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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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- Takagi, H, et al.
(författare)
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USP40 gene knockdown disrupts glomerular permeability in zebrafish
- 2017
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Ingår i: American journal of physiology. Renal physiology. - : American Physiological Society. - 1522-1466 .- 1931-857X. ; 312:4, s. F702-F715
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Tidskriftsartikel (refereegranskat)abstract
- Unbiased transcriptome profiling and functional genomics approaches have identified ubiquitin-specific protease 40 (USP40) as a highly specific glomerular transcript. This gene product remains uncharacterized, and its biological function is completely unknown. Here, we showed that mouse and rat glomeruli exhibit specific expression of the USP40 protein, which migrated at 150 kDa and was exclusively localized in the podocyte cytoplasm of the adult kidney. Double-labeling immunofluorescence staining and confocal microscopy analysis of fetal and neonate kidney samples revealed that USP40 was also expressed in the vasculature, including in glomerular endothelial cells at the premature stage. USP40 in cultured glomerular endothelial cells and podocytes was specifically localized to the intermediate filament protein nestin. In glomerular endothelial cells, immunoprecipitation confirmed actual protein-protein binding of USP40 with nestin, and USP40-small-interfering RNA transfection revealed significant reduction of nestin. In a rat model of minimal-change nephrotic syndrome, USP40 expression was apparently reduced, which was also associated with the reduction of nestin. Zebrafish morphants lacking Usp40 exhibited disorganized glomeruli with the reduction of the cell junction in the endothelium and foot process effacement in the podocytes. Permeability studies in these zebrafish morphants demonstrated a disruption of the selective glomerular permeability filter. These data indicate that USP40/Usp40 is a novel protein that might play a crucial role in glomerulogenesis and the glomerular integrity after birth through the modulation of intermediate filament protein homeostasis.
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- Asanuma, H., et al.
(författare)
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Projection of individual pyramidal tract neurons to lumbar motor nuclei of the monkey
- 1979
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Ingår i: Experimental Brain Research. - 0014-4819 .- 1432-1106. ; 34, s. 73-89
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Tidskriftsartikel (refereegranskat)abstract
- The projection of individual pyramidal tract (PT) neurons from the hindlimb area in the precentral gyrus of the cerebral cortex to the lumbar spinal cord was studied in the monkey by systematically searching for sites within identified regions of the spinal gray from which the PT neurons could be antidromically activated by local stimulation. All investigated neurons belonged to the fast conducting fraction of PT neurons. The following results were obtained. 1. Each PT neuron could be activated from more than one region of the spinal gray matter, including identified spinal motor nuclei and areas dorsomedial to these nuclei, but not the intermediate nucleus or regions dorsal to it. "Passage areas" and "termination areas" were defined. 2. Half of the PT neurons with termination areas within motor nuclei had these areas in more than one nucleus. There were thus strong suggestions for synaptic contacts of some PT neurons with motoneurons of more than one muscle. 3. Four groups of three or four neurons were recorded simultaneously by the same cortical electrode. Comparisons of passage and termination areas within groups revealed both similarities and differences in projections of neighboring neurons. Every neuron was activated from some region(s) where others of the group were not. Common passage areas, or passage and termination areas, for two or three neurons of a group within at least one motor nucleus were found for all groups. Termination areas in the same motor nucleus have been found for the majority of the neurons of only one group. These common projection areas are compatible with, but do not prove, that a group of adjacent PT neurons has common target cells in the spinal cord. © 1979 Springer-Verlag.
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