasitoses/veterinária , Doenças dos Peixes/parasitologia , Metalotioneína/metabolismo , Qualidade da Água , Animais , Cromatografia em Gel/veterinária , Copépodes/patogenicidade , Ectoparasitoses/parasitologia , Biomarcadores Ambientais , Brânquias/química , Brânquias/parasitologia , Fígado/química , Metalotioneína/análise , Músculos/química , Coelhos
4.
J Dairy Sci ; 104(10): 10628-10639, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34304873

RESUMO

The effect of ultrasound (US) pretreatment (0, 200, 400, 600, and 800 W) on the physicochemical, emulsification, and gelatinization characteristics of citric acid (CA)-treated whey protein isolate (WPI) was investigated. Size exclusion chromatography demonstrated that when compared with untreated WPI, US pretreatment promoted production of more molecular polymers in the CA-treated WPI. There was a reduction in particle size of CA-treated WPI with the increase of US power (0-800 W), whereas its free sulfhydryl content, surface hydrophobicity, and intrinsic fluorescence strength increased. Furthermore, compared with untreated WPI, emulsifying ability index and emulsifying stability index of CA-treated WPI were increased by 14.04% and 10.10%, respectively, at 800 W. Accordingly, US pretreatment promoted the gel formation of CA-treated WPI, and its gel hardness was increased by 28.0% with US power ranging from 0 to 800 W. Therefore, US and CA treatment can be considered as an effective way to improve the emulsifying and gelatinization characteristics of WPI.


Assuntos
Ácido Cítrico , Animais , Cromatografia em Gel/veterinária , Emulsões , Interações Hidrofóbicas e Hidrofílicas , Tamanho da Partícula , Proteínas do Soro do Leite
5.
J Dairy Sci ; 104(6): 6463-6471, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33714584

RESUMO

Extracellular vesicles (EV) are important for delivering biologically active substances to facilitate cell-to-cell communication. Milk-derived EV are widely known because of their potential for immune enhancement. However, procedures for isolating milk-derived EV have not been fully established. To obtain pure milk-derived EV and accurately reveal their function, such procedures must be established. The aim of the present study was to compare methods using commercially available kits for isolating milk-derived EV. Initially, we investigated procedures to remove casein, which is the major obstacle in determining milk-derived EV purity. We separated whey using centrifugation only, acetic acid precipitation, and EDTA precipitation. Then, we isolated milk-derived EV by ultracentrifugation, membrane affinity column, size exclusion chromatography (SEC), polymer-based isolation, or phosphatidylserine-affinity isolation. Using EV count per milligram of protein, which is a good indicator of purity, we determined that acetic acid precipitation was the best method for removing casein. Using nanoparticle tracking analysis, protein quantity analysis, and RNA quantity analysis, we comprehensively compared each isolation method for its purity and yield. We found that SEC-based qEV column (Izon Science) could collect purer milk-derived EV at higher quantities. Thus, a combination of acetic acid precipitation and qEV can effectively isolate high amounts of pure extracellular vesicles from bovine milk.


Assuntos
Vesículas Extracelulares , Leite , Animais , Caseínas , Bovinos , Cromatografia em Gel/veterinária , Feminino , Ultracentrifugação/veterinária
6.
Vet Parasitol ; 275: 108932, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31600614

RESUMO

This study describes the in vitro anthelmintic activity of a hydroalcoholic extract from the fruit of Piper cubeba and its major isolated components against the eggs and larvae of gastrointestinal nematodes obtained from naturally-infected ovines. In vitro anthelmintic activity was evaluated using the egg hatch test (EHT), larval development test (LDT) and L3 migration inhibition test (LMT). The extract showed ovicidal and larvicidal activity, with an EC50 of 200 µg/mL and 83.00 µg/mL in the EHT and LDT, respectively. The extract inhibited 100% of larval migration at the lowest tested concentration (95 µg/mL). The crude extract was purified using successive silica gel chromatographic columns, which revealed the lignans hinokinin, cubebin and dihydrocubebin as the major compounds that were present, which were then used in in vitro tests. Cubebin, dihydrocubebin and hinokinin showed higher activity than the crude extract, with an EC50 for ovicidal activity of 150.00 µg/mL, 186.70 µg/mL and 68.38 µg/mL, respectively. In the LDT, cubebin presented an EC50 of 14.89 µg/mL and dihydrocubebin of 30.75 µg/mL. Hinokinin inhibited 100% the larval development at all concentrations evaluated. In the LMT, dihydrocubebin inhibited 100% the larval migration in all concentrations evaluated while cubebin and hinokinin showed EC50 values of 0.89 µg/mL and 0.34 µg/mL, respectively. P. cubeba extract is rich in several classes of active compounds, but here we demonstrate that the described anthelmintic activity may be related to the presence of these lignans, which are present in larger concentrations than other components of the extract. Our results demonstrate for first time the anthelmintic activity against gastrointestinal nematodes in sheep for this class of special metabolites that are present in P. cubeba fruit. However, future detailed studies are needed to evaluate the effectiveness of P. cubeba fruits extract and active lignans in in vivo tests.


