Search PubMed⌕ Search

Biomedical subjects

D M Lee

Publications and source records attributed to D M Lee.

At least 91 records · Page 5Linked to original sources
PubMed1988

Comparison of the toxicity of orally administered L-triiodothyronine (T3) in rat and cynomolgus monkey.

Oral administration of L-triiodothyronine (L-T3) (0.015-1 mg/kg) for 30 days to mature rats or cynomolgus monkeys resulted in both species in a high mortality at 1 mg/kg (after 2 weeks of treatment) and a progressive loss in body weight. Dose-related elevations in plasma marker enzymes occurred, mainly after 1-2 weeks of treatment. The approximate no-effect dose for these changes was around 0.015-0.020 mg/kg for both rat and primate. The large elevations of leucine aminopeptidase (LAP) at 1 mg/kg L-T3 in monkey indicated hepatocellular toxicity although in the rat such large increases in alanine aminotransferase (ALT) and glutamate dehydrogenase (GLDH) were not seen. L-T3 also showed little toxicity to rat hepatocytes in vitro. High concentrations of L-T3 (7 x 10(-9) to 7 x 10(-7) M) had minimal effects on parameters of cell viability such as lactate dehydrogenase (LDH) leakage, chromium-51 release and [3H]leucine incorporation. Urinary enzymes in the rat showed a similar profile to those in plasma. Large rises in alkaline phosphatase (AKP) and N-acetyl glucosaminidase (NAG) at 1 mg/kg indicated possible proximal tubular damage although this was not supported histologically. Clinically, in both species L-T3 appeared more toxic to males than females but this was not supported histologically. The histological lesions observed were different in the 2 species. In the monkeys there was extensive lipid vacuolation of hepatocytes and changes in thyroid and adrenal cortex. In the rat there was fine, non-lipid vacuolation of hepatocytes and thyroid changes. In the rat, 2 previously unreported lesions were also noted. There were multinucleated cells in the renal distal tubular epithelium, and focal fibroplasia of serosal surfaces of abdominal viscera.

Acetylglucosaminidase↗
PubMed1988

Apolipoprotein B-100 is the major form of this apolipoprotein secreted by human intestinal Caco-2 cells.

Although the discovery of stop codon has explained the mechanism for the formation of the intestinal marker, apolipoprotein B-48, the dispute regarding the presence of apolipoprotein B-100 in the intestine is still unsettled. To further investigate the characteristics of intestinal apolipoprotein B, the newly developed human colonic adenocarcinoma Caco-2 cells which express functional properties of the differentiated enterocytes, were used. SDS-polyacrylamide gel electrophoresis analyses of the intact culture medium or its lipoproteins of d less than 1.23 g/ml showed the presence of only a single protein band of apolipoprotein B-100 with no detectable apolipoprotein B-48. After immunoblotting with oligoclonal antibodies to the amino-terminal peptide of apolipoprotein B, a trace amount of apolipoprotein B-48 was observed in the isolated lipoproteins, but not in the intact culture medium. These results suggest that apolipoprotein B-100 is the major form of apolipoprotein B secreted by human intestinal cells.

Adenocarcinoma↗
PubMed1988

Degradation of apolipoprotein B-100 in human chylomicrons.

The purpose of this study was to investigate the molecular forms of apolipoprotein B (ApoB) in human chylomicrons under well-preserved conditions. To this end, plasma and serum were collected from the same normal subjects after ingestion of a fatty meal. The samples were divided into three or four aliquots before the addition of various preservative mixtures, including antibiotics, antioxidants and proteinase inhibitors. The chylomicrons were isolated immediately, and all steps were carried out at or below 4 degrees C. Changes in the molecular weight of ApoB in chylomicrons were followed by a time study using 3.3% polyacrylamide gel electrophoresis containing SDS. ApoB from chylomicrons analyzed within 5 h of blood collection showed a single band with mobility identical to that of ApoB (ApoB-100) in low-density lipoproteins. When analyzed after 1-2 days, satellite bands smaller than ApoB-100 were observed, and a very faint band with Mr 200,000 appeared, which comigrated with intestinal ApoB (ApoB-48). Upon storage, the molecular weight of ApoB was smaller in chylomicrons subjected to a higher number of reflotations than those in chylomicrons washed less frequently, suggesting that purified chylomicrons degrade faster. A longer storage time at 4 degrees C (i.e., 7 or 14 days) revealed a stepwise degradation of ApoB, yielding Mr 200,000 band as the prominent form. The degradation of ApoB-100 was slower when both proteinase inhibitors, leupeptin and epsilon-amino caproic acid, were employed, and the appearance of Mr 200,000 band was quicker when the chylomicrons were processed at higher temperature (15-25 degrees C) in the absence of a proteinase inhibitor. Immunoblotting shows that the segment removed from ApoB-100 was the carboxyl-terminal portion. These results suggest the possible presence of a proteinase(s), which copurified with chylomicrons, and which converts ApoB-100 from a large to a smaller molecular form. Although the stop codon has been discovered recently in intestinal ApoB mRNA, which explains the mechanism for direct synthesis of ApoB-48, apparently ApoB-100 is also synthesized in the intestine of all eight subjects studied here, and the ApoB-100 degrades to a form which is ApoB-48-like.

