PubMed Health⌕ Search

Biomedical subjects

J W Barlow

Publications and source records attributed to J W Barlow.

At least 19 recordsLinked to original sources
PubMed2004

Persistency of adenoviral-mediated lysostaphin expression in goat mammary glands.

Gene therapy has great potential to enable synthesis of protein molecules in targeted cells of an animal. One application may be the production of antibacterial enzymes by the mammary gland as a means of preventing or treating mastitis. We have previously demonstrated that goat mammary cells are capable of producing lysostaphin, an antistaphylococcal enzyme, after being transduced in vivo with a recombinant adenoviral vector containing a modified lysostaphin gene (Ad-lys). The current study examined duration of expression, and antibody response to lysostaphin and the adenoviral vector. Following intramammary infusion into nonlactating goats (n = 4), recovery of transducible adenoviral vector in mammary secretions persisted for 11 d. Transducible vector was not detected in serum, saliva, urine, or feces. Peak lysostaphin concentrations (< 20 microg/mL) in mammary secretions of infused udders were detected approximately 1 wk postinfusion, and generally returned to undetectable levels after an additional 1 to 2 wk. The poor persistency of expression was likely due to the very potent immune response to both the adenovirus and the expressed lysostaphin. Serum IgG antibodies recognizing the adenoviral vector developed within 7 d of the infusion, and titers rose dramatically to greater than 1:1 x 10(5). Similar titers of serum IgG antibodies to lysostaphin developed in 3 goats, with more moderate titers in the fourth goat. The antibody response to lysostaphin was delayed by approximately 4 d in comparison to the response to the adenovirus. Serum IgG antibody profiles were reflected in mammary secretions. No IgA antibodies to adenovirus or lysostaphin were detected in sera or mammary secretion. We demonstrate that while the lysostaphin gene can be introduced to the mammary gland using an adenoviral-mediated gene transfer technique, the strong immune response that it provokes makes the approach unsuitable for combating mastitis.

Adenoviridae↗
PubMed2002

Adenoviral-mediated transfer of a lysostaphin gene into the goat mammary gland.

As a step toward preventing and curing Staphylococcus aureus mastitis, an adenoviral-mediated gene transfer technique was used to enable mammary cells to synthesize and secrete lysostaphin, an anti-staphylococcal protein. A lysostaphin gene, modified for eukaryotic expression of the bioactive variant, Gln125,232-lysostaphin, was inserted into a replication deficient adenovirus by homologous recombination in 293 cells. The resulting adenoviral vector containing the modified lysostaphin gene (Ad-lys) was used to infect bovine mammary epithelial cells in vitro and caprine mammary cells in vivo. A similar adenoviral vector containing the Escherichia coli gene encoding beta-galactosidase (Ad-lacZ) was also evaluated. Transduction of cultured bovine cells by Ad-lacZ was confirmed by the presence of beta-galactosidase in fixed cells 48 h postinfection. Bovine cells transduced by Ad-lys secreted immunoreactive Gln125,232-lysostaphin (0.8 microg/ml) into media that had approximately 20% bioactivity compared with native lysostaphin. To evaluate transduction in vivo, udder halves of four nonlactating goats were exposed to 10(10) plaque-forming units (pfu) ofAd-lacZ by two intramammary infusions given 48 h apart. The animals were euthanized 24 h later, and extensive expression of beta-galactosidase was detected in cells lining the teat canals, with more moderate expression observed in adjoining mammary parenchyma. Udder halves of two other nonlactating goats were infused with 10(10) pfu of Ad-lys while contralateral udder halves received Ad-lacZ. The animals were euthanized 48 h postinfusion. In both animals, extensive expression of beta-galactosidase was detected in Ad-lacZ exposed teats. Immunoreative Gln125,232-lysostaphin was detectable in secretions from Ad-lys exposed glands 24 h postinfusion, increasing to approximately 1 microg/ml at 48 h postinfusion. As with cultured bovine mammary epithelial cells, the bioactivity of goat-derived Gln125,232-lysostaphin was approximately 20% of native lysostaphin. These results demonstrate that an adenoviral vector can be used to introduce a gene into the ruminant mammary gland, enabling the secretion of a bioactive form of lysostaphin.

Adenoviridae↗
PubMed2000

Identification and analysis of error types in high-throughput genotyping.

