Characterization of the Ah receptor-associated protein, ARA9.

The unliganded aryl hydrocarbon receptor (AHR) is found in a complex with other proteins including the 90-kDa heat shock protein (Hsp90) and a 37-kDa protein we refer to as ARA9. We found that the three tetratricopeptide repeats found in the COOH terminus of ARA9 are necessary and sufficient for interaction with the AHR complex. Conversely, the AHR's "repressor"/Hsp90 binding domain is required for interaction with ARA9. Because ARA9 closely resembles the 52-kDa FK506-binding protein (FKBP52), found in the unliganded glucocorticoid receptor (GR) complex, we compared the binding specificities of ARA9 and FKBP52 for AHR and GR. In co-immunoprecipitation experiments, ARA9 specifically associated with AHR-Hsp90 complex but not with GR-Hsp90 complexes. In addition, ARA9 showed a greater capacity than FKBP52 to associate with AHR-Hsp90 complexes. The biological importance of this interaction was suggested by the observation that in a yeast expression system ARA9 expression enhanced the response of AHR to the agonist beta-napthoflavone, decreasing the EC50 by greater than 5-fold and increasing the maximal response 2.5-fold. In contrast, co-expression of FKBP52 had no effect on AHR signaling. In addition, although ARA9 contains a domain similar to that found in other FK506-binding proteins, ARA9 binding to 3H-FK506 could not be detected. Finally, we have characterized the developmental and expression pattern of ARA9 in the developing mouse embryo and mapped the ARA9 locus to human chromosome 11q13.3.

Run-on gene transcription in human neocortical nuclei. Inhibition by nanomolar aluminum and implications for neurodegenerative disease.

The incorporation of [alpha-32P]-uridine triphosphate into DNA transcription products was examined in short post-mortem interval (PMI) human brain neocortical nuclei (n, 22; PMI, 0.5-24 h) using run-on-gene transcription. Reverse Northern dot-blot hybridization of newly synthesized RNA against either total cDNA or Alu repetitive DNA indicated that human brain neocortical nuclei of up to 4-h PMI were efficient in incorporating radiolabel into new transcription products, after which there was a graded decline in de novo RNA biosynthetic capacity. To test the effects of 0-3000 nM concentrations of ambient aluminum on RNA polymerase I (RNAP I) and RNA polymerase II (RNAP II) transcription, dot blots containing 0.5, 1.0, 2.0, and 5.0 micrograms of DNA for (1) the human-specific Alu repetitive element (2) the neurofilament light (NFL) chain, and (3) glial fibrillary acidic protein (GFAP) were Northern hybridized against newly synthesized radiolabeled total RNA. These DNAs represent heterogeneous nuclear RNA (hnRNA), neuronal-, and glial-specific markers, respectively. We report here a dose-dependent repression in the biosynthetic capabilities of brain RNAP II in the range of 50-100 nM aluminum, deficits similar to those previously described using a rabbit neocortical nuclei transcription system and at concentrations that have been reported in Alzheimer's disease (AD) euchromatin. Transcription from RNAP II and the neuron-specific NFL gene in the presence of aluminum was found to be particularly affected. These findings support the hypothesis that brain gene transcription in the presence of trace amounts of ambient aluminum impairs mammalian brain DNA to adequately read out genetic information.

Three-dimensional mapping of norepinephrine and serotonin in human thalamus.

Detailed quantitative information on catecholamines and 5-hydroxytryptamine (serotonin) in the human thalamus is much needed because of increasing interest in norepinephrine and serotonin as modulators of thalamic behavioral state control and overall information processing. This study provides three-dimensional distribution patterns of these monoamines in postmortem thalami from 13 normal subjects (no known neurological or psychiatric histories). The patterns come from a relatively fine-grained grid mapping procedure on successive coronal sections. Samples were analyzed by high performance liquid chromatography with electrochemical detection. The highest endogenous concentrations of norepinephrine are found in a ventromedial core that includes a number of the medial and intralaminar sub-nuclei but extends only slightly into the sensory regions of the lateral tier. The posterior portion of the thalamus, the pulvinar, contains low levels of norepinephrine. The distribution of 5-hydroxytryptamine is quite similar to that of norepinephrine in the rostral two-thirds of thalamus; however, in the pulvinar region, levels of serotonin are considerably increased and differ markedly between individual thalami. The study provides the first definitive mapping of serotonin levels in human thalamus. Consistent with many animal studies, there is no evidence for major dopaminergic innervation of human thalamus. By emphasizing the pattern distribution of the monoamines rather than the absolute values, it can be shown that the ambiguities of postmortem degradation frequently associated with biochemical assays are largely avoided. The terminal field distribution of norepinephrine is an essentially constant neurochemical signature in all thalami examined. The utility of the biochemical grid mapping procedure may be especially significant in terms of matching with data from functional neuroimaging techniques.

