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F Svec

Publications and source records attributed to F Svec.

At least 145 records · Page 8Linked to original sources
PubMed1984

Glucocorticoids inhibit the growth of AtT-20 mouse pituitary tumor cells.

The effect of steroids on the growth of AtT-20 mouse pituitary tumor cells was investigated. It was found that under certain conditions glucocorticoids inhibit growth. Specificity studies indicated that only glucocorticoids caused this effect. Biopotency studies indicated that dexamethasone had its mid-maximal effect around 5 nM. At least part of this inhibition was caused by a decrease in the cell's ability to synthesize DNA; glucocorticoids inhibit the rate at which tritiated thymidine is incorporated into TCA-precipitable material. The magnitude of the growth-inhibiting effect depended upon the prior culture history of the cell. The effect was least on cells derived from exponentially growing cultures and most effective on cells derived from cultures that were approaching their density limit. It is concluded that glucocorticoids inhibit AtT-20 cell growth by interfering with their ability to transform from confluent cultures to ones that grow exponentially. The significance of this finding is that now two mechanisms must be considered when investigating the pathways through which glucocorticoids decrease the ACTH production of the AtT-20 cell.

Adrenocorticotropic Hormone↗
PubMed1984

The relationship between glucocorticoid biopotency and receptor binding in the AtT-20 cell.

The AtT-20 cell is a commonly used model for investigations of glucocorticoid agonist action. As yet, however, no reports quantify the relationship between the magnitude of agonist binding to the intact cell's glucocorticoid receptor and the magnitude of response to agonists. Because this correlation may illuminate the molecular mechanism of hormone action, the abilities of triamcinolone acetonide, dexamethasone, and corticosterone to inhibit the AtT-20 cell's production of ACTH were determined and compared with their binding affinities for the intact cell's glucocorticoid receptor. Both variables were assayed under similar conditions. Each of the three steroids caused a dose-dependent suppression of ACTH production. To compare the potencies of the three, the concentration of agonist that caused a 50% reduction in ACTH secretion was determined and was found to be 0.12 nM for triamcinolone acetonide, 0.58 nM for dexamethasone, and 4.8 nM for corticosterone. The affinities of these steroids for the intact cell's glucocorticoid receptor were determined by Scatchard analysis. The Kd values were 3.4 nM for triamcinolone acetonide, 8.5 nM for dexamethasone, and 51 nM for corticosterone. Comparison of the biopotency and binding data suggests that the agonists are more potent than one would predict from the binding studies and that only a fraction of the cell's receptors need be filled for the agonist to be fully active. Control studies showed that this quantitative discrepancy between steroid binding and biopotency was not caused by misdilution of steroids, steroid metabolism, or steroid-binding components in serum. Thus, this discrepancy leads to the conclusion that the AtT-20 cell has a large number of "spare" glucocorticoid receptors.

Adrenocorticotropic Hormone↗
PubMed1983

Characterization of the glucocorticoid receptor translocated to the nucleus in intact AtT-20 mouse pituitary tumor cells at low temperatures.

Although low temperatures retard the nuclear translocation of glucocorticoid-receptor complexes, they do not completely inhibit it. To gain insight into the mechanism of nuclear translocation, we determined the Stokes radius and surface charge density of the receptors bound to the nucleus at 0 C and compared them with the values obtained from standard preparations of activated and unactivated glucocorticoid receptors. Specifically, intact, viable AtT-20 cells were incubated at 0 C for 3 h with 10 nM tritiated triamcinolone acetonide and the nuclear receptor extracted with 0.3 M KCl/20 mM sodium molybdate. These nuclear receptors were found to elute from an Agarose A 1.5 m column with a Stokes radius identical to that of an "activated" receptor (6.4 nm) but eluted from a DEAE column at the same position as an "unactivated" species (0.17 M KCl). From these results we hypothesize that the change in receptor structure that leads to nuclear binding is a multistep process, and although the change in molecular size may be a prerequisite for nuclear binding, the change in surface charge is not.

Animals↗
PubMed1982

Interactions of antiglucocorticoids with glucocorticoid receptors.

