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Biomedical subjects

G Castro

Publications and source records attributed to G Castro.

At least 55 records · Page 3Linked to original sources
PubMed1996

Transgenic rabbits expressing human apolipoprotein A-I in the liver.

Human apolipoprotein A-I (apo A-I) transgenic rabbits were created by use of an 11-kb genomic human apo A-I construct containing a liver-specific promoter. Five independent transgenic lines were obtained in which human apo A-I gene had integrated and was expressed. Plasma levels of human apo A-I ranged from 8 to 100 mg/dL for the founder and up to 175 mg/dL for the progeny. Rabbit apo A-I levels were substantially decreased in the transgenic rabbits. HDL cholesterol (HDL-C) levels were higher in two of the five transgenic rabbit lines than in controls (line 20 versus nontransgenic littermate, HDL-C = 80 +/- 7 versus 37 +/- 6 mg/dL; line 8 versus nontransgenic littermate, HDL-C = 54 +/- 16 versus 35 +/- 6 mg/dL). This resulted in less atherogenic lipoprotein profiles, with very low (VLDL + LDL-C)/HDL-C ratios. HDL size and protein and lipid compositions were similar between transgenic and littermate nontransgenic rabbits. However, a large amount of pre-beta apo A-I-containing lipoproteins was observed in the plasma of the highest human apo A-I expressor. Cell cholesterol efflux was evaluated with the incubation of whole serum from transgenic and control rabbits. Cell cholesterol efflux was highly correlated with HDL cholesterol, with apo A-I, and with the presence of pre-beta apo A-I-containing lipoproteins. These rabbits will be an extremely useful model for the evaluation of the effect of increased hepatic apo A-I expression on atherosclerosis.

Animals↗
PubMed1995

Apolipoprotein A-I conformation in reconstituted discoidal lipoproteins varying in phospholipid and cholesterol content.

The effects of the size and cholesterol content on the conformation of apolipoprotein A-I (apoA-I) have been studied in reconstituted discoidal lipoproteins containing two apoA-I per particle (Lp2A-I). The immunoreactivity of a series of 13 epitopes distributed along the apoA-I sequence has been evaluated in Lp2A-I with a phospholipid/apoA-I molar ratio ranging from 31 to 156 and in Lp2A-I with constant phospholipids but varying in cholesterol content from 0 to 22 molecules. The results are compatible with a three domain structure in apoA-I in which the central domain is located between residues 99 and 143 and postulated to be a hinged domain that responds differentially to changes in phospholipid and cholesterol contents. Increasing the phospholipid content results in significant changes of epitope immunoreactivity throughout the N-terminal and central domains of apoA-I with fewer modifications in the C-terminal domain. In contrast, increasing Lp2A-I of two central epitopes, A11 (residues 99-132) and 5F6 (residues 118-148), and an extreme N-terminal epitope, 4H1 (residues 2-8). Interestingly, the effects of increasing cholesterol or phospholipids on these epitopes are opposite. This suggests a specific effect of cholesterol on the central domain tertiary structure between residues 99 and 143. Competition binding assays among pairs of antibodies binding to apoA-I on Lp2A-I are best explained by invoking inter- as well as intramolecular competitions. The specificity of the intermolecular competitions suggests an N to C termini arrangement of the two apoA-I molecules around the disc. Increasing the phospholipid content of Lp2A-I mainly increases the competitions between 3G10 and antibodies binding to most adjacent epitopes. Simultaneously as Lp2A-I enlarges, several of these antibodies also enhance the binding of 3G10. This has been interpreted as evidence of a structural rearrangement of apoA-I as a result of the size increase where the alpha-helix (residues 99-121) that contains the 3G10 epitope is increasingly interacting with lipids resulting in the enhanced expression of this epitope. The increasing interactions of apoA-I helices with lipids in the enlarging disc are compatible with previous reports of a greater apoA-I stability in the large discs. By contrast, cholesterol has limited but specific effects on antibody competitions and decreases the interaction of the N-terminal domain with the domain containing 3G10, either by direct cholesterol protein interaction or by modification of the lipid phase packing.

