Is microsomal triglyceride transfer protein the missing link in abetalipoproteinemia?
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Abetalipoproteinemia↗
Biomedical subjects
R L Hamilton
Publications and source records attributed to R L Hamilton.
Effect of root conditioning on periodontal wound healing with and without guided tissue regeneration: a pilot study. 1. Histologic evaluation.
Closed fenestration wounds in four mongrel dogs were used to study the source of fibroblast proliferation and extracellular matrix production during healing; the arrangement and attachment of newly formed collagenous fibers; and the cementogenesis and osteogenesis at healing sites. Fenestration wounds were made through the alveolar bone, periodontal ligament, cementum, and dentin, and citric acid, tetracycline, or sterile water was applied to the dentinal walls for 3 minutes. Nonresorbable membranes were randomly placed over half of the defects. Animals were killed at 1, 3, 7, or 21 days and routine histologic examinations with hematoxylin and eosin staining followed. Results of this pilot study suggest that the periodontal ligament and/or alveolar bone are the main source of fibroblast proliferation and migration as well as extracellular matrix formation at the initial stages of healing, and that at 21 days, citric acid stimulated more cementogenesis than tetracycline or sterile water. Also, while the tetracycline influenced the maximal deposition of alveolar bone, no differences in healing were found between the citric acid, tetracycline, and sterile water with and without the use of membrane barriers.
Acid Etching, Dental↗
Site-directed mutagenesis and structure-function analysis of the human apolipoprotein A-I. Relation between lecithin-cholesterol acyltransferase activation and lipid binding.
We have mutagenized the human apoA-I gene and have generated cell lines which express normal and mutant apoA-I forms. Point mutations were introduced which changed Gln-1, Gln-2 to Arg,Arg, Pro99 to His, and Pro121 to His. In addition, the following amino acid deletions (delta) were generated: delta 113-124, delta 148-186, delta 212-233, and delta 213-243. The apoA-I form isolated from the culture medium of C127 cells was analyzed for its ability to activate lecithin-cholesterol acyltransferase (LCAT) and to bind to phospholipid vesicles and high density lipoprotein (HDL). Compared with the wild type (WT) apoA-I, the relative activation of LCAT achieved by the point mutations Gln-1, Gln-2----Arg,Arg, Pro99----His, and Pro121----His were 106 +/- 7, 92 +/- 6, and 77 +/- 9%, respectively. Kinetic analysis of one mutant apoA-I form showed similar Vmax but a 15-fold increase in the Km of the mutant apoA-I form. Furthermore, the activation achieved by the internal deletion mutants delta 113-124, delta 148-186, delta 212-233, and delta 213-243 was 47 +/- 3, 0.5 +/- 0.4, 28 +/- 4 and 13 +/- 5%, respectively. Mutants deficient in their ability to activate LCAT displayed alterations in liposome and HDL binding, compared with WT as determined by density gradient ultracentrifugation analysis of the culture medium. Thus, the peak recovery (approximately 50%) of apoA-I bound to HDL was at density 1.14 g/ml for the WT apoA-I, at 1.18 g/ml for the mutants delta 113-124 and delta 148-186, and at d greater than 1.21 g/ml for the delta 212-233 and delta 213-243. Electron microscopy of the proteoliposome LCAT substrate generated by WT and mutant apoA-I forms showed that the carboxyl-terminal deletion mutants which displayed aberrant binding to HDL also displayed reduced ability to convert the spherical lecithin-cholesterol vesicles into discs compared with WT. The findings suggest that (a) the importance of the carboxyl terminus of apoA-I for LCAT activation is related to its ability to bind to lipid and/or to form discoidal substrate for LCAT, and (b) the interaction of several domains of apoA-I are required for the activation of LCAT.
Algorithms↗
Binding of transition metals by apolipoprotein A-I-containing plasma lipoproteins: inhibition of oxidation of low density lipoproteins.
