Analysis of the glucocorticoid antagonist action of dexamethasone 21-mesylate in HeLa S3 cells

Several properties of human glucocorticoid receptors complexed to the synthetic glucocorticoid agonists dexamethasone (DEX) and triamcinolone acetonide (TA) and the antagonist dexamethasone 21-mesylate (DM) are compared in an attempt to define the mode of action of DM. Both DEX and TA induce an increase in alkaline phosphatase activity in HeLa S3 cells. Not only is DM without effect on alkaline phosphatase activity at concentrations as great as 10(-7) M, it blocks the action of DEX and TA on enzyme induction, thus acting as a pure antagonist in this system. DM-receptor complexes, like agonist-receptor complexes, are recovered in the cytosol when cells are incubated with ligand at 0 degrees C but are recovered from the nucleus when incubation is shifted to 37 degrees C, suggesting that activation of the antagonist-receptor complex occurs in vivo. The molecular species that undergoes this temperature-dependent shift from the cytosolic compartment to the nuclear compartment exhibits saturable binding to the antagonist. Both the cytosolic and nuclear species exhibit a relative molecular mass of approximately equal to 94,000 Daltons when analysed by SDS-polyacrylamide gel electrophoresis. Receptors labeled in intact cells with [3H]DM at 0 degrees C sediment at approximately 8S in sucrose gradients, shifting to 4S when the gradients contain 0.4 M KCl. DEX- and TA-labeled receptors show the same sedimentation behavior, which has been accepted as one criterion of receptor subunit dissociation, or activation.     

DNA damage production in CHO cells at elevated temperatures

Induction of DNA lesions in the nucleus of Chinese hamster ovary (CHO) cells was observed at hyperthermic temperatures using the alkaline filter elution and the alkaline sucrose gradient sedimentation methods. These lesions were observed principally at temperatures greater than 45 degrees C with an activation energy of 140 kcal/mole. On alkaline sucrose gradients the cell genome was reduced to a 140 S or 2 X 10(8) dalton subunit of DNA independent of increasing exposure time at temperatures above 45 degrees C. The large thermal activation energy and the limited DNA size reduction suggest the possible involvement of thermal denaturation of a nuclear polypeptide in the production of these nuclear lesions.     

Relationship of crystallization to the nature of polyhedron protein,with reference to a nuclear-polyhedrosis virus of the silkworm, Bombyx mori

When the silkworm nuclear polyhedrosis inclusion bodies were exposed to a drop of weak alkaline solution, the protein part of the bodies dissolved, and subsequently recrystallized out with the gradual evaporation of water from the preparation under ordinary room condition. The crystals assumed a variety of crystalline forms. These crystalline forms could be demonstrated by a simulation of folding paper strips along the edge or diagonal lines of rhombi which are to be formed on the strips serially by passing parallel lines with an inclination of 60° against the strip edge. Polyhedron protein was very homogeneous and simple in nature, the sedimentation constant being around 6.26 Svedberg units (S). These protein particles were easily associated to form large complexes which gave 13.6, 19.7, 29.7, and 38.0 S in neutral solutions. Associated polyhedron protein complexes were separable into various sizes by ultracentrifuge and disc electrophoresis.     

Stoichiometry of large T antigen and pp53 in complexes isolated from simian virus 40-transformed rat cells.

Simian virus 40-transformed cells synthesize high-molecular-weight protein complexes (22 to 30S) that consist of the virus-coded large T antigen (81,500 daltons) and the cellular antigen pp53. These complexes were partially purified from lysates of transformed rat cells by sucrose velocity sedimentation. The stoichiometry of the two proteins in the complex was studied by direct enzyme-linked immunosorbent assays, using alkaline phosphatase-conjugated anti-T and anti-pp53 monoclonal antibodies. The results from these experiments indicate that the T antigen-to-pp53 ratio in the complex is 0.87 +/- 0.27. No statistically significant differences were found in this ratio for faster- and slower-sedimenting complexes. These results from enzyme-linked immunosorbent assays and previous molecular weight estimates of the complex suggest that this complex is composed, on the average, of four molecules of T antigen and four or five molecules of pp53.     

Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver: lack of sensitivity to alkaline phosphatase when compared with that of glucocorticoid receptor

Rat hepatic cytosol was treated with alkaline phosphatase in order to determine if dephosphorylation altered the ability of Ah receptor to bind 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD). Glucocorticoid receptor was studied for comparison. As previously had been shown in other laboratories, treatment of cytosol with purified alkaline phosphatase dramatically reduced the subsequent ability of glucocorticoid receptor to bind hormone. However, alkaline phosphatase had no effect on the ability of Ah receptor to bind [3H]TCDD. If either glucocorticoid receptor or Ah receptor was occupied by its ligand prior to exposure to alkaline phosphatase there was no loss in ligand binding capacity. Crude alkaline phosphatase (containing some protease activity) substantially reduced the ability of glucocorticoid receptor to bind hormone and shifted the sedimentation position of the glucocorticoid receptor from approximately 8 S to approximately 2 S. Crude alkaline phosphatase did not reduce the ability of Ah receptor to bind [3H]TCDD and did not alter sedimentation of the 9 S [3H]TCDD. Ah receptor complex. Although the Ah receptor appears to be a member of the steroid receptor superfamily, the lack of effect of alkaline phosphatase on Ah receptor (compared to the sensitivity of glucocorticoid receptor) highlights another significant difference in molecular characteristics between the Ah receptor and the receptors for steroid hormones.     

Tobacco mosaic virus protein: sedimentation equilibrium studies of the initial stages of polymerization.

The lowest stages of polymerization of tobacco mosaic virus protein were studied by means of high-speed sedimentation equilibrium experiments. Several distinct modes of polymerization were found. At pH 7.1 the expected monomer-trimer-higher polymer equilibrium was observed--very little dimer was detected at this pH. At pH 7.5, however, a strong dimerization was observed--neither monomer nor trimer was detected at this pH. An octamer appeared to be the only species present other than the dimer. When 0.01 M beta-mercaptoethanol was added to the solvent pH 7.5, the dimer was dissociated, resulting in a monomer-trimer association. The dimerization may be the basis for the larger "doubled" polymers formed by the protein at alkaline pH, while the octamer may correspond to the 8S peak frequently observed in sedimentation velocity experiments at alkaline pH. On the other hand, the monomer-trimer-higher polymer equilibrium may correspond to the single helix formed by the protein at slightly acid pH and to the combination of 4S and 20S peaks seen in sedimentation velocity experiments at slightly acid pH.     

Sedimentation Analysis of DNA from Irradiated and Unirradiated L Cells

DNA, released from unirradiated mouse L-cells gently lysed in a thin layer of 2% sucrose on top of an alkaline sucrose gradient, was found to sediment in a narrow band with a sedimentation coefficient of about 500S. Exposure of cells to increasing doses of X-rays (89-712 rads) continuously reduced the DNA sedimentation velocity until, after about 890 rads, the DNA appeared in a narrow peak with a sedimentation coefficient of approximately 180S. As the dose given to cells was increased beyond 890 rads, the sedimentation coefficient of the DNA released continued to decrease and the sedimentation profiles now broadened in a manner consistent with the random production of single-strand breaks in the DNA. The DNA released from unirradiated cells (500S) is thought to be loosely aggregated and only partially single stranded. It is presumed that cells exposed to low doses of radiation release DNA with marked reductions in sedimentation coefficient because single-strand breaks produced in the DNA aid the alkaline denaturation process. By using the system to be described, it has been possible to demonstrate DNA repair (rejoining of X-ray-induced single-strand breaks) during postirradiation incubation of cells given doses as low as 400 rads.     

Role of Genes 46 and 47 in Bacteriophage T4 Reproduction: I. In Vivo Deoxyribonucleic Acid Replication

Functional proteins coded by genes 46 and 47 are required for (i) continuation of deoxyribonucleic acid (DNA) synthesis in the late period of T4 infection and (ii) production of normal, late replicating DNA which contains strands with a sedimentation coefficient in alkaline sucrose greater than that of mature DNA (73S). Continued DNA synthesis in the late period in the absence of functional genes 46 or 47 can be achieved by inhibiting late protein synthesis either by using bacterio-phage with a second mutation in gene 55 or by adding chloramphenicol to the culture before the decline in the rate of DNA synthesis. However, when functional 46/47 proteins are absent throughout infection, no strands with a sedimentation coefficient greater than 73S (in alkaline sucrose) are produced. This is the case even when DNA synthesis is allowed to continue. DNA arrest is accompanied by conversion of rapidly sedimenting, replicating DNA to slower sedimenting forms. When 46/47 is absent from the beginning of infection, the conversion product has a smaller sedimentation coefficient than mature DNA both in neutral and alkaline sucrose. When DNA arrest occurs midway in infection by heat-inactivating the ts46 enzyme, the conversion product has a sedimentation coefficient (i) the same as mature DNA in both neutral (63S) and alkaline sucrose if capsid assembly is allowed to take place and (ii) close to 63S in neutral sucrose but heterogenous and relatively greater (up to 100S) in alkaline sucrose if capsid assembly is inhibited. The structure of this DNA is unknown.     

