[Autophagy in differentiation and development].
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Animals↗
Biomedical subjects
Akiko Kuma
Publications and source records attributed to Akiko Kuma.
Organelle degradation during the lens and erythroid differentiation is independent of autophagy.
Autophagy is a bulk degradation system within cells through which cytoplasmic components are degraded within lysosomes. Primary roles of autophagy are starvation adaptation and intracellular protein quality control. In contrast to the ubiquitin-proteasome system, autophagy can also degrade organelles. Here we examined a possible role of autophagy in organelle degradation during lens and erythroid differentiation. We observed that autophagy occurs in embryonic lens cells. However, organelle degradation in lens and erythroid cells occurred normally in autophagy-deficient Atg5(-/-) mice. Our data suggest that degradation system(s) other than autophagy play major roles in organelle degradation during these processes.
Animals↗
Delivery advantage to the unilateral kidney by direct drug application to the kidney surface in rats and pharmacokinetic verification based on a physiological model.
The objective of this study was to evaluate the drug delivery advantage to the unilateral kidney by direct drug application to the rat kidney surface based on a physiological pharmacokinetic model. Under anesthesia, a cylindrical diffusion cell (i.d. 6 mm, area 0.28 cm(2)) was attached to the right kidney surface in rats. Phenolsulfonphthalein (PSP), an organic anion chosen as a model compound, was added into the diffusion cell. The free PSP concentration in the right (applied) kidney after application to the right kidney surface at a dose of 1 mg was significantly higher than that of the left (non-applied) kidney until 60 min after application. Similarly, the urinary excretion rate of free PSP from the applied kidney was much faster than that from the non-applied kidney, with a 2.6 times larger excreted amount in 240 min. These results imply the possibility that a considerable drug delivery advantage to the unilateral kidney could be obtained after direct absorption from the kidney surface. This tendency was also observed at the other application doses of 0.3 and 1.5 mg. On the other hand, fluorescein isothiocyanate dextran (Mw 4400, FD-4) was equally excreted into the urine from each kidney and the renal concentrations in the applied and non-applied kidneys were almost the same, possibly due to the involvement of passive transport for the absorbed FD-4, i.e. glomerular filtration. The computer simulations of free PSP concentrations in the plasma and each kidney based on a physiological model after kidney surface application were consistent with the respective experimental data. Moreover, the delivery advantage of kidney surface application of PSP was verified by its comparison with other routes such as i.v. and i.a. administrations.
Absorption↗
Absorption characteristics of model compounds from the small intestinal serosal surface and a comparison with other organ surfaces.
We examined the absorption of phenolsulfonphthalein (PSP) and fluorescein isothiocyanate dextrans (FD-4, MW 4400; FD-10, MW 9500; FD-40, MW 40 500) as model compounds through the small intestinal serosal surface. After application to the rat small intestinal serosal surface using a cylindrical diffusion cell, each compound was absorbed at different rates. The absorption ratios in 6 h after PSP, FD-4, FD-10 and FD-40 application were calculated to be 89.2, 34.6, 14.9 and 2.1% of dose, respectively. Elimination profiles of PSP, FD-4 and FD-10 from the small intestinal serosal surface obeyed first-order kinetics. Moreover, we calculated the apparent permeability coefficient P(app) for comparison to other organ surfaces. The kidney had the highest absorption efficiency, as shown by having more than 1.5 times significantly higher P(app) values of PSP, FD-4 and FD-10. Similar to the other organ surfaces, a correlation was observed between the P(app) of the small intestine and the molecular weight of these hydrophilic compounds. In addition, the small intestine is likely to contribute largely to hydrophilic compound absorption from the peritoneal cavity, judging from absorption clearance, CL(a), calculated using the peritoneal organ surface area.
Animals↗
The role of autophagy during the early neonatal starvation period.
At birth the trans-placental nutrient supply is suddenly interrupted, and neonates face severe starvation until supply can be restored through milk nutrients. Here, we show that neonates adapt to this adverse circumstance by inducing autophagy. Autophagy is the primary means for the degradation of cytoplasmic constituents within lysosomes. The level of autophagy in mice remains low during embryogenesis; however, autophagy is immediately upregulated in various tissues after birth and is maintained at high levels for 3-12 h before returning to basal levels within 1-2 days. Mice deficient for Atg5, which is essential for autophagosome formation, appear almost normal at birth but die within 1 day of delivery. The survival time of starved Atg5-deficient neonates (approximately 12 h) is much shorter than that of wild-type mice (approximately 21 h) but can be prolonged by forced milk feeding. Atg5-deficient neonates exhibit reduced amino acid concentrations in plasma and tissues, and display signs of energy depletion. These results suggest that the production of amino acids by autophagic degradation of 'self' proteins, which allows for the maintenance of energy homeostasis, is important for survival during neonatal starvation.
