3-Methyladenine (5 mM) inhibits endogenous protein degradation in isolated rat hepatocytes by about 60%, while having no adverse effect on the degradation of an exogenous protein (asialofetuin), on protein synthesis, or on intracellular ATP levels. 3-Methyladenine appears to act specifically upon the autophagic/lysosomal pathway of degradation, as judged from its lack of effect in the presence of amino acids or a lysosomotropic amine (propylamine). The effect of the purine is not mediated by amino acids because the inhibition of protein degradation is accompanied by a significant depression of intracellular amino acid levels. The ability of 3-methyladenine to suppress the formation of electron microscopically visible autophagosomes suggests that it may be regarded as a specific inhibitor of autophagy.Intracellular protein degradation takes place by both lysosomal and nonlysosomal mechanisms (1-3). The lysosomes play a major role in (i) the degradation of exogenous proteins (4-7) and (ii) the degradation of endogenous proteins under conditions of amino acid starvation (step-down) (3,8,9). Amino acids apparently exert their regulatory effect by suppressing the first step in the autophagic/lysosomal pathway-i.e., the formation of autophagosomes (10, 11). Nothing is known, however, about the biochemical mechanisms involved in autophagy.We have found that, like the amino acids, certain N6-methylated adenosine derivatives (6-dimethylaminopurine riboside and puromycin aminonucleoside) could specifically inhibit the degradation of endogenous protein by the autophagic/lysosomal pathway in isolated rat hepatocytes (12). In a subsequent screening of a large number of purines and related substances (unpublished data), one compound-3-methyladenine-showed a unique selectivity in being able to inhibit endogenous protein degradation strongly without affecting protein synthesis. In the present report, the effects of 3-methyladenine are described in detail, and it is shown that this purine is capable of suppressing autophagosome formation independently of amino acids.MATERIALS AND METHODS Chemicals. 3-Methyladenine (6-amino-3-methyl-purine) was purchased from Fluka, Buchs, Switzerland. 3-Methyladenosine (p-toluene sulfonate salt) was a gift from T. Fujii (Kanazawa University, Kanazawa, Japan). The other 3-substituted adenine derivatives (identified by their NSC code numbers) were donated by the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD). Propylamine was purchased from Koch-Light Laboratories (Colnbrook, England), and gentamicin (Garamycin) was The degradation of an exogenous protein, "25I-labeled asialofetuin, was measured as the net release ofacid-soluble 125I after a 15-min ingestion period, as described by Tolleshaug et al (5). ATP. The intracellular ATP level was determined in neutralized perchloric acid extracts of the cells by means of a bioluminescence monitor (Luminometer; LKB) by using the luciferin/luciferase assay and the reagents accompanying th...
Colon cancer prevention currently relies on colonoscopy using white light to detect and remove polyps, but small and flat polyps are difficult to detect and frequently missed when using this technique. Fluorescence colonoscopy combined with a fluorescent probe specific for a polyp biomarker may improve polyp detection. Here we describe GE-137, a water-soluble probe consisting of a 26-amino acid cyclic peptide that binds the human tyrosine kinase c-Met conjugated to a fluorescent cyanine dye. Intravenous administration of GE-137 leads to its accumulation specifically in c-Met-expressing tumors in mice, and it is safe and well tolerated in humans. Fluorescence colonoscopy in patients receiving intravenous GE-137 enabled visualization of all neoplastic polyps that were visible with white light (38), as well as an additional nine polyps that were not visible with white light. This first-in-human pilot study shows that molecular imaging using an intravenous fluorescent agent specific for c-Met is feasible and safe, and that it may enable the detection of polyps missed by other techniques.
Abstract. Seven cytosolic enzymes with varying halflives (ornithine decarboxylase, 0.9 h; tyrosine aminotransferase, 3.1 h; tryptophan oxygenase, 3.3 h; serine dehydratase, 10.3 h, glucokinase, 12.7 h; lactate dehydrogenase, 17.0 h; aldolase, 17.4 h) were found to be autophagically sequestered at the same rate (3.5%/h) in isolated rat hepatocytes. Autophagy was measured as the accumulation of enzyme activity in the sedimentable organdies (mostly lysosomes) of electrodisrupted cells in the presence of the proteinase inhibitor leupeprin. Inhibitors of lysosomal fusion processes (vinhlasrine and asparagine) allowed accumulation of catalytically active enzyme (in prelysosomal vacuoles) even in the absence of proteolyric inhibition, showing that no inactivation step took place before lysosomal proteolysis. The completeness of protection by leupeptin indicates, furthermore, that a lysosomal cysteine proteinase is obligatorily required for the initial proteolytic attack upon autophagocytosed proteins. The experiments suggest that sequestration and degradation of normal cytosolic proteins by the autophagic-lysosomal pathway is a nonselective bulk process, and that nonautophagic mechanisms must be invoked to account for differential enzyme turnover.
Sequestration of the inert cytosolic marker [ 14C]sucrose by sedimentable organelles was measured in isolated rat hepatocytes made transiently permeable to sucrose by means of electropermeabilization . Lysosomal integrity, protein degradation, autophagic sequestration, and other cellular functions were not significantly impaired by the electric treatment .Hepatocytes sequestered sucrose at an initial rate of -10%/h, which is threefold higher than the estimated rate of auto phagic-lysosomal protein degradation . Almost one-third would appear to represent mitochondrial fluid uptake ; the rest was nearly completely and specifically inhibited by 3-methyladenine (3MA) and can be regarded as autophagic sequestration . A complete amino acid mixture was somewhat less inhibitory than 3MA, and partially antagonized the effect of the latter . This paradoxical effect, taken together with the high sequestration rate, may suggest heterogeneity as well as selectivity in autophagic sequestration . There was no detectable recycling of sequestered [ 14C]sucrose between organelles and cytosol .Studies of individual amino acids revealed histidine as the most effective sequestration inhibitor . Leucine may have a regulatory function, as indicated by its unique additive/ synergistic effect, and a combination of Leu + His was as effective as the complete amino acid mixture . Asparagine inhibited sequestration only 20%, i .e., its very strong effect on overall (long-lived) protein degradation must partially be due to post-sequestrational inhibition.The lysosomal (amine-sensitive) degradation of short-lived protein was incompletely inhibited by 3MA, indicating a contribution from nonautophagic processes like crinophagy and endocytic membrane influx. The ability of an amino acid mixture to specifically antagonize the inhibition of short-lived protein degradation by AsN + GIN (but not by 3MA) may suggest complex amino acid interactions at the level of fusion between lysosomes and other vesicles in addition to the equally complex interactions at the level of autophagic sequestration .Rat hepatocytes offer a particularly suitable material for the study of intracellular protein degradation . These cells are in positive protein balance in well-fed animals (13) or in a culture medium supplied with insulin and high amino acid concentrations (20, 26), but upon starvation in vivo (or amino acid deprivation in vitro), the hepatocytes rapidly turn on their autophagic-lysosomal degradation pathway and enter a state of negative protein balance in an attempt to supply the organism (or medium) with amino acids (13,14,26,27,33). The overall rate of protein degradation may be as high as 4-5%/h, of which the autophagic-lysosomal pathway accounts for -3%/h on the basis of the extent of inhibition seen with amino acids and lysosome inhibitors (32, 35). The remaining -1 .5% (corresponding to the "basal" degradation generally THE JOURNAL OF CELL BIOLOGY " VOLUME 99 AUGUST 1984 435-444 0 The Rockefeller University Press -0021-9525/84/08/435/10 $1 .00 ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.