Antibodies, Monoclonal↗
PubMed1995

Modulation of cholesterol efflux from Fu5AH hepatoma cells by the apolipoprotein content of high density lipoprotein particles. Particles containing various proportions of apolipoproteins A-I and A-II.

The influence of apolipoproteins (apo) A-I and A-II on the ability of high density lipoproteins (HDL) to remove cholesterol from cultured Fu5AH rat hepatoma cells was studied independently on alterations in the overall structure and lipid composition of the lipoprotein particles. To this end, apoA-I was progressively replaced by apoA-II in ultracentrifugally isolated HDL3 without inducing changes in other remaining lipoprotein components. As apoA-II was progressively substituted for apoA-I in HDL3 (A-II:A-I+A-II percentage mass: 29.5, 47.6, 71.5, 97.4, and 98.9%), the rate of cholesterol efflux from Fu5AH hepatoma gradually and significantly decreased after 2 or 4 h of incubation at 37 degrees C (cholesterol efflux: 30.4 +/- 0.8, 24.1 +/- 1.0, 19.8 +/- 1.2, 15.7 +/- 1.4, and 13.4 +/- 1.3%/2h, respectively; 38.4 +/- 1.5, 29.2 +/- 0.9, 27.0 +/- 0.2, 20.4 +/- 0.4, and 17.5 +/- 1.0%/4h, respectively) (p < 0.01 with all A-II-enriched HDL3 fractions as compared with non-enriched homologues). In agreement with data obtained with total HDL3, increasing the A-II:A-I+A-II percentage mass in HDL3 particles containing initially only apoA-I (HDL3-A-I) progressively reduced cellular cholesterol efflux. After 2 h of incubation, cholesterol efflux correlated negatively with A-II:A-I+A-II percentage mass (r = -0.86; p < 0.0001; n = 20), but not with either free cholesterol:phospholipid ratio, A-I+A-II:total lipid ratio or mean size of HDL3. As determined by using Spearman rank correlation analysis, the A-II:A-I+A-II% mass ratio correlated negatively with the apparent maximal efflux (Vmax(efflux)) (rho = -0.68; p < 0.05, n = 10), but not with the HDL3 concentration required to obtain 50% of maximal efflux (Km(efflux)) (rho = -0.08; not significant, n = 10). It was concluded that the apoA-I and apoA-II content of HDL3 is one determinant of its ability to promote cholesterol efflux from Fu5AH rat hepatoma cells.

Animals↗
PubMed1995

Tissue-specific expression of the human gene for lecithin: cholesterol acyltransferase in transgenic mice alters blood lipids, lipoproteins and lipases towards a less atherogenic profile.

Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme in the reverse cholesterol pathway but its role in lipid metabolism is still unclear. We have generated mice transgenic for a 7-kb genomic DNA fragment comprising the 6 exons and 5 introns of the LCAT gene with 1932 bp of 5' flanking and 908 bp of 3' flanking sequences. One line had integrated about 30 copies and expressed about 40-fold increased LCAT activity in a human test system. The expression showed correct tissue specificity of the human LCAT gene. Increased LCAT activity resulted in a decrease of plasma triacylglycerols below 50% of fasting controls. This reduction was seen in all lipoprotein fractions. Lipoprotein lipase activity did not change significantly, whereas hepatic triacylglycerol lipase increased markedly. Plasma total cholesterol was similar in fasting transgenic and control mice, but low-density lipoprotein and very low-density lipoprotein cholesterol were reduced to about 50%. High-density lipoprotein cholesterol increased about 20%, accompanied by a correspondingly increased size and a higher cholesterol efflux-stimulating activity of transgenic LCAT high-density lipoprotein. Both apolipoprotein A-I and A-II plasma concentrations increased in transgenic mice. Plasma triacylglycerol and cholesteryl ester fatty acid distribution showed an increased proportion of palmitic acid, whereas oleic, linoleic and arachidonic acid decreased, thus resembling more closely the human situation. Overexpression of the human LCAT gene provokes major changes in plasma lipoprotein and apolipoprotein concentrations, resulting in a less atherogenic plasma lipoprotein profile through a reduction in atherogenic and an increase in anti-atherogenic lipoproteins.