We have found transition metals tightly bound to apolipoprotein A-I-containing lipoproteins [Lp(A-I)] isolated by selected affinity immunosorption from human serum. Prominent among the metal ions detected were iron and copper. By immunoblotting the proteins of Lp(A-I), we detected both transferrin and ceruloplasmin. The transferrin-containing Lp(A-I) particles, isolated by selected affinity immunosorption against transferrin, were larger (mean diameter of 14.2 nm) and had a higher protein content than most high density lipoproteins (HDL). Ultracentrifugally isolated HDL were found to contain much less transferrin, whereas transferrin was found associated with apolipoprotein A-I from the greater than 1.21-g/ml ultracentrifugal fraction. This suggests that the complex is not recovered in the classic HDL density interval because of its very high density. HDL inhibit copper-catalyzed oxidation of low density lipoproteins (LDL) in vitro. We have found that immunoisolated Lp(A-I) are an order of magnitude more effective in inhibiting the oxidation of LDL than ultracentrifugally isolated HDL, on the basis of protein mass. When the Lp(A-I) particles containing transferrin and ceruloplasmin were removed from the bulk of Lp(A-I), inhibition of the in vitro oxidation of LDL was significantly decreased.
Apolipoprotein A-I↗
Acute effects of topical methyl tert-butyl ether or ethyl propionate on gallbladder histology in animals: a comparison of two solvents for contact dissolution of cholesterol gallstones.
Experiments were performed in anesthetized rabbits and piglets to assess gallbladder mucosal injury during irrigation with methyl tert-butyl ether, a C5 ether, or ethyl propionate, a C5 ester--two organic solvents used in the contact dissolution of cholesterol gallstones. In 44 New Zealand White rabbits, the gallbladder was exposed to individual solvents or saline solution through a transhepatic catheter for 2 hr. Gallbladders were then harvested and fixed immediately or after a recovery period of 1, 4 or 8 days. Tissue sections were examined under light microscopy, and severity of injury was graded with predefined criteria by two pathologists blinded to the animals' treatment regimens. Histological assessment showed severe mucosal injury such as necrosis of the cells at the villus tips immediately after 2 hr of exposure to either solvent. After 4 days, injury had decreased significantly; after 8 days, complete mucosal healing had taken place. A similar study was performed in 32 piglets. Solvent or saline solution was oscillated in and out of the gallbladders of these piglets with a computer-controlled syringe pump at a pressure less than the leakage pressure of the gallbladder. Histological assessment was performed on tissue samples obtained immediately after the procedure or 8 days later. Both solvents caused severe mucosal injury; however, after 8 days complete mucosal healing had occurred, so that gallbladders exposed to solvent were indistinguishable from gallbladders exposed to saline solution, which was used as control. We conclude that both methyl tert-butyl ether and ethyl propionate cause moderate to severe epithelial injury but that the gallbladder epithelium regenerates within a few days.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Alkyl and aryl neoalkanamides: highly effective insect repellents.
Alkyl and aryl neoalkanamides with a total carbon number between 11 and 14, or within a molecular weight range between 185 and 227, were highly effective repellents of male German cockroaches, Blattella germanica (L.). Comparison with known repellents showed that members of this unique family of secondary amides are among the most effective and long-lasting repellents of cockroaches examined to date. In assays with females and nymphs of the German cockroach, male American cockroaches, Periplaneta americana (L.), and carpenter ant workers, Camponotus pennsylvanicus (De Geer) methyl neodecanamide, propyl neodecanamide, and methyl neotridecanamide were found highly repellent. Because of their broad spectrum of activity, longevity, and safety, these compounds, along with several other members of this family, have important applications as repellents of nuisance pests and of arthropods of public health importance.
Amides↗
Effects of surfactant apolipoproteins on liposome structure: implications for tubular myelin formation.
