Radioimmunoassay of rat apolipoprotein B peptides in lipoproteins and tissues

Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) separates rat apolipoprotein B (apoB) into one lower and two higher molecular weight components. Of the latter, peptide I (PI) corresponds to human B-100, while the slightly faster-migrating peptide II (PII) lacks a human counterpart; the smaller species peptide III (PIII) corresponds to human B-48. We describe here a competitive radioimmunoassay which separately measures the amounts of total (i.e., PI + PII + PIII) and larger (i.e., PI + PII) rat apoB peptides, with the amounts of PIII obtained by difference. Standard rat PIII and combined PI + PII (PI,II) were isolated by high-pressure gel filtration liquid chromatography in the presence of SDS, and the PI,II was used as an immunogen to raise rabbit antisera which were capable of binding all three forms of rat apoB. However, Scatchard analysis showed this binding to represent two distinct types of antibodies: one high-affinity class which bound only PI,II and a second class which bound all apoB peptides with equal but lower affinity. Thus, since 125I-labeled PIII was displaced equally effectively by PI,II and PIII, but 125I-labeled PI,II was displaced only by PI,II, the unabsorbed antiserum could be used to measure either total apoB or PI,II alone, depending on the choice of labeled ligand. The validity of the assay for apoB peptides in very-low-density and low-density lipoproteins and in liver microsomes was verified by comparison with peptide determinations by SDS-PAGE.     

Rat McA-RH7777 cells efficiently assemble rat apolipoprotein B-48 or larger fragments into VLDL but not human apolipoprotein B of any size

Studies of truncated apoB peptides in human subjects with familial hypobetalipoproteinemia, as well as of puromycin-generated spectra of nascent apoB peptides in rat and hamster liver, suggest that a minimum size is required for N-terminal fragments of apoB to be efficiently assembled into full-sized VLDL. We report here results of experiments undertaken to examine this phenomenon in greater detail by expressing individual carboxyl-truncated human apoB constructs in McArdle cells. Thus, apoB-29, -32, -37, -42, -47, -53, -70 and full length apoB-100 were transiently expressed in rat McA-RH7777 hepatoma cells, or human apoB-31 and apoB-53 were stably expressed in the same cells, and the secreted VLDL particles were characterized by kinetic gradient ultracentrifugal flotation. Calibration with rat plasma VLDL subfractions showed that about 90 and 50%, respectively, of lipoprotein particles containing endogenous rat B-100 and B-48 floated between fractions 2;-8 of the 11-fraction gradient. This corresponds to the normal VLDL diameter range of about 47 to 28 nm, with the remaining half of rat B-48 recovered as HDL particles in the 1.1 g/ml range. In contrast, regardless of their size, only 2;-5% of any of the truncated human apoB peptides expressed in these cells was recovered in the VLDL region of the gradient. The remaining 95+% of the lipoproteins were found as high density particles; as previously found in other systems the densities of the latter were inversely related to their peptide chain-length. Furthermore, transiently expressed full-length human apoB-100 was inefficiently secreted as VLDL by these cells, with the remainder appearing as LDL-sized particles. Thus, although we showed that McA-RH7777 cells secreted endogenous rat apoB as normal-sized VLDL, we found them unsuitable for our original purpose of using human apoB fragments to further define effects of apoB size on VLDL assembly. These cells appeared unable to efficiently use any size of human apoB for that process. Pulse-labeled untransfected McA-RH7777 cells chased in the presence of puromycin did, however, show a sharp decline in VLDL assembly efficiency for endogenous nascent rat apoB peptides shorter than B-48, similar to that originally found in normal rat liver.     