Animals↗
PubMed1995

Lipoproteins containing apolipoprotein B isolated from patients with abetalipoproteinemia and homozygous hypobetalipoproteinemia: identification and characterization.

Abetalipoproteinemia (ABL) and homozygous hypobetalipoproteinemia (HBL) are inherited disorders which are classically characterized by progressive retinal and spinocerebellar disease, fat-soluble vitamin deficiency, and absence of apolipoprotein (apo) B from the plasma. Using immunoaffinity chromatography with an anti-apo B antiserum, we isolated apo B-containing lipoprotein (LpB) particles from the plasma of 4 ABL and 2 HBL patients. The LpB particles were characterized and compared with low density lipoprotein (LDL) and LpB isolated from normal plasma. The ABL/HBL LpB particles were similar in size and charge to normal LpB particles but were relatively enriched in several other apolipoproteins. They contained alpha-tocopherol in a ratio to cholesterol that was proportionately much higher than the very low ratio of alpha-tocopherol to cholesterol in plasma. They bound saturably to fibroblasts and were internalized and degraded similarly to LDL. Hence, the molecular defects in ABL and HBL permit the secretion of a very small number of apo B-containing lipoproteins which may be important for transport of alpha-tocopherol to peripheral tissues.

Abetalipoproteinemia↗
PubMed1995

Abnormal reverse cholesterol transport in controlled type II diabetic patients. Studies on fasting and postprandial LpA-I particles.

The high incidence and prevalence of coronary heart disease in diabetes mellitus is clearly established. The usual lipid pattern found in type II diabetic patients is a moderate increase in fasting triglyceride levels associated with low HDL cholesterol levels. These abnormalities are further amplified in the postprandial state. To study the effect of these alterations on reverse cholesterol transport, we isolated lipoprotein containing apoA-I but not apoA-II (LpA-I) particles by immunoaffinity chromatography from the plasma of well-controlled type II diabetic patients and nondiabetic matched control subjects. Different parameters involved in this antiatherogenic pathway were measured in both fasting and postprandial states. Diabetic patients had reduced levels of LpA-I particles that were protein enriched and phospholipid depleted. Gradient gel electrophoresis showed that control LpA-I particles had five distinct populations, whereas diabetic particles lacked the largest one. LpA-I isolated from diabetic plasma exhibited a decreased capacity to induce cholesterol efflux from Ob 1771 adipose cells both in fasting (15.1 +/- 10.0% versus 7.5 +/- 2.7%, P < .05) and postprandial (17.7 +/- 11.2% versus 7.7 +/- 3.9%, P < .05) states, whereas only control particles showed significantly higher ability to promote cholesterol efflux after the test meal (P = .02). Lecithin:cholesterol acyltransferase activity measured with an exogenous substrate showed a 54% increase and an 18% decrease postprandially for control subjects and patients, respectively. Thus, the different abnormalities found in the fasting state were further amplified in the postprandial situation. This resulted in LpA-I particles with aberrant size and composition and decreased ability to accomplish their antiatherogenic role in type II diabetic patients.

Adipose Tissue↗
PubMed1994

Lipoproteins containing apolipoprotein A-IV: composition and relation to cholesterol esterification.

