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Biomedical subjects

P O Seglen

Publications and source records attributed to P O Seglen.

At least 127 records · Page 7Linked to original sources
PubMed1981

The role of protein accumulation in the cell cycle control of human NHIK 3025 cells.

The cell cycle kinetics of NHIK 3025 cells, synchronized by mitotic selection, was studied in the presence of cycloheximide at concentrations (0.125-1.25 microM) which inhibited protein synthesis partially and slowed down the rate of cell cycle traverse. The median cell cycle duration was equal to the protein doubling time in both the control cells and in the cycloheximide-treated cultures at all drug concentrations. This conclusion was valid whether protein synthesis was continuously depressed by cycloheximide throughout the entire cell cycle, or temporarily inhibited during shorter periods at various stages of the cell cycle. These results may indicate that cell division does not take place before the cell has reached a critical size, or has completed a protein accumulation-dependent sequence of events. When present throughout the cell cycle, cycloheximide increased the median G1 duration proportionally to the total cell cycle prolongation. However, the entry of cells into S, once initiated, proceeded at an almost unaffected rate even at cycloheximide concentrations which reduced the rate of protein synthesis 50%. The onset of DNA synthesis seemed to take place in the cycloheximide-treated cells at a time when the protein content was lower than in the control cells. This might suggest that DNA synthesis in NHIK 3025 cells is not initiated at a critical cell mass.

Cell Cycle↗
PubMed1981

Effects of antibiotics on protein synthesis and degradation in primary cultures of rat hepatocytes.

In primary hepatocyte cultures, maintained in a protein-free medium, streptomycin, penicillin, and Garamycin (gentamicin) all inhibited protein synthesis at concentrations above 0.1 mM. Some inhibition was also observed with the fungicide Mycostatin at 100 U/ml. Hepatocytic protein degradation was markedly inhibited by penicillin fat concentrations above 0.1 mM, whereas streptomycin and Garamycin only showed slight inhibition at concentrations in excess of 1 mM. None of the antibiotics had any detectable effect on the structural integrity (viability) of the cells.

Animals↗
PubMed1981

Effects of insulin and anchorage on hepatocytic protein metabolism and amino acid transport.

Insulin partially inhibits endogenous protein degradation in isolated hepatocytes. The inhibition seems to specifically affect the lysosomal pathway of degradation, since it is not additive to the effects of lysosome inhibitors such as propylamine and leupeptin. The insulin effect is potentiated by intermediate concentrations of amino acids, but is largely abolished at high amino acid concentrations which suppress degradation maximally, suggesting that the hormone may exert its effect indirectly by acting upon the more basal amino acid control mechanism. Glucagon, which stimulates protein degradation, similarly displays its effect only in the presence of intermediate amino acid concentrations. The insulin inhibition is not affected by the aminotransferase inhibitor, aminooxyacetate, indicating that it is not due to interference with amino acid metabolism. Protein synthesis furthermore does not seem to be required, since a significant insulin effect can be seen in the presence of the protein synthesis inhibitor, cycloheximide. The issue is, however, complicated by the fact that cycloheximide itself inhibits protein degradation to approximately the same extent as does insulin. Insulin stimulates uptake of the amino acid alpha-aminoisobutyrate (AIB), but not the uptake of valine, indicating a specific stimulation of 'A'-type transport. Cycloheximide similarly stimulates AIB uptake, without completely obfuscating the transport effect of insulin. Neither protein synthesis, protein degradation, amino acid transport, nor the effects of insulin were affected by cell-to-substratum anchorage (attachment and spreading) in any detectable way.

Amino Acids↗
PubMed1981

Role of microtubuli in the lysosomal degradation of endogenous and exogenous protein in isolated rat hepatocytes.

Colchicine and vinblastine, two well known inhibitors of microtubular function, inhibited endogenous protein degradation as well as protein secretion and the degradation of an exogenous, internalized protein (asialo fetuin), in isolated rat hepatocytes. The inhibitors appeared to selectively affect the lysosomal pathway of degradation, since their effects were not additive to that of ammonia, a lysosomotropic degradation inhibitor.

Ammonium Chloride↗
PubMed1981

Effects of amino acids and amino acid analogues on lysosomal protein degradation in isolated rat hepatocytes.

An amino acid mixture, specially designed to improve the protein balance in isolated hepatocytes, inhibited lysosomal (propylamine-sensitive) degradation of endogenous proteins by 80-90%. The amino acids had no effect on the degradation of the endocytosed protein asialofetuin, the conclusion being that amino acids are involved in the regulation of autophagy. Furthermore, the effect of two amino acids was tested individually, at high concentrations, in an electron microscopic analysis. Leucine caused a 79% decrease in the early forms of autophagic vacuoles, while asparagine had no effect. Leucine appears to be important in the regulation of the bulk autophagy observable in the electron microscope but this type of autophagy possibly contributes only about one-half of the total amount of protein degraded in lysosomes, since leucine only inhibits some 30% of the lysosomal protein degradation. Certain branched-chain amino acids related to leucine inhibited lysosomal protein degradation to the same extent as did leucine.

Amino Acids↗
PubMed1981

Inhibitors and pathways of hepatocytic protein degradation.

