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1.
Front Vet Sci ; 7: 594, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33195496

RESUMO

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that causes severe economic losses in the livestock industry. Currently available vaccines are based on the inactivated FMD virus (FMDV). Although inactivated vaccines have been effective in controlling the disease, they have some disadvantages. Because of these disadvantages, investigations are being made to produce vaccines in low containment facilities. The use of recombinant empty capsids (also referred as Virus Like Particles, VLPs) has been reported to be a promising candidate as a subunit vaccine because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. Mignaqui and collaborators have produced recombinant FMDV empty capsids from serotype A/ARG/2001 using a scalable technology in mammalian cells that elicited a protective immunity against viral challenge in a mouse model. However, further evaluation of the immune response elicited by these VLPs in cattle is required. In the present work we compare the effect that VLPs or inactivated FMDV has on bovine dendritic cells and the humoral response elicited in cattle after a single vaccination.

2.
Front Vet Sci ; 7: 601, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33173790

RESUMO

Inactivated Foot-and-Mouth Disease (FMD) vaccine has proven to be effective in the control of the disease. However, its production has some disadvantages, including the costly biosafety facilities required for the production of huge amounts of growing live virus, the need of an exhaustive purification process to eliminate non-structural proteins of the virus in the final formulations in order to differentiate infected from vaccinated animals and variable local regulatory restrictions to produce and commercialize the vaccine. Thus, a novel vaccine against FMD that overcome these restrictions is desirable. Although many developments have been made in this regard, most of them failed in terms of efficacy or when considering their transferability to the industry. We have previously reported the use of transient gene expression in mammalian cells to produce FMD virus-like particles (VLPs) as a novel vaccine for FMD and demonstrated the immunogenicity of the recombinant structures in animal models. Here, we report the optimization of the production system by assaying different DNA:polyethylenimine concentrations, cell densities, and direct and indirect protocols of transfection. Also, we evaluated the reproducibility and scalability of the technology to produce high yields of recombinant VLPs in a cost-effective and scalable system compatible with industrial tech-transfer of an effective and safe vaccine.

3.
Eukaryot Cell ; 9(8): 1262-71, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20581295

RESUMO

The Saccharomyces cerevisiae UGA4 gene encodes a permease capable of importing gamma-aminobutyric acid (GABA) and delta-aminolevulinic acid (ALA) into the cell. GABA-dependent induction of this permease requires at least two positive-acting proteins, the specific factor Uga3 and the pleiotropic factor Uga35/Dal81. UGA4 is subjected to a very complex regulation, and its induction is affected by the presence of extracellular amino acids; this effect is mediated by the plasma membrane amino acid sensor SPS. Our results show that leucine affects UGA4 induction and that the SPS sensor and the downstream effectors Stp1 and Stp2 participate in this regulation. Moreover, we found that the Uga3 and Uga35/Dal81 transcription factors bind to the UGA4 promoter in a GABA-dependent manner and that this binding is impaired by the presence of leucine. We also found that the Leu3 transcription factor negatively regulates UGA4 transcription, although this seems to be through an indirect mechanism.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Leucina/farmacologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologia , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos/genética , Modelos Genéticos , Mutação/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos
4.
Microbiology (Reading) ; 153(Pt 11): 3677-3684, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17975075

RESUMO

The Saccharomyces cerevisiae UGA4 gene, which encodes the gamma-aminobutyric acid (GABA) and delta-aminolaevulinic acid (ALA) permease, is well known to be regulated by the nitrogen source. Its expression levels are low in the presence of a rich nitrogen source but are higher when a poor nitrogen source is used. In addition, GABA can induce UGA4 expression when cells are grown with proline but not when they are grown with ammonium. Although vast amounts of evidence have been gathered about UGA4 regulation by nitrogen, little is known about its regulation by the carbon source. Using glucose and acetate as rich and poor carbon source respectively, this work aimed to shed light on hitherto unclear aspects of the regulation of this gene. In poor nitrogen conditions, cells grown with acetate were found to have higher UGA4 basal expression levels than those grown with glucose, and did not show UGA4 induction in response to GABA. Analysis of the expression and subcellular localization of the transcription factors that regulate UGA4 as well as partial deletions and site-directed mutations of the UGA4 promoter region suggested that there are two parallel pathways that act in regulating this gene by the carbon source. Furthermore, the results demonstrate the existence of a new factor operating in UGA4 regulation.


Assuntos
Carbono/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Acetatos/metabolismo , Carbono/química , Meios de Cultura , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Glucose/metabolismo , Mutação , Nitrogênio/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica
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