Steroid hormone receptor studies in melanoma model systems

The Transplantable B-16 melanotic melanoma carried in syngeneic C57B1/6J female mice and the Syrian hamster melanoma cell line, RPMI 3460, were utilized to determine whether steroid-hormone receptors are present in animal melanomas. In the B-16 melanoma, a cytoplasmic-estrogen receptor is detectable, but there is no evidence for androgen or progestin receptors. Some tumors contain a glucocorticoid-binding macromolecule. Sucrosedensity gradient centrifugation of cytosol after incubation with [³H]-estradiol revealed an 8S peak that was suppressed by excess radioinert diethylstilbesterol. Binding varied from 5–35 fmoles per mg cytosol protein. Scatchard analysis of [³H]-estradiol binding in cytosol yielded a single class of high-affinity binding sites; the dissociation constant is 6 × 10^?10 M. The receptor molecule is shown to be estrogen-specific by ligand competition assays. In contrast to B-16 melanoma, no estrogen, androgen, or progestin receptor can be found in the Syrian hamster melanoma cell line. However, a substantial level of specific binding is observed using [³H]-dexamethasone. Sucrose-gradient centrifugation of cytosol from this cell line after incubation with [³H]-dexamethasone revealed a 7S peak that was suppressed by excess radioinert dexamethasone. Scatchard analysis indicated a single class of high affinity sites with a dissociation constant of 2 × 10^?9 M. Binding levels from 70–610 fmoles per mg cytosol protein were observed. The Syrian hamster melanoma cells also exhibit a biological response to glucocorticoids: Dexamethasone causes both an inhibition of growth and a decrease in final-cell density in these cells.     

The antagonistic effect of dexamethasone and insulin on alpha-fetoprotein secretion by cultured H4-II-E-C3 cells derived from the Reuber H-35 hepatoma.

Non-confluent monolayers of H4-II-E-C3 cells were maintained in serum-free media. Dexamethasone alone (5 × 10^?7M) stimulated α-fetoprotein secretion 2- to 4-fold while insulin alone (8.7 × 10^?8M) inhibited α-fetoprotein secretion by 20%. When dexamethasone (5 × 10^?7 to 5 × 10^?9M) and insulin (8.7 × 10^?8 to 8.7 × 10^?11M) were added simultaneously, insulin diminished the stimulatory effect of dexamethasone. When α-fetoprotein secretion was elevated by dexamethasone and the medium was replaced by media containing either insulin or no hormones, the rate of α-fetoprotein secretion diminished more rapidly with the insulin-supplemented medium. Alone or in combination, insulin and dexamethasone had little effect on albumin secretion.     

Effect of glucocorticoids on fetoprotein production by an established cell line from Morris hepatoma 8994

Glucocorticoids at concentrations equal to or higher than 10^?7M lead to an increase of alpha-fetoprotein production by an established cell line from Morris hepatoma 8994. These cells also secreted alpha_M-fetoprotein into the culture medium but only after addition of at least 4×10^?7M hydrocortisone or 5×10^?8M dexamethasone. The effects on both fetoproteins were observed in spite of a decrease of cell multiplication and an increase of cell detachment.     

Effect of salicylic acid on glucucorticoid receptor in cultured fibroblasts derived from rat carrageenin granuloma

The binding activity of [³H]dexamethasone to the specific receptor was studied in the cytoplasmic fraction of a established fibroblast line derived from rat carrageenin granuloma in culture condition. Specific receptor to dexamethasone was demonstrated. Scatchard analysis revealed a single class of binding sites with a dissociation constant for [³H]dexamethasone of 3.64 · 10^?8 M and a concentration of binding sites of 0.825 pmol per mg cytosol protein. The number of cytoplasmic binding sites per cell was calculated at 1.15 · 10⁵.Total binding activity to [³H]dexamethasone of the cytoplasmic fraction was enhanced when the cells were cultured in a medium containing salicylic acid at 37°C. The maximum enhancement was seen at the concentration of 10^?3 M and in 3 h treatment of salicylic acid. This enhancement by salicylic acid was lost when cycloheximide was added to the culture medium at the same time. If salicylic acid was added to the cell free system, it showed no effect on the binding activity. The other non-steroidal anti-inflammatory drugs; phenylbutazone and indomethacin, also enhanced the total binding activity to [³H]dexamethasone of the cytoplasmic fraction at the concentration of 2 · 10^?5 M and 2 · 10^?7 M, respectively.     

