Biomedical subjects

J W Harney

Publications and source records attributed to J W Harney.

15 recordsLinked to original sources

PubMed1992

Substitution of cysteine for selenocysteine in type I iodothyronine deiodinase reduces the catalytic efficiency of the protein but enhances its translation.

Type I iodothyronine 5' deiodinase (5'DI) contains selenocysteine, encoded by a UGA codon, and this amino acid is essential for maximum catalytic efficiency in this enzyme. We recently showed that translation of UGA as selenocysteine in this protein requires a specific sequence of about 250 nucleotides in the 3' untranslated region of the messenger RNA. Translation of a 5'DI cysteine mutant does not require the 3' untranslated region. To examine both the efficiency of UGA codon recognition and the relative catalytic efficiency of selenocysteine vs. cysteine in 5'DI, we used bromoacetyl 125I-T3 labeling to quantitate transiently expressed selenocysteine (wild type) and cysteine containing type I iodothyronine deiodinases in transfected COS-7 and JEG-3 cell lines. Kinetic analyses of the same cell sonicates were performed to determine the apparent maximum velocity and Michaelis-Menten constant values for reverse T3 5' deiodination. COS-7 cells express the cysteine mutant protein at about 20-fold and JEG-3 cells about 400-fold higher levels than the selenoenzyme. However, in both cell types, the apparent catalytic constant values were at least 100-fold higher for the wild-type enzyme, compared with the cysteine mutant. These results indicate that cell lines differ markedly in their capacity to translate UGA-containing messenger RNAs. The much higher catalytic constant values for the selenium-containing enzyme illustrate the biochemical advantage of this element as compared with sulfur in the catalysis of iodothyronine deiodination.

Catalysis↗

PubMed1992

Cloning and in vitro expression of the human selenoprotein, type I iodothyronine deiodinase.

The type I 5' iodothyronine deiodinase (5' DI) catalyzes the deiodination of T4 to the biologically active hormone T3 and accounts for a significant fraction of its production. We have recently cloned the complementary DNA (cDNA) for the rat 5' DI, which contains the rare amino acid selenocysteine, and used this to screen human liver and kidney cDNA libraries to identify a human 5' DI cDNA clone. From these, we constructed a cDNA encoding a functional 5' DI. The 2222 base pair human 5' DI cDNA is approximately 200 nucleotides shorter than the 2.4-kilobase hybridizing band in Northern blots of human liver, kidney, and thyroid, because of missing 5' untranslated sequence and the poly A tail. The deduced amino acid sequence codes for a protein of 28.7 kilodaltons assuming the UGA codon at position 382 encodes selenocysteine, and is highly homologous (88% similarity) to the rat. We transiently expressed the 5' DI in COS-7 cells to establish that it encodes a functional enzyme and to study its kinetics. These show saturable deiodination of rT3 (Ka 0.52 +/- 0.04 mumol/L and Vmax 63.2 +/- 16.4 pmol min-1 mg-1). T4 and gold thioglucose are competitive inhibitors of rT3 deiodination. 6-n-Propylthiouracil (PTU) is an uncompetitive inhibitor (with rT3) and competitive inhibitor (with dithiothreitol) of rT3 deiodination. 6-n-Propylthiouracil inhibits T4 to T3 conversion. Labeling of COS-7 cells transiently transfected with the human 5' DI cDNA with bromoacetyl-125I-T3 demonstrates a 28-kilodalton protein. This indicates that in the human, as well as in the rat messenger RNA, the UGA encodes selenocysteine and translation terminates at the UAA codon at nucleotides 754 to 756. Reverse T3 and gold thioglucose (100 nmol/L) block bromoacetyl-125I-T3 labeling of the transiently expressed human and rat 5' DI proteins. These results demonstrate that the human 5' DI is a selenoprotein, analogous to the rat enzyme. Given the previously demonstrated critical role of the selenium atom in catalyzing deiodination by this protein, we conclude that this trace element is essential for normal thyroid hormone action in man.

Affinity Labels↗

PubMed1992

Differential capacity of wild type promoter elements for binding and trans-activation by retinoic acid and thyroid hormone receptors.

