Search PubMed⌕ Search

Biomedical subjects

J F Dice

Publications and source records attributed to J F Dice.

At least 73 records · Page 4Linked to original sources
PubMed1985

Red blood cell-mediated microinjection: methodological considerations.

Red blood cell-mediated microinjection is a powerful approach to introducing proteins into the cytoplasm of cultured cells. In the course of our microinjection studies of intracellular protein degradation, we have encountered several potential problems with certain proteins. The microinjection procedure may be accompanied by denaturation of protein by radiolabeling procedures, binding of protein to red cell ghosts during loading, degradation of protein by the red cell ghost prior to microinjection, and adsorption of protein that leaks from red cell ghosts in the presence of fusogen to the fibroblast monolayer. We conclude with a list of points that must be considered prior to use of red cell-mediated microinjection to study a particular protein.

Cells, Cultured↗
PubMed1985

Regulation of catabolism of ribonuclease A microinjected into human fibroblasts.

We are using ribonuclease A (RNase A) as a model protein to study how the degradative rates of proteins are regulated within cells. RNase A and several derivatives can be microinjected into confluent cultures of human fibroblasts using red cell-mediated microinjection. The half-life of RNase A is 80-100 hrs in cells maintained in the presence of serum, and the degradative rate is enhanced approximately two-fold upon serum withdrawal. The ability of fibroblasts to regulate breakdown of this protein depends on a small peptide region within the amino terminal twenty amino acids. This amino terminal peptide from RNase A can be covalently attached to unrelated proteins and will cause their catabolism to become serum responsive. The mechanism of degradation of RNase A involves lysosomal pathways both in the presence and absence of serum, and the enhanced catabolism during serum deprivation results from a two-fold increase in the rate of uptake of the protein by lysosomes. These findings suggest that autophagy, or some other process occuring in serum-deprived cells, can be highly selective.

Fibroblasts↗
PubMed1984

Intracellular protein degradation in cultures of dystrophic muscle cells and fibroblasts.

We have analysed protein degradation in primary cultures of normal and dystrophic chick muscle, in fibroblasts derived from normal and dystrophic chicks, and in human skin fibroblasts from normal donors and from patients with Duchenne muscular dystrophy (DMD). Our results indicate that degradative rates of both short- and long-lived proteins are unaltered in dystrophic muscle cells and in dystrophic fibroblasts. Longer times in culture and co-culturing chick fibroblasts with the chick myotubes do not expose any dystrophy-related abnormalities in protein catabolism. Furthermore, normal and dystrophic muscle cells and fibroblasts are equally able to regulate proteolysis in response to serum and insulin. We conclude that cultures of chick myotubes, chick fibroblasts, and fibroblasts derived from humans afflicted with DMD are not appropriate models for studying the enhanced protein degradation observed in dystrophy.

Animals↗
PubMed1984

Altered degradation of intracellular proteins in aging human fibroblasts.

Confluent cultures of fibroblasts at different population doubling levels were incubated with [14C]leucine for 2 days and with [3H]leucine for 2 h to label long-lived and short-lived proteins, respectively. Proteolysis was then measured in the presence of excess unlabeled leucine to prevent reutilization of the isotope. Catabolism of long-lived proteins was reduced in senescent cells when measured in media without fetal bovine serum, insulin, fibroblast growth factor, or dexamethasone. In contrast, degradation of short-lived proteins was increased in senescent cells but only when measured in the presence of serum, hormones, and growth factors. Further experiments with cells of varying ages indicate that in unsupplemented medium half-lives of long-lived proteins lengthened by as much as 20 min per population doubling and in supplemented media half-lives of short-lived proteins decreased by 4 min per population doubling. The reduced catabolism of long-lived proteins in senescent cells cannot be explained by age-related changes in protein secretion or cell death during degradation measurements. These alterations in proteolysis may have major effects on protein content and composition in senescent cells.

Cell Survival↗
PubMed1983

Erythrocyte-mediated microinjection, a technique to study protein degradation in muscle cells.

The technique of erythrocyte-mediated microinjection has been successfully adapted for use with cultured muscle cells. Erythrocytes were fused with primary chick myotube cultures with poly(ethylene glycol), and fluorescent antibodies to haemoglobin demonstrated that this protein was injected into the sarcoplasm of myotubes. The microinjection treatment did not significantly alter protein metabolism in the muscle cells as monitored by rates of synthesis and degradation of muscle proteins. 125I-labelled ribonuclease A and bovine serum albumin were degraded with the expected exponential decay kinetics after microinjection into muscle cells, and the half-life of ribonuclease A (40 h) was approximately twice that of bovine serum albumin (17 h). The degradation of ribonuclease A in the muscle cells was enhanced 1.6-fold in the absence of horse serum and chick-embryo extract, whereas the degradation of bovine serum albumin was not altered during deprivation. These results are characteristic of the breakdown of microinjected ribonuclease A and bovine serum albumin in other cell types. Therefore, our experiments indicate the erythrocyte-mediated microinjection is a valid technique to study protein degradation in primary chick muscle cultures.

