Evidence for the Importance of Puromycyl Peptides in the Inhibition by Puromycin of Cell Aggregation In Vitro

1973 ◽  
Vol 12 (2) ◽  
pp. 641-653
Author(s):  
M. J. DUNN ◽  
E. OWEN ◽  
R. B. KEMP
Keyword(s):  
Carbon Dioxide ◽  
Protein Synthesis ◽  
Cell Aggregation ◽  
Experimental Period ◽  
Respiratory Metabolism ◽  
Inhibitory Effects ◽  
Carbon Dioxide Evolution ◽  
Dissociated Cells ◽  
Cellular Oxygen

Trypsin-dissociated cells from the muscle tissue of 9-day-old chick embryos were employed to investigate the effects of cycloheximide and a puromycin-cycloheximide mixture on cell aggregation, protein synthesis and respiratory metabolism. Cycloheximide when introduced at a concentration of 10 µg/ml into a suspension of cells in Eagle's MEM inhibited aggregation by 25% at 24 h. At this time an inhibition of 40% was apparent in the presence of a mixture of cycloheximide and puromycin both at a concentration of 10 µg/ml. Both cycloheximide and the cycloheximide-puromycin mixture arrested protein synthesis of rotated cells by 90% within 15 min of introducing the antibiotics into cell suspensions. The antibiotics retained their inhibitory effects on protein synthesis for the 24-h period of rotation. Cycloheximide inhibited cellular oxygen uptake and carbon dioxide evolution of rotated cells by 25% at the end of the 24-h experimental period. At this time an inhibition of 30% was observed in the presence of the cycloheximide-puromycin mixture. The release of radioactive carbon dioxide by cycloheximide-treated cells was inhibited by 46% at 24 h. In the presence of the antibiotic mixture, 14CO2 release was inhibited by 30% at 4 h, but after 8 h very little further 14CO2 was evolved. As a control, puromycin (10 µg/ml) inhibited cell aggregation and respiration to an extent similar to that previously reported. The results are discussed in terms of puromycyl peptides producing a metabolic effect on cell aggregation. It is considered that this is additional to the effect of puromycin inhibiting aggregation through the arrest of protein synthesis.

Studies on the Mechanism Underlying the Inhibition by Puromycin of Cell Aggregation In Vitro

1970 ◽  
Vol 7 (2) ◽  
pp. 557-573
Author(s):  
M. J. DUNN ◽  
E. OWEN ◽  
R. B. KEMP
Keyword(s):  
Carbon Dioxide ◽  
Protein Synthesis ◽  
Cellular Metabolism ◽  
Cell Aggregation ◽  
Experimental Period ◽  
Inhibitory Effect ◽  
Dissociated Cells ◽  
Gyratory Shaker ◽  
Reduced Rate

Cells dissociated with 0.25% crude trypsin from the muscle tissue of 9-day-old chick embryos were employed to investigate the effect of puromycin on cellular metabolism. Parallel studies were also made, using the gyratory shaker, to confirm the effectiveness of puromycin in inhibiting cell aggregation and protein synthesis. Puromycin when introduced at a concentration of 10µg/ml into a suspension of cells in Eagle's MEM did not completely inhibit cell aggregation. Small aggregates were formed in the first 4 h of the experiment. Protein synthesis of the rotated cells, as measured by the incorporation of L-[α-14C]leucine into proteins, was arrested by 91.7% within 15 min of introducing puromycin into a cell suspension. The antibiotic retained its inhibitory effect on protein synthesis for the 24-h period of rotation. Puromycin inhibited the cellular oxygen uptake and carbon dioxide evolution of the rotated cells by 40% within 4 h of its introduction. However, treated cells were still respiring, though at a much reduced rate, at the end of the 24-h experimental period. The release of radioactive carbon dioxide by puromycin-treated cells was also inhibited by 40% at the 4-h stage but after 8 h no further 14CO2 was evolved. The presence of the antibiotic markedly inhibited the uptake of glucose by trypsin-dissociated cells. The level of glycogen and lactate in cells suspended in Eagle's MEM was reduced very considerably over a 24-h period. The presence of puromycin accelerated glycogen utilization over the first 6 h of rotation but at 24 h there was a difference of only 0.6% between the glycogen content of treated cells and controls. At 24 h 11.3% less lactate remained in the puromycin-treated cells than in the controls. The ATP/ADP ratio of trypsin-dissociated cells decreased from an initial value of 2.59 to 1.45 after rotation for 24 h. In the presence of puromycin the ATP/ADP ratio was 0.62 at 4 h and had further declined to 0.48 by 24 h. The effects of puromycin on the aggregation, protein synthesis and cellular metabolism of trypsin-dissociated cells are discussed in relation to cellular adhesive mechanisms.

