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C Parkison

Publications and source records attributed to C Parkison.

At least 19 recordsLinked to original sources
PubMed1994

Tissue-dependent developmental expression of a cytosolic thyroid hormone protein gene in Xenopus: its role in the regulation of amphibian metamorphosis.

We have cloned the cDNA encoding the Xenopus laevis homolog of mammalian cytosolic thyroid hormone binding protein (CTHBP). We found that while its mRNA level varies little in whole animals during development, the expression of CTHBP is inversely correlated with tissue-specific transformations during metamorphosis. A high level of its mRNA was observed in the tail of premetamorphic tadpoles. However, the expression is dramatically repressed with the onset of rapid tail resorption. In the hindlimb, the expression of CTHBP is very low during morphogenesis. Subsequently, its expression continuously increases during the period of limb growth. In contrast, a low level of CTHBP expression was detected in the intestine throughout metamorphosis. These results suggest that CTHBP could function to modulate the metamorphic process by regulating the level of intracellular thyroid hormones.

Amino Acid Sequence↗
PubMed1994

Stimulation of proliferation by 3,3',5-triiodo-L-thyronine in poorly differentiated human hepatocarcinoma cells overexpressing beta 1 thyroid hormone receptor.

To understand the role of thyroid hormone nuclear receptors (TRs) in hepatocarcinogenesis, we characterized the TRs in nine human hepatocarcinoma cell lines. The expression of TR proteins is receptor subtype- and cell type-dependent. TR alpha 1 protein expresses similarly at a low level in each of the nine cell lines. In contrast, TR beta 1 is overexpressed in hepatocarcinoma cells which are poorly differentiated. Furthermore, thyroid hormone was found to stimulate the proliferation of cells in which TR beta 1 is overexpressed. These results suggest that TR beta 1 is most likely involved in the differentiation and proliferation of hepatocarcinoma cells. Our studies have shed new light in the understanding of the role of TRs in liver carcinogenesis.

Biomarkers↗
PubMed1993

Conformational changes of human beta 1 thyroid hormone receptor induced by binding of 3,3',5-triiodo-L-thyronine.

To understand the structural basis in the hormone-dependent transcriptional regulation of human beta 1 thyroid hormone receptor (h-TR beta 1), we studied the conformational changes of h-TR beta 1 induced by binding of 3,3',5-triiodo-L-thyronine (T3). h-TR beta 1 was treated with trypsin alone or in the presence of T3, thyroid hormone response element (TRE) or T3 together with TREs. Without T3, h-TR beta 1 was completely digested by trypsin. Binding of TREs had no effect on the tryptic digestion pattern. However, T3-bound h-TR beta 1 became resistant to tryptic digestion and yielded trypsin-resistant peptide fragments with molecular weight of 28,000 and 24,000. Chymotryptic digestion also yielded a T3-protected 24 Kd peptide fragment. Using anti-h-TR beta 1 antibodies and amino acid sequencing, the 28 Kd fragment was identified to be Ser202-Asp456. The 24 Kd tryptic fragments were found to be Lys239-Asp456 and Phe240-Asp456. The 24 Kd chymotryptic fragment was identified to be Lys235-Asp456. The structural changes as a result of T3 binding could serve as a transducing signal to modulate the gene regulating activity of h-TR beta 1.

Amino Acid Sequence↗
PubMed1993

Structure of the hormone binding domain of human beta 1 thyroid hormone nuclear receptor: is it an alpha/beta barrel?