Functional and structural characteristics of polyribosomes associated with chick embryo cell nuclei

Polyribosomes bound to the outer nuclear membrane was isolated from purified preparations of chicken embryo cell nuclei. These polyribosomes were shown to consist fractions forming unstable complexes with the nuclear membrane which can be separated from the latter by treatment with high ionic strength buffer solutions. Using sedimentation and gradient density analyses, the nuclei-bound RNP complexes were shown to be predominantly composed of 80S monosomes which take an active part in collagen polypeptide synthesis in cell-free protein-synthesizing systems. A comparison of sedimentation properties and collagen-synthesizing activity of nuclei-bound polyribosomes and cytoplasmic polyribosomes forming unstable complexes with endoplasmic membranes, it was concluded that the nuclei-bound 80S monosomes are an early step in the formation of cytoplasmic polyribosomes.     

Sedimentation velocity of DNA in isokinetic sucrose gradients: calibration against molecular weight using fragments of defined length.

The relationship between sedimentation coefficient and molecular weight for DNA sedimenting in preformed alkaline and neutral sucrose gradients was determined using absolute molecular weight standards (restriction fragments of plasmid pBR322 and phage lambda DNA). The range of calibration for alkaline gradients was extended to small DNA fragments (652 base-pairs) for the first time. The exponent b in the equation S20 degrees, w = aMb was found to be 0.380 in neutral gradients and 0.410 in alkali. The latter value differs significantly from previous estimates. The gradients were isokinetic, and the distance sedimented was shown to be directly proportional to the sedimentation coefficient at all times.     

Physical measurements of the liver glucocorticoid receptor.

Physical measurements were made on the cytosolic form of the liver [3H]dexamethasone receptor. These include a Stokes radius of 3.5 nm, determined by gel filtration, and sedimentation coefficients of 5.1 and 7-8S, by sucrose-density-gradient centrifugation. From these measurements, the following physical properties were calculated: apparent mol. wt. 78000 (the 5.1 S form); D app. 6.1 X 10(-7) cm2-S-1; f/fo 1.25; axial ratio 4.7; these indicate a globular protein. Measurements of sedimentation coefficient of cytosol steroid-receptor complexes previously subjected to various activating conditions gave different values and lead to the conclusion that the mechanism of activation in vitro enabling the steroid-receptor complex to bind to DNA is more complex than simple disaggregation to a uniform size.     

Non-identical subunits of 7S globulin (gossipulin-1) from cotton seeds]

7S globulin is isolated from cotton seeds and is designated as "Gossipulin-1". Three different amino acids (glycine, aspartic acid and leucine) are found to follow the N-terminal amino acid which is indicative of a non-identity of its subunits. Gossipulin-1 dissociated in 0.1 M HCl, depending on the time of incubation, into 3.87, 2.39 and 1.35 subunits. It had a sedimentation constant of 0.37S in alkaline medium and of 1.24S on 8 M urea. A sedimentation peak of 1.1S was discovered in reduced and alkylated Gossipulin-1.     

Platelet activation induces the formation of a stable gelsolin-actin complex from monomeric gelsolin

We have studied the interactions between gelsolin and actin in crude extracts from activated and unactivated platelets and in mixtures of purified platelet gelsolin and muscle actin. Extracts were prepared using 10 mM EGTA from human platelets treated either with 100 microM aspirin and 2.5 mM tetracaine to retard activation or with the calcium ionophore A23187 to effect activation. The extracts were fractionated by gel filtration on Sephadex G-150 or by sedimentation on sucrose gradients and then analyzed using anti-gelsolin immunoblots and actin filament nucleation assays. The nucleation activity in both extracts was associated with gelsolin. The activity in the extracts from unactivated platelets sedimented with an S value of 5.2 and had an Mr = 90,000. The activity in the extracts prepared with EGTA from activated platelets sedimented at 6.8 S and had an Mr = 130,000. We have shown previously that the Mr = 130,000 species is an EGTA-stable binary complex of one actin and one gelsolin. Transient exposure of the extracts from unactivated platelets to 100 microM Ca2+ and subsequent fractionation in EGTA-containing buffers demonstrated that the formation of the binary complex occurs in the presence of Ca2+. Fractionation in the presence of 100 microM Ca2+ demonstrated higher order complexes including a ternary complex with a sedimentation constant of 8.2 S and an Mr = 165,000. Sedimentation and gel filtration experiments using purified platelet gelsolin and rabbit skeletal muscle actin demonstrated that formation of the EGTA-stable binary complex required Ca2+. At least one additional actin is bound to the binary complex in the presence of Ca2+, but is not sufficiently stable to be purified when EGTA is added. The results suggest that gelsolin exists either as a monomer or perhaps as a weak complex with actin in unactivated platelets but complexes tightly with actin during the transient Ca2+ rise that occurs during activation.     

