The inability to tan properly after sun exposure strongly correlates with increased incidence of skin cancer. The melanocortin 1 receptor (MC1R) is a transmembrane Gs-coupled cell surface receptor found on epidermal melanocytes that transmits pro-survival and pro-differentiation signals mediated by the second messenger cAMP. Humans carrying loss-of-function polymorphisms in MC1R signaling exhibit higher incidences of skin cancers including melanoma. This study focused on the physiologic effects of topical application of forskolin, an adenylate cyclase activator, in extension (Mc1re/e) K14-SCF animals, which model the fair-skinned UV-sensitive human. Twice daily application of the drug promoted accelerated pigmentation, increased skin darkening due to epidermal deposition of melanin pigment, and induced epidermal melanin, which protected the skin against UV injury as judged by “minimal erythematous dose” (MED). Moreover, MC1R signaling regulated the expression of antioxidant enzymes at the transcriptional level. The human melanoma cell line A375, known to harbor a loss-of-function signaling mutation in MC1R, was used to determine effects of cAMP stimulation on the expression of antioxidant enzymes. We observed increases in expression of genes that control the biosynthesis and regulation of glutathione including the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2), glutathione peroxidase, GPX, and glutathione reductase GSR. In addition, there is an increase in manganese superoxide dismutase (MnSOD) at the protein level. There was accumulation of MnSOD in the mitochondria after pharmacologic induction of cAMP with forskolin. Addition of the oxidative agent H2O2 enhanced the expression of MnSOD at the protein level as early as one hour after MC1R stimulation. Oxygen consumption rate on mitochondria was measured using Seahorse analysis; pharmacologic activation of MC1R/cAMP signaling did not affect mitochondrial metabolism. In addition, topical application of a crude extract of Solidago inhibited UV-induced inflammation in K14-SCF mice. Several UV-induced cytokines, including TNF-α, were down-regulated at the transcriptional level after topical application of Solidago extract. Together, these results indicate that MC1R signaling protects melanocytes from UV damage by regulating antioxidant enzyme expression and suggest that pharmacologic cAMP induction may be a useful preventive mechanism against UV-mediated skin sunburn and oxidative injury.

Made available in DSpace on 2014-07-29T15:07:50Z (GMT). No. of bitstreams: 1 dissertacao final CORRIGIDA POS DEFESA bruno.pdf: 1600495 bytes, checksum: cbb62e60517748529f04ae54537c61a1 (MD5) Previous issue date: 2009-03-30 / Considering the in vitro method of embryo production, field results have shown a lower resistance to cryopreservation for B. indicus, when compared to B. taurus embryos. A possible explanation for this is a great concentration of lipid droplets in the cytoplasm of cells from B. indicus embryos. The objective of this study was to compare two cryoprotectants (Propanediol and DMSO) to vitrification and evaluate the effect of adding 10&#956;M Forskolin to the SOF medium for embryo culture prior to cryopreservation. For all the experiments, ovaries from slaughtered Nelore Bos indicus donors were recovered and maintained at 30-35°C in NaCl solution until recovery of the cumulus-oocyte complexes. Embryos submmited to vitrification were expanded blastocysts at day seven of in vitro culture. In the first experiment embryos were first incubated in 10% Ethylene Glycol (EG) plus 10% DMSO dissolved in holding medium (TCM-Hepes with 20% calf serum) for 1 min and then transferred to droplet of 20% EG plus 20% DMSO in holding medium and 0.5M sucrose for 20 seconds before immersing in liquid nitrogen (n = 107; group EG+DMSO). For the group EG+Propanediol (EG+PRO; n = 96) blastocysts were placed in 10% EG plus 10% PRO in holding medium for 1 min, and then transferred to a droplet of 20% EG plus 20% PRO in holding medium and 0.5M sucrose for 20 seconds before immersing in liquid nitrogen. Both treatments were performed using the Cryotop system. Results were compared with embryos (n = 118) not submitted to cryopreservation. The evaluation was done by the hatching rate of blastocysts at Day 9, being higher (86.4%) for embryos not cryopreserved, when comparing to 77.1% for group EG+PRO and 72.9% for group EG+DMSO (P<0.05). In the second experiment, day 5 embryos obtained in vitro from Nelore donors were cultured using SOF medium with 10&#956;M Forskolin (n = 112) or not (control; n = 101), being all submitted to cryopreservation using Cryotop and the same vitrification method for group EG+DMSO. Results were compared with embryos cultured with SOF medium and not submitted to cryopreservation (n = 96). The evaluation was performed by considering hatching rate at Day 9, being higher - 85.4% - for not cryopreserved, when compared with 63.3% for control and 70.5% for Forskolin group (P<0.05). Considering embryos submitted to cryopreservation, the hatching