Tubular myelin is one of several forms of lung surfactant and may play an important role in its surface activity. To determine possible mechanisms of tubular myelin formation, we studied the effects of purified surfactant proteins (SP-A, SP-B, and SP-C) on large unilamellar dipalmitoylphosphatidylcholine-egg phosphatidylglycerol (7/3; wt/wt) liposomes. We studied different types of membrane interaction induced by the apolipoproteins and correlated these with the observed changes in ultrastructure. Aggregation was assessed by measurement of light absorbance, lysis, and fusion by measurement of the fluorescence emitted by water-soluble and lipid-soluble probes, respectively. Mixtures of the apolipoproteins and liposomes were examined in ultrastructural studies by negative staining and by thin sectioning. We found that each protein had a pronounced and distinct effect on liposome structure. SP-A caused aggregation, whereas SP-B and SP-C also caused extensive leakage of liposome contents (lysis) and some degree of lipid mixing (fusion). The disruptive effects of SP-B and to a lesser extent those of SP-C were correlated by negative staining with the appearance of bilayer disks, which tended to aggregate into large sheets. There was a marked synergy between SP-A and SP-B in the process of membrane fusion in the presence of calcium, which correlated with an early (10 min) and extensive rearrangement of the structures seen by electron microscopy followed by a delayed (24 h) appearance of small amounts of tubular myelin.
Animals↗
Two apolipoprotein B gene defects in a kindred with hypobetalipoproteinemia, one of which results in a truncated variant, apoB-61, in VLDL and LDL.
We report the presence of two distinct defects of the gene for apolipoprotein B, one resulting in a new truncated variant, apoB-61, in a kindred with familial hypobetalipoproteinemia (FHB). The proband (age 33) and a sister (age 36) are both compound heterozygotes with total cholesterol levels of 39 mg/dl and 50 mg/dl, and apoB levels of 1 mg/dl and 2 mg/dl in plasma, respectively. Both appear to be asymptomatic. The apoB-61 mutation, present in a total of five individuals and inherited from the proband's father, is a 37 bp deletion in exon 26 starting with nucleotide 8525. This results in an apoB of 2784 amino acids with 12 novel carboxy-terminal residues. The apoB-61 is present to a considerable degree, relative to apoB-100, in the proband's very low (VLDL) and low density (LDL) lipoprotein fractions. Both lipoprotein fractions have abnormal particle size distribution by electron microscopy. The LDL contain cuboidal particles. Total cholesterol, LDL cholesterol, and apoB levels in the family display three phenotypic patterns: normal, low, and extremely low. ApoB haplotyping indicates the presence of another defective apoB allele in a total of seven individuals. This allele leads to low levels of apoB-100. The second apoB gene-linked defect occurring together with the apoB-61 mutation explains the 3-phenotype pattern. The severe hypocholesterolemia seen in the proband and a sister result from the genetic compound state involving both alleles. This study shows that severe hypolipidemia in an individual heterozygous for a truncation in apoB is likely to involve a second genomic defect.
Adult↗
Differences in local conformation in human apolipoprotein B-100 of plasma low density and very low density lipoproteins as identified by cathepsin D.
The conformational changes of human apolipoprotein (apo) B-100 which accompany the conversion of plasma very low density lipoproteins (VLDL) to low density lipoproteins (LDL) were investigated by studying the accessibility of apoB-100 in LDL and VLDL to limited proteolysis with cathepsin D, an aspartyl proteinase involved in intracellular protein degradation. We characterized the proteolytic products of apoB-100 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by NH2-terminal sequence analysis to locate cleavage sites. The results identified at least 10 cleavage products generated from apoB-100 and showed differential accessibility of cleavage sites for cathepsin D in apoB-100 between LDL and VLDL. We identified a specific peptide region (residues 2660-2710), which is preferentially accessible to limited proteolysis by cathepsin D but inaccessible to limited proteolysis by 12 other enzymes tested. Within this peptide region, cathepsin D cleaved apoB-100 of LDL and VLDL preferentially at different sites, separated by 33-36 amino acids (2665-2666 or 2668-2669 (LDL) and 2701-2702 (VLDL]. In addition, we identified a cleavage site, located at residues 3272-3273, specific for cathepsin D, which is contained within the COOH-terminal enzyme-accessible peptide region (residues 3180-3280), which we have demonstrated using 12 endoproteases with various specificities. The previously identified NH2-terminal region (residues 1280-1320) appears to be resistant to limited cleavage by cathepsin D. However, a new site was revealed only approximately 66 kDA from the NH2 terminus. We conclude that differential accessibility and the shift of the novel scission site for cathepsin D by 33-36 amino acids indicate significant differences in local conformation at these sites in apoB-100 as VLDL are converted to LDL.