Regulation of high- and low-affinity epidermal growth factor receptors by glucocorticoids

It is reported that receptors for epidermal growth factor (EGF) in HeLa S₃ cells exist in two forms, which differ in both affinity and capacity. Both the number of receptors and their distribution into low- and high-affinity forms are modulated by glucocorticoids. Scatchard analysis of saturation binding assays performed at 0 °C indicates that there is a low-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 1.5}\text{nm}$), which contains approximately 6 × 10⁴ binding sites per cell, and a second, high-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 0.16}\text{nm}$) containing approximately 5 × 10³ binding sites per cell. Exposure of HeLa S₃ cells to 10^?7m dexamethasone for 24 h increased EGF binding to whole cells by increasing the numbers of low- and high-affinity receptors by 20 and 114%, respectively. The increase in EGF binding depends upon the dose of dexamethasone, being raised from 10^?11 to 10^?6m. EGF binding is half-maximal near 2–4 × 10^?9m, a concentration equal to the K_d of dexamethasone for the glucocorticoid receptor in these cells. The increase in EGF binding is specific for glucocorticoids, occurring when the HeLa S₃ cells are exposed to 10^?7m cortisol or dexamethasone for 24 h, but not when the cells are similarly treated with testosterone, 5α-dihydroxytestosterone, 17β-estradiol, or progesterone. The effect on EGF binding appears to be biphasic; the initial rapid increase occurs between 8 and 12 h, is blocked by both 10^?6m cyclohexamide and 0.1 μg/ml actinomycin D, and is followed by a more gradual increase thereafter. These data indicate that glucocorticoids are able to regulate both the number of EGF receptors and their distribution into high- and low-affinity components. Press, Inc.     

Activation and inhibition of the action potential Na+ ionophore of cultured rat muscle cells by neurotoxins.

Both myoblasts and myotubes in cultures of clonal rat muscle cells have action potential Na⁺ ionophore activity. The ionophore is activated by batrachotoxin (K_0.5 = 3 to 5 × 10^?7 M) and veratridine (K_0.5 = 4 to 6 × 10^?5 M) which compete for the same activation site. As in denervated rat muscle, the ionophore of cultured muscle is 100 fold more resistant to inhibition by tetrodotoxin (K_0.5 = 1.5 to 3 × 10^?6 M) and 20 fold more resistant to inhibition by saxitoxin (K_0.5 = 1.5 to 3 × 10^?7 M) than in nerve, innervated muscle, or cultured neuroblastoma cells.     

Trihalomethanes in desalinated water: Human exposure and risk analysis

Desalinated seawater is used to satisfy domestic water demands in many countries. The treated freshwater is blended with desalinated water to increase the water supply. The desalinated and blended water contains disinfection byproducts (DBPs), some of which may induce cancer risk to human. In this study, concentrations of trihalomethanes (THMs) in desalinated and blended water in Saudi Arabia were investigated, and human exposure and risk were predicted. The intakes of THMs (chloroform, bromodichloromethane, dibromochloromethane, and bromoform) were predicted to be 8.38 × 10^?5, 7.57 × 10^?5, 2.54 × 10^?5, and 4.32 × 10^?4 mg/kg-d, respectively. The overall cancer risk and hazard index were estimated to be 1.78 × 10^?5 (range: 7.40 × 10^?7–9.26 × 10^?5) and 3.49 × 10^?2 (range: 1.20 × 10^?3–2.34 × 10^?1), respectively. The probabilities of cancer risk exceeding the risk levels of 1 × 10^?6, 1 × 10^?5, and 5.0 × 10^?5 were 1.0, 0.775, and 0.012, respectively. The loss of disability adjusted life years (DALYs) was predicted to be 25.1 per year while the cancer risk represented 8.48 × 10^?7 DALY per person per year. The financial burden from such risk was estimated to be US$ 2.72 (range: US$ 2.52–2.91) million per year. The findings may assist in better understanding and reducing cancer risk from DBPs in desalinated and blended water.     