In order to investigate the relationship of lipid and apolipoprotein composition to cholesterol esterification in lipoproteins containing apolipoprotein (apo) A-IV, apo A-containing lipoprotein particles were isolated from fresh human plasma using a system of sequential immunoaffinity chromatography. Plasma was first depleted of apo B- and apo E-containing lipoproteins. Four major subpopulations of apo A-containing lipoprotein particles were separated: Lp A-I, Lp A-I: A-II, Lp A-IV and Lp A-I: A-IV: A-II. Lp A-IV and Lp A-I: A-IV: A-II contained less total lipid, less cholesterol and more triacylglycerol than Lp A-I and Lp A-I: A-II. Lp A-IV and Lp A-I: A-IV: A-II contained more sphingomyelin and less phosphatidylcholine than Lp A-I and Lp A-I: A-II and were richer in (16:0 + 18:0) saturated fatty acids. Among these isolated lipoprotein particles, Lp A-IV contained the highest lecithin: cholesterol acyltransferase (LCAT) activity per micrograms of protein. Cholesterol esterification rates were 2.6 +/- 0.5, 5.3 +/- 0.4 and 0.8 +/- 0.2 mumol of cholesterol per hour per mg of lipoproteins for Lp A-IV, Lp A-I and Lp A-I: A-II, respectively. The apolipoprotein and lipid composition and LCAT activity of Lp A-IV suggest that this lipoprotein may be a source of cholesterol esterification in plasma.

Adult↗
PubMed1994

Intestinal anaphylaxis: radiation-induced suppression.

The gastrointestinal tract is highly sensitive to ionizing radiation. Some of the most radiosensitive cells in this system are mast cells and epithelium. This article describes experiments that test the hypothesis that irradiation suppresses mucosal immune responses in which mast cells and epithelium are involved. The hypothesis was tested by examining the impact of ionizing radiation on anaphylactically mediated Cl- secretion in jejunum of rats sensitized by Trichinella spiralis infection and challenged with antigen derived from the parasite. Antigen-induced Cl- secretion was measured electrophysiologically in vitro. Rats were immunized by inoculation with 3 x 10(3) T. spiralis larvae and, 30-50 days later, exposed to total abdominal irradiation from a cobalt-60 gamma source. Doses were 5, 7, and 9 Gy. At 1, 3, 5, 7, 14, and 21 days postirradiation (DPI), jejunal segments were assessed for immune responsiveness. (Duration of suppression to antigenic challenge was directly related to radiation dose). Recovery of response to antigenic challenge after irradiation with 5 Gy was complete by 5 DPI. At 7 Gy, responsiveness was totally suppressed from 1 to 5 DPI, was partially expressed from 5 to 14 DPI, and was completely restored by 21 DPI. A dose of 9 Gy completely suppressed immune responsiveness throughout the 21-day period. Full responsiveness of jejunum to exogenous Cl- secretagogues at 1-5 DPI indicates that the immunosuppressive effect of radiation was not due to a breakdown in the secretory process at the epithelium level.(ABSTRACT TRUNCATED AT 250 WORDS)

Anaphylaxis↗
PubMed1994

[Hemodynamic effects of propafenone in dogs anesthesized with halothane or isoflurane].