Amino Acid Sequence↗
Changes in lipid structure produced by surfactant proteins SP-A, SP-B, and SP-C.
Pulmonary surfactant phospholipids may assume several different structures including tubular myelin, unilamellar and multilamellar vesicles, and others. These populations of materials appear to have similar phospholipid compositions but may differ in their association with surfactant proteins SP-A, SP-B, or SP-C. We have used electron microscopy to determine the changes in structure of simple lipid mixtures (phosphatidylglycerol, dipalmitoylphosphatidylcholine) produced by adding one or combinations of the three proteins. Adding SP-A to lipids generated multilamellar structures composed of membranes with fuzzy or particulate surfaces. In contrast, SP-B or SP-C generated discoidal particles and structures that appeared to be sheets of membrane formed by associated particles. Used together, SP-A and SP-B reorganized some of the lipid into tubular myelin, a structure that was not observed in SP-A, SP-C recombinants. These observations confirm the in vitro formation of tubular myelin reported by others and support the possibility that surfactant materials with defined structure can be produced in vitro for analyses of their molecular organizations.
Animals↗
Molecular and metabolic basis for the metabolic disorder normotriglyceridemic abetalipoproteinemia.
We have previously described a disorder, normotriglyceridemic abetalipoproteinemia, that is characterized by the virtual absence of plasma low density lipoproteins and complete absence of apoB-100, but with apparently normal secretion of triglyceride-rich lipoproteins containing apoB-48. The patient's plasma lipoproteins were shown on polyacrylamide gels and by antibody mapping to have a new truncated apoB variant, apoB-50, circulating along with her apoB-48. We have found this individual to be homozygous for a single C-to-T nucleotide substitution at apoB codon 2252, which produces a premature in-frame stop codon. Thus, this is a rare example of homozygous hypobetalipoproteinemia. Electron photomicrographs revealed that the diameters of particles in the d less than 1.006 g/ml lipoprotein fraction, in both the postprandial and postabsorptive state, are bimodally distributed. The molar ratio of apoE to apoB in these particles is 3.5:1, similar to normal VLDL. The plasma LDL interval contains both spherical and cuboidal particles. Autologous reinfusion of labeled d less than 1.006 g/ml lipoproteins showed exponential disappearance from plasma, with an apparent half-removal time of 50 min, somewhat slower than for normal chylomicrons but within the normal range for VLDL. The calculated production rate for apoB was within the normal range in this subject. A very small amount of label was found briefly in the IDL fraction, but none at any time in LDL or HDL. Therefore, because LDL particles that contain apoB-50 lack the putative ligand domain of the LDL receptor, we conclude that the very low level of LDL is due to the rapid removal of the abnormal VLDL particles before their conversion to LDL can take place.
Abetalipoproteinemia↗
Isolation and characterization of three endosomal fractions from the liver of normal rats after lipoprotein loading.
In earlier research we isolated and characterized three endosomal fractions from livers of estradiol-treated rats (1987. Belcher et al. Proc. Natl. Acad. Sci. USA. 84: 6785; 1989. Jäckle et al. Proc. Natl. Acad. Sci. USA. 86: 1880). We now describe the isolation of comparable endosomal fractions from untreated rats. The fraction of lowest density was composed almost exclusively of lipoprotein-filled multivesicular bodies (MVBs); the intermediate density fraction was composed of smaller lipoprotein-filled vesicles that were also multivesicular and had more numerous membranous appendages; the fraction of highest density was composed of membranes resembling the appendages of the two vesicular fractions. The paucity of contamination of these fractions with nonendosomal organelles is supported by their ultrastructural characteristics and by the proteins, lipids, and marker enzymes of their membranes. Only small amounts of MVBs could be separated from untreated rats not given a load of lipoproteins. However, after injection of large amounts of beta-very low density lipoproteins (beta-VLDL) from cholesterol-fed rabbits, the mass of MVBs increased dramatically. Under these conditions radiolabeled beta-VLDL and epidermal growth factor taken up by the liver accumulated in isolated endosomes at rates similar to those found for LDL in estradiol-treated rats. Although chylomicrons and chylomicron remnants were rapidly taken up by the liver of normal rats, chylomicrons and chylomicron remnants accumulated in endosomes at a lower rate than beta-VLDL. These findings, which differ from earlier data in estradiol-treated rats (1989 Jäckle et al., Proc. Natl. Acad. Sci. USA. 86: 1880) that showed equivalent rates of processing of chylomicron remnants and beta-VLDL, suggest that extracellular processing of chylomicron remnants in the liver normally ; precedes endocytosis.