Apolipoprotein B-100↗
PubMed1988

Identification of the thiol ester linked lipids in apolipoprotein B.

Human plasma low-density lipoproteins of 1.032-1.043 g/mL density were totally delipidized. The reduced and carboxymethylated apolipoprotein B was incubated with 50 mM [14C]methylamine at pH 8.5 at 30 degrees C. Covalent incorporation of [14C]methylamine was observed with concomitant generation of new sulfhydryl groups, which could be blocked with [3H]- or [14C]iodoacetic acid. One type of the [14C]methylamine-modified products was separated from the protein and was found to be lipid in nature. Its Rf on thin-layer chromatography (TLC) was similar to that of the synthetic N-methyl fatty acyl amides. After purification with TLC and transesterification in 3 N methanolic HCl, methyl esters of C16 and C18 fatty acids at 1:1 ratio were identified by gas-liquid chromatography. The transesterification method was verified with the known N-methyl fatty acyl amides. These results suggest the presence of labile thiol ester linked palmitate and stearate in apolipoprotein B. Under mild alkaline conditions, the thiol ester bonds are broken by methylamine and form N-methyl fatty acyl amides and release new-SH groups. Intramolecular thiol ester bonds linked between cysteine side chains and acidic amino acid residues were also found present, which will be reported separately.

Apolipoproteins B↗
PubMed1987

Selective isolation of human plasma low-density lipoprotein particles containing apolipoproteins B and E by use of a monoclonal antibody to apolipoprotein B.

A monoclonal antibody to human plasma apolipoprotein B was used in a single-step immunoaffinity chromatography procedure to isolate a subpopulation of low-density lipoprotein particles from normolipidemic human plasma. The isolated particles were homogeneous in terms of size (20 nm), flotation coefficient (Sf = 9.5), and electrophoretic mobility (beta band). Their protein moiety consisted of apolipoproteins B and E in a molar ratio close to 2. The lipid moiety consisted of 47.3% cholesterol, 4.7% triglycerides, and 48.0% phospholipids. To indicate its characteristic apolipoprotein composition and hydrated density properties, this family of particles was named LP-B:EL2. In most normolipidemic subjects, LP-B:EL2 particles accounted for less than 10% of the total plasma apolipoprotein B content. The LP-B:EL2 particles bound to the membranes of the human hepatoma HepG2 cells in a specific and saturable manner indicative of receptor-mediated binding. Their binding was significantly higher than that of low-density lipoprotein particles containing only apolipoprotein B.

Antibodies, Monoclonal↗
PubMed1987

Apolipoprotein B is a globular protein--morphological studies by electron microscopy.

Water-soluble apolipoprotein B was prepared from fresh plasma by quick isolation of low density lipoproteins and immediate delipidization under non-oxidative conditions. The denatured protein in 6 M guanidine X HCl was reduced and carboxymethylated, dialyzed through 6 M urea/preservatives and to 1% ammonium acetate/0.05% EDTA/0.13% epsilon-amino caproic acid, pH 7.3 under N2 at 4 degrees C. The morphological studies were carried out by electron microscopy with negative staining and freeze fracture. Both these techniques showed that apolipoprotein B is a globular protein with average diameter of 11.48 +/- 1.25 nm (n = 978). The M.W. of apolipoprotein B calculated from this particle size was comparable to that from amino acid sequence.

