scholarly journals Effect of microtubular or translational inhibitors on general cell protein degradation. Evidence for a dual catabolic pathway

1977 ◽  
Vol 168 (2) ◽  
pp. 223-227 ◽  
Author(s):  
J S Amenta ◽  
M J Sargus ◽  
F M Baccino
Keyword(s):  
Protein Degradation ◽  
Trichloroacetic Acid ◽  
Fold Increase ◽  
Cell Protein ◽  
Inhibitory Effects ◽  
Minimal Essential Medium ◽  
Catabolic Pathway ◽  
Rat Embryo ◽  
Step Down ◽  
Observation Period

Rat embryo fibroblasts were grown in Eagle's minimal essential medium with 10% serum and cell proteins prelabelled with L-[1-(14)C]leucine, followed by a 24h chase. When transferred to medium deprived of serum these cells showed a 2—3-fold increase in the production of trichloroacetic acid-soluble radioactivity during a 4h observation period. The microtubular poisons vinblastine, vincristine and colchicine partially inhibited this induced proteolysis, but had no effect on the proteolytic rate of cells maintained in medium with 10% serum. A similar discriminating effect on induced proteolysis was observed with cycloheximide, puromycin and insulin. The inhibitory effects of cycloheximide and vinblastine were not additive. These data support the hypothesis that, in addition to the basal turnover of cell proteins, a second mechanism of protein degradation involving cytoplasmic autophagy can be activated by nutritional step-down and is selectively inhibited by agents that interfere with microtubular function and protein synthesis.

scholarly journals Mechanisms of protein degradation in growing and non-growing L-cell cultures

1979 ◽  
Vol 182 (3) ◽  
pp. 847-859 ◽  
Author(s):  
J S Amenta ◽  
M J Sargus
Keyword(s):  
Stationary Phase ◽  
Protein Degradation ◽  
Media Analysis ◽  
Cell Protein ◽  
Minimal Essential Medium ◽  
Buffer Solution ◽  
L Cells ◽  
Effective Inhibition ◽  
Processing Procedure

L-cells prelabelled with [14C]leucine and [3H]thymidine were placed in either fresh growth medium (minimal essential medium with 10% serum) or stepdown medium (minimal essential medium) for 3 days. The 14C/3H ratio remained constant in the growing cultures and decreased in the stationary-phase cultures, indicating no protein turnover in growing cultures and a degradative rate of 0.6%/h in the stationary-phase cultures. Media analysis, however, indicated that 14C-labelled proteins were being degraded at approx. 1.2%/h in growing cultures and 1.7%/h in stationary-phase cultures. Additional studies indicated that a subpopulation of L-cells in the monolayer, comprising approx. 20–30% of the total, were lost in the original processing procedure. Experiments in which recoveries approached 100% by fixation of the monolayer in situ indicated that a protein-degrading subpopulation accounted for all the observed proteolysis in the growing cultures. Proteolysis in these cultures was only partially inhibited with NH4Cl, indicating that only a small part of the protein degradation was occurring in an activated lysosomal-autophagic system. NaF produced a more effective inhibition of proteolysis, but we were not able to distinguish whether this effect was on an ATP-requiring basal-turnover mechanism or a direct effect on unregulated activity of proteinases in the cell hyaloplasm. However, NH4Cl inhibited the proteolysis induced when cells were placed in stepdown medium, suggesting that the induced proteolysis was occurring via the autophagic system. We conclude that L-cells exist in at least two states with respect to protein degradation: (a) a subpopulation that is actively replicating and does not degrade cellular proteins, and (b) a second subpopulation of cells, derived from the preceding one, which degraded most of their labelled proteins, are not capable of further replication, and are not sedimented in an iso-osmotic EDTA buffer solution. In addition, proliferating L-cells, when placed in stepdown medium, begin to degrade cell protein through a mechanism involving autophagolysosomes.

