Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos

Myostatin is described as a negative regulator of the skeletal muscle growth. Genetic engineering, in order to produce animals with double the muscle mass and that can transmit the characteristic to future progeny, may be useful. In this context, the present study aimed to analyse the feasibility of lentiviral-mediated delivery of short hairpin RNA (shRNA) targeting of myostatin into in vitro produced transgenic bovine embryos. Lentiviral vectors were used to deliver a transgene that expressed green fluorescent protein (GFP) and an shRNA that targeted myostatin. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR. The lentiviral vector was microinjected into the perivitellinic space of in vitro matured oocytes. Non-microinjected oocytes were used as the control. After injection, oocytes were fertilized and cultured in vitro. Blastocysts were evaluated by epifluorescence microscopy. Results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells, as the transducted group had a less amount of myostatin mRNA after 72 h of differentiation (p < 0.05) and had less myotube formation than the non-transduced group (p < 0.05). There was no difference in cleavage and blastocyst rates between the microinjected and control groups. After hatching, 3.07% of the embryos exhibited GFP expression, indicating that they expressed shRNA targeting myostatin. In conclusion, we demonstrate that a lentiviral vector effectively performed shRNA myostatin gene knockdown and gene delivery into in vitro produced bovine embryos. Thus, this technique can be considered a novel option for the production of transgenic embryos and double muscle mass animals.

Protein Assembly onto Cationic Supported Bilayers

Cationic supported bilayers on latex are useful to isolate and immobilize oppositely charged proteins as a monomolecular layer over a range of low protein concentrations and particle number densities. Cholera toxin (CT) from Vibrio cholerae, an 87 kDa AB(5) hexameric protein and bovine serum albumin (BSA) self-assembled on dioctadecyldimethylammonium bromide (DODAB) supported bilayers with high affinity yielding highly organized and monodisperse particulates at 5 x 10(9) particles/mL, over a range of low protein concentrations (0-0.025 mg/mL BSA or CT). Protein association onto the bilayer-covered polystyrene sulfate (PSS) was determined from adsorption isotherms, dynamic light scattering for size distributions and zeta-potential analysis revealing a monomolecular, thin and highly organized protein layer surrounding each particle with potential for biospecific recognition such as antigen-antibody, receptor-ligand, hybridization of oligonucleotide sequences, all of them important in immunodiagnosis, selective biomolecular chromatographic separations, microarrays design and others.

Protein Adsorption onto Polyelectrolyte Layers: Effects of Protein Hydrophobicity and Charge Anisotropy

Ellipsometry was used to investigate the influence of ionic strength (I) and pH on the adsorption of bovine serum albumin (BSA) or beta-lactoglobulin (BLG) onto preabsorbed layers of two polycations: poly(diallyldimethylammonium chloride) (PDADMAC) or poly(4-vinylpyridine bromide) quaternized with linear aliphatic chains of two (QPVP-C2) or five (QPVP-C5) carbons. Comparisons among results for the three polycations reveal hydrophobic interactions, while comparisons between BSA and BLG-proteins of very similar isoelectric points (pI)-indicate the importance of protein charge anisotropy. At pH close to pI, the ionic strength dependence of the adsorbed amount of protein (Gamma) displayed maxima in the range 10 < I < 25 mM corresponding to Debye lengths close to the protein radii. Visualization of protein charge by Delphi suggested that these ionic strength conditions corresponded to suppression of long-range repulsion between polycations and protein positive domains, without diminution of short-range attraction between polycation segments and locally negative protein domains, in a manner similar to the behavior of PE-protein complexes in solution.(1-4) This description was consistent with the disappearance of the maxima at pH either above or below pI. In the former case, Gamma values decrease exponentially with I(1/2), due to screening of attractions, while in the latter case adsorption of both proteins decreased at low I due to strong repulsion. Close to or below pI both proteins adsorbed more strongly onto QPVP-C5 than onto QPVP-C2 or PDADMAC due to hydrophobic interactions with the longer alkyl group. Above pI, the adsorption was more pronounced with PDADMAC because these chains may assume more loosely bound layers due to lower linear charge density.