Apolipoproteins B↗
PubMed1986

Apolipoproteins B, C-III and E in two major subpopulations of low-density lipoproteins.

To determine the concentration and distribution of apolipoproteins C-III and E in low density lipoproteins (LDL) of d 1.025-1.043 g/ml, fresh human plasma was fractionated by single-spin density gradient ultracentrifugation into five layers. Two major subpopulations including layer 2 (d 1.025-1.029 g/ml) and layer 3 (d 1.032-1.043 g/ml) were isolated and characterized by determination of flotation coefficient, neutral lipids and apolipoproteins B, C-III and E. The apolipoprotein B/C-III/E ratio of layer 2 was 100/(3.3 +/- 2.0)/(5.1 +/- 2.9) (wt/wt) and that of layer 3 was 100/(0.61 +/- 0.32)/(0.58 +/- 0.29) (wt/wt). These weight ratios corresponded to molar ratios of 1.0/(1.90 +/- 1.16)/(0.74 +/- 0.42) and 1.0/(0.34 +/- 0.18)/(0.08 +/- 0.04), respectively. Layer 2 contained 6-23% of the total plasma apolipoprotein B or 7-27% of total LDL2 (d 1.019-1.063 g/ml) apolipoprotein B. Layer 3 contained 41-65% of plasma apolipoprotein B or 62-86% of LDL2 apolipoprotein B. About 5-17% of apolipoprotein C-III and 8-30% of apolipoprotein E in plasma are distributed in layers 2 and 3 with the majority present in layer 2. These results show an evident apolipoprotein heterogeneity of LDL2 isolated from normolipidemic subjects. Moreover, they show that the relatively small amounts of apolipoprotein C-III and apolipoprotein E in lower-density segments of LDL2 take on a greater significance when presented in molar rather than weight concentrations. The existence of different ratios of apolipoprotein C-III/apolipoprotein E in layer 2 and layer 3 suggest the presence in LDL2 of varying amounts of several discrete apolipoprotein B- and/or apolipoprotein C-III- and apolipoprotein E-containing lipoprotein particles.

Adult↗
PubMed1986

Apolipoprotein B is a calcium binding protein.

Human hepatocarcinoma Hep G2 cells were grown in culture medium containing [45Ca2+]. The secreted lipoproteins of d less than 1.063 g/ml and d 1.063-1.21 g/ml were isolated from the culture media and analyzed by 3.3% and 7% SDS-polyacrylamide gel electrophoresis. Radioactivity profiles of [45Ca] from the gels showed that the peak of radioactivity corresponded to the apolipoprotein B band. The molar ratio of the incorporated [45Ca2+] and apolipoprotein B was close to unity. No radioactivity was found associated with any other secreted apolipoproteins. To confirm these findings, apolipoprotein B-containing lipoproteins were precipitated with anti-apolipoprotein B and high density lipoproteins were precipitated with anti-apolipoprotein A-I. Only the former precipitate was radioactive. These results suggest that apolipoprotein B is a calcium binding protein.

Animals↗
PubMed1986

Conformational studies of lipoprotein B and apolipoprotein B: effects of disulfide reducing agents, sulfhydryl blocking agent, denaturing agents, pH and storage.

The purposes of this study were to establish the role of disulfide linkages in the secondary structure of apolipoprotein B, to investigate the effects of sulfhydryl blocking agents, denaturing agents, pH and storage on the conformation of apolipoprotein B and lipoprotein B, and to compare the conformation of water-soluble apolipoprotein B in the presence and absence of its lipids by using circular dichroism. Fresh lipoprotein B examined in Tris/EDTA at pH 9.0, 7.3 and 2.7 exhibited alpha-helical content of 24.4, 26.7 and 26.9%, and beta-pleated sheet 25.1, 15.4 and 18.0%, respectively. The carboxymethylated (CM-) lipoprotein B had similar alpha-helical contents, and lower contents of beta-sheets. Storage of lipoprotein B resulted in marked change of beta-sheets and gradual decrease in alpha-helical structure, in spite of the preventive measures taken for lipid peroxidation and proteolytic degradation. Exposure of apolipoprotein B to 6 M guanidine X HCl led to a complete disappearance of the alpha-helix with an increase in the beta-sheets to 35-40%, irrespective of the use of disulfide-reducing agents. By substituting 6 M urea for guanidine X HCl, the alpha-helical contents for both CM- and reduced CM-apolipoprotein B increased up to 7-9% with a concomitant decrease in beta-structure. When urea was replaced with aqueous buffers, these apolipoprotein B preparations regained their alpha-helical contents (25-27%) to the full extent originally present in the parent lipoprotein samples. No difference was observed between the secondary structure of CM- and reduced CM-apolipoprotein B. Furthermore, the conformation of apolipoprotein B did not vary with pH when pH was changed from 2.7 to 9.0. These results suggest that (1) the conformation of apolipoprotein B is more stable with respect to pH in the absence of lipids than in their presence, (2) intramolecular disulfide linkages play an insignificant role in the conformation of apolipoprotein B, and (3) the changes in alpha-helix structure of lipoprotein B or CM-lipoprotein B due to delipidization and denaturation are reversible.