Assuntos
Enteropatias Parasitárias/veterinária , Lignanas/farmacologia , Nematoides/efeitos dos fármacos , Infecções por Nematoides/veterinária , Piper/química , Extratos Vegetais/farmacologia , 4-Butirolactona/análogos & derivados , 4-Butirolactona/química , 4-Butirolactona/isolamento & purificação , 4-Butirolactona/farmacologia , Animais , Benzodioxóis/química , Benzodioxóis/isolamento & purificação , Benzodioxóis/farmacologia , Cromatografia em Gel/veterinária , Dioxolanos/química , Dioxolanos/isolamento & purificação , Dioxolanos/farmacologia , Fezes/parasitologia , Frutas/química , Enteropatias Parasitárias/tratamento farmacológico , Enteropatias Parasitárias/parasitologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/fisiologia , Lignanas/química , Lignanas/isolamento & purificação , Microscopia Eletrônica de Varredura/veterinária , Nematoides/crescimento & desenvolvimento , Nematoides/fisiologia , Infecções por Nematoides/tratamento farmacológico , Infecções por Nematoides/parasitologia , Óvulo/efeitos dos fármacos , Óvulo/fisiologia , Extratos Vegetais/química , Ovinos , Doenças dos Ovinos/parasitologia
7.
Reprod Domest Anim ; 52(4): 585-595, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28326614

RESUMO

AWN is a porcine (Sus scrofa domestica) seminal plasma protein and has been linked to a variety of processes related to fertilization. To acquire the protein in sufficient amount and purity for functional studies, we established its recombinant expression in E. coli and a three-step purification protocol based on different chromatographies. The test for AWN-phospholipid interaction revealed phosphatidic acid and cardiolipin as potential binding partners. As phosphatidic acid is surmised to play a role in cation-induced membrane destabilization and fusion events, we propose a membrane protective function of the presented binding affinity. Further studies with recombinant AWN will allow new insights into the mechanism of sperm-spermadhesin interaction and might provide new approaches for artificial reproduction techniques.


Assuntos
Proteínas de Transporte/biossíntese , Proteínas de Transporte/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas de Plasma Seminal/biossíntese , Proteínas de Plasma Seminal/isolamento & purificação , Suínos , Animais , Cardiolipinas/metabolismo , Cromatografia em Gel/veterinária , Escherichia coli/metabolismo , Masculino , Ácidos Fosfatídicos/metabolismo , Proteínas Recombinantes/isolamento & purificação , Sêmen
8.
Fish Shellfish Immunol ; 45(1): 124-31, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25703710

RESUMO

This study was undertaken to isolate active secondary metabolites from immunostimulatory Bacillus Licheniformis XY-52 and evaluate their activities at 0%, 0.5%, 1.0%, and 2.0% doses supplementation with feed on immune response in common carp at weeks 1, 2, and 3. By applying chromatography techniques and successive recrystallization, two purified metabolites were obtained and identified by spectral data (mass spectrometry and nuclear magnetic resonance) as: Cyclo-(Phe-Tyr) and Cyclo-(Phe-Gly). The results revealed that humoral innate immune parameters (lysozyme activity, phagocytic activity and bactericidal activity) were significantly (P < 0.05) increased after feeding on the two active compounds-supplemented diet. Furthermore, administration of the two active compounds significantly (P < 0.05) up regulated IL-1ß, Type 1 IFN, IFN g2b, IL10 and TNF-α gene expression. Heat shock protein 70 (HSP70) gene expression was significantly (P < 0.05) lower as compared to control group at the end of trial. Common carp fed with the two compounds had higher survival rates (69.3%) compared to the controls (32.0%) after challenged with the pathogen, Aeromonas hydrophila. The present study indicates that the two isolated active compounds could positively influence immune response and enhance disease resistance of common carp against A. hydrophila infection.


