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S Benvenga

Publications and source records attributed to S Benvenga.

At least 73 records · Page 4Linked to original sources
PubMed1993

Characterization of thyroid hormone binding to apolipoprotein-E: localization of the binding site in the exon 3-coded domain.

Apolipoprotein-E (apoE) has been shown by noncovalent binding and photoaffinity labeling with [125I]T4 to possess a single L-T4 binding site with a K5 of about 3 x 10(7) M-1 and a relative affinity for analogs of L-T4 = D-T4 = rT3 = triiodothyroacetic acid > L-T3. T4 binding was not affected by the flavonoid EMD 21388 or heparin, but was inhibited by diclofenac = mefenamic acid > furosemide. Localization of the T4 site to the N-terminal 62-amino acid region of the mature peptide coded by exon 3 was deduced from the following evidence. 1) The N-terminal 15- to 26-kilodalton (kDa) fragments (within residues 1-160 to 210), but not the approximately 10- to 11-kDa fragments (within residues approximately 220-299), were labeled by [125I]T4. 2) Variants apoE2 and apoE4, with nonconservative mutations at positions 112 and 158 (the latter unable to interact with the apoB/E receptor), maintained the ability to bind T4. 3) Monoclonal antibodies MAb 1D7 and 3H1 (epitopes at positions 139-169 and 243-272, respectively) failed to inhibit T4 binding, but MAb 6H7 (epitope at 1-125) decreased labeling by about 24%. 4) Polymers of apoE were specifically labeled despite the interaction between amphipathic alpha-helices of the exon 4-encoded region (63-299). We conclude that apoE, as previously observed with apoA-I and apoB, possesses a T4-binding domain separate from the lipid-binding domain and distinct from both the heparin- and the cell receptor-binding sites. Thyroid hormone binding by apoE may facilitate uptake of the hormone by cells through apoB/E receptors, which are widely distributed in tissues.

Affinity Labels↗
PubMed1992

Thyroxine binding to the apolipoproteins of high density lipoproteins HDL2 and HDL3.

Four preparations of high density lipoprotein HDL2, five of HDL3, and purified apolipoproteins apoA-I, apoA-IV, and apoE were photoaffinity labeled with [125I]T4 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were also immunoblotted with antiserum against apoA-I, apoA-II, apoA-IV, apoE, or apo(a), and the immunostained membrane was then autoradiographed. In HDL2, the two major radioactive bands migrated near the origin of the resolving gel and at 28-31 kilodaltons (kDa). The first band, stained by anti-apo(a) and anti-apoB-100, accounted for 40-96% of the total radioactivity and was attributed to lipoprotein(a), which is isolated in the same density range as HDL2. The second band, stained by anti-apoA-I, accounted for 1-57% [41-95% after correction for contaminating lipoprotein(a)] of the [125I]T4 in the resolving gel. In HDL3, the major radioactive band was identified as apoA-I and contained 93-94% of the [125I]T4 in the resolving gel. Minor radioactive bands in both HDL2 and HDL3 were identified as apoA-II (17-18 kDa), apoA-II monomer (7-10 kDa), apoE (36-38 kDa), and apoAII-apoE heterodimer (46 kDa). In addition, HDL3 contained apoA-IV (43 kDa). Photoaffinity labeling of isolated apoA-IV and apoE showed that each protein interacted with [125I]T4. In both HDL2 and HDL3, photoaffinity labeling in the presence of unlabeled L-T4 (1-10 microM) showed inhibition, suggesting a Kd in the micromolar range. This inhibition varied among different apo bands of the same HDL2 or HDL3 preparation and among the same bands of different preparations. Labeling in the presence of heparin or other inhibitors of T4 binding to plasma proteins (furosemide, diclofenac, and mefenamic acid) showed that HDL2-associated apoA-I was more sensitive to inhibition than HDL3-associated apoA-I. In conclusion, 1) HDL2 and HDL3 carry T4 mainly through apoA-I and secondarily through apoA-II and apoE. The inter- and intrasubclass variations in T4 binding and sensitivity to inhibitors can be explained by the known heterogeneity of HDL particles and possible differences in conformation of the apo. The findings reported here, that apo other than apoA-I and apoB exhibit saturable binding of T4, suggest that thyroid hormone-lipoprotein interactions may have even wider physiological implications than previously appreciated.

