Ah receptor for 2,3,7,8- tetrachlorodibenzo-p-Dioxin: Ontogeny in chick embryo liver

Aryl hydrocarbon hydroxylase (AHH, cytochrome P₁-450) is induced in chick liver very early during embryonic development if embryos are treated with 3-methylcholanthrene–type compounds such as 3,4,3′4′-tetrachlorobiphenyl. In mammals, AHH induction is known to be mediated by the Ah receptor. Liver from embryonic and newly hatched chicks was found to contain a cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) which has properties that are very similar to properties of the Ah receptor previously characterized in mammalian tissues. In chick embryo liver, cytosolic binding sites for TCDD were of high affinity (K_d for [3-H]-TCDD = 0.2 nM) and were specific for 3-methylcholanthrene–type inducers. The specific binding component sedimented at about 9S on sucrose density gradients prepared at low ionic strength. A high level of Ah receptor was detected in chick embryo liver by the fifth day of incubation (5 DI); this is at least 24 hours prior to the onset of AHH inducibility. The Ah receptor concentration increased from 5 DI to 8 DI, the period when chick liver is undergoing early morphological differentiation. After 8 DI, Ah receptor levels dropped substantially and remained low into the posthatching period. In contrast, AHH inducibility was high by 7 DI and remained high throughout embryonic development and into the posthatching period. The discrepancy between Ah receptor levels and the degree of AHH inducibility suggests that only a small fraction of the Ah receptor population is required for maximal AHH induction.     

2,3,7,8-tetrachlorodibenzo-p- dioxin reduces high-affinity binding of epidermal growth factor to cell surface receptors in C3H 10T1/2 cells

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on epidermal growth factor (EGF)-binding characteristics was studied in a cultured embryonic fibroblast cell line, C3H 10T1/2. At very low concentrations, TCDD was found to cause a persistent decline in EFG binding, the median effective concentration (EC-50) being 10^?12M. This particular effect was most conspicuous when TCDD was added at the time of medium change with fresh Dulbecco's modified Eagle's medium. Cells at an early stage of confluency were more responsive to TCDD than those at a later stage. Although most reported TCDD-evoked biological changes are recognized to occur slowly during the course of a few days to weeks, the response of C3H 10T1/2 cells to TCDD was swift, showing a sign of decline of EGF binding as early as three hours after TCDD addition. C3H 10T1/2 cells appear to be an excellent in vitro model to study TCDD's biochemical action mechanisms.     

Protamine: a unique and potent inhibitor of oligopeptidase B.

Oligopeptidase B is a serine endopeptidase found in prokaryotes, unicellular eukaryotes and higher plants. The enzyme has been shown recently to play a central role in the pathogenesis of several parasitic diseases such as African trypanosomiasis, and to be a potential therapeutic target. This study reports that protamine, a basic peptide rich in arginine, is a potent inhibitor at the nanomolar level of oligopeptidase B from E. coli and wheat. Protamines 1B, 2C, 3A and TP17 displayed similar inhibitory activities and were capable of binding strongly to oligopeptidase B without proteolytic cleavage. The concentration of protamine needed for 50% inhibition (IC50) of oligopeptidase B was 10(4)-fold lower than the IC50 of trypsin. Oligopeptidase B was highly sensitive to inhibition by protamines even in the presence of serum (IC50, 1 microM). These data indicate that protamines might provide information useful for the design of more specific synthetic oligopeptidase B inhibitors.     

Heterogeneity of rat hepatic Ah Receptor: Identification of two receptor forms which differ in their biochemical properties

In cytosol, the rat hepatic Ah receptor (AhR) appears to exist in two distinct forms (AhR^α, AhR^β) in similar concentration. The binding of ligand (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)) to AhR^α requires the receptor be in its oligomeric 8–10 to S conformation (bound to other protein subunits), while ligand binding to AhR^β can occur with the dissociated 5–6 S form. Occupancy of AhR^β by ligand (TCDD) protects it from salt-dependent inactivation; AhR^β is not inactivated by high salt conditions. The addition of molybdate to cytosol during tissue homogenization stabilized AhR^α against salt-dependent inactivation and subunit dissociation but did not prevent dissociation of AhR^β by high salt. Although the presence of molybdate appears to stabilize AhR^α in its oligomeric 8–10 S, it had no significant effect on the overall amount of TCDD:AhR complex which bound to its specific DNA recognition site, the dioxin responsive element (DRE). These results suggest that AhR^α, unlike AhR^β, is either unable to transform or bind to the DRE with high affinity.     