Expression, Purification, Bioactivity, and Partial Characterization of a Recombinant Human Bone Morphogenetic Protein-7 Produced in Human 293T Cells

Bone morphogenetic protein-7 (BMP-7) is a secreted multifunctional growth factor of the TGF-beta superfamily, which is predominantly known for its osteoinductive properties and emerging potential for treatment of kidney diseases. The mature 34-38 kDa disulfide-linked homodimer protein plays a key role in the differentiation of mesenchymal cells into bone and cartilage. In this study, the full-length sequence of hBMP-7 was amplified and, then, cloned, expressed, and purified from the conditioned medium of 293T cells stably transfected with a lentiviral vector. The mature protein dimer form was properly secreted and recognized by anti-BMP-7 antibodies, and the protein was shown to be glycosilated by treatment with exoglycosidase, followed by western blotting. Moreover, the activity of the purified protein was demonstrated both in vitro, by alkaline phosphatase activity in C2C12 cells, and in vivo by induction of ectopic bone formation in Balb/c Nude mice after 21 days, respectively. This recombinant protein platform may be very useful for expression of different human cytokines and other proteins for medical applications.

Interaction of bovine serum albumin (BSA) with ionic surfactants evaluated by electron paramagnetic resonance (EPR) spectroscopy

EPR spectra of 5- and 16-doxyl stearic acid nitroxide probes (5-DSA and 16-DSA, respectively) bound to bovine serum albumin (BSA) revealed that in the presence of ionic surfactants, at least, two label populations coexist in equilibrium. The rotational correlation times (tau) indicated that component I displays a more restricted mobility state, associated to the spin labels bound to the protein; the less immobilized component 2 is due to label localization in the surfactant aggregates. For both probes, the increase of surfactant concentration leads to higher motional levels of component 1 followed by a simultaneous decrease of this fraction of nitroxides and its conversion into component 2. For 10 mM cethyltrimethylammonium chloride (CTAC), the nitroxides are 100% bound to the protein, whereas at 10mM N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) and sodium dodecyl sulfate (SDS) the fractions of bound nitroxides are reduced to 18% and 86%, respectively. No significant polarity changes were observed in the whole surfactant concentration range for component 1. Moreover, at higher surfactant concentration, component 2 exhibited a similar polarity as in the pure surfactant micelles. For 16-DSA the surfactant effect is different: at 10mM of HPS and CTAC the fractions of bound nitroxides are 76% and 49%, respectively, while at 10 mM SDS they are present exclusively in a micellar environment, consistent with 100% of component 2. Overall, both SDS and HPS are able to effectively displace the nitroxide probes from the protein binding sites. while CTAC seems to affect the nitroxide binding to a significantly smaller extent. (C) 2008 Elsevier B.V. All rights reserved.

Isoelectric Point Determination for Glossoscolex paulistus Extracellular Hemoglobin: Oligomeric Stability in Acidic pH and Relevance to Protein-Surfactant Interactions