A specific glucocorticoid binding macromolecule of rabbit uterine cytosol

A high affinity (K_d=2.7 × 10^?10M at 0°) dexamethasone binding macro-molecule has been identified in the cytosol fraction of rabbit uteri. Competition studies show high specificity for glucocorticoids since binding of labeled dexamethasone is inhibited by cortisol and corticosterone but not by progesterone, testosterone, or estradiol 17β. The binding component has a sedimentation coefficient of 8S and its concentration in uterine cytosol is about 0.2 pmoles per mg protein. Uptake of labeled dexamethasone by isolated uterine nuclei requires the presence of cytosol and is temperature dependent. The KCl-extractable nuclear complex sediments at 4S. Thus the dexamethasone binding components of the rabbit uterus have properties similar to those described for steroid hormone receptors present in target tissues. Specific dexamethasone binding could not be demonstrated in rat uterine cytosol.     

Puromycin resistance in Chinese hamster cells: Genetic and biochemical studies of partially resistant,unstable clones

Resistance to 10 μg/ml of puromycin has been analyzed in V79 Chinese hamster cells. Clones that were isolated in 10 μg/ml of puromycin and subsequently cultivated in its absence consistently lost their resistance. One clone was analyzed in detail by recloning in the presence and absence of puromycin, and it was found that non-puromycin cultivated sublones also lost their resistance and regained inhibition profiles similar to the V79 parent. Reconstruction experiments between sensitive and resistant cells demonstrated that the yield of mutants was not affected by metabolic cooperation. The mutation rate was calculated to be 1 × 10^?7 per cell per generation, and was the same within the limits of statistical error in a colchicine-produced polyploid derivative of the V79 line. Although a number of resistant clones were found to have polyploid karyotypes, the polyploid V79 line was not more resistant to puromycin, nor did it possess a higher frequency of puromycin resistant cells. Studies employing radiolabeled puromycin established that resistance was due to a lowered uptake of puromycin and that an inverse relationship existed between resistance level and uptake rate.     

Studying the interaction between three synthesized heterocyclic sulfonamide compounds with hemoglobin by spectroscopy and molecular modeling techniques

The interaction between synthesized heterocyclic benzene sulfonamide compounds, N-(7-benzyl-56-biphenyl-2m-tolyl-7H-pyrrolo[23-d]pyrimidine–4–yl)-benzene sulfonamide (HBS₁), N-(7-benzyl-56-biphenyl-2-m-tolyl-7H-pyrrolo[23-d] pyrimidine-4-yl)-4-methyl- benzene sulfonamide (HBS₂), and N-(7-benzyl-56-biphenyl-2-m-tolyl-7H-pyrrolo[23-d]pyrimidine-4-yl)-4-chloro-benzene sulfonamide (HBS₃) with Hb was studied by fluorescence quenching, zeta potentional, circular dichroism, and molecular modeling techniques. The fluorescence spectroscopy experiments were performed in order to study the conformational changes, possibly due to a discrete reorganization of Trp residues during binding between HBS derivatives and Hb. The variation of the K_SV value suggested that hydrophobic and electrostatic interactions were the predominant intermolecular forces stabilizing the complex. The K_SV1 ans K_SV2 values of HBS derivatives with Hb are .6 × 10¹³ and 3 × 10¹³ M^?1 for Hb–HBS₁, 1 × 10¹³ and 4 × 10¹³ M^?1 for Hb–HBS₂, .9 × 10¹³, and 6 × 10¹³ M^?1 for Hb–HBS₃, respectively. The molecular distances between Hb and HBS derivatives in binary and ternary systems were estimated according to Förster’s theory of dipole–dipole non-radiation energy transfer. The quantitative analysis data of circular dichroism spectra demonstrated that the binding of the three HBS derivatives to Hb induced conformational changes in Hb. Changes in the zeta potential of the Hb–HBS derivatives complexes demonstrated a hydrophobic adsorption of the anionic ligand onto the surface of Hb as well as both electrostatic and hydrophobic adsorption in the case of the complex. The modeling data thus confirmed the experimental results. This study is expected to provide important insight into the interaction of Hb with three HBS derivatives to use in various toxicological and therapeutic processes.     

