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Biomedical subjects

D S Goodman

Publications and source records attributed to D S Goodman.

At least 91 records · Page 5Linked to original sources
PubMed1983

Ultrastructural visualization of low-density lipoproteins during receptor binding and cellular endocytosis.

Human fibroblasts possess surface receptors which have a high affinity for low-density lipoproteins (LDL). However, previous studies have not provided direct ultrastructural visualization of LDL bound to the receptor. To permit direct observation of unlabeled LDL during receptor binding and cellular endocytosis, we examined several fixative regimens which employ lipophilic stains. Staining with tannic acid, an oxidized form of ruthenium red, or potassium ferrocyanide imparted sufficient contrast to individual molecules of LDL to permit high-resolution electron microscopy of receptor binding and endocytosis. The LDL molecule was observed in immediate contact with the receptor and the coated vesicle, indicating that receptor-ligand binding occurs by short-range interactions.

Cell Membrane↗
PubMed1983

Presence of an abnormal transthyretin (prealbumin) in Portuguese patients with familial amyloidotic polyneuropathy.

In summary, these studies have demonstrated the following: 1) plasma TTR levels are significantly decreased in Portuguese patients with FAP; 2) FAP-TTR is indistinguishable from normal TTR with regard to a wide range of physiochemical properties; 3) RBP levels and vitamin A transport appear to be normal in FAP patients; 4) an abnormal TTR with a methionine for valine substitution at position 30 is found as amyloid protein in tissues of patients with FAP; and 5) this abnormal TTR is present in small amounts in plasma of patients with FAP. Based on these observations, we postulate that this abnormal form of TTR selectively deposits in tissues as the amyloid characteristic of the disease.

Adult↗
PubMed1983

Studies on plasma transthyretin (prealbumin) in familial amyloidotic polyneuropathy, Portuguese type.

Amyloid deposits in several heredofamilial forms of amyloidosis are known to be chemically related to transthyretin (TTR, the plasma protein usually referred to as prealbumin). A genetic mutation, leading to an abnormal TTR, may be involved. Studies were conducted to investigate whether or not Portuguese patients with familial amyloidotic polyneuropathy (FAP) have an abnormal species of TTR circulating in their plasma and, if so, whether this might have any impact on vitamin A transport and retinol-binding protein (RBP) metabolism in these patients. The initial studies examined the plasma concentrations of TTR, RBP, and retinol in patients with FAP. Significantly reduced (p less than 0.005) levels of TTR were found in patients with FAP. The TTR levels in 24 FAP patients were approximately two thirds of those of a group of 18 control subjects from the same geographic area. In contrast, RBP levels were not reduced, nor was there an abnormality in the ratio of retinol to RBP, in the patients with FAP. Thus there does not appear to be an abnormality in vitamin A transport in Portuguese patients with FAP. There does, however, appear to be an abnormality of TTR metabolism, accounting for the reduced plasma levels of TTR. TTR was isolated from pooled sera of the FAP patients and was characterized in detail. FAP-TTR was indistinguishable from normal TTR with regard to a variety of parameters, including (1) electrophoretic mobility, (2) chromatographic behavior, (3) molecular weight, (4) stability of the TTR tetramer, (5) immunoreactivity in TTR radioimmunoassays using antisera prepared against both normal and FAP-TTR, (6) ability to form a protein-protein complex with RBP and affinity for RBP as assessed by polarization of fluorescence, and (7) overall amino acid composition. The possible explanations are as follows: (1) an abnormal TTR molecule is not produced in this form of FAP; (2) the abnormal molecule is present in only trace amounts (not detectable in the present study) in FAP plasma; or (3) the abnormal TTR is structurally almost identical to normal TTR and does not differ from normal TTR with regard to the various physical and chemical properties investigated in this study. Preliminary observations suggest that FAP patients do produce an abnormal form of TTR that selectively deposits in tissues as amyloid protein.

Adult↗
PubMed1983

Cholesterol turnover and metabolism in two patients with abetalipoproteinemia.

Total body turnover of cholesterol was studied in two patients with abetalipoproteinemia, a 32-year-old man and a 31-year-old woman. The patients received [14C]cholesterol intravenously, and the resulting specific activity-time curves (for 40 and 30 weeks, respectively) were fitted with a three-pool model. Parameters were compared with those from studies of cholesterol turnover in 82 normal and hyperlipidemic subjects. A three-pool model gave the best fit for the abetalipoproteinemic patients, as well as for the 82 previously studied subjects, suggesting general applicability of this model. Cholesterol production rates in the two abetalipoproteinemic subjects (0.82 and 0.89 g/day) were close to values predicted for persons of their body weight. Thus, total body turnover rate of cholesterol was quite normal in abetalipoproteinemia, confirming previous reports. Very low values (9.2 and 8.4 g) were found for M1, the size of the rapidly exchanging compartment pool 1, in the two abetalipoproteinemic subjects. These values were well below the values predicted (from the comparison study population) for normal persons of this size with low plasma cholesterol levels. For one patient, total body exchangeable cholesterol was very low, although not significantly below the predicted values for a person of his size. In the second patient, the observed estimate for total body exchangeable cholesterol was well within the range of values predicted for persons of her size with low to extremely low cholesterol levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Abetalipoproteinemia↗
PubMed1982

Effect of retinol status on retinol-binding protein biosynthesis rate and translatable messenger RNA level in rat liver.