Administration, Oral↗
Isolation and properties of nascent lipoproteins from highly purified rat hepatocytic Golgi fractions.
Two procedures were used to isolate hepatocytic Golgi fractions from rat liver. One procedure yields a light Golgi fraction (GF1 + 2) and the other "intact" stacks of cisternae. Triglyceride fatty acids in nascent very low density lipoproteins (VLDL) were labeled by injection of [3H]palmitate intravenously, and radiolabeled lipoproteins were injected as markers of potentially contaminating endosomes. GF1 + 2 fractions were enriched manyfold in the endosomal markers, indicative of substantial endosomal contamination, whereas intact Golgi fractions from the same livers were about 7% as contaminated. By electron microscopy, GF1 + 2 fractions contained mainly multivesicular bodies (MVBs), together with some Golgi-derived secretory vesicles. The small endosomal contamination of intact Golgi fractions was further reduced by a simple modification of the procedure, which removed most entrained endosomes. The surface constituents of Golgi VLDL (d less than 1.010 g/ml) released from these highly purified intact Golgi fractions differed from those of plasma VLDL. Golgi VLDL contained fivefold less unesterified cholesterol than plasma VLDL, but twofold more phospholipids. Golgi VLDL and plasma VLDL contained similar amounts of cholesteryl esters and triglycerides. The protein content of Golgi VLDL was substantially lower than that of plasma VLDL. ApoB-100 and apoB-48 were similarly represented, but nascent VLDL contained less of the C apolipoproteins. ApoA-I was present mainly as the proprotein in Golgi VLDL, but was virtually lacking in plasma VLDL. ApoE comprised about 22% of the protein mass of Golgi VLDL as well as plasma VLDL; the distribution of apoE isoforms was also similar. Apolipoproteins E and pro A-I released from ruptured Golgi cisternae were largely bound to the Golgi VLDL or were associated with Golgi membranes. Particles resembling low density lipoproteins (LDL) and high density lipoproteins (HDL) were not seen by electron microscopy in contents of intact Golgi fractions. These observations indicate that nascent Golgi VLDL are the primary particulate precursors of rat plasma lipoproteins of hepatocytic origin, and suggest that particles with the density of plasma HDL and LDL do not exist within the secretory pathway of normal hepatocytes. Thus, the results of this research on the properties of nascent plasma lipoprotein precursors contained within uncontaminated hepatocytic Golgi fractions differ substantially from previous published work.
Animals↗
Sublethal effects of chlorpyrifos-methyl on reproduction in female German cockroaches (Dictyoptera: Blattellidae).
Longevity and lifetime fecundity were significantly reduced after topical application of LC10, LC20, and LC60's of chlorpyrifos-methyl to 2-d-old female German cockroaches, Blattella germanica (L.). Low concentrations (LC10 and LC20) often resulted in failure of the first ootheca to hatch. This effect is temporary, and greater than or equal to 90% of subsequent oothecae hatched normally. Application of an LC60 also resulted in a high percentage of abortions of first oothecae, but also caused greater than 15% inviability in subsequent oothecae.
Animals↗
Integrated suppression of synanthropic cockroaches.
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Animals↗
Apolipoprotein E localization in rat hepatocytes by immunogold labeling of cryothin sections.