Animals↗
PubMed1983

Regulation of intracellular protein degradation in IMR-90 human diploid fibroblasts.

Human diploid fibroblasts (IMR-90) regulate their overall rates of proteolysis in response to the composition of the culture medium and the ambient temperature. The magnitude and, in some cases, the direction of the response depend on the half-lives of the cellular proteins that are radioactively labeled and the time chosen for measurements of protein degradation. Fetal calf serum, insulin, fibroblast growth factor, epidermal growth factor, and amino acids selectively regulate catabolism of long-lived proteins without affecting degradation of short-lived proteins. Fetal calf serum reduces degradative rates of long-lived proteins and is maximally effective at a concentration of 20%, but the effect of serum on proteolysis is evident only for the first 24 hr. Insulin inhibits degradation of long-lived proteins in the presence or absence of glucose and amino acids in the medium, but is maximally effective only at high concentrations (10(-5) M). Amino acid deprivation increases degradative rates of long-lived proteins for the first 6 hr, but then decreases their catabolism for the subsequent 20 hr. Lowered temperature is the only condition tested that significantly alters degradative rates of short-lived proteins. Although cells incubated at 27 degrees C have reduced rates of degradation for both short-lived and long-lived proteins compared to cells at 37 degrees C, lowered temperature reduces catabolism of long-lived proteins to a greater extent.

Amino Acids↗
PubMed1983

Regulation of catabolism of microinjected ribonuclease A requires the amino-terminal 20 amino acids.

RNase A introduced into the cytoplasm of IMR-90 human diploid fibroblasts by erythrocyte-mediated microinjection is degraded with a half-life of approximately equal to 75 hr in the presence of fetal bovine serum. In response to serum deprivation the degradative rate of microinjected RNase A is enhanced 2-fold. RNase S protein (amino acids 21-124) is degraded with a half-life similar to that of RNase A in the presence of serum, but its catabolism is not increased during serum withdrawal. Reconstitution of RNase S protein with RNase S peptide (amino acids 1-20) restored full enzymatic activity to the S protein as well as the ability of fibroblasts to increase its catabolism during serum deprivation. Finally, RNase S peptide microinjected alone shows the full 2-fold increase in degradative rate during serum withdrawal. These results show that recognition of RNase A for enhanced breakdown during serum deprivation is based on some feature of its amino-terminal 20 amino acids. Furthermore, our results indicate that the enhanced protein catabolism during serum deprivation can be highly selective.

Amino Acid Sequence↗
PubMed1982

Altered degradation of proteins microinjected into senescent human fibroblasts.

Ribonuclease A was introduced into the cytoplasm of IMR-90 human diploid fibroblasts by red cell-mediated microinjection. Early passage fibroblasts degraded ribonuclease A with a half-life of approximately 90 h in the presence of 10% fetal calf serum and enhanced the degradative rate 1.6-fold upon serum withdrawal. Senescent cells degraded ribonuclease A more slowly with half-lives ranging between 125 and 250 h and had diminished capacities to enhance the catabolism of this protein during serum starvation. Decreased protein degradation in senescent cells was also evident for microinjected RNase S-protein, RNase B, aldolase, lysozyme, and the synthetic copolymer polyglutamate: tyrosine:alanine (1:1:1). These alterations in the mechanisms and regulation of intracellular protein degradation may contribute to several biochemical abnormalities characteristic of aging cells and organisms.

Cell Line↗
PubMed1981

Selective depletion of small basic non-glycosylated proteins in diabetes.

Degradative rates of small basic non-glycosylated proteins are preferentially enhanced in rat liver cytosol during severe streptozotocin-induced diabetes. Synthetic rates of these classes of proteins are not selectively enhanced in diabetes, so small basic non-glycosylated proteins should be depleted from liver cytosol as a consequence of this disease. To test this hypothesis, proteins were analysed from normal animals, from diabetic animals receiving insulin and from diabetic animals after insulin withdrawal for 3 days. The proteins were separated according to subunit molecular weight by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, according to isoelectric point by isoelectric focusing and according to carbohydrate content by affinity chromatography with concanavalin A linked to agarose. Severe uncontrolled diabetes is associated with the predicted depletion of small basic non-glycosylated proteins from liver cytosol. The preferential degradation and loss of these protein classes may be of considerable physiological importance to the animal.

Animals↗
PubMed1981

Degradation of proteins microinjected into IMR-90 human diploid fibroblasts.