OBJECTIVE: Several studies suggest that anaesthetic drugs influence the haemodynamic effects of the antiarrhythmic drugs. The aim of this study was to compare the haemodynamic effects of a standard dose of propafenone (1.5 mg/kg) on dogs anaesthetized with halothane or with isoflurane. DESIGN: Randomized laboratory animal study. MATERIAL AND METHODS: Six dogs were anaesthetized with 1% of halothane-Group I, and six dogs with an equianaesthetic dosage of isoflurane (1.5%)-Group II. Dogs breathed in spontaneous ventilation. Haemodynamic monitoring was performed with a femural arterial catheter and a flow-directed pulmonary artery catheter and cardiac output was measured by thermodilution. After a haemodynamic assessment considered as initial a bolus of 1.5 mg/kg of propafenone was given during a period of 30 seconds and similar assessments were made 5, 15, 30 and 60 minutes after. RESULTS: Before propafenone, heart rate was significantly higher in Group II-isoflurane (p < 0.05). After propafenone we found: In both Groups, a decrease in the cardiac output (p < 0.05) with the mean arterial pressure maintained; in Group I (halothane) a decrease in the stroke volume (p < 0.05) which was not found in Group II (p = ns); In Group II (isoflurane) a decrease in heart rate (p < 0.05) not found in Group I (p = ns). All the changes were higher at the 5th minute values. 30th minute and 60th minute values were not significantly different from initial values. CONCLUSION: In dogs anaesthetized with halothane 1.2 MAC a reduction in the stroke volume, resulting in a cardiac output decrease, was observed, suggesting that propafenone increases the negative inotropic action of halothane. In dogs anaesthetized with isoflurane 1.2 MAC the decrease in cardiac output was similar to the decrease in heart rate, and therefore no reduction in the stroke volume was observed. The decrease in the heart rate found in this group but not in the halothane group was probably related with the beta-blocker action of the propafenone. Looking to the systemic vascular resistances, our study suggested that propafenone didn't have any vasodilator effect during halogenated anaesthesia.

Anesthesia↗
PubMed1994

Transfer of cholesterol from Ob1771 cells or LDL to reconstituted, defined high density lipoproteins.

We used defined, reconstituted high density lipoproteins (rHDL) to study the effects of structure and composition of these particles on their role as cholesterol acceptors from cell membranes or from low density lipoproteins (LDL). Three discoidal rHDL and one spherical rHDL with distinct apolipoprotein A-I conformations, diameters and compositions were used in conjunction with Ob1771 cells to measure the rate of [3H]cholesterol efflux from the cells, direct binding to the cells, and competition with native HDL3 for binding. In addition, the same rHDL particles were used to study the kinetics of cholesterol mass transfer from LDL. The results show that the rates of cholesterol transfer depend on the nature of the donor (t1/2 11-19 min from LDL, and t1/2 5 h from the cells), on the phosphatidylcholine/cholesterol ratio in the acceptors (the closer this ratio is to the equilibrium value, the slower is the rate), and on the diameter of the acceptors (the smallest particles have the lowest t1/2 for cholesterol uptake from LDL, and are the most effective acceptors of [3H]cholesterol from cells after their phospholipid content is taken into account). The cholesterol uptake by the rHDL, both from the cells and from LDL, is determined mostly by the phospholipid pool available in the acceptors. Binding to the cells was equivalent for all the rHDL (Kd = 38-67 micrograms/ml) and comparable to HDL3, suggesting that the differences in apoA-I conformation have no effect on the binding to cells. Finally we observed that exposure of rHDL to cells may lead to remodeling of some of the lipoprotein particles.

Adipocytes↗
PubMed1993

Biochemical characterization of the three major subclasses of lipoprotein A-I preparatively isolated from human plasma.

Apolipoprotein (apo) A-I is the major protein constituent of plasma high-density lipoproteins (HDL). HDL consist of two major classes of apoA-I-containing lipoproteins: LpA-I and LpA-I:A-II. LpA-I includes heterogeneous lipoprotein particles that differ in size and hydrated density. LpA-I was isolated by immunoaffinity chromatography from the fasting plasma of 24 normal human subjects and separated by gel filtration chromatography. Three major subclasses of LpA-I were eluted: large (Lg-LpA-I), medium (Md-LpA-I), and small LpA-I (Sm-LpA-I). By nondenaturing gradient PAGE, Lg-LpA-I, Md-LpA-I, and Sm-LpA-I had mean Strokes diameters of 10.8 +/- 0.5, 8.9 +/- 0.5, and 7.5 +/- 0.3 nm, respectively. The lipid/protein ratios were 1.25 +/- 0.12 for Lg-LpA-I, 0.75 +/- 0.10 for Md-LpA-I, and 0.38 +/- 0.08 for Sm-LpA-I. Lg-LpA-I was relatively lipid and cholesteryl ester rich compared with Md-LpA-I and Sm-LpA-I. Sm-LpA-I contained phospholipids as the major lipid component. ApoA-I was the major apolipoprotein in all LpA-I subfractions, whereas apoE was present only in Lg-LpA-I and apoA-IV was associated with both Md-LpA-I and Sm-LpA-I. All three LpA-I subclasses exhibited predominantly alpha mobility on agarose electrophoresis. Lg-LpA-I migrated as a diffuse band in the fast alpha position, whereas Md-LpA-I and Sm-LpA-I migrated to the slow alpha position.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Interaction of reconstituted high density lipoprotein discs containing human apolipoprotein A-I (ApoA-I) variants with murine adipocytes and macrophages. Evidence for reduced cholesterol efflux promotion by apoA-I(Pro165-->Arg).

