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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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| 5. |
- Bucher, Denis, et al.
(författare)
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The elusive 5'-deoxyadenosyl radical in coenzyme-B12-mediated reactions
- 2012
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society. - 0002-7863 .- 1520-5126. ; 134:3, s. 1591-1599
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Tidskriftsartikel (refereegranskat)abstract
- Vitamin B12 and its biologically active counterparts possess the only examples of carbon-cobalt bonds in living systems. The role of such motifs as radical reservoirs has potential application in future catalytic and electronic nanodevices. To fully understand radical generation in coenzyme B12 (dAdoCbl)-dependent enzymes, however, major obstacles still need to be overcome. In this work, we have used Car-Parrinello molecular dynamics (CPMD) simulations, in a mixed quantum mechanics/molecular mechanics (QM/MM) framework, to investigate the initial stages of the methylmalonyl-CoA-mutase-catalyzed reaction. We demonstrate that the 5'-deoxyadenosyl radical (dAdo(center dot)) exists as a distinct entity in this reaction, consistent with the results of extensive experimental and some previous theoretical studies. We report free energy calculations and first-principles trajectories enzymes catalyze coenzyme activation and control highly reactive radical intermediates. that help understand how B12 enzymes catalyze coenzyme activation and control highly reactive radical intermediates.
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| 6. |
- Durbeej, Bo, et al.
(författare)
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On the importance of ribose orientation in the substrate activation of the coenzyme B12-dependent mutases
- 2009
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Ingår i: Chemistry - A European Journal. - : Wiley. - 0947-6539 .- 1521-3765. ; 15:34, s. 8578-8585
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Tidskriftsartikel (refereegranskat)abstract
- The degree to which the corrin ring portion of coenzyme B-12 can facilitate the H-atom-abstraction step in the glutamate mutase (GM)-catalyzed reaction of (S)-glutamate has been investigated with density functional theory. The crystal structure of GM identifies two possible orientations of the ribose portion of coenzyme B-12. In one orientation (A), the OH groups of the ribose extend away from the corrin ring, whereas in the other orientation (B) the OH groups, especially that involving O3', are instead directed towards the corrin ring. Our calculations identify a sizable stabilization amounting to about 30kJ mol(-1) in the transition structure (TS) complex corresponding to orientation B (TS(B)Corlm). In the TS complex where the ribose instead is positioned in orientation A, no such effect is manifested. The observed stabilization in TS(B)CorIm appears to be the result of favorable interactions involving O3' and the corrin ring, including a C-H center dot center dot center dot O hydrogen bond. We find that the degree of stabilization is not particularly sensitive to the Co-C distance. Our calculations show that any potential stabilization afforded to the H-atom-abstraction step by coenzyme B12 is sensitive to the orientation of the ribose moiety.
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- Kabosova, Andrea, et al.
(författare)
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Compositional differences between infant and adult human corneal basement membranes
- 2007
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Ingår i: Investigative Ophthalmology & Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 1552-5783. ; 48:11, s. 4989-4999
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE. Adult human corneal epithelial basement membrane ( EBM) and Descemet's membrane ( DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development. METHODS. Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components. RESULTS. Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from alpha 1-alpha 2 to alpha 3-alpha 4 chains after 3 years of life; in the adult, alpha 1-alpha 2 chains were retained only in the limbal BM. Laminin alpha 2 and beta 2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, beta 2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin gamma 3 chain. In the infant DM, type IV collagen alpha 1-alpha 6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years. CONCLUSIONS. The distribution of laminin gamma 3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.
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