Although it is clear that errors in genotyping data can lead to severe errors in linkage analysis, there is as yet no consensus strategy for identification of genotyping errors. Strategies include comparison of duplicate samples, independent calling of alleles, and Mendelian-inheritance-error checking. This study aimed to develop a better understanding of error types associated with microsatellite genotyping, as a first step toward development of a rational error-detection strategy. Two microsatellite marker sets (a commercial genomewide set and a custom-designed fine-resolution mapping set) were used to generate 118,420 and 22,500 initial genotypes and 10,088 and 8,328 duplicates, respectively. Mendelian-inheritance errors were identified by PedManager software, and concordance was determined for the duplicate samples. Concordance checking identifies only human errors, whereas Mendelian-inheritance-error checking is capable of detection of additional errors, such as mutations and null alleles. Neither strategy is able to detect all errors. Inheritance checking of the commercial marker data identified that the results contained 0.13% human errors and 0.12% other errors (0.25% total error), whereas concordance checking found 0.16% human errors. Similarly, Mendelian-inheritance-error checking of the custom-set data identified 1.37% errors, compared with 2.38% human errors identified by concordance checking. A greater variety of error types were detected by Mendelian-inheritance-error checking than by duplication of samples or by independent reanalysis of gels. These data suggest that Mendelian-inheritance-error checking is a worthwhile strategy for both types of genotyping data, whereas fine-mapping studies benefit more from concordance checking than do studies using commercial marker data. Maximization of error identification increases the likelihood of linkage when complex diseases are analyzed.

Alleles↗
PubMed2000

Displaced supracondylar fractures of the humerus in children. Audit changes practice.

We performed an audit of 71 children with consecutive displaced, extension-type supracondylar fractures of the humerus over a period of 30 months. The fractures were classified according to the Wilkins modification of the Gartland system. There were 29 type IIA, 22 type IIB and 20 type III. We assessed the effectiveness of guidelines proposed after a previous four-year review of 83 supracondylar fractures. These recommended that: 1) an experienced surgeon should be responsible for the initial management; 2) closed or open reduction of type-IIB and type-III fractures must be supplemented by stabilisation with Kirschner (K-) wires; and 3) K-wires of adequate thickness (1.6 mm) must be used in a crossed configuration. The guidelines were followed in 52 of the 71 cases. When they were observed there were no reoperations and no malunion. In 19 children in whom they had not been observed more than one-third required further operation and six had a varus deformity. Failure to institute treatment according to the guidelines led to an unsatisfactory result in 11 patients. When they were followed the result of treatment was much better. We have devised a protocol for the management of these difficult injuries.

Bone Wires↗
PubMed1997

Modulation of T3-induced sex hormone-binding globulin secretion by human hepatoblastoma cells.

We have shown previously that tri-iodothyronine (T3)-induced sex hormone-binding globulin (SHBG) secretion by the human hepatoblastoma cell line, HepG2, can be modulated by retinoids. We have now used this model to study a range of other compounds that are known to influence T3 responsiveness in various cell systems. HepG2 cells were incubated for 4 days in serum-free medium containing T3, together with insulin, dexamethasone, phorbol myristate (PMA), sodium butyrate or estradiol. T3 (10 nmol/l) alone induced a concentration of SHBG secreted by HepG2 cells that was 187 +/- 20% (mean +/- S.D., n = 9) of control. Insulin (100 nmol/l) reduced basal SHBG secretion from 24.7 +/- 5.2 nmol/l to 16.1 +/- 1.7 nmol/l (P < 0.01). This effect was dose responsive, half-maximal at 3.4 +/- 3.0 nmol/l (approximately 600 mU/l) and maximal with 100 nmol/l insulin. Co-incubating 0-10 nmol/l T3 with 100 nmol/l insulin resulted in a downward shift in the dose-response curve without a change in the half-maximal response to T3. Conversely, 0-100 nmol/l insulin reduced SHBG production induced by 10 nmol/l T3. In contrast; while dexamethasone alone was without effect on SHBG secretion, 100 nmol/l dexamethasone induced a shift to the left in half-maximal T3 stimulation from 0.37 nmol/l to 0.10 nmol/l. The effect of PMA on SHBG secretion was reminiscent of the previously observed retinoid effect. PMA 100 nmol/l abolished maximal T3 stimulation. This effect was dose responsive, with a threshold at 1 nmol/l PMA. Sodium butyrate, up to 1 mmol/l was without effect; with greater concentrations, SHBG secretion was reduced. T3 responsiveness was virtually abolished by 3 mmol/l sodium butyrate; higher concentrations were cytotoxic and secretion was reduced to less than 20% of basal. Lack of an effect of estradiol on SHBG secretion by HepG2 cells was confirmed. These studies suggest that T3-induced SHBG secretion by HepG2 cells is independently influenced by insulin, potentiated by dexamethasone, and modulated by PMA. Detailed molecular analysis of this model will increase our understanding of the mechanism of action of T3, specifically in human liver cells.