Comparison of 7S nerve growth factor and nerve growth factor I from mouse submandibular glands

7S nerve growth factor (7S NGF) and nerve growth factor I (NGFI) are NGF-containing protein complexes isolated from mouse submandibular glands by different protocols, and reports suggest that the molecules differ chemically. In this study, we compared the molecular properties and subunit compositions of the two proteins. Purified 7S NGF and NGFI electrophoresed to identical positions on polyacrylamide gels in nondissociating buffers, with electrophoretic mobilities indistinguishable from that of unpurified NGF in salivary gland extracts. Ultraviolet absorption curves were identical, and sedimentation coefficients were similar (7.3 +/- 0.25 S for 7S NGF; 7.2 +/- 0.2 S for NGFI) as determined by sedimentation velocity analysis. By sedimentation equilibrium analysis, molecular weights of 135 000-140 000 were obtained for both complexes at protein concentrations in the centrifuge cell greater than 85 micrograms/mL; when protein concentrations within the centrifuge cell ranged from approximately 30 to 100 micrograms/mL at equilibrium, both complexes dissociated. Molecular weight values determined by gel filtration on Bio-Gel P300 and Sephadex G200 resins were similar for both proteins, and the values determined on Sephadex agreed with those obtained by ultracentrifugation. The subunit compositions of the complexes were also similar as determined by nonequilibrium isoelectric focusing, NGFI being composed of proteins that migrated to positions identical with those of the alpha, beta, and gamma subunits of 7S NGF. Furthermore, the stoichiometry of the subunits was similar in the two complexes as determined by radioimmunoassays to each of the subunits and by densitometric analysis of electrophoretic gels. Both methods showed that the complexes contain approximately 2 mol of the alpha and gamma subunits per mole of beta-NGF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of kinetoplast DNA and RNA of Phytomonas davidi

Kinetoplast DNA networks were isolated from stationary-phase culture forms of Phytomonas davidi. The networks banded in CsCl at a density of 1.699 g/ml and consisted of covalently closed circular molecules. The networks were sensitive to shear forces and exhibited several discrete sedimenting components in neutral and alkaline sucrose. Closed monomeric minicircles were isolated from sonicated networks by alkaline band sedimentation. Closed monomers showed a heterogeneous banding pattern on electrophoresis in acrylamide-agarose gels and had sedimentation coefficients of 20.5 S in alkaline sucrose and 11 S in neutral sucrose. The mean minicircle molecular weight as measured by cospreading with φXRF II was 0.70 × 10⁶ or 1064 nucleotide pairs. Minicircles exhibited a sequence microheterogeneity as evidenced by restriction enzyme analysis, melting analysis, and renaturation kinetics. Network maxicircles were evidenced by the appearance of high molecular weight fragments after restriction with several enzymes and by the existence of supertwisted “edge loops” extending out from the periphery of networks. The maxicircle molecular weight was estimated to be approximately 24 × 10⁶. A purified kinetoplast-mitochondrion fraction was found to contain 9 and 12 S RNA species that comigrated with L. tarentolae 9 and 12 S kinetoplast RNAs.     