Six known or potential antiglucocorticoids were used as steroid probes in a competitive binding assay to elucidate the binding specificity of the agonist sites of four different rat glucocorticoid receptors. The object was to determine whether the binding of this class of steroids was particularly sensitive to subtle differences between these sites. Cytosolic extracts of heart, pancreas, kidney and liver were evaluated. The order of competitive potency for the first three preparations was found to be medroxyprogesterone greater than deoxycorticosterone greater than progesterone greater than R-5020 greater than cortexolone greater than 17-hydroxyprogesterone. The order for the glucocorticoid receptor of liver, on the other hand, was R-5020 greater than progesterone greater than deoxycorticosterone greater than medroxyprogesterone greater than cortexolone greater than 17-hydroxyprogesterone. Although this partial reversal of specificity could reflect a difference in the agonist site of the liver receptor, mixing experiments, in which cytosolic extracts of liver were incubated with kidney cytosol extracts, demonstrated that the liver cytosol contained an additional factor that could change the apparent specificity of the kidney glucocorticoid receptor. This factor was stable at 0 degrees C for at least 18 h, heat-labile and non-dialyzable. These results suggest that the liver receptor's specificity may actually be the same as the other three, but appears to be different in this type of assay because of this additional factor. It is similar that their agonist sites are most likely identical.

Animals↗
PubMed1982

Dissociation between the magnitude of nuclear binding and the biopotency of glucocorticoids.

After 5 days of incubation with 10 nM tritiated dexamethasone, the amount of glucocorticoid displaceably-bound by the ACTH-secreting, glucocorticoid-responsive AtT-20 mouse pituitary tumor cell decreases by 70-80%. This decrease takes place in both the cytosolic and nuclear fractions. Although one might predict that there should be a decrease in agonist efficacy associated with this loss of nuclear binding, there was not; long-term incubations with dexamethasone revealed that the AtT-20 cells showed no tendency to resume secreting ACTH. Hence, although there was a dramatic decrease in the amount of nuclear-bound steroid, there was not a resulting decrease in the steroid's biopotency. These results suggest that the agonist-induced decrease in receptor content is either a means of removing redundant nuclear receptors, or a new, previously unrecognized, step in the mechanism of glucocorticoid hormone action.

Adrenocorticotropic Hormone↗
PubMed1981

Glucocorticoids regulate the glucocorticoid receptor in the AtT-20 cell.

Incubation of AtT-20/D-1 mouse pituitary tumor cells with glucocorticoids leads to a progressive diminution in the number of cellular glucocorticoid receptors. Control incubations demonstrate that non-glucocorticoids don't cause this depletion and that the decrease is not due to deleterious alterations in the incubation medium or degradation of the steroid. Incubation of cells with 0.5 micrograms/ml of cycloheximide alone does not dramatically diminish receptor content. However, incubation of cells with both cycloheximide and dexamethasone results in receptor depletion, suggesting that depletion involves enhanced receptor degradation. The cell's ability to bind glucocorticoids can be regenerated by a 24- to 48-h incubation in steroid-free medium. However, unlike the depletion step, the repletion process is sensitive to cycloheximide.

Animals↗
PubMed1981

Glucocorticoid hormone receptors in rat pancreas.

Although glucocorticoids influence pancreatic function, it has not been established whether they act directly at the level of the pancreas, or indirectly by causing metabolic changes in other target tissues. As a step in elucidating the actions of glucocorticoids on the pancreas, a search was conducted for glucocorticoid hormone receptors in this tissue. Uptake and binding studies indicated that there were glucocorticoid hormone receptors in the high-speed cytosolic extract of rat pancreas. These receptors appear to be similar to other rat glucocorticoid receptors: they bind glucocorticoids rapidly in a reversible manner at 0 degrees C, competitive binding analysis studies show that they have a preference for glucocorticoids and, like other receptors, bind the synthetic steroids triamcinolone acetonide and dexamethasone with a higher affinity than corticosterone. Scatchard analysis demonstrated that there are 1.37 . 10(-13) mol glucocorticoid-binding sites/mg cytosolic protein. This demonstration of a glucocorticoid hormone receptor in pancreatic cytosol suggests that some of the effects glucocorticoids exert on pancreatic function are a consequence of their direct actions on this target tissue.

Animals↗
PubMed1981

Conditions affecting AtT-20 cell glucocorticoid uptake.

Glucocorticoid uptake by intact AtT-20/D-1 cells was studied to determine if the extent of uptake was enhanced or retarded by binding components in serum. The results demonstrate that the uptake of corticosterone, which binds to transcortin, was reduced by addition of serum while uptake of triamcinolone acetonide, which is not bound by transcortin, was unaffected. Neither heat denatured serum nor bovine serum albumin affected corticosterone uptake, further emphasizing the specificity of the inhibition. The presence of serum also affected the apparent binding specificity, since steroids able to bind to transcortin became less effective competitors when serum was present in the incubation medium. In the absence of serum, the specificity of glucocorticoid uptake was qualitatively similar to that of the isolated cytosol receptor. These results emphasize that the selective inhibitory effect of serum transcortin on whole cell uptake of certain steroids should be considered when assaying steroid potency in intact cellular systems.