Assuntos
Bacillus/química , Carpas , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica , Infecções por Bactérias Gram-Negativas/veterinária , Imunidade Inata , Aeromonas hydrophila/fisiologia , Ração Animal/análise , Animais , Cromatografia em Gel/veterinária , Dieta/veterinária , Suplementos Nutricionais/análise , Doenças dos Peixes/genética , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Imunidade Humoral
9.
Fish Physiol Biochem ; 41(1): 107-17, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25389068

RESUMO

A novel incomplete vitellogenin (VgC) was purified from the plasma of estradiol-treated male murrel, Channa punctatus, by gel filtration chromatography. The native mass of VgC protein was 180 kDa, and it resolved as a single peptide of 100 kDa on SDS-PAGE. The peptide on subjecting to matrix-assisted laser desorption/ionization-time of flight produced a peptide mass fingerprint. On tandem mass spectrometry, some of these peptides showed mass to charge (m/z) ratio and amino acid sequence similarity with VgC peptides of other teleosts. Phylogenetic analysis revealed a similarity of murrel VgC with fish species of the order Perciformes. Semi-quantitative RT-PCR assay was developed to study expression of vgc gene at variable levels of estradiol exposure. Presence of VgC in males indicates that fish has been exposed to estrogens; hence, it can be used as a biomarker for estrogenic exposure.


Assuntos
Biomarcadores/sangue , Perciformes/genética , Filogenia , Vitelogeninas/genética , Sequência de Aminoácidos , Animais , Cromatografia em Gel/veterinária , Eletroforese em Gel de Poliacrilamida/veterinária , Estradiol/farmacologia , Funções Verossimilhança , Masculino , Modelos Genéticos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Homologia de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Vitelogeninas/sangue , Vitelogeninas/isolamento & purificação
10.
Glycoconj J ; 31(5): 387-99, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24906475

RESUMO

Structural characterizations of marsupial milk oligosaccharides have been performed in only three species: the tammar wallaby, the red kangaroo and the koala. To clarify the homology and heterogeneity of milk oligosaccharides among marsupials, 21 oligosaccharides of the milk carbohydrate fraction of the common brushtail possum were characterized in this study. Neutral and acidic oligosaccharides were separated from the carbohydrate fraction of mid-lactation milk and characterized by (1)H-nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The structures of the 7 neutral oligosaccharides were Gal(ß1-3)Gal(ß1-4)Glc (3'-galactosyllactose), Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc (3", 3'-digalactosyllactose), Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I), Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (galactosyl lacto-N-novopentaose I), Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)Gal(ß1-4)Glc (galactosyl lacto-N-novopentaose II). The structures of the 14 acidic oligosaccharides detected were Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-4)Glc (sialyl 3'-galactosyllactose), Gal(ß1-3)(O-3-sulfate)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I sulfate a) Gal(ß1-3)[Gal(ß1-4)(O-3-sulfate)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I sulfate b), Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Neu5Ac(α2-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (sialyl lacto-N-novopentaose a), Gal(ß1-3)(-3-O-sulfate)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)(-3-O-sulfate)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)[Neu5Ac(α2-6)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (sialyl lacto-N-novopentaose b), Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)(-3-O-sulphate)Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)(-3-O-sulphate)Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)(-3-O-sulphate)GlcNAc(ß1-6)]Gal(ß1-4)Glc and Gal(ß1-3)Gal(ß1-3)[Neu5Ac(α2-6)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (galactosyl sialyl lacto-N-novopentaose b). No fucosyl oligosaccharides were detected. Galactosyl lacto-N-novopentaose II, lacto-N-novopentaose I sulfate a, lacto-N-novopentaose I sulfate b and galactosyl sialyl lacto-N-novopentaose b are novel oligosaccharides. The results are compared with those of previous studies on marsupial milk oligosaccharides.