Affinity Labels↗
PubMed1991

The thyroxine-binding site of human apolipoprotein-A-I: location in the N-terminal domain.

We tested the ability of nine monoclonal antibodies (MAb) against human apolipoprotein-A-I (apoA-I), the 28.3-kDa major apoprotein of high density lipoproteins (HDL), to inhibit its photoaffinity labeling with [125I]T4. Two forms were evaluated: isolated lipid-free apoA-I (Sigma or Calbiochem) and lipid-complexed apoA-I [HDL2, (density, 1.063-1.125 g/ml) and HDL3 (density, 1.125-1.210 g/ml)]. After labeling with 0.5 nM [125I]T4 in the presence of MAb or normal mouse IgG, the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent densitometric quantitation of radioactivity associated with the 28.3-kDa band. Group I MAbs, namely those having epitopes in the N-terminal portion of apoA-I, include MAb 16 (epitopes at residues 1-16), 4 and 14 (residues 1-86), and 18 (residues 98-105); group II includes MAbs 7,10, 15, and 17 (epitopes at residues 87-148); group III includes MAb 9 (residues 149-243). All group I MAbs inhibited [125I]T4 binding to isolated apoA-I with this order of potency: MAb 16 (-50% to -61%) greater than MAb 14 (-37% to -41%) greater than MAb 4 (-27% to -33%) greater than MAb 18 (-19% to -27%). In the case of lipid-associated apoA-I, the pattern of hierarchy was variable, presumably related to the known markedly polydisperse nature of HDL, but a constant feature, in contrast to the case of isolated apoA-I, was that MAb 4 was more potent than MAb 14. Group II MAbs gave less than 3% inhibition in both isolated and lipid-complexed apoA-I. Group III MAb 9 either failed to inhibit or gave 18-27% inhibition (one preparation each of HDL2 and HDL3). We conclude that the T4 site of apoA-I is in the N-terminal domain of apoA-I, closer to the epitope for MAb 16 than to that for MAb 18, and that conformational changes occurring when apoA-I is associated with lipids in the HDL particle alter the spatial relationship between some epitopes and the T4 site. Our definition of the T4 site of apoA-I is consistent with another set of data showing that heparin failed to inhibit [125I]T4 binding to isolated apoA-I. Heparin is known to interact with clusters of basic residues, and these residues are concentrated in the midregion of apoA-I.

Antibodies, Monoclonal↗
PubMed1990

Enhancement of thyroxine entry into low density lipoprotein (LDL) receptor-competent fibroblasts by LDL: an additional mode of entry of thyroxine into cells.