Studies were conducted to explore the role of retinol in the control of the rate of synthesis of plasma retinol-binding protein (RBP) in the liver of the rat. Previous studies have shown that nutritional retinol status strongly influences RBP secretion from the liver cell. Both the in vivo relative rate of RBP synthesis and the in vitro translatable level of RBP-specific mRNA were examined in normal vitamin A, retinol-depleted, and retinol-repleted rats. The relative rate of RBP synthesis was estimated by measuring the extent of incorporation of [3H]leucine, [3H]lysine, and [3H]phenylalanine after a 12-min pulse label into immunoprecipitable RBP, relative to the incorporation of these amino acids into total liver trichloroacetic acid-precipitable protein. The level of translatable RBP-specific mRNA was quantitated in vitro by translation of rat liver poly(A+)RNA in the rabbit reticulocyte lysate protein-synthesizing system. The amount of newly synthesized RBP was determined relative to the amount of newly synthesized total protein. Both the relative rate of RBP synthesis (approximately 0.26%) and the translatable level of RBP-specific mRNA (approximately 0.14%) were found to be constant regardless of the retinol status of the rats. These results indicate that retinol, the molecule that RBP specifically binds and transports, does not appear to control the rate of synthesis of RBP or the translatable level of RBP-specific mRNA in the liver of the rat. Regulation of plasma RBP levels by retinol must be exercised at a locus beyond that of RBP synthesis.

Animals↗
PubMed1982

Effect of platelet-derived and endothelial cell-derived growth factors on the low density lipoprotein receptor pathway in cultured human fibroblasts.

Human platelet-derived growth factor (PDGF) has been previously shown to stimulate low density lipoprotein (LDL) receptor activity in cultured cells. Studies were conducted to delineate in detail the effects of PDGF on the LDL receptor pathway in normal human fibroblasts and to explore relationships between the effects of PDGF on LDL metabolism, on cholesterol metabolism, and on DNA synthesis. Increasing concentrations of PDGF stimulated parallel increases in both DNA synthesis and 125I-LDL cell surface binding. The effect of PDGF was due entirely to an increase (up to 4.3-fold) in the number of receptor sites per cell, and not to a change in receptor affinity (Kd approximately 2.0 nM). Parallel PDGF concentration-dependent increases in 125I-LDL binding, internalization, and degradation at 37 degrees C were observed. The results indicate that PDGF-stimulated cells metabolize receptor-bound LDL in a manner that is identical with that seen with quiescent cells. A single study with highly purified PDGF demonstrated that it was PDGF itself, and not some other component in the partially purified PDGF preparation used in most of this work, that was responsible for the observed effects. Studies were conducted on the effects of PDGF on hydroxymethylglutaryl CoA reductase activity, on cholesterol esterification, and on down-regulation by LDL of the LDL receptor. These studies indicated that LDL cholesterol taken into the PDGF-stimulated cell via the receptor pathway, appeared to become available normally and to have metabolic effects within the cell similar to those seen in quiescent cells. Fibroblasts from subjects with familial hypercholesterolemia showed a normal mitogenic response to PDGF, despite the absence or near absence of an effect on the LDL receptor pathway. Finally, studies were also conducted with endothelial cell-conditioned medium (ECCM), used as a source of the endothelial cell-derived growth factor. ECCM was similar to PDGF in stimulating LDL binding, but differed strikingly from PDGF in that the degradation of internalized LDL was inhibited. As a result, ECCM-treated cells did not effectively increase cholesterol esterification or suppress hydroxymethylglutaryl CoA reductase activity when LDL was present. These findings with substances produced by endothelial cells may have important implications for atherogenesis.

Binding, Competitive↗
PubMed1982

Retinoid affinity label for the binding site of retinol-binding protein.