Ah receptor involvement in mediation of pyruvate carboxylase levels and activity in mice given 2,3,7,8-tetrachlorodibenzo-p-dioxin

The arylhydrocarbon receptor (AhR) plays a central role in mediating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity in animals. The investigations described here provide evidence that support a role for the AhR in TCDD-mediated pyruvate carboxylase (PC) level/activity reductions in mice. Pyruvate carboxylase plays a pivotal role in gluconeogenesis and in supplying carbon units for the citric acid cycle. Delivered ip in a corn oil carrier, TCDD suppresses PC activity/amount at doses as low as 1 μg/kg in responsive C57BL/6J(Ah^b/b) mice. Corn oil alone injected ip into mice at 4 mL/kg appears to be an inducer that increases the amount and activity of PC. However, TCDD suppresses this induction. In the Ah^b/b mouse, PC levels and activity are reduced to 10% of control values at a dose of 75 μg/kg. A time-course experiment shows that the PC reductions are apparent within 16 hours post-TCDD exposure. Here we report investigations on the PC/TCDD response using a congenic C57BL/6J(Ah^d/d) mouse strain having an AhR with a low affinity for TCDD. If the PC/TCDD response is AhR mediated, the congenic mouse strain (Ah^d/d) would require much higher doses of TCDD to suppress PC. In the Ah^d/d mice, we observe that an approximately 60-fold increase in TCDD dose is necessary to produce a PC/TCDD effect. We also find that in Ah^d/d mice, corn oil does not induce an increase in PC activity/amounts, as reported for Ah^b/b mice.     

Evaluation of Three Measures of Exposure Concentration: A Case Study of Surface Sediment Concentrations in the Passaic River

Arithmetic and lognormal means historically have been used as estimates of exposure concentration for both human health and ecological risk assessment. Past risk assessment guidance has emphasized the need to include all available informa tion, particularly regarding the spatial and temporal aspects of data and exposure. One estimate of exposure concentration that includes spatial information is the 95% upper confidence limit (UCL) of the area weighted mean. An area weighted mean is calculated using weightings based on the ratio of the area associated with a sample location and the total area of the study area. Currently, U.S. Environmental Protection Agency (USEPA) risk assessment guidance recommends the use of the 95% UCL of the arithmetic mean as the most appropriate measure of exposure concentration for soil and sediment in human health and ecological risk assess ments. The purpose of this study was two fold: (1) to evaluate the use of the 95% UCL of the area weighted mean vs. the 95% UCL of the arithmetic mean for the lognormal distribution and the normal distribution using a large data set from a recent riverine sediment characterization study, and (2) to evaluate the potential associated with the different approaches for reduction in surface sediment exposure concentrations for 2,3,7,8 TCDD and coplanar polychlorinated biphenyls (PCBs) for several subsets of the data. The results of this evaluation indicated that there was no significant reduction in exposure concentrations of the study area surface sediments after any of the selected higher concentration data points had been removed from the various data subsets. In addition, this evaluation indicated that, due to its dependence on the variance of the data, the 95% UCL of the arithmetic mean for a lognormal distribution produced subset exposure concentrations that were higher than the calculated concentrations of the full data set and produced the smallest percent changes in concentration compared to the other two measures. Based on this analysis, the 95% UCL of the area weighted mean was determined to be the most stable and appropriate estimator of exposure concentration for surface sediments.     

