RESUMEN
The objective was to investigate effects of progesterone (P4) dose on abundance of luteinizing hormone receptor (LHCGR), aromatase (CYP19A1), 3ß-hydroxysteroid dehydrogenase (HSD3B1), and other steroidogenic mRNA transcripts in granulosa cells from dominant follicles. Nellore heifers were assigned to one of six groups: new, first-use controlled internal drug release device (CIDR1) inserted for 5 days (Large-P4-dose-D5; n = 7) or 6 days (Large-P4-dose-D6; n = 8), prostaglandin (PG)F2α administered on D0 and 1 previously-used CIDR (CIDR3) inserted for 5 days (Small- P4-dose-D5; n = 8) or 6 days (Small-P4-dose-D6; n = 8), CIDR1 inserted on D0 and removed plus PGF2α on D5 (Large-P4-dose-proestrus (PE); n = 7), and CIDR3 and PGF2α on D0 and 1, CIDR3 removed plus PGF2α on D5 (Small-P4-dose-PE; n = 7). Duration of P4 treatment (D5 compared to D6) affected abundances of CYP19A1 mRNA transcripts, with there being greater abundances on D6 than D5 (P ≤ 0.05). Heifers treated with the large dose of P4 had a smaller dominant follicle, less serum and intra-follicular estradiol (E2) concentrations (P ≤ 0.05) and lesser LHCGR, CYP19A1, and HSD3B1 transcript abundances (P ≤ 0.05). Heifers treated to induce PE had a larger follicle diameter (P = 0.09), greater intra-follicular E2 concentrations and larger abundances of CYP19A1 mRNA transcript (P ≤ 0.05) than heifers of the D6 group. Overall, treatment with larger doses of P4 resulted in lesser abundances of LHCGR, HSD3B1, and CYP19A1 mRNA transcripts; thus, potentially leading to development of smaller dominant follicles and lesser E2 concentrations.
Asunto(s)
Bovinos , Sincronización del Estro/efectos de los fármacos , Progesterona/farmacología , Receptores de HL/metabolismo , Animales , Aromatasa/genética , Aromatasa/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Dinoprost/administración & dosificación , Dinoprost/análogos & derivados , Dinoprost/farmacología , Estradiol/administración & dosificación , Estradiol/análogos & derivados , Estradiol/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Progesterona/administración & dosificación , Progesterona Reductasa/genética , Progesterona Reductasa/metabolismo , Receptores de HL/genética , Esteroide Isomerasas/genética , Esteroide Isomerasas/metabolismoRESUMEN
BACKGROUND: Making peptide pharmaceuticals involves challenging processes where many barriers, which include production and manufacture, need to be overcome. A non common but interesting research area is related to peptides with intracellular targets, which opens up new possibilities, allowing the modulation of processes occurring within the cell or interference with signaling pathways. However, if the bioactive sequence requires fusion to a carrier peptide to allow access into the cell, the resulting peptide could be such a length that traditional production could be difficult. The goal of the present study was the development of a flexible recombinant expression and purification system for peptides, as a contribution to the discovery and development of these potentially new drugs. RESULTS: In this work, a high throughput recombinant expression and purification system for production of cell penetrating peptides in Escherichia coli has been designed and implemented. The system designed produces target peptides in an insoluble form by fusion to a hexahistidine tagged ketosteroid isomerase which is then separated by a highly efficient thrombin cleavage reaction procedure. The expression system was tested on the anticancer peptides p53pAnt and PNC27. These peptides comprise the C-terminal region and the N-terminal region of the protein p53, respectively, fused by its carboxyl terminal extreme to the cell penetrating peptide Penetratin. High yields of purified recombinant fused peptides were obtained in both cases; nevertheless, thrombin cleavage reaction was successful only for p53pAnt peptide release. The features of the system, together with the procedure developed, allow achievement of high production yields of over 30 mg of highly pure p53pAnt peptide per g of dry cell mass. It is proposed that the system could be used for production of other peptides at a similar yield. CONCLUSIONS: This study provides a system suitable for recombinant production of peptides for scientific research, including biological assays.