The extracellular hemoglobin from Glossoscolex paulistus (HbGp) has a molecular mass of 3.6 M Da, It has a high oligomeric stability at pH 7.0 and low autoxidation rates, as compared to vertebrate hemoglobins. In this work, fluorescence and light scattering experiments were performed with the three oxidation forms of HbGp exposed to acidic pH. Our focus is on the HbGp stability at acidic pH and also on the determination of the isoelectric point (pI) of the protein. Our results show that the protein in the cyanomet form is more stable than in the other two forms, in the whole range. Our zeta-potential data are consistent with light scattering results. Average values apt obtained by different techniques were 5.6 +/- 0.5, 5.4 +/- 0.2 and 5.2 +/- 0.5 for the oxy, met, and cyanomet forms. Dynamic light scattering (DLS) experiments have shown that, at pH 6.0, the aggregation (oligomeric) state of oxy-, met- and cyanomet-HbGp remains the same as that at 7.0. The interaction between the oxy-HbGp and ionic surfactants at pH 5.0 and 6.0 was also monitored in the present study. At pH 5,0, below the protein pI, the effects of sodium dodecyl sulfate (SDS) and cetyltrimethylammonium chloride (CTAC) are inverted when compared to pH 7.0. For CTAC, in acid pH 5.0, no precipitation is observed, while for SDS an intense light scattering appears due to a precipitation process. HbGp interacts strongly with the cationic surfactant at pH 7.0 and with the anionic one at pH 5.0. This effect is due to the predominance, in the protein surface, of residues presenting opposite charges to the surfactant headgroups. This information can be relevant for the development of extracellular hemoglobin-based artificial blood substitutes.

Serum protein concentrations, including acute phase proteins, in calves with hepatogenous photosensitization

Foram examinados 100 bezerros da raça Nelore com 6 a 12 meses de idade, distribuídos em: grupo controle (G1; 50 bezerros sadios) e grupo fotossensibilização (G2; n= 50). As amostras de sangue foram coletadas 12 a 24 horas após o início da dermatite (M1) e 15 a 30 dias após (M2), época da cura das lesões cutâneas. O proteinograma sérico foi obtido por eletroforese em gel de acrilamida. em todos os bezerros foram identificadas 18 proteínas com pesos moleculares (PM) entre 16.000 a 189.000 dáltons (Da). em M1 e M2, as concentrações séricas das proteínas de PM 115.000Da (ceruloplasmina), 61.000Da (1-antitripsina), 45.000Da (haptoglobina) e 40.000Da (glicoproteína ácida) foram significativamente maiores em bezerros com fotossensibilização hepatógena em comparação com aquelas dos animais do grupo-controle. A determinação dos teores séricos de proteínas de fase aguda pode ser útil no monitoramento da progressão da fotossensibilização hepatógena em bovinos, inclusive orientando possíveis alterações em procedimentos terapêuticos.

Aberrant expression of E-cadherin and beta-catenin proteins in placenta of bovine embryos derived from somatic cell nuclear transfer

Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and beta-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n - 6) and control pregnancies (n - 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or beta-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total beta-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-beta-catenin protein in SCNT tissues compared with controls. However, qRT-PCR confirmed that the wingless (WNT)/beta-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and beta-catenin proteins, along with defective beta-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus.

The effect of offering an energy and protein supplement to grazing canchim beef cows either postpartum or both pre- and postpartum on lipid blood metabolites and folliculogenesis