Regulated biosynthesis and divergent metabolism of three forms of hepatic apolipoprotein B in the rat

Studies using rat livers perfused with recycled, serum-containing medium plus [3H]leucine revealed that secreted VLDL contain three forms of apolipoprotein B (apoB), B-48, B-95, and B-100, all synthesized by the liver. The B-48/(B-95 + B-100) [3H]leucine incorporation ratio ranged from 0.22 to 3.25 with livers of rats fed different diets, and the ratio was positively correlated with the triglyceride secretion rate in most of the livers. Generally, as more triglyceride was secreted, a greater proportion was packaged with B-48, which is the apoB form most rapidly cleared from the circulation. Together, these findings suggest a mechanism for regulating plasma triglyceride levels. [3H]Leucine incorporation into apoA-I also was positively correlated with the triglyceride secretion rate. Secretion of newly synthesized B-48 was delayed relative to all other apolipoproteins. There was little segregation of any of the three apoB forms into any of five subfractions of secreted VLDL separated on the basis of Sf value; only the smallest VLDL (Sf 20-100) were slightly enriched in B-95 and B-100. Less than 5% of newly synthesized apoB appeared in perfusate LDL. The B-100/B-95 [3H]leucine incorporation ratio was 3.3 with perfused livers of fed rats but only 1.6 in post-surgical, relatively fasted rats in vivo, suggesting physiologic regulation also of the relative amounts of the two large apoBs produced. With recycled serum-free perfusate, as opposed to serum-containing medium, there was hepatic reuptake of nascent VLDL, indicated by the reuptake of newly synthesized apoE and all three forms of apoB, and not other apolipoproteins. Divergent metabolism of B-100 and B-95 in the rat was evident from the following results: a) B-95 disappeared more rapidly from recycled, serum-free liver perfusate; b) B-100 disappeared more rapidly from the circulation in vivo; c) plasma lipoprotein fractions of increasing density between d less than 1.019 and d 1.072 g/ml contained increasing proportions of B-95 over B-100. In summary, these results show that hepatic VLDL production in the rat involves the biosynthesis of three forms of apoB, that the relative amounts produced are regulated by physiologic variables, and that there is divergent metabolism of the VLDL particles into which these different apoB forms, either individually or in combination, become incorporated.     

Plasma very low density lipoproteins contain a single molecule of apolipoprotein B

Rat and human very low density lipoproteins (VLDL) were fractionated by zonal ultracentrifugation, yielding sharply defined fractions with narrow sedimentation limits. Sedimentation coefficients for the individual fractions were determined at two densities with the analytical ultracentrifuge, and the results were analyzed to yield buoyant densities and molecular weights for the particles in each fraction. For the rat lipoproteins, the weight concentrations of triglycerides, cholesterol, phospholipid, and protein were determined for each fraction, and their molar concentrations of apolipoprotein B were measured with a radioimmunoassay. For the human lipoproteins the corresponding values were taken from Patsch et al. (Patsch, W., J. R. Patsch, G. M. Kostner, S. Sailer, and H. Braunsteiner. 1978. Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation. J. Biol. Chem. 253:4911-4915). From these data, a ratio of the number of apoB peptides to the number of lipoprotein particles was calculated for each fraction. This ratio was close to 1 for all VLDL fractions, ranging in particle diameter from about 40 to 80 mm and 30 to 50 mm, respectively, for rat and human VLDL. The majority rat VLDL contain B-48 rather than B-100 as their (single) apoB peptide. Based on these data, we proposed that only a single copy of B-48 is required for VLDL assembly in rat liver, unless nascent hepatic VLDL contain additional apoB peptides which are uniformly lost from the plasma VLDL particles when they are analyzed.     