Assuntos
Leite/química , Oligossacarídeos/química , Trichosurus/fisiologia , Animais , Sequência de Carboidratos , Cromatografia em Gel/veterinária , Cromatografia Líquida de Alta Pressão/veterinária , Feminino , Concentração de Íons de Hidrogênio , Lactação , Ressonância Magnética Nuclear Biomolecular , Fisiologia Comparada/métodos , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária , Estereoisomerismo , Trissacarídeos/química
11.
Poult Sci ; 93(6): 1495-502, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24879699

RESUMO

Avian bile is rich in matrix metalloproteinases (MMP), the enzymes that cleave extracellular matrix proteins such as collagens and proteoglycans. Changes in bile MMP expression have been correlated with hepatic and gall bladder pathologies, but the significance of their expression in normal, healthy bile is not understood. We hypothesized that the MMP in bile may aid the digestion of native collagens that are resistant to conventional gastric proteases. Hence, the objective of this study was to characterize the bile MMP and check its regulation in association with dietary factors. We used substrate zymography, azocoll protease assay, and gelatin affinity chromatography to identify and purify the MMP from chicken bile. Using zymography and SDS PAGE, 5 bands at 70, 64, 58, 50, and 42 kDa were detected. The bands corresponding to 64, 50, and 42 kDa were identified as MMP2 using trypsin in-gel digestion and matrix-assisted laser desorption time-of-flight mass spectrometry and peptide mass fingerprinting. Chickens fed diets containing gelatin supplements showed higher levels of MMP expression in the bile by both azocoll assay and zymography. We conclude that the bile MMP may be associated with the digestion of collagens and other extracellular matrix proteins in avian diets.


Assuntos
Ração Animal/análise , Bile/efeitos dos fármacos , Bile/metabolismo , Galinhas/metabolismo , Suplementos Nutricionais/análise , Metaloproteinases da Matriz/metabolismo , Animais , Compostos Azo/metabolismo , Cromatografia de Afinidade/veterinária , Cromatografia em Gel/veterinária , Colágeno/metabolismo , Dieta/veterinária , Eletroforese em Gel de Poliacrilamida/veterinária , Masculino , Espectrometria de Massas/veterinária , Metaloproteinases da Matriz/isolamento & purificação , Distribuição Aleatória
12.
Exp Parasitol ; 134(2): 190-4, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23474203

RESUMO

The objective of the present study was to study the characteristics of lactate dehydrogenase (LDH) from Hypoderma sinense larva. H. sinense larvae were collected from yak (Bos grunniens) and identified by a PCR-RFLP method. Analysis of LDH activity showed that the total LDH activity in H. sinense larva was negatively correlated with the length of larva. Polyacrylamide gel electrophoresis of the extracts of H. sinense larvae revealed one band of LDH, which was then purified by affinity chromatography and gel filtration. This enzyme showed an approximately 36 kDa band on SDS-gel under both reducing and non-reducing conditions, in addition, size exclusion chromatography analysis showed that its molecular weight was smaller than bovine serum albumin (67 kDa), indicating that it contains only one subunit. Michaelis constants (Km) values assay revealed that LDH from H. sinense larva showed significantly lower Km for lactate than other animals. LDH of H. sinense larva was stable at 60 °C for 15 min, and also exhibited high catalytic efficiency in a wide range of pH. HgCl2 at the concentration of 0.1mM significantly decreased the activity of LDH from H. sinense larva but not at the concentration of 0.01 mM. The results of the present study demonstrate that LDH from H. sinense larva is a thermal stable and pH insensitive enzyme suitable for catalyzing both forward and reverse reactions.


Assuntos
Doenças dos Bovinos/parasitologia , Dípteros/enzimologia , L-Lactato Desidrogenase/isolamento & purificação , Miíase/veterinária , Animais , Bovinos , China , Cromatografia de Afinidade/veterinária , Cromatografia em Gel/veterinária , Dípteros/classificação , Dípteros/genética , Eletroforese em Gel de Poliacrilamida/veterinária , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/metabolismo , Larva/classificação , Larva/enzimologia , Larva/genética , Cloreto de Mercúrio/farmacologia , Peso Molecular , Miíase/parasitologia , NAD/metabolismo , Reação em Cadeia da Polimerase/veterinária , Polimorfismo de Fragmento de Restrição , Piruvatos/metabolismo , Temperatura
13.
Exp Parasitol ; 133(3): 327-33, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23266486