Having demonstrated that plasma low density lipoproteins (LDL) bind T4 through a specific interaction with their sole apolipoprotein, apoB-100, we tested the hypothesis that cells could internalize the LDL-T4 complex via cell surface LDL receptors. These receptors are down-regulated by cholesterol loading and up-regulated by cholesterol deficiency. We, therefore, studied the uptake of [125I]T4 or [125I]T3 by human skin fibroblasts grown in 10% lipoprotein-deficient serum in the absence or presence of LDL. At concentrations of LDL (12.5 and 22 micrograms protein/ml) that gave significant binding of T4 but did not exceed LDL receptor capacity, both the initial rate of saturable T4 uptake and the uptake at equilibrium increased by 27-63%. No significant increase occurred at a LDL concentration of 1.6 micrograms protein/ml (less than 3% occupancy), whereas there was a 20 to 31% reduction at 125 micrograms/ml (approximately 5 times the saturation dose for the LDL receptor). These changes were confirmed with several different LDL preparations and were mimicked by isolated apoB-100 and apoE, the sole ligands for the LDL receptors (apoB/E receptors). T4 uptake did not increase in normal fibroblasts with down-regulated LDL receptors or in LDL receptor-deficient fibroblasts from a patient with familial hypercholesterolemia. In the latter cell line the uptake of T4 (and T3) in the absence of LDL was indistinguishable from that of normal fibroblasts. T3 uptake in normal fibroblasts was not enhanced by LDL. The specificity of the LDL effect was shown by the finding that T4-binding globulin, prealbumin, or serum albumin, at concentrations giving 10-90% T4 bound, failed to increase T4 uptake. Instead, each of these major thyroid hormone-binding plasma proteins caused a dose-dependent decrease in T4 entry. It is concluded that at least two modes of entry into fibroblasts are available for T4. The first is the cell surface thyroid hormone-binding sites, which recognize the free hormone and are present in both normal and LDL receptor-negative fibroblasts. The second, and additional, mode of entry is via the LDL receptors, which recognize the T4-LDL complex, are absent in LDL receptor-negative fibroblasts, are reduced in down-regulated fibroblasts, and are unavailable for T3, owing to the low affinity of T3 for LDL.

Cell Line↗
PubMed1990

Localization of the thyroxine binding sites in apolipoprotein B-100 of human low density lipoproteins.

By photoaffinity labeling human low density lipoproteins (LDL) with [125I]T4 we confirmed our previous observation that of the three T4 binding sites of apolipoprotein B-100 (apoB-100) one is in its 26% NH2-terminal portion [apoB-26, the 140 kDa fragment, residues 1-1297] and two in the remaining 74% COOH portion (apoB-74, 410 kDa, residues 1298-4536). We now show that of these two sites one is in the NH2 portion of apoB-74 (apoB-44, 240 kDa, residues 1298-3249) and the other in the nonoverlapping COOH portion (apoB-30, 170 kDa, residues 3250-4536). ApoB-100 contains 13 binding sites for heparin, a known inhibitor of T4 binding to the major T4 carrier plasma proteins; however, heparin failed to inhibit T4 binding to apoB-100 and fragments thereof. The same failure was seen with three monoclonal antibodies (MAbs), 4G3, 5E11, and 43 that block totally or partially LDL binding to the LDL receptor [respective epitopes at residues 2980-3084 (apoB-44), 3441-3569, and 4027-4081 (apoB-30)]. Of the other 3 MAbs, all without effect on LDL binding to the LDL receptor, [1D1, 2D8, and 16, respective epitopes at residues 474-539 (apoB-26), 1438-1481 (apoB-44), and 4154-4189 (apoB-30)], only two (MAbs 1D1 and 2D8) inhibited T4 binding (21 to 39%). We conclude that the three T4-binding sites of apoB-100 are outside the LDL receptor binding domain, distant from the heparin binding sites and, assuming no allosteric effects, in the vicinity of residues 474-539 (T4 site of apoB-26), 1438-1481 (T4 site of apoB-44), and in the C terminal quarter of apoB-30.

Antibodies, Monoclonal↗
PubMed1990

Defective neuromotor and cognitive ability in iodine-deficient schoolchildren of an endemic goiter region in Sicily.