Apolipoproteins B↗
PubMed1986

Characterization of a monoclonal antibody that binds equally to all apolipoprotein and lipoprotein forms of human plasma apolipoprotein B. I. Specificity and binding studies.

A stable mouse hybridoma cell line has been developed that produces monoclonal antibody to human plasma apolipoprotein B. This antibody was proven to be specific for apolipoprotein B immunoblotting and an enzyme immunoassay using apolipoprotein B and other apolipoproteins. The antibody bound with comparable affinities to soluble apolipoprotein B, chylomicrons, very-low-density (VLDL) and low-density lipoproteins (LDL). Coupled to agarose, this antibody allowed complete removal of apolipoprotein B-containing lipoproteins from normolipidemic, hypertriglyceridemic and hypercholesterolemic plasma. Desialyzation and deglycosylation had no effect on its binding to LDL. The described antibody had no effect on the receptor-mediated binding of radiolabeled LDL to the human hepatoma cells (HepG2) in culture. Analysis of 25 different samples of human plasma indicated identical expression of the corresponding epitope in these individuals. The described monoclonal antibody, most likely, binds to a rather stable domain of apolipoprotein B that is not altered by the interaction with lipids or polymorphism of the apolipoprotein B. We propose that this antibody be called 'Pan B' antibody.

Animals↗
PubMed1986

Escape from serial stimuli leading to food.

If the functional relations governing the strength of a conditioned reinforcer correspond to those obtained with other Pavlovian procedures (e.g., Kaplan, 1984), the termination of stimuli appearing early in the interval between successive food deliveries should be reinforcing. During initial training we presented four key colors, followed by food, in a recurrent sequence to each of 6 pigeons. This established a baseline level of autoshaped pecking. In later sessions, we terminated each of these colors or only the first color for a brief period following each peck, replacing the original color with a standard substitute to avoid darkening the key. Pecking decreased in the presence of the last color in the sequence but increased in the presence of the first. In accord with contemporary models of Pavlovian conditioning, these and other data suggest that the behavioral effects of stimuli in a chain may be better understood in terms of what each stimulus predicts, as measured by relative time to the terminal reinforcer, than in the exclusively positive terms of the traditional formulation (Skinner, 1938). The same model may also account for the initial pause under fixed-interval and fixed-ratio schedules of reinforcement.

Journal Article↗
PubMed1985

Evaluation of immunoaffinity chromatography for isolating human lipoproteins containing apolipoprotein B.

Because of high specificity, immunoaffinity chromatography is the most suitable procedure for the isolation of lipoprotein (LP) particles defined by their apolipoprotein (Apo) composition. The purpose of the present study was to describe immunosorber methodology and its application to the isolation of ApoB-containing lipoproteins from either plasma or isolated lipoprotein density classes. The exploration of various coupling procedures demonstrated that immunosorbers of highest capacity were obtained by cyanogen bromide activation of Sepharose. Among various dissociating agents tested, 3 M sodium thiocyanate was found to be the most effective desorbent for bound lipoproteins. Studies on the non-specific binding of serum albumin to several different immunosorbers showed a negligible retention (1.9%) of albumin. Good recoveries (80-98%) were obtained with all apolipoproteins tested with both anti-ApoA-I and anti-LP-B immunosorbers. By using the optimal experimental conditions, it was shown that the ApoB-containing lipoproteins retained by immunosorbers with antibodies to LP-B had chemical, physical and immunological properties similar, if not identical, to those of their corresponding parent density classes. The application of an alternative immunoaffinity chromatography procedure with serially connected immunosorbers with antibodies to apolipoproteins other than ApoB resulted in the isolation of LP-B, a lipoprotein containing ApoB as its sole protein constitutent. LP-B had chemical and physical properties very similar to those of subclass 2 of low-density lipoproteins (density 1.019-1.063 g/ml, flotation coefficient 0-12). Based on these studies, we suggest that immunoaffinity chromatography in combination with microanalytical procedures for quantification of lipids and apolipoproteins offers a powerful tool for the isolation and functional characterization of lipoprotein particles defined by their apolipoprotein composition.