Butyrates↗
PubMed1996

Preferential inhibition of cytoplasmic T3 binding is associated with reduced nuclear binding in cultured cells.

Previous studies from our laboratory have suggested that the nonsteroidal antiinflammatory drug, diclofenac (DCF), is a more potent competitor for T3 binding sites in cytoplasm than for those in the nucleus. In the present study we have examined the competitive potency for DCF and its effect on nuclear binding of T3 in cultured cells. DCF was a weak competitor for T3 binding sites in cytosol and nuclear extracts prepared from HepG2 cells with a potency of 21 and 295 microM, respectively. When expressed relative to T3, DCF was 135-fold more potent in cytosol than in nuclear extract. In intact cells, T3 was bound by nuclei with an affinity, Kd of 0.22 +/- 0.07 nM whereas in nuclear extract the affinity was 0.60 +/- 0.21 nM. DCF was a competitive inhibitor in both preparations but reduced the apparent affinity 4-fold in intact cells but only 2-fold in nuclear extract. In whole-cell experiments, DCF increased the rate of dissociation of T3 from cells prelabeled with hormone for 30 min. When these prelabeled cells were incubated with DCF, 0.1 mM, cell-associated T3 was significantly lower at 30 and 60 min than in cells reincubated without the drug. These data show that cellular transport mechanisms precede nuclear binding by T3 and suggest that there is a critical role for nonnuclear binding proteins in thyroid hormone action.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1996

Concentrations of thyroid hormones and iodothyronine binding proteins in serum of the koala (Phascolarctos cinereus).

OBJECTIVE: To examine circulating total and free thyroid hormone (T3 and T4) concentrations, determine serum iodothyronine binding characteristics and estimate thyroid stimulating hormone (TSH) activity in sera of coastal and inland koalas. DESIGN: A prospective study. PROCEDURE: Koala serum T3 and T4 were measured by radioimmunoassay. T4 binding parameters were determined by radioligand binding and electrophoresis. Koala TSH values were determined by bioassay. RESULTS: Mean total T4 concentrations were 3.2 +/- 2.1 nM although values were significantly higher in inland-dwelling females in comparison to coastal-dwelling males. Free T4 was 3.3 +/- 2.1 pM. Total and free T3 were 0.4 +/- 0.2 nM and 1.4 +/- 0.9 pM respectively, although these values were at the lower end of the assay detection limit and should be viewed with reservation. Electrophoresis of [125I]-T4-labelled serum revealed only two proteins of electrophoretic mobility similar to human transthyretin (TTR) and albumin. Scatchard analysis of T4 binding to serum gave a curvilinear plot, which could be resolved into two binding sites with affinities identical to that of TTR and albumin but both of low concentration. The bioactivity of the TSH present in the sera was measured using a cell line (JP09) transfected with the human TSH receptor. The mean level of stimulation found in the sera corresponded to a bovine TSH activity of less than 10 mU/L. CONCLUSION: These results suggest that the serum concentrations of free and total thyroid hormones in koalas are low compared to other marsupials and very low compared to eutherian mammals. The mechanism of maintenance of euthyroidism in this species remains to be determined.

Animals↗
PubMed1996

Differential modulation of thyroid hormone responsiveness by retinoids in a human cell line.