Oncogene-induced transformation of a rat embryo fibroblast cell line is enhanced by tumor promoters

1986 ◽  
Vol 6 (6) ◽  
pp. 1943-1950
Author(s):  
W L Hsiao ◽  
T Wu ◽  
I B Weinstein
Keyword(s):  
Cell Line ◽  
Fold Increase ◽  
Fibroblast Cell ◽  
Tumor Promoters ◽  
Fibroblast Cell Line ◽  
Ras Oncogene ◽  
Rat Embryo ◽  
Synergistic Interactions ◽  
Multistage Carcinogenesis ◽  
System Tumor

Rat embryo fibroblast cell line 6 was transfected with plasmid pT24, which contains the activated human bladder c-Ha-ras oncogene, and the cells were grown continuously in the absence or presence of the tumor promoters 12-O-tetradecanoyl phorbol-13-acetate (TPA) or teleocidin. The presence of TPA or teleocidin led to a 6- to 14-fold increase in the number of morphologically transformed foci. No transformed foci were seen when rat 6 cells were transfected with the normal c-Ha-ras oncogene in the absence or presence of TPA, or in cells simply treated with TPA or teleocidin. Enhancement of pT24-induced foci was seen even when the addition of TPA was delayed until day 16. In transfection studies with the drug resistance genes gpt and neo, TPA and teleocidin did not increase the number of Gpt+ or Neo+ colonies. When rat 6 cells were cotransfected with pT24 and neo genes and grown in the absence or presence of TPA, the presence of TPA did not increase the yield of Neo+ colonies but caused a fivefold increase in the number of Neo+ colonies that displayed a transformed morphology. Southern blot analyses of DNAs obtained from these clones indicated that TPA treatment did not influence the extent of integration of either the pT24 or neo gene. DNA samples from all of the morphologically transformed cells displayed a characteristic 2-kilobase SacI fragment homologous to pT24 DNA and expressed relatively high levels of the corresponding mRNA. Our findings indicate that in this system tumor promoters do not simply enhanced the process of DNA transfection per se. Thus, this model system may be useful for analyzing synergistic interactions between tumor promoters and activated oncogenes during multistage carcinogenesis. It may also serve as a simple screening test for detecting new tumor promoters.

Endotoxin and thrombin elevate rodent endothelial cell protein C receptor mRNA levels and increase receptor shedding in vivo

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1687-1693 ◽  
Author(s):  
Jian-Ming Gu ◽  
Yasuhiro Katsuura ◽  
Gary L. Ferrell ◽  
Paula Grammas ◽  
Charles T. Esmon
Keyword(s):  
Cell Culture ◽  
Septic Shock ◽  
Endothelial Cell ◽  
Protein C ◽  
Fold Increase ◽  
Cell Protein ◽  
Thrombin Inhibitor ◽  
Mrna Levels ◽  
Soluble Epcr

The endothelial cell protein C receptor (EPCR) facilitates protein C activation by the thrombin-thrombomodulin complex. Protein C activation has been shown to be critical to the host defense against septic shock. In cell culture, tumor necrosis factor- (TNF-) down-regulates EPCR expression, raising the possibility that EPCR might be down-regulated in septic shock. We examined EPCR mRNA and soluble EPCR levels in mice and rats challenged with lethal dose 95 levels of endotoxin. Toxic doses of TNF- failed to alter EPCR mRNA levels in mice. Rather than EPCR mRNA levels falling in response to endotoxin, as predicted from cell-culture experiments, they rose approximately 3-fold 6 hours after exposure to endotoxin before returning toward baseline levels at 24 hours after exposure. Soluble EPCR levels rose approximately 4-fold. Infusion of hirudin, a specific thrombin inhibitor, before endotoxin exposure almost completely blocked the increase in EPCR mRNA and soluble EPCR. Consistent with the idea that the responses were mediated by thrombin, thrombin infusion (5 U/kg of body weight for 3 hours) resulted in an approximately 2-fold increase in EPCR mRNA and soluble EPCR. Incubation of rat endothelial cells with thrombin or murine protease-activated receptor 1 agonist peptide resulted in a 2-fold increase in EPCR mRNA. These results indicate that thrombin plays a major role in up-regulating EPCR mRNA and shedding in vivo.

