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Biomedical subjects

P A Harper

Publications and source records attributed to P A Harper.

At least 19 recordsLinked to original sources
PubMed1999

Physicochemical differences in the AH receptors of the most TCDD-susceptible and the most TCDD-resistant rat strains.

Long-Evans rats (strain Turku AB; L-E) are at least 1000-fold more sensitive (LD50 about 10 microg/kg) to the acute lethal effects of 2, 3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) than are Han/Wistar (Kuopio; H/W) rats (LD50 > 9600 microg/kg). The AH receptor (AHR) is believed to mediate the toxic effects of TCDD and related halogenated aromatic hydrocarbons. We compared the AHRs of L-E and H/W rats to determine if there were any structural or functional receptor differences that might be related to the dramatic difference in the sensitivity of these two strains to the lethal effects of TCDD. Cytosols from liver and lung of the sensitive L-E rats contained about twofold higher levels of specific binding sites for [3H]TCDD than occurred in H/W rats; the Kd for binding of [3H]TCDD to AHR in hepatic cytosols was similar between the two strains. Addition of the oxyanions, molybdate or tungstate (20 mM), had little effect upon ligand binding to AHR in hepatic cytosols from L-E rats whereas in cytosols from H/W rats these agents substantially diminished or totally abolished TCDD binding. The AHR in H/W cytosols also lost ligand-binding function when NaCl (20 to 400 mM) was added to the buffer whereas, in cytosols from L-E rats, the addition of 400 mM NaCl caused the receptor complex to shift from 9S to 6S during velocity sedimentation but did not destroy ligand binding function. AHR from hepatic cytosol of both the L-E and H/W rats could be transformed to the DNA-binding state in the presence of TCDD or other dioxin congeners as assessed by gel mobility shift assays. The most dramatic difference in AHR properties between L-E and H/W rats is molecular mass. Immunoblotting of cytosolic proteins revealed that the AHR in L-E rats has an apparent mass of approximately 106 kDa, similar to the mass of the receptor previously reported in several other common laboratory rat strains. In contrast, the mass of the AHR in H/W rats is approximately 98 kDa, significantly smaller than the mass of receptor reported in any other rat strains. F1 offspring of a cross between L-E and H/W rats expressed both the 106- and the 98-kDa protein. There was no apparent difference in the mass of the AHR nuclear translocator protein (ARNT) between the two strains, but the hepatic concentration of ARNT was about three times as high in L-E as in H/W rats. It will be interesting to find out how the altered structure of the AHR in H/W rats is related to their remarkable resistance to the lethal effects of TCDD.

Animals↗
PubMed1999

Comparison of the biological characteristics of two isolates of Neospora caninum.

This study compared the biological and genetic properties of a bovine (NC-SweB1) and a canine (NC-Liverpool) isolate of Neospora caninum. A mouse model for CNS infection demonstrated marked differences in pathogenicity between the isolates. NC-Liverpool induced severe clinical signs of neosporosis in 57/58 mice including discoordinated movement, hindlimb paralysis and coat ruffling with severe weight loss. In contrast NC-SweB1 induced similar but less severe symptoms in a much smaller proportion of mice over the same time-period. Statistically significant differences were observed between the isolates in the response (mean weight loss) of mice through time to the different doses inoculated. Histopathological effects on brain tissue reflected the isolate-based differences described above. NC-Liverpool infection resulted in intense inflammatory infiltrates and highly necrotic lesions whereas NC-SweB1 induced a milder meningoencephalitis. Passage in cell-culture over a period of 14 months did not affect the pathogenicity of NC-Liverpool. Immunoblots showed that antibodies to N. caninum appeared earlier in mice inoculated with NC-Liverpool than with NC-SweB1. Finally, RAPD-PCR analysis of NC-Liverpool DNA generated profiles distinct from that observed with DNA from NC-SweB1 or Toxoplasma gondii. In summary this study provides evidence for significant biological and genetic differences between 2 isolates of N. caninum.

Animals↗
PubMed1998

Regulation of cytochrome P450 enzymes by aryl hydrocarbon receptor in human cells: CYP1A2 expression in the LS180 colon carcinoma cell line after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin or 3-methylcholanthrene.