Ligand-dependent interaction of the aryl hydrocarbon receptor with a novel immunophilin homolog in vivo.

In an effort to identify regulators of aryl hydrocarbon receptor (AHR) signaling, we have employed the yeast two-hybrid system to screen for human proteins that interact in a ligand-dependent manner with the AHR. After screening 1.4 x 10(6) clones from a human B cell library, two distinct clones were identified that associated specifically with the liganded receptor. No clones were identified that interacted preferentially with the unliganded AHR. One of the ligand-dependent clones, ARA9, encodes a novel 330-amino acid protein with regions of amino acid sequence similarity to the 52-kDa FK506-binding protein known to be associated with the glucocorticoid receptor. Yeast two-hybrid experiments with ARA9 demonstrated a strong interaction with the AHR that is enhanced 11-fold in the presence of the ligand beta-naphthoflavone. In vitro experiments using proteins generated in reticulocyte lysates confirmed this interaction and indicated that ARA9 can be co-immunoprecipitated with the AHR using antisera raised specifically for either the AHR or the 90-kDa heat shock protein. The observation that ARA9 has a high affinity for both the 90-kDa heat shock protein-associated and ligand-activated forms of the AHR suggests that ARA9 is a component of the AHR-signaling pathway in vivo.

The 90-kDa heat shock protein is essential for Ah receptor signaling in a yeast expression system.

In an effort to provide a more powerful system to study the Ah receptor (AHR) signaling pathway, we expressed the AHR, its dimerization partner ARNT, and a beta-galactosidase (lacZ) reporter gene, driven by two dioxin-responsive enhancers, in the yeast Saccharomyces cerevisiae. In this system, the agonists beta-naphthoflavone and alpha-naphthoflavone induced transcription of the lacZ gene, with EC50 values of 7.9 x 10(-8) and 3.0 x 10(-7) M, respectively, while the nonagonist dexamethasone was without effect. As a first application of this system, we examined the relationship between the 90-kDa heat shock protein (hsp90) and AHR function. To accomplish this in a manner that was independent of the ARNT protein, we constructed a chimeric receptor in which the DNA binding and primary dimerization domains of the AHR were swapped with analogous domains from the LexA protein. Coexpression of this AHR-LexA chimera and a lacZ reporter gene driven by eight LexA operator sites in a yeast strain with regulatable levels of hsp90, yielded pharmacology that closely mirrored that of the AHR/ARNT/dioxin-responsive enhancer system described above, but only when hsp90 levels were held near their wild type levels. When hsp90 levels were reduced to approximately 5% of normal, AHR signaling in response to agonist was completely blocked despite normal cell growth. These results provide the first genetic evidence for the role of hsp90 in AHR signaling and provide the basis for a powerful new system in which to study this pathway.

Tissue specific expression of the rat Ah-receptor and ARNT mRNAs.

The Ah-receptor (AHR) is a ligand activated transcription factor that mediates the biological effects of agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Upon binding agonists, the AHR dimerizes with a structurally related protein known as ARNT and this heterodimer then binds cognate enhancer elements and activates the expression of target genes. In this report we describe the cloning of the rat AHR cDNA and a fragment of the rat ARNT cDNA for use as probes in ribonuclease protection analysis. Ribonuclease protection analysis indicated that the rat AHR mRNA is expressed at the highest levels in the lung > thymus > kidney > liver while lower levels were expressed in heart and spleen. The rat AHR and ARNT mRNAs were expressed in a largely coordinate manner across the eight tissues examined with the exception of the placenta where AHR levels were relatively low compared to ARNT. In these experiments, a rare splice variant of the AHR was cloned that encoded a protein with a deletion in the ligand binding domain. In vitro expression studies demonstrated that in contrast to the full length AHR, the splice variant did not bind ligand nor did it bind to a cognate enhancer element in the presence of ARNT.

Epstein-Barr virus infection in Desert Storm reservists.

Approximately 150 U.S. Army reservists from Indiana reported symptoms consistent with chronic fatigue syndrome after returning stateside from the tour of duty in Saudi Arabia. A psychiatric team confirmed the diagnosis, evaluated possible etiology, and treated the service members when appropriate. Those available service members who met the study's diagnostic criteria for chronic fatigue syndrome (n = 37) received an Epstein-Barr virus panel. Seventy-three percent of these selected service members were positive either for an acute or reactivated Epstein-Barr viral infection. These data suggest that service members who suffer from chronic fatigue syndrome may have their symptoms increased and prolonged by secondary viral infections.

Chromosomal localization of the human AHR locus encoding the structural gene for the Ah receptor to 7p21-->p15.