Interaction of cells with both native and reconstituted high density lipoproteins (rHDL) containing apolipoprotein (apo) A-I mediates efflux of cellular cholesterol. To characterize structural requirements for this activity in apoA-I, six different genetic apoA-I variants and the corresponding normal allele products were isolated from heterozygous carriers, reconstituted with dimyristoylphosphatidylcholine (DMPC) into discoidal HDL and compared with regard to their ability to release intracellular cholesterol from murine adipocytes and peritoneal macrophages. In previous studies we determined the amino acid changes of these variants: Pro3-->Arg, Pro4-->Arg, Lys107-->0, Lys107-->Met, Pro165-->Arg, and Glu198-->Lys (von Eckardstein, A., Funke, H., Walter, M., Altland, K., Benninghoven, A., and Assmann, G. (1990) J. Biol. Chem. 265, 8610-8617) and demonstrated that all apoA-I variants except apoA-I(Lys107-->0) form discoidal HDL particles with phosphatidylcholine analogues indistinguishable from normal apoA-I (Jonas, A., von Eckardstein, A., Kezdy, K. E., Steinmetz, A., and Assmann, G. (1991) J. Lipid Res. 32, 95-106). In the present study, all apoA-I.DMPC complexes except those containing apoA-I(Pro165-->Arg) promoted cholesterol efflux as effectively as those containing normal apoA-I. Cholesterol efflux from adipocytes obtained after 180 min of incubation with apoA-I(Pro165-->Arg).DMPC complexes was decreased by 30% although this variant was bound to adipocytes with normal affinity. By contrast, apoA-I(Lys107-->Met).DMPC complexes were decreased in their binding affinity compared to normal apoA-I.DMPC complexes but normally promoted cholesterol efflux. Incubation of mouse peritoneal macrophages with apoA-I(Pro165-->Arg).DMPC complexes did also result in a 30% decreased efflux of radiolabeled cholesterol if compared to rHDL with the normal allele product from the same donor. Together the observations suggest that the substitution of proline at residue 165 interferes with the formation of a structural domain in apoA-I that is crucial for cellular cholesterol efflux stimulation. Furthermore, our results suggest that binding of HDL to adipocytes and cholesterol efflux promotion by HDL requires different structural domains.

Adipose Tissue↗
PubMed1993

Aneurysmal bone cyst versus hemangioma of the mandible. Report of a long-term follow-up of a self-limiting case.

The aneurysmal bone cyst is an enigmatic and misnamed lesion displaying variable etiopathogenic and histologic characteristics. The aneurysmal bone cyst may appear as a primary lesion or as a secondary lesion associated with other pathologic entities. This article presents a probable self-limiting lesion that provides an opportunity to present an approach to classifying aneurysmal bone lesions.

Aged↗
PubMed1993

Characterization of apoA-IV-containing lipoprotein particles isolated from human plasma and interstitial fluid.