Salt-induced activation of 1,25-dihydroxyvitamin D3 receptors to a DNA binding form

In this report we examine the DNA-cellulose binding and sedimentation properties of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptors from rat intestine and cultured human mammary cancer cells (MCF-7) extracted in nonactivating (low salt) buffers. Receptors prepared in hypotonic buffer had low DNA binding (13%) compared to receptors extracted with 0.3 M KCl (50%). Treatment of low salt receptor preparations with KCl significantly increased (approximately 3-fold) DNA-binding (activation), demonstrating that receptors can be "activated" in vitro. Activated receptors eluted from DNA-cellulose at 0.18 M KCl. Sedimentation analysis followed by DNA-cellulose binding indicated that activated receptors are approximately 3.2 S and unactivated receptors 5.5 S in size. These results suggest that dissociation of an aggregated moiety may lead to receptor activation. Treatment of unactivated receptor with RNase did not alter DNA binding or sedimentation properties of the aggregated receptor. Treatment of unactivated receptor complexes with heat did not increase DNA binding, and molybdate did not block subsequent salt activation. In summary these results suggest that 1,25(OH)2D3 receptors undergo a salt-induced activation step similar to that described for other steroid receptor systems. However, 1,25(OH)2D3 receptors differ from other steroid receptors in not exhibiting heat activation nor having salt activation blocked by molybdate.     

Recombinant models of lipoproteins. Apolipoprotein A-I/phosphatidylcholine/cholesterol complexes formed in a 2-chloroethanol-water mixture

Apolipoprotein A-I can spontaneously associate with phosphatidylcholine and cholesterol in 2-chloroethanol-water mixture. It was demonstrated, using a spin label technique, that dissolved molecules participate in complex formation. The apolipoprotein A-I/phosphatidylcholine/cholesterol complexes were isolated by gel chromatography. Complexes of three types were prepared and characterized: type A, large heterogeneous aggregates with molecular weight 600 000, sedimentation coefficient 10 S and the following molar composition - protein/phosphatidylcholine/cholesterol, 1:(70-100):(10-12); types B and C, with weight average molecular weights 140 000 and 110 000, average sedimentation coefficients 3.6 S and 1.7 S, respectively. Both types have the same molar composition - protein/phosphatidylcholine/cholesterol, 1:25:8. The dissimilar sedimentation coefficients between complexes B and C may be explained by the difference in the monomer/tetramer ratio (monomer molecular weight 50 000). The spin label sn-1-O-stearoyl-2-O-9'-spiro(4',4'-dimethyloxazolidine-3'-oxyl) heptadecanoylglycero-3-phosphocholine introduced into the complexes A and B showed different thermal properties of these complexes, which may be due to differences in the lipid-protein interactions.     

The structure of SV 40 chromatin.

Simian virus 40 (SV40) nucleoprotein complexes were studied with the electron microscope. Depending on the isolation procedure, SV40 chromatin has two different conformations: complexes isolated in the presence of 0.15 M NaCl appeared as very compact globular structures, while those isolated in the presence of 0.6 M NaCl had the typical 'beads-on-a-string' appearance of the primary nucleofilament. Concomitant with this structural change was a variation in the histone pattern and sedimentation behaviour of the complexes: with NaCl at 0.15 mol 1(-1) the isolated complexes contained both the nucleosomal histones and histone H1, and sedimented in sucrose gradients at 70S. Increasing the ionic strength to 0.6 M NaCl resulted in the removal of histone H1 from the complexes and in a decrease of the sedimentation coefficient to 40S. DNA relaxing enzyme is associated with the SV40 nucleoprotein complexes. The numbers of superhelical turns in DNA from compact and open types of complexes were found to be the same. Therefore the transition from the condensed to the open structure of viral chromatin does not require a change in the topological winding number of its DNA.     

Protein-RNA crosslinking in Escherichia coli 30S ribosomal subunits. Identification of a 16S rRNA fragment crosslinked to protein S12 by the use of the chemical crosslinking reagent 1-ethyl-3-dimethyl-aminopropylcarbodiimide.

1-ethyl-3-dimethyl aminopropylcarbodiimide (EDC) was used to cross-link 30S ribosomal proteins to 16S rRNA within the E. coli 3OS ribosomal subunit. Covalently linked complexes containing 30S proteins and 16S rRNA, isolated by sedimentation of dissociated crosslinked 30S subunits through SDS containing sucrose gradients, were digested with RNase T1, and the resulting oligonucleotide-protein complexes were fractionated on SDS containing polyacrylamide gels. Eluted complexes containing 30S proteins S9 and S12 linked to oligonucleotides were obtained in pure form. Oligonucleotide 5'terminal labelling was successful in the case of S12 containing but not of the S9 containing complex and led to identification of the S12 bound oligonucleotide as CAACUCG which is located at positions 1316-1322 in the 16S rRNA sequence. Protein S12 is crosslinked to the terminal G of this heptanucleotide.