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Kosmos
|
2013
|
vol. 62
|
issue 2
171-180
PL
Doświadczenia na żywych organizmach od lat były podstawą badań w naukach biologicznych. Pozwalają one na bezpośrednią obserwację mechanizmów biologicznych zachodzących w różnego rodzaju komórkach, mikrobach czy nawet w żywych zwierzętach. Dzięki rozwojowi różnego rodzaju technik fluorescencyjnych i wykorzystaniu tradycyjnej mikroskopii fluorescencyjnej czy mikroskopii konfokalnej badania przyżyciowe prowadzi się obecnie w laboratoriach na całym świecie. Rozwój technik obrazowania przyżyciowego na wielu płaszczyznach technologicznych, zaczynając od różnego rodzaju komponentów optycznych, mechanicznych czy elektronicznych, poprzez koncepcyjne podejścia do tego typu badań, a kończąc na tworzeniu nowych znaczników fluorescencyjnych, których właściwości chemiczne są dostosowane do konkretnych doświadczeń, pozwolił na obserwacje i śledzenie nawet pojedynczych molekuł czy badanie interakcji pomiędzy białkami. Artykuł ten przybliża podstawowe techniki badań przyżyciowych z wykorzystaniem mikroskopu szerokiego pola oraz mikroskopu konfokalnego, jak również zalety i wady stosowania obu rodzajów mikroskopów.
EN
Experiments on living organisms has always been one of the most important research in the life sciences. They allow direct observation of biological mechanisms occurring in different types of cells, microbes, or even in living animals. With the development of the various techniques for measuring fluorescence signals, using either wide field or confocal microscopy, are by now extremely well established and routine in many laboratories in the world. This resulted in development of live techniques in many areas of technology, starting with the various components (optical components, mechanical or electronic) by a conceptual approach to this type of research, ending with the creation of new fluorescent dyes which chemical properties are adjusted to the specific experiments. Thanks to the current available techniques, it is possible to observe and track even single molecules or study the interactions between proteins. This article introduces the basic techniques of live experiments using a wide field microscopy and confocal microscopy as well as the advantages and disadvantages of those two microscopes.
EN
Studies on insulin resistance of liver cells are often performed with the use of various hepatoma cell lines. Such an approach allows investigating selected biochemical pathways at the cellular level. However, possible modifications of metabolic processes due to the neoplastic nature of such cells must be considered. Expanding the diversity of hepatoma cell lines used in metabolic studies could deliver new data for comparison with those obtained for other cell lines and should reduce the risk of misleading conclusions. In this study rat hepatoma AS-30D cells were tested as a potential model for studies on palmitate-induced insulin resistance. It was found that insulin-induced Akt kinase phosphorylation was substantially reduced in cells incubated with palmitate at a concentration as low as 75 µM. This effect was not accompanied by excessive reactive oxygen species (ROS) generation or increased Jun N-terminal kinase (JNK) phosphorylation. Moreover, preincubation of AS-30D cells with rosiglitazone, an antidiabetic agonist of peroxisome proliferator-activated receptor gamma (PPARγ), efficiently prevented the palmitate-induced insulin resistance. We conclude that AS-30D hepatoma cells may be used as a model sensitive to insulin and vulnerable to palmitate-induced insulin resistance.
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