On the basis of experiments using amino acids and various inhibitors (lysosomotropic amines, leupeptin, chymostatin, vanadate, vinblastine, anoxia, methylaminopurines), five different modes of endogenous protein degradation in isolated rat hepatocytes can be distinguished. The two non-lysosomal (amine-resistant) mechanisms preferentially degrade relatively labile (short-lived) proteins: one of these mechanisms is energy-dependent and chymostatin-sensitive, the other is not. Of the three lysosomal (amine-sensitive) mechanisms, one--quantitatively minor--is amino acid-resistant and preferentially degrades labile proteins. The two amino acid-sensitive mechanisms each seen account for about one-half of the degradation of relatively stable (long-lived) proteins; one of them is suppressed by leucine and apparently corresponds to the formation of electron microscopically visible autophagosomes; the other may represent a different type of autophagy, inhibited by asparagine and glutamine. A new class of inhibitors, the purine derivatives (methylated 6-aminopurines, and 6-mercaptopurines) appear to specifically suppress autophagic/lysosomal protein degradation, and may help to further elucidate the mechanisms of autophagy.

Adenine↗
PubMed1980

Differential effects of proteinase inhibitors and amines on the lysosomal and non-lysosomal pathways of protein degradation in isolated rat hepatocytes.

Ammonia, which like other lysosomotropic amines inhibits protein degradation in isolated rat hepatocytes by 70---80%, was utilized as a diagnostic tool to distinguish between the relative effects of various proteinase inhibitors on the lysosomal and non-lysosomal pathways of intracellular protein degradation. Leupeptin was found to inhibit lysosomal protein degradation by 80---85%, and non-lysosomal degradation by about 15%. Antipain had a similar, but somewhat weaker effect. Pepstatin, bestatin and aprotinin (Trasylol) produced minor inhibitory effects (possibly on both degradation pathways), whereas bacitracin and soybean trypsin inhibitor were ineffective. Chymostatin inhibited lysosomal protein degradation by about 45%, whereas the non-lysosomal pathway was inhibited by more than 50%. Chymostatin was unique among the inhibitors tested in causing such a pronounced effect on non-lysosomal protein degradation, and appeared to selectively inhibit the energy-dependent portion of this pathway. The effects of the various inhibitors were additive to the extent expected on the basis of their known actions only sosomal and non-lysosomal protein degradation. Thus, a combination of methylamine, leupeptin and chymostatin inhibited overall protein degradation by about 90%, resulting in a substantial improvement of the cellular nitrogen balance. The degradation inhibitors caused a partial inhibition of protein synthesis, apparently mainly by shutting down the supply of amino acids from the lysosomes. The inhibitory effects of leupeptin and antipain were completely reversed by amino acid addition, whereas some inhibition remained in the case of chymostatin and the lysosomotropic amines, possibly reflecting a certain nonspecific toxicity.

Amines↗
PubMed1980

Amino acid inhibition of the autophagic/lysosomal pathway of protein degradation in isolated rat hepatocytes.

Protein degradation in isolated rat hepatocytes, as measured by the release of [14C]valine from pre-labelled protein, is partly inhibited by a physiologically balanced mixture of amino acids. The inhibition is largely due to the seven amino acids leucine, phenylalanine, tyrosine, tryptophan, histidine, asparagine and glutamine. When the amino acids are tested individually at different concentrations, asparagine and glutamine are the strongest inhibitors. However, when various combinations are tested, a mixture of the first five amino acids as well as a combination of leucine and asparagine inhibit protein degradation particularly strongly. The inhibition brought about by asparagine plus leucine is not additive to the inhibition by propylamine, a lysosomotropic inhibitor; thus indicating that the amino acids act exclusively upon the lysosomal pathway of protein degradation. Following a lag of about 15 min the effect of asparagine plus leucine is maximal and equal to the effect of propylamine, suggesting that their inhibition of the lysosomal pathway is complete as well as specific. Degradation of endocytosed 125I-labelled asialofetuin is not affected by asparagine plus leucine, indicating that the amino acids do not affect lysosomes directly, but rather inhibit autophagy at a step prior to the fusion of autophagic vacuoles with lysosomes. The aminotransferase inhibitor, aminooxyacetate, does not prevent the inhibitory effect of any of the amino acids, i.e. amino acid metabolites are apparently not involved.

Amino Acids↗
PubMed1980

Subcellular distribution of proteolytically generated valine in isolated rat hepatocytes.

1. A small fraction of the intracellular acid-soluble radioactivity in [14C]valine-labelled hepatocytes remained non-extracted even after repeated incubations in isotope-free medium at 37 degrees C. This non-extracted radioactivity was only present when the cells had been labelled under conditions allowing protein synthesis. 2. Approximately two-thirds of the non-extracted radioactivity was recovered in material with a molecualr weight larger than free valine, i.e. presumably acid-soluble peptides. The intracellular contents of these peptides were unaffected by inhibitors of hepatocytic protein degradation, and they would therefore seem to represent primary synthesis products rather than products of proteolysis. 3. The remaining one-third of the radioactivity was free [14C]valine, derived from intracellular protein degradation as indicated by the reduced levels seen in the presence of inhibitors of lysosomal (ammonia, methylamine, leupeptin, chymostatin) and non-lysosomal (chymostatin) proteolysis. The intracellular levels of twelve other amino acids were similarly reduced upon inhibition of protein degradation. 4. The contents of free [14C]valine in subcellular fractions were compatible with a uniform distribution of the amino acid throughout the cell. There was no evidence for any enrichment of [14C]valine in a purified lysosomal fraction, separated from mitochondria by means of isotonic metrizamide gradients. 5. It can be concluded that the non-extracted valine in hepatocytes may represent a steady-state level, maintained by intracellular protein degradation, of amino acid in transit through the cell. The non-extractability, i.e. the maintenance of a concentration gradient towards the extracellular medium, can be calculated to be compatible with a limitation of valine efflux by the concentration-dependent valine transport system. Since no specific subcellular compartmentation is indicated, intracellular valine can provisionally be regarded as a single, uniform pool.

Amino Acids↗