12-O-tetradecanoylphorbol-13-acetate upregulates the Ah receptor and differentially alters CYP1B1 and CYP1A1 expression in MCF-7 breast cancer cells

Elevated expression of cytochrome P450 1B1 (CYP1B1) and estradiol 4-hydroxylation have been reported to be biomarkers of tumorigenesis in humans. The aromatic hydrocarbon receptor (AhR) regulates expression of human cytochrome P450 1A1 (CYP1A1) and CYP1B1, 17β-estradiol (E₂) 2- and 4-hydroxylases, respectively. There is also evidence that expression of estrogen receptor α (ERα) potentiates CYP1A1 inducibility in breast cancer cells. To characterize these relationships further, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), which downregulates ERα, and the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the expression of AhR, ERα, CYP1A1, and CYP1B1 in MCF-7 human breast cancer cells. Treatment with TPA, which suppressed ERα mRNA levels, caused a greater than fourfold elevation of AhR mRNA and protein levels, whereas treatment with TCDD caused a decrease in AhR protein but no change in ERα or AhR mRNA levels. In MCF-7 cells treated with TPA prior to treatment with TCDD, the AhR mRNA level was elevated, the ERα mRNA level remained suppressed, and the ratio of CYP1B1 to CYP1A1 mRNA was increased compared with treatment with TCDD alone. A corresponding increase in the ratio of the rates of 4- to 2-hydroxylation pathways of E₂ metabolism was also observed in response to pretreatment with TPA prior to the addition of TCDD. These results demonstrate differential regulation of the human CYP1A1 and CYP1B1 genes and provide a cellular model to investigate further the mechanisms that may be involved in the elevated expression of CYP1B1 in tumorigenesis. J. Cell. Biochem. 70:289–296, 1998. © 1998 Wiley-Liss, Inc.     

精核蛋白对小鼠1-细胞期受精卵转录的影响

应用一种非常敏感的、新的荧光方法 ,进行了精核蛋白对小鼠 1 细胞期受精卵转录影响的观察 .hCG注射后 18h的受精卵作为精核蛋白抗体显微注射对象 .镜下观察 ,非抗体注射与抗体注射的卵细胞荧光强度有明显差异 .根据荧光分光光度计测得的结果 ,抗体注射卵细胞的平均荧光强度值相当于抗体非注射的 2 2 8%,高 1 3倍 ,经t检验 (n =30 )得P <0 0 1,差别有显著性意义 .而非BSA注射和BSA注射卵细胞的镜下观察 ,则没有多大差异 ;测两组卵细胞的相对荧光强度值分别为 10 0 %和 115 %,t检验 (n =30 )得p >0 0 5 ,差别无统计学意义 .将α 鹅膏蕈碱与精核蛋白抗体等量混合后一起注射 ,卵细胞组间荧光的显著性差别不复存在 .实验结果表明 ,精核蛋白在小鼠 1 细胞期受精卵中起着转录抑制作用 .     

Ammonium sulfate precipitation of the glucocorticoid receptor from rat liver

The transformed glucocorticoid receptor (GR) from rat liver precipitated at 30% saturation of ammonium sulfate and sedimented at 4.3 S on glycerol gradient centrifugation, whereas the nontransformed GR precipitated at higher concentrations of ammonium sulfate (40-50% saturation) and sedimented at 8.6 S on a gradient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that heat shock protein 90 (hsp 90) precipitated at 40-50% saturation of ammonium sulfate. Moreover, hsp 90 and the nontransformed GR were eluted from DEAE high performance ion-exchange chromatography at similar salt concentrations (0.22-0.23 M NaCl), whereas the transformed GR was eluted at 0.1 M NaCl. Therefore, hsp 90 seems to be responsible for the surface charge characteristics of the nontransformed GR.     

Protamine immobilization and heparin adsorption on the protamine-bound cellulose fiber membrane

Immobilization of protamine to the inner lumen of cellulose hollow fibers has been shown useful in preventing both heparin- and protamine-induced complications during an extracorporeal blood circulation procedure. The current study examined the effects of variables on the immobilization of protamine to cyanogen bromide (CNBr)-activated cellulose hollow fibers. The degree of protamine immobilization was controlled by three independent parameters: the amount of CNBr used during the activation process, the duration of the coupling process, and the protamine concentration in the coupling solution. By the adjustment of these parameters, cellulose fibers containing desired amounts of immobilized protamine (ranging from 1 to 20 mg of immobilized protamine per gram of dry fibers) were readily prepared.Heparin adsorption to the protamine-bound cellulose fibers was also examined. The adsorption isotherm followed a Langmuir adsorption model. The amount of heparin adsorbed was dependent on both the heparin concentration in the substrate solution and the protamine loading on the fibers. The Langmuir adsorption constant K was estimated to be 0.37 +/- 0.06 mL/mg, whereas the saturation capacity Q(s) of the protamine-bound fibers increased with increasing the protamine loading.     