Animals↗
Alfy, a novel FYVE-domain-containing protein associated with protein granules and autophagic membranes.
Phosphatidylinositol-3-phosphate [PtdIns(3)P] regulates endocytic and autophagic membrane traffic. In order to understand the downstream effects of PtdIns(3)P in these processes, it is important to identify PtdIns(3)P-binding proteins, many of which contain FYVE zinc-finger domains. Here, we describe a novel giant FYVE-domain-containing protein, named autophagy-linked FYVE protein (Alfy). Alfy is ubiquitously expressed, shares sequence similarity with the Chediak-Higashi-syndrome protein and has putative homologues in flies, nematodes and fission yeast. Alfy binds PtdIns(3)P in vitro and partially colocalizes with PtdIns(3)P in vivo. Unlike most other FYVE-domain proteins, Alfy is not found on endosomes but instead localizes mainly to the nuclear envelope. When HeLa cells are starved or treated with a proteasome inhibitor, Alfy relocalizes to characteristic filamentous cytoplasmic structures located close to autophagic membranes and ubiquitin-containing protein aggregates. By electron microscopy, similar structures can be found within autophagosomes. We propose that Alfy might target cytosolic protein aggregates for autophagic degradation.
Adaptor Proteins, Signal Transducing↗
Binding of 14-3-3beta but not 14-3-3sigma controls the cytoplasmic localization of CDC25B: binding site preferences of 14-3-3 subtypes and the subcellular localization of CDC25B.
The dual specificity phosphatase CDC25B positively controls the G2-M transition by activating CDK1/cyclin B. The binding of 14-3-3 to CDC25B has been shown to regulate the subcellular redistribution of CDC25B from the nucleus to the cytoplasm and may be correlated with the G2 checkpoint. We used a FLAG-tagged version of CDC25B to study the differences among the binding sites for the 14-3-3 subtypes, 14-3-3beta, 14-3-3epsilon and 14-3-3sigma, and the relationship between subtype binding and the subcellular localization of CDC25B. All three subtypes were found to bind to CDC25B. Site-directed mutagenesis studies revealed that 14-3-3beta bound exclusively near serine-309 of CDC25B1, which is within a potential consensus motif for 14-3-3 binding. By contrast, 14-3-3sigma bound preferentially to a site around serine-216, and the presence of serine-137 and -309 enhanced the binding. In addition to these binding-site differences, we found that the binding of 14-3-3beta drove CDC25B to the cytoplasm and that mutation of serine-309 to alanine completely abolished the cytoplasmic localization of CDC25B. However, co-expression of 14-3-3sigma and CDC25B did not affect the subcellular localization of CDC25B. Furthermore, serine-309 of CDC25B was sufficient to produce its cytoplasmic distribution with co-expression of 14-3-3beta, even when other putative 14-3-3 binding sites were mutated. 14-3-3epsilon resembled 14-3-3beta with regard to its binding to CDC25B and the control of CDC25B subcellular localization. The results of the present study indicate that two 14-3-3 subtypes can control the subcellular localization of CDC25B by binding to a specific site and that 14-3-3sigma has effects on CDC25B other than the control of its subcellular localization.
14-3-3 Proteins↗
Absorption of phenolsulfonphthalein as a model across the mesenteric surface in rats to determine the drug absorption route after intraperitoneal administration.