Visual perceptual integrative motor ability was investigated in 719 6- to 12-yr-old, presumably normal, primary schoolchildren living in 2 iodine-deficient endemic goiter areas in Sicily, identified on the basis of the presence (area A) or absence (area B) of endemic cretinism, by administrating the Bender Gestalt test. All of these clinically euthyroid schoolchildren were also examined neurologically by an investigator unaware of the result of the Bender test. Ninety-nine (13.76%) schoolchildren were found to be defective by the Bender test; this prevalence was significantly higher than that (3.0%) found in an iodine-sufficient goiter-free control area (area C) lying at sea level (chi 2 = 36.25; P less than 0.000001). No difference in the prevalence of Bender abnormalities was apparent if the children were divided according to the area of provenience (area A, 14.4%; area B, 13.1%). A high percentage of children falling in the lower range of normality was found in both area A (15.5%) and area B (19.0%); this was significantly higher than that in area C (3.8%; chi 2 = 77.55; P less than 0.000001). Neuromuscular and neurosensorial abnormalities, including increased tendon reflexes, clonus of the foot, Babinski sign, minor disturbances in balance, and gait, and minor defects in hearing and speech, were apparent in 19.3% (area A) and 18.5% (area B) of the children. These disorders were significantly more frequent in defective children identified by the Bender test (33.3%) than in normal children (15.3%; (chi 2 = 17.29; P less than 0.00005). The general intellectual aptitude in Bender deficient subjects was evaluated by the Terman Merrill test and was found to be impaired in 95%, thus confirming the existence of an endemic cognitive deficiency (ECD), distinct from the endemic mental deficiency previously found in other endemic goiter, iodine-deficient areas. ECD seems to be epidemiologically independent of the existence of endemic cretinism. Further clinical auxological and biochemical studies in a selected group of ECD children suggested the epidemiological and, possibly, pathogenic association of cognitive impairment with iodine deficiency.

Bender-Gestalt Test↗
PubMed1989

Binding of thyroxine to human plasma low density lipoprotein through specific interaction with apolipoprotein B (apoB-100).

Human plasma low density lipoprotein (LDL), which binds 0.2% of plasma T4, was shown to interact with the hormone through its protein moiety, apolipoprotein B-100. LDL and LDL2, the major subfraction of LDL, were found to have 3 equivalent binding sites for T4 with Ka = 2.5 x 10(6) M-1. Photoaffinity labeling of LDL with inner ring-labeled [125I]T4, followed by SDS-PAGE or agarose-SDS-PAGE of the labeled products, revealed that apoB-100 and its proteolytic cleavage products, apoB-74 and apoB-26, bound [125I]T4. In the presence of 1 or 10 microM T4, labeling was decreased in 7 separate experiments by 40-53% or 65-86%, respectively, consistent with a Ka of approximately 10(6) M-1. Binding of T4 to apoB-100 associated with VLDL was also demonstrated by photoaffinity labeling. The observed thyroid hormone binding property of lipid-complexed apoB-100 and the knowledge that receptors for the apolipoprotein exist in various tissues suggest a possible physiological role in thyroid hormone transport.

Apolipoproteins B↗
PubMed1989

The 27-kilodalton thyroxine (T4)-binding protein is human apolipoprotein A-I: identification of a 68-kilodalton high density lipoprotein that binds T4.

We have identified a previously described 66K T4-binding protein as a lipoprotein composed of two molecules of apolipoprotein A-I, five of cholesteryl esters, nine of phosphatidylcholine, and two of sphingomyelin. This 68.4K high density lipoprotein (HDL) corresponds to the minor approximately 67K HDL subfraction that we recently demonstrated as binding most of the HDL-associated T4. Since we have recently found that lipids inhibit the binding of T4 to apolipoprotein A-I, the very low lipid content (16 mol/mol) of this 68K HDL relative to that (greater than 100 mol/mol) of high mol wt HDL subfractions may account for the preferential binding of T4 to the low mol wt subfraction.

Apolipoprotein A-I↗
PubMed1989

Characterization of the binding of thyroxine to high density lipoproteins and apolipoproteins A-I.