Apolipoprotein (apo) A-IV has been proposed to play a role in reverse cholesterol transport. ApoA-IV-containing lipoprotein particles (A-IVLp) were isolated from human plasma and interstitial fluid and characterized by immunoaffinity chromatography. Two major A-IVLp subpopulations, lipoprotein particles containing apoA-IV with apoA-I (LpA-I:A-IV) and lipoprotein particles containing apoA-IV without apoA-I (LpA-IV), were identified. The larger subpopulation of A-IVLp is the LpA-IV that represents 70% (protein mass) of the initial particles. Only 5.8% of apoA-IV was recovered in the retained fraction after affinity chromatography with an anti-apoA-I immunosorbent. ApoA-I, apoA-II, apoA-IV, apoB, apoC-III, apoD, apoE, apoH, lecithin: cholesterol acyltransferase (LCAT), cholesteryl ester transfer (CET) protein, proline-rich protein, and a protein of Mr 59,000 were detected in the A-IVLp. These particles contain more than 20% triglycerides (lipid mass). ApoA-IV-containing particles that were isolated from plasma are heterogeneous in size, consisting of two major populations with Stokes' diameters of 10.3 nm and 9.3 nm. Both subpopulations of A-IVLp contain LCAT and CET activities and promote cholesterol efflux from cholesterol-preloaded adipose cells. These data support the hypothesis that A-IVLp particles may be involved in reverse cholesterol transport.

Adipose Tissue↗
PubMed1992

Apolipoprotein A-I-containing particles and reverse cholesterol transport in IDDM.

Lipoprotein particles containing apoA-I but not apoA-II are, among high-density lipoproteins, effective protectors against atherosclerosis that act by promoting the efflux of cellular cholesterol and the reverse cholesterol transport process. Because previous studies showed that in vitro nonenzymatic glycosylation of HDL impairs HDL receptor-mediated cholesterol efflux, we isolated Lp A-I from two poorly controlled insulin-dependent diabetic patients and compared the chemical composition and ability to promote cholesterol efflux with the same particles purified from two matched nondiabetic control subjects. No differences in lipid composition or in the ability to promote cholesterol efflux from cultured adipose cells were noted between the two types of Lp A-I preparations. However, when we separated Lp A-I from diabetic subjects by degree of glycosylation, the specifically glycosylated subfractions were about 50% less effective in producing cholesterol efflux than the nonglycosylated particles.

Adult↗
PubMed1992

Thrombin cleavage of apolipoprotein Bh of rabbit LDL: structural comparisons with human apolipoprotein B-100.

Rabbit plasma low density lipoprotein (LDL) contains one major apolipoprotein of apparent molecular weight of 320 kDa, designated apolipoprotein (apo) Bh, while another component termed apoB1 of apparent molecular weight of 220 kDa is found in chylomicrons. The fragments generated by thrombin digestion of the protein moieties of rabbit and human LDL were separated by polyacrylamide gradient gel electrophoresis and compared. As in the human species, the enzyme produced limited cleavage patterns of rabbit LDL apoB. Within the first 2 h, two fragments (Tr1 and Tr2, with apparent molecular weights 280,000 and 44,000, respectively) appeared. Longer incubations led to the production of two additional peptides, Tr3 and Tr4 (apparent molecular weights 180,000 and 96,000, respectively). Ten monoclonal antibodies, developed against rabbit LDL and designated P01 to P10, were found to react with rabbit apoB. Some also cross-reacted with human apoB. Epitope mapping, performed with these antibodies, showed that Tr3 and Tr4 were derived from the further degradation of Tr1. The rabbit is one of the most frequently used animals in atherosclerosis research. Its LDL receptor has been characterized and there exists a strain of homozygous LDL receptor-deficient rabbits referred to as WHHL rabbits. Despite this, little has been done to characterize the structure of rabbit apoB; only a short region has been sequenced and shown to be the carboxyl-terminal region, the rabbit apoB1. The molecular weight of human apoB (550,000) is much larger than rabbit apoBh. In both species, a primary and secondary thrombin cleavage occur, but the size of the fragments produced is very different between the two species. Identification of the thrombolytic fragments of the rabbit apoB have afforded the opportunity to compare the structures of both apoB species.

Animals↗