scholarly journals Inhibition of cardiac proteolysis by colchicine. Selective effects on degradation of protein subclasses

1983 ◽  
Vol 210 (1) ◽  
pp. 63-71 ◽  
Author(s):  
J S Crie ◽  
J M Ord ◽  
J R Wakeland ◽  
K Wildenthal
Keyword(s):  
Protein Degradation ◽  
Total Protein ◽  
Protein Turnover ◽  
Branched Chain Amino Acids ◽  
Inhibitory Effects ◽  
Similar Extent ◽  
Specific Effects ◽  
Protein Pool ◽  
Branched Chain ◽  
Mitochondrial Cytochromes

1. The effect of colchicine (2.5 microM) on cardiac protein turnover was tested with foetal mouse hearts in organ culture. 2. Colchicine had no effect on protein synthesis, but inhibited total protein degradation by 12-18%. Lumicolchicine, which lacks colchicine's ability to disaggregate microtubules, but shares its non-specific effects, did not alter protein degradation. 3. The colchicine-induced inhibition of protein degradation was accompanied by significant changes in cardiac lysosomal enzyme activities and distribution. 4. Colchicine inhibited the degradation of organellar proteins, including mitochondrial cytochromes, more than that of cytosolic proteins. 5. Colchicine decreased the rate of myosin degradation and the rate of proteolysis of the total protein pool to a similar extent. Since the regulation of myosin degradation does not involve lysosomes, this suggests that colchicine affects non-lysosomal as well as lysosomal pathways. 6. Release of branched-chain amino acids from colchicine-treated hearts was disproportionately decreased, suggesting that colchicine increased their metabolism. 7. It is concluded that colchicine, via its actions on microtubules, exerts important inhibitory effects on cardiac proteolysis. Colchicine is especially inhibitory to the degradation of organellar proteins, including mitochondrial cytochromes. Its inhibitory effects may be mediated in part via lysosomal mechanisms, but non-lysosomal mechanisms are probably involved as well.

THE DETERMINATION OF TERMINAL PROTEIN-BOUND FUCOSE

1965 ◽  
Vol 43 (11) ◽  
pp. 1807-1811 ◽  
Author(s):  
G. Gyorky ◽  
J. C. Houck
Keyword(s):  
Protein Degradation ◽  
Spectrophotometric Determination ◽  
Trichloroacetic Acid ◽  
Degradation Products ◽  
Dilute Acid ◽  
Terminal Protein ◽  
Residual Protein ◽  
Color Yield ◽  
Fucose Content

The spectrophotometric determination of protein-bound fucose is badly compromised by spurious chromagens developed from protein degradation products. To minimize the contribution of these spurious products to the color yield of fucose, the glycoprotein was partially hydrolyzed in dilute acid, thus releasing the terminal carbohydrate from the protein moieties, and the residual protein was removed with trichloroacetic acid. That the fucose content of this supernatant was real was confirmed by paper chromatography and spectral studies.The spurious chromagens were shown to result from the interaction of protein degradation products and galactose.

Kinetics of Human and Murine Mobilization of Acute Myeloid Leukemia in Response to AMD3100.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 867-867 ◽  
Author(s):  
Geoffrey L. Uy ◽  
Michael P. Rettig ◽  
Pablo Ramirez ◽  
Bruno Nervi ◽  
Camille N. Abboud ◽  
...  
Keyword(s):  
Fusion Gene ◽  
Fold Increase ◽  
Genotoxic Stress ◽  
Peripheral Circulation ◽  
Cathepsin G ◽  
Cxcr4 Antagonist ◽  
Leukocyte Counts ◽  
Kinetics Of ◽  
Refractory Aml ◽  
Observation Period