We studied binding of T4 to the lipid-complexed apolipoproteins (apo) of high density lipoproteins (HDL), the major lipoprotein carrier of thyroid hormones in human plasma, and to lipid-free apoA-I. HDL isolated from fresh normal plasma by ultracentrifugation (density, 1.063-1.210 g/mL) was photoaffinity labeled with [3,5-(125)I]T4 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands corresponding to apoA-I (28.3K) and apoC-II or apoC-III (8.6-9.2K) were seen, and their radioactivity decreased by 50-60% when labeled in the presence of 1 mumol/L T4. Photoaffinity labeling of isolated apoA-I also was demonstrated and was decreased 74% by 1 mumol/L T4, suggesting a higher affinity of the lipid-free protein for T4. T4 binding of isolated apoA-I was optimal at pH 7-8, reached a maximum after 1 h at 23 C, and decreased after incubation at 37 C. Scatchard analysis revealed a single T4-binding site with a Ka of 7.5 x 10(7) L/mol at 23 C, pH 8.2. The potency of T4 analogs as inhibitors of T4 binding to isolated apoA-I was L-T4 = D-T4 = triiodothyroacetic acid = L-rT3 much greater than L-T3 much greater than L-thyronine. The binding of T4 to apoA-I was reduced by known inhibitors of T4 binding to serum proteins (diclofenac = mefenamic acid = furosemide = 8-anilinonaphthalene sulfonic acid much greater than dilantin greater than heparin greater than barbital) and by lipids (unsaturated fatty acids greater than cholesterol = cholesterol esters = phospholipids greater than saturated fatty acids = diglycerides = triglycerides). We conclude that the binding of T4 to HDL is mediated by a specific interaction of the hormone with apoA-I and with apoC-II and/or apoC-III. Since the lipid constituents of HDL inhibit T4 binding to apoA-I, the HDL subfraction in plasma that carries most of the HDL-bound T4 should be one with a low lipid content.

Apolipoprotein A-I↗
PubMed1988

Binding of thyroid hormones to human plasma lipoproteins.

The binding of T4, T3, and rT3 to plasma lipoproteins was investigated in normal subjects and patients with abnormal lipoprotein metabolism. Gel filtration on Sepharose CL-6B demonstrated iodothyronine binding to all lipoprotein classes. In the total lipoprotein fraction (density less than 1.210 g/mL), high density lipoproteins (HDL) were the major binders, accounting for 92% of lipoprotein-bound T4, 99% of lipoprotein-bound T3, and 55% of lipoprotein-bound rT3. The estimated iodothyronine binding in normal plasma to HDL, low density lipoproteins (LDL), and very low density lipoproteins (VLDL) was 3%, 0.2%, and 0.03% for T4, 6%, 0.05%, and 0.02% for T3, and 0.1%, 0.1%, and 0.01% for rT3, respectively. These estimates may be low owing to possible dissociation during chromatography and the short incubation period used to avoid changes in lipoprotein structure. In VLDL and LDL deficiency (abetalipoproteinemia), HDL deficiency (Tangier disease), LDL excess (type IIa hyperlipoproteinemia), and VLDL excess (type III, IV, and V hyperlipoproteinemia), the distribution of iodothyronines reflected the lipoprotein abnormality. Variations resulting from altered distribution within HDL subclasses were also found. Binding was saturable, with approximate dissociation constants for VLDL, LDL, and HDL of 10(-5)-10(-6) mol/L. We conclude that thyroid hormones bind specifically to apolipoproteins, although additional binding by solubilization in the lipid components of the lipoproteins may also occur.

Abetalipoproteinemia↗
PubMed1988

Autoantibodies to thyroxin and triiodothyronine in the immunoglobulin G fraction of serum.

Thyroid hormone antibodies (THAbs)--i.e., antibodies to thyroxin (T4) and triiodothyronine (T3)--are detected rarely in human serum, where they are searched for, possibly because of a quantitatively minimal interaction between thyroid hormones (the haptens) and serum IgGs (the antibodies). The weak binding could result from these facts: (a) there are already six physiological carrier proteins for thyroid hormones; (b) THAbs usually account for a very small fraction of the total serum IgGs; (c) THAbs may have--as reported in the literature--a relatively low affinity. To ascertain whether THAbs could pass undetected in serum, we measured antibodies to T3 and T4 in both the serum and the corresponding IgG fraction of six normal persons and 45 patients with various thyroid diseases (Graves' disease, idiopathic myxedema, Hashimoto's thyroiditis, subacute thyroiditis, tumors), using radioimmunoprecipitation. The prevalence of antibodies to T4 was 0/51 in both the sera and the IgG fractions; the prevalence of antibodies to T3 was 1/51 in both materials. Because all of the sera that tested THAb negative were confirmed to be so in the THAb assay of the IgG fraction, we conclude that the prevalence of serum THAbs is not underestimated and that autoimmunization against thyroid hormones is really a rare phenomenon.