Adsorption↗
PubMed1985

Glucose tolerance and plasma lipid distributions in rats fed a high-sucrose, high-cholesterol, low-chromium diet.

Male Sprague-Dawley rats were fed either a low-chromium (60 to 100 micrograms per kg of diet) or chromium-supplemented (5 mg per kg of diet), high-sucrose, high-cholesterol diet from weaning until age 18 months. Rats that were pair- and meal-fed the low-chromium diet had higher one-hour postprandial plasma glucose concentrations than their supplemented partners at ages 4 and 8 months (P less than 0.05), but not at age 12 months. One-hour postgavage (250 mg glucose/100 g body wt) glucose concentrations did not differ between dietary groups at 12 months. Plasma cholesterol concentrations increased up to age 12 months, but neither they nor postprandial triglyceride concentrations differed significantly between dietary groups. Ad libitum feeding of the low-chromium and chromium-supplemented diets was initiated at age 14 months in order to determine whether there were differences due to dietary chromium content which might not be manifest on the pair-feeding regimen. Animals of both dietary groups had significant weight gain by age 16 months, but their one-hour postgavage plasma glucose concentrations did not differ significantly. Plasma cholesterol concentrations increased significantly following institution of ad libitum feedings, but neither they nor lipoprotein cholesterol or triglyceride distributions differed significantly between dietary groups. Experimental conditions, methods, and results from this study and previous studies are compared and critically examined. We suggest that other factors in addition to dietary chromium content may contribute to the differences in glucose tolerance and plasma cholesterol concentrations described in such studies and that there is a need for improved documentation of glucose intolerance and tissue chromium concentrations in this animal model.

Animals↗
PubMed1985

Kinetic properties of human milk bile salt-activated lipase: studies using long chain triacylglycerol as substrate.

Studies on the hydrodynamic properties of human milk bile salt-activated lipase (BAL) indicated that it is a monomer with molecular weight of 107,000. The presence of taurocholate (1 mM) did not lead to an association of the enzyme. The enzyme had a basal activity with trioctanoylglycerol and with shorter chain, but not with longer chain, monoacid triacylglycerols. Based on kinetic analyses, we suggest that the BAL-catalyzed lipolysis of long-chain triacylglycerol can be described to follow a compulsory sequential mechanism. The initial interaction of BAL with the activator (taurocholate) leads to a conformational change of the enzyme which facilitates the further interaction with the long chain triacylglycerol substrate in forming the enzyme-bile salt-substrate ternary complex. We also suggest that the binding of BAL with substrate involves direct interaction of the active site with the fatty acyl-chain of the triacylglycerol rather than with nonspecific hydrophobic interactions at the emulsion interface.

Bile Acids and Salts↗
PubMed1984

Presence of B-100 in rat mesenteric chyle.

Molecular forms of apolipoprotein B (ApoB) were studied in the rat intestinal chyle by SDS-polyacrylamide gel electrophoresis, immunoblotting and immunodiffusion. Time studies on intestinal chyle showed the presence of B-100 in all the samples analyzed within 3 hr after drawing. However, the analyses repeated on day 2 or day 3 revealed disappearance of B-100 and appearance of B-48. Addition of 3 mM EDTA, 10 mM diisopropylfluorophosphate, 5 mM chloroquine and 10 mM epsilon-amino caproic acid slowed down but could not prevent the disappearance of B-100. Chylomicrons isolated from chyle in the presence of preservatives immediately after drawing displayed B-100 as a major and B-48 as a minor ApoB form. However, repeatedly washed chylomicrons or those isolated from chyle 18-24 hr after drawing showed B-48 as the only ApoB present. These results suggest that rat intestine synthesizes B-100 which is quickly converted to smaller molecular form.

Animals↗