RESUMEN
The overly zinc sensitive Arabidopsis thaliana mutant ozs3 shows reduced growth of the primary root, which is exacerbated by an excess specifically of Zn ions. In addition, ozs3 plants display various subtle developmental phenotypes, such as longer petioles and early flowering. Also, ozs3 seedlings are completely but reversibly growth-arrested when shifted to 4°C. The causal mutation was mapped to a gene encoding a putative substrate-recognition receptor of cullin4 E3 ligases. OZS3 orthologous genes can be found in almost all eukaryotic genomes. Most species from Schizosaccharomyces pombe to Homo sapiens, and including A. thaliana, possess one ortholog. No functional data are available for these genes in any of the multicellular model systems. CRISPR-Cas9-mediated knockout demonstrated that a complete loss of OZS3 function is embryo-lethal, indicating essentiality of OZS3 and its orthologs. The OZS3 protein interacts with the adaptor protein DAMAGED DNA BINDING1 (DDB1) in the nucleus. Thus, it is indeed a member of the large yet poorly characterized family of DDB1-cullin4 associated factors in plants. Mutant phenotypes of ozs3 plants are apparently caused by the weakened DDB1-OZS3 interaction as a result of the exchange of a conserved amino acid near the conserved WDxR motif.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Cullin/genética , Zinc/toxicidad , Arabidopsis/fisiología , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Respuesta al Choque por Frío , Secuencia Conservada/genética , Proteínas Cullin/metabolismo , Proteínas Cullin/fisiología , Genes de Plantas/genética , Genes de Plantas/fisiología , Mutación/genética , Estrés Fisiológico , Rayos Ultravioleta/efectos adversosRESUMEN
BACKGROUND: Ultraviolet radiation (UVR) is known to be harmful to normal human epidermal keratinocytes (NHEKs) of the epidermal skin layer, as well as to hair-follicle-associated keratinocytes. An oral formulation containing l-cystine, thiamin, calcium d-pantothenate, medicinal yeast, keratin and p-aminobenzoic acid (Panto[vi]gar®) has demonstrated clinical efficacy for the treatment of diffuse telogen effluvium; however, its mode of action at the cellular level, and in particular whether protective mechanisms are involved, has yet to be elucidated. OBJECTIVES: To assess the capacity of ingredients of this oral formulation, both separately and in combination, to modulate the effects of UVR in growth-limited NHEKs in vitro. METHODS: NHEKs were incubated in keratinocyte basal medium, keratinocyte basal medium lacking cystine, thiamin, calcium d-pantothenate, folic acid and biotine (minimal growth medium [MGM]) or MGM plus test compound. Test compounds comprised the following four ingredients related to the oral formulation: l-cystine, thiamin, calcium d-pantothenate and folic acid (a proposed metabolite of p-aminobenzoic acid), and a combination of these (Panto[vi]gar®-in vitro correlate; P-IC). The effect of different doses of these compounds on the metabolic activity and proliferation of NHEKs was tested, as well as their influence on the impact of UV light on NHEKs assessed by monitoring metabolic activity, cell number and apoptosis induction. RESULTS: Compared with basal medium, MGM reduced the proliferation of NHEKs in a time-dependent manner. Reduced proliferation is a characteristic of the multifactorial and complex phenotype associated with diffuse hair loss. l-cystine (50⯵M) increased metabolic activity and proliferation 3-fold versus MGM (pâ¯<â¯0.05). Thiamin also had a significant effect (pâ¯<â¯0.05) on proliferation and metabolic activity of NHEKs, but calcium d-pantothenate and folic acid did not when tested individually in this in vitro model. In the presence of P-IC, metabolic activity increased 4-fold and proliferation 3-fold compared with MGM alone (pâ¯<â¯0.05 for both). Following UV irradiation, cells in MGM showed a 72% reduction in metabolic activity, while P-IC-treated cells showed only a 12-18% reduction. The observed prevention of the UV-induced reduction in metabolic activity was not simply due to filtering UVR by the P-IC components, as P-IC-mediated reduction of this effect persisted even when P-IC was washed out during UV irradiation. CONCLUSION: This study demonstrated that l-cystine and thiamin are essential for proliferation of epidermal keratinocytes and suggests a novel, UV-protective potential of formulations combining l-cystine and thiamin in growth-limited inter-follicular NHEKs in vitro.
Asunto(s)
Cistina/farmacología , Cabello/crecimiento & desarrollo , Queratinocitos/citología , Tiamina/farmacología , Rayos Ultravioleta/efectos adversos , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Células Epidérmicas/efectos de los fármacos , Células Epidérmicas/efectos de la radiación , Cabello/efectos de los fármacos , Cabello/efectos de la radiación , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/efectos de la radiaciónRESUMEN
Genome sequencing and annotation studies clearly highlight the impact of transcriptional regulation in plants. However, functional characterization of the majority of transcriptional regulators remains elusive. Hence, high-throughput techniques are required to facilitate their molecular analysis. Here, we provide a detailed protocol to conduct a high-throughput protoplast trans-activation (PTA) screening, which enables simultaneous analysis of up to 95 individual transcription factor activities on a customizable promoter:LUCIFERASE reporter. This system is well suited to decipher complex transcriptional networks such as that triggered by the phytohormone auxin.