Differential labeling of rat hepatic Golgi and serum very low density lipoprotein apoprotein B variants

The synthesis of apoB-100 and apoB-48 by rat liver was investigated by studying the apoB complement of very low density lipoproteins (VLDL) from hepatic perfusates and Golgi fractions. The relative amounts of apoB-100 and apoB-48 in perfusate and Golgi VLDL as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis were similar to those in serum VLDL. To investigate the relative rates of synthesis of the VLDL B proteins, rats were injected intraportally with tritiated amino acid, and hepatic Golgi and serum VLDL were isolated from 7.5 to 120 min later. In hepatic Golgi VLDL, apoB-100 and apoE were maximally labeled at 15 min after the tritiated amino acid pulse. In contrast, VLDL apoB-48 attained maximum radioactivity at 30 min after isotope injection. In serum VLDL, apoB-100 and apoE were maximally labeled at 30 min post-isotope injection, while activity in apoB-48 peaked at 60 min. The data suggest that the synthesis of the B proteins and incorporation into rat liver nascent VLDL are independently regulated. The differential labeling patterns of the VLDL B proteins may be explained by an intracellular pool of apoB-48 that is larger than that of apoB-100. An alternative explanation of the results is that apoB-100 is a precursor to apoB-48.     

Expression of apolipoprotein B-100 in isolated human small intestine epithelium.

Apolipoprotein B-48 (apoB48) is synthesized in the small intestine and becomes a component of chylomicrons (CM). Apolipoprotein B-100 (apoB100) is synthesized in liver and becomes a component of both very low density lipoprotein (VLDL) and low density lipoprotein (LDL). To evaluate whether apoB100 is present in the human small intestine, we performed immunohistochemical staining using anti-apoB100 monoclonal antibody (mAb). Jejunal samples stained positive and the granular staining was noted in the supranuclear region of epithelial cells. We also identified apoB100 expression in the epithelial cells by immunoblotting and dot-blotting of PCR-amplified cDNA. In order to exclude submucosal stroma contaminated with blood, we used isolated epithelium from human small intestine obtained by a crypt isolation technique. The results indicate that not only apoB48, but also apoB100 are expressed in human small intestine epithelium. The expression of apoB100 suggests that the dietary VLDL may be synthesized in human small intestine epithelium and converted into LDL, which might play an important role in atherosclerosis.     

The disappearance rate of human versus rat intermediate density lipoproteins from rat liver perfusion.

The aim of this work was to compare the disappearance rate of human and rat intermediate density lipoproteins (IDL) using the rat liver perfusion system. Human and rat IDL were produced in vitro by incubating human or rat very low density lipoproteins (VLDL) with either rat post-heparin plasma (method I) or a resolubilized isopropanol precipitate of rat post-heparin plasma (method II). With both methods, the degree of triacylglycerol lipolysis was approximately 55%. The different preparations of IDL were labelled with 125I and added to perfusates of rat livers. The disappearance rates of 125I-labelled IDL were monitored by measuring the radioactivity associated with apolipoprotein (apo) B in the perfusate during a 15-min period. Both human and rat IDL prepared with method I had an increased apoE to apoC ratio as compared with their native counterparts. Furthermore, human IDL had a significantly higher apoE to apoC ratio than rat IDL. However, when IDL were produced in the absence of exchangeable apolipoproteins (method II), no change in the apoE to apoC ratios was observed for the transformation of VLDL to IDL and the ratios were similar for human and rat IDL. Despite these differences, human IDL were always removed at a lower rate than rat IDL. The only striking difference between the two types of IDL made by method II was that the apoB100 to apoB48 ratio was considerably higher in human than in rat IDL. These results suggest that the apoB100 to apoB48 ratio is likely to be responsible for the observed differences in liver uptake between rat and human IDL.     

Microsomal membrane-associated apoB is the direct precursor of secreted VLDL in primary cultures of rat hepatocytes