RESUMO

The immunogenic components of adult Paramphistomum cervi excretion-secretion (ES) fraction were revealed by SDS-PAGE and immunoblotting technique using sera from cattle naturally infected with P. cervi, Fasciola gigantica, strongylids, Trichuris sp., and Strongyloides sp. By SDS-PAGE, it was found that the ES fraction comprised 13 distinct protein bands. Immunoblotting analysis of these proteins exhibited nine prominent antigenic bands which were recognized by paramphistomosis antisera. These antigenic proteins had molecular weights ranging from 10-170 kDa. One antigenic protein band of 40 kDa was found to give a consistent reaction with sera from all infected cattle. Its diagnostic sensitivity, specificity and accuracy using this test were 100%, 98.9% and 99.3%, respectively. The positive and negative predictive values were 98% and 100%, respectively. The 40 kDa antigen was partially purified by gel filtration and ion-exchange chromatography. The antigenicity of 40 kDa protein for diagnosis of P. cervi infection was confirmed by immunoblotting and indirect ELISA (at 1:78,125 dilution) using a pool of sera and individual serum samples from infected cattle. The present findings suggest that the 40 kDa protein may be used as a diagnostic antigen for paramphistomosis.


Assuntos
Antígenos de Helmintos/análise , Doenças dos Bovinos/parasitologia , Proteínas de Helminto/análise , Paramphistomatidae/imunologia , Infecções por Trematódeos/veterinária , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Antígenos de Helmintos/isolamento & purificação , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/imunologia , Cromatografia em Gel/veterinária , Cromatografia por Troca Iônica/veterinária , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Proteínas de Helminto/imunologia , Proteínas de Helminto/isolamento & purificação , Helmintíase Animal/imunologia , Helmintíase Animal/parasitologia , Immunoblotting/veterinária , Peso Molecular , Paramphistomatidae/isolamento & purificação , Paramphistomatidae/metabolismo , Valor Preditivo dos Testes , Rúmen/parasitologia , Sensibilidade e Especificidade , Coloração pela Prata/veterinária , Infecções por Trematódeos/diagnóstico , Infecções por Trematódeos/imunologia , Infecções por Trematódeos/parasitologia
14.
Fish Physiol Biochem ; 39(2): 373-90, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22918791

RESUMO

Elasmobranchs (sharks and rays) exhibit unique reproductive characteristics and, in contrast to the situation in teleosts, very little is known about the identity, structure and physical characteristics of their egg yolk proteins. The aims of this study were to (1) detect and purify the vitellogenin (Vtg; egg yolk precursor) and yolk proteins (YPs) of the cloudy catshark (Scyliorhinus torazame), (2) examine the relationships between Vtg and YPs and (3) characterize and classify the deduced primary structure of the Vtg transcript (vtg). The apparent molecular weights of purified Vtg and putative Vtg-related YPs (lipovitellin: Lv, phosvitin: Pv) were determined by gel filtration and were ~560, >669 and ~58 kDa, respectively. Following SDS-PAGE, these purified products (i.e., Vtg, Lv and Pv) appeared as bands of ~210, ~110 and ~22 kDa, respectively. On Western blots, antisera against purified Vtg, Lv and Pv recognized the ~210 kDa Vtg band. Catshark Pv, in contrast to teleost Pvs, had a very low serine content. The catshark Vtg cDNA sequence (vtg) appeared to contain an open-reading frame consisting of domains encoding Lv, Pv and ß'-component (ß'-c). A phylogenetic analysis, with a consideration of genome duplication events, placed catshark vtg into the 'vtgAB type.' It is concluded that at least a single major type of Vtg protein, which is transcribed and translated from catshark vtgAB gene, is the precursor of three egg yolk proteins (Lv, Pv and ß'-c) in catshark.