Previous studies have suggested that there is an interrelationship between responses mediated by retinoic acid (RA) and those to thyroid hormone (T3). These experiments have used transfected gene constructs, often in receptor-negative cells. To study the relationship between RA- and T3-mediated responses in intact human cells, we incubated HepG2 cells for 4 days in serum-free medium with T3 and/or RA or 9-cis-RA. Measured responses were stimulation of secreted sex hormone-binding globulin (SHBG) or inhibition of secreted T4-binding globulin (TBG). T3 induced a dose-responsive increase in SHBG secretion that was maximal at 10nM (206 +/- 24% of untreated value) and half-maximal at 0.36 +/- 0.16 nM T3. RA and 9-cis-RA, up to 100 nM, induced a slight fall in SHBG secretion to 79 +/- 9% and 88 +/- 9%, respectively. T3 induction of SHBG secretion was significantly attenuated in cells coincubated with T3(0-10nM) and RA. With T3 (10 nM) together with RA (3, 10, or 100 nM), the maximal SHBG responses were reduced to 193 +/- 24%, 151 +/- 5% and 132 +/- 30%, respectively. With T3 and 9-cis-RA (100 nM), maximal stimulation was 169 +/- 20%. Importantly, the effective half-maximal stimulatory concentration of T3 in the presence of either retinoid (3-100 nM) was unchanged at 0.3 nM T3. In addition, the inhibitory effect of 9-cis RA could not be overcome even with 300 nM T3. The threshold for the RA effect was between 0.3-1 nM, with half-maximal inhibition at 30 nM. 9-cis-RA was approximately 10-fold less potent than RA. Preliminary studies suggested that changes in SHBG messenger RNA levels were similar to those in secreted SHBG. No effect was observed with vitamin D or clofibrate, either alone or combined with T3. Conversely, T3 reduced TBG secretion, with maximal suppression to 74 +/- 5% of the control value at a T3 concentration of 10 nM. RA alone reduced TBG secretion to 76% of the control value. RA did not attenuate the effect of T3, and the two agents combined showed no synergism. Neither T3 nor RA, alone or in combination, influenced secreted total protein or albumin. RA did not alter the concentration of nuclear T3-binding sites. These data suggest that retinoids act via a gene-dependent mechanism to modulate maximal, but not half-maximal, responses to T3 in HepG2 cells with the specificity of RA greater than that of 9-cis-RA.

Carcinoma, Hepatocellular↗
PubMed1995

Down-regulation of thyroxine-binding globulin messenger ribonucleic acid by 3,5,3'-triiodothyronine in human hepatoblastoma cells.

A sensitive [125I]-T4 binding assay was used to measure serum T4-binding globulin (TBG) in 60 individuals selected on the basis of their total circulating T3 concentrations, and a relationship between TBG and circulating thyroid hormone levels in humans was confirmed. There was a significant correlation between serum TBG and T3 or free T4 index. TBG secretion and TBG messenger ribonucleic acid (mRNA) production were studied with a continuous culture of the human hepatoblastoma cell line, HepG2. Cells were maintained in serum-free media for experimental manipulations. The addition of 100 nmol/L T3 to the cell medium resulted in a time-dependent down-regulation of TBG mRNA to 33 +/- 6% (+/- SD, n = 4) of untreated control levels by 24 h. Suppression of TBG mRNA was first detectable at 8 h (57% of untreated control levels). The effect of T3 was dose-responsive, with half-maximal suppression of TBG mRNA occurring at a bioavailable T3 concentration of approximately 30 pmol/L. The effect of T3 on TBG mRNA was not caused by a change in mRNA stability. Proteins secreted by HepG2 cells bound T4 with an affinity identical to that of normal circulating TBG. Cell secretion of TBG was parallel to total protein secretion and consistent with a TBG secretion rate of 50 ng/10(6) cells per day. Variations in the concentration of secreted binding protein in the presence of T3 corresponded to the changes observed in TBG mRNA. These data show that circulating TBG concentration is negatively correlated with total serum T3 in vivo. The corresponding down-regulation observed between TBG mRNA and secreted protein in HepG2 cells suggests that this effect is the result of the action of T3 on cellular TBG mRNA synthesis.

Cell Line↗
PubMed1994

Thyroxine binding by human transthyretin variants: mutations at position 119, but not position 54, increase thyroxine binding affinity.

