Biomedical subjects

O Bakker

Publications and source records attributed to O Bakker.

15 recordsLinked to original sources

PubMed1996

Structure-function relationship of the inhibition of the 3,5,3'-triiodothyronine binding to the alpha1- and beta1-thyroid hormone receptor by amiodarone analogs.

Desethylamiodarone (DEA), the major metabolite of the potent antiarrhythmic drug amiodarone (A), acts as a competitive inhibitor of T3, binding to the alpha1-thyroid hormone receptor (alpha1-T3R), but as a noncompetitive inhibitor with respect to the beta1-T3R. To gain insight into the structure- function relationship of the interaction between A metabolites and T3Rs, we investigated the effects of several A analogs on T3 binding to the alpha1-T3R and beta1-T3R in vitro. The analogs tested were: 1) compounds obtained by deethylation of A, DEA, and desdiethylamiodarone (DDEA); 2) compounds obtained by deiodination of A, monoiodoamiodarone and desdiiodoamiodarone (DDIA); and 3) benzofuran derivatives with various iodination grades, 2-butyl-3-(4-hydroxy-3,5-diiodo-benzoyl)benzofuran (L3373, two iodine atoms), L6424 (L3373 with one iodine atom), and L3372 (L3373, no iodine atoms). IC50, values of inhibition of T3 binding to alpha1-T3R and beta1-T3R, respectively, were as follows (mean +/- SD, expressed x 10(-5) M): DEA, 4.7 +/- 0.9 and 2.7 +/- 1.4 (P < 0.001); DDEA, 3.7 +/- 0.9 and 1.9 +/- 0.3 (P < 0.001); monoiodoamiodarone, more than 20 and more than 20; DDIA, 16.2 +/- 5.6 and 9.1 +/- 2.1 (P < 0.01); L3373, 3.8 +/- 1.0 and 3.6 +/- 0.5 (P = NS); L6424, 11.3 +/- 5.7 and 10 +/- 2.0 (P = NS); and L3372, no inhibition. Scatchard analyses in the presence of DDEA, DDIA, and L3373 demonstrated a dose-dependent decrease in Ka, but no change in the maximum binding capacity (MBC) of T3 binding to alpha1-T3R. Langmuir plots clearly indicated competitive inhibition of T3 binding to alpha1-T3R by DDEA, DDIA, and L3373. In contrast, these three analogs acted differently with respect to the beta1-T3R. DDEA and DDIA decreased both Ka and MBC in Scatchard plots using beta1-T3R, demonstrating noncompetitive inhibition. L3373 decreased dose-dependently Ka, but not MBC, values of T3 binding to the beta1-T3R and clearly acted as a competitive inhibitor. Ki plots indicated that DDEA, DDIA, and L3373 do not interfere significantly with occupied T3Rs. KI (inhibition constant for the unoccupied receptor) plots demonstrated increasing inhibition of the T3 binding to unoccupied receptors with increasing analog concentrations. In summary, 1) removal of one or two ethyl groups of A results in compounds with strong but almost equal potency of inhibiting T3R binding, whereas removal of one or two iodine atoms of A has a lower potency in this respect. The strong inhibitory potency of the benzofuran derivative L3373 (equalling that of the deethylated compounds) is lost upon deiodination. 2) All tested A analogs acted as competitive inhibitors to the alpha1-T3R. The behavior to the beta1-T3R was different; deethylation or deiodination of A resulted in noncompetitive inhibition, whereas L3373 was a competitive inhibitor. The potency of deethylated and deiodinated compounds (but not of the benzofuran derivatives) for inhibiting T3 binding was twice as high for the beta1-T3R as for the alpha1-T3R. 3) All tested A analogs preferentially interfere with T3 binding to unoccupied receptors. The implications of these findings for the structure-activity relationship are the following: 1) the size of the diethyl-substituted nitrogen group and of the two bulky iodine atoms in the A molecule hamper the binding of A at the T3 binding site of T3Rs; and 2) differences in the hormone-binding domain of alpha1- and beta1-T3Rs are likely to account for the competitive or noncompetitive nature of inhibition of T3 binding by A analogs.

