| 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. |
- Cardell, Monika, et al.
(författare)
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Protein kinase C is translocated to cell membranes during cerebral ischemia
- 1990
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Ingår i: Neuroscience Letters. - : Elsevier BV. - 0304-3940. ; 119:2, s. 228-232
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Tidskriftsartikel (refereegranskat)abstract
- The subcellular distribution of PKC(α) and PKC(γ) was studied in homogenates of cerebral cortex from rats subjected to 10 and 15 min of ischemia and 15 min of ischemia followed by 1 h, 6 h, 24 h, 48 h, and 7 days of reperfusion. During ischemia no significant changes in the levels of PKC (α) were seen. During the first hour of reperfusion, a transient 2.5-fold (P < 0.05) increase in PKC(α) levels was observed in the particulate fraction. In contrast, a three-fold increase of PKC(γ) in the particulate fraction concomitant with a 40% decrease in the cytosol was noted during ischemia. In the postischemic phase the levels in the cytosol decreased to 35% of control values at 2 days following ischemia, with a concomitant decrease in the particulate fraction to control levels. The redistribution of PKC to the cell membranes during and following ischemia could be due to ischemia induced receptor activation, increased levels of diacylglycerols, arachidonate and intracellular calcium, and may be of importance for the development of ischemic neuronal damage.
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| 3. |
- Hu, Bing‐Ren, et al.
(författare)
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Persistent Translocation and Inhibition of Ca2+/Calmodulin‐Dependent Protein Kinase II in the Crude Synaptosomal Fraction of the Vulnerable Hippocampus Following Hypoglycemia
- 1995
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Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042. ; 64:3, s. 1361-1369
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Tidskriftsartikel (refereegranskat)abstract
- Abstract: Alterations in the levels and activity of Ca2+/calmodulin‐dependent protein kinase II (CaM‐kinase II) were studied in the rat hippocampus during and after insulin‐induced hypoglycemic coma. A permanent loss of CaM‐kinase II immunohistostaining in the neuronal layer begins at 10 min of isoelectricity in the tip of the dentate gyrus and at 30‐min isoelectricity in the CA1 region. The reduction in immunohistostaining in the neurites is less pronounced. Immunoreactivity of CaM‐kinase II on western blots increases in the crude synaptosomal fractions and decreases in cytosolic fraction, indicative of a translocation of CaM‐kinase II. The translocation persists for at least 1 day of recovery after 30 min of isoelectricity in the vulnerable hippocampus (dorsomedial hippocampus) but not in the resistant hippocampus (dorsolateral hippocampus). Calmodulin binding to western blots shows changes similar to the immunoblots. Ca2+/calmodulin‐dependent activity of CaM‐kinase II in the crude synaptosomal fraction is elevated immediately before isoelectricity and is then inhibited during and after 30 min of isoelectricity, despite the increase of CaM‐kinase II immunoreactivity. This was seen in the vulnerable hippocampus. The data indicate that stimulus of translocation and inhibition of CaM‐kinase II persist during the recovery phase, preceding neuronal degeneration in the vulnerable hippocampus. This may be of significance for hypoglycemia‐induced neuronal death.
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| 4. |
- Hu, Bing‐Ren ‐R, et al.
(författare)
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Persistent Translocation of Ca2+/Calmodulin‐Dependent Protein Kinase II to Synaptic Junctions in the Vulnerable Hippocampal CA1 Region Following Transient Ischemia
- 1995
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Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 64:1, s. 277-284
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Tidskriftsartikel (refereegranskat)abstract
- Abstract: The influence of brain ischemia on the subcellular distribution and activity of Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) was studied in various cortical rat brain regions during and after cerebral ischemia. Total CaM kinase II immunoreactivity (IR) and calmodulin binding in the crude synaptosomal fraction of all regions studied increase but decrease in the microsomal and cytosolic fractions, indicative of a translocation of CaM kinase II to synaptosomes. The translocation of CaM kinase II to synaptic junctions occurs but not to synaptic vesicles. The translocation in neocortex and CA3/DG (dentate gyrus) is transient, whereas in the hippocampal CA1 region, it persists for at least 1 day of reperfusion. The Ca2+/calmodulin‐dependent activity of CaM kinase II in the subsynaptosomal fractions of neocortex is persistently decreased by up to 85%, despite the increase in CaM kinase II IR. The decrease in activity is more pronounced than the decline in IR, suggesting that CaM kinase II is covalently modified in the postischemic phase. The persistent translocation of CaM kinase II in the vulnerable ischemic CA1 region indicates that a pathological process is sustained in the area after the reperfusion phase and this may be of significance for ischemic brain injury.
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| 5. |
- Hu, Bing‐Ren, et al.
(författare)
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Tyrosine Phosphorylation and Activation of Mitogen‐ Activated Protein Kinase in the Rat Brain Following Transient Cerebral Ischemia
- 1994
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Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042. ; 62:4, s. 1357-1367
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Tidskriftsartikel (refereegranskat)abstract
- Abstract: Activation of trophic factor receptors stimulates tyrosine phosphorylation on proteins and supports neuronal survival. We report that in the recovery phase following reversible cerebral ischemia, tyrosine phosphorylation increases in the membrane fraction of the resistant hippocampal CA3/dentate gyrus (DG) region, whereas in the sensitive CA1 region or striatum, tyrosine phosphorylation is less marked or decreases. In the cytosolic fractions, a 42‐kDa protein, identified as mitogen‐activated protein (MAP) kinase, is markedly phosphorylated and activated immediately following ischemia, in particular in CA3/DG, but not in striatum. In the CA1 region, phosphorylation of MAP kinase is less intense and decreases later during re‐ perfusion, which could explain the delay of neuronal degeneration in this structure. The data suggest that in ischemia‐resistant neurons the growth factor receptor‐coupled signaling cascade is stimulated and, through its effects on DNA transcription and mRNA translation, supports neuronal survival.
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| 6. |
- Kurihara, Junichi, et al.
(författare)
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Changes in the Tyrosine Phosphorylation of Mitogen‐Activated Protein Kinase in the Rat Hippocampus During and Following Severe Hypoglycemia
- 1994
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Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 63:6, s. 2346-2348
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Tidskriftsartikel (refereegranskat)abstract
- Abstract: The changes in the levels of tyrosine‐phosphorylated proteins in the cytosolic fraction of the rat hippocampus subjected to severe hypoglycemia were analyzed. A marked increase in tyrosine phosphorylation of a 43‐kDa protein was observed at 30 min of isoelectric EEG and 30 min and 1 h of recovery. Immunostaining of the same blot with antibody against mitogen‐activated protein (MAP) kinase demonstrated a double band of ∼42 and 43 kDa. The increased tyrosine phosphorylation of MAP kinase during hypoglycemic coma and the early recovery period suggests that MAP kinase may be involved in neuronal degeneration and repair.
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