The purpose of this study was to clarify the absorption characteristics of a drug across the mesenteric surface, which occupies a large area of absorption in the peritoneal cavity, in order to determine the drug absorption route after intraperitoneal administration. Absorption of phenolsulfonphthalein (PSP) as a model after application to the mesenteric surface was investigated in rats by employing a cylindrical diffusion cell attached to the mesentery with or without blood vessels. PSP was absorbed from the rat mesenteric surface, followed by its appearance in the plasma and bile, regardless of blood vessel existence. The absorption ratios of PSP in 6 h were calculated to be 92.1 and 83.6% from the mesenteric surface with and without blood vessels, respectively. We then employed an experimental system in which a polyethylene (PE) cap was stuck on the surface of the other side to exclude the influence of absorption of the drug from the other organ surfaces that penetrated across the mesentery. The PE cap decreased the appearance of PSP in the plasma from the mesenteric surface with blood vessels and eliminated the PSP absorption completely from the mesenteric surface without blood vessels. Accordingly, blood vessels on the mesenteric surface must actually play an important role in drug absorption, but the contribution of the mesenteric surface to drug absorption from the peritoneal cavity is unlikely to be significant because there is a small effective area of blood vessels.
Animals↗
Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate.
Macroautophagy is the major intracellular degradation system delivering cytoplasmic components to the lysosome/vacuole. We have shown that, in yeast and mammalian cells, the Apg12-Apg5 protein conjugate, which is formed by a ubiquitin-like system, is essential for autophagosome formation. In yeast, the Apg12-Apg5 conjugate interacts with a small coiled-coil protein, Apg16, to form a approximately 350 kDa multimeric complex. We demonstrate that the mouse Apg12-Apg5 conjugate forms a approximately 800 kDa protein complex containing a novel WD-repeat protein. Because the N-terminal region of this novel protein shows homology with yeast Apg16, we have designated it mouse Apg16-like protein (Apg16L). Apg16L, however, has a large C-terminal domain containing seven WD repeats that is absent from yeast Apg16. Apg16L interacts with both Apg5 and additional Apg16L monomers; neither interaction, however, depends on the WD-repeat domain. In conjunction with Apg12-Apg5, Apg16L associates with the autophagic isolation membrane for the duration of autophagosome formation. Because these features are similar to yeast Apg16, we concluded Apg16L is the functional counterpart of the yeast Apg16. We also found that membrane targeting of Apg16L requires Apg5 but not Apg12. Because WD-repeat proteins provide a platform for protein-protein interactions, the approximately 800 kDa complex is expected to function in autophagosome formation, further interacting with other proteins in mammalian cells.
Amino Acid Sequence↗
Formation of the approximately 350-kDa Apg12-Apg5.Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for autophagy in yeast.
Autophagy, responsible for the delivery of cytoplasmic components to the lysosome/vacuole for degradation, is the major degradative pathway in eukaryotic cells. This process requires a ubiquitin-like protein conjugation system, in which Apg12 is covalently bound to Apg5. In the yeast Saccharomyces cerevisiae, the Apg12-Apg5 conjugate further interacts with a small coiled-coil protein, Apg16. The Apg12-Apg5 and Apg16 are localized in the cytosol and pre-autophagosomal structures and play an essential role in autophagosome formation. Here we show that the Apg12-Apg5 conjugate and Apg16 form a approximately 350-kDa complex in the cytosol. Because Apg16 was suggested to form a homo-oligomer, we generated an in vivo system that allowed us to control the oligomerization state of Apg16. With this system, we demonstrated that formation of the approximately 350-kDa complex and autophagic activity depended on the oligomerization state of Apg16. These results suggest that the Apg12-Apg5 conjugate and Apg16 form a multimeric complex mediated by the Apg16 homo-oligomer, and formation of the approximately 350-kDa complex is required for autophagy in yeast.
Autophagy↗
Absorption characteristics of model compounds with different molecular weights from the serosal caecal surface in rats.
The purpose of this study was to clarify the absorption characteristics of drugs across the serosal caecal surface membrane, occupying a large absorption area in the peritoneal cavity in rats. Absorption of phenolsulfonphthalein (PSP) and fluorescein isothiocyanate dextrans (FDs) as model drugs after application to the rat serosal caecal surface was investigated using a cylindrical diffusion cell. PSP was absorbed from the rat serosal caecal surface, followed by appearance in the plasma and bile. The time course of the remaining PSP amount in the diffusion cell obeyed first-order kinetics, and the rate constant, Ka, was calculated to be 8.01 x 10(-3) min(-1). No significant difference was seen in the absorption ratio of PSP, which was approximately 90% in 6 h for three doses (0.3, 0.5 and 1 mg), suggesting linear absorption. Moreover, the absorption ratios of FDs from the rat serosal caecal surface at 3 h decreased with an increase in the molecular weight (24.7% for FD4, 12.8% for FD-10 and 3.4% for FD-40).
Absorption↗