To understand the structure of the hormone binding domain (HBD) of human beta 1 thyroid hormone nuclear receptor (h-TR beta 1), truncated h-TR beta 1 fragments, MD32 (M169-D456), KD29 (K201-D456), DD28 (D211-D456), KD25 (K235-D456), and KP28 (K201-P448), were analyzed by circular dichorism (CD). MD32 and KD29 show intense CD spectra with double minima at 222 and 208-210 nm, indicating the presence of extensive regions of alpha-helix. DD28 and KD25 have spectra which are reduced in intensity with minima around 215 nm, characteristic of a beta-sheet. The observed spectra are compatible with sequence analysis which predicts that HBD contains alternating stretches of alpha-helix and beta-strand. These extensive decreases in secondary structure in DD28 and KP28 in which the predicted first beta-strand or last alpha-helix was deleted, respectively, were accompanied by the loss of hormone binding activity. On the basis of these results, we suggest a new model for h-TR beta 1 consisting of the known DNA binding domain linked by an alpha-helical hinge to the HBD, with the tertiary structure of an alpha/beta barrel. The model is compatible with previous chemical and genetic studies on the structure of this protein.

Amino Acid Sequence↗
PubMed1993

One-step immunoaffinity purification of human beta 1 thyroid hormone receptor with DNA and hormone binding activity.

An efficient and versatile method to purify large amounts of active human beta 1 thyroid hormone receptor (h-TR beta 1) was developed. Using a T7 expression system, h-TR beta 1 was overexpressed in Escherichia coli. Approx. 80% of the expressed receptor protein was concentrated in the insoluble inclusion bodies and approximately 20% was in the soluble form (h-TR beta 1-S). h-TR beta 1-S was conveniently purified by one immunoaffinity chromatographic step. From 1 l of cell culture, approx. 0.1 mg of purified h-TR beta 1-S was obtained. The purified h-TR beta 1-S binds to 3,3',5-triiodo-L-thyronine with a Ka = 2 x 10(9) M-1 and exhibits analog specificity. The purified h-TR beta 1-S also binds to T3 response elements (TRE) with different orientation in the half-sites with differential activity. In addition, binding of h-TR beta 1-S to TREs was enhanced by retinoid X receptor. These results indicate that the purified h-TR beta 1-S retains its hormone and DNA binding activity. The purified h-TR beta 1-S is suitable for structural and functional studies. This method could be used to purify h-TR beta 1 or rat TR beta 1 expressed in insect cells or yeast.

Base Sequence↗
PubMed1993

Interaction of human beta 1 thyroid hormone receptor and its mutants with DNA and retinoid X receptor beta. T3 response element-dependent dominant negative potency.

Mutations in the human beta thyroid hormone receptor (h-TR beta) gene are associated with the syndrome of generalized resistance to thyroid hormone. We investigated the interaction of three h-TR beta 1 mutants representing different types of functional impairment (kindreds ED, OK, and PV) with different response elements for 3,3',5-triiodothyronine (T3) and with retinoid X receptor beta (RXR beta). The mutant receptors showed an increased tendency to form homodimers on a palindromic T3-response element (TREpal), a direct repeat (DR + 4), and an inverted palindrome (TRElap). On TRElap, wild type TR binding was decreased by T3, while the mutant receptors showed a variably decreased degree of dissociation from TRElap in response to T3. The extent of dissociation was proportional to their T3 binding affinities. RXR beta induced the formation of h-TR beta 1:RXR beta heterodimers equally well for mutants and the wild type h-TR beta 1 on these T3 response elements. However, the T3-dependent increase in heterodimerization with RXR beta was absent or reduced for the mutant TRs. Transient transfection studies indicated that the dominant negative potency was several-fold more pronounced on the TRElap as compared to TREpal or DR + 4. In CV-1 and HeLa cells, transfection of RXR beta could not reverse the dominant negative action. These results demonstrate that the binding of mutant h-TRs to DNA, as well as their dominant negative potency, are TRE dependent. In addition, competition for DNA binding, rather than for limiting amounts of RXR beta, is likely to mediate the dominant negative action.

Animals↗
PubMed1991

The monomer of pyruvate kinase, subtype M1, is both a kinase and a cytosolic thyroid hormone binding protein.