Assuntos
Proteínas do Ovo/genética , Fosvitina/genética , Tubarões/metabolismo , Vitelogeninas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting/veterinária , Cromatografia/veterinária , Cromatografia em Gel/veterinária , Análise por Conglomerados , Biologia Computacional , Primers do DNA/genética , DNA Complementar/genética , Proteínas do Ovo/química , Eletroforese em Gel de Poliacrilamida/veterinária , Dados de Sequência Molecular , Peso Molecular , Fosvitina/química , Filogenia , Análise de Sequência de DNA/veterinária , Vitelogeninas/análise , Vitelogeninas/química
15.
Fish Physiol Biochem ; 39(2): 191-200, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22878544

RESUMO

A study was conducted to isolate, partial characterize Asian sea bass (Lates calcarifer) vitellogenin (vtg). Two-year-old juvenile L. calcarifer (n = 10) were given three intraperitoneal injections of 17-ß estradiol (E2) at a dose of 2 mg/kg body weight to induce vitellogenesis. Blood was collected 3 days after the last injection, and plasma was purified through gel filtration chromatography. A broad single symmetrical peak consisting of vtg molecule was produced. Protein concentration was 0.059 mg/ml as determined by Bradfrod assay using bovine serum albumin as a standard. The protein appeared as one circulating form in Native PAGE considering the dimeric form of putative vtg with molecular weight of 545 kDa. In SDS-PAGE under reducing conditions, two major bands appeared at 232.86 and 118.80 kDa and minor bands at 100.60, 85.80 and 39.92 kDa, respectively. The purified vtg was used to generate a polyclonal antibody, and the specificity of antibody was assessed by Western blot analysis. Two major bands were immunoreacted, but no cross-reactivity was observed with plasma from non-induced males. The protein was characterized as phosphoglycolipoprotein as it positively stained for the presence of lipid, phosphorus and carbohydrate using Sudan Black B, methyl green and periodic acid/Schiff reagent solution, respectively. The amino acid composition was analyzed by high sensitivity amino acid analysis that showed high percentage of non-polar amino acids (~48 %). The results suggest the potential utilization of vtg as a basis tool to further study about reproductive physiology of this important economical species.


Assuntos
Bass/genética , Vitelogeninas/isolamento & purificação , Aminoácidos/análise , Animais , Bass/metabolismo , Western Blotting/veterinária , Cromatografia em Gel/veterinária , Cromatografia Líquida de Alta Pressão/veterinária , Eletroforese em Gel de Poliacrilamida/veterinária , Estradiol/administração & dosagem , Estradiol/farmacologia , Malásia , Vitelogênese/efeitos dos fármacos , Vitelogeninas/sangue
16.
Anim Reprod Sci ; 136(4): 289-95, 2013 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-23206437

RESUMO

The camel seminal plasma contains a diverse array of components including lipids, carbohydrates, peptides, ions and proteins. These are essential for maintaining normal physiology of spermatozoa and are secreted mainly from the prostrate, epidydimis and bulbo-urethral glands of reproductive system. The protein profiles of camel seminal plasma were resolved by two-dimensional gel electrophoresis (2D-PAGE). The majority of the protein was found in acidic regions below pI 7.0 and the 19 brightly stained proteins were identified by MALDI-TOF/MS analysis. On the basis of proteomic profiles, ß-nerve growth factor (ß-NGF) was purified by ion-exchange and gel filtration chromatography and identified by SDS-PAGE and MALDI-TOF/MS analysis. It was further confirmed by western blotting experiments using rabbit anti-ß-NGF primary antibody.


Assuntos
Camelus/fisiologia , Fator de Crescimento Neural/isolamento & purificação , Proteômica/métodos , Sêmen/fisiologia , Animais , Western Blotting/métodos , Western Blotting/veterinária , Camelus/genética , Cromatografia em Gel/métodos , Cromatografia em Gel/veterinária , Cromatografia por Troca Iônica/métodos , Cromatografia por Troca Iônica/veterinária , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel Bidimensional/veterinária , Masculino , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária
17.
Theriogenology ; 78(7): 1548-56, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22925635