It has been difficult to study the regulation of cytochrome P4501A2 (CYP1A2) because expression of this enzyme is reported to be limited or absent in cell culture. We found that CYP1A2 can be induced significantly by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (MC), or benz[a]anthracene in the human colon carcinoma cell line LS180. TCDD and MC each caused a dramatic elevation of CYP1A2 mRNA, as assessed by reverse transcription-polymerase chain reaction or by northern blot analysis. TCDD also increased immunoreactive CYP1A2 protein and the activity of phenacetin-O-deethylase, a diagnostic catalytic marker for CYP1A2. The induction of CYP1A2 at all levels (mRNA, protein, catalytic activity) was concentration- and time-dependent: the EC50 for mRNA induction by TCDD = 0.5 nM, and by MC = 1.4 microM. Inducible CYP1A2 mRNA also was detected at lower levels in two other human cell lines, the hepatoma cell line HepG2 and the breast carcinoma cell line MCF-7. CYP1A1 and CYP1B1, additional CYP1 enzymes regulated by the aryl hydrocarbon receptor (AHR), also were inducible by TCDD and MC in LS180 cells; their concentration-dependent induction was highly correlated with induction of CYP1A2 at mRNA, protein, and catalytic levels. CYP1B1 was constitutively expressed and inducible in the LS180, MCF-7, and HepG2 cell lines as well as in the human choriocarcinoma cell line JEG-3 and the squamous cell carcinoma line A431. CYP1A2 was neither constitutively expressed nor inducible in A431 or JEG-3 cells. The expression of mRNAs encoding the regulators of CYP1 enzymes-the AHR and its heterodimerization partner, the ARNT (AH receptor nuclear translocator) protein-was not altered by treatment with TCDD or MC. However, the cytosolic content of AHR protein and ARNT protein was depleted substantially following treatment with TCDD. The LS180 cell line should constitute a good model for further mechanistic studies on AHR-regulated CYP1A2 expression.

Aryl Hydrocarbon Receptor Nuclear Translocator↗
PubMed1998

Comparison of the large subunit ribosomal DNA of Neospora and toxoplasma and development of a new genetic marker for their differentiation based on the D2 domain.

The latest release of the large subunit ribosomal database contains 429 sequences, yet only 10 (six nuclear and four mitochondrial) are derived from parasites of the phylum Apicomplexa. Three of these (all Toxoplasma gondii) were previously contained in the 1994 release of the database. As an initiative towards an understanding of ribosomal gene organization in the Apicomplexa, the primary sequence of the large subunit (LSU) rDNA of Neospora caninum is presented, and compared with a consensus sequence derived for the LSU rDNA of T. gondii. Nucleotide differences observed between these two taxa in the D2 expansion segment (or domain) (also called the C1/C1' region) of the LSU rDNA were incorporated into a primer that forms the basis of a species-specific polymerase chain reaction (PCR) for N. caninum. The D2 domain of the LSU rDNA, therefore, represents a new genetic marker that can be used for the differentiation and identification of Neospora from other cyst-forming coccidia.

Animals↗
PubMed1998

Chondrodysplasia in Australian Dexter cattle.

OBJECTIVE: To describe the occurrence of chondrodysplasia in Australian Dexter cattle. DESIGN: A pathological and genetic case report. PROCEDURE: Congenital lethal chondrodysplasia was studied in two female Dexter foetuses aborted mid to late gestation. Clinicopathological findings including histological changes in limb bones, and analysis of pedigree information were evaluated. RESULTS: Characteristic features of congenital lethal chondrodysplasia (Dexter bulldog) include abortion, disproportionate dwarfism, a short vertebral column, marked micromelia, a relatively large head with retruded muzzle, cleft palate and protruding tongue and a large abdominal hernia. Histological changes in limb bones are consistent with failure of endochondral ossification. Dexter chondrodysplasia is considered to be inherited in an incompletely dominant manner with the homozygous form producing the congenital lethal condition. A preliminary minimum estimate of heterozygote frequency is 19% within the registered Australian Dexter herd, based on analysis of the contribution of three obligate heterozygotes whose semen has been widely used by artificial insemination in Australia. CONCLUSION: Dexter chondrodysplasia is present in Australian cattle and further cases of the homozygous form, congenital lethal chondrodysplasia, are likely to occur. RECOMMENDATION: It is requested that spleen and liver tissue from bulldog foetuses and blood from their parents be collected to assist research into Dexter chondrodysplasia.

