RESUMO
Normal human epidermal keratinocytes were isolated and cultivated in serum-free medium. The expression of the integrin subunits alpha6 and beta1 indicated that a high number of keratinocytes from the stem cell system was present. These cells were transfected with complexes made of different cationic lipids and marker genes. Effectene showed a 20-fold higher transfection efficiency, compared to Lipofectin and Lipofectamine, and a similar low toxicity. The transfection protocol was optimised. A DNA/lipid ratio of 0.133 showed the highest transfection efficiency. Keratinocytes expressed the marker gene luciferase for 20 days. The maximum expression occurred after 3-4 days, where individual patches of fluorescent keratinocytes were detected. Transfected keratinocytes, cultivated at the air-liquid interface, expressed the marker gene beta-galactosidase for at least 7 weeks.
Assuntos
Técnicas de Cultura de Células/métodos , Técnicas de Transferência de Genes , Genes Reporter/genética , Queratinócitos/metabolismo , Transfecção/normas , Resinas de Troca de Cátion/metabolismo , Resinas de Troca de Cátion/normas , DNA/metabolismo , Expressão Gênica , Humanos , Indicadores e Reagentes/metabolismo , Indicadores e Reagentes/normas , Integrina alfa6beta1 , Integrina beta1/genética , Integrina beta1/metabolismo , Integrinas/genética , Integrinas/metabolismo , Queratinócitos/citologia , Metabolismo dos Lipídeos , Lipídeos/normas , Lipossomos/metabolismo , Luciferases/genética , Luciferases/normas , Fosfatidiletanolaminas/metabolismo , Fosfatidiletanolaminas/normas , Células-Tronco/citologia , Fatores de Tempo , Transfecção/métodos , beta-Galactosidase/genética , beta-Galactosidase/normasRESUMO
Genetically prepared protein A fusion proteins, having retained antibody binding capacity, were used to design different well-defined standard molecular weight marker proteins for Western blotting. The blotted marker proteins are developed at the same time and with the same reagents as the protein sample of interest.
Assuntos
Western Blotting/normas , Proteínas/normas , Proteína Estafilocócica A/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/normas , Escherichia coli/genética , Luciferases/genética , Luciferases/normas , Peso Molecular , Proteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/normas , Padrões de Referência , Proteína Estafilocócica A/químicaRESUMO
1. Flash height recorded following the injection of firefly into the external calibration medium depends on the concentration of K-glutamate used, e.g. 120 mM K glutamate reduces flash height by approximately 20 percent. 2. Suspending the fibre in air instead of artificial sea water (ASW) or replacement of NaCl in the bathing medium with Na-glutamate fails to alter flash height. 3. Firefly preparations from DuPont, Packard and SAI give similar myoplasmic ATPMg values viz. 1.1 mM. 4. Analysis of 36 fibres shows the following: myoplasmic ATP = 1.03 +/- 0.06 mM; total ATP (firefly method) = 5.26 +/- 0.12 mmol/kg water; total ATP (enzymic fluorimetry) = 6.27 +/- 0.13 mmol/kg water and ArP = 20.76 +/- 0.59 mmol/kg water. 5. Measurement of ATPMg in samples of myoplasmic aspirate gives a value that is greater than that obtained in situ. 6. Iodoacetate, whether applied externally or internally, reduces resting luminescence in a dose-dependent manner. It also reduces myoplasmic ATP and total ATP. 7. 2-Deoxy-d-glucose fails to reduce myoplasmic ATP but reduces total ATP. 8. Diethylpyrocarbonate, whether applied externally or internally, reduces myoplasmic ATP. It also causes a slow decline in ArP but little change in total ATP. 9. Injection of L-arginine causes a fall in resting luminescence in some fibres while in others it causes a prompt transitory rise. Injection of L-arginine also causes a fall in total ATP. 10. Collectively, these results suggest that the immediate buffering system in the myoplasm is ArP and that ATP supplied by glycolysis lies in a compartment, presumably the interfibrillar space, which is inaccessible to injected firefly.