Brefeldin A (BFA) added to primary cultures of rat hepatocytes, at a concentration of 0.2 microg/ml, prevented the assembly of newly synthesized apolipoprotein B (apoB) into mature, secretory VLDL but did not prevent the secretion of apoB as denser particles (HDL apoB), or of albumin. The unassembled apoB remained associated with the membranes of the cellular microsomal fraction. There was no effect of BFA on the removal of apoB from the lumen of these vesicles. VLDL apoB formed only a minor component of the total apoB in the microsomal lumen. Higher (5 microg/ml) concentrations of BFA were required to prevent the secretion of HDL apoB and albumin. Under these conditions apoB accumulated in the microsomal lumen, as well as in the membranes of these vesicles. Again, apoB VLDL formed only a minor proportion of the total lumenal apoB. ApoB-48 VLDL and apoB-100 VLDL assembly could be restored by removing BFA from the medium. This reactivation of VLDL assembly was accompanied by an increased removal of apoB from the microsomal membranes, but there was no detectable increase in the small quantity of VLDL apoB that was recovered from the microsomal lumen. In the absence of BFA, during pulse-chase experiments the pattern of change in the specific radioactivity of microsomal membrane apoB was similar to that of the secreted VLDL apoB whereas that of the lumenal apoB resembled that of the secreted HDL apoB. The results suggest that membrane-associated apoB is the main direct precursor of secreted VLDL apoB in primary cultures of rat hepatocytes and that VLDL assembly does not involve primarily microsomal lumenal apoB as an intermediate.     

Insulin-Stimulated Degradation of Apolipoprotein B100: Roles of Class II Phosphatidylinositol-3-Kinase and Autophagy

Both in humans and animal models, an acute increase in plasma insulin levels, typically following meals, leads to transient depression of hepatic secretion of very low density lipoproteins (VLDL). One contributing mechanism for the decrease in VLDL secretion is enhanced degradation of apolipoprotein B100 (apoB100), which is required for VLDL formation. Unlike the degradation of nascent apoB100, which occurs in the endoplasmic reticulum (ER), insulin-stimulated apoB100 degradation occurs post-ER and is inhibited by pan-phosphatidylinositol (PI)3-kinase inhibitors. It is unclear, however, which of the three classes of PI3-kinases is required for insulin-stimulated apoB100 degradation, as well as the proteolytic machinery underlying this response. Class III PI3-kinase is not activated by insulin, but the other two classes are. By using a class I-specific inhibitor and siRNA to the major class II isoform in liver, we now show that it is class II PI3-kinase that is required for insulin-stimulated apoB100 degradation in primary mouse hepatocytes. Because the insulin-stimulated process resembles other examples of apoB100 post-ER proteolysis mediated by autophagy, we hypothesized that the effects of insulin in autophagy-deficient mouse primary hepatocytes would be attenuated. Indeed, apoB100 degradation in response to insulin was significantly impaired in two types of autophagy-deficient hepatocytes. Together, our data demonstrate that insulin-stimulated apoB100 degradation in the liver requires both class II PI3-kinase activity and autophagy.     

A 40 kilodalton rat liver nuclear protein binds specifically to apolipoprotein B mRNA around the RNA editing site.

Apolipoprotein (apo) B-48 mRNA is the product of RNA editing which consists of a C----U conversion changing a CAA codon encoding Gln-2153 in apoB-100 mRNA to a UAA stop codon in apoB-48 mRNA. In the adult rat, RNA editing occurs both in the small intestine and the liver. We have studied the ability of rat liver nuclear extracts to bind to synthetic apoB mRNA segments spanning the editing site. Using an RNA gel mobility shift assay, we found the sequence-specific binding of a protein(s) to a 65-nucleotide apoB-100 mRNA. UV crosslinking followed by T1 ribonuclease digestion and SDS-polyacrylamide gel electrophoresis demonstrated the formation of a 40 kDa protein-RNA complex when 32P-labeled apoB-100 mRNA was incubated with a rat liver nuclear extract but not with HeLa nuclear extract. Binding was specific for the sense strand of apoB mRNA, and was not demonstrated with single-stranded apoB DNA, or antisense apoB RNA. The complex also failed to form if SDS was present during the UV light exposure. Binding experiments using synthetic apoB mRNAs indicate that the 40 kDa protein would also bind to apoB-48 mRNA but not apoA-I, apoA-IV, apoC-II or apoE mRNA. Experiments using deletion mutants of apoB-100 mRNA indicate efficient binding of wildtype 65-nucleotide (W65), 40-nucleotide (W40) and 26-nucleotide (W26) apoB-100 mRNA segments, but not 10-nucleotide (or smaller) segments of apoB-100 mRNA to the 40 kDa protein. In contrast, two other regions of apoB-100 mRNA, B-5' (bases 1128-3003) and B-3' (bases 11310-11390), failed to bind to the protein. The 40 kDa sequence-specific binding protein in rat liver nuclear extract may play a role in apoB-100 mRNA editing.     