Erythrocyte ghosts loaded with 125I-labeled proteins were fused with confluent monolayers of IMR-90 fibroblasts using polyethylene glycol. Erythrocyte-mediated microinjection of 125I-proteins did not seriously perturb the metabolism of the recipient fibroblasts as assessed by measurements of rates of protein synthesis, rates of protein degradation, or rates of cellular growth after addition of fresh serum. A mixture of cytosolic proteins was degraded after microinjection according to expected characteristics established for catabolism of endogenous cytosolic proteins. Furthermore, withdrawal of serum, insulin, fibroblast growth factor, and dexamethasone from the culture medium increased the degradative rates of microinjected cytosolic proteins, and catabolism of long-lived proteins was preferentially enhanced with little or no effect on degradation of short-lived proteins. Six specific polypeptides were degraded after microinjection with markedly different half-lives ranging from 20 to 320 h. Degradative rates of certain purified proteins (but not others) were also increased in the absence of serum, insulin, fibroblast growth factor, and dexamethasone. The results suggest that erythrocyte-mediated microinjection is a valid approach for analysis of intracellular protein degradation. However, one potential limitation is that some microinjected proteins are structurally altered by the procedures required for labeling proteins to high specific radioactivities. Of the four purified proteins examined in this regard, only ribonuclease A consistently showed unaltered enzymatic activity and unaltered susceptibility to proteolytic attack in vitro after iodination.

Cell Fusion↗
PubMed1979

Studies on the relationship between the degradative rates of proteins in vivo and their isoelectric points.

Acidic proteins tend to be degraded more rapidly than neutral or basic proteins in rat liver, skeletal muscle, kidney and brain and in mouse liver and skeletal muscle. We now report a similar relationship among soluble proteins from rat lung, heart and testes, and from human fibroblasts and mouse-embryo cells grown in culture. These findings indicate that the correlation between protein net charge and degradative rate is a general characteristic of intracellular protein degradation in mammals. This relationship between isoelectric point and half-life appears to be distinct from the previously reported correlation between subunit molecular weight and protein half-lives. The more rapid degradation of acidic proteins does not result from their being of larger molecular weight than neutral or basic proteins. Furthermore, proteins within specific isoelectric point ranges still exhibit a relationship between subunit size and half-life. Finally, a group of membrane or organelle-associated proteins that are insoluble in phosphate-buffered saline and water but soluble in 1% Triton X-100 exhibit a correlation between size and half-life, but not between net charge and half-life. The biochemical reasons for the relationship between protein isoelectric point and half-life are unclear, although several possible explanations are presented. It is not due to a greater sensitivity of acidic proteins to proteolytic attack since experiments with a variety of endoproteinases, including trypsin, chymotrypsin, Pronase, papain, chymopapain, Staphylococcus aureus V8 proteinase, pepsin and lysosomal cathepsins from rat liver, have failed to demonstrate more rapid digestion of acidic proteins.

Animals↗
PubMed1979

Protein degradation in metabolic and nutritional disorders.

The increased protein degradation associated with diabetes appears to be different in many respects from protein catabolism in normal, well-nourished cells. In all normal eukaryotic cells examined, degradation of cytosolic proteins exhibits several striking features. Large proteins tend to be degraded more rapidly than small proteins, acidic proteins tend to be degraded more rapidly than neutral or basic proteins, and glycoproteins are degraded more rapidly than non-glycoproteins. Furthermore, a general correlation exists between protein half-life in vivo and susceptibility to proteolytic attack in vitro. In streptozotocin-diabetic rats the relationships between degradative rate and protein size, net charge, and carbohydrate content are absent or markedly reduced among cytosolic proteins of the liver. However, the correlation between protein half-life and susceptibility to proteinase in vitro is unaltered. Therefore, the enhanced protein degradation in diabetes shows little or no selectivity towards large, acidic, glycoproteins but does show specificity for proteins than tend to be sensitive to proteinases. Similar studies using other tissues from diabetic rats are reported and general characteristics of the enhanced liver protein catabolism in starvation and hyperthyroidism are briefly discussed. The biochemical reasons for the increased protein catabolism in diabetes are unclear although several possible explanations are presented. The enhanced breakdown is probably not due to cellular proteins becoming more proteinase sensitive in diabetes since experiments with a variety of endoproteinases including pronase, chymotrypsin, pepsin, and lysosomal cathepsins have failed to demonstrate more rapid digestion of liver proteins from diabetic animals.

Animals↗
PubMed1978

General characteristics of protein degradation in diabetes and starvation.

The enhanced protein degradation associated with diabetes and starvation is fundamentally different from normal protein catabolism. In normal eukaryotic cells large molecular weight proteins tend to be degraded more rapidly than small proteins, acidic proteins tend to be degraded more rapidly than neutral or basic proteins, and glycoproteins tend to be degraded more rapidly than nonglycoproteins. All three of these general correlations are absent or markedly reduced in liver and muscle of diabetic and starved rats. In contrast, the correlations between proteins size and half-life, between protein net charge and half-life, and between protein carbohydrate content and half-life are not affected in brain of diabetic or starved animals. These results suggest that diabetes and starvation alter the general characteristics of intracellular protein degradation in target tissues of insulin. Degradation of serum proteins is also affected in diabetes and starvation. In normal animals a general correlation exists between isoelectric points of serum proteins and their degradative rates. This relationship is abolished in diabetes and starvation, as it is among liver and muscle proteins. The implications of our findings are discussed with regard to possible mechanisms of the enhanced protein breakdown.

Animals↗