The distribution of apolipoprotein (apo) E in rat hepatocytes was investigated with an affinity-purified polyclonal antibody raised against apoE isolated from hepatogeneous very low density lipoproteins (VLDL). The distribution of this antibody was visualized with colloidal gold complexed to anti-rabbit IgG. By epipolarization microscopy, apoE was found uniformly along the basolateral surfaces of all hepatic parenchymal cells, showing a striking intensity along the sinusoidal front. Punctate deposits of colloidal gold appeared to be randomly distributed within all hepatocytes. Widely scattered Kupffer cells also stained for apoE. Electron microscopic examination of immunogold-labeled cryothin sections showed that hepatocytic microvilli projecting into the space of Disse consistently contained clusters of immunogold. The gold particles were variably associated with evident lipoprotein particles, raising the possibility that apoE alone may bind to receptors or other macromolecules at the surface of hepatocytes. Endosomes near the sinusoidal front and multivesicular bodies in the Golgi/biliary area labeled intensely for apoE, consistent with a high content of apoE associated with triglyceride-rich lipoprotein remnants contained within these organelles. Some but not all nascent VLDL particles within putative forming Golgi secretory vesicles were labeled, but many other Golgi vesicles and cisternae that lacked evident VLDL particles were also labeled. These results suggest that at least some apoE associates with nascent VLDL in forming Golgi secretory vesicles. Unexpectedly, the matrix of all hepatocytic peroxisomes was heavily labeled. Immunoblots with the affinity-purified anti-rat apoE IgG against proteins from highly purified peroxisomes isolated from rat hepatocytes revealed a protein with an apparent molecular mass of 34.5 kDa, similar to that of rat apoE in rat blood plasma. In addition, gold was sometimes found in the area either adjacent to peroxisomes or between multivesicular bodies and the bile canaliculus not evidently associated with a membranous compartment. These observations suggest that apoE may participate in interorganellar cholesterol transport within hepatocytes.
Animals↗
Nascent very low density lipoproteins from rat hepatocytic Golgi fractions are enriched in phosphatidylethanolamine.
The phospholipid composition of nascent very low density lipoproteins (VLDL) of rat hepatocytic Golgi fractions differs greatly from that of plasma VLDL. The phospholipids of nascent VLDL contain about four times more phosphatidylethanolamine (PE) than plasma VLDL, whereas plasma VLDL contain considerably more sphingomyelin. Thus, the ratio of PE to sphingomyelin differs by a factor of about 12 between nascent Golgi VLDL and circulating plasma VLDL. It is evident from these data that the PE/sphingomyelin ratio of VLDL can be used to estimate endosomal contamination of hepatocytic Golgi fractions.
Animals↗
Distribution of lipid-binding regions in human apolipoprotein B-100.
The distribution of lipid-binding regions of human apolipoprotein B-100 has been investigated by recombining proteolytic fragments of B-100 with lipids and characterizing the lipid-bound fragments by peptide mapping, amino acid sequencing, and immunoblotting. Fragments of B-100 were generated by digestion of low-density lipoproteins (LDL) in the presence of sodium decyl sulfate with either Staphylococcus aureus V8 protease, pancreatic elastase, or chymotrypsin. Particles with electron microscopic appearance of native lipoproteins formed spontaneously when detergent was removed by dialysis from enzyme digests containing fragments of B-100 and endogenous lipids, or from incubation mixtures of delipidated B-100 fragments mixed with microemulsions of exogenous lipids (cholesteryl oleate and egg phosphatidylcholine). Fractionation of the recombinant particles by isopycnic or density gradient ultracentrifugation yielded complexes similar to native LDL with respect to shape, diameter, electrophoretic mobility, and surface and core compositions. Circular dichroic spectra of these particles showed helicity similar to LDL but a somewhat decreased content of beta-structure. Most of the fragments of B-100 were capable of binding to lipids; 12 were identified by direct sequence analysis and 14 by reaction with antisera against specific sequences within B-100. Our results indicate that lipid-binding regions of B-100 are widely distributed within the protein molecule and that proteolytic fragments derived from B-100 can reassociate in vitro with lipids to form LDL-like particles.
Apolipoprotein B-100↗