RESUMO

The objectives were to evaluate postthaw sperm quality and the response to an inducer of in vitro sperm capacitation in boar sperm, cryopreserved with (T) or without (C) α-tocopherol. Boar sperm frozen in 0.2-mL pellets were thawed and washed (W) or selected by three methods: Percoll discontinuous gradient (PS) or Sephadex (Sigma-Aldrich, St. Louis, MO, USA) (neutral [S] or with ion exchange [S+IO] columns). All separation methods enhanced sperm motility, plasma membrane integrity, and functionality and acrosome integrity for both C and T samples (P < 0.05). The best results were obtained with S and ionic Sephadex column. There was a decrease (P < 0.05) in capacitation-like changes in C samples separated with Sephadex (W: 19 ± 0.9%, PS: 22 ± 2.5%, S: 17 ± 1.2%, and S+IO: 17 ± 2.0%). Cryopreservation with α-tocopherol decreased (P < 0.05) the percentage of cryocapacitated sperm (W: 14 ± 0.7%, PS: 14 ± 1.0%, S: 13 ± 1.0%, and S+IO: 14 ± 0.9%) compared with C samples, without differences among selection techniques. Freezing with α-tocopherol and subsequent selection decreased lipid peroxidation (W: 20.79 ± 2.64 nmol thiobarbituric acid reactive substances (TBARS)/10(8) sperm; PS: 13.15 ± 2.39 nmol TBARS/10(8) sperm; S: 13.20 ± 2.18 nmol TBARS/10(8) sperm, and S+IO: 13.62 ± 2.76 nmol TBARS/10(8) sperm), with respect to washed and selected C samples (W: 37.69 ± 5.34 nmol TBARS/10(8) sperm, PS: 25.61 ± 5.85 nmol TBARS/10(8) sperm, S: 19.16 ± 3.28 nmol TBARS/10(8) sperm, and S+IO: 22.16 ± 6.09 nmol TBARS/10(8) sperm). In vitro capacitation levels were significantly higher for neutral Sephadex-selected T samples in comparison with C and unselected samples. These results were confirmed with a follicular fluid-induced acrosome reaction. In conclusion, cryopreserved sperm with α-tocopherol and subsequent Sephadex selection, improved postthaw quality and functionality of boar sperm, which could be useful for assisted reproductive techniques.


Assuntos
Cromatografia em Gel/veterinária , Criopreservação/veterinária , Preservação do Sêmen/veterinária , Espermatozoides/fisiologia , Suínos , alfa-Tocoferol , Animais , Membrana Celular/fisiologia , Separação Celular/métodos , Separação Celular/veterinária , Centrifugação com Gradiente de Concentração , Cromatografia por Troca Iônica/veterinária , Criopreservação/métodos , Dextranos , Masculino , Povidona , Preservação do Sêmen/métodos , Dióxido de Silício , Capacitação Espermática , Motilidade dos Espermatozoides , Espermatozoides/ultraestrutura
18.
Vet Immunol Immunopathol ; 145(1-2): 143-50, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22209367

RESUMO

Mucus immunoglobulin (Ig) of flounder (Paralichthys olivaceus) was purified by the combination of salting-out, Sephacryl S-300 gel filtration chromatography and DEAE Sepharose chromatography. According to the SDS-PAGE and native-PAGE, the purified mucus Ig showed apparent molecular weights of 72 kDa (heavy chain) and 26 kDa (light chain), and a total molecular weight of 798 kDa, which indicated mucus IgM was in tetrameric form. Purified mucus Ig was used to immunize the Balb/C mice, nineteen hybridomas secreting monoclonal antibodies (mAbs) against flounder mucus Ig were obtained by indirect enzyme-linked immunosorbent assay, and three of them designated as 1A-M2, 1C-M10 and 3F-M9 were cloned by limiting dilution. In Western blotting, the three mAbs specifically reacted to the heavy (H) chain of mucus Ig, but not reacted with serum Ig of flounder, whereas mAb 2D8 against serum Ig previously produced could react with the H chain of both mucus and serum Ig, indicating the composition of the mucus and serum Ig H chains was different. Meanwhile, surface Ig positive (sIg+) lymphocytes in the peripheral blood, spleen, skin and gills of healthy flounder, were analyzed by flow cytometry using mAb 1A-M2 and mAb 2D8, and the results revealed that both mAbs were reactive with the sIg+ lymphocytes. The positive reactivity rates for mAb 1A-M2 were 38.64% in the peripheral blood, 23.6% in the spleen, 16.56% in the skin and 6.26% in the gills, while the positive reactivity rates for mAb 2D8 were 48.89%, 33.7%, 15% and 6.02%, respectively, suggesting mucus Ig was similar, but not identical, to serum Ig. These results generated important mucosal immunological information and gave a valuable insight into understanding the mucosal immunity in flounder.