Using a T7 expression system, the monomer of rat pituitary pyruvate kinase, subtype M1 (PKM1), was overexpressed in Escherichia coli and purified to homogeneity. The monomeric p58-M1 has intrinsic enzymatic activity with a Vmax of 79 +/- 20 units/mg and Km's for ADP and PEP of 1.43 +/- 0.76 and 0.14 +/- 0.07 mM, respectively. The monomer binds 3,3',5-triiodo-L-thyronine (T3) with Ka = 1.5 x 10(7) M-1. The order of analog specificity is L-T3 greater than L-thyroxine greater than D-T3 greater than 3'-isopropyl-3,5-diiodo-L-thyronine greater than or equal to 3',5',3-triiodo-L-thyronine. In contrast, tetrameric PKM1 lacks T3 binding activity. The kinase activity of p58-M1 is inhibited by T3 and its analogs in a concentration-dependent manner with the order of inhibitory activity similar to that of binding activity. This inhibition, however, is reversed by the addition of fructose 1,6-bisphosphate. p58-M1 is the second PK isoenzyme monomer to be identified as having thyroid hormone binding activity.

Animals↗
PubMed1991

An essential role of domain D in the hormone-binding activity of human beta 1 thyroid hormone nuclear receptor.

By analogy with steroid receptors, human placental thyroid hormone nuclear receptor (hTR beta 1) could be divided into four functional domains: A/B (Met1-Leu101), C (Cys102-Ala170), D (Thr171-Lys237), and E (Arg238-Asp456). The E domain was thought to bind thyroid hormone. To evaluate whether domain E alone is sufficient to bind T3 or requires the presence of other domains for functional T3-binding activity, a series of deletion mutants was constructed. The mutants were expressed in Escherichia coli, and the expressed proteins were purified. Analysis of the T3-binding affinity and analog specificity of the purified truncated hTR beta 1 indicated that domain E alone did not have T3-binding activity. Extension of the amino-terminal sequence of domain E to include part of domain D yielded a mutant (Lys201-Asp456) with a Ka for T3 of 0.5 +/- 0.2 x 10(9) M-1. Further extension to include the entire domain D (Met169-Asp456) yielded a mutant with T3-binding activity with a Ka of 0.8 +/- 0.1 x 10(9) M-1. Further extension of the amino-terminal sequence to include domain C increased the affinity for T3 by nearly 2-fold (Ka = 1.5 +/- 0.4 x 10(9) M-1). The Ka for the wild-type hTR beta 1 is 1.5 +/- 0.2 x 10(9) M-1. Furthermore, mutant (Met169-Asp456) binds to 3',5',3-triiodo-L-thyropropionic acid, D-T3, L-T4, and L-T3 with 307%, 37%, 7%, and 0.1%, respectively, of the activity of L-T3. This order of analog affinity is similar to that of the wild-type hTR beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1989

Cytosolic thyroid hormone-binding protein is a monomer of pyruvate kinase.

A cDNA clone encoding a human cytosolic thyroid hormone-binding protein (p58) has been isolated. The human sequence was found to be homologous to that of rat pyruvate kinase (EC 2.7.1.40) subtype M2. p58 is a monomer that has approximately 5% the enzymatic activity of the tetrameric pyruvate kinase M2. The tetrameric M2 does not bind 3,3',5-triiodo-L-thyronine (T3). Binding of p58 to T3 and its analogs resulted in the inhibition of its pyruvate kinase activity. The apparent Ki values of T3, L-thyroxine, and D-T3 are 30 nM, 100 nM, and 2 mM, respectively. L-Thyronine and 3,3',5'-triiodo-L-thyronine had no effect. This order of activity correlates with the thermogenic effects reported for T3 and its analogs. Conversion of p58 to the tetramer is reversible and is under the control of fructose 1,6-bisphosphate. The conversion is inhibited by T3 in a dose-dependent manner. Since pyruvate kinase is a key enzyme in regulating cellular ADP, ATP, and pyruvate, our findings suggest that p58 may be involved in mediating some of the cellular metabolic effects induced by thyroid hormones.

Amino Acid Sequence↗
PubMed1987

The nucleotide sequence of a human cellular thyroid hormone binding protein present in endoplasmic reticulum.