Reduced hepatic release of apoprotein B after enteral nutrition in rats

To investigate the effects of enteral and parenteral alimentation on VLDL release from the liver, a lipid-free liquid nutriment was continuously administered to free-moving rats via the oral cavity (oral group), stomach (enteral group) or superior caval vein (parenteral group). After 1-week of nutrition, the plasma VLDL concentrations were significantly lowered in the enterally-fed group. By immunoblotting assay using a specific antiserum, plasma contents of both apoprotein B-100 and B-48, the major components of rat apoprotein B, were found to be decreased in the enteral group, whereas only that of apoprotein B-48 was reduced in the parenteral group as compared with the oral group. Sucrose gradient centrifugation of the lipid droplets in the liver from the enteral group showed an increase of the free-triacylglycerol fraction with a concomitant increase of the apoprotein B-48-rich triacylglycerol fraction. These results suggest that enteral nutrition causes triacylglycerol accumulation in the liver, at least in part by impairment of lipoprotein release from the liver.     

Modulation of apolipoprotein B-100 mRNA editing: effects on hepatic very low density lipoprotein assembly and intracellular apoB distribution in the rat

We have studied the consequences of alterations to hepatic apoB mRNA editing on the biosynthesis and intracellular distribution of newly synthesized apoB variants together with their mass distribution in nascent Golgi very low density lipoproteins (VLDL). Radiolabeled liver membrane fractions were prepared from control or hypothyroid animals and separated by discontinuous sucrose gradient centrifugation. Hepatic apoB-100 synthesis in these groups accounted for 93-100% of total newly synthesized apoB species of Golgi fractions recovered from the sucrose gradients (G1 and G2). The analogous fractions isolated from the livers of hyperthyroid (treated with 3,3',5-triiodo-L-thyronine, T3) animals revealed that newly synthesized apoB-100 accounted for only 46 +/- 10% (G1) and 24 +/- 11% (G2), respectively, of total newly synthesized apoB. ApoB-100 mass in nascent Golgi VLDL from control and hypothyroid G1 fractions represented 70-78% total apoB as determined by Western blot analysis. By contrast, Golgi VLDL from hyperthyroid animals contained predominantly (greater than 78%) apoB-48 as the apoB species. Electron microscopy revealed that the morphology and size distribution of hyperthyroid G1 VLDL were similar to particles isolated from control animals. Thus, despite a profound reduction in the proportion of apoB-100 mRNA species containing an unmodified codon (CAA, B-GLN) at position 2153 in hyperthyroid animals (6 +/- 1% vs 50-61% in control and hypothyroid animals) apoB-100 biosynthesis was detectable in a defined membrane fraction isolated by discontinuous sucrose gradient centrifugation. However, no apoB-100 synthesis was detectable in liver samples prepared by Polytron disruption in Triton-containing buffers. These data suggest that effective hepatic VLDL assembly and secretion in the T3-treated rat continues despite a profound reduction in apoB-100 biosynthesis and implies that apoB-48 contains the requisite domains to direct this process, a situation analogous to that in the intestine.     

Suppression of cytosolic triacylglycerol recruitment for very low density lipoprotein assembly by inactivation of microsomal triglyceride transfer protein results in a delayed removal of apoB-48 and apoB-100 from microsomal and Golgi membranes of primary rat hepatocytes.

