The role of vitamin C in the gene expression of oxidative stress markers in fibroblasts from burn patients

Abstract Purpose: To assess the action of vitamin C on the expression of 84 oxidative stress related-genes in cultured skin fibroblasts from burn patients. Methods: Skin samples were obtained from ten burn patients. Human primary fibroblasts were isolated and cultured to be distributed into 2 groups: TF (n = 10, fibroblasts treated with vitamin C) and UF (n = 10, untreated fibroblasts). Gene expression analysis using quantitative polymerase chain reaction array was performed for comparisons between groups. Results: The comparison revealed 10 upregulated genes as follows: arachidonate 12-lipoxygenase (ALOX12), 24-dehydrocholesterol reductase (DHCR24), dual oxidase 1 (DUOX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 5 (GPX5), microsomal glutathione S-transferase 3 (MGST3), peroxiredoxin 4 (PRDX4), phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-REX1), prostaglandin-endoperoxide synthase 1 (PTGS1), and ring finger protein 7 (RNF7). Conclusion: Cultured fibroblasts obtained from burn patients and treated with vitamin C resulted in 10 differentially expressed genes, all overexpressed, with DUOX1, GPX5, GPX2 and PTGS1 being of most interest.

Aldefluor protocol to sort keratinocytes stem cells from skin

Abstract Purpose: To investigate the use Aldefluor® and N, N - Dimethylaminobenzaldehyde (DEAB) to design a protocol to sort keratinocyte stem cells from cultured keratinocytes from burned patients. Methods: Activated Aldefluor® aliquots were prepared and maintained at temperature between 2 to 8°C, or stored at -20°C. Next, the cells were collected following the standard protocol of sample preparation. Results: Best results were obtained with Aldefluor® 1.5µl and DEAB 15 µl for 1 x 106 cells, incubated at 37°C for 15 minutes. Flow cytometer range for keratinocyte stem cells separation was evaluated. There were 14.8% of stem cells separated in one sample of keratinocyte culture used to pattern the protocol. After being defined the ideal concentration, the same test pattern was performed in other keratinocyte samples. We observed a final mean of 10.8%. Conclusion: Aldefluor® has been shown as a favorable marking of epidermal keratinocyte stem cells for subsequent separation on a flow cytometer, with detection of 10.8% of epidermal keratinocyte stem cells, in this protocol.

Keratinocyte growth factor and the expression of wound-healing-related genes in primary human keratinocytes from burn patients

ABSTRACT PURPOSE: To evaluate the effect of keratinocyte growth factor (KGF) treatment on the expression of wound-healing-related genes in cultured keratinocytes from burn patients. METHODS: Keratinocytes were cultured and divided into 4 groups (n=4 in each group): TKB (KGF-treated keratinocytes from burn patients), UKB (untreated keratinocytes from burn patients), TKC (KGF-treated keratinocytes from controls), and UKC (untreated keratinocytes from controls). Gene expression analysis using quantitative polymerase chain reaction (qPCR) array was performed to compare (1) TKC versus UKC, (2) UKB versus UKC, (3) TKB versus UKC, (4) TKB versus UKB, (5) TKB versus TKC, and (6) UKB versus TKC. RESULTS: Comparison 1 showed one down-regulated and one up-regulated gene; comparisons 2 and 3 resulted in the same five down-regulated genes; comparison 4 had no significant difference in relative gene expression; comparison 5 showed 26 down-regulated and 7 up-regulated genes; and comparison 6 showed 25 down-regulated and 11 up-regulated genes. CONCLUSION: There was no differential expression of wound-healing-related genes in cultured primary keratinocytes from burn patients treated with keratinocyte growth factor.

Modulação da função do receptoe AT1 da angiotensina II em células ovarianas de hamster chinês expressando o receptor AT1 selvagem ou mutante: mecanismos moleculares / Modulation of the angiotensin II AT1 recptor funcion in chines hamster ovary cells expressing wild and mutants AT1 receptors: molecular mechanisms

Durante o mestrado obtivemos mutantes (L265D e L262D) que apresentaram redução na afinidade, expressão funcional, e significante redução no ritmo de crescimento celular com baixa ativação da fosfolipase C, que foi correlacionado com um aumento significativo nos níveis basais de AMPc. Portanto foi nosso interesse reavaliar o fenótipo produzido pela expressão destes mutantes que apresentaram propriedades diferentes daquelas do receptor AT, selvagem. No doutorado novas mutações foram planejadas, e, para examinar a possível participação da segunda ponte dissulfeto na manutenção e conformação funcional do receptor ligado ao peptídio, os resíduos de cisteína no domínio EC da membrana foram substituídos pela serina e produzidos dois mutantes, C18S e o C18, 274S para estudar as consequências funcionais destas mutações. Também foi investigado o papel de uma inserção, isto é, uma sequência de 9 aminoácidos (de GIn26' a LyS171 ) a mais da terceira alça EC que não consta na maioria dos GPCRs, deletando esse segmento para verificar a expressão e a função do receptor. E, o papel do resíduo de asparagina 176 da EC-2 do receptor AT, em promover a interação intramolecular ou mesmo com a AII, através da produção do mutante N176A. Essas mutações foram testadas em células CHO transfectadas com os vetores de expressão contendo os cDNAs do receptor AT, selvagem (WT) com as devidas substituições introduzidas. O estudo com os mutantes previamente obtidos, L262D e 1-265D, mostraram elevados valores basais de cAMP e IP, que, foram bem correlacionados com a inibição do crescimento celular e com a concentração reduzida de DNA genômico em comparação ao receptor WT. A ação mediada pelo AMPc pareceu não ser limitada à proliferação celular, pois foi também observada transformação morfológica das células CHO que foi atribuída à ação do AMPc…(au)