Retinoic acid receptor (RAR) and thyroid hormone receptor (T3R) are structurally similar and can bind as homodimers or T3R-RAR heterodimers to a single synthetic DNA response element. The interaction of these two types of receptors with wild type elements, however, has not been systematically investigated. Promoter elements from genes regulated by retinoic acid (RA) or thyroid hormone (T3) were tested for response to T3 and RA in transient transfections in both JEG and COS cells. The elements were classified as primarily responsive to RA or to T3 or responsive to both ligands. Binding of highly purified RAR alpha and T3R alpha to the various elements was assessed using the gel shift assay. Those elements predominantly responsive to one ligand showed preferential binding to the appropriate receptor. A series of point mutations were introduced into the rat GH T3 response element to further define sequence requirements for response to both RA and T3. Down-mutations in any of the three hexamers (previously demonstrated to be required for full response to T3 and full binding of T3R) also decreased RA induction and RAR binding. However, only one of two sets of up-mutations for T3 response also increased RA induction, demonstrating differences in hexamer preference between RAR and T3R. Variation in spacing of the three hexamers did not influence RA vs. T3 induction or RAR vs. T3R binding according to the predictions of a simple hexamer spacing model. There was a strong correlation between the extent of T3R dimer binding and strength of T3 induction for a subset of elements studied in JEG cells (r = 0.97, P < 0.01) and a weaker but significant correlation in COS cells (r = 0.65, P < 0.05)). In contrast, RAR dimer binding by the wild type elements did not quantitatively correlate with RA induction in either JEG (r = 0.13, P > 0.05) or COS cells (r = 0.21, P > 0.05). These results suggests that RAR interacts with a heterodimer partner(s) which influences binding site specificity, whereas T3R heterodimer partner(s) is less likely to alter binding site recognition. The observed difference in COS and JEG cells as well as the weak T3R binding-function relationship of the malic enzyme element, however, suggest that the influence of T3R heterodimer partner(s) on binding site specificity is likely to vary with cell type and the specific element tested.

Animals↗

PubMed1992

Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements.

Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common feature which defines the capacity of these elements to confer T3 induction.

Animals↗

PubMed1991

Oligomeric binding of T3 receptor is required for maximal T3 response.

Receptors in the thyroid-steroid hormone superfamily bind preferentially as dimers to palindromic response elements containing two hexameric half-sites. The 23-base pair rat growth hormone (rGH) T3 response element (T3RE), however, contains three hexameric binding domains, all of which are required for maximal T3 response. We examined the binding of purified T3 receptor alpha (T3R alpha), overexpressed in Escherichia coli, to wild-type and up and down mutations of the rGH T3RE to evaluate whether transcriptional potency correlates with changes in T3R binding. T3R binds to the rGH T3RE as a monomer, dimer, or higher order oligomer. Cooperative T3R dimer binding was demonstrated to two hexameric domains of the rGH T3RE arranged as either direct or inverted repeats. Decreased binding was seen with point mutations in each domain as well as with mutations which altered hexamer orientation and spacing within the site. These results demonstrate that all three hexamers of the rGH T3RE are involved in binding T3R. Occupancy of all three hexamers by T3R in the gel shift assay was observed with functional up mutations of the T3RE, increasing receptor concentration or addition of nuclear extract. The transcriptional response potencies of T3RE up or down mutants in a transient transfection assay correlated closely with T3R binding. These results confirm our earlier hypothesis that all three hexamers of the rGH T3RE bind T3R in a novel binding arrangement and provide a model for the interaction of T3R and other nuclear proteins with the DNA sequences of thyroid hormone-regulated genes.

Animals↗

PubMed1991

Recognition of UGA as a selenocysteine codon in type I deiodinase requires sequences in the 3' untranslated region.