Autoantibodies↗
PubMed1987

Abnormal daily periodicity of serum thyrotropin (TSH) and evidence for defective TSH suppression in a case of non-neoplastic syndrome of inappropriate TSH secretion.

A non-neoplastic syndrome of inappropriate secretion of TSH (ITSHS) was diagnosed in a hemithyroidectomized and clinically euthyroid 44-yr-old man, who also exhibited limping (Perthes' disease), genu valgum, pes supinatus and lateral nystagmus. Computed tomography demonstrated an enlarged sella turcica due to empty sella. Baseline serum T3, T4, free T3, free T4 and TSH fluctuated between 179 and 274 ng/dl, 6.0 and 13.2 micrograms/dl, 4.2 and 6.0 pg/ml, 7.6 and 15.3 pg/ml, and 4.3 and 33.0 microU/ml, respectively. Serum alpha-TSH subunit was repeatedly normal (0.36-0.69 ng/ml) over the follow-up period (greater than 3 yr). No changes in serum liver enzymes and lipids were observed after thyroid hormone administration, whereas red blood cell glucose-6-phosphate dehydrogenase (G-6-PD) and urinary OH-proline were slightly enhanced during 120 micrograms/day L-T3 regimen. This also resulted in an inappropriately normal glucagon-stimulated cAMP levels. Tachycardia was experienced only during L-T3 and very high L-T4 dose treatments. Therefore, the patient showed some evidence for thyroid hormone peripheral refractoriness. Patient's TSH was physiologically responsive to agents (thyrotropin releasing hormone, methimazole, the dopamine antagonists domperidone and sulpiride) known to elicit its release into circulation, while it responded paradoxically to those which normally inhibit TSH secretion. In fact, the infusion of somatostatin (320 micrograms/h) or dopamine (4 micrograms/Kg/min), and the oral administration of bromocriptine or nomifensine (two dopamine agonists) or corticosteroids (dexamethasone) provoked an unexpected elevation of both unstimulated and TRH-stimulated TSH levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1987

Abnormalities in pituitary thyroid axis function tests in patients with paroxysmal supraventricular arrhythmias.

The study was carried out on 60 consecutive patients (23 males and 37 females) aged between 20 and 83 years (means +/- SD, 40.7 +/- 16) who arrived at our Cardiologic Unit with paroxysmal supraventricular arrhythmias (PSVA) including junctional paroxysmal tachycardia (n = 32), atrial fibrillation (n = 13), atrial flutter (n = 1), premature beats (n = 13) and with no obvious cardiovascular causes. Serum thyroxine and triiodothyronine were normal in all patients and thyroid scintiscan revealed normal shape and size thyroids without autonomously functioning nodule(s). Thyrotropin (TSH) response to thyrotropin releasing hormone (TRH) was normal in 44 subjects in whom normal serum free T4 (FT4) and free T3 (FT3) levels were measured. Six patients with normal FT4 and FT3 levels did not respond to TRH. Abnormalities in thyrotropin response to TRH were observed in 10 patients all exhibiting increased FT4 or also FT3 levels. Among these, 5 patients did not respond to TRH, whereas the remaining 5 exhibited a blunted TSH response to TRH. These results suggest that only in a small proportion (5/60) of consecutive patients with PSVA it is possible to recognize a status of "occult thyrotoxicosis" on the basis of the combined evaluation of free thyroid hormones and TSH response to TRH.

Adult↗