Electron microscopy of human interphase nuclei

Quantitative electron microscopy was used to analyze surface-spread, critical-point-dried human interphase nuclei and chromatin. The following information is presented: (1) Unstimulated interphase nuclei of lymphocytes from peripheral blood have a mean dry mass of 50.30×10^?12 g. The mean dry mass of stimulated nuclei of lymphocytes was determined to be 59.34×10^?12 g, a significant statistical difference from the unstimulated ones. (2) Mean diameter of chromatin fibers and mean fiber mass per micron were 199ű15% coefficient of variation (C.V.) and 5.95×10^?16g×29% C.V., respectively. (3) A line of regression of fiber mass on fiber diameter for 83 fibers indicated that a 200-Å fiber has a mass of 5.86×10^?16g/μ, or almost the same as the mean fiber mass of 5.95× 10^?16g/μ. (4) With the value 7×10^?12g for the DNA content of an unstimulated lymphocyte nucleus, a total length of 215 cm is calculated for the DNA double helix. When this length is compared to the mean length of chromatin fiber per nucleus (7.59 cm), a ratio of 28.3 to 1 results, which is called the DNA-packing ratio. (5) This DNA-packing ratio of 28.3 is reasonably close to the packing ratio of 26.9 suggested from model calculations for the second DNA supercoil in a 200-Å chromatin fiber.     

Purification and some properties of γ-conglycinin in soybean seeds

A 7S globulin (γ-conglycinin) which was one of four major antigenic components in soybean globulins was purified and found to be homogeneous on ultracentrifugation, disc electrophoresis, immunoelectrophoresis and disc electrofocusing by gel filtration, preparative-scale disc electrophoresis and two kinds of affinity chromatography. Subsequently, some physico-chemical properties of the protein were determined. The sedimentation coefficient, isoelectric point, MW and diffusion constant were 6·55S, pH 5·80, 104000 and 5·80 × 10^?7 cm²/sec, respectively. The protein was a glycoprotein which contained 5·49% total carbohydrate per protein. The protein did not aggregate and dissociate with a change of ionic strength from 0·1 to 0·5.     

Biochemical properties of the 1 alpha, 25-dihydroxyvitamin D3 cytoplasmic receptors from human and chick parathyroid glands

Cytoplasmic receptors for 1α, 25-dihydroxyvitamin D₃ from human parathyroid adenoma tissue and rachitic chick parathyroid glands have been characterized with regard to a number of physical, chemical, and ligand binding properties. Both receptors are 3.6–3.7 S proteins with molecular weights of approximately 75,000 and Stoke's molecular radii of 36 Å. It was found that the receptors possess a cysteine residue in or near the 1α, 25-dihydroxyvitamin D₃ binding site which is critical for ligand binding activity. The receptors both have equilibrium dissociation constants for 1α, 25-dihydroxyvitamin D₃ in the range of 2 to 5 × 10^?10m at 4 °C and second-order association rate constants for their seco-steroid ligand of 1 × 10⁷, m^?1 min^?1 (0 °C). The dissociation rate constants were found to be 5.3 × 10^?4 min^?1 (4 °C) for the human receptor and 1.3 × 10^?5 min^?1 (4 °C) for the chick receptor. The great deal of similarity which exists between the cytoplasmic 1α, 25-dihydroxyvitamin D₃ receptors from avian and mammalian parathyroid glands suggests a homologous function for these molecules in the two tissues.     