Amiodarone↗

PubMed1995

Desethylamiodarone is a competitive inhibitor of the binding of thyroid hormone to the thyroid hormone alpha 1-receptor protein.

Desethylamiodarone (DEA), the major metabolite of the potent antiarrythmic drug amiodarone, is a non-competitive inhibitor of the binding of thyroid hormone (T3) to the beta 1-thyroid hormone receptor (T3R). In the present study, we investigated whether DEA acts in a similar way with respect to the alpha 1-T3R. The chicken alpha 1-T3R, expressed in an E. coli system, was incubated in the presence or absence of DEA with [125I]T3 in buffer containing 0.05% Triton X-100, 0.05% BSA and 1% ethanol (v/v) in order to solubilise DEA. DEA, but not amiodarone, inhibited T3 binding in a dose-dependent manner; the IC50 value was 3.5 x 10(-5) M. Scatchard analyses in the presence of DEA demonstrated a dose-dependent decrease in Ka values, but no change in MBC. Lineweaver-Burk plots clearly indicated competitive inhibition by DEA. Pre-incubation of the alpha 1-receptor with DEA decreased maximal [125I]T3 binding, which was independent of the duration of pre-incubation. In conclusion, in contrast to the beta 1-T3R, where DEA acts as a non-competitive inhibitor, we now report as a new finding the competitive action of DEA to the alpha 1-T3R.

Amino Acid Sequence↗

PubMed1995

Importance of the content and localization of tyrosine residues for thyroxine formation within the N-terminal part of human thyroglobulin.

Thyroxine (T4) is formed by coupling of iodinated tyrosine residues within thyroglobulin (TG). In mature TG, some iodinated tyrosine residues are involved preferentially in T4 formation. In order to investigate the specific role of various tyrosine residues in T4 formation, N-terminal TG fragments with mutated tyrosine residues were constructed. An N-terminal TG fragment 198 amino acids in size and containing seven tyrosine residues at amino acid positions 5, 29, 89, 97, 107, 130 and 192 was expressed in a baculovirus system. Using site-directed mutagenesis, eight mutant TG fragments were constructed in which different tyrosine residues were replaced by phenylalanine. In the first four TG mutants, one single tyrosine residue (5, 89, 97 or 130) was mutated. In the mutant Y(5,89,97,130)F all of these four tyrosine residues were replaced. The sixth mutant Y(29,89,107,130,192)F contained only tyrosine residues 5 and 97 and the seventh (Y(29,89,97,192)F) contained only tyrosine residues 5, 107 and 130. A TG fragment (Y(5,29,89,97,107,130,192)F) in which all tyrosine residues were replaced by phenylalanine was used as a negative control. After in vitro iodination with lactoperoxidase, specific T4 formation was established in the non-mutated wild-type N-terminal TG fragment. In general the T4 formation in the mutant TG constructs decreased when the total number of tyrosine residues in the 198 amino acid fragment decreased, except fragment Y(29,89,97,192) containing three tyrosine residues, two of them being 5 and 130. Although the rate of T4 formation in this mutated N-terminal TG fragment was lower, the ultimate T4 generation was the same as in the wild-type fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

The role of cytokines in the lipopolysaccharide-induced sick euthyroid syndrome in mice.