This study was designed to evaluate the effect of nutritional supplementation offered during the pre- and postpartum periods on serum cholesterol, triglycerides and total lipids of Canchim beef cows and their relationship with folliculogenesis. Thirty cows with predicted calving date between September and October, kept in pastures of Brachiaria brizantha cv. Marandu together with their calves, were randomly distributed into three experimental groups: the first received only a mineral mixture (Control Group, CG); the second group received a concentrate with 16% crude protein/kg dry matter (DM) and 3000 kcal digestible energy/kg DM offered for 45 days prepartum and 120 days postpartum (PREG); the third group received the concentrate from parturition until the 120th day postpartum (POSG). Consumption was estimated at 1% of body weight, and each cow received approximately 4.0 kg/day (fresh weight) of supplement. Blood samples were taken and an ultrasound examination of the ovaries was performed twice a week until the 60th day postpartum. The body condition score (BCS) and the weight of the cows were recorded at 15-day intervals from calving until the 60th day postpartum. Data are presented as mean +/- SEM. Mean weight and BCS at calving were, respectively, 448 +/- 54.9 kg and 6.2 +/- 0.25 (PREG); 432 +/- 71.1 kg and 5.5 +/- 0.69 (POSG); and 434 +/- 66.4 kg and 5.5 +/- 0.69 (CG). Total cholesterol (TC), triglycerides (TRIG) and total lipids (TLIP) were measured using colorimetry until the 60th day postpartum. TC averages were PREG 186 +/- 62.6 mg/dL, POSG 159 +/- 43.1 mg/dL and CG 133 +/- 35.1 mg/dL (P < 0.05). For TRIG, the means were PREG 29 +/- 11.3 mg/dL (P < 0.05), POSG 24 +/- 8.1 mg/dL and CG 26 +/- 12.1 mg/dL (P > 0.05). Serum concentrations of TLIP were PREG 588 +/- 145.6 mg/dL, POSG 512 +/- 137.6 mg/dL and CG 452 +/- 122.4 mg/dL (P < 0.05). The first dominant follicle (DF) was identified on Day 21 +/- 10.3 (PREG), 36 +/- 28.5 (POSG) and 51 +/- 32.8 (CG) after calving. The difference between PREG and CG was significant (P < 0.05). TC was positively correlated with the calving to first estrus interval (P < 0.05). Results showed that nutritional supplementation before parturition assured good body condition at calving and suggested that it was effective at increasing cholesterol availability to maintain ovarian follicle function and to favor earlier resumption of ovarian activity. (C) 2010 Published by Elsevier B.V.

Growth and antimicrobial activity of lactic acid bacteria from rumen fluid according to energy or nitrogen source

Objetivou-se avaliar os efeitos da suplementação com diferentes fontes de energia e de compostos nitrogenados sobre o crescimento e produção de bacteriocinas de bactérias ácido-láticas in vitro. As incubações foram conduzidas utilizando-se fluido ruminal originado de um novilho Holandês-Zebu fistulado no rúmen. O animal foi mantido em pastagem de Brachiaria decumbens recebendo 200 g/dia de proteína bruta suplementar. Os substratos e o inóculo foram acondicionados em frascos de vidro considerando-se oito tratamentos: celulose, celulose e caseína, celulose e peptona de soja, celulose e ureia, amido, amido e caseína, amido e peptona de soja e amido e ureia. Incubações sucessivas foram conduzidas para seleção dos microrganismos em função das fontes energéticas e de compostos nitrogenados. O amido favoreceu o crescimento de bactérias ácido-láticas em comparação à celulose. A suplementação com proteína verdadeira (peptona de soja e caseína) estimulou o crescimento dessas bactérias em comparação ao controle (sem suplementação com compostos nitrogenados). A adição de ureia não estimulou o crescimento de bactérias ácido-láticas. Nenhuma atividade antimicrobiana foi detectada a partir das colônias de bactérias ácido-láticas isoladas. Fontes de proteína verdadeira incrementam a competição entre bactérias fermentadoras de carboidratos estruturais e não-estruturais.

Rumen fermentation and rumen microbes in Nellore steers receiving diets with different lipid contents

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Degradabilidade ruminal e digestibilidade intestinal da proteína de capim-elefante com três idades de corte

Determinaram-se a degradabilidade potencial (DP) e a digestibilidade intestinal da proteína não degradada no rúmen (DIPNDR) do capim-elefante em diferentes idades de rebrote (30, 45 e 60 dias) e comparou-se a técnica do saco de náilon móvel (in situ) com o método de três estádios (in vitro). Para tanto, utilizaram-se seis novilhos mestiços canulados no rúmen e duodeno alimentados exclusivamente com capim-elefante picado. O ensaio de degradabilidade foi realizado com amostras do capim incubadas no rúmen por 3, 6, 9, 12, 24, 48, 72, 96 e 120h. A digestibilidade intestinal foi determinada utilizando-se os resíduos de incubação por 24 horas. Na técnica in situ os resíduos em sacos de náilon foram colocados no duodeno e recuperados nas fezes. No método in vitro, os resíduos foram submetidos à digestão com HCl-pepsina-pancreatina. em amostras de capim com idades de 30, 45 e 60 dias foram observados valores de DP da proteína de 87,5; 87,8 e 83,8%, respectivamente. A DIPNDR variou com a idade do capim e foi semelhante entre os métodos in situ e in vitro somente para o capim com 60 dias. O método in situ apresentou estimativa de digestibilidade intestinal mais coerente com as mudanças na composição química do capim-elefante decorrentes do envelhecimento.