The AHR locus encodes the structural gene for the Ah receptor, a ligand activated transcription factor that regulates the expression of a number of enzymes involved in the metabolism of chemical carcinogens and that appears to mediate the tumor promoting properties of compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using polymerase chain reaction (PCR), we amplified exon-10 and intron-D of the AHR gene from human genomic DNA. By using PCR analysis of somatic cell hybrids and fluorescence in situ hybridization of metaphase cells, we localized AHR to human chromosome 7, bands p21-->p15. This mapping data should prove useful in determining the role that the AHR locus plays in human cancer incidence and in the identification of human populations with altered susceptibilities to the toxic/carcinogenic effects of planar aromatic hydrocarbons.

Cloning and expression of a human Ah receptor cDNA.

In this report, we describe the cloning and expression of a cDNA encoding a human Ah receptor, a basic/helix-loop-helix protein that mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin. A comparison of this human cDNA with a murine homologue (Ahb1 allele) indicates that the molecular mass variation observed between the receptors found in these two species results from hypervariability of amino acid sequences in the carboxyl termini (< 60% conserved over 450 amino acids). Differential usage of stop codons generates proteins with molecular masses that differ by 6 kDa. In contrast, the amino-terminal halves of these proteins are highly conserved and show 90% amino acid sequence identity. Northern blot analysis indicates that the human Ah receptor mRNA is expressed at its highest levels in placenta and is also highly expressed in lung, heart, pancreas, and liver, with lower levels of expression found in brain, kidney, and skeletal muscle. Expression of the human cDNA in a rabbit reticulocyte lysate system allowed functional analysis of ligand binding, agonist-induced and Ah receptor nuclear translocator-dependent DNA binding, and receptor stabilization by sodium molybdate.

Molecular characterization of the murine Ahr gene. Organization, promoter analysis, and chromosomal assignment.

The AH receptor is a ligand-activated transcription factor that mediates the biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin. The AH receptor has primary sequence homology to its dimerization partner the AH receptor nuclear translocator, and to the Drosophila proteins Sim and Per. Characterization of the gene encoding the murine AH receptor (Ahr gene) reveals that its structural organization is also conserved with respect to the sim gene, since 6 of 11 Ahr exons are spliced at homologous sites. Interestingly, little splicing homology was observed between the Ahr and per genes. The promoter of the Ahr gene is GC-rich and contains no TATA or CCAAT boxes; however, sequence analysis has shown several binding sites for the transcription factor Sp1 (GC boxes). Additionally we have identified a potential cAMP response element, AP-1 and E box sites, and two elements demonstrated in other genes to confer placenta-specific expression. Using a restriction fragment length polymorphism in exon 7 and recombinant inbred mouse lines, the Ahr gene was found to be concordant with the phenotypically defined Ahr locus, supporting the identity of these two genetic elements.

Isolation and characterization of doxorubicin-resistant Lewis lung carcinoma variants.

A series of drug-resistant variants of the murine Lewis lung carcinoma (3LL-CK) cell line has been isolated using stepwise selection in increasing concentrations of doxorubicin. These variants exhibited classical multidrug resistance as evidenced by decreased doxorubicin accumulation, cross-resistance to vinblastine, reversibility of resistance by verapamil, and overexpression of P-glycoprotein. When the doxorubicin-resistant 3LL-CK cell populations were injected into the tail veins of B6D2F1 mice, their metastatic abilities were consistently reduced compared with that of the parental line.

Antihypertensive drugs: their postural hypotensive effect and their blood pressure lowering activity in conscious normotensive rats.

The postural hypotensive (PH) response to various antihypertensive agents was examined as an independent pharmacologic activity in conscious normotensive rats. Full PH dose-response curves for standard antihypertensive drugs were explored and were compared to their hypotensive dose-response curves. In untreated control rats, only a negligible change in blood pressure occurred in response to a 2 min 90 degrees head-up tilt. However, treatment with an agent acting on the nervous system, pentolinium, resulted in a profound and dose-dependent drop of blood pressure in response to tilt. A comparable hypotensive effect was also induced. Alpha blockers (e.g., prazosin) induced only moderate dose-dependent PH effects while producing profound hypotensive effects. Direct vasodilators (e.g., minoxidil), calcium antagonists (e.g., nifedipine) and a converting enzyme inhibitor, captopril, were virtually free of PH effects despite moderate or profound hypotensive effects. Clonidine exhibited greater PH than hypotensive effects. Propranolol and hydrochlorothiazide did not exhibit PH effects and did not lower blood pressure. With the exception of clonidine, these findings are generally in agreement with human data. It is suggested that the normotensive rat may be a useful model for gaining an insight into a drug's potential for producing postural hypotension in man and that the full PH dose-response curve should be explored.