Abortion, Veterinary↗
PubMed1998

Point mutation in intron sequence causes altered carboxyl-terminal structure in the aryl hydrocarbon receptor of the most 2,3,7,8-tetrachlorodibenzo-p-dioxin-resistant rat strain.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent dioxin. There are exceptionally wide inter- and intraspecies differences in sensitivity to TCDD toxicity with Han/Wistar (H/W) (Kuopio) rats being the most resistant mammals tested. A peculiar feature of H/W rats is that despite their unresponsiveness to the acute lethality of TCDD, their sensitivity to other biological impacts of TCDD (e.g., CYP1A1 induction) is preserved. The biological effects of TCDD are mediated by the aryl hydrocarbon receptor (AhR). We recently found that the AhR of H/W rats (about 98 kDa) is smaller than the receptor in other rat strains (106 kDa). In the present study, molecular cloning and sequencing of the H/W rat AhR revealed that the reason for its smaller size is a deletion/insertion-type change at the 3' end of exon 10 in the receptor cDNA. This change emanates from a single point mutation at the first nucleotide of intron 10, resulting in altered mRNA splicing. At the protein level, the mutation leads to a total loss of either 43 or 38 amino acids (with altered sequence for the last seven amino acids in the latter case) toward the carboxyl-terminal end in the trans-activation domain of the AhR. H/W rats also harbor a point mutation in exon 10 that will cause a Val-to-Ala substitution in codon 497, but this occurs in a variable region of the AhR. These findings suggest that there is a relatively small region in the AhR trans-activation domain that may be capable of providing selectivity to its function.

Animals↗
PubMed1996

Inherited disorders: the comparative picture.

When confronted with a novel familial disorder, veterinarians should consult McKusick's catalogue of inherited disorders in humans, called Mendelian Inheritance in Man (MIM), or its online version (OMIM), to see whether a similar disorder has been reported in humans. They should also consult the other readily available sources of comparative information on mice and domesticated species. Increasingly, such consultations can be conducted on the Internet via the World Wide Web. If it is thought that an animal disorder is homologous with a human disorder, publications describing the animal disorder should include the MIM number(s) for that disorder. Future research can then test the hypothesis of homology, until a consensus is reached.

Animals↗
PubMed1995

Characterization of polyclonal antibodies to the aromatic hydrocarbon receptor.

The aromatic hydrocarbon receptor (AHR) is a soluble intracellular protein that mediates most, if not all, the toxic effects of polycyclic aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene. Initial binding of specific AHR ligands occurs in the cytoplasm; after a "transformation" step the ligand.receptor complex translocates to the cell nucleus and binds to specific DNA sequences, which act as transcriptional enhancers. We used a synthetic peptide--KLH conjugate corresponding to a 20 amino acid sequence at the N-terminal of the AHR to generate rabbit polyclonal anti-AHR antibodies. The antiserum was affinity purified, using the synthetic peptide conjugated to ovalbumin, and screened by western blot analyses, using [3H]TCDD photoaffinity labeled AHR. Specificity of the antiserum was confirmed by co-migration of photolabeled AHR with the major immunoreactive band identified by western blot. Further characterization showed that the antipeptide antibodies recognized equally both mouse and human AHR, which differ significantly in molecular mass (mouse Hepa-1 cells approximately 95 kDa; human LS180 cells approximately 110 kDa). The affinity-purified antibodies also recognized undenatured TCDD.AHR complexes, as determined by a shift in sedimentation of the [3H]TCDD.AHR complex on a sucrose gradient. The high specificity and sensitivity of this antibody were used to determine the fate of the AHR in cells exposed to [3H]TCDD. Western blot analysis revealed that TCDD exposure caused a dramatic decrease in total cellular AHR to about 20% pre-TCDD levels within 2 h after TCDD, which persisted up to 20 h after initial TCDD exposure. However, in the presence of actinomycin D or cycloheximide, nuclear AHR remained elevated in cells exposed to TCDD, at levels similar to or greater than the maximum previously observed after 1-h incubations. These data suggest that ligand-dependent downregulation of the AHR is the result of protein degradation by a short-lived protease.

