| 1. |
- 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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| 2. |
- Li, He, et al.
(författare)
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Enhanced production of trehalose in Escherichia coli by homologous expression of otsBA in the presence of the trehalase inhibitor, validamycin A, at high osmolarity
- 2012
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Ingår i: Journal of Bioscience and Bioengineering. - : Elsevier. - 1389-1723 .- 1347-4421. ; 113:2, s. 224-232
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Tidskriftsartikel (refereegranskat)abstract
- Trehalose production in Escherichia coli DH5α was explored by overexpressing otsBA operon encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase. Production and subsequent degradation of trehalose resulted in low production of trehalose in engineered cells overexpressing otsBA, which was primarily due to the concomitant expression of endogenous trehalase. Through an in vitro enzyme assay and flask cultures of engineered cells, trehalase expression was shown to be directly related to the expression of otsBA rather than osmotic stress. Validamycin A effectively inhibited E. coli trehalase and the intracellular accumulation of trehalose was markedly enhanced in the presence of validamycin A at an optimal concentration in the medium. The trehalose production was further increased upon growth in a hypertonic medium in the presence of validamycin A, with most trehalose accumulating as an intracellular product. The highest titer was obtained when otsBA expression was induced by a medium-copy vector, ptrc99A, with 0.5mM of isopropyl β-d-1-thiogalactopyranoside. Trehalose titer was 1.7 g/L in controlled bioreactor cultures using synthetic M9 medium supplemented with 40 g/L glycerol, 0.1mM validamycin A, and 300 mM NaCl.
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| 3. |
- Kim, Chang Joon, et al.
(författare)
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발리올아민의 UV 분석방법 (A method for analysis by UV detector of valiolamine)
- 2011
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Patent (populärvet., debatt m.m.)abstract
- A UV analysis method of valiolamine is provided to maximize detection sensitivity of valiolamine by making valiolamine to phenylisocyanate, and to enable accurate and quick HPLC analysis. CONSTITUTION: A UV analysis method of valiolamine comprises the steps of: deriving valiolamine in a microbial culture medium with a chromophore reagent to increase the detection sensitivity; and analyzing the valiolamine with increased detection sensitivity with a high performance liquid chromatography(HPLC)/UV detector. The microbial culture medium uses at least one of (i) glucose 50 g/L, soybean flour 36 g/L, peptone 5 g/L, CaCO3 4 g/L; (ii) glucose 20 g/L, corn starch 40 g/L, corn steep liquor 20 g/L, corn gluten meal 40 g/L, NH4Cl 5 g/L, NaCl 15 g/L, CaCO3 15 g/L; or (iii) glucose 10 g/L, soluble starch 50 g/L, peptone 15 g/L, corn gluten powder 30 g/L, NaCl 5 g/L, CaCO3 10 g/L. COPYRIGHT KIPO 2010
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