Functional assay for GABA receptor subtypes of a cockroach giant interneuron.

gamma-Aminobutyric acid (GABA) receptors were examined in the cockroach central nervous system (CNS) using the single fiber-oil gap method applied to an identified giant interneuron. Short-lasting pressure application of 10 mM GABA developed a multiphasic response composed of a fast hyperpolarization followed by a transient depolarizing component and a stable hyperpolarization. This triphasic characteristic shape of the response was modified according to the dose of GABA injected or bath-applied and to the precise localization of the injection within the dendritic area. The transient depolarizing phase showed a negative reversal potential of -70 mV. Both hyperpolarizing phases reversed at a more negative level ranging to -80 mV. A positive shift of these values was caused by a decrease in external chloride concentration. Bath-application of 0.1 mM picrotoxin (Ptx) decreased the depolarizing phase which was progressively replaced by a stable hyperpolarization. The transient depolarizing component desensitized quickly and was the most sensitive phase to Ptx action. The Ptx-resistant response reversed at a mean value of -100 mV close to the equilibrium potential for potassium ions (EK+), suggesting that it was generated by a K(+)-channel coupled receptor. Although baclofen was unable to mimic the Ptx-resistant GABA response, the compound CGA 147823, known to bind with a high specificity to vertebrate GABAB receptors, has been successfully used to reproduce the Ptx-resistant GABA response. It is suggested that, in addition to GABA receptors linked to chloride channels, the insect CNS possesses GABA receptors sharing ionic characteristics of GABAB receptors especially those located in the vertebrate CNS, although they are insensitive to baclofen.     

Enzyme-linked immunosorbent assay for the epidermal growth factor receptor

An enzyme-linked immunosorbent assay (ELISA) for the epidermal growth factor (EGF) receptor was developed using three different antibody preparations, one of which is commercially available. Using one of the antisera (986), the assay could detect as few as 200 × 10⁶ receptors. This is equal to 0.332 fmol. This sensitivity means that a minimum of 100 A-431 cells (human carcinoma) or 5,000 normal cells are needed to quantitate the number of EGF receptors. Two of the antisera (986 and 451) recognized EGF receptors from placental tissue. EGF receptors from as little as 667 ng of placental membrane protein were detectable. The assay is highly species specific, with the sensitivity for the EGF receptor from different species dependent on the antiserum used. The commercial antibody, 29.1, had especially strong reactivity against pig and dog EGF receptors. An ELISA using this antibody had the capacity to detect the number of EGF receptors in 10 μg of liver membrane protein. The assay is sensitive to receptor conformation. The binding of antisera 986 and 451 to 1% sodium dodecyl sulfate (SDS)-denatured receptor was reduced. The binding of antibody 29.1 was impaired by the presence of 1% Triton X-100 but not the same levels of Tween-20 or SDS. In addition to being a sensitive technique for the quantitation of the EGF receptor, this assay is very rapid, taking a total of 4 h. The microtiter dish format also allows hundreds of samples to be assayed at once. By using the appropriate antiserum and standards, the EGF receptor can be quantitated in tissues from humans, dogs, pigs, and mice.     