The CXCR4-SDF-1 axis possesses a central role in the trafficking and retention of both normal and malignant stem cells in the bone marrow. Previous work from our laboratory established that in a murine model, a single dose of the CXCR4 antagonist, AMD3100, sensitizes AML blasts to chemotherapy supporting the premise that the interaction between AML blasts and the marrow microenvironment confers resistance to genotoxic stress (Nervi et al., ASH 2006). Here we examine the effects of repetitive dosing of AMD3100 on the kinetics of normal and leukemic mobilization. Following SQ injection of AMD3100 5mg/kg into B6/129 F1 mice daily for 5 days (n=8), we observed a 2.4 fold increase in total leukocyte counts with a 12.4 increase in CFU-GM when compared to 3 hours post injection (Fig 1A). No differences were seen in the degree of mobilization between d1 and d5 with WBC and CFU-GM counts returning to baseline after 24 hours. We next tested repetitive doses of AMD3100 in our mouse model of AML in which 106 blasts derived from leukemic mice carrying the PML-RARα fusion gene in the murine cathepsin G locus are adoptively transferred into genetically compatible secondary recipients. AMD3100 at 5mg/kg was then administered to these AML mice for 4 consecutive days. At 3 hrs post AMD3100 injection, we observed a 1.8 fold increase in peripheral leukocyte counts with a 4.5 fold increase in circulating blasts compared to baseline (n=3). Again, no significant differences are seen in the degree of mobilization from d1 to d4 (Fig 1B). Based on these preclinical data, we have initiated a phase I/II trial of AMD3100 plus mitoxantrone, etoposide and cytarabine (MEC) in relapsed or refractory AML in which AMD3100 is administered 4 hours prior to MEC daily for 5 consecutive days. To study the kinetics of human AML mobilization, we administered AMD3100 by SQ injection followed by 24hr observation period prior to chemotherapy. Two patients have been treated at the first dose level of AMD3100, 80 μg/kg. In pt #1 following AMD3100 mobilization, total WBC increased from 3 × 103/mm3 to a peak of 17 × 103/mm3 at 6 hours post-AMD3100 representing a 5.7 fold increase in total white count (Fig 2). In addition, the blasts (CD45dim, SSlow) increased by 7.3 fold. Similarly in pt #2, we observed a 2 fold increase in the total WBC from 2.5 to 5.1 × 103/mm3 with a 2.3 fold increase in blasts (CD45dim, SSlow). Mobilization of AML was confirmed in both patients through informative FISH for 11q23 (MLL). No adverse events have been observed during mobilization. These data provide the preclinical rationale for repetitive dosing of AMD3100 and direct clinical evidence that AMD3100 mobilizes human AML blasts into the peripheral circulation. Our trial of AMD3100 plus MEC in relapsed or refractory AML is ongoing. Figure 1. AMD3100 induced mobilization of (A) normal progenitors and (B) AML blasts Figure 1. AMD3100 induced mobilization of (A) normal progenitors and (B) AML blasts Figure 2. AMD3100 mobilization of human AML Figure 2. AMD3100 mobilization of human AML

scholarly journals Decreased protein and puromycinyl-peptide degradation in livers of senescent mice

1982 ◽  
Vol 202 (1) ◽  
pp. 47-51 ◽  
Author(s):  
L Lavie ◽  
A Z Reznick ◽  
D Gershon
Keyword(s):  
Protein Degradation ◽  
Proteolytic Activity ◽  
Trichloroacetic Acid ◽  
Liver Protein ◽  
Native Proteins ◽  
Age Related ◽  
Degradation Rates ◽  
Time Required ◽  
Old Mice ◽  
Related Increase

Liver protein-degradation rates were determined in young and old C57B1 mice by the method of Swick & Ip [(1974) J. Biol. Chem. 249, 6836-6841]. The results indicated a marked age-related increase in the half-lives of short-lived proteins in the nuclear, mitochondrial, lysosomal and 100000 g-supernatant cellular fractions and in total trichloroacetic acid-precipitable proteins. The efficiency of the degradation system in removing aberrant proteins from livers of young and old mice was tested. The time required for 50% disappearance of puromycinyl-peptides changed from about 20 min in 6-month-old mice to approx. 150 min in 24-month-old animals. These findings suggest that in old animals the proteolytic activity involved in degradation of aberrant proteins, and presumably of "native proteins, is markedly defective.

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