Cellular apoB in primary rat hepatocyte cultures was pulse-labeled with [(35)S]methionine for 1 h. Cells were then chased with excess unlabeled methionine for periods of up to 16 h in the presence or absence of BMS-200150, an inhibitor of microsomal triglyceride transfer protein (MTP). The secretion of apoB-48-VLDL was more sensitive to MTP inhibition than was apoB-100-VLDL. Inhibition of MTP had no inhibitory effect on the secretion of denser particles (apoB-48 HDL and apoB-100 HDL). BMS-200150 delayed the net removal of newly synthesized apoB-48 and apoB-100 from the microsomal and Golgi membranes, but not from the corresponding lumenal compartments. Only minor proportions of the microsomal lumen apoB-48 and apoB-100 (12-16% and 17-19%, respectively) were present as VLDL irrespective of whether MTP was inactivated or not. The HDL fraction contained most of the lumenal apoB-48 (67-73%) and a somewhat smaller proportion of apoB-100 (44-47%). The remainder of the lumenal apoB was associated with the IDL/LDL fraction. These proportions were unaffected by MTP inactivation. Excess labeled apoB which accumulated in the membranes in the presence of BMS-200150 was degraded. Inhibition of MTP prevented the removal of pre-synthesized triacylglycerol (TAG) from the hepatocytes as apoB-VLDL. Under these conditions intracellular TAG accumulated mainly in the cell cytosol, but also, to a lesser extent, in the microsomal membranes. The results suggest that inactivation of MTP inhibits a pathway of VLDL assembly which does not involve the bulk lumenal compartments of the microsomes. Suppression of this pathway ultimately prevents the net transfer of cytosolic TAG into mature apoB-VLDL.     

Manipulation of cholesterol and cholesteryl ester synthesis has multiple effects on the metabolism of apolipoprotein B and the secretion of very-low-density lipoprotein by primary hepatocyte cultures.

Inhibition of esterified and non-esterified cholesterol synthesis by lovastatin in primary rat hepatocytes suppressed the net synthesis and very-low-density lipoprotein (VLDL) secretion of apolipoprotein B (apoB)-48 and apoB-100. Lovastatin did not alter the rates of apoB-48 and apoB-100 post-translational degradation. 25-Hydroxycholesterol, which inhibited non-esterified cholesterol synthesis but increased the synthesis of cholesteryl ester, showed differential effects on the metabolism of apoB-48 and apoB-100. Whereas the secretion of apoB-48 VLDL was suppressed there was no effect on the secretion of apoB-100 VLDL. The post-translational degradation of apoB-48, but not of apoB-100, was enhanced by 25-hydroxycholesterol. The net synthesis rates of apoB-48 and apoB-100 were unaffected by 25-hydroxycholesterol. The inhibitory effect of lovastatin alone on the net synthesis of apoB-48 and apoB-100 was reversed by the simultaneous presence of 25-hydroxycholesterol, suggesting a role for newly synthesised cholesteryl ester. Prevention of the reversal effect by the acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor YM 17E supported this interpretation. In the presence of lovastatin, restoration of the net synthesis of apoB by 25-hydroxycholesterol was not accompanied by an increased VLDL output of apoB-48 and apoB-100. However, under these conditions there was an increased post-translational degradation of apoB-48 and apoB-100. These results suggest that interference with intracellular cholesterol and cholesteryl ester metabolism interrupts VLDL assembly at sites of both apoB net synthesis and post-translational degradation.     