Gluconeogenesis in rat liver parenchymal cells in primary culture: Permissive effect of the glucocorticoids on glucagon stimulation of gluconeogenesis

Primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion procedure and maintained as a monolayer in a serum-free culture medium were used to study glucoeogenesis and the role that the glucocorticoids play in the control of this pathway. These cells carried out gluconeogenesis from three-carbon precursors (alanine and lactate) in response to glucagon and dexamethasone added alone or in combination. Maximum glucose production was observed with cells pretreated for several hours with dexamethasone and glucagon prior to addition of substrate and glucagon (8- to 12-fold increase over basal glucose production). Half-maximum stimulation of gluconeogenesis was seen with 3.6 × 10^?10 M glucagon and 3.6 × 10^?8 M dexamethasone. Maximum stimulation was oberved with 10^?7 M glucagon and 10^?6 M dexamethasone. The length of time of dexamethasone pretreatment was found to be important in demonstrating the effect of glucocorticoids on glucagon-stimulated gluconeogenesis. Treeatment of cells with dexamethasone for 2 hours did not result in an increase in glucose production over identical experimental conditions in the absence of dexamethasone, wherease pretreatment for 5 hours (1.2-fold increase) or 15 hours (1.7-fold increase) did result in an increase in glucose production. The results establish that the adult rat liver parenchymal cells in primary culture are a valid model system to study hepatic gluconeogenesis. In addition, we have established directly that the glucocorticoids amplify the glucagon stimulation of gluconeogenesis.     

Epidermal growth factor receptors in fetal and maternal rabbit lung

The pattern of morphologic and functional development of lung during intrauterine period is influenced by several endogenous compounds. Recently Epidermal Growth Factor (EGF), when administered in vivo, has been shown to accelerate pulmonary maturation in fetal rabbit and sheep. We sought evidence for EGF receptor occurrence in fetal and maternal rabbit lung plasma membranes. The percent specific binding (mean ± S.E.M.) (125-I) EGF to LPM in the mother (n=5) and the fetus at term (n=7) was 1.08 ± 0.08 and 2.25 ± 0.12 per 175 μg of LPM protein respectively. The number of receptor sites per mg of LPM protein in the mother were significantly less than that in the fetus (44 ± 11 and 250 ± 24 × 10^?10, p < 0.001) with no apparent differences in Kd (2.10 ± 0.39 and 2.47 ± 0.24 × 10⁹). Presence of high affinity receptors for EGF in fetal and maternal lung plasma membranes suggests a direct role of EGF in fetal lung maturation.     

Induction of glucokinase by insulin under the permissive action of dexamethasone in primary rat hepatocyte cultures.

Glucokinase, the organ specific key enzyme of glucose metabolism in liver, was studied in primary cultures of adult rat hepatocytes during the first two days after cell preparation. In the presence of dexamethasone low concentrations of insulin (10^?9 mol/l) prevented the observed time dependent decrease of glucokinase activity while higher insulin concentrations (10^?8 and 10^?7 mol/l) led to a twofold increase of enzyme activity. The enhancement of glucokinase activity was completely blocked by either actinomycin D or cycloheximide. The degree of this insulin dependent induction was correlated with the concentration of added dexamethasone, which seemed to perform a permissive function. The induction of glucokinase activity could be prevented by addition of glucagon (2 × 10^?7 mol/l).     

Abundance and Diversity of Sulfur-Oxidizing Bacteria along a Salinity Gradient in Four Qinghai-Tibetan Lakes,China

