RESUMO
The phospholipase A(2) (PLA(2)) superfamily of lipolytic enzymes is involved in a number of essential biological processes, such as inflammation, development, host defense, and signal transduction. Despite the proven involvement of plant PLA(2)s in many biological functions, including senescence, wounding, elicitor and stress responses, and pathogen defense, relatively little is known about plant PLA(2)s, and their genes essentially remain uncharacterized. We characterized three of four Arabidopsis thaliana PLA(2) paralogs (PLA(2)-ß, -γ, and -δ) and found that they (1) are expressed during pollen development, (2) localize to the endoplasmic reticulum and/or Golgi, and (3) play critical roles in pollen development and germination and tube growth. The suppression of PLA(2) using the RNA interference approach resulted in pollen lethality. The inhibition of pollen germination by pharmacological PLA(2) inhibitors was rescued by a lipid signal molecule, lysophosphatidyl ethanolamine. Based on these results, we propose that plant reproduction, in particular, male gametophyte development, requires the activities of the lipid-modifying PLA(2)s that are conserved in other organisms.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Germinação , Fosfolipases A2/metabolismo , Pólen/crescimento & desenvolvimento , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Retículo Endoplasmático/enzimologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Complexo de Golgi/enzimologia , Lisofosfolipídeos/metabolismo , Mutação , Fosfolipases A2/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Pólen/genética , Pólen/ultraestrutura , Interferência de RNA , RNA de Plantas/genéticaRESUMO
Newcastle disease virus (NDV) is not only one of the most economically important pathogen of poultry but also has a potential as anticancer virotherapy. The role of NDV V protein in virus-production kinetics was investigated using DF-1 cell-based production system. The presence of an anti-interferon (IFN)-alpha antibody resulted in enhanced NDV production kinetics in a dose-dependent manner by blocking binding of NDV-induced IFN to its receptor. To prepare DF-1 cell whose cellular IFN signaling is blocked efficiently, stable cell lines expressing either lentogenic or velogenic NDV V protein known as an IFN antagonist were established. The overexpression of NDV V protein enhanced NDV production kinetics and expedited the rate of NDV production, while it had no effect on Japanese encephalitis virus production. NDV V protein functions as an IFN antagonist by inhibiting the increase in type I IFNs by NDV infection. The IFN signals in cells expressing NDV V protein were weakened by decreased activation or expression of the dsRNA-activated enzymes. These IFN antagonist activities enhance rapid virus replication and spread in the early phase of viral infection and will be useful in improving the production of viral vaccine strains.
Assuntos
Fibroblastos/virologia , Interferon-alfa/antagonistas & inibidores , Vírus da Doença de Newcastle/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Cultura de Vírus/métodos , Replicação Viral , Animais , Linhagem Celular , Embrião de Galinha , Chlorocebus aethiops , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Fibroblastos/imunologia , Regulação Viral da Expressão Gênica , Imunidade Inata , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon-alfa/metabolismo , Cinética , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/imunologia , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais , Transfecção , Células VeroRESUMO
Most animal cell culture media can be buffered using bicarbonate and high pressure CO(2) in a closed system. However, in an open system, the pH of the culture media increases continuously due to the marked difference in CO(2) pressure between the culture media and the atmosphere. Therefore, it is important to measure the exact pH of the culture media in an intact closed system. In this study, a pH measurement method was developed using visible light. The pH was calculated from light absorbance by the cells and by the culture media. This method was successfully applied to both suspension and anchorage-dependent cell cultures.
Assuntos
Colorimetria/métodos , Meios de Cultura/química , Animais , Técnicas de Cultura de Células , Linhagem Celular , Concentração de Íons de HidrogênioRESUMO
A method for the rapid detection and quantification of Newcastle disease virus (NDV) produced in an animal cell culture-based production system was developed to enhance the speed of the NDV vaccine manufacturing process. A SYBR Green I-based real-time RT-PCR was designed with a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The validation results satisfied the predetermined acceptance criteria. The validated method was used to quantify virus samples produced in an animal cell culture-based production system. The method was able to quantify the NDV samples from mid- or late-production phases, but not effective on samples from the early-production phase. For comparison with other quantifiable methods, immunoblotting, plaque assay, and tissue culture infectious dose 50 (TCID50) assay were also performed with the NDV samples. The results demonstrated that the real-time RT-PCR method is suitable for the rapid quantification of virus particles produced in an animal cell-culture-based production system irrespective of viral infectivity.