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- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 4. |
- Zhang, Yao, et al.
(författare)
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Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: A study on the enzyme knockout mice.
- 2010
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Ingår i: Journal of Lipid Research. - 1539-7262. ; 52:4, s. 771-781
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Tidskriftsartikel (refereegranskat)abstract
- Alkaline sphingomyelinase (alk-SMase) hydrolyses sphingomyelin (SM) to ceramide in the gut. To evaluate physiological importance of the enzyme, we generated alk-SMase knockout (KO) mice by Cre-LoxP system and studied SM digestion. Both wild type (WT) and the KO mice were fed 3H-palmitic acid labeled SM together with milk SM by gavage. The lipids in intestinal content, intestinal tissues, serum and liver were analysed by TLC. In KO mice, non-digested 3H-SM in the intestinal content increased by 6 fold and the formation of 3H-ceramide decreased markedly, resulting in 98% reduction of 3H-ceramide/3H-SM ratio 1 h after gavage. The absorbed 3H-palmitic acid portion was decreased by 95%. After three hours, a small increase in 3H-ceramide was identified in distal intestine in KO mice. In feces 3H-SM was increased by 243% and ceramide decreased by 74% in the KO mice. The KO mice also showed significantly decreased radioactivity in liver and serum. Furthermore alkaline phosphatase activity in the mucosa was reduced by 50%, and histological comparison of two female littermates preliminarily suggested mucosal hypertrophy in KO mice. The study provides definite proofs for crucial roles of alk-SMase in SM digestion and points to possible roles in regulating mucosal growth and alkaline phosphatase function.
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