Animals↗
PubMed1979

Enzyme immobilization techniques on poly(glycidyl methacrylate-co-ethylene dimethacrylate) carrier with penicillin amidase as model.

Two types of bead-form macroporous carriers based on glycidyl methacrylate with ethylene dimethacrylate copolymers were used for the immobilization of penicillin amidase either directly or after chemical modification. Direct binding through oxirane groups, which is equally efficient at pH 4.2 and 7, is relatively slow and brings about an activity loss at low enzyme concentrations. The most efficient immobilization was achieved on glutaraldehyde-activated amino carrier, irrespective of whether the amino groups were formed by ammonia or 1,6-diaminohexane treatment of the original oxirane carrier. Hydrazine treatment gave lower immobilization yields. The same is true of the azide method independent of the length of the spacer. Most enzyme activity was preserved by coupling the carbodiimide-activated enzyme to the carrier with alkyl or arylamino groups at the end of a longer substituent. Immobilization on diazo-modified carrier gave average results. Rapid immobilization by a lysine-modified phosgene-treated carrier resulted in an activity loss. It is suggested that multipoint and very tight attachment of the enzyme molecule to the matrix decreased the activity. The immobilized activity is quite stable in solution and very stable upon lyophilization with sucrose.

Amidohydrolases↗
PubMed1979

Heterogeneity of AtT-20 cell glucocorticoid binding sites: evidence for a membrane receptor.

In previous studies we have found that intact AtT-20 cells contained two glucocorticoid binding sites with distinctly different affinities and specificity. In this paper, the nature of these sites was investigated by studying glucocorticoid binding to cytosol and to plasma membranes isolated from AtT-20 mouse pituitary tumor cells. Plasma membrane vesicles were isolated from AtT-20 cells and found to take up alpha-aminoisobutyric acid, indicating that they were properly oriented and functionally intact. Corticosterone bound to these vesicle in a time- and temperature-dependent manner. The binding exhibited a glucocorticoid preference since non-glucocorticoids such as progesterone, testosterone or estradiol were unable to inhibit binding. In addition, binding specificity differed from that of the cytoplasmic receptor since the synthetic glucocorticoids were also ineffective competitors. The major inhibitors of binding were corticosterone greater than 11-dehydrocorticosterone greater than 11-ketoprogesterone greater than cortisol. In other complementary studies, AtT-20 cell cytosol was tested to determine whether heterogenous soluble sites exhibiting a preference for the natural vs the synthetic steroid could also be identified. We found that binding sites for both steroid classes were approximately similar in number, specificity and behavior on ion-exchange chromatography. We conclude that, in addition to a classical soluble cytoplasmic glucocorticoid receptor, AtT-20 cells contain plasma membrane glucocorticoid binding sites. The affinity and specificity of these sites for the natural ligand, corticosterone, suggest that they play an important role in the subcellular mechanism of glucocorticoid action.

Aminoisobutyric Acids↗
PubMed1978

Methacrylate gels with epoxide groups as supports for immobilization of enzymes in pH range 3-12.

Glycidyl methacrylate gels are carriers suitable for attachment of enzymes and for use in affinity chromatography. Experiments on the coupling of glycyl-L-leucine and acetyl-L-leucine to these gels have shown a high pH-dependence of the bond formation between the support and the alpha-amino group (pH optimum 9.7); the coupling reaction between the epoxide group and the carboxyl group is practically pH-independent. Serum albumin and trypsin were attached to a greater extent in acidic than in alkaline media. The effects of time and temperature were also studied. The catalytic action of immobilized trypsin, as well as its use for affinity chromatography of trypsin inhibitor, were studied.

Dipeptides↗
PubMed1977

Experimental chemotherapy of rat leukemia RBA-Le with cis-diamminedichloroplatinum.

The antileukemic activity of cis-diamminedichloroplatinum (PDD) was studied in rats bearing myelogenous leukemia RBA-Le. Clinical picture, changes in life-span, hematological indices and weight changes were used to assess the effectiveness of the therapy. Maximum effectiveness was noted when PDD was given in combination with Methotrexate (MTX) and Poly I:C, respectively. The mean life-span of the rats treated with PDD and MTX was prolonged to 37 days that is an increase of 147 percent of the control level. Furthermore 20 percent of the treated animals survived symptom-free for more than 60 days. Out of 20 animals receiving PDD in combination with Poly I:C 6 rats survived 60 days symptom-free. In remaining 14 animals who finally died on leukemia an 85 percent increase in life-span was noted. Only slight increase in life-span was recorded in those groups treated with PDD alone and in combination with Cyclophosphamide regardless of dosage schedule. The general toxicity of PDD therapy is discussed.

Animals↗