Selenocysteine is incorporated cotranslationally at UGA codons, normally read as stop codons, in several bacterial proteins and in the mammalian proteins glutathione peroxidase (GPX), selenoprotein P and Type I iodothyronine 5' deiodinase (5'DI). Previous analyses in bacteria have suggested that a stem-loop structure involving the UGA codon and adjacent sequences is necessary and sufficient for selenocysteine incorporation into formate dehydrogenase and glycine reductase. We used the recently cloned 5'DI to investigate selenoprotein synthesis in eukaryotes. We show that successful incorporation of selenocysteine into this enzyme requires a specific 3' untranslated (3'ut) segment of about 200 nucleotides, which is found in both rat and human 5'DI messenger RNAs. These sequences are not required for expression of a cysteine-mutant deiodinase. Although there is little primary sequence similarity between the 3'ut regions of these mRNAs and those encoding GPX, the 3'ut sequences of rat GPX can substitute for the 5'DI sequences in directing selenocysteine insertion. Computer analyses predict similar stem-loop structures in the 3'ut regions of the 5'DI and GPX mRNAs. Limited mutations in these structures reduce or eliminate their capacity to permit 5'DI translation. These results identify a 'selenocysteine-insertion sequence' motif in the 3'ut region of these mRNAs that is essential for successful translation of 5'DI, presumably GPX, and possibly other eukaryotic selenocysteine-containing proteins.

Animals↗

PubMed1991

Selenocysteine confers the biochemical properties characteristic of the type I iodothyronine deiodinase.

The conversion of thyroxine to 3,5,3'-triiodothyronine (T3) is the first step in thyroid hormone action, and the Type I iodothyronine deiodinase supplies most of this extrathyroidal T3 in the rat. We found that the cDNA coding for this enzyme contains an in-frame UGA encoding the rare amino acid selenocysteine. Using site-directed mutagenesis, we have converted selenocysteine to cysteine and expressed the wild-type and cysteine mutant enzymes in JEG-3 cells by transient transfection. The kinetic properties of the transiently expressed wild-type enzyme are nearly identical to those reported for rat liver Type I deiodinase. Substitution of sulfur for selenium causes a 10-fold increase in the Km of the enzyme for the favored substrate 3,3',5'-triiodothyronine (rT3), a 100-fold decrease in the sensitivity of rT3 deiodination to competitive inhibition by gold and a 300-fold increase in the apparent Ki for uncompetitive inhibition by 6-n-propylthiouracil. These results demonstrate that selenium is responsible for the biochemical properties which characterize Type I iodothyronine monodeiodination.

Animals↗

PubMed1991

Effects of varying the position of thyroid hormone response elements within the rat growth hormone promoter: implications for positive and negative regulation by 3,5,3'-triiodothyronine.

The thyroid hormone response element (T3RE) of the rat GH (rGH) promoter is located at -188 to -165 relative to the mRNA start site (TSS). Similar sites have been identified in other genes regulated by T3. We have investigated some of these T3REs in positions within the rGH promoter to assess the relative influences of DNA-binding site and position on positive and negative regulation by T3. Synthetic oligonucleotides were used with sequences from the rGH T3RE and proposed negative T3REs (nT3RE) from the rat and human alpha-subunit and rat beta TSH genes. The nT3REs were placed in the background of the wild-type rGH promoter in two positions, at -55 and down-stream of the TSS, with up- and down-mutations of the rGH T3RE. Rat GH T3RE elements were placed 700 basepairs up-stream of a basal rGH promoter and some also at the -55 and TSS positions. Constructions were tested in a transient transfection assay in rat pituitary tumor cells. Two copies of the rGHPAL (palindromic T3RE) placed 700 basepairs up-stream of the rGH promoter conferred 10-fold T3 induction. In the -55 position, the rGHPAL increased T3 induction compared to that in controls, whereas a fragment from the rat and human alpha-subunit gene in the same position reduced induction. Negative T3REs from rat beta TSH and human alpha-subunit reduced T3 induction 50% when placed at the TSS position of a rGH promoter containing an up-mutant T3RE. The T3REPAL placed at the same site increased T3 induction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Functional characterization of the rat growth hormone promoter elements required for induction by thyroid hormone with and without a co-transfected beta type thyroid hormone receptor.