Assuntos
Anticorpos Monoclonais/imunologia , Linguado/imunologia , Imunoglobulinas/imunologia , Muco/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Western Blotting/veterinária , Cromatografia em Gel/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Cadeias Pesadas de Imunoglobulinas/imunologia , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular
19.
Dev Comp Immunol ; 36(2): 418-32, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21911003

RESUMO

Pangasianodon hypophthalmus serum was fractionated by affinity chromatography on 12 different Sepharose-carbohydrate columns and proteins eluted by the corresponding sugar. Binding to the affinity matrices is dependent on Ca(2+) ions. Upon gel filtration using Superose-12, essentially one fraction was obtained, eluting as a protein with a molecular mass of about 900 kDa. SDS-PAGE in reducing conditions revealed the presence of large (72 kDa) subunits (H-chains) and one up to three small (24, 26 and/or 28-29 kDa) subunits (L-chains). The isolated proteins were shown to be IgM since they bind monoclonal anti-P. hypophthalmus IgM antibodies. Rabbit polyclonal anti-galactose-binding IgM only cross-react with some sugar-binding IgM. The H-chains of the anti-carbohydrate IgM are glycosylated. Circular dichroism studies revealed that the IgMs have an "all-ß" type of structure, and that Ca(2+) ions, though essential for carbohydrate-binding activity, are not required for the structural integrity of the molecules. In non-reducing SDS-PAGE, only monomers and halfmers were obtained, showing that there are no disulfide bonds linking the monomers, and that a disulfide bond connecting both H-chains within one monomer is only present in 45% of the molecules. Both the monomers and the halfmers display molecular mass heterogeneity which is indicative for redox forms at the level of the intradomain disulfide bonds. The native carbohydrate-binding IgMs agglutinate erythrocytes from different animals, as well as fish pathogenic bacteria. Similar proteins could not be isolated from another catfish, Clarias gariepinus.


Assuntos
Peixes-Gato/imunologia , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Imunoglobulina M/isolamento & purificação , Lectinas/isolamento & purificação , Testes de Aglutinação/veterinária , Animais , Western Blotting/veterinária , Cálcio/imunologia , Peixes-Gato/sangue , Cromatografia de Afinidade/veterinária , Cromatografia em Gel/veterinária , Dicroísmo Circular/veterinária , Eletroforese em Gel de Poliacrilamida/veterinária , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/ultraestrutura , Imunoglobulina M/imunologia , Imunoglobulina M/ultraestrutura , Lectinas/imunologia , Lectinas/ultraestrutura , Estrutura Secundária de Proteína , Espectrofotometria Ultravioleta/veterinária
20.
Fish Shellfish Immunol ; 31(6): 911-8, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21871568

RESUMO

Leukocyte cell-derived chemotaxin 2 (LECT2) is reported to be an immunorelevant protein in ayu (Plecoglossus altivelis). In this study, ayu LECT2 mature peptide (aLECT2m) was expressed as insoluble inclusion bodies in Escherichia coli. The denatured recombinant aLECT2m (raLECT2m) was refolded by a size-exclusion chromatography refolding process achieved by using arginine-containing mobile phase and a decreasing urea gradient. The in vitro chemotactic activity assay showed that the refolded raLECT2m had the bioactivity. By using suppression subtractive hybridization (SSH) method, we further identified up-regulated genes in ayu macrophages treated with refolded raLECT2m. These genes were tightly involved in endocytosis, hydrolysis, transcriptional regulation, signal transduction, and so on. Moreover, real-time quantitative PCR (RT-qPCR) results confirmed that selected 10 genes expression was significantly up-regulated in refolded raLECT2m-treated ayu macrophages. This study provides a basis for further studies of the mechanism of cytokine LECT2 in fish immune responses.


Assuntos
Fatores Quimiotáticos/metabolismo , Regulação da Expressão Gênica/imunologia , Osmeriformes/imunologia , Animais , Quimiotaxia/genética , Cromatografia em Gel/veterinária , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Rim Cefálico/citologia , Corpos de Inclusão/metabolismo , Macrófagos/metabolismo , Osmeriformes/metabolismo , Redobramento de Proteína , Reação em Cadeia da Polimerase em Tempo Real/veterinária
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