Effects of Cytochalasins on the Initial Aggregation in Vitro of Embryonic Chick Cells

1974 ◽  
Vol 14 (1) ◽  
pp. 187-196
Author(s):  
J. C. APPLETON ◽  
R. B. KEMP
Keyword(s):  
Cytochalasin B ◽  
Cell Aggregation ◽  
Cellular Injury ◽  
Cellular Motility ◽  
Carbon Dioxide Evolution ◽  
Site Of Action ◽  
Dissociated Cells ◽  
Cytochalasin A ◽  
Final Confirmation

The initial aggregation of trypsin-dissociated cells from the skeletal muscle tissue of 9-day-old chick embryos in the presence of cytochalasins A and B was studied in order to discover the effects of these agents on contact and adhesion. Cytochalasin B (3 µg/ml) had a negligible effect on the rate of aggregation of cells over an 8-h period, but cytochalasin A at concentrations between 3 and 20 µg/ml markedly inhibited aggregation. Both agents altered the shape and size of aggregates and caused cells at their periphery to appear more spherical. The oxygen uptake of the treated cells was not noticeably different from that of the controls, despite the severe inhibition of isotopic carbon dioxide evolution. The effect of cytochalasin B on cell aggregation was reversible and although the cytochalasin A effect could not be abolished on return to medium free of A, the unaltered oxygen consumption was taken as an indication that permanent cellular injury did not occur. The effect of the cytochalasins on aggregate structure was interpreted on the basis of arrested cellular motility, but the singular inhibition by cytochalasin A of the rate of aggregation must await final confirmation of its site of action.

Abolition by Myosin and Heavy Meromyosin of the Inhibitory Effect of Smooth-Muscle Actomyosin Antibodies on Cell Aggregation In Vitro

1973 ◽  
Vol 12 (2) ◽  
pp. 631-639
Author(s):  
R. B. KEMP ◽  
B. M. JONES ◽  
U. GRÖSCHEL-STEWART
Keyword(s):  
Smooth Muscle ◽  
Striated Muscle ◽  
Cell Aggregation ◽  
Inhibitory Effect ◽  
Regulatory Function ◽  
Rabbit Serum ◽  
Heavy Meromyosin ◽  
Γ Globulins ◽  
Dissociated Cells

The ability of anti-chicken smooth-muscle actomyosin γ-globulins (anti-GAM) to inhibit the aggregation of dissociated cells from the skeletal muscle and liver of chick embryos was abolished by pretreatment of the anti-GAM with either myosin or heavy meromyosin (HMM). When the same cells were treated with HMM at a concentration of 1 mg per 2 x 106 cells/ml Eagle's MEM they aggregated as readily as untreated cells. The negative electrophoretic mobility of the embryonic chick fibroblastic cells was significantly reduced by the globulin fraction of anti-GAM but not of HMM-treated anti-GAM or non-immunized rabbit serum. Anti-chicken striated muscle actomyosin γ-globulins slightly reduced negative mobility but HMM had no effect. The experiments show that the inhibitory effect on cell aggregation of anti-GAM preparations is produced by the anti-myosin antibodies. They also provide support for the theory that a surface-localized myosin-like protein has a regulatory function in cell adhesion.

ASPECTS OF DECOMPOSITION OF CELLULOSE IN CANADIAN SOILS: II. NITRATE NITROGEN LEVELS AND CARBON DIOXIDE EVOLUTION

1960 ◽  
Vol 6 (3) ◽  
pp. 317-323 ◽  
Author(s):  
H. T. Tribe
Keyword(s):  
Carbon Dioxide ◽  
Nitrogen Mineralization ◽  
Nitrate Nitrogen ◽  
Experimental Period ◽  
Cellulose Film ◽  
Carbon Dioxide Evolution ◽  
Nitrogen Levels ◽  
Canadian Soils ◽  
Humus Soil

The effect of addition of cellulose film on the level of nitrate in a mull humus soil was studied over a period of 16 weeks. During the early stages of decomposition, nitrate was taken up from the soil, leaving it deficient in nitrate for up to 7 weeks. In later stages of decomposition, some of this nitrate was released again. Carbon dioxide was produced from the cellulose film over the whole experimental period. The results were broadly correlated with previous observations on the succession of microorganisms and fauna on cellulose film. The role of the fauna in nitrogen mineralization is discussed.