To evaluate the role of cytokines in the sick euthyroid syndrome, we tried to establish an animal model of non-thyroidal illness in mice by the administration of a sub-lethal dose of bacterial endotoxin (lipopolysaccharide; LPS) which induces a variety of cytokines, including tumour necrosis factor (TNF alpha), interleukin-1 (IL-1 alpha), interleukin-6 (IL-6) and interferon-gamma (IFN gamma). When compared with pair-fed controls, a single dose of LPS resulted in (a) systemic illness, (b) induction of TNF alpha and IL-6 and (c) a decrease of liver 5'-deiodinase mRNA from 4 h onwards followed by a decrease of serum tri-iodothyronine (T3) and thyroxine (T4) at 8 h and of serum free T3 (fT3) and free T4 (fT4) at 24 h; serum TSH remained unchanged. We then studied whether a single dose or a combination of IL-1 alpha, TNF alpha, IL-6 or IFN gamma could induce the sick euthyroid syndrome in mice, again using pair-fed controls. None of the cytokines except IL-1 alpha caused systemic illness, and IL-1 alpha was the only cytokine that decreased liver 5'-deiodinase mRNA transiently. IL-1 alpha, TNF alpha or IL-6 did not decrease serum T3, T4 and TSH, but administration of IFN gamma decreased serum T4, T3 and fT3 in a dose-dependent manner without changes in serum TSH. Administration of all four cytokines together had no synergistic effects; observed changes were of a smaller magnitude than after LPS. The following conclusions were reached.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Amiodarone-induced hypercholesterolemia is associated with a decrease in liver LDL receptor mRNA.

Amiodarone decreases plasma and tissue triiodothyronine (T3) and increases plasma cholesterol levels resembling changes seen during hypothyroidism. To elucidate the mechanism of amiodarone-induced hypercholesterolemia we investigated gene expression of three key proteins in cholesterol metabolism (cholesterol 7 alpha-hydroxylase, LDL receptor, HMG-CoA reductase) in livers of rats. Animals were treated with amiodarone or propylthiouracil (to induce mild hypothyroidism). The LDL receptor mRNA was downregulated (approximately 50%) in both amiodarone-treated and hypothyroid animals, while the other mRNA remained unchanged after 14-day treatment. The results suggest that amiodarone-induced hypercholesterolemia is associated with decreased LDL receptor mRNA levels.

Amiodarone↗

PubMed1994

Desethylamiodarone is a noncompetitive inhibitor of the binding of thyroid hormone to the thyroid hormone beta 1-receptor protein.

It has been hypothesized that amiodarone (A), a potent antiarrythmic and antianginal drug, induces a local hypothyroid-like condition in extrathyroidal tissues. This might be related to competitive antagonism of A for the thyroid hormone receptor reported in some studies but denied in others. These conflicting results are presumably due to the poor solubility of A in a hydrophilic environment. We, therefore, studied the effect of the drug and its major metabolite, desethylamiodarone (DEA), on the in vitro binding of thyroid hormone (T3) to its receptor protein using the rat beta 1-thyroid hormone receptor expressed in Escherichia coli. A and DEA stayed in solution up to 10(-4) M when 0.05% Triton X-100 was added to the incubation buffer, as evidenced by a recovery of 80-90% for both chemicals, as measured by HPLC. DEA, but not A, had a clear inhibitory effect on the binding of T3 to its receptor (IC50, 1-3 x 10(-5) M). Scatchard analysis in the presence of DEA demonstrated a dose-dependent decrease in the Ka as well as the maximum binding capacity. Lineweaver-Burke analysis indicated noncompetitive inhibition. Plots of the intercepts of Lineweaver-Burke plots vs. DEA concentration were linear (y = 0.334 + 0.098x), giving a Ki of 30 microM for the binding of DEA to the occupied receptor. Plots of the slopes vs. inhibitor concentration were parabolic (y = 3.01 + 0.06x + 0.16x2), indicating a progressively stronger effect of DEA on the unoccupied receptor as concentrations rise. This preference for the unoccupied receptor is reflected in experiments that show a progressive loss of T3 binding when the receptor was incubated for increasing periods with DEA before adding T3. We conclude that DEA is a noncompetitive inhibitor of the binding of T3 to the beta 1-thyroid hormone receptor protein, interacting preferably with the unoccupied T3 receptor.

Amiodarone↗

PubMed1993

Expression of a functional thyroglobulin fragment in a baculovirus system.