Protamine inhibits platelet derived growth factor receptor activity but not epidermal growth factor activity

Protamine sulfate blocked 125I-PDGF binding to its specific physiological receptor on Swiss mouse 3T3 cells. Reduced 125I-PDGF binding in the presence of protamine sulfate correlated directly with a protamine sulfate dose-dependent decrease in the PDGF-dependent incorporation of [3H]-thymidine into 3T3 cells and a decreased PDGF-stimulated tyrosine-specific protein kinase activity in isolated membrane preparations of 3T3 cells. Protamine sulfate blocked 125I-PDGF binding to simian sarcoma virus transformed cells (SSV-NIH 3T3 and SSV-NP1 cells) and to nontransformed cells in a manner qualitatively identical to unlabelled PDGF. In contrast, protamine sulfate enhanced the specific binding of 125I-EGF by increasing the apparent number of EGF receptors on the cell surface. The increase in 125I-EGF receptor binding was not prevented by cycloheximide nor by actinomycin D. Protamine sulfate did not affect 125I-EGF binding to membranes from 3T3 cells or the EGF-stimulated 3T3 cell membrane tyrosine specific protein kinase activity, suggesting that protamine sulfate may have exposed a population of cryptic EGF receptors otherwise not accessible. Protamine sulfate was fractionated into four active fractions by Sephadex G-50 gel filtration columns; the half maximum inhibition concentration of 125I-PDGF binding to 3T3 cells of protamines I and II (MW approximately 11,000 daltons and 7,000 daltons, respectively) is approximately 0.4 microM. Protamine II (MW approximately 4,800 daltons) was equally active (half maximum inhibition concentration approximately 0.4 microM); protamine IV (MW approximately 3,300 daltons) was substantially less active (half maximum inhibition concentration approximately 2.8 microM). These investigations have extended previous observations that protamine sulfate is a potent inhibitor of PDGF binding and establish that protamine sulfate blocks PDGF binding at the physiological receptor, preventing PDGF initiated biological activities. Protamine sulfate can be used as a reagent to separate the influence of PDGF and EGF on cells with high specificity and has been used to demonstrate that the receptors on simian sarcoma virus transformed 3T3 cells qualitatively respond identically to protamine sulfate as to unlabelled PDGF and are likely identical to those on nontransformed 3T3 cells.     

A high-throughput fluorescent polarization assay for nuclear receptor binding utilizing crude receptor extract.

A homogenous high-throughput assay has been developed to measure the binding between nuclear receptors and test compounds. This assay applies a fluorescence polarization (FP) detection method using human glucocorticoid receptor (GR) as a model system. Crude receptor extract, which requires no additional purification, is used in the assay. The binding conditions (i.e., DMSO tolerance, temperature, stability, and variability) have been investigated and validated. At the optimized conditions, a signal-to-background ratio of 2:1 and a Z'-factor of 0.7 was achieved in a 384-well format. Several known strong and weak GR ligands have been evaluated in this system. Possible interference of fluorescent compounds and methods to identify false positives are also discussed. This FP-based assay system can potentially be used for many soluble nuclear receptors in high-throughput binding assays.     

A new role for the P2X7 receptor: a scavenger receptor for bacteria and apoptotic cells in the absence of serum and extracellular ATP

The P2X7 receptor is widely recognized to mediate the proinflammatory effects of extracellular ATP. However this receptor in the absence of ATP may have a function unrelated to inflammation. Our data show that P2X7 expressed on the surface of monocyte/macrophages or on epithelial HEK-293 cells greatly augments the engulfment of latex beads and live and heat-killed bacteria by effector phagocyte in the absence of ATP and serum. The expression of P2X7 on the effector also confers the ability to phagocytose apoptotic target cells and an accumulation of P2X7 can be seen at the attachment point to the target. Activation of the P2X7 receptor by ATP causes a slow dissociation (over 10–15 min) of nonmuscle myosin from the P2X7 membrane complex and abolishes further P2X7-mediated phagocytosis of these targets. The recent crystal structure of the homologous zebrafish P2X4 receptor shows an exposed “nose” of the ectodomain (residues 115–162) which contains three of the five disulfide bonds conserved in all P2X receptors. Three short biotin-labeled peptides mimicking sequence of this exposed region bound to apoptotic target cells but not to either viable cells or to other target particles. All three peptides contained one or two cysteine residues and their replacement by alanine abolished peptide binding. These data implicate thiol-disulfide exchange reactions in the initial tethering of apoptotic cells to macrophage and establish P2X7 as one of the scavenger receptors involved in the recognition and removal of apoptotic cells in the absence of extracellular ATP and serum.