rate was higher (P<0.05) for Forskolin group. / Os embriões bovinos de raças zebuínas produzidos in vitro (PIV) são mais sensíveis ao congelamento, devido, em parte, ao acúmulo de gotas lipídicas no citoplasma das células embrionárias. A criopreservação de embriões é um passo fundamental para a aplicação prática de outras técnicas de embriologia nos animais domésticos. Além disso, o sucesso da criopreservação de embriões Nelore PIV é fundamental para o desenvolvimento da comercialização de material genético nos mercados interno e externo. Neste trabalho, foram conduzidos dois experimentos: no primeiro, o objetivo foi avaliar a taxa de eclosão de embriões bovinos da raça Nelore PIV vitrificados no estágio de blastocisto expandido (Bx) empregando a metodologia CRYOTOP com o uso das soluções Etileno Glicol (EG) + Dimetil Sulfóxido (DMSO) e EG + 1,2 Propanediol (PRO). No segundo experimento, objetivou-se determinar a taxa de eclosão de embriões bovinos da raça Nelore PIV cultivados ou não em meio contendo 10 &#956;M do agente lipolítico forskolin, vitrificados no estágio de BX. No experimento 1, a taxa de eclosão dos embriões do grupo não vitrificado (G3) foi de 86,4%, superior (P<0,05) aos grupos vitrificados, os quais apresentaram taxas de eclosão de 77,1% e 72,9% nos grupos G1 (EG+PRO) e G2 (EG+DMSO), respectivamente. No experimento 2 observou-se maior viabilidade (P<0,05) do G3-F (grupo não vitrificado; 85,4%) em relação ao G1-F (cultivo com SOF controle; 63,3%) e ao G2-F (cultivo com forskolin; 70,5%). Entre os grupos vitrificados, os embriões cultivados com forskolin obtiveram taxa de eclosão superior ao grupo não tratado (P<0,05). Sendo assim, conclui-se que as soluções de EG+PRO e EG+DMSO apresentaram resultados semelhantes na vitrificação de embriões PIV de bovinos e o uso do agente lipolítico forskolin no cultivo de embriões PIV de bovinos da raça Nelore pode melhorar a criotolerância dos embriões.

Carrier mediated nutrient import is vital for cell and tissue homeostasis. Structural insights of carrier mediated transport, particularly the human glucose transporter GLUT1, are essential for understanding the mechanisms of human metabolic disease, and provide model systems for cellular processes as a whole. GLUT1 function and expression is characterized by a complexity unexplained by the current hypotheses for carrier-mediated sugar transport (9). It is possible that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae(RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1) to characterize its functional properties. Identical protein sequences generated different kinetic parameters when expressed in RE700A yeast, erythrocytes, and HEK293 cells. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific. Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 sugar export site. Paradoxically, very low concentrations of these inhibitors produce a modest stimulation of sugar transport (16). This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, intracellular binding sites for e1 ligands CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogs, asks what structural features of exit site ligands determine binding site affinity and cooperativity. Our findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of GLUT1 quaternary structure and evaluate the major determinants of ligand binding affinity and cooperativity. Cytochalasin B (CB) inhibits GLUT1 substrate transport at or near the endofacial sugar binding site. N-bromosuccinamide analysis combined with 3H-CB photolabeling implicates the region between Trp388 and Trp412 in ligand binding. Although its structure has been modeled(5), the specific residues comprising the sugar binding site are unknown. A series of alanine point mutants were made, and mutant protein 2-deoxy glucose transport was tested in the presence of increasing [CB]. Arg126Ala and Cys421Ala GLUT1 mutations altered CB affinity but were determined not to be in the e1 site. The Arg400Ala mutation decreased binding affinity for CB, and may comprise part of the e1 binding site. Because point mutations were individually insufficient to abrogate CB binding, Trp388 to Trp412 chimeras were made. GLUT1/GLUT4388-412/GLUT1 and GLUT1/GLUT5388-412/GLUT1 chimeras showed moderately less sensitivity to CB inhibition of transport; these amino acids likely comprise regions determinant of CB binding affinity. Furthermore GLUT1/GLUT5388-412/GLUT1 shows enhancement of 2-DG uptake at 50nM CB, but an overall dose response indistinguishable from WT GLUT1. A multisite fit of the data suggested GLUT1/GLUT5388-412/GLUT1 chimera possesses strong first site affinity for CB but slight negative second-site cooperativity. We conclude that point mutants were insufficient to abrogate CB binding and that the Trp388 to Trp412 sequence is necessary for CB binding affinity but is not the sole determinant of inhibition of 2 deoxyglucose uptake by CB. We discuss these results with their implications for structure-function sequence localization of the CB binding site, and by extension, the e1 sugar binding site.