Inhibitory effects of histidine analogues on growth and protein synthesis by Plasmodium falciparum in vitro

1986 ◽  
Vol 35 (9) ◽  
pp. 1589-1596 ◽  
Author(s):  
Russell J. Howard ◽  
Annette T. Andrutis ◽  
James H. Leech ◽  
William Y. Ellis ◽  
Louis A. Cohen ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Synthesis ◽  
Inhibitory Effects

Reconstruction of Mammalian Heart Tissue from Isolated Embryonic Heart Cells

1977 ◽  
Vol 35 ◽  
pp. 580-581
Author(s):  
Asish C. Nag ◽  
Debra S. Buszke
Keyword(s):  
Basal Medium ◽  
Cell Aggregation ◽  
Heart Tissue ◽  
Cell Suspensions ◽  
Heart Cells ◽  
Mammalian Heart ◽  
Dissociated Cells ◽  
Gyratory Shaker ◽  
And Function

Although monolayer cultures are useful in various cell studies, they are not always adequate for examining the nature of cellular interrelationships and interactions in the formation, differentiation, and function of tissues. The procedures of aggregation in vitro of dissociated cells (Moscona & Moscona, 1966) enable one to study in detail the formation of cell contacts, the assembly of cells into multicellular systems, and cell cooperation in forming organized and differentiating tissues. We adapted the techniques of cell aggregation by rotation to studies on embryonic mammalian heart cells. Cell suspensions from the 18-day-old embryonic rat were prepared by dissociation with 0. 5% trypsin. The cells were dispersed in a culture medium which consisted of Eagle's basal medium with 10% fetal bovine serum, 1% glutamine solution, and 1% penicillin-streptomycin mixture. Cultured in 25ml Erlenmeyer flasks on a gyratory shaker at 70 rpm at 37°C were 3ml aliquots of the cell suspensions.

A Biochemical Investigation into the Inhibitory Effects of Glucosamine and N-Acetylglucosamine on the Aggregation In Vitro of Embryonic Chick Muscle Cells

1971 ◽  
Vol 9 (1) ◽  
pp. 85-101
Author(s):  
C. W. LLOYD ◽  
R. B. KEMP
Keyword(s):  
Lactic Acid ◽  
Oxygen Uptake ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Muscle Cells ◽  
Cell Aggregation ◽  
Experimental Period ◽  
Metabolic Effects ◽  
In Cells

