RESUMEN
Betaine has important roles in preimplantation mouse embryos, including as an organic osmolyte that functions in cell volume regulation in the early preimplantation stages and as a donor to the methyl pool in blastocysts. The origin of betaine in oocytes and embryos was largely unknown. Here, we found that betaine was present from the earliest stage of growing oocytes. Neither growing oocytes nor early preantral follicles could take up betaine, but antral follicles were able to transport betaine and supply the enclosed oocyte. Betaine is synthesized by choline dehydrogenase, and female mice lacking Chdh did not have detectable betaine in their oocytes or early embryos. Supplementing betaine in their drinking water restored betaine in the oocyte only when supplied during the final stages of antral follicle development but not earlier in folliculogenesis. Together with the transport results, this implies that betaine can only be exogenously supplied during the final stages of oocyte growth. Previous work showed that the amount of betaine in the oocyte increases sharply during meiotic maturation due to upregulated activity of choline dehydrogenase within the oocyte. This betaine present in mature eggs was retained after fertilization until the morula stage. There was no apparent role for betaine uptake via the SIT1 (SLC6A20) betaine transporter that is active at the 1- and 2-cell stages. Instead, betaine was apparently retained because its major route of efflux, the volume-sensitive organic osmolyte - anion channel, remained inactive, even though it is expressed and capable of being activated by a cell volume increase.
Asunto(s)
Betaína , Blastocisto , Oocitos , Animales , Betaína/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Femenino , Ratones , Blastocisto/metabolismo , Blastocisto/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Embrionario/fisiología , Folículo Ovárico/metabolismo , Folículo Ovárico/efectos de los fármacos , Colina-Deshidrogenasa/metabolismoRESUMEN
In Dunaliella tertiolecta, a microalga renowned for its extraordinary tolerance to high salinity levels up to 4.5 M NaCl, the mechanisms underlying its stress response have largely remained a mystery. In a groundbreaking discovery, this study identifies a choline dehydrogenase enzyme, termed DtCHDH, capable of converting choline to betaine aldehyde. Remarkably, this is the first identification of such an enzyme not just in D. tertiolecta but across the entire Chlorophyta. A 3D model of DtCHDH was constructed, and molecular docking with choline was performed, revealing a potential binding site for the substrate. The enzyme was heterologously expressed in E. coli Rosetta (DE3) and subsequently purified, achieving enzyme activity of 672.2 U/mg. To elucidate the role of DtCHDH in the salt tolerance of D. tertiolecta, RNAi was employed to knock down DtCHDH gene expression. The results indicated that the Ri-12 strain exhibited compromised growth under both high and low salt conditions, along with consistent levels of DtCHDH gene expression and betaine content. Additionally, fatty acid analysis indicated that DtCHDH might also be a FAPs enzyme, catalyzing reactions with decarboxylase activity. This study not only illuminates the role of choline metabolism in D. tertiolecta's adaptation to high salinity but also identifies a novel target for enhancing the NaCl tolerance of microalgae in biotechnological applications.
Asunto(s)
Betaína , Colina-Deshidrogenasa , Tolerancia a la Sal , Betaína/metabolismo , Tolerancia a la Sal/genética , Colina-Deshidrogenasa/metabolismo , Colina-Deshidrogenasa/genética , Colina/metabolismo , Chlorophyceae/genética , Chlorophyceae/fisiología , Chlorophyceae/enzimología , Chlorophyceae/metabolismo , Microalgas/genética , Microalgas/enzimología , Microalgas/metabolismo , Simulación del Acoplamiento Molecular , Cloruro de Sodio/farmacologíaRESUMEN
Pathogenic Acanthamoeba spp. cause granulomatous amoebic encephalitis and keratitis. Acanthamoeba keratitis (AK) is a rare but serious ocular infection that can result in permanent visual impairment or blindness. However, pathogenic factors of AK remain unclear and treatment for AK is arduous. Expression levels of proteins secreted into extracellular space were compared between A. castellanii pathogenic (ACP) and non-pathogenic strains. Two-dimensional polyacrylamide gel electrophoresis revealed 123 differentially expressed proteins, including 34 increased proteins, 7 qualitative increased proteins, 65 decreased proteins, and 17 qualitative decreased proteins in ACP strain. Twenty protein spots with greater than 5-fold increase in ACP strain were analyzed by liquid chromatography triple quadrupole mass spectrometry. These proteins showed similarity each to inosine-uridine preferring nucleoside hydrolase, carboxylesterase, oxygen-dependent choline dehydrogenase, periplasmic-binding protein proteinases and hypothetical proteins. These proteins expressed higher in ACP may provide some information to understand pathogenicity of Acanthamoeba.
Asunto(s)
Acanthamoeba castellanii , Queratitis por Acanthamoeba , Acanthamoeba , Ceguera , Carboxilesterasa , Colina-Deshidrogenasa , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Encefalitis , Espacio Extracelular , Infecciones del Ojo , Queratitis , Espectrometría de Masas , Péptido Hidrolasas , Virulencia , Trastornos de la VisiónRESUMEN
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