A mutation at codon 119 in the transthyretin (TTR) gene leads to a substitution of methionine for threonine at this position in the circulating protein. As the amino acid at position 119 is located in the T4 binding channel, mutations here may affect the binding of T4 by TTR. A previous study has shown an increase in the amount of hormone carried by the TTRMet119 variant. To determine whether this increase in binding was due to a change in affinity or capacity, TTR was partially purified from normal individuals and those with the TTRMet119 mutation. The isolation procedure was a rapid, single step passage through Blue Sepharose. With normal serum, the resulting protein bound T4 with a single site of intermediate affinity (Ka, 1.63 +/- 0.36 x 10(7) L/mol). No sites of higher or lower affinity were detected. Comparisons of binding capacity and immunoreactive TTR concentrations showed that the preparations bound T4 with a molar ratio between 1-2. With TTRMet119 serum, the T4 affinity was approximately doubled [Ka, 3.40 +/- 0.76 x 10(7) L/mol (+/- SD); P < 0.001] with no change in binding capacity. This doubling in affinity explains the observed T4 levels of about 120 nmol/L in individuals with this mutation. Binding of rT3 to TTRMet119 was increased approximately 5-fold over normal. Identical experiments with TTRGly54, in which glycine is substituted for glutamine, showed that the T4 affinity of this variant was unchanged from normal. These results suggest that the TTRMet119 mutation leads to secretion of a normal concentration of TTR that has a raised affinity for T4. Depending on their location, mutations in the TTR gene may lead to an increase or no change in T4 binding by the secreted protein.

Genetic Variation↗
PubMed1994

Synthesis and characterization of anti-idiotypic anti-T4 antibodies.

We injected rabbits with purified monoclonal murine immunoglobulin (IgG1) or polyclonal antithyroxine antibodies (anti-T4) and polyclonal anti-triiodothyroacetic acid (anti-Triac) antibodies to stimulate the production of anti-idiotypic antibodies. Purified immunoglobulins from all five rabbits immunized with monoclonal primary antibodies were able to inhibit the interaction between [125I]T4 and the primary antibody. The preimmune sera were inactive. This effect was not due to endogenous T4 contamination or contamination with the injected primary antibody. Half-maximal inhibition of binding of primary antibody with anti-idiotype was between 1.6 and 30 micrograms of total immunoglobulins. Addition of normal mouse IgG1 did not alter the inhibitory effect of the anti-idiotypic antibody, suggesting that this effect is specific. These anti-idiotypic antibodies reacted differently with different polyclonal antibodies, reflecting the heterogeneous nature of polyclonal antibody populations. Polyclonal antibodies were less effective in stimulating anti-idiotypic antibody production. One polyclonal anti-T4 and one anti-Triac antibody produced weak anti-idiotypic antibody that had to be used at a concentration of > 600 micrograms of total immunoglobulins to be inhibitory. Both inhibited the binding of T4 to the monoclonal anti-T4 antibody. However, they were ineffective in inhibiting the function of their own antigen, the polyclonal anti-T4 or anti-Triac antibody. We tested the most potent anti-idiotypic antibodies for their ability to compete with T4 for other T4-binding proteins. Specific inhibition of T4 binding to thyroid-binding globulin was observed with half-maximal effect at approximately 450 micrograms of total IgG.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Stimulation of sex hormone-binding globulin mRNA and attenuation of corticosteroid-binding globulin mRNA by triiodothyronine in human hepatoma cells.

We examined the time course and dose response of the triiodothyronine (T3) effect on mRNAs for sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) in cells of the human hepatoma line HepG2. After 7 h of exposure to a saturating dose of T3, SHBG mRNA was unchanged but increased to 1.5 +/- 0.1 times the unstimulated control at 22 h. Maximal stimulation (2.3 +/- 0.6) was observed at 2-3 days. Corticosteroid-binding globulin mRNA was unchanged for 22 h after exposure to T3 but diminished thereafter to 64% by day 3. At 3-4 days of exposure, the changes in both SHBG mRNA and CBG mRNA were dose-responsive to the T3 concentration. For both mRNAs, half-maximal response occurred between 10 and 20 pmol/l bioavailable T3. Cortisol-binding proteins secreted by HepG2 cells after 3 days in culture also were T3 dose-responsive. No re-uptake of secreted CBG by the cells was observed, suggesting that the T3 effect on CBG secretion occurs during production of the mature protein. These data suggest that T3 stimulates the expression of the SHBG gene and attenuates the expression of the CBG gene. The effects of T3 on these genes are consistent with the increase in circulating SHBG and decrease in circulating CBG observed in hyperthyroidism. The HepG2 cells may be a useful human cell line in which to study the diversity of the molecular mechanisms of T3 action.

Carcinoma, Hepatocellular↗
PubMed1994

Drug competition for intracellular triiodothyronine-binding sites.