Performance and carcass characteristics of Nellore young bulls fed different sources of oils, protected or not from rumen degradation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Consumo e Digestibilidade Aparente Total e Parcial de Rações com Cana-de-açúcar, Casca e Raspa de Mandioca Ensiladas com Polpa Cítrica

O objetivo deste estudo foi avaliar o consumo e a digestibilidade aparente total (DAT) e parcial dos nutrientes das silagens de cana-de-açúcar (SCnPc), de raspa (SRpPc) e de casca de mandioca (SCcPc) ensiladas com polpa cítrica peletizada (PCP). A dieta basal foi constituída de silagem de milho com farelo de soja, participando com 60% da matéria seca (MS) dos tratamentos com SRpPc e SCcPc. Foram utilizados novilhos mestiços, fistulados no rúmen e duodeno, num delineamento experimental em quadrado latino (4x4). A determinação do coeficiente de digestibilidade total e parcial dos nutrientes, a produção fecal e o fluxo duodenal de MS foram estimados a partir da fibra detergente ácido indigestível. Os consumos de MS e proteína bruta (PB) (% PV) foram superiores para a SMi (2,47 e 0,33), SCcPc (2,12 e 0,30) e SRpPc (1,88 e 0,27) em relação à SCnPc (1,38 e 0,19), respectivamente. As DAT da matéria seca (65,0%), matéria orgânica (66,9%) e energia bruta (64,3%) para a SRpPc foram superiores em relação às demais dietas, menos para a FDN (39,4%). Os coeficientes de digestibilidade ruminal (CDR) e intestinal (CDI) da MS, MO e PB não apresentaram diferenças significativas entre as dietas, somente o CDR da FDN (% ingerido) apresentou valores superiores para SMi (43,5), SRpPc (39,3) e SCnPc (37,0), sobre a SCcPc (20,0). A utilização da silagem de raspa de mandioca adicionada da PCP mostrou-se uma alternativa de boa qualidade na alimentação de bovinos.

Enhanced stabilization of aerosol-OT surfactant monolayer upon interaction with small amounts of bovine serum albumin at the air-water interface

An investigation is made of the influence from small amounts of the protein bovine serum albumin (BSA) on the lateral organization of low molecular weight surfactant sodium bis-2-ethylhexyl sulfosuccinate (AOT) at the air-water interface. Surface pressure (pi - A), surface potential (DeltaV - A) and Brewster angle microscopy (BAM) experiments were carried out, with particular emphasis on the monolayer stability under successive compression-expansion cycles. AOT monolayer is not stable at the air-water interface, which means that the majority of AOT molecules go into the aqueous subphase as monomers and/or normal micelles. When a waiting time elapses between spreading and compression, the surfactant monolayer tends to reorganize partially at the air-water interface, with a monolayer expansion being observed for waiting times as large as 12 h. The incorporation of very small amount of BSA (10(-9) M) at the interface, also inferred from BAM, increases the monolayer stability as revealed by pi - A and DeltaV - A results. For a waiting time of circa 3 h, the mixed monolayer reaches its maximum stability. This must be related to protein (and/or protein-surfactant complexes) adsorbed onto the AOT monolayer, thus altering the BSA conformation to accommodate its hydrophobic/hydrophilic residues. Furthermore, the effects from such small amounts of BSA in the monolayer formation and stabilization mean that the AOT monolayer responds cooperatively to BSA. (C) 2004 Elsevier B.V. All rights reserved.