The synthesis is described of an N-terminal thyroglobulin (Tg) polypeptide of 27 kDa, which is capable of hormonogenesis, in a baculovirus system. This polypeptide was made using a 657 bp Tg cDNA cloned from human thyroid RNA by a polymerase chain reaction method. The cDNA contained the information for the Tg signal peptide, the N-terminally located site for thyroid hormone formation and, at the 3' end, a sequence coding for six histidine residues. The fragments produced were purified using a nickel-nitrilotriacetic acid column using these six histidine residues. The products were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and showed two glycosylated fragments of 32 and 34 kDa, both of which started with asparagine. Iodination of the fragments with lactoperoxidase in vitro resulted in the formation of thyroxine (T4). The formation rate of T4 in the fragments was about five times lower than that of the mature Tg dimer of 660 kDa, but ten times more rapid than in bovine serum albumin under the same conditions.

Amino Acid Sequence↗

PubMed1991

CAAT/enhancer binding protein is able to bind to ATF/CRE elements.

The transcription factor C/EBP is known to be able to bind to two different classes of sites, a CAAT-box and one present in a number of viral enhancers. In this paper we show using band-shift assays, methylation interference and footprinting that C/EBP is also able to bind with high affinity to ATF/CRE sites. Competition with mutant ATF sites and methylation interference indicate that C/EBP may be able to bind to the ATF/CRE sites by virtue of their homology to the enhancer core elements. Furthermore, we show that C/EBP is able to direct transcription from this site in transient transfection experiments and that mutations in the ATF binding site that impair DNA binding also effect the ability of C/EBP to stimulate transcription.

Activating Transcription Factors↗

PubMed1990

The progesterone receptor can regulate transcription in the absence of a functional TATA box element.

We have investigated the importance of the TATA box element in the induction of transcription by the progesterone receptor. Transcription was analyzed from promoters containing a steroid response element upstream of a wild-type or mutated TATA box. Mutation of the TATA box resulted in a loss of correctly initiated transcripts and abolished binding of TATA factor to the TATA box in vitro but did not inhibit transcriptional activation by the progesterone receptor. Thus we conclude that the receptor is able to stimulate the rate of transcription in the absence of a functional TATA box.

Animals↗

PubMed1990

Tissue-specific differences in the binding of nuclear proteins to a CCAAT motif in the promoter of the androgen-regulated C3 gene.

The expression of the gene for the C3 polypeptide is confined to the ventral prostate of the male rat and is regulated by androgens. To study the mechanism of this tissue-specific and hormone-dependent regulation we have used DNase-I footprinting and band-shift assays to locate binding sites for nuclear proteins isolated from different tissues. In this paper we present evidence that there are tissue-specific differences in the nuclear proteins that are able to bind to a CCAAT motif and a nuclear factor-I consensus that are present in the promoter of the rat C3 gene. Using competition assays and heat denaturation we show that the CAAT box/enhancer binding protein itself may be one of the transcription factors involved.

Androgen-Binding Protein↗

PubMed1988

Turnover products of the apo very low density lipoprotein II messenger RNA from chicken liver.

The mature apo Very Low Density Lipoprotein II (apo VLDLII) mRNA appears in chicken liver within a few hours after estrogen administration. Apart from this mRNA species, shorter RNA molecules hybridizing to apo VLDLII sequences have been detected in rooster liver upon estrogen stimulation. These molecules are present in the non-polyadenylated fraction of the total cellular- and polysomal RNA. Northern blotting and electron microscopy of R-loops were employed to show that these shorter RNA molecules are truncated at their 3'-end. The 3'-termini were further characterized by nuclease S1 analyses, and are located predominantly in the 3' untranslated region of the mRNA. Using a secondary structure model (Shelness and Williams, J. Biol. Chem. 260, 8637-8646, 1985), we show that the 3' termini map mainly in unpaired regions of the structure.

Animals↗

PubMed1988

Protein-DNA interactions in vitro with 5'-flanking DNA fragments from the chicken vitellogenin gene.

The expression of the vitellogenin gene in the liver of oviparous animals is under strict control of estrogen. We have studied the interaction of proteins extracted from nuclei of different estrogen responsive tissues with two fragments (-728 to -470 and -625 to -470) of the upstream region of the chicken vitellogenin gene, using the gel-retardation technique. We found a complex pattern of retarded bands using nuclear extracts from laying hen liver, rooster liver and MCF-7 cells. The patterns observed display differences in the position and intensities of some of the bands, depending on the source of the extract used. The possible significance of these findings will be discussed.