The effect of glucosamine and N-acetylglucosamine on aggregation and energy metabolism was investigated over an 8-h period in cells dissociated by 0.25% (w/v) trypsin from the skeletal muscle of 9-day-old chick embryos. At 8 h, 0.023 M glucosamine and N-acetylglucosamine inhibited the aggregation of cells suspended in Eagle's minimal essential medium by 18.9% and 16.4% respectively, as judged on a basis of aggregate size. Glucosamine and N-acetylglucosamine reduced the cellular ATP level by a mean of 36% and 27% respectively; values reflected in the 32% and 19% loss of total adenine nucleotides caused by these sugars. The adenine nucleotide balance was also changed from a mean control ATP/AMP ratio of 13.7 to 8.75 by N-acetylglucosamine and to 8.0 by glucosamine. Intracellular lactate/pyruvate ratios were similarly disrupted in cells incubated with 0.023 M hexosamine. Although the hourly values fluctuated, it was seen that the amount of lactic acid relative to pyruvic acid, considered as an average for the 8-h period, was raised from 6:1 in controls to 8:1 in N-acetylglucosamine-treated and to 10:1 in glucosamine-treated cell preparations. Compared to controls at 8 h, glucosamine enhanced the production of lactate into the suspension medium by 99%. The N-acetyl analogue caused cells to produce more lactic acid than did controls for 4-5 h only, for by 8 h 25% less of this metabolite was assayed in the culture medium. The incorporation of D-[U-14C]glucose into glycogen paralleled the results of extracellular lactic acid assays. N-acetylglucosamine inhibited the incorporation by 30% at 4 h, although by 6 h, and for the remainder of the experimental period, there was more 14C-labelled glycogen in these cells than in controls. By contrast, glucosamine inhibited the incorporation of radioactive glucose into glycogen by 42% at 4 h and, unlike N-acetylglucosamine, consistently thereafter. Glucosamine also enhanced cellular oxygen uptake throughout the experimental period, to the extent of 59% at 8 h. The oxygen uptake of N-acetylglucosamine-treated cells was similar to controls until about the 5th hour, when there was a subsequent inhibition which had accumulated to 13% by the end of the experiment. The release of 14CO2 by cells was inhibited by glucosamine. This hexosamine depressed production by 19% at 12 h whereas N-acetylglucosamine inhibited this evolution by 9% at this time. The metabolic effects of these hexosamines on chick muscle cells in vitro are mainly attributed to a central alteration of the adenine nucleotide balance although certain other documented effects of glucosamine are considered to be involved. An inhibition of cell aggregation by glucosamine and N-acetylglucosamine is discussed in terms of a depressed cellular metabolic economy.

Effects of immunosuppressive drugs on secondary antibody response in vitro

1968 ◽  
Vol 14 (1) ◽  
pp. 7-11 ◽  
Author(s):  
F. C. Leung ◽  
S. I. Vas
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Immunosuppressive Agent ◽  
Immunosuppressive Drugs ◽  
Tissue Cultures ◽  
Secondary Antibody ◽  
Inhibitory Effects ◽  
Antibody Synthesis ◽  
Relative Inhibition

The inhibitory effects of 6-mercaptopurine, Imuran, and chloramphenicol on the in vitro incorporation of radioactive amino acid into antibody have been investigated. Inhibition of antibody synthesis by these drugs was compared to inhibition of protein synthesis within the same tissue cultures. The relative inhibition of antibody synthesis compared to that of protein synthesis provided an index which indicated the specificity and toxicity of the immunosuppressive agent. Imuran, at concentrations of 10–20 μg/ml inhibited antibody synthesis primarily, while at higher concentrations, protein synthesis was similarly affected. 6-Mercaptopurine at 177–250 μg/ml inhibited antibody production to a certain degree, while chloramphenicol at 300 μg/ml showed no preferential inhibition of antibody synthesis.

scholarly journals Effects of Progesterone on the Oestrogen-Stimulated Uterus: a Comparative Study of the Mouse, Guinea Pig, Rabbit and Sheep

1979 ◽  
Vol 32 (6) ◽  
pp. 549 ◽  
Author(s):  
BG Miller ◽  
Jennifer Wild ◽  
GM Stone
Keyword(s):  
Protein Synthesis ◽  
Guinea Pig ◽  
Comparative Study ◽  
Peroxidase Activity ◽  
Species Differences ◽  
Inhibitory Effects ◽  
Dna And Rna ◽  
Wet Weight ◽  
Vitro Protein

To examine more closely the anti-oestrogenic action of progesterone (P), its effect on various parameters in the 17 fl-oestradiol (E2)-primed uterus of the mouse, guinea pig, rabbit and ewe was studied. Changes in uterine wet weight, rate of in vitro protein synthesis, protein: DNA and RNA :. DNA ratios, peroxidase activity and the level of cytosol receptors for E2 and P were measured. Considerable between-species differences in the effect of P on these parameters were observed. The anti-uterotrophic action was greater in the mouse than in the guinea pig and was not seen in the rabbit or ewe. P inhibited protein synthesis in the mouse, was without significant effect in the guinea pig and was mildly stimulatory in the rabbit and ewe. Inhibitory effects on protein: DNA and RNA: DNA ratios were substantial in the mouse, minor in the guinea pig and absent in the rabbit and ewe. Peroxidase activity was decreased in the mouse and guinea pig, essentially lacking in the rabbit and not detectable in the ewe. In all species the level of both oestrogen and progesterone cytosol receptors was decreased, although the effect on the E2 receptor was less marked in the ewe.

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