A variety of substances, including frusemide, non-esterified fatty acids (NEFAs) and non-steroidal anti-inflammatory drugs (NSAIDs), can compete for triiodothyronine (T3)-binding sites in serum and at the cell surface. We examined the competitive potency of these agents at intracellular T3-binding sites in order to assess their potential to act as T3 antagonists. Competition for [125I]T3 binding was determined using hydroxyapatite separation in cytosols and nuclear extracts prepared from livers of Macaca fascicularis. The T3 affinities were 15.8 +/- 1.2 nmol/l in cytosol and 0.23 +/- 0.02 nmol/l in nuclear extract. Dose-response curves were analysed by a four-parameter sigmoid curve-fitting program to determine competitor potency. The nineteen agents tested included various NSAIDs, NEFAs, non-bile acid cholephils (NBACs), frusemide, amiodarone and the flavonoid EMD 21388. In nuclear extract the most active competitors were linoleic acid (8.5 mumol/l) and linolenic acid (7.8 mumol/l). Potencies of NSAIDs varied between 66 mumol/l (meclofenamic acid) and 525 mumol/l (diclofenac). In cytosol, NEFAs were less potent but NSAIDs were stronger competitors than in nuclear extract. Half-inhibitory potencies in cytosol were between 13.2 mumol/l (meclofenamic acid) and 63.1 mumol/l (flufenamic acid). The NBAC bromosulphthalein was one of the most potent inhibitors in both cytosol and nuclear extract. When expressed relative to T3, diclofenac was a more effective competitor in cytosol than it was in nuclear extract. Amiodarone and EMD 21388 were without effect both in cytosol and nuclear extract. Frusemide (759 mumol/l) was weakly active in cytosol only.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiodarone↗
PubMed1993

Influence of calmodulin antagonists and calcium channel blockers on triiodothyronine uptake by rat hepatoma and myoblast cell lines.

The influence of calcium-related mechanisms on cellular uptake of triiodothyronine (T3) has not yet been defined, although it is known that T3 can stimulate cellular entry of calcium. We therefore investigated the saturable uptake of [125I]-T3 (10(-11) mol/L) from serum-free medium in vitro by hepatoma (H4) cells and skeletal myoblast (L6) cells to establish the calcium-dependency of this process. We studied the effects of the following three structurally distinct types of calmodulin antagonists in H4 cells: the naphthalene sulfonamides W7, W12, and W13, calmidazolium, and trifluoperazine. Uptake of [125I]-T3 as a percentage of control values (n = 4, 10(-4) mol/L antagonist) was as follows: W7, 42.0% +/- 3.3% (P < .001); W12, 87.5% +/- 4.5% (NS); W13, 79.5% +/- 2.5% (P < .05); calmidazolium (10(-6) mol/L, n = 8), 55.1% +/- 2.2% (P < .001); and trifluoperazine (10(-5) mol/L, n = 6), 65.7% +/- 4.1% (P < .001). To investigate whether the calmodulin sensitivity of uptake was mediated via transmembrane calcium flux, we also studied the effects of three structurally distinct types of organic calcium channel blockers in both H4 and L6 cells. [125I]-T3 uptake as a percent of control values (10(-4) mol/L blocker, n = 4) was as follows: nifedipine, 8.6% +/- 0.9% (H4) and 16.7% +/- 7.2% (L6); verapamil, 24.6% +/- 3.2% (H4) and 61.9% +/- 4.2% (L6); diltiazem, 62.7% +/- 3.6% (H4) and 36.1% +/- 5.4% (L6); all P < .001. Eadie-Hofstee analysis indicated competitive inhibition of T3 uptake for both calmidazolium and nifedipine.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1993

A naturally occurring furan fatty acid enhances drug inhibition of thyroxine binding in serum.