A cDNA clone encoding a human cellular thyroid hormone binding protein (p55) has been isolated. The cDNA sequence contains a single open reading frame of 1524 nucleotides which encodes a polypeptide of 491 amino acids and a signal sequence of 17 amino acids. Two partial protein sequences determined from the purified protein correspond to the deduced amino acid sequence. In vitro translation of mRNA prepared by transcription of the cDNA clone yielded a protein of the size of an unprocessed precursor (Mr 57,000) as well as smaller fragments which were immunoprecipitated by antibodies against p55. The isolation of the cDNA clone should allow elucidation of the cellular function of this thyroid hormone binding protein which is present in the endoplasmic reticulum and nuclear envelope.

Amino Acid Sequence↗
PubMed1981

Freeze-fracture cytochemistry: replicas of critical point-dried cells and tissues after fracture-label.

Applications of the new fracture-labeling techniques for the observation of cytochemical labels on platinum-carbon replicas are described. Frozen cells, embedded in a cross-linked protein matrix, and frozen tissues are fractured with a scalpel under liquid nitrogen, thawed, labeled, dehydrated by the critical point drying method, and replicated. This method allows direct, high-resolution, two-dimensional chemical and immunological characterization of the cellular membranes in situ, as well as detection of sites within cross-fractured cytoplasm and extracellular matrix.

Animals↗
PubMed1981

Fracture-label:O cytochemistry of freeze-fracture faces in the erythrocyte membrane.

A method--"fracture label"--is described for the cytochemical labeling of the membrane faces produced by freeze-fracture. Human erythrocytes embedded in a crosslinked matrix are frozen, fractured in liquid nitrogen, thawed, labeled, and cut into thin sections. Electron microscope observation of the fracture faces shows preferential partition of concanavalin A binding sites with the inner half of the membrane. This signifies that, during freeze-fracture, binding sites are dragged from the outer surface across the outer ("exoplasmic") half of the membrane and retained on the protoplasmic fracture face (face P). The fracture process results in exposure of new anionic sites on face P. Fracture-label can be applied to the cytochemical characterization of the cellular components exposed by freeze-fracture of isolated cells and tissues.

Colloids↗
PubMed1981

Freeze-fracture cytochemistry: thin sections of cells and tissues after labeling of fractures faces.

Experimental details of a new method for the cytochemical characterization of the membrane faces and cytoplasm produced by freeze-fracture of isolated cells and tissues are presented. This new method-"fracture-label"-involves grinding of frozen samples immersed in liquid nitrogen, thawing, cytochemical labeling of the fractured faces, and processing for thin section electron microscopy. Cationized ferritin (at pH. 7.5 and 4.0), colloidal iron, as well as concanavalin A are used to label the fractures faces of leukocytes and Hela cells embedded in a cross-linked matrix of bovine serum albumin and of liver and spleen tissues. Our results show the presence of numerous anionic binding sites on the fracture faces of all plasma and cytoplasmic membranes, and of concanavalin A binding sites preferentially associated to the exoplasmic fracture faces of plasma and nuclear envelope membranes. A proportion of the anionic sites appears to be revealed by, or during, the freeze-fracture process. Colloidal iron labeling also shows preferential association with the chromatin areas of cross-fractured nuclei. The results show that "fracture-label", i.e., the combined application of freeze-fracture and cytochemical labeling techniques, can be used to study the surface chemistry of the fractures faces of biological membranes as well as of cross-fractured cytoplasm.

Animals↗
PubMed1980

Fusion of human erythrocytes induced by Sendai virus: freeze-fracture aspects.

Analysis of the freeze-fracture morphology of human erythrocytes during Sendai virus-induced fusion indicates that it occurs in several spots, with the formation of membrane-bound cytoplasmic connexions. Radial expansion of these connexions causes the formation of a polymorphic network of membrane tubules with a lumen continuous with the external space. Our results are best explained assuming that the viral envelopes do not necessarily participate directly in inter-membrane fusion but, instead, indirectly through conditioning and modification of the erythrocyte membranes by viral membrane components.

Cell Fusion↗