Genotypic associations of the hepatic secretion of VLDL apolipoprotein B-100 in obesity

We examined the effect of genetic polymorphisms of proteins regulating intrahepatic processing of apolipoprotein B-100 (apoB) and the supply of neutral lipids to the liver on the hepatic secretion of very low density lipoprotein (VLDL) apoB in obesity. Hepatic secretion of very low density apolipoprotein B-100 (VLDL apoB) was measured using an infusion of [1-(13)C]leucine in 29 obese men. Isotopic enrichment and turnover of VLDL apoB was determined using gas chromatography-mass spectrometry and multi-compartmental modelling, respectively. Visceral fat was measured by magnetic resonance imaging. Genotypes for the apoB signal peptide (SP27/SP24 alleles), microsomal triglyceride transfer protein promoter (MTP, -493 G/T alleles), apoE (E2, E3, E4 alleles), hepatic lipase promoter (-514 C/T alleles), and cholesteryl ester transfer protein (CETP, Taq1B B1/B2 alleles) were determined using polymerase chain reaction. Statistically significant associations were found between hepatic secretion of apoB and allelic combinations of i) apoB SP with apoE (P = 0.02), hepatic lipase (P = 0.02), and CETP (P = 0. 006) genes, ii) MTP promoter with CETP genes (P = 0.03); the association with apoBSP/MTP promoter allelic combinations just failed to reach significance (P = 0.06), however. The CETP/apoBSP allelic combination was the most significant predictor of apoB secretion, and this was independent of visceral fat, plasma lathosterol and insulin levels, and dietary fat. SP24 carriers who were homozygous for CETP B1 had 60% lower apoB secretion than B2 heterozygotes who were non-carriers of SP24 (10.5 +/- 1.74 mg/kg fat free mass/day, n = 7 vs. 26.1 +/- 3.16, n = 22). The data suggest that variation in both the apoB and CETP genes may be a major genetic determinant of the hepatic secretion of apoB in men with visceral obesity.     

Metabolism of apoB lipoproteins of intestinal and hepatic origin during constant feeding of small amounts of fat

We aimed to identify mechanisms by which apolipoprotein B-48 (apoB-48) could have an atherogenic role by simultaneously studying the metabolism of postprandial apoB-48 and apoB-100 lipoproteins. The kinetics of apoB-48 and apoB-100, each in four density subfractions of VLDL and intermediate density lipoprotein (IDL), were studied by stable isotope labeling in a constantly fed state with half-hourly administration of almond oil in five postmenopausal women. A non-steady-state, multicompartmental model was used. Despite a much lower production rate, VLDL and IDL apoB-48 shared a similar secretion pattern with apoB-100: both were directly secreted into all fractions with similar percentage mass distributions. Fractional catabolic rates (FCRs) of apoB-48 and apoB-100 were similar in VLDL and IDL. We identified a fast turnover compartment of light VLDL that had a residence time of <30 min for apoB-48 and apoB-100. Finally, a high secretion rate of apoB-48 was associated with a slow FCR of VLDL and IDL apoB-100. In conclusion, the intestine secretes a spectrum of apoB lipoproteins, similar to what the liver secretes, albeit with a much lower secretion rate. Once in plasma, intestinal and hepatic triglyceride-rich lipoproteins have similar rates of clearance and participate interactively in similar metabolic pathways, with high apoB-48 production inhibiting the clearance of apoB-100.     

Aphid transmission of cauliflower mosaic virus requires the viral PIII protein

The open reading frame (ORF) III product (PIII) of cauliflower mosaic virus is necessary for the infection cycle but its role is poorly understood. We have used in vitro protein binding ('far Western') assays to demonstrate that PIII interacts with the cauliflower mosaic virus (CaMV) ORF II product (PII), a known aphid transmission factor. Aphid transmission of purified virions of the PII-defective strain CM4-184 was dependent upon added PII, but complementation was efficient only in the presence of PIII, demonstrating the requirement of PIII for transmission. Deletion mutagenesis mapped the interaction domains of PIII and PII to the 30 N-terminal and 61 C-terminal residues of PIII and PII, respectively. A model for interaction between PIII and PII is proposed on the basis of secondary structure predictions. Finally, a direct correlation between the ability of PIII and PII to interact and aphid transmissibility of the virus was demonstrated by using mutagenized PIII proteins. Taken together, these data argue strongly that PIII is a second 'helper' factor required for CaMV transmission by aphids.