Sulfur-oxidizing bacteria (SOB) play important roles in the sulfur cycle and are widespread in a number of environments, but their occurrence and relationship to geochemical conditions in (hyper)saline lakes are still poorly understood. In this study, the abundance and diversity of SOB populations were investigated in four Qinghai-Tibetan lakes (Erhai Lake, Gahai Lake 1, Gahai Lake 2 and Xiaochaidan Lake) by using quantitative polymerase chain reaction (qPCR) and soxB gene- (encoding sulfate thiohydrolase) based phylogenectic analyses. qPCR analyses showed that in the studied lakes, the total bacterial 16S rRNA and soxB gene abundances in the sediments were distinctly higher than in the overlying waters. The 16S rRNA gene abundance in the waters ranged 5.27 × 10⁶–6.09 × 10⁸ copies per mL and 7.39 × 10¹⁰–2.9 × 10¹¹ copies per gram sediment. The soxB gene abundance in the waters ranged from 1.88 × 10⁴ to 5.21 × 10⁵ per mL and 4.73 × 10⁶–2.65 × 10⁷ copies per gram sediment. The soxB gene in the waters of the two hypersaline lakes (Gahai Lake 2 and Xiaochaidan Lake) was more abundant (2.97 × 10⁵ and 5.21 × 10⁵ copies per mL) than that in the two low-salinity lakes (1.88 × 10⁴ and 3.36 × 10⁴ copies per mL). Phylogenetic analysis showed that Alpha- and Betaproteobacteria were dominant SOB in the investigated lakes, and the composition of proteobacterial subgroups varied with salinity: in freshwater Erhai Lake and low-salinity Gahai Lake 1, the SOB populations were dominated by the Betaproteobacteria, whereas in hypersaline Lake Gahai 2 and Xiaochaidan Lake, the SOB populations were dominated by Alphaproteobacteria. Overall, salinity played a key role in controlling the diversity and distribution of SOB populations in the investigated Qinghai-Tibetan lakes.     

Distribution,sources, and health risk assessment of polycyclic aromatic hydrocarbons in dust from urban environment in the Niger Delta,Nigeria

The concentrations of the 16 U.S. Environmental Protection Agency polycyclic aromatic hydrocarbons (PAHs) were measured in urban street dust with a view to understanding the potential risk to urban residents exposed to street dust, sources, and distribution of the PAHs. Gas chromatography–mass spectrometry was used to measure the concentrations of the PAHs after ultra-sonication with hexane/dichloromethane and clean up. The total concentrations of the ?16 PAHs in the urban street dust from Warri and its environs ranged from 165.1 to 1012 µg kg^?1. The isomeric ratios and the PCA indicated that combustion of petroleum and biomass are the major sources of PAHs in Warri and its environs. The total BaP carcinogenic and mutagenic potency concentrations (?BaP_TEQ and ?BaP_MEQ) were in the range of 0.03 to 219 µg kg^?1 and 0.52 to 182 µg kg^?1, respectively. The incremental life cancer risk from exposure of the residents of Warri and its environs ranged from 4.07 × 10^?7 to 3.11 × 10^?3 and 2.13 × 10^?7 to 1.49 × 10^?3 for the children and adults, respectively, which were higher than the baseline value of acceptable risk of 10^?6 (i.e., one case per million people).     

UV-induced mutagenesis at the hypoxanthine—guanine phosphoribosyl transferase locus in two L5178Y mouse lymphoma cell strains with different UV sensitivities

Two strains of L5178Y mouse lymphoma cells, L5178Y-R (LY-R) and L5178Y-S (LY-S), differ markedly in their sensitivity to 254 nm UV radiation (D₀ = 0.7 and 5.5 J/m²; n = 6.0 and 2.0 for LY-R and LY-S cells, respctively). In this study, the frequency o hypoxanthine-guanine-phosporibosyl-transferase-deficient mutants was determined, using 6-thioguanine (TG) as a selective agent, in populations of LY-R and LY-S cells exposed to various fluences of UV radiation. The spontaneous mutation frequency for LY-R cells was (3.7 ± 0.6) × 10^?5 TG^r mutants per viable cell, and the UV induction rate was (2.2 ± 0.8) × 10^?4 TG^r mutants per viable cell, per J/m². Both spontaneous and induced mutantion frequencies were much lower for LY-S cells. The sopntaneous mutation frequency for these cells were too low to make its measurement practicable ( < 0.0013 × 10^?5 TG^r mutants per viable cell). Mutation induction rate was (4.2 ± 2.2) × 10^?7 TG^r mutants per viable cell, per J/m². These differences in mutability do not appear to be due to gene duplication in LY-S cells, or to selective growth disadvantage of LY-S-derived TG-resistant mutants. Possible mechanisms underlying the differences in mutability of LY-R and LY-S cells are considered.