We studied the thyroxine (T4)-displacing effects of a naturally occurring, highly albumin-bound furanoid acid that accumulates in serum in renal failure to concentrations in excess of 0.2 mmol/L. This substance, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), has been shown to displace acidic drugs from albumin binding. The effects of CMPF on ligand binding were assessed in the following systems: (1) T4 binding to T4-binding globulin (TBG) and transthyretin (TTR), (2) T4 binding in undiluted serum, (3) T4-displacing potency of fenclofenac, furosemide, diflunisal, and aspirin in undiluted serum, (4) serum binding of [14C]-drug preparations, and (5) serum binding of [14C]-oleic acid. CMPF had a minor direct effect on T4 binding to TBG comparable in relative affinity to that of aspirin, ie, almost 7 orders of magnitude less than T4 itself. CMPF alone at a concentration of 0.3 mmol/L, which produced only a 10% to 14% increase in free T4 augmented the T4-displacing effects of high therapeutic concentrations of the various drugs in undiluted serum as follows: furosemide by 180%, fenclofenac by 160%, diflunisal by 130%, and aspirin by 40%. In the presence of fenclofenac, increments of CMPF from 0.075 to 0.3 mmol/L progressively augmented the T4-displacing effect of this drug, associated with a progressive increase in its calculated free concentration. CMPF also inhibited the binding of [14C]-oleic acid, suggesting that in some situations CMPF could also indirectly influence thyroid hormone binding by increasing the unbound concentration of nonesterified fatty acids (NEFA), as previously described.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1992

Characterization of cytoplasmic T3 binding sites by adsorption to hydroxyapatite: effects of drug inhibitors of T3 and relationship to glutathione-S-transferases.

To facilitate studies of thyroid hormone (T3) binding to cytoplasmic proteins, we prepared monkey (M. fascicularis) liver cytosol (100,000g supernatant) and examined T3 binding using hydroxyapatite (HAP) separation. HAP adsorbs cytoplasmic and nuclear binding sites but not serum T4 binding proteins. Cytosol was incubated with [125I]T3 for 30 min at 4 degrees C and separated by adding an equal volume of HAP (15 g/100 mL). After a further incubation of 10 min, the HAP pellet was washed three times in buffer containing Triton X-100, 0.5%. With this method, a single class of T3 binding site was observed with Kd 15.8 +/- 1.2 nM, concentration 0.62 +/- 0.17 pmol/mg protein (n = 3, mean +/- SD). We used this assay to assess potential drug inhibitors of cytoplasmic binding and to evaluate the proposal that glutathione-S-transferases (GST) and cytoplasmic T3 binding proteins are identical. Displacement of [125I]T3 by unlabeled iodothyronines relative to T3 (100) was T4 58, Triac 7, rT3 7, Tetrac less than or equal to 1. This hierarchy indicates that this binding site is distinct from nuclear or serum binding sites. T3 binding was displaceable by nonsteroidal anti-inflammatory drugs (NSAID) and nonbile acid cholephils (NBAC). Half-inhibitory concentrations (microM, mean +/- SD, n greater than or equal to 3) were diclofenac 4.9 +/- 1.3, mefenamic acid 13.6 +/- 0.6, bromosulphthalein 45 +/- 3, iopanoic acid approximately 200. Amiodarone and furosemide were inactive up to 100 microM. No displacement was observed with cortisol or the bile acid taurocholate, up to 100 microM. Dithiothreitol, 5 mM, did not change binding affinity or capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiodarone↗
PubMed1992

Effects of thyroid hormone on sex hormone-binding globulin gene expression in human cells.

We have used a human hepatoblastoma cell line to establish a model system for thyroid hormone (T3) action in human cells. HepG2 cells were grown for 3 days in Dulbecco's Modified Eagle's Medium containing fetal calf serum and were maintained in serum-free medium for experimental manipulations. [125I]T3 incubated with cells was bound by newly secreted protein and degraded. After 24-h exposure to HepG2 cells in Dulbecco's Modified Eagle's Medium, only 35-40% of the radioactivity was recovered as authentic T3. Degradation of hormone was neither time nor concentration dependent, and occurred to a greater degree in the absence of cells, suggesting an interaction between the hormone and the plastic culture dish. After 4 days, in the absence of fetal calf serum and considering hormone binding and degradation, the concentration of free T3 available to cells was approximately 15% of that added initially. Sex hormone-binding globulin (SHBG) was secreted by HepG2 cells in the absence of T3 and was specifically stimulated by the addition of T3. After 4 days, maximum stimulation occurred with added T3 concentrations of 10(-8) M or greater, and half-maximal stimulation of SHBG secretion was observed at about 3 x 10(-11) M free T3. No significant changes in total secreted protein or cellular DNA content were observed under similar conditions. Northern analysis of RNA extracted from HepG2 cells revealed a SHBG mRNA of 2 kilobases, which was stimulated in a dose-responsive manner by T3. No stimulation of corticosteroid-binding globulin mRNA was seen. Stimulation of the SHBG gene in HepG2 cells may be a useful model for investigation of T3 action in human cells.

Blotting, Northern↗