We have extensively characterized the sequences of the rat growth hormone (rGH) promoter required for induction by T3 (thyroid hormone, 3,5,3'-L-triiodothyronine) in a transient transfection system. Oligonucleotides containing portions of the rGH promoter sequence with various deletions and point mutations were placed upstream of the first 137 base pairs of the rGH promoter or the heterologous herpes virus thymidine kinase promoter in chloramphenicol acetyltransferase expression vectors. The rGH137 and thymidine kinase promoters show no or minimal response to T3 in the basal state. The constructs were tested in GH4C1 rat pituitary cells and COS cells (functionally deficient in thyroid hormone receptor) with and without a co-transfected plasmid expressing a beta type c-erbA gene coding for a functional T3 receptor. Oligonucleotides containing the T3 receptor binding site confer hormone-dependent induction in a manner that is independent of either orientation or variation in position on the helix relative to the promoter. Point mutations in the sequence -189 to -173 result in loss of T3 induction, and bases between -173 and -167 were also required for a full T3 response. The minimal length to confer T3 induction to the rGH promoter was 23 base pairs (-190 to -167). Point mutations creating a perfect duplication of 7 base pairs within the receptor binding site conferred 12-fold T3 response to the rGH137 promoter, 3-fold greater than the wild type rGH237 construct. T3 inductibility was also transferred to the thymidine kinase promoter by an oligonucleotide containing the sequence -200 to -157, demonstrating that cell type specific elements located 3' to 157 of the rGH promoter are not required for thyroid hormone responsiveness.

Animals↗

PubMed1989

Mutations of the rat growth hormone promoter which increase and decrease response to thyroid hormone define a consensus thyroid hormone response element.

We have previously identified sequences required for thyroid hormone (T3) induction of the rat GH (rGH) promoter, which lie in a region from -188 to -164 upstream of the mRNA start site. Within this region, Domains A, -189 to -184 and B, -179 to -174, are imperfect direct repeats, and domain C, -172 to -167, is a divergent inverted copy that matches the A domain at 4/6 positions. A series of synthetic mutant versions of this sequence were inserted upstream of a truncated rGH promoter, or as a replacement for wild-type sequences in a synthetic 237 base pair rGH promoter or upstream of the heterologous thymidine kinase promoter. Mutations changing the B domain to a perfect copy of the A domain significantly increased T3 induction (21.3-fold) relative to the wild type (3.6-fold). A single point mutation making the C domain a better match to the A domain also increased T3 induction to 16.2-fold. Combining this up-mutation with any of three down-mutations in the A, B, or C domains strongly decreased response, showing that all three domains contribute to the amplified T3 response. Binding affinity of the various mutant oligonucleotides was assessed using in vitro translated receptor and affinity paralleled the functional responses for most binding site mutations. Requirements for in vitro binding were, however, less rigorous than those for functional T3 induction. Based on these results, we propose a consensus T3 receptor binding half-site, AGGT(C/A)A, at least two copies of which are required for a T3 response.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Thyroid hormone aporeceptor represses T3-inducible promoters and blocks activity of the retinoic acid receptor.

In the presence of its ligand, thyroid hormone receptor (T3R) binds specifically to DNA sequences near a number of genes and induces their expression. We show that in the absence of the hormone, a T3R binding site acts in cis to decrease expression from such genes. The endogenous T3 receptors in rat pituitary cell lines are sufficient to mediate this effect, as shown by comparisons of basal levels of expression directed by transiently transfected plasmids containing the rat growth hormone promoter with wild-type or point-mutated T3 response elements (T3RE). The magnitude of the negative effect is increased by increasing the strength of the T3RE or by raising intracellular levels of T3R by appropriate transfections. T3REs exert a similar negative effect on the herpes virus thymidine kinase (TK) promoter; this effect is dependent on expression of functional T3 aporeceptor (apoT3R). Analysis of a set of T3REs of increasing strength inserted upstream of the TK promoter showed a strong correlation between the level of induced expression in the presence of hormone and the level of repressed expression in the absence of hormone. These results show that, unlike other members of the nuclear hormone receptor family, T3R binds to specific DNA sequences in the absence of hormone and exerts a negative effect on expression of linked genes. The apparent affinity of apoT3R and hormone-bound T3R for a T3RE was assessed by using varying amounts of T3R expression vector in a transfection dose response assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Isolation of a cDNA clone encoding a biologically active thyroid hormone receptor.

We have isolated a c-erbA cDNA clone from a GH3 cell library. The clone, denoted erb62, is 4.5 kilobases long and encodes a 461-amino acid beta-type c-erbA protein. This c-erbA protein binds 3,5,3'-triiodothyronine (T3) and T3 analogs with affinities similar to those of the authentic T3 receptor. By RNA gel blot analysis, erb62 hybridizes to a 6-kilobase RNA found in organs that express T3 receptors--e.g., heart, kidney, and brain. A COS-cell transient cotransfection system was used to show that erb62 encodes a biologically active T3 receptor. An oligonucleotide, corresponding to a portion of the rat growth hormone gene 5'-flanking region that contains a T3 response element, was inserted on the 5' side of the herpes simplex virus thymidine kinase promoter in a chloramphenicol acetyltransferase-expressing plasmid. Reporter gene expression directed by this hybrid promoter was T3 inducible only if this plasmid was cotransfected with an erb62-expressing plasmid.