Adjuvants, Immunologic↗
PubMed1995

Aromatic hydrocarbon receptor in cultured fetal cells from C57BL/6J and DBA/2J mice: similarity in molecular mass to receptors in adult livers.

In liver of adult responsive C57BL/6J (B6) mice the aromatic hydrocarbon receptor (AHR) has high affinity for specific halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as well as nonhalogenated aromatic hydrocarbons (PAHs), such as benz[a]anthracene (BA) or 3-methylcholanthrene (MC). In livers of adult nonresponsive DBA/2J (D2) mice TCDD binds to a low-affinity variant form of AHR. Both TCDD and MC induce aryl hydrocarbon hydroxylase (AHH) in adult B6 mice, whereas adult D2 mouse liver is nonresponsive to MC. In fetal cell cultures derived from D2 mice AHH is induced by PAHs such as MC or BA, and these PAHs bind to cytosolic AHR (P.A. Harper, C.L. Golas, and A.B. Okey. Mol. Pharmacol. 40: 818-826, 1991). We compared AHR from fetal cell cultures with AHR from adult livers to determine whether there was some structural differences in receptors expressed in fetal cell culture that might permit cells from "nonresponsive" mice to respond to PAHs. The apparent molecular mass of AHR from cells cultured from 18-day fetuses is identical with that from adult liver within each strain of inbred mice tested (M(r) approximately 95 kDa in B6 and approximately 105 kDa in D2 mice). The AHR in D2 fetal cells was able to activate a transfected chloramphenicol acetyltransferase linked to a dioxin-responsive element nucleotide sequence (DRE-CAT) when the cells were treated with TCDD or MC. The potency of CAT expression in D2 fetal cells was similar to that in B6 fetal cells. Our data suggest that the responsiveness of fetal cells from "nonresponsive" mice is likely mediated by AHR in these cells but is not due to expression of a different allelic form of AHR ligand-binding subunit in fetal cells versus adult liver.

Affinity Labels↗
PubMed1994

2,3,7,8-Tetrachlorodibenzo-p-dioxin versus 3-methylcholanthrene: comparative studies of Ah receptor binding, transformation, and induction of CYP1A1.

Halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (MC) cause transcriptional activation of the CYP1A1 gene via their interaction with the aromatic hydrocarbon (Ah) receptor. Direct radioligand binding and competitive binding studies demonstrated that the cytosolic Ah receptor from the mouse hepatoma cell line Hepa-1 bound TCDD with an affinity approximately 3-4-fold greater than that for MC. However, TCDD was approximately 1,000-fold more potent than MC as an inducer of CYP1A1-mediated aryl hydrocarbon hydroxylase activity in cultured Hepa-1 cells as assessed at 14 h following exposure to inducer. To understand the basis for this quantitative discrepancy between Ah receptor binding affinity and CYP1A1 induction potency, we systematically compared TCDD and MC for their abilities to activate sequential events in the CYP1A1 induction mechanism that occur subsequent to initial binding to the cytosolic Ah receptor. Using a gel retardation assay, TCDD and MC were shown to be equipotent in causing in vitro transformation of the cytosolic Ah receptor to its DNA-binding form. In addition, the transformed Ah receptor bound to a specific dioxin-responsive enhancer sequence with the same apparent affinity when MC was the ligand as when TCDD was the ligand. At an early time point (i.e. 2 h) in the CYP1A1 induction process, TCDD was only approximately 4-25-fold more potent than MC in stimulating the nuclear uptake of the ligand-Ah receptor complex, and the two ligands displayed a relatively small difference (> or = 10-fold) in CYP1A1 mRNA induction potency. When assessed at 4 h following ligand treatment, TCDD was only approximately 10-fold more potent than MC as an aryl hydrocarbon hydroxylase inducer, suggesting a time-dependent reduction in the potency of MC in intact cells. Exposure of Hepa-1 cells to MC over a 16-h time course resulted in an increased ability of these cells to convert [3H]MC to alkali-extractable metabolites. Our data are consistent with the idea that TCDD and MC display relatively small differences in their intrinsic abilities to activate Ah receptor-mediated events. The reduced biological potency of MC observed in intact cells and whole animals is at least partially due to the more rapid metabolic inactivation of this ligand compared with the poorly metabolized TCDD. By extension, the extraordinary toxicity of TCDD may not be explained solely by its high affinity for the cytosolic Ah receptor.

Animals↗
PubMed1994

Molecular biology of the aromatic hydrocarbon (dioxin) receptor.

The aromatic hydrocarbon (AH) (dioxin) receptor was discovered almost 20 years ago and achieved notoriety as the front-line site of action of highly toxic environmental chemicals such as halogenated dioxins and polychlorinated biphenyls. Increasing evidence suggests that the AH receptor plays a key role in proliferation and differentiation of cells exposed to dioxins and, perhaps, to endogenous ligands. Recent cloning of the AH receptor and its indispensable partner, the AH-receptor-nuclear-translocator protein, has opened new opportunities to determine how the AH receptor functions, how it evolved and what its multiple roles might be in normal physiology as well as in toxicology. This review by Allan Okey, David Riddick and Patricia Harper aims to provide a brief history of AH receptor research and gives a timely summary of what is known and what is not known about the structure and function of this fascinating protein.

Animals↗
PubMed1994

The Ah receptor: mediator of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds.

A considerable body of research over the past fifteen years establishes that in laboratory animals the Ah (aromatic hydrocarbon) receptor (AhR) mediates most, if not all, toxic effects of halogenated aromatic hydrocarbons such as polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and polyhalogenated biphenyls. More recently the AhR has been shown to also exist in a wide variety of human tissues and human cell lines. In general the AhR in humans appears to function very much like the AhR in rodents. However, the affinity with which toxic HAHs such as 2,3,7,8-tetrachlorodibenzo-p-dioxin bind to the AhR from human sources generally is lower than the affinity with which these HAHs bind to the Ah receptors from rodent tissues. This lower affinity may explain, in part, why the human species seems less sensitive than many laboratory animals to the effects of HAHs. The AhR enhances transcription of genes encoding cytochrome P450 enzymes in the CYP1A subfamily, but most of the toxic effects of HAHs do not seem to require P450 induction per se. Recent molecular approaches to the mechanism of HAH toxicity indicate that the AhR also may mediate expression of several other genes, including genes that regulate cell growth and differentiation. Despite the expanding repertoire of cellular responses known to be altered by HAHs (potentially through the AhR) it is not yet clear which AhR-mediated actions are the key events in HAH toxicity. Within the past year two subunits of the AhR have been cloned; this cloning, along with other molecular investigations, should greatly expand our opportunity to understand the specific mechanisms and pathways by which HAHs cause toxicity.

Animals↗
PubMed1993

The mechanism of the verapamil-digoxin interaction in renal tubular cells (LLC-PK1).

Verapamil, usually given as a racemic mixture, decreases in vivo and in vitro digoxin renal tubular secretion, which is suggested to be mediated by P-glycoprotein, an ATP-dependent multidrug efflux pump. Importantly, the two enantiomers of verapamil have been reported to similarly inhibit P-glycoprotein-mediated transport of chemotherapeutic agents. In this study, we examined effects of enantiomers of verapamil on digoxin transport across an LLC-PK1 cell monolayer, a model of proximal renal tubular cells. The results indicate that verapamil inhibition of digoxin transport is non-stereospecific. Furthermore, the verapamil-digoxin interaction is not competitive. The two drugs may not share a common initial step in the P-glycoprotein-mediated transport.

Animals↗
PubMed1993

P-glycoprotein-mediated renal tubular secretion of digoxin: the toxicological significance of the urine-blood barrier model.

We provide direct evidence that verapamil inhibits active digoxin secretion in renal tubular cells (LLC-PK1), and that verapamil increases cellular accumulation of digoxin. These findings suggest that verapamil inhibits the digoxin active secretory transport at the apical membranes, supporting the theory that P-glycoprotein mediates digoxin secretion in the renal tubular cells. Based on existing data on digoxin transport, we present a hypothetical model for the renal handling of digoxin, implying that P-glycoprotein functions as a driving mechanism of a unidirectional "urine-blood" barrier.

ATP Binding Cassette Transporter, Subfamily B, Mem↗