Animals↗

PubMed1988

Estrogen-inducible binding of a nuclear factor to the vitellogenin upstream region.

The estrogen-dependent binding of a protein to the upstream region of the chicken vitellogenin gene was detected by using in vivo dimethyl sulfate, genomic DNase I, and in vitro exonuclease III footprinting. The site is located between base pairs -848 and -824, and its sequence resembles that of the nuclear factor I binding site. The results suggest that a nuclear factor binding to this site is involved in the regulation of the vitellogenin gene.

Animals↗

PubMed1986

Differential estrogen responsiveness of the vitellogenin and apo very low density lipoprotein II genes in the rooster liver.

The primary transcript of the chicken apo Very Low Density Lipoprotein II (apoVLDL-II) gene is formed almost immediately after a first estrogen administration, contrary to the appearance of the vitellogenin primary transcript which has a lag of at least 4 h. However, after a second estrogen administration the vitellogenin gene transcription shows no detectable lag (memory effect). After estrogen withdrawal, the primary transcripts of both genes rapidly decline to undetectably low levels. In the presence of estrogen, the half-lives of the mRNAs of apoVLDL-II and vitellogenin are 15 and at least 70 h, respectively, whereas in the absence of hormone they are only 3.5 and 5.5 h, respectively. Thus estrogen not only controls the transcription of both genes, but also the turnover of their mRNAs. In short, there appears to be a quantitative difference in the response of both genes.

Animals↗

PubMed1984

Induction of ornithine decarboxylase and augmentation of tyrosine aminotransferase activity by N-hydroxy-2-acetylaminofluorene and 2-acetylaminofluorene in rat liver. Influence of sex, retinylacetate, indomethacin, and pentachlorophenol.

IP injection in rats of 2-acetylaminofluorene (AAF) or N-hydroxy-2-acetylaminofluorene (N-OH-AAF) resulted in a transient increase of hepatic ornithine decarboxylase (ODC) and tyrosine aminotransferase (TAT) activity. Maximal activity of ODC was observed 4 hr and of TAT 3 hr after administration of either AAF or N-OH-AAF. A lag-time of 2 hr preceded the increase of ODC and TAT activity. N-OH-AAF dependent ODC induction displayed an almost linear dose-response in the dose range up to 94.1 mumol/kg bw (body weight) when the ODC activity was measured at its maximum 4 hr after administration. Elevation of the dose N-OH-AAF to 126 mol/kg bw resulted in a lower ODC induction. Administration of doses AAF to 31.4 mumol did not change ODC activity. At doses up to 126 mumol/kg bw ODC induction increased linear. TAT induction increased linear in the dose range 15.7-94.1 mumol N-OH-AAF and 31.4-94.1 mumol AAF/kg. Lowering the dose of AAF did not result in a lower ODC or TAT activity. Judged by the effects of actinomycin D or cycloheximide administered 1 hr prior to AAF or N-OH-AAF, the in vivo induction of rat liver ODC activity by AAF and N-OH-AAF appeared to be under transcriptional control, whereas augmentation of TAT activity under influence of AAF or N-OH-AAF appeared the result of (post) translational events. Induction of ODC by AAF or N-OH-AAF was not significantly changed by indomethacin, was slightly increased by pentachlorophenol (PCP) and was synergistically enhanced by retinylacetate (RA). The increase of TAT activity was stimulated by PCP and RA. The effect of PCP indicates that N-sulfonoxy-2-acetylaminofluorene is most probably not involved in the induction of ODC. AAF appeared more effective hepatic ODC inducer in females than males and moreover more effective than N-OH-AAF in females. N-OH-AAF had stronger ODC inducing capacity in males than females. Similar observations were made with respect to TAT activity. When induction of ODC is indicative for a tumor promoting property then the data presented here suggest that tumor promotion of the complete carcinogens AAF and N-OH-AAF is not mediated by N-O-sulfation; this might be due to other metabolic conversions.

2-Acetylaminofluorene↗