Amino Acid Sequence↗

PubMed1988

Multihormonal regulation of the human, rat, and bovine growth hormone promoters: differential effects of 3',5'-cyclic adenosine monophosphate, thyroid hormone, and glucocorticoids.

We have analyzed the effects of a variety of hormones on activity of the rat GH (rGH), human GH, (hGH), and bovine GH (bGH) promoters. After transient transfection of rat pituitary tumor cells, all three promoters are induced by addition of 8-bromo-cAMP. Sequences required for the cAMP responsiveness of the hGH and rGH promoter lie within 183 base pairs of the mRNA start site. Although the rGH promoter is thyroid hormone (T3) responsive in this system, a construct containing 2.7 kilobases of the hGH promoter 5'-flanking sequences is not. Since we also found that the bGH promoter is T3 responsive in these cells, the hGH results are not likely to be due to a species specific factor required for induction in rat pituitary cells. The hGH promoter is weakly induced by dexamethasone whereas the rGH promoter does not respond to glucocorticoids. The hGH and rGH promoters are not responsive to TRH. These results illustrate the potential heterogeneity in hormonal responses of the same gene in different species.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1978

Impairments in lactation in the rat following destruction of the median raphe nucleus.

Lesions of the median raphe (MR) nucleus were placed in cycling female rats and their ability to lactate was evaluated following subsequent pregnancies. Pups from MR-lesioned (MRL) animals grew more slowly and had greatly impaired survival rates compared to pups from sham-lesioned animals. Chronic treatment of MRL mothers with oxytocin (Oxy; 1 IU, s.c., once or twice/day) did not increase the growth rates of their litters. Acute responses to exogenous Oxy (1 IU, i.p.) in MRL mothers, measured by the weight gain of litters during 1/2-h suckling intervals before and after injection, were marginally significant. Milk yield during the total hour suckling period (stomach contents of pups) was clearly less in the MRL animals (p less than 0.01). Treatment with either prolactin (Prl; 250 microgram, twice/day), Prl + GTC (4 mg/kg gorwth hormore, 30 microgram/kg thyroxine, 0.5 mg/rat cortisol, once/day), or 5-HTP (75 mg 5-hydroxytryptophan/kg, twice/day) did not improve the growth rates of litters from MRL animals. However, when milk yield (stomach contents after 1 h) following a 14-h non-suckling interval was measured, lactogenic hormones (Prl or Prl + GTC) restored milk yield in MRL animals to control levels. This response was clearly not dependent upon exogenous Oxy. These results suggest that deficits in the release of lactogenic hormones are involved in the impairments in lactation following lesions of the MR nucleus.

Animals↗

PubMedDate not supplied

Behavioral and toxicological studies of cyclopentanoid monoterpenes from Nepeta cataria.

Two samples of catnip oil were analyzed by tic, gc, and hplc; the results indicated the presence of 23 components. Fractionation of the commercial sample of catnip oil by either distillation or gc yielded 40% nepetalactone and 43% nepetalic acid. Catnip oil, nepetalic acid, and a nepetalactone-enriched fraction were evaluated for toxicological and behavioral effects in mice and rats. The LD50 of catnip oil, the nepetalactone-enriched fraction, and nepetalic acid were found in mice to be: 1300 mg/kg, 1550 mg/kg and 1050 mg/kg, respectively. Catnip oil (500 mg/kg) and nepetalic acid (62.5 mg/kg) were found to significantly increase hexobarbital sleeping time in mice. Rats trained on a Sidman avoidance schedule showed a significant decrease in performance following intraperitoneal injections of catnip oil (500--750 mg/kg), nepetalic acid (125--250 mg/kg), and the nepetalactone-enriched fraction (500--750 mg/kg). Rats trained on the same avoidance schedule developed behavioral tolerance after daily injections of 750 mg/kg catnip oil.

Animals↗