Three radioactive retinoid bronoacetates were synthesized as potential retinoid affinity labels for the retinol binding site of human plasma retinol-binding protein (RBP). The compounds synthesized were beta-[9-3H]ionyl bromoacetate (IBA), beta-[11-3H]ionylideneethyl bromoacetate (IEBA), and [15-3H]retinyl bromoacetate (RBA). When excess ligand was incubated with RBP for 5 h at 37 degrees C, IBA and IEBA formed nearly 1:1 molar complexes with RBP, whereas RBA bound only approximately one-third as well. Subsequent addition of retinol to the retinoid-RBP complex resulted in complete displacement of IBA and RBA from the protein, whereas a large proportion (37%) of the [3H]IEBA remained bound to the retinol binding site of RBP. For maximization of covalent bonding of IEBA to RBP, IEBA was incubated with RBP for varying lengths of time, followed, in each instance, by addition of retinol to displace noncovalently bound IEBA. The amount of IEBA remaining bound to RBP increased with increasing incubation time, reaching a maximum of about 0.66 mol/mol of RBP at 18 h. Moreover, at each time point, the binding of retinol to RBP was inhibited to an extent that was equivalent to the amount of [3H]IEBA that was not displaced from RBP by retinol. Only a very small proportion of the bound [3H]IEBA that was not displaced with retinol could be extracted from the protein with chloroform-methanol. Taken together, these several lines of evidence strongly suggest that the IEBA was bound in the retinol binding site of RBP and was attached to the protein in a covalent manner. Thus, IEBA appears to be an effective affinity label for the retinol binding site of RBP.

Affinity Labels↗
PubMed1982

Retinoid-binding proteins.

Much has been learned during the past decade about the specific retinoid-binding proteins that exist in plasma and in the intracellular compartment in a number of tissues. Vitamin A is mobilized from liver stores and transported in plasma in the form of the lipid alcohol retinol, bound to a specific transport protein, retinol-binding protein (RBP). A great deal is now known about the chemical structure, metabolism, and biologic roles of RBP. A number of tissues of rats, humans, and other species contain soluble binding proteins with specificity for either retinol (cellular retinol-binding protein, CRBP) or retinoic acid (cellular retinoic acid-binding protein, CRABP). These intracellular proteins have been purified from several tissues and partly characterized. This paper provides a concise review of our current knowledge about the three retinoid-binding proteins, RBP, CRBP, and CRABP.

Animals↗
PubMed1982

Studies on the mechanism of the inhibition of platelet aggregation and release induced by high levels of arachidonate.

We have previously reported that arachidonic acid induced a biphasic pattern of platelet aggregation and the release of both dense and alpha-granule components. Low levels of arachidonate (0.025--0.1 mM) specifically induced aggregation and release, while high concentrations (0.15--0.35 mM) caused a progressive inhibition of these platelet responses in human gel-filtered platelets (GFP). We now report studies of the mechanism(s) responsible for this arachidonate-induced turn-off of platelet function. Electron micrographic studies demonstrated that there was no gross damage to the platelets during the turn-off. Active synthesis of malondialdehyde and thromboxane A2 was seen at the high arachidonate levels, despite the inhibition of aggregation. Furthermore, GFP inhibited by 0.25 mM arachidonate were capable of undergoing aggregation and serotonin release in response to other stimuli, such as collagen or thrombin. Thus, GFP appeared to be metabolically intact and functional during the inhibiton by high arachidonate levels. Thin-layer chromatographic studies revealed that prostaglandin metabolism was not changed at the high arachidonate levels. In addition, indomethacin (20 microM) did not abolish the arachidonate-induced inhibition of platelet function. Therefore, the inhibitory effect of high arachidonate did not depend on its conversion to other prostaglandin products. Platelet cyclic AMP levels increased twofold at the high arachidonate concentrations (1.3 +/- 0.3 pmole/10(8) platelets at peak aggregation, compared with 2.9 +/- 0.4 pmole/10(8) platelets at inhibition by 0.25 mM arachidonate, p less than 0.001). Prostaglandin-D2, a platelet inhibitor known to increase cyclic AMP, generated a similar rise (to 2.4 +/- 0.2 pmole/10(8) platelets). Thus, the magnitude of the arachidonate-induced increase in platelet cyclic AMP levels can account for the inhibition of aggregation and release.

Arachidonic Acid↗
PubMed1982

Cholesterol turnover in lipid phases of human atherosclerotic plaque.

The turnover of free cholesterol in atheromatous plaque lipid phases was studied in a patient undergoing peripheral vascular surgery. [14C]Cholesterol was injected intravenously 139 days prior to surgery, and [3H]cholesterol was injected 12 days pre-op. The plasma cholesterol specific radioactivity decay curves were determined from the times of isotope injection until surgery. At surgery, atheroma, skin, muscle, and tendon were obtained. Lipid phases of plaque homogenate were isolated by density gradient centrifugation. The top layer of the gradient, layer 1, contained the cholesteryl ester oil droplet phase, layer 2 was enriched in phospholipid bilayer phase, layer 3 contained cholesterol monohydrate crystals and the pellet, layer 4 had more dense plaque components such as collagen and elastin. The tissue:plasma specific radioactivity ratios on days 12 and 139 respectively were muscle, 0.86, 2.47; skin, 0.74, 1.20; tendon, 0.18, 1.45; total plaque, 0.22, 1.39; plaque layer 1, 0.31, 1.50; layer 2, 0.22, 1.53; layer 3, 0.08, 0.61; and layer 4, 0.20, 0.88. Thus, plaque atheroma, which contains physically distinct forms of cholesterol, had correspondingly different rates of cholesterol turnover. Cholesterol solubilized in liquid oil droplets (layer 1) and liquid crystalline phospholipid bilayers (layer 2) had specific radioactivity values similar to those of tendon cholesterol, and represented tissue cholesterol that was undergoing slow equilibration with the plasma cholesterol pool. Pellet cholesterol (layer 4), which is probably connective tissue-associated, had lower specific radioactivity values, well below those of plasma cholesterol even after 5 months. Crystalline cholesterol (layer 3) had the lowest specific radioactivity values of all tissues and plaque fractions. Therefore, cholesterol in the crystalline state is relatively inert. Since crystalline cholesterol can account for over 40% of plaque free cholesterol, resistance to mobilization of this lipid may be an important obstacle to plaque regression.

Aged↗
PubMed1981

Biosynthesis of plasma retinol-binding protein in liver as a larger molecular weight precursor.

A study was performed to identify the first translated product of messenger RNA for retinol-binding protein (RBP), the specific plasma transport protein for vitamin A. Poly(A)+RNA was isolated from rat liver and translated in the rabbit reticulocyte in vitro protein-synthesizing system. RBP was identified and separated from other translated products by immunoprecipitation with specific rabbit anti-rat RBP antiserum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of the immunoprecipitate consistently revealed one major product which migrated more slowly than purified rat serum RBP. The protein (preRBP) had an approximate molecular weight of 24,000. When dog pancreas microsomal membranes were cotranslationally present, the newly synthesized preRBP was processed to a protein which migrated coincidentally with purified rat serum RBP, approximately 20,500 daltons. These results indicate that RBP is initially synthesized as a larger molecular weight precursor (preRBP) which is rapidly processed by the removal of a peptide of approximately 3,500 daltons to the size of the final RBP molecule that circulates in the plasma.

Animals↗
PubMed1981

Retinoid-binding proteins in plasma and in cells.

Much has been learned during the past decade about the specific retinoid-binding proteins that exist in plasma, and in the intracellular compartment in a number of tissues. Vitamin A is mobilized from liver stores and transported in plasma in the form of the lipid alcohol retinol, bound to a specific transport protein, retinol-binding protein (RBP). A great deal is now known about the chemical structure, metabolism, and biological roles of RBP. Vitamin A mobilization from the liver is highly regulated by factors that control the rates of RBP production and secretion. Retinol deficiency specifically blocks the secretion of RBP, which can then be rapidly stimulated by intravenous retinol repletion. The cellular and molecular mechanisms that mediate these phenomena are under investigation. Delivery of retinol to peripheral tissues appears to involve specific cell surface receptors for RBP. The retinol so delivered enters the target cell, where it may become associated with the intracellular binding protein for retinol (CRBP). A number of tissues of rats, humans, and other species contain soluble proteins with binding specificity for retinol (CRBP) or for retinoic acid (CRABP). These proteins have been purified from several tissues and partly characterized. They differ in a number of ways from plasma RBP, and differ from each other in regard to binding specificity and immunoreactivity. It has been suggested that these intracellular proteins may play a direct role in the biological expression of vitamin A activity in the cell. Studies are in progress to explore this and other possibilities.

Humans↗
PubMed1981

Comparison of the effects of colestipol hydrochloride and clofibrate on plasma lipids and lipoproteins in the treatment of hypercholesterolemia.

The effects of colestipol HCl resin and clofibrate on plasma lipid and lipoprotein levels were compared in 65 patients with primary hypercholesterolemia. Patients were randomly assigned to treatment with colestipol (in progressive doses of 15, 20, and 30 g/day), clofibrate (2 g/day), or placebo resin; lipoprotein levels were determined at months 0, 2, 4, 6, and 9. The colestipol group received both colestipol and clofibrate during months 7 through 9 of the study. After 6 months of treatment, mean plasma total cholesterol fell from 333 to 266 (P less than 0.01) on colestipol, and from 329 to 270 (P less than 0.05) on clofibrate. More patients responded, however, to colestipol than to clofibrate. Both drugs also produced significant reductions in LDL cholesterol levels, and clofibrate lowered plasma triglycerides as well. HDL cholesterol level did not change significantly on either medication. The placebo group showed no change in any of the parameters studied. A significant difference was not observed between the effects of 15 g/day of colestipol and those of the higher doses studies. Addition of clofibrate to